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Review article, sustaining protein nutrition through plant-based foods.

• 1 Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources, Pusa, New Delhi, India
• 2 Division of Plant Physiology, Indian Agricultural Research Institute, Pusa, New Delhi, India
• 3 Department of Biotechnology, Jamia Milia Islamia, New Delhi, India
• 4 Dryland Agricultural Research Station, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Srinagar, India

Proteins are essential components of the human diet. Dietary proteins could be derived from animals and plants. Animal protein, although higher in demand, is generally considered less environmentally sustainable. Therefore, a gradual transition from animal- to plant-based protein food may be desirable to maintain environmental stability, ethical reasons, food affordability, greater food safety, fulfilling higher consumer demand, and combating of protein-energy malnutrition. Due to these reasons, plant-based proteins are steadily gaining popularity, and this upward trend is expected to continue for the next few decades. Plant proteins are a good source of many essential amino acids, vital macronutrients, and are sufficient to achieve complete protein nutrition. The main goal of this review is to provide an overview of plant-based protein that helps sustain a better life for humans and the nutritional quality of plant proteins. Therefore, the present review comprehensively explores the nutritional quality of the plant proteins, their cost-effective extraction and processing technologies, impacts on nutrition, different food wastes as an alternative source of plant protein, and their environmental impact. Furthermore, it focuses on the emerging technologies for improving plant proteins' bioavailability, digestibility, and organoleptic properties, and highlights the aforementioned technological challenges for future research work.

Introduction

Since the beginning of life, plants have been utilized for human benefits, providing food, therapeutics, wood, fibers, and many others. Moreover, plants were considered the bioproduction system for valuable substances and provide many primary and secondary metabolites having therapeutic effects. Primary metabolites (protein, carbohydrates, fats, and nucleic acid) are the building blocks of life. Besides these, the secondary metabolites are produced by plants to protect them from predators and pathogens, cope with environmental stress, attract pollinators, and work as their defense system ( 1 ). Proteins are molecules with great complexity and diversity that play an important role in maintaining the structure and function of the living form ( 2 ). Therefore, it is being used for many applications such as medicine, food, and feed.

By 2050, the world's total population is expected to grow or might exceed 9 billion, and, hence, the demand for food, feed, and fiber around the globe is expected to increase by 70% ( 3 ). To meet this increasing demand, new sources must be explored. Nowadays, food derived from plants plays a vital role in the human diet as an important source of bioactive components, such as vitamins, phenolic compounds, or bioactive peptides. Hence, these components benefit human health and protect against various disease conditions ( 4 ). For meeting protein requirements, generally, animals are considered perfect. However, due to many diseases in animals, their consumption is not safer for human health. Also, it replaces animal-based proteins with plant-based proteins due to various limitations, such as increased cost, limited supply of nutrients, hazard for human health, freshwater depletion, and susceptibility to climate change ( 5 – 7 ). Plant-based proteins are considered vegan food, provide an ample number of amino acids, are directly absorbed by the body, and help in treating various disease ailments. Moreover, the proteins derived from plant-based foods are rich in fiber, polyunsaturated fatty acids, oligosaccharides, and carbohydrates. Hence, they are mainly associated with a reduction in cardiovascular diseases, low-density lipoprotein (LDL) cholesterol, obesity, and type II diabetes mellitus ( 8 ). Different sources of plant-based protein that include cereals (wheat, rice, millet, maize, barley, and sorghum), legumes (pea, soybean, bean, faba bean, lupin, chickpea, and cowpea), pseudocereals (buckwheat, quinoa, and amaranth), nuts, almonds, and seeds (flaxseed, chia, pumpkin, sesame, and sunflower) were well-explored ( 5 , 9 – 11 ) ( Figure 1 ). However, the demand for the supply of protein is continuously increasing with the rise of the global population ( 12 ), hence the need to search for new sources.

Figure 1 . Plant-based proteins derived from different crops.

It is hard and expensive to extract an adequate amount of animal proteins; therefore, an alternative for improving the nutritional status of humans is mainly received from plant proteins. Hence, attention has been paid to evaluating the nutritional quality of proteins from different plant species. The best way to increase the supply of proteins is to improve the protein expression and efficiency of protein production in natural resources. The advancement of recombinant technologies of protein production, such as engineering of expression hosts, upstream cultivation optimization (e.g., nutritional, bioreactor design, and physical parameters), and development of methods of protein extraction, as well as purification, supported the growth of the market ( 13 ). Also, improving the protein functionality in foods through modification, enhancing the plant proteins proportion in human diets, and improving the bioavailability and digestibility of food proteins in the digestion process ( 14 , 15 ) could be helpful to increase the overall utilization of plant-based protein.

Along with providing amino acids in food, proteins play a significant role in food formulations due to their diverse properties, such as emulsification, gelling, thickening ability, water holding, foaming, and fat absorption capacity ( 16 , 17 ). Therefore, several thermal techniques (such as cooking, autoclaving, microwave heating, irradiation, germination, fermentation, extrusion, and drying) used during food processing could be optimized to improve the quality of plant proteins ( 2 ). Also, they can be isolated from sustainable and cheap sources such as plant-derived wastes from agriculture and by-products of crop and oil industries, which can also regulate food waste reduction ( 2 , 7 , 18 ).

To provide an overview of plant-based protein that helps sustain a better life for humans and the nutritional quality of plant proteins, this review mainly focuses on the current state of using plants to produce proteins for human health. It mainly focuses on various sources and their alternatives with high-quality protein, factors affecting the nutritional value of plant-based protein, bioactivity and functionality, and its modifications. Also, the information on the nutritional quality of proteins derived from plants and potential health issues linked with plant protein will be elaborated. Finally, the issues and challenges of plant-based proteins from availability, consumption, processing, and functionality will be elaborated, and recommendations were made for sustainable production and better utilization of plant-based proteins for meeting human health requirements.

Plant-Based Sources and Demand of Dietary Protein

Plant-based protein sources.

Among all the existing sources of dietary proteins, plant-based sources dominate the supply of proteins (57%), with the remaining 43% consisting of dairy products (10%), shellfish and fish (6%), meat (18%), and other products from animals (9%) ( 19 ).

To provide dietary protein supply and overcome the challenges of feeding the population, several sources of proteins from plants have been searched recently ( 10 , 20 – 22 ). Based on sources, proteins from plant origin might lack some essential amino acids. For instance, cereals generally contain less lysine, whereas legumes are deficient in sulfur-containing amino acids like cysteine and methionine ( 23 ). However, a good amount of lysine is present in pseudocereals (e.g., quinoa and amaranth). Sometimes, the same plants have different nutrients due to differences in soil diversity, climatic conditions, precipitation levels, geographic latitude and altitude, agricultural practices, and different varieties/cultivars ( 24 , 25 ). Some traditional plants have been utilized by human beings as protein sources, including beans, pea, and soybean. Also, new sources (such as proteins from insects and algae) ( 14 ) and unconventional and alternative protein sources (like agro-industry by-products from the extraction of edible oil and those discarded by fruit processing) have been discovered ( 7 ). In addition, different meat, milk, and egg analogs from plant-based protein sources have also been identified ( Figure 1 ) ( 26 ).

A diet rich in legumes provides various health beneficial effects for humans ( 26 ). Legumes are considered the best dietary options due to their abundant carbohydrates, protein, energy, vitamins, minerals, and fibers. Various commonly known legume crops for protein and other nutritional sources include soybean, common beans, peas, and chickpea. The protein obtained from soybean has been widely studied ( 22 ). Common beans are considered the primary source of vegetable protein in developing countries ( 27 ). Highly nutritious legumes such as peas can be utilized for different food product formulations to improve the human intake of protein. Food products from chickpea are the major dietary protein source of high-quality protein ( 22 ). The protein isolates and defatted flour from lupin fulfill the requirements of essential amino acids ( 28 ). Moreover, pigeon pea and its derived isolates of protein are the potential sources rich in sulfur-containing amino acids suitable for the consumption of human beings ( 29 ).

Cereal consumption, such as wheat, rice, barley, and corn, are the most common staple food throughout the world ( 30 ). Globally, in developed and developing countries, rice is one of the most widely consumed cereal crops. Amagliani et al. ( 30 ) analyzed the amino acid composition of proteins present in the rice and found that lysine content is highest in albumin, while sulfur-containing amino acids are majorly present in the globulin. Some studies have also been conducted to improve rice protein's extracted yield by using different isolation techniques ( 31 ). In one of the studies, it has also been found that lysine is present in significantly less rice protein isolates ( 32 ). Mainly consumed in developing countries, millet, and its concentrates of protein are a mostly nutritious source of proteins. It usually contains a high amount of essential amino acids, including lysine. Nutritional profiles of cereal-based proteins have also been extensively used in industrial applications and bakery products. In a study, faba bean flour, and wheat flour bread products showed an increased amount of essential amino acids after fermentation. The mixture of legumes and cereal helps improve the overall nutritional quality ( 33 ).

Pseudocereals

Pseudocereals like amaranth, buckwheat, and quinoa are mainly the dicotyledonous plants that are considered false cereals ( 34 ). Recently, more interest has been paid to utilize pseudocereals protein, like amaranth and quinoa, to fulfill the high demand for proteins. These sources mainly contain high-quality protein, unsaturated fatty acids, fibers, vitamins, and minerals. They also have a high quality of essential amino acids and increased bioavailability of proteins. Along with these qualities, they are also gluten free, being an alternative in the diet of patients with celiac disease ( 35 ). One of the studies also showed that amaranth and quinoa contain a high quantity of lysine, useful as dietary supplements ( 35 ).

The consumption of plant-derived food components increases continuously, and seeds are an important source that provides good quality of nutrition ( 30 ). Flaxseed, one of the richest sources of high-quality protein, also contains phenolic compounds, fibers, and essential amino acids; however, some studies argued that lysine is limiting in flaxseed ( 36 ). In their study, Lugo et al. ( 37 ) observed that the composition of essential amino acids in chia lacks lysine, whereas the watermelon seeds were found to contain a good amount of leucine and arginine ( 38 ). One of the studies has also been identified that the flour of paprika seed mainly contains aromatic amino acids like threonine, lysine, and tryptophan but poor in sulfur-containing amino acids and isoleucine ( 39 ).

Almond and Nuts

Almonds and nuts are generally known for their high-quality lipid and fatty acids content and also contain high-quality protein content. The species known as pequi and baru from Brazilian Savanna are non-traditional almonds that are good protein sources and have a complete profile of amino acids ( 40 ). Baru almond contains all essential amino acids, whereas pequi almonds are rich in sulfur amino acids and lack lysine, similar to cashew nut ( Anacardiumothonianum ). Peanuts are limited in valine and lysine and are considered as the inferior source of protein.

Meat Analogs From Plant Proteins

Currently, commercial plant-based meat analogs revolutionize the modern food industry. In the US, the market price of plant-based products was ~$940 million in 2019, which will increase by 38% in recent years ( 41 ). Currently, the food industry helps produce high-quality plant-based meat analogs, such as sausages, burgers, ground meat, and nuggets. However, it is more challenging to make the products that match the properties of whole muscle tissues like connective tissue, muscle fibers, and adipose tissue that form hierarchical structures ( 42 ). The arrangement of tissue structure plays a significant role in determining meat products' sensory and physicochemical properties. Plant-based whole muscle products of high quality first require the most suitable ingredients and processing techniques to stimulate muscle fiber, adipose, and connective tissue.

Many reviews have been published on meat analogs from plant proteins ( 41 – 44 ). Ideally, meat analogs should provide adequate structural similarity besides nutrient composition. Meat analogs are mainly produced from plants' macronutrients, including polysaccharides, proteins, and fats, and some micronutrients and other ingredients, such as minerals, vitamins, flavoring, and color agent preservatives, and binders. The components and processing techniques utilized to produce these analogs should be optimized for each meat product. The appearance of the meat analogs' surface should be of opaque texture like real meat. Food industries have used several techniques to maintain the color of plant-based meat alternatives. For instance, Meat™ uses beet juice extract that contains a natural pigment called betalain to recreate the suitable color of meat. Also, Impossible Foods™ uses leg hemoglobin (plant-based heme protein) extracted from soybeans roots to color its products. Various technological and scientific methods, like processing and physicochemical approaches, are being searched to create potential structures of plant-based meat that aim to accurately mimic the texture of real meat. It should also be noted that meat analogs usually simulate the fluid-holding capacity like real meat during cooking. Knowledge about the essential constituents of flavor present in products of real meat is helpful to identify plant-based ingredients that give the meaty flavors in plant-based meat analogs. However, developing plant-based meat analogs is challenging and providing a similar nutritional profile to real meat.

Milk Analogs From Plant Proteins

One of the most consumed food products from plant origin is plant-based milk analogs. Various attributes, such as processing methods, sensory quality, raw materials, physicochemical properties, and nutritional profiles of plant-based milk analogs, have been presented and described in many articles ( 45 , 46 ). Milk analogs are colloidal dispersions consisting of several particles, such as fat droplets, oil bodies, plant tissue fragments, protein aggregates, and insoluble calcium carbonate particles dispersed in an aqueous solution containing soluble proteins, sugars, salts, and polysaccharides ( 46 ). For the formation of high-quality milk analogs, there should be correct information on light scattering theory, techniques of particle reduction, as well as mechanisms of particle instability. Two approaches have been used for producing milk analogs, such as disruption of plant tissue (including soaking, mechanical disruption, enzymatic hydrolysis, separation, formulation, homogenization, and thermal treatment for breaking of plants materials into small particles) and homogenization (including blending of plant-based components that are isolated, such as emulsifiers, oils, and thickeners) ( 46 ). The components and processing techniques are generally optimized for creating milk analogs that mimic cow's milk's functional and desirable properties ( 46 ). For developing a better quality product, the plant-based milk analogs have been extensively analyzed for features, such as appearance, flavor, color, bio-availability, and nutrition profile.

Egg Analogs From Plant Proteins

The hen's egg consists of 75% water, 12% proteins, and 12% lipids. Also, it includes a variety of constituents that help in different food applications like foaming, emulsification, and gelation ( 47 ). Eggs are mainly used in various ways, such as boiled, fried, poached, or scrambled, and part of many other foods, including dressings, mayonnaise, desserts, and baked goods. Generally, plant-based egg analogs should have desirable functional and physicochemical properties. For example, eggs analogs should have the functional ability to transform a solution into a gel when heat is supplied, just like that of real eggs. Plant proteins used in egg analogs have solution temperature in the range of 63–93°C, which shows that higher temperature is needed to mimic the structure and texture of real eggs. Various methods, such as dynamic shear rheometry and differential scanning calorimetry, have been utilized, which provide information on gelation temperatures and denaturation of proteins. The gel nature of plant-based egg analogs depends on the type of protein (e.g., chickpea, pea, sunflower, bean, and soybean), the concentration of protein, and environmental conditions (e.g., pH, ionic strength, and thermal history). Plant-based egg analogs should have the best emulsifying solubility, segregation, separation, and stabilization properties. Like real eggs, they also have a better appearance, flavor, color, bioavailability, and a nutrition profile to produce a better quality of plant-based milk analogs.

Food Waste/By-Products as a Protein Source

Increasing population and industrialization also negatively affected the environmental conditions. Eco-innovation is the term that addresses the essential changes for sustainable development. It is an approach where by-products and waste from plants become an important resource. Food waste/by-products have also been utilized for the extraction of proteins. These mainly include oil meals/press cakes, by-products of cereals, and legume processing.

Oil Meals/Press Cakes

During oil processing, the by-products, such as oil meals/press cakes, have been released from oil-bearing fruits and seeds ( 48 ). Oil meals contain 15–50% of protein content and are, hence, considered valuable sources for the extraction of proteins ( 48 ). Soybean, cottonseed, peanut, sunflower seed, sesame seed, pumpkin seed, hazelnut, grape seed, walnut, hemp seed, and rapeseed are the major oilseed crops containing a high proportion of protein meal. Also, oil-bearing crops, such as coconut, palm, and olive, have oil in their fruit pulp, and their residues are useful to isolate proteins. The protein content varies depending on the processing of hulled and dehulled meals of oilseed. Usually, the dehulled meals have higher protein content and lower fiber content, while dehulled meals require an additional fractionation step before they have been used for protein extraction.

By-Products of Cereal and Legume Processing

By-products after cereal and legume processing are important raw materials for the extraction and isolation of proteins. The high content of protein in legumes makes them most important, followed by cereals. Rice bran is the most important protein source among cereals. Along with the rice, several other crops by-products have been used as promising protein sources, such as wheat bran, broken rice, brewer's spent grains, and defatted wheat germ. Commercial milling of pulses also produces ~25% of by-products consisting of powder, husk, broken, shriveled, and unprocessed seeds. With high nutritional value and a well-balanced profile of amino acids and also various bioactivities, the cereal crops and their by-products are of major attention. Hence, these are considered as appropriate materials for protein extraction due to their quantities, availability, and composition of amino acids ( 30 ).

Demand of Dietary Protein

Proteins are molecules with great complexity and diversity that have played an important role in maintaining the structure and function of living cells ( 29 , 49 ). It is being applied in a number of applications, such as medicine, nutraceuticals, industries, food, feed, etc., and the demand for protein is continuously increasing with the rise of the global population ( 12 ). Globally, protein requirements are fulfilled by both plants (80%) (such as cereal grains, beans, soy, pulses, nuts, vegetables, and fruits) and animals (~20%) (such as meats, milk, eggs, fish, yogurt, and cheese) ( 50 ). Along with the increasing nutritious food demand, the protein demand is continuously increasing globally by changing socioeconomic status. Increased urbanization, as well as economic development, has led to various transitions in dietary patterns in the population of low- and middle-income countries, especially the demand for foods derived from animals, which is noticed in developing countries ( 51 ). Protein from animal origin causes emissions of greenhouse gases from livestock as well as loss of terrestrial biodiversity by human interventions ( 52 ). Therefore, plant-based protein requirements are continuously increasing.

Plant-based proteins play a major role in the human diet as they are rich in a large number of other nutrients, vitamins, and minerals ( 53 ). Foods obtained from plants enhance the content of protein that contains various essential amino acids and may also improve the nutritional status of human diets. From the last few decades, interest has been drawn for the search of protein sources with high nutritional quality and functionality and industrial applications (like emulsification, solubility, gelation, foaming, viscosity, oilholding, and water-holding capacities). Furthermore, the development and utilization of novel techniques of food processing enhance the nutritional quality of traditional sources of plant protein. According to the overall status of health, human nutrition is considered an important issue that provides the methods for prevention or development of a number of diseases resulting from excessive, unbalanced, or insufficient nutrient intake ( 15 ). Generally, the daily intake of protein is provided by animal-based foods. However, changes in the consumers' requirement led to adoption of alternative sources of proteins for human consumption. And, also, the protein produced from animal sources is costly and environmentally non-sustainable and requires more water (about 100 times) during production than plant protein. Emerging factors in animal proteins, like the growth of world population, climate change, and occurrence of animal diseases, more research is now dedicated to finding various new sources and technologies to produce proteins from plants with high content and resilient to changing climate and thus provide balanced nutrition in humans' diet ( 51 ).

The proteins and their amino acid composition play a major role in human health. For instance, sulfur-containing amino acids, such as methionine and cysteine, play a vital role in maintaining the immune system functioning ( 54 ) and also the peroxidative protection mechanism in muscles, nervous, and cardiovascular systems ( 55 ). Lysine is important for bones calcification, liver activities, nitrogen balance inside the body, and muscle and blood synthesis. While valine helps in the coordination of motor cells, and aspartate and glutamate are essential for hormonal regulation and immunological stimulation, respectively ( 35 ). Leucine and isoleucine are assisting as building blocks of other proteins ( 36 ). Generally, it has been recommended that, for adults, the protein intake should be in-between 0.8 and 1 g/Kg body-weight/day ( 56 , 57 ). Pregnant, lactating women, and infants need higher protein ingestion than adults as 1.1, 1.3, and 1.2–1.52 g/kg/day, respectively ( 57 ). The intake requirement of proteins and amino acids is determined by various factors linked with genotypic as well as phenotypic characters (age, gender, body weight, lifestyle habits, physical exercises, health conditions, and metabolic capacities) ( 5 ).

Factors Affecting the Nutritional Value of Plant Proteins

The protein's nutritional quality can be identified in different ways, but, in a simple way, it is the balance and relative amounts of essential amino acids, as well as digestibility, bioavailability, and bioactivity, which mainly identify its nutritional value. Compared with animal-based protein, the proteins derived from plants are easier to produce; however, when utilized as dietary sources for human consumption, most of the plant proteins are deficient in essential amino acids and are, therefore, nutritionally incomplete. For example, some cereal proteins are low in tryptophan, lysine, and threonine content, while vegetable proteins and legumes have a lower amount of sulfur-containing amino acids, such as methionine and cysteine ( 58 ). Due to this deficiency, these essential amino acids become the limiting factor in legumes and cereals. Practically, neither legumes nor cereals can compensate for the deficiency of amino acids for other crops, and, hence, diet feeding regularly provides supplementary amino acids. There are also other factors that affect the nutritional quality of crops, including soil condition, crop maturity, postharvest handling, storage, use of fertilizers and pesticides, crop variety, and climatic conditions ( Figure 2 ).

Figure 2 . Various factors that affect the nutritional quality of crops.

It is important in terms of nutritional as well as economic value to increase the essential amino acids content in plant-based proteins ( 59 ). In the past decades, plant breeders and geneticists have done much research for the improvement of the quality and characteristics of plant proteins. For instance, natural mutations, like the high content of lysine in barley and corn, have been recognized and made as elite genotypes ( 60 ). But, unfortunately, undesirable characters, like lower yields and susceptibility to pests and diseases, were also linked with these types of natural mutations. Nowadays, the techniques of modern biotechnology as alternative methods help to solve these problems. The method known as the protein digestibility-corrected amino acid score (PDCAAS) is an effective tool for the quality evaluation of protein ( 49 , 61 ). One of the new methods recommended by FAO in 2013, digestible indispensable amino acid score (DIAAS), has also been used to evaluate protein quality, and, in terms of scientific knowledge, it is considered more accurate than PDCAAS ( 62 ).

Bioactive and Functional Properties of Plant-Based Proteins

Bioactive properties of plant-based proteins.

Several reports have shown the health effects of plant-based proteins as antitumor, antioxidant, hypoglycemic, ACE inhibitory, antimicrobial, and hypolipidemic effects ( Figure 3 ) ( 63 , 64 ). It has been observed that in countries where a high number of pulses are consumed, risk diseases, such as type-2 diabetes, cardiovascular diseases, colorectal cancer, and different types of chronic diseases, have been reduced ( 65 , 66 ). The bioactivity of small peptides that are mainly released from enzymatic hydrolysis by various proteases, such as pepsin, trypsin, chymotrypsin, alcalase, papain, pancreatin, thermolysin, and flavorzyme, are present in different pulse proteins ( 67 ). These peptides exert various bioactivities, such as antioxidant, antifungal, antitumoral, and ACE inhibition activity ( 67 , 68 ), and are also used for different purposes, like food supplements, functional food ingredients, and nutraceuticals ( 63 ) ( Table 1 ).

Figure 3 . The major bioactivities of plant-based proteins.

Table 1 . The commonly used physical modification methods of protein and their applications.

Plant-Based Proteins Against Cardiovascular Disease and Metabolic Risk Factors

A large number of studies showed the potential impact of dietary proteins derived from plants against cardio-metabolic risk factors. The first study for the synthesis and intake of plant proteins as an alternative to animal protein was reported and published in 2017 ( 83 ). In this study, the authors reviewed and demonstrated biomarkers for cardiovascular disease from plant proteins consumption ( 83 ). They also studied and reported a decrease in the concentration of blood lipids (such as lower apolipoprotein B, low-density lipoprotein cholesterol, and non-high-density lipoprotein cholesterol). The authors also conducted randomized trials, which proved that plant protein is effective in reducing the risk factors associated with cardiovascular diseases in adults. In another study, the impact of proteins derived from plants (mostly soy products) on hypercholesterolemic patients was found superior in lowering the lipid profiles compared with the animal proteins ( 84 ). In populations, the adolescent stage, most of the benefits of plant-based proteins and metabolic health concerns have been discussed. Several studies to examine the benefits of plant-based proteins intake have been done for metabolic syndrome, weight management, and obesity, as these are the serious and growing health issues globally among adolescents. However, the regulation of protein intake is critical to many physiological development and functions. Therefore, enhancing the proteins derived from plants in adolescent diets as a substitute for animal-based proteins helps in controlling obesity and other cardio-metabolic factors ( 85 ). The authors in different studies concluded that there should be the addition of more proteins of plant origin in the human diet for reducing the risks associated with cardiovascular disease as well as metabolic risk factors ( 86 ). Also, it was found that consumption of plant-based proteins lowers blood pressure in patients with hypertension (including elderly patients) as compared to animal protein ( 87 , 88 ).

Most of the studies were also associated with the intake of plant protein sources and mortality. In a recent cohort from the NIH-AARP Diet and Health Study, the authors also observed the effect of choice of dietary protein on mortality ( 89 ). In this study, more than six lakh individuals from the U.S. in the age group of 50–71 years were followed from 1996 till December 2011. It was noticed that plant protein intake has led to inverse the mortality rate as well as from stroke in both males and females and cardiovascular disease. They observed the replacing of animal protein with only 3% of plant protein reduced 10% risk of overall mortality in both males and females ( 89 ). Therefore, it is beneficial to substitute plant proteins into the diet instead of animal proteins in terms of mortality and longevity. In a recently published review of the 32 cohort studies, it has been interpreted that the plant-based protein diet lowers the risk of all-cause and cardiovascular-associated mortality. Replacement of foods containing animal proteins with plant protein improves longevity ( 90 ).

Plant-Based Proteins and Diabetes

Although plant-based diets are mainly linked with reducing the risk of diabetes ( 91 ), it is still not clear that substituting the plant-based proteins for animal proteins helps in reducing the risk of diabetes in the population. After studying and analyzing using the dataset from the Nurses' Health Study II, Malik et al. ( 92 ) observed that 5% substitution of vegetable protein for animal protein was linked to the 23% reduction in type 2 diabetes risk. In a meta-analysis conducted in 2015, the sources of animal protein were replaced with plant-based protein for ~35% of the intake of dietary protein for 8-week randomized controlled trials. From this study, the authors found that there are significant improvements in the levels of fasting glucose, fasting insulin, and HbA1c in patients with diabetes (individuals with both type 1 and type 2 diabetes) ( 93 ). In a cohort study, individuals were provided a protein-based diet and found that higher protein intake is associated with a lower risk of diabetic and pre-diabetic incidences, and plant-based proteins are the main determinant ( 94 ). The plant-based protein diet also contains a variety of bioactive components, which provide beneficial health effects as compared to processed meat products. In another randomized crossover trial, substituting red meat with legumes (lentils, chickpeas, peas, and beans) significantly decreased fasting blood glucose, insulin, and the triglyceride level in patients with diabetes type-2, suggesting the potential role of plant-based proteins over animals ( 95 ).

Plant-Based Proteins Against Cancer

Generally, a large number of factors, such as environmental, genetic, dietary, and other habitual features, are associated with the development of cancer. One research group has studied and examined the risk factor of colorectal cancer in individuals with the help of analyzing gene-environment interaction, including other factors, such as genetic, lifestyle, and cancer risk factors ( 96 ). The authors reported the linkage between colorectal cancer and the genetic diversity of fatty acid metabolism, which are mainly associated with a higher intake of meat, and concluded that those who consume a high diet of meat have a high risk of colorectal cancer ( 96 ). Therefore, plant-based protein substitution for animal protein is a better way to reduce the risk of colorectal cancer in humans with certain genetic polymorphisms.

Plant-Based Proteins and Their Renoprotective Effect

The diet, which is lower in vegetables, fruits, healthy oils, and dairy food, but higher in total protein foods, total grains, saturated fats, sodium, and added sugar, has been under trials to know the differences that help to cure chronic disease, especially chronic kidney disease (CKD) ( 97 ). Recent studies have suggested that, along with the amount of protein, protein's origin (for example, plant vs. animal) might be a crucial factor that affects the function of the kidney ( 98 ). For individuals with chronic kidney disease, on the consumption of plant-based protein, a significant 23% lower mortality rate was reported ( 99 ). In a randomized control trial in diabetic adults with macro-albuminuria, the animal protein diet was substituted with soy protein diet (by 50%) and found that it significantly improved proteinuria, cholesterol, and the glucose level ( 100 ).

In a crossover study, a diet rich in soy protein reduces glomerular hyperfiltration in individuals having type 1 diabetes with early-stage nephropathy ( 101 ). With the increase in glomerular hyperfiltration and the glomerular filtration rate, the incidence of kidney injury has been decreased ( 102 ). The plant-based proteins mainly extracted from rice endosperm and soybean have also shown renal protective function in diabetic rat models ( 103 ). Also, other factors, like phytochemicals and fiber, also played a significant role in renal protection by consuming whole food from plant-based diets as well as other components of plants. Thus, it is recommended to incorporate high-quality plant proteins for renoprotective effects.

Functional Properties of Plant-Based Proteins

Plant proteins have also been utilized as functional foods. A large number of studies have been done to examine and reduce the risk factors of cardiovascular disease, modulating inflammation and immune system by functional analysis and bioactive properties of soy protein ( 104 ). The recent systematic review has focused on the bioactive properties of sources of plant proteins, such as rice, lentil, fava bean, pea, lupin, hemp, and oat ( 105 ). Various trials have been done to test the benefits of proteins derived from plants by observing the concentrations of insulin, blood glucose, and hormones regulating the appetite. However, conflicts in results were seen when the study was conducted for determining the beneficial effects of plant proteins on postprandial glycemia regulation. A number of components present in plants, like flavonoids and carotenoids, also confer the benefits of bioactive functionality on human health.

In addition to the nutritional quality of plant proteins and their bioactive properties, these compounds also have functional properties. They play a major role in food processing and formulation, i.e., the production of gluten-free and protein-rich food ( 106 ). Chemical and physical properties of protein help during the storage, consumption, processing, and preparation of food products. Properties like solubility of the protein, foaming capacity, absorbing capacity of water and fat, foam stability, gel-forming, and emulsifying activity are involved in protein interaction by combining with other molecules, like proteins, carbohydrates, salts, lipids, water, and volatiles. These functional properties are largely affected by the molecular size of peptides and/or proteins, charge distribution, and structure of the protein. Additionally, different environmental conditions that affect the structural changes of protein during food processing will also affect the functional properties of plant proteins ( 107 ). For improvement of nutritional quality and potential health benefits, different protein formulations can be added, such as isolates, concentrates, and protein flours. However, the functional properties of various plant-based proteins were utilized in the industrial production of food products. Briefly, various functional properties such as protein solubility during beverage production lead to solvation of protein; absorption of water molecules and their binding allows entrapment of water in bread, meat, cakes, sausages, etc.; absorption of fat is linked with binding of free fat in meats, doughnuts, and sausages; emulsifying properties of proteins lead to the production and stabilization of emulsions of fats in pasta, cakes, sausages, soups, etc.; protein's foaming properties permit the entrapping of gasses by forming stable films in whipped toppings, bakery products, cakes, and desserts; gelation properties are linked with the formation and maintaining of protein matrix in meats, cheese, and curds ( 106 ).

Applications of Plant-Based Proteins in Food and Non-Food Industries

Proteins are the important ingredients of the human diet with great complexity and diversity that play an important role in structural and functional development ( 29 , 49 ). Plant protein provides many essential amino acids, vital macronutrients and is sufficient to achieve full protein nutrition. Moreover, plants have a high demand for the supply of protein to the increasing population ( 12 ). Thus, instead of animals, plants were considered the bioproduction system for useful substances, especially in medicine, which usually provide a large number of secondary metabolites having therapeutic effects. These substances produced by plants mainly help to protect from predators and pathogens, attract pollinators, and have properties like anti-inflammatory, wound-healing, anti-microbial, psychoactive, etc. ( 1 ), and hence utilized for protecting and maintaining human and animal health.

Different sources of plants have been widely used as supplements of protein, such as cereals (wheat, rice, millet, maize, barley, and sorghum), legumes (pea, soybean, bean, faba bean, lupin, chickpea, and cowpea), pseudocereals (buckwheat, quinoa, and amaranth), nuts and almonds, and seeds (flaxseed, chia, pumpkin, sesame, and sunflower). Along with providing health benefits, proteins also play a significant role in food formulations because of their diverse functions, such as emulsifying, gelling, and thickening agents, and also have water-holding, foaming, and fat absorption ability ( 16 , 17 ). In addition, these crops have number of beneficial effects on health and have technological and functional properties with industrial applications in development of food. Thus, these proteins play an important role in circular production systems.

Food derived from plants plays a vital role in human health as an important source of bioactive components, minerals, vitamins, and bioactive peptides ( 4 ). In addition, protein obtained from plants provides essential amino acids and improves the overall nutritional status of human diets.

From the last few years, much interest has been paid to search for protein sources with high nutritional quality and functionality in food processing and industrial applications (emulsification, solubility, gelation, foaming, and viscosity oil-holding and water-holding capacities). Recently, the importance and benefits of proteins derived from plants have been trending to provide various health benefits. Many studies have been conducted on the potential impact of dietary proteins derived from plants on reducing cardio-metabolic risk factors, metabolic syndrome, weight management, and obesity ( 86 – 88 ). Most of these studies concluded that there should be an addition of proteins of plant origin in the human diet for reducing the risks associated with cardiovascular disease and metabolic diseases ( 86 ). Another interesting area of research to examine the benefits of intake of plant proteins instead of animal protein is reducing cancer risk factors.

Food products containing plant proteins have also been known as functional foods. Various trials have been conducted to test the health benefits of plant-based proteins by observing the concentrations of insulin, blood glucose, and hormones regulating the appetite. Most of the studies were also associated with the intake of plant protein sources and mortality. In a recent cohort from the NIH-AARP (National Institutes of Health-American Association of Retired Persons) Diet and Health Study, the authors also observed the effect of choice of dietary protein on mortality ( 89 ). The diet, which is lower in vegetables, fruits, healthy oils, and dairy food, but higher in total protein foods, total grains, saturated fats, sodium, and added sugar, has been under trials to know the differences that help to cure chronic disease, especially chronic kidney disease (CKD) ( 97 ). Recent studies have suggested that, along with the amount of protein, protein's origin (for example, plant vs. animal) might be a factor that affects the function of the kidney ( 98 ).

In addition to the nutritional quality of plant proteins and their bioactive properties, they play a major role in food processing and formulation, i.e., the production of gluten-free (GF) and protein-rich foods ( 106 ). In addition, however, the functional properties of various plant-based proteins were utilized in the industrial production of food products. Various applications, like protein solubility (bread, meat, cakes, sausages, doughnuts, and sausages; emulsifying properties emulsions of fats in pasta, cakes, sausages, soups, etc.; protein's foaming properties, bakery products, cakes, and desserts; and gelation properties provide stability to the protein matrix in meats, cheese, and curds ( 106 ).

Some traditional proteins from plant origin have been utilized by humans as a protein source, such as beans, pea, and soybean. Still, various recent studies have been done for novel (such as proteins from insects and algae) ( 2 ) and unconventional and alternative protein sources (like agroindustry by-products from extraction of edible oil) ( 7 ).

Gluten-free pseudocereals help in curing of patients with celiac disease ( 35 ). The food industry helps produce high-quality plant-based milk, egg, and meat analogs, such as sausages, burgers, ground meat, and nuggets. The proteins derived from plants are considered important and functional ingredients with different roles in food formulations, including gelling and thickening agents, foams and emulsions stabilizers, and binding material for water and fat. Most of the proteins have biological activities, like ACE inhibitory, antioxidant, antimicrobial, and stimulating characteristics ( 70 ), and the protein from vegetables is also utilized for synthesizing and extracting bioactive peptides.

Health Issues Linked With Plant-Based Proteins

Antinutrients.

There are many health concerns linked with a large intake of dietary proteins derived from plants. Antinutrients, such as tannins, phenolics, saponins, phytates, glucosinolates, and erucic acid, are naturally produced by plants and further interfere with absorption, digestion, and utilization of nutrients present in food, with other side effects as well ( 108 ). The adverse effects of antinutrients might be maldigestion of proteins (protease and trypsin inhibitors), carbohydrates (alpha-amylase inhibitors), autoimmune and leaky gut (e.g., some saponins and lectins), malabsorption of minerals (oxalates, phytates, and tannins), inflammation and interfering in thyroid iodine uptake (goitrogens), behavioral effects, and gut dysfunction (when converting cereal gliadins to exorphins) ( 108 ). These adverse effects of antinutrients are generally seen in animals when consumed unprocessed proteins of plant origin. However, these antinutrients also showed beneficial health effects. For instance, at a lower level of lectins, phytates, enzyme inhibitors, saponins, and phenolic compounds, there is a reduction in plasma cholesterol, triglycerides, and blood glucose levels ( 108 ). Saponins may play a significant role in liver functioning and decrease platelets agglutination. In contrast, some of the saponins and also protease inhibitors, phytates, phytoestrogens, and lignans might help in reducing cancer risk ( 108 ). Additionally, tannins also have antimicrobial effects ( 108 ). To reduce the concentration of antinutrients in plant proteins and their adverse effects, various treatment processes, such as fermentation, soaking, gamma irradiation, sprouting (germination), heating, and genomic technologies, have been adopted ( 108 ). Food processing techniques also remove most of the antinutrients like phytates, glucosinolates, erucic acid, and also insoluble fiber from canola proteins that further improve and increase the digestibility and bioavailability ( 109 ).

Isoflavones and Soy Protein

Soy protein is associated with both positive and negative health concerns. The adverse effect on health is due to the presence of isoflavones in soy protein, which are chemically similar to estrogen and could also be bound to estrogen receptors ( 110 ). Due to soy isoflavones, the issue of endocrine-disrupting effect is seen on thyroid and reproductive hormones at higher doses in rodent and in vitro cell culture studies ( 111 – 113 ). The isoflavones content of different ingredients of soy protein has been reported; for example, isolates of soy protein (88–164 mg/100 g), defatted and whole soy flours (120–340 mg/100 g), textured soy protein isolates that are commercially used (66–183 mg/100 g), and soy hypocotyl and flours' commercial isolates (542–851 mg/100 g) ( 114 ). Therefore, consumers mainly avoid taking soy proteins due to various adverse effects on thyroid and reproductive hormones. The study conducted by the European Food Safety Authority in 2015 showed that 35–150 mg daily doses of isoflavones in pre- and postmenopausal women resulted in no significant enhancement in breast cancer risk, uterus's histopathological changes or thickness in the endometrial lining of the uterus, and thyroid hormonal status for about 30 months ( 115 ). A meta-analysis has also been done on 15 men of different ages and found that intake of 60 g/day of soy protein has not been linked with sex hormone-binding globulin, changes in testosterone, free androgen index, or free testosterone ( 116 ). Also, it did not influence the parameters of semen quality, such as sperm concentration, semen volume, sperm mobility, sperm count, sperm percent motility, sperm morphology, and total motile sperm count in healthy men ( 117 ). It has also been reported in the meta-analysis that intake of soy protein might be linked with reducing breast cancer risk in women ( 118 – 120 ).

Plant-Based Proteins and Their Association in Allergenicity

There is an increasing trend of consuming plant proteins, which indicates that different sources of protein from plants influence our health. Such dietary proteins may also have some adverse effects, including allergenicity. An allergy from food is basically an adverse effect that results inactivation of immune response when exposed to a food. According to the literature review, food allergy is found to affect up to 10% of the population ( 121 ). It has been identified that more than 170 foods in the United States of America are responsible for food allergies. Foods commonly causing allergy are tree nuts, soy, wheat, fish, peanuts, milk, shellfish, and egg. Other common food allergens based on the countries are lupines (European Union); sesame seeds (Canada, European Union, and Australia); buckwheat (Japan and Korea), and mustard (European Union and Canada) ( 122 ). A higher number of children than adults are sensitive to dietary proteins that mainly cause allergy ( 123 ).

Food allergens from plants are mainly categorized into four families, the cupin superfamily, the prolamin superfamily, profilins, and the Bet v 1 family. More than 50% of allergens of plant proteins fall into two categories, i.e., the cupin and prolamin superfamilies ( 124 ). The prolamin family has 8 cysteine residues of amino acid that is conserved with pattern CXnCXnCCXnCXCXnCXnC, which mainly stabilizes the structure of protein and contributes proteins allergenicity. The most commonly found allergens are cereal prolamins, α-amylase, 2S albumins, non-specific lipid transfer proteins, and trypsin inhibitor, protein families.

Comparison Between Animal and Plant-Based Proteins

Dietary proteins could be derived from animals and plants. Animal protein, although higher in demand, is generally considered less environmentally sustainable. A gradual transition from animal to plant-based protein food may be desirable to maintain environmental stability, ethical reasons, food affordability, greater food safety, fulfilling higher consumer demand, and combating of protein-energy malnutrition. Since the last 20 years, among the alternative sources of protein, the scientific research team and private companies have mainly focused on algae, earthworm or earthworm meal, insects, and other invertebrates ( 52 , 53 ). Nowadays, food derived from plants plays a vital role in the human diet as an important source of bioactive components, such as vitamins, phenolic compounds, or bioactive peptides. Hence, these components are very helpful to human health and protect against various pathogens ( 4 ). Instead of animals, plants were considered the bioproduction system for useful substances, especially in medicine, which usually provide a large number of secondary metabolites having therapeutic effects. These substances produced by plants mainly help protect from predators and pathogens, attract pollinators, and have properties like anti-inflammatory, wound healing, anti-microbial, psychoactive, etc. ( 1 ), and hence utilized for protecting and maintaining human and animal health.

The proteins derived from plant-based foods are increasingly used as a health-promoting and economical alternative source in place of animal proteins in human nutrition. However, various limitations, such as increased cost, limited supply, biodiversity loss, hazard for human health in different diseases, freshwater depletion, and susceptibility to climate change, replace animal-based proteins ( 5 – 7 ). Moreover, it is hard and expensive to extract an adequate amount of animal proteins; therefore, an alternative for improving the nutritional status of humans is mainly received from plant proteins.

Globally, protein is produced from both plants (80%), such as cereal grains, beans, soy, pulses, nuts, vegetables, and fruits, as well as animals (~20%) in the form of meats, milk, eggs, fish, yogurt, and cheese ( 50 ). Compared to animal-based proteins, the proteins derived from plant-based foods are rich in fiber, polyunsaturated fatty acids, oligosaccharides, and carbohydrates. Therefore, they reduce the cardiovascular diseases and type II diabetes ( 8 ). Increased urbanization and economic development have led to various transitions in dietary patterns in the population of low- and middle-income countries, especially the demand for foods derived from animals, which was seen in developing countries.

Recently, plant-based sources of protein have dominated the supply of proteins throughout the world (57%), with the remaining 43% consisting of dairy products (10%), shellfish and fish (6%), meat (18%), and other products from animals (9%) ( 3 , 19 ). Generally, the daily intake of protein is provided by animal-based foods. However, changes in the consumers' requirement led to adoption of alternative sources of proteins for human consumption. Therefore, emerging factors for animal proteins like growth of world population, climate change, and production of protein sources that are economically and environmentally sustainable need more research focus, and that is mainly dedicated to proteins from plants with high content, resilient to changing of climate and providing balance nutrition in humans' diet.

Compared with animal-based protein, the proteins derived from plants are easier to produce. Still, when utilized as dietary sources for human consumption, most plant proteins are deficient in essential amino acids and are, therefore, nutritionally incomplete. For example, some cereal proteins are low in tryptophan, lysine, and threonine content. In contrast, vegetable proteins and legumes have lower sulfur-containing amino acids, such as methionine and cysteine ( 58 ). Due to this deficiency, these essential amino acids become the limiting factor in legumes and cereals. Practically, neither legumes nor cereals can compensate for the deficiency of amino acids for other crops, and, hence, feed diets regularly provide supplementary amino acids.

Many studies have been done on the potential impact of dietary proteins derived from plants and serve as reducing cardio-metabolic risk factors. The first study for the synthesis and intake of plant proteins as an alternative to animal protein was reported ( 15 ). However, the regulation of protein intake is critical to many physiological development and functions. Therefore, enhancing the proteins derived from plants in adolescent diets as a substitute for animal-based proteins help in controlling obesity and other cardio-metabolic factors ( 85 ). Although plant-based diets are mainly linked with reducing the risk of diabetes ( 91 ), it is not clear that substituting the plant-based proteins for animal proteins helps in reducing the risk of diabetes in the population. After studying and analyzing the Nurses' Health Study II dataset, Malik et al. ( 92 ) observed that 5% substitution of vegetable protein for animal protein was linked with the 23% reduction of type 2 diabetes risk. Another interesting area of research to examine the benefits of the intake of plant proteins instead of animal protein is reducing cancer risk factors ( 96 ).

The Modification Approaches of Plant-Based Food Proteins

Protein modification is the process of alteration of the chemical groups or molecular structure of a protein by specific methods for improving the bioactivity and functionality of proteins. The modification approaches for plant-based proteins help them to make multifunctional food products. The modification of proteins can be classified into physical ( 18 , 62 , 69 – 82 ), chemical ( 125 – 130 ), biological ( 131 , 132 ), and other novel methods ( 133 – 137 ) as briefly described in Tables 1 – 3 . The physical modification approaches include heat treatment (such as conventional thermal treatment, ohmic heating, microwave heating, radio frequency treatment, infrared irradiation), gamma irradiation, electron beam irradiation, ultraviolet radiation, pulsed-electric field, high-pressure treatment (such as high hydrostatic pressure, dynamic high-pressure fluidization), sonication, extrusion, ball mill treatment, cold atmospheric plasma processing, and ultrafiltration. The chemical modification approaches include glycation, phosphorylation, acylation, deamidation, cationization, and pH shifting treatment. The biological modification approaches include enzymatic modification and fermentation. Instead of physical, chemical, and biological modifications, various other modification approaches have been identified, which include complexation (such as protein-polysaccharide, protein-protein, protein-phenolic, and protein-surfactant) and amyloid fibrillization ( Tables 1 – 3 ) ( 138 ).

Table 2 . The commonly used chemical modification methods of protein and their applications.

Table 3 . The commonly used biological and some other modification methods of protein and their applications.

Protein Extraction Technologies

The advancement in recombinant technologies of protein production, such as engineering of expression hosts, upstream cultivation optimization (e.g., nutritional, bioreactor design, and physical parameters), and development of protein extraction methods supported the growth of the market ( 7 , 13 ). The use of protein extraction technologies can help improve the yield of extracted protein and its nutritional and functional properties. Hence, a suitable type of protein extraction method should be selected ( Figure 4 ).

Figure 4 . Extraction technologies for plant-based proteins.

Dry Protein Extraction Technique

Sieving and/or air classification techniques, majorly a part of novel dry protein extraction techniques, have been widely used to prepare fiber or protein-rich fractions. Although a high protein yield was generated, however, it utilized more energy than wet protein extraction. Also, the disadvantage of these processes includes the presence of impurity and particle agglomeration ( 7 ).

Wet Protein Extraction Technique

In wet protein extraction techniques, the process starts with protein solubilizing in a medium with the pH far from the isoelectric point and then precipitating in that medium where pH is close to the isoelectric point. Several protocols for acidic and alkaline extraction of protein have been reported ( 7 ).

Enzyme-Assisted Extraction of Proteins

This method is based on the principle of cell wall disruption with specific enzymes that degrade celluloses, hemicelluloses, and/or pectin, and also proteases that help in the hydrolyzation of protein for solubility enhancement. With the degradation of cell walls, protein bodies released are enabled. This method needs more processing time, high cost, more energy consumption, and suitable conditions like temperature and pH. Still, this method is mostly used with lower environmental impact and superior quality of products for human consumption ( 7 , 139 ).

Subcritical Water Extraction

In this technique, hot water in the range of 100–374°C with high pressure (for maintaining it into the liquid state) has been used. Biomaterials like carbohydrates and proteins have been hydrolyzed by this method without using an additional number of catalysts. For example, when soy meals were heat denatured, soy protein extraction yield was significantly increased with this method by 59.3% ( 139 ).

Reverse Micelles Extraction

This method applies reverse micelles—surfactant molecules aggregate of the nano-meter size that generally contains inner cores of water molecules inside non-polar solvents. The polar molecules of water present in reverse micelles help in solubilizing hydrophilic biomolecules like proteins. The three-phase system called a water–surfactant–organic solvent system has been formed by reverse micelles to protect the denaturation of proteins by organic solvents inside the polar water pools, using forward extraction or backward extraction ( 7 ).

Aqueous Two-Phase Systems Extraction

This extraction method is formed when two polymers like two salts or one salt and one polymer are mixed in a suitable concentration at a particular temperature. This method has been considered as the environment-friendly method of protein extraction. It was first reported by Zeng et al. ( 140 ) for extracting proteins by an ionic liquid aqueous two-phase system, resulting in proteins extraction with a yield of 99.6% ( 7 ).

Novel-Assisting Cell Disruption Techniques

Cell disruption is the initial process in both dry and wet techniques of protein extraction, which helps release protein from protein bodies. Previously, cell disruption was done by mechanical methods like milling, grinding, etc., or chemical or thermal treatments.

Microwave-Assisted Extraction of Proteins

This technology utilizes electromagnetic radiations having a frequency between 300 MHz and 300 GHz, which helps in hydrogen bond disruption, dissolved ions migration, and enhancement of porosity of the biological matrix, which leads to the extraction of protein. For example, one study reported the utilization of this technique to extract proteins from rice bran ( 141 ).

Ultrasound-Assisted Extraction of Proteins

This technology utilized sound waves, having a frequency of 20 kHz that induces the phenomenon of cavitation, which enhances the matrix porosity and improves solvent permeation into the matrix. This method has the advantage of effective mixing, selective extraction, faster energy transfer, reduced extraction temperature and thermal gradients, faster response, reduced equipment size, and increased production. Yet, denaturation of protein structure and disruption of functional properties of proteins are reported ( 7 ).

Pulsed Electric Energy-Assisted Extraction of Proteins

Several pulsed electric energy technologies for proteins extraction have emerged. This method uses electric pulses of short duration (from several nanoseconds to several milliseconds) of high-pulse amplitude (from 100 to 300 V/cm to 10–50 kV/cm) for the induction of structural changes of the compound of interest. Among a large number of PEE techniques, pulsed ohmic heating (POH), pulsed electric fields (PEF), and high-voltage electrical discharges (HVED) have been widely used in the food industry ( 7 ).

High Hydrostatic Pressure-Assisted Extraction of Proteins

High hydrostatic pressure-assisted extraction of proteins is mostly used in the food industry for large-scale microbial cell disruption, meat tenderization, and emulsification. This method is only restricted to bioactive compounds instead of proteins. However, with the application of several HHP iterations, the efficiency of separation and extraction yield has been reduced due to swelling of the cell wall, increase in dynamic viscosity, and size of the particle ( 7 ).

Issues, Challenges, and Future Prospects of Plant-Based Proteins and Their Utilization in Food Products

The proteins derived from plants are considered as important and functional ingredients, having different roles in food formulations as gelling and thickening agents, foams and emulsion stabilizers, and binding material for water and fat ( 142 – 145 ). Most of the proteins have biological activities, like antihypertensive, antioxidant, antimicrobial, and stimulating characteristics ( 146 , 147 ), and the protein from vegetables is also utilized for synthesizing and extraction of bioactive peptides ( 148 , 149 ). However, most of the proteins from plant origin are interactable because of their susceptibility and complexity of ionic strength, pH, and temperature, and also have poor water solubility that mainly limits the applications of plant-based proteins ( 150 ). Most of the plant-based proteins, like flaxseed, soy, and pea proteins, have the combined nature of various proteins with different fractions, and, hence, they have a wide range of isoionic point (pI). Therefore, modulating the properties of plant-based proteins for improving their functions and formulation characteristics is essential. A deep understanding of the functional and physicochemical properties of proteins derived from plants is necessary for improving their utilization in food formulation and nutritional value ( 151 – 153 ). The presence of some particular plant residues considered as antinutrients is another challenge of plant-based proteins. These compounds are produced in plants having various biological properties, such as they protect the plants and seeds from insects, fungus, viruses, and other microbes. Therefore, some of the modification approaches discussed have been used to reduce or eliminate the adverse effects of antinutrients ( 18 ). Furthermore, some plant-based proteins have challenges in food applications due to their bitter taste, which can be masked by various modulation techniques. The methods of modification for plant-based proteins should be carefully chosen, especially in pharmaceutical and food applications, because these methods have effects on the organoleptic and functional characteristics and nutritional value of plant proteins.

The bio-efficacy of any active compounds generally depends on various factors, like digestibility, solubility, bioaccessibility, food matrix, transporters, metabolizing enzymes, and molecular structures. Therefore, identifying the bioavailability of food constituents is challenging. There are many challenges associated with sustainability and food availability that needs to be solved with different methods of protein modification. The higher amount of essential nutrients found in animal products (meat, milk, egg, etc.) was important and provided a large number of nutrients in the daily diet compared with plant-based proteins ( 154 ). Although animal-meat-based products provide a large nutrient component, however, the disease associated with animals, unhygienic conditions, and environmental impact will all provide more attention to the plant-based proteins. Because of that, consumers are also more focused on the health and environmental benefits of plant-based diets, promote the food guidelines on the basis of health and sustainability criteria, produce more attractive plant-based alternative products, and realign their fiscal policy along with environmental and efficiency criteria ( 155 – 159 ).

Conclusions

People are facing protein and mineral deficiency in their diet throughout the world, especially in developing countries. This challenge is due to lower consumption of pulses and cereals in their diets and other foods that are rich in zinc, iron, calcium, and magnesium. These foods derived from plants also contain higher levels of antinutritional factors that bind to the minerals ions and reduce bioavailability and absorption of plant minerals as well as proteins. Animal protein, although higher in demand, is generally considered less environmentally sustainable and prone to disease conditions, which negatively impact health. A gradual transition from animal- to plant-based protein may be desirable in order to maintain environmental stability, ethical reasons, affordability of food, greater food safety, fulfilling higher consumer demand, and combating of protein-energy malnutrition. Nowadays, products made with proteins from plant origin gain popularity throughout the world. Plant-based proteins have been linked with a number of health-related functionalities. Plant-based proteins are becoming innovative and fast-growing ingredients in various food application industries due to a large number of benefits over animal-derived proteins. Various technologies help in improving the functional and nutritional properties of plant-based proteins. Generally, plant-based proteins have inferior functionality as compared with animal proteins, and also various factors affect their nutrient quality; hence, modification approaches have been required. Different physical, chemical, biological, and other approaches were also mentioned for modification of proteins that induce the structural, chemical, and biophysical changes in protein from plant origins.

This review mainly focuses on the current state of using plants for the production of protein. The potential plants offering various sources and their alternative with high-quality protein demand for future consumption were discussed. Factors that affect protein consumption, bioavailability, and also protein production techniques were covered. Various bioactive and functional properties of plant-based proteins, as well as the factors affecting the nutritional quality of plant-based proteins and the future research strategies, were explained. The modification approaches, protein extraction, purification technologies, along with digestibility, absorption, and bioavailability of plant-based proteins, were discussed. Finally, it gave an idea of issues and challenges as well as future prospects in this emerging area.

Author Contributions

SL conceived the idea. SL and FK wrote the manuscript. PY, ZD, RS, and AK edited the manuscript. All authors contributed to the article and approved the submitted version.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

The authors are thankful to the director, ICAR-NBPGR, for all the support.

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Keywords: proteins, plants, nutrition, extraction, sustainability

Citation: Langyan S, Yadava P, Khan FN, Dar ZA, Singh R and Kumar A (2022) Sustaining Protein Nutrition Through Plant-Based Foods. Front. Nutr. 8:772573. doi: 10.3389/fnut.2021.772573

Received: 08 September 2021; Accepted: 13 December 2021; Published: 18 January 2022.

Reviewed by:

Copyright © 2022 Langyan, Yadava, Khan, Dar, Singh and Kumar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Sapna Langyan, singh.sapna06@gmail.com ; Pranjal Yadava, pranjal.yadava@icar.gov.in

This article is part of the Research Topic

Sustaining Protein Nutrition through Plant-Based Foods: A Paradigm Shift

• Open access
• Published: 13 April 2022

The effect of animal versus plant protein on muscle mass, muscle strength, physical performance and sarcopenia in adults: protocol for a systematic review

• Rachel J. Reid-McCann   ORCID: orcid.org/0000-0003-4080-9050 1 ,
• Sarah F. Brennan 1 ,
• Michelle C. McKinley 1 &
• Claire T. McEvoy 1  

Systematic Reviews volume  11 , Article number:  64 ( 2022 ) Cite this article

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The evidence base for the role of dietary protein in maintaining good muscle health in older age is strong; however, the importance of protein source remains unclear. Plant proteins are generally of lower quality, with a less favourable amino acid profile and reduced bioavailability; therefore, it is possible that their therapeutic effects may be less than that of higher quality animal proteins. This review aims to evaluate the effectiveness of plant and animal protein interventions on muscle health outcomes.

A robust search strategy was developed to include terms relating to dietary protein with a focus on protein source, for example dairy, meat and soy. These were linked to terms related to muscle health outcomes, for example mass, strength, performance and sarcopenia. Five databases will be searched: MEDLINE, Scopus, Cochrane Central Register of Controlled Trials, Embase and Web of Science. Studies included will be randomised controlled trials with an adult population (≥ 18) living in the community or residential homes for older adults, and only English language articles will be included. Two independent reviewers will assess eligibility of individual studies. The internal validity of included studies will be assessed using Cochrane Risk of Bias 2.0 tool. Results will be synthesised in narrative format. Where applicable, standardised mean differences (SMD) (95% confidence interval [CI]) will be combined using a random-effects meta-analysis, and tests of homogeneity of variance will be calculated.

Dietary guidelines recommend a change towards a plant-based diet that is more sustainable for health and for the environment; however, reduction of animal-based foods may impact protein quality in the diet. High-quality protein is important for maintenance of muscle health in older age; therefore, there is a need to understand whether replacement of animal protein with plant protein will make a significant difference in terms of muscle health outcomes. Findings from this review will be informative for sustainable nutritional guidelines, particularly for older adults and for those following vegan or vegetarian diets.

Systematic review registration

PROSPERO CRD420201886582

Peer Review reports

Sarcopenia is a debilitating condition that is characterised by loss of muscle mass and strength and is associated with a range of other health outcomes including reduced physical functional performance, weakness, frailty, falls, hospitalisation and death [ 1 , 2 ]. It has been estimated that 30% of over 60s and 50% of over 80s have sarcopenia [ 3 ]. With the over 85s population in the UK expected to double in the next three decades, sarcopenia will be a greater public health concern than ever before [ 4 ].

An inadequate protein intake is a core modifiable risk factor for sarcopenia due to the role of dietary protein in supplying essential amino acids for muscle protein synthesis and therefore maintenance of muscle mass [ 3 , 5 ]. Numerous longitudinal studies have indicated that a higher dietary protein intake is protective of muscle mass, strength and physical performance [ 6 , 7 , 8 , 9 ]. Likewise, there is good experimental evidence that protein supplementation is effective in improving muscle mass and strength in sarcopenic populations [ 10 ]. It is for these reasons that protein supplementation, alongside resistance training, is currently the standard treatment for sarcopenic patients [ 11 ]. Encouragement of adequate dietary protein intake as part of a healthy diet is also an important preventive measure. What is considered to be an adequate dietary protein intake for older adults is likely to be higher than that of younger populations due to age-related anabolic resistance of muscle protein synthesis [ 12 ]. For this reason, expert consensus suggests that older adults should consume an additional 0.2–0.7 g of dietary protein per kg body weight than younger adults daily in order to protect against muscle atrophy [ 13 ].

While the evidence base for the role of dietary protein in maintaining good muscle health in older age is strong, the importance of protein source remains unclear. There is evidence that equal amounts of protein from different sources are not met with an equal postprandial response in terms of amino acid absorption and metabolic utilisation. For example, modelling studies have found that soy protein experiences greater splanchnic extraction and nitrogen losses compared to milk protein [ 14 , 15 ]. This is especially pertinent for older adults, given the increase in splanchnic extraction of amino acids associated with ageing and therefore the reduced free amino acid pool available for muscle protein synthesis [ 16 ]. These age-related changes in protein digestion combined with the varied postprandial response to different protein sources indicate that there may be important differences for the anabolic potential of different protein sources between younger and older adults.

There may also be important differences between male and female populations in terms of their anabolic response to different dietary proteins. There is evidence of sex dimorphism in protein metabolism and muscle protein synthesis, which is particularly evident during periods of life in which significant hormonal changes take place, e.g. menopause [ 17 ]. This suggests that the choice of protein source for conservation of muscle health will be particularly important in older age especially as later life is an important period of hormonal change for men and women alike.

Proteins also inherently differ in their quality, i.e. their amino acid profile combined with their bioavailability. Proteins from animal food sources are referred to as high-quality proteins due to the presence of all nine essential amino acids (EAA) in high quantities as well as the greater bioavailability of these EAA. In comparison, plant proteins often have very little of one or several of the EAAs, for example many legumes lack methionine, cysteine and tryptophan [ 18 ]. They are also less bioavailable due to the structure of plant proteins and high concentration of compounds that bind protein, for example tannins and phytic acid [ 19 ]. A greater proportion of dietary fibre in plant protein food matrices is also expected to reduce protein digestibility [ 20 ]. Protein quality can be summarised using the protein digestibility-corrected amino acid score (PDCAAS) [ 21 ]. See Fig. 1 for an overview of PDCAAS for different protein sources.

Protein digestibility-corrected amino acid score (PDCAAS) for 12 protein sources (source: Berrazaga et al. [ 19 ])

Animal protein sources such as meat, fish and dairy have a consistently high protein quality, whereas the quality of plant protein sources is more variable (Fig. 1 ). This suggests that animal sources will be more effective for preserving muscle health during ageing. However, the encouragement of a greater consumption of animal protein sources for healthy muscle ageing may not be appropriate for optimising all outcomes related to diet. Animal products such as dairy are nutritionally rich, important dietary sources of calcium and protective of musculoskeletal health [ 22 ]. However, on the other hand, a plant-based diet has repeatedly shown to be associated with improved cardiovascular health outcomes and all-cause mortality [ 23 , 24 ]. The optimum proportion of plant to animal food items in the diet in terms of optimising health outcomes is not currently known, and consideration must include the environmental impact of any recommendation to increase animal protein intake. The EAT-Lancet Commission, “Our Food in the Anthropocene: Healthy Diets from Sustainable Food Systems”, aims to develop global scientific targets based on evidence available for healthy diets and sustainable food production in order to meet the UN Sustainable Development Goals (SDGs) and Paris Agreement [ 25 ]. The lack of scientific targets to date is thought to have hindered efforts to transform the global food system, and it has been stated that current targets for carbon emissions will not be met if the current Westernised dietary pattern does not change in favour of a more plant-based diet [ 25 ].

Previous systematic reviews have attempted to distinguish the effects of different protein sources on muscle health outcomes including muscle mass and strength (Appendix 3 ). However, to our knowledge, previous reviews have not extended the scope to include important physical performance or sarcopenia outcomes for the ageing muscle [ 26 , 27 , 28 ]. Furthermore, reviews have been limited either by the sole inclusion of younger adults (< 40 years) [ 26 ] or by focusing primarily on soy plant proteins [ 27 ] rather than the comprehensive range of plant proteins that have been studied. Previous reviews also did not consider the effects of sex in analyses, yet there may be important sex differences in the impact of different protein sources on muscle health. Furthermore, energy deficit can impair muscle protein synthesis [ 29 ]; however, previous meta-analyses did not conduct separate subgroup analyses for the pooled effects of protein interventions with and without energy deficit [ 28 ].

This protocol for a systematic review outlines methodology that aims to add to the current knowledge base by introducing novel factors to address the aforementioned gaps: a wider scope in terms of muscle health outcomes, a comparison of effects by sex and independent statistical analyses of studies featuring energy deficits in the intervention.

Hypotheses and research questions

We hypothesise that a similar weight of high quality, plant protein isolate (i.e. soy) is as effective as animal protein isolate (e.g. whey) for preserving muscle health during ageing. We hypothesise that interventions substituting whole animal protein foods (e.g. red meat) with plant proteins (e.g. soybeans) or whole plant diets (e.g. vegan diets) are not as effective owing to a potentially lower ratio of protein in plant protein foods.

The primary research question for this review is as follows:

What is the effect of animal versus plant protein on muscle mass, muscle strength, physical performance and sarcopenia in adults?

Secondary research questions are as follows:

Does the effect of animal versus plant proteins on muscle health differ between males and females?

Does the effect of animal versus plant proteins on muscle health vary at different life stages (e.g. younger or older than 60)?

How does the effect of different plant proteins (e.g. soy, wheat) compare to animal proteins for muscle health?

Methods and design

The methods for this systematic review have been developed according to the recommendations from the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) 2015 statement [ 30 ]. The protocol has been registered with PROSPERO: CRD42020188658.

Inclusion criteria

Participants.

Adults over the age of 18 are eligible for inclusion if they are either living in the community or in residential care homes for older adults. Hospitalised populations are excluded. Those with a disease that affects the normal absorption, metabolism or requirements of dietary protein are excluded, for example patients with cancer, chronic kidney disease or malnutrition (see Table 1 ).

Intervention(s)

The intervention in included studies is consumption of plant protein. This may be presented in various forms:

Supplementation of diet with a whole food source of protein, e.g. tofu or beans

Supplementation of diet with an isolated or concentrated form of plant protein, e.g. soy protein isolate powder

A whole diet intervention in which protein sources are predominantly from plant sources, e.g. a vegan diet or a plant-based diet low in animal source foods

The intervention should have a minimum duration of 4 weeks as this time period has been shown to be sufficient for measurable hypertrophy to take place when combined with resistance training [ 33 ]. Studies that include such physical activity components can be included if the intervention and comparator follow the same training programme. Likewise, studies that provide micronutrients alongside both plant and animal interventions can be included provided these are identical, i.e. vitamin D supplementation in both arms.

Comparator(s)

The comparator will be a parallel intervention of animal protein. The comparator and intervention will have similar quantities or protein content in order for treatments to be comparable. As with the intervention, the comparator may be supplementation with a single animal protein source, for example isolated whey protein powder or a whole food such as a portion of chicken. Similarly, an animal-based/omnivorous diet may be compared to a diet based on plant protein sources given the quantities are comparable.

The outcomes of interest are mean change in muscle mass, muscle strength, physical performance and sarcopenia from baseline. These may be measured by a range of methods as listed in Table 2 . Each outcome is equal in importance, and no additional outcomes are under investigation. The rationale for outcome choices is that muscle mass, strength and physical performance are altogether important determinants of sarcopenia and are each a component of the sarcopenia case definition.

Report characteristics

This review will include randomised controlled trials (RCTs) published in the English language before July 2020. Only full papers will be considered; conference abstracts are excluded as extraction of sufficient data and quality assessment may not be possible from the limited information given (Table 1 ).

Information sources and search strategy

An initial scoping review was conducted on MEDLINE using key search terms such as ‘dietary protein’ and ‘muscle’. This scoping exercise identified a sufficient number of randomised trials focusing on plant versus animal effects on muscle outcomes, particularly for optimising sports performance, for measuring effects of soy on menopause symptoms or as a part of a weight loss intervention. Relevant words or terms used in the titles and abstracts of these papers were identified and contributed to construction of a comprehensive search algorithm with the guidance of an information specialist. The final search algorithm is a combination of the reviewer’s own terms combined with standardised medical subject headings (MeSH). Two examples of this search strategy, tailored to the CENTRAL and Scopus databases, can be seen in Appendices 1 and 2 .

Five databases will be searched in total: MEDLINE, Scopus, Embase, Web of Science and Cochrane Central Register of Controlled Trials (CENTRAL). A manual search of reference lists and recently published papers will be undertaken prior to data extraction to ensure any relevant papers not captured by the search will be included. Study authors will be contacted in any case of unclear or missing data.

Study records

Screening and selection.

Once searches are complete, all references will be downloaded to Endnote [version X9 3.2, Clarivate Analytics, PA, USA] and duplicates removed. Following this, studies will be uploaded to Rayyan [Qatar Computing Research Institute, Doha, Qatar] where titles and abstracts will be screened. Two reviewers (RRM, SB) will screen abstracts against inclusion criteria seen in Table 1 while blinded to each other’s decisions, and conflicts will be resolved through discussion between the other members of the review team (CME, MMK). Studies that meet the inclusion criteria at this stage will subsequently undergo blinded full-text screening by two reviewers (RRM, SB) using Rayyan. A PRISMA flow diagram will be developed to show the progress from the initial search to final selection of studies to be included in review.

Data extraction

A predefined template will be used for data extraction. A summary of variables to be extracted from each included study is provided in Table 3 . One reviewer (RRM) will contact authors in the event of missing data or unclear reporting. Studies will be grouped based on their methodological similarities.

Risk of bias in individual studies

Two reviewers (RRM, SB) will assess the methodological quality and internal validity of eligible trials at the study level using the Cochrane Risk of Bias 2.0 tool (RoB 2) [ 34 ]. For each trial that meets eligibility criteria, risk of bias will be assessed across five domains: the randomisation process, deviations from intended interventions, missing outcome data, outcome measurement and the reporting of results. For each domain, the signalling questions listed in the RoB 2.0 will be applied to the individual study, and a risk of bias judgement will be made, either high risk, some concerns or low risk. The overall risk of bias will be determined as follows:

Overall low risk of bias only if all independent domains are found to have low risk of bias

Overall high risk of bias if at least one domain presents high risk of bias or if multiple domains raise some concerns

Overall, some concerns if at least one domain gives this result and no domains give a high risk of bias

Discrepancies will be resolved through discussion between two reviewers (RRM, SB) and a third reviewer if required (CME).

Data synthesis

Summary tables will be presented to show key information for each paper including study and participant characteristics, intervention and comparator characteristics, outcomes and RoB 2 category. All studies will be then discussed in a narrative synthesis, and meta-analyses will be performed for each outcome. All analyses will be conducted using RevMan software [Review Manager, version 5.3, 2014].

Where data permits, we will quantify the effect of plant versus animal protein interventions on muscle health in adults by calculating between-group standardised mean difference (SMD) and 95% confidence intervals (95% CI) for each of the muscle outcomes. Results will be presented in a forest plot for each outcome.

Statistical heterogeneity will be assessed using several methods. Each forest plot will be visually assessed for inter-study heterogeneity. The chi-squared test for heterogeneity and the I 2 statistical test will also be conducted, with levels of heterogeneity for the I 2 test defined as follows: low, 0–25%; moderate, 25–50%; high, 75–100% [ 35 ]. Later subgroup analyses will be interrogated to explain any heterogeneity found at this stage.

If significant heterogeneity is detected, a meta-analysis will be conducted for each outcome using a random-effects model to account for such inter-study and between-study heterogeneity [ 36 ]. Studies with greater than one intervention/plant protein group will be presented as follows: plant protein group 1 vs comparator and associated mean difference and plant protein group 2 vs comparator and associated mean difference. Any studies such as these with > 1 result presented in a meta-analysis will receive a smaller weight in any pooled analysis.

If possible, subgroup analyses will be conducted for the following:

Male and female populations

Different life stages, i.e. young adults, midlife and older adults

Different plant protein sources, i.e. pea protein, soy protein

Sensitivity analyses

A sensitivity analysis of studies with a low to medium risk of bias will be undertaken to examine whether studies with a high risk of bias are likely to have affected the result. If possible, another set of analyses separating industry- and nonindustry-funded studies will be undertaken to reveal any potential funding outcome biases.

Risk of meta-biases

Several methods will be used to interrogate risk of meta-bias in this review. Funnel plot asymmetry will be interrogated by two reviewers (RRM, SB) who will come to a joint conclusion as to the risk of publication bias in the review. Egger’s test will also be performed to statistically analyse funnel plot asymmetry [ 37 ]. However, conclusions drawn regarding publication bias are likely to be tentative based on the small number of studies expected for each separate outcome and thus the limited capacity of these tests to detect publication bias, as well as the potential that Egger’s test has limitations when assessing continuous outcomes [ 38 ]. Risk of reporting bias should be limited as any relevant results that are not explicitly reported in studies will be requested from study authors. However, if no response is received from study authors, the risk of reporting bias will be discussed in the review manuscript.

Confidence in cumulative evidence

The GRADE framework will be used to assess the certainty of evidence [ 39 , 40 ]. A separate GRADE assessment will be conducted for each RCT by one reviewer (RRM), and consensus agreement will be sought from the entire review team.

The completed systematic review manuscript is intended to be published in a suitable peer-reviewed journal. Any amendments or deviation from this protocol will be outlined in the later manuscript. Results from this review will be a valuable addition to the area of plant-based and sustainable nutrition, providing a quantitative summary of any muscle health-related trade-offs between plant proteins as a more sustainable protein source, and animal proteins which are of higher quality but less sustainable. With increasing numbers of people adhering to flexitarian, vegetarian and vegan diets, it is necessary to know how plant protein sources compare to traditional animal sources, especially as these populations age and muscle atrophy and disability become a greater concern.

Availability of data and materials

Data sharing is not applicable to this article as no datasets were generated or analysed at this stage of the review.

Abbreviations

Asian Working Group for Sarcopenia

Bioelectrical Impedance

Computed tomography

Dual-energy X-ray absorptiometry

Essential amino acids

European Working Group on Sarcopenia

Mean difference

Magnetic resonance imaging

Protein digestibility-corrected amino acid score

Randomised controlled trial

Risk of Bias 2

Standardised mean difference

Short physical performance battery

Timed Up-and-Go

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Acknowledgements

Thank you to Angela Thompson, information specialist at Queen’s University Belfast, for guidance in constructing search algorithms.

This review is funded by the Northern Ireland Department for the Economy as a part of RRM’s PhD programme and had no involvement in protocol development.

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Rachel J. Reid-McCann, Sarah F. Brennan, Michelle C. McKinley & Claire T. McEvoy

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RRM conducted the scoping review, RRM and CME developed the search strategy, RRM, CME and MMK contributed to the development of systematic review methods, and RRM and CME developed the protocol manuscript. All authors contributed to proofreading and final comments for protocol manuscript. RRM is the review guarantor. The author(s) read and approved the final manuscript.

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Search strategy formatted for use in Cochrane Central Register of Controlled Trials (CENTRAL) database

[mh "dietary proteins"] OR [mh ^"grain proteins"] OR [mh ^"soybean proteins"] OR [mh "dairy products"] OR [mh ^eggs] OR [mh ^"food, fortified"] OR [mh ^"functional food"] OR [mh ^"meat proteins"] OR [mh meat] OR [mh ^nuts] OR [mh "soy foods"] OR [mh "diet, high-protein"] OR [mh ^"diet, vegetarian"] OR [mh ^vegans] OR (diet* NEAR/2 protein*):ti,ab,kw OR (plant NEAR/2 protein*):ti,ab,kw OR (animal NEAR/2 protein*):ti,ab,kw OR (soy NEAR/2 protein*):ti,ab,kw OR (nut NEAR/2 protein*):ti,ab,kw OR (protein NEAR/2 source):ti,ab,kw OR (soy*):ti,ab,kw OR (tofu):ti,ab,kw OR (pea):ti,ab,kw OR (pulses):ti,ab,kw OR (legumes):ti,ab,kw OR (lentil*):ti,ab,kw OR (chickpea*):ti,ab,kw OR (quinoa):ti,ab,kw OR (nut):ti,ab,kw OR (mycoprotein):ti,ab,kw OR (egg):ti,ab,kw OR (dairy):ti,ab,kw OR (milk):ti,ab,kw OR (whey):ti,ab,kw OR (fish):ti,ab,kw OR (poultry):ti,ab,kw OR (chicken):ti,ab,kw OR (meat):ti,ab,kw OR (plant-based):ti,ab,kw OR (vegetarian*):ti,ab,kw OR (vegan*):ti,ab,kw AND ("muscle mass"):ti,ab,kw OR ("fat-free mass"):ti,ab,kw OR (strength):ti,ab,kw OR ("body composition"):ti,ab,kw OR (sarcopenia):ti,ab,kw OR (physical NEAR/2 performance):ti,ab,kw OR (lean NEAR/2 mass):ti,ab,kw OR (muscle NEAR/2 function*):ti,ab,kw OR (physical NEAR/2 function*):ti,ab,kw OR [mh ^"muscle, skeletal"] OR [mh ^"muscle weakness"] OR [mh ^"body composition"] OR [mh ^sarcopenia] OR [mh "muscle strength"] OR [mh ^anthropometry] OR [mh ^"gait analysis"] OR [mh ^gait] OR [mh ^"walking speed"] OR [mh ^"physical functional performance”].

Search strategy formatted for use in Scopus database

( TITLE-ABS-KEY ( "muscle mass" ) OR ( "fat-free mass" ) OR strength OR ( "body composition" ) OR sarcopenia OR ( physical W/2 performance ) OR ( lean W/2 mass ) OR ( muscle W/2 function* ) OR ( physical W/2 function* ) ) AND ( ( TITLE-ABS-KEY ( diet* W/2 protein* ) OR ( plant W/2 protein* ) OR ( animal W/2 protein* ) OR ( soy W/2 protein* ) OR ( nut W/2 protein* ) OR ( protein W/2 source ) ) OR ( TITLE-ABS-KEY ( soy* OR tofu OR pea OR pulses OR legumes OR lentil* OR chickpea* OR quinoa OR nut OR mycoprotein OR egg OR dairy OR milk OR whey OR fish OR poultry OR chicken OR meat OR plant-based OR vegetarian* OR vegan* ) ) ) ) AND ( TITLE-ABS-KEY ( rct ) OR ( "RANDOMI?ED TRIAL" ) OR ( "RANDOMI?ED CONTROLLED TRIAL" ) AND ( LIMIT-TO ( DOCTYPE , "ar" ) ) AND ( LIMIT-TO ( LANGUAGE , "English" ) ) AND LIMIT-TO ( EXACTKEYWORD , "Randomized Controlled Trial” AND ( LIMIT-TO ( SUBJAREA , "MEDI" ) OR LIMIT-TO ( SUBJAREA , "NURS" ) OR LIMIT-TO ( SUBJAREA , "HEAL" ) )

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Reid-McCann, R.J., Brennan, S.F., McKinley, M.C. et al. The effect of animal versus plant protein on muscle mass, muscle strength, physical performance and sarcopenia in adults: protocol for a systematic review. Syst Rev 11 , 64 (2022). https://doi.org/10.1186/s13643-022-01951-2

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DOI : https://doi.org/10.1186/s13643-022-01951-2

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Dietary intake of total, animal, and plant proteins and risk of all cause, cardiovascular, and cancer mortality: systematic review and dose-response meta-analysis of prospective cohort studies

• Related content
• Peer review
• Sina Naghshi , masters student of nutrition 1 2 ,
• Omid Sadeghi , Ph.D of nutrition 3 ,
• Walter C Willett , professor of epidemiology and nutrition 4 5 ,
• Ahmad Esmaillzadeh , professor of nutrition 6 7 8
• 1 Students’ Scientific Research Center, Tehran University of Medical Sciences, Tehran, Iran
• 2 Department of Clinical Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, Iran
• 3 Department of Community Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, Iran
• 4 Departments of Nutrition and Epidemiology, Harvard TH Chan School of Public Health, Boston, MA, USA
• 5 Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
• 6 Department of Community Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, PO Box 14155-6117, Tehran, Iran
• 7 Obesity and Eating Habits Research Centre, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
• 8 Department of Community Nutrition, School of Nutrition and Food Science, Isfahan University of Medical Sciences, Isfahan, Iran
• Correspondence to: A Esmaillzadeh a-esmaillzadeh{at}sina.tums.ac.ir
• Accepted 20 May 2020

Objective To examine and quantify the potential dose-response relation between intake of total, animal, and plant protein and the risk of mortality from all causes, cardiovascular disease, and cancer.

Design Systematic review and meta-analysis of prospective cohort studies.

Data sources PubMed, Scopus, and ISI Web of Science until December 2019, and references of retrieved relevant articles.

Study selection Prospective cohort studies that reported the risk estimates for all cause, cardiovascular, and cancer mortality in adults aged 18 or older.

Data synthesis Random effects models were used to calculate pooled effect sizes and 95% confidence intervals for the highest versus lowest categories of protein intake and to incorporate variation between studies. Linear and non-linear dose-response analyses were done to evaluate the dose-response relations between protein intake and mortality.

Results 32 prospective cohort studies were included in the systematic review and 31 in the meta-analysis. During the follow-up period of 3.5 to 32 years, 113 039 deaths (16 429‬ from cardiovascular disease and 22 303‬ from cancer) occurred among 715 128 participants. Intake of total protein was associated with a lower risk of all cause mortality (pooled effect size 0.94, 95% confidence interval 0.89 to 0.99, I 2 =58.4%, P<0.001). Intake of plant protein was significantly associated with a lower risk of all cause mortality (pooled effect size 0.92, 95% confidence interval 0.87 to 0.97, I 2 =57.5%, P=0.003) and cardiovascular disease mortality (pooled hazard ratio 0.88, 95% confidence interval 0.80 to 0.96, I 2 =63.7%, P=0.001), but not with cancer mortality. Intake of total and animal protein was not significantly associated with risk of cardiovascular disease and cancer mortality. A dose-response analysis showed a significant inverse dose-response association between intake of plant protein and all cause mortality (P=0.05 for non-linearity). An additional 3% energy from plant proteins a day was associated with a 5% lower risk of death from all causes.

Conclusions Higher intake of total protein was associated with a lower risk of all cause mortality, and intake of plant protein was associated with a lower risk of all cause and cardiovascular disease mortality. Replacement of foods high in animal protein with plant protein sources could be associated with longevity.

Introduction

Cardiovascular disease and cancer are two leading causes of death, contributing to 26.9 million deaths worldwide in 2016. 1 Diet has an important role in these conditions. The optimal macronutrient composition of a diet for supporting longevity remains uncertain, 2 3 particularly for protein intake. A global transition towards higher protein diets has occurred in recent decades. 4 In addition, adherence to a high protein diet has recently become popular because of its possible effects on weight loss, preservation of muscle mass, and increased strength. 5 6

High protein diets have also been linked to improvements in cardiometabolic biomarkers, including blood glucose and blood pressure levels. Increasing evidence suggests that diets rich in protein, particularly protein from plants, significantly decrease serum concentrations of blood lipids, without any significant effect on concentrations of high density lipoprotein cholesterol and the risk of cardiovascular disease. 7 These effects may be related to bioactive peptides and the amino acid composition of plant proteins, but other components in the same foods could also contribute. A significant positive association between animal protein intake and an increased incidence of cardiovascular disease and some cancers has also been reported, 8 which could be attributed to the content of high sulfur amino acids in animal proteins.

Findings on the association between total protein intake and longevity are still controversial. Total protein intake was associated with a decreased risk of mortality in some investigations, 9 10 but others failed to find such evidence. 11 12 The same findings have also been reported for animal or plant proteins. 11 13 14 Several studies found that consumption of animal proteins was associated with a higher risk of mortality, 15 16 17 whereas others reported no significant association between intake of animal or plant proteins and risk of all cause and cause specific mortality. 11 13 18 A recent meta-analysis showed that intake of soy protein was associated with a reduced risk of breast cancer mortality, but it was not associated with all cause and cardiovascular disease mortality. 19 No information is available for the strength and shape of a dose-response relation between consumption of proteins and risk of mortality. We conducted a systematic review and dose-response meta-analysis of prospective cohort studies to summarise the association between intake of dietary protein and risk of mortality from all causes, cardiovascular disease, and cancer.

Findings from this systematic review and meta-analysis were reported based on the preferred reporting items for systematic review and meta-analysis (PRISMA) guideline. 20

Search strategy

We conducted a systematic search of all articles published up to 31 December 2019 of online databases, including PubMed/Medline, ISI Web of Science, and Scopus, with no limitation on language or time of publication. Supplementary table 1 provides details of the search terms. To avoid missing any publication, we also checked the reference lists of extracted papers and recent reviews. Unpublished studies were not included because they could have been of lower methodological quality than published studies owing to the absence of peer review. 21 Duplicate citations were removed.

Inclusion and exclusion criteria

Published studies were included if they were observational prospective studies conducted on human adults, or studies that reported effect sizes including hazard ratios or relative risks or odds ratios with the corresponding 95% confidence intervals for the association between intake of total protein, animal protein, or plant protein as the exposure of interest and mortality from all causes, cardiovascular disease, total or specific cancers as the outcome of interest. All outcomes were classified based on the World Health Organization’s ICD-10 (international classification of diseases, 10th revision). 22 If the same dataset had been published in more than one publication, we included the one with more complete findings or the greatest number of participants.

We excluded letters, comments, reviews, meta-analyses, and ecological studies. We also excluded studies performed on children or adolescents and on patients with chronic kidney disease or who were undergoing haemodialysis, end stage cancer, or critical illness. In addition, studies that considered urine urea nitrogen, as a surrogate index of protein intake, and those that considered individual dietary sources of protein as the exposure, rather than total protein, were excluded. If a study reported the effect sizes for risk of disease and mortality combined, we did not include it in the analysis. Moreover, studies with insufficient data were excluded, as were studies on protein intake from specific sources such as soy or legumes.

Data extraction

Two researchers (SN and OS) conducted data extraction independently and resolved any disagreements in consultation with the principal investigator (AE). From each eligible article we extracted the name of the first author, publication year, study design, location of study, age range and health status at study entry, sex, cohort size, incidence of death, duration of follow-up, exposure, method used for assessment of exposure, comparison categories, and relevant effect sizes of comparison categories together with 95% confidence intervals and confounding variables adjusted for in the statistical analysis. When the data were reported for men and women separately, we considered each part as a distinct study. If an included study reported several risk estimates, we extracted the fully adjusted effect sizes. Numerical estimates were extracted from graphs using Plot Digitizer ( http://plotdigitizer.sourceforge.net/ ).

Risk of bias assessment

Risk of bias was assessed using the non-randomised studies of exposures (ROBINS-E) tool. 23 This tool comprises seven domains—bias due to confounding, departure from intended exposures, and missing data, and bias in the selection of participants, classification of exposures, measurement of outcomes, and selection of reported results. Studies were categorised as low risk, moderate risk, serious risk, and critical risk of bias under each domain. Supplementary table 2 presents the results of the risk of bias assessment.

Statistical methods

Odds ratios, relative risks, and hazard ratios (along with 95% confidence intervals) for comparison of the highest versus lowest categories of total, animal, and plant protein intake were used to calculate log odds ratios, relative risks, and hazard ratios with standard errors. A random effects model was used for analyses, in which we calculated both the Q statistic and I 2 as indicators of heterogeneity. 24 25 26 27 I 2 values greater than 50% were considered as significant heterogeneity between studies. 21 A random effects model can account for variation between studies, and thus it can provide more conservative results than a fixed effects model. 28 29

For studies that reported effect sizes separately for intake of animal and plant protein, we first combined the estimates by using the fixed effects model to obtain an overall estimate and then included the pooled effect size in the meta-analysis. Studies that investigated only cancer or cardiovascular disease mortality in relation to protein intake were also considered in the meta-analysis of all cause mortality. If an estimate was reported for the lowest category of protein intake compared with the highest category, we computed the highest versus lowest estimates using the Orsini method. 30 When significant heterogeneity between studies was found, we performed a subgroup analysis to examine possible sources of heterogeneity. These analyses were based on study location, duration of follow-up, sex, dietary assessment tools, health status of study participants, high versus low/middle income countries, single/repeated measurements of protein intake, effect size type, and statistical controlling for confounders (body mass index (BMI), total energy intake, and macronutrients (fat and carbohydrate)). Heterogeneity between subgroups was examined with a fixed effects model.

Publication bias was examined by visual inspection of funnel plots. Formal statistical assessment of funnel plot asymmetry was also done with Egger’s regression asymmetry test and Begg’s test. A trim and fill method was used to detect the effect of probable missing studies on the overall effect. We also conducted a sensitivity analysis using a fixed effects model, in which each prospective cohort study was excluded in turn to examine the influence of that study on the overall estimate.

A method suggested by Greenland 31 and Orsini 30 was used to compute the trend from the odds ratios, relative risks, or hazard ratios estimates and their respective 95% confidence intervals across categories of protein intake. In this method, the distribution of cases and the odds ratios, relative risks, or hazard ratios with the variance estimates for three or more quantitative categories of exposure were required. We considered the midpoint of dietary protein intake in each category. For studies that reported the protein intake as a range, we estimated the midpoint in each category by calculating the mean of the lower and upper bound. When the highest and lowest categories were open ended, we assumed the length of these open ended intervals to be the same as those of the adjacent intervals.

A two stage, random effects dose-response meta-analysis was applied to examine a possible non-linear association between protein intake and mortality. This meta-analysis was done through modelling of protein intake and restricted cubic splines with three knots at fixed centiles of 10%, 50%, and 90% of the distribution. Based on the Orsini method, 30 we calculated restricted cubic spline models by a generalised least squares trend estimation method, which takes into account the correlation within each set of reported odds ratios, relative risks, or hazard ratios. The study specific estimates were then combined by the restricted maximum likelihood method in a multivariate random effects meta-analysis. 32 A probability value for non-linearity was estimated by null hypothesis testing, in which the coefficient of the second spline was considered equal to zero. A linear dose-response association between an additional 3% of energy from proteins and mortality was investigated by use of the two stage generalised least squares trend estimation method. Study specific slope lines were first estimated and then these lines were combined to obtain an overall average slope. 30 Study specific slope lines were combined by a random effects model. Statistical analyses were conducted using STATA version 14.0. A P value of less than 0.05 was considered significant for all tests, including Cochran’s Q test.

Patient and public involvement

No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for design, or implementation of the study. No patients were asked to advise on interpretation or writing up of results. There are no plans to disseminate the results of the research to study participants or the relevant patient community.

Literature search

Overall, 18 683 articles were identified in the initial search. After exclusion of duplicate papers and those that did not meet the inclusion criteria, 57 full text articles of potentially relevant studies were identified. After full text review, an additional 25 articles were excluded: seven that enrolled patients with chronic renal diseases or who were undergoing haemodialysis, six that were conducted in the intensive care unit or on critically ill patients, one that was conducted on patients with end stage cancer, and four that reported associations with dietary sources of protein, rather than intake of total protein. 34 35 36 37 One article that combined mortality and ischaemic heart disease as the outcome was also excluded. 38 Another paper that had considered urine urea nitrogen as a surrogate index of protein intake and reported the hazard ratio for mortality across categories of overnight urine urea nitrogen was excluded. 39 One article that had considered total dietary patterns 40 and three with insufficient data 41 42 43 were also excluded. In one study, the type of protein intake was assessed rather than amount in relation to mortality and was therefore excluded. 44

Finally, 32 papers of cohort studies were included in the systematic review, 7 9 10 11 12 13 14 15 16 17 18 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 and 31 papers were included in this meta-analysis. 7 9 10 11 12 13 14 15 16 17 18 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 63 64 65 Twenty two papers reported effect sizes for all cause mortality, 7 9 10 11 12 13 14 15 16 17 18 46 47 49 50 51 52 54 55 59 63 65 17 for cardiovascular disease mortality, 9 10 11 13 14 15 16 17 18 47 49 50 51 53 57 58 64 and 14 for cancer mortality. 7 9 13 14 15 16 17 18 45 46 48 56 60 61 Of these publications, 26 had reported effect sizes for intake of total protein, 7 9 10 12 14 16 17 18 45 46 47 48 49 50 51 52 53 54 55 56 59 60 61 63 64 65 16 for intake of animal protein, 7 9 10 12 13 14 15 16 18 45 49 56 58 61 62 64 and 18 for intake of plant protein. 7 9 10 11 12 13 14 15 16 18 45 49 56 57 58 61 62 64 Figure 1 shows a flow diagram of study selection.

Flow diagram of study selection

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Of 32 included publications in the systematic review, some studies were done on the same populations. The study by Song et al 15 was conducted on the Nurses’ Health Study and the Health Professionals Follow-up Study datasets, and the study by Preis et al 64 was conducted on the Health Professionals Follow-up Study dataset. In the current study, the study by Song et al 15 was included in the main analysis because it was more complete than the study by Preis et al, but it lacked the required data for the dose-response analysis between intake of total protein and mortality. The study by Preis et al 64 had reported such information, however, so was included in the dose-response analysis. The study by Song et al 15 was considered in the calculation of total number of participants and cases of mortality.

In addition, two papers (Papanikolaou et al 2019 13 and Levine et al 2014 17 ) were published based on the dataset of the Third National Health and Nutrition Examination Survey (NHANES III). The study by Papanikolaou et al 13 was included in the main analysis owing to its comprehensiveness; however, because of lack of required data for the dose-response analysis in that study, 13 we also used the study by Levine et al. 17

Three additional studies ((Holmes et al 1999 and 2017, 55 56 Song et al 2018 7 ) were also published, based on the Nurses’ Health Study or Health Professionals Follow-up Study datasets. All three studies were on patients with cancer, who were excluded from the other studies published from these datasets. Therefore these three studies were included. The two studies by Holmes et al 55 56 were performed on patients with breast cancer in the Nurses’ Health Study dataset; one had reported the effect size for cancer mortality and the other the effect size for all cause mortality. Therefore both were included. To calculate total number of participants and cases of mortality, one of the duplicate publications (Holmes et al 2017, 56 Song et al 2016, 15 Papanikolaou et al 2019 13 ) was considered.

Characteristics of included studies

Tables 1-3 show the characteristics of the included prospective cohort studies. The number of participants in these studies ranged from 288 to 135 335, with an age range between 19 and 101 years. In total 715 128 participants were included in the 32 publications considered in this systematic review. During the follow-up periods ranging from 3.5 to 32 years, the total number of deaths from all causes was 113 039, from cardiovascular disease was 16 429,‬ and from cancer was 22 303. The sample size from the most comprehensive report was considered when it was published more than once. 13 15 56 Three articles included only men, 12 61 64 and seven publications included only women. 11 14 48 55 56 60 63 Of the remaining studies, three papers had reported hazard ratios for men and women separately. 9 52 58 In total, 14 publications described studies in the United States, 7 11 13 14 15 17 52 53 55 56 61 62 63 64 17 in non-US countries, 9 10 12 16 18 45 46 47 48 49 50 54 57 58 59 60 65 and 1 the populations from 18 different countries. 51

Characteristics of included studies for association between protein intake and all cause mortality in adults aged 19 or older

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Characteristics of included studies for the association between protein intake and cardiovascular disease mortality in adults aged over 18

Characteristics of included studies for the association between protein intake and cancer mortality in adults aged >18 years

To examine protein intake, 11 publications had used dietary records or recalls 10 12 13 17 47 52 53 57 59 61 65 and 19 had used a food frequency questionnaire. 7 9 11 14 15 16 18 45 46 48 49 51 55 56 58 60 62 63 64 In the studies by Halbesma et al 54 and Courand et al, 50 intake of total protein was estimated with the use of overnight urine urea nitrogen. In total, 31 publications used baseline data of protein intake in their analysis (single measurement), whereas one article considered the average protein intake throughout the follow-up (repeated measurements) as the main exposure. 15 All studies except for one 60 adjusted the associations for age.

Most cohorts controlled for some conventional risk factors, including BMI (n=24), smoking (n=22), and alcohol consumption (n=14). Others also adjusted for physical activity (n=14), energy intake (n=25), other dietary variables (n=14), and macronutrients (fat or carbohydrate; n=12). Based on the ROBINS-E tool, 15 articles had a low risk of bias in all components (supplementary table 2). 7 11 12 13 14 15 16 17 18 46 51 55 62 63 64 Nine papers provided effect sizes for mortality from cardiovascular disease and cancer, without reporting any effect size for all cause mortality. 10 45 48 53 56 57 58 60 61 The reported effect sizes in these studies were combined and the overall effect size was considered in the meta-analysis of all cause mortality.

Systematic review

Of 29 articles on the association between intake of total protein and all cause mortality, six reported an inverse association, 9 10 47 48 54 55 one showed a positive association, 7 and the others reported no significant association. 12-18 45 46 49-53 56 58-61 63-65 For the association between intake of animal protein and all cause mortality, two studies showed an inverse association 10 16 and the others indicated no significant association. 7 9 12-15 18 45 49 58 61 62 64 Moreover, seven publications showed an inverse association between intake of plant protein and all cause mortality. 9 11 16 18 49 57 64 For cardiovascular disease mortality, two studies reported a protective association with intake of total protein, 10 47 one study with animal protein, 10 and six articles with plant protein. 11 14 15 18 57 64 One study indicated an inverse association between intake of total protein and cancer mortality. 48 One study also showed an inverse association between intake of plant protein and cancer mortality. 7

Meta-analysis on protein intake and all cause mortality

Of 29 papers on intake of total protein and all cause mortality, 21 presented sufficient data for comparison of the highest versus lowest categories of total protein intake. 9 10 12 13 14 15 16 18 45 46 48 50 51 54 55 58 59 60 61 63 65 Of 480 304 participants included in these articles, 72 261 died. The summary effect size for all cause mortality comparing the highest and lowest intakes of total protein was 0.94 (95% confidence interval 0.89 to 0.99, P=0.02), indicating a significant inverse association between total protein intake and all cause mortality ( fig 2 ). Significant heterogeneity was seen between studies (I 2 =58.4%, P<0.001).

Forest plot for association between protein intake and risk of all cause mortality in adults aged 19 or older, expressed as comparison between highest and lowest categories of protein intake. Diamonds represent pooled estimates from random effects analysis

When the association between consumption of animal protein and all cause mortality was examined in 11 publications, 10 11 13 14 15 16 17 19 56 58 61 including a total of 304 100 participants and 60 495 deaths, no significant association was found (pooled effect size comparing highest and lowest intakes was 1.00, 95% confidence interval 0.94 to 1.05, P=0.86), with moderate heterogeneity among the studies (I 2 =45.2%, P=0.04; fig 2 ). Consumption of plant protein, however, which was examined in 13 articles 10 11 12 13 14 15 16 17 19 56 57 58 61 with a total of 439 339 participants and 95 892 deaths, was inversely associated with all cause mortality (pooled effect size comparing the highest and lowest intakes was 0.92, 0.87 to 0.97, P=0.002), with significant heterogeneity among the studies (I 2 =57.5%, P=0.003; fig 2 ).

Meta-analysis on protein intake and cardiovascular disease mortality

Ten publications 9 10 13 14 15 16 18 50 51 58 examined the association between intake of total protein and risk of cardiovascular disease mortality. These studies included a total of 427 005 participants and 15 518 deaths. The summary effect size for cardiovascular disease mortality, comparing the highest and lowest protein intakes, was 0.98 (95% confidence interval 0.94 to 1.03, P=0.51), indicating no clear significant association between total protein intake and cardiovascular disease mortality ( fig 3 ). No significant heterogeneity was seen among the studies (I 2 =16.4%, P=0.28).

Forest plot for association between protein intake and risk of cardiovascular disease mortality in adults aged 19 or older, expressed as comparison between highest and lowest categories of protein intake. Diamonds represent pooled estimates from random effects analysis

The association between consumption of animal protein and cardiovascular disease mortality was examined in eight papers, 9 10 13 14 15 16 18 58 which included 290 542 participants and 13 667 deaths. No significant association was found (pooled effect size comparing the highest and lowest intakes was 1.02, 95% confidence interval 0.94 to 1.11, P=0.56), with no significant heterogeneity among the studies (I 2 =31.7%, P=0.16; fig 3 ). For plant protein consumption, however, which was examined in 10 articles 9 10 11 13 14 15 16 18 57 58 with a total of 425 781 participants and 14 021 deaths, an inverse association was found with cardiovascular disease (pooled effect size comparing the highest and lowest intakes was 0.88, 0.80 to 0.96, P=0.003; fig 3 ). No significant heterogeneity was found between studies (I 2 =63.7%, P=0.001).

Meta-analysis on protein intake and cancer mortality

Twelve papers, 9 13 14 15 16 18 45 46 48 56 60 61 with a total of 292 629 participants and 22 118 deaths, examined the association between intake of total protein and cancer mortality. The summary effect size for cancer mortality comparing the highest and lowest protein intakes was 0.98 (95% confidence interval 0.92 to 1.05, P=0.63), indicating no clear association; however, evidence of moderate heterogeneity was found between studies (I 2 =40.9%, P=0.06; fig 4 ). The same findings were obtained for animal protein consumption and cancer mortality based on nine publications 9 13 14 15 16 18 45 56 61 with a total of 274 370 participants and 21 759 deaths (pooled effect size comparing the highest and lowest protein intakes was 1.00, 95% confidence interval 0.98 to 1.02, P=0.88), with no significant heterogeneity among the studies (I 2 =0%, P=0.46; fig 4 ). This was also the case for plant protein consumption, which was examined in nine articles 9 13 14 15 16 18 45 56 61 with a total of 274 370 participants and 21 759 deaths (pooled effect size comparing the highest and lowest protein intakes was 0.99, 0.94 to 1.05, P=0.68). Moreover, no significant heterogeneity among the studies was found in this case (I 2 =12.2%; P=0.33; fig 4 ).

Forest plot for association between protein intake and risk of cancer mortality in adults aged 19 or older, expressed as comparison between highest and lowest categories of protein intake. Diamonds represent pooled estimates from random effects analysis

Linear and non-linear dose-response analysis

Eight 9 10 16 17 18 46 51 64 of 21 publications on the association between total protein intake and all cause mortality were included in the dose-response analysis ( fig 5 ). No significant non-linear association was found (P=0.40 for non-linearity). Furthermore, linear dose-response meta-analysis showed no significant association between total protein intake and all cause mortality by an additional 3% of energy from protein a day (pooled effect size 0.99, 0.97 to 1.00, P=0.10; supplementary fig 1). Combining data from five 10 15 16 18 of 11 papers in the dose-response analysis of animal protein intake and all cause mortality, no significant non-linear association was seen (P=0.54 for non-linearity; fig 5 ). Moreover, the linear association between an increase of 3% of energy from animal proteins a day and all cause mortality was not significant (pooled effect size 0.99, 0.96 to 1.02, P=0.61; supplementary fig 1). In the dose-response analysis of plant protein intake and all cause mortality, based on six articles 9 10 15 16 18 57 of 13 publications, a significant non-linear association was found (P=0.05 for non-linearity; fig 5 ). Based on linear dose-response analysis, an additional 3% of energy from plant proteins a day was associated with a 5% lower risk of death from all causes (pooled effect size 0.95, 95 0.93 to 0.98, P<0.001; supplementary fig 1).

Non-linear dose-response association of intakes of total, animal, and plant protein (based on percentage of kcal/day ( 1 kcal=4.18 kJ=0.00418 MJ ) with risk of mortality from all causes, cardiovascular disease (CVD), and cancer in adults aged 19 or older. Dietary intake of protein was modelled with restricted cubic splines in a multivariate random effects dose-response model. Black line indicates the linear model; solid purple line indicates the spline model; dashed lines represent 95% confidence intervals. ES=effect size

Non-linear dose-response analysis of seven of 10 papers 9 10 16 17 18 51 64 showed no significant association between intake of total protein and cardiovascular disease mortality (P=0.07; fig 5 ). Findings from a linear dose response meta-analysis showed no significant association between total protein intake and cardiovascular disease mortality (pooled effect size 0.98, 0.97 to 1.00, P=0.08; supplementary fig 2). No significant non-linear association was found between animal protein intake and cardiovascular disease mortality based on five publications 9 10 15 16 18 (P=0.37 for non-linearity; fig 5 ). As with non-linear dose-response meta-analysis, linear dose-response analysis showed no significant association between animal protein intake and cardiovascular disease mortality based on an additional 3% of energy from animal proteins a day (pooled effect size 0.98, 0.94 to 1.02, P=0.32; supplementary fig 2). An inverse association between plant protein intake and cardiovascular disease mortality was found in the non-linear dose-response analysis based on six articles 9 10 15 16 18 57 (P<0.001 for non-linearity; fig 5 ). Linear dose-response analysis showed no significant association between an additional 3% of energy from plant protein intake and cardiovascular disease mortality (pooled effect size 0.96, 95 0.89 to 1.04, P=0.30; supplementary fig 2).

Of 13 papers on the association between intake of total protein and cancer mortality, five 9 16 17 18 46 were included in the non-linear dose-response analysis. No significant association was found between intake of total protein and cancer mortality (P=0.84; fig 5 ). This was also the case for animal protein (P=0.93) and plant protein intakes (P=0.52) based on four papers 9 15 16 18 ( fig 5 ). Linear dose-response analysis showed that an additional 3% of energy from total protein intake (pooled effect size 0.98, 0.94 to 1.03, P=0.39), animal protein intake (0.99, 0.96 to 1.02, P=0.50), and plant protein intake (0.94, 0.85 to 1.03, P=0.19) was not associated with cancer mortality (supplementary fig 3).

Subgroup and sensitivity analyses, and publication bias

To test the robustness of the findings and investigate possible sources of heterogeneity between studies, subgroup analyses were conducted. These analyses were performed based on predefined criteria, including study location, duration of follow-up, sex, dietary assessment tools, health status of study participants, high versus low or middle income countries, single or repeated measurements of protein intake, effect size type, and statistical controlling for confounders (BMI, total energy intake, and macronutrients (fat and carbohydrate)). Supplementary table 3 presents findings for the different subgroups.

A significant inverse association was seen between total protein intake and all cause mortality in women, in studies that used a food frequency questionnaire for assessment of total protein intake, among those studies that did not control for total energy intake and macronutrients intake, those with a follow up duration of less than 15 years, and those that were performed on people with comorbidities. For cardiovascular disease mortality, a significant inverse association with total protein intake was seen in studies that did not control for total energy intake and macronutrients intake, and among those with a follow-up of less than 15 years.

For animal protein intake, a significant inverse association was seen with all cause mortality in studies with a follow up duration of less than 15 years. In addition, inverse associations between animal protein intake and mortality from cardiovascular disease were observed in studies that did not control for macronutrients intake.

Plant protein intake was inversely associated with all cause mortality in both men and women, in studies that were performed in US and non-US countries, in studies with a follow-up of more than 15 years and less than 15 years, in studies that applied a food frequency questionnaire for dietary assessment, among studies that controlled their analysis for energy and macronutrients intakes and BMI, in studies that were done on individuals without comorbidities, in studies performed in high income countries, and in studies that reported a hazard ratio for their analysis. The same findings were also seen between plant protein intake and cardiovascular disease mortality, but this association was not significant in men and women in studies that were performed in the US and those with a follow-up of more than 15 years.

Findings from the sensitivity analysis using a fixed effects model showed that exclusion of the studies by Song et al, 15 Kurihara et al, 57 Budhathoki et al, 18 and Sun et al 11 resulted in a change in the significant inverse association between plant protein intake and cardiovascular disease mortality to a marginally significant inverse association. Sensitivity analysis for the other associations examined showed that exclusion of any single study from the analysis did not appreciably alter the pooled effect sizes. No missing studies were imputed in regions of the contour enhanced funnel plots. No publication bias was found based on Begg’s rank correlation test. For the association between total protein intake and mortality from all causes and from cardiovascular diseases, and between plant protein intake and mortality from cardiovascular diseases, Egger’s linear regression test indicated possible publication bias. Application of the trim and fill method, however, did not result in a change in the average effect size, further suggesting that the results were not affected by publication bias.

In this systematic review and meta-analysis, we found a significant inverse association between intake of total protein and all cause mortality; no clear significant association was seen between total or animal protein intake and cardiovascular disease and cancer mortality. Intake of plant protein was associated with a lower risk of all cause and cardiovascular disease mortality. The inverse associations between plant protein intake and mortality from all causes and cardiovascular disease remained significant in studies that controlled for energy, BMI, and macronutrients intake, and in studies with follow-up of less than 15 years, and those that applied a food frequency questionnaire for dietary assessment.

Comparison with other studies

We systematically and quantitatively summarised earlier investigations on the association between intake of total, animal, and plant proteins and mortality. A recent systematic review and meta-analysis showed that consumption of soy protein was significantly associated with a decreased risk of mortality from breast cancer, but it was not associated with mortality from all causes and cardiovascular disease. 19 In addition, high intake of legumes, grains, and nuts as major sources of plant proteins was associated with a lower risk of all cause and cardiovascular disease mortality. 66 67 Long term observational studies indicated that high consumption of total and animal proteins was associated with an increased risk of cancer and diabetes. 17 68 69 Substitution of non-meat proteins for meat proteins has been favourably associated with fasting insulin levels and reduced insulin resistance. 70 Consumption of low carbohydrate, high protein, and fat diets was not associated with increased risk of coronary heart disease in women. When vegetable sources of fat and protein were chosen, however, these diets were associated with a lower risk of coronary heart disease. 71 Overall, all available studies support the beneficial effects of plant proteins on human health.

In this meta-analysis, no significant association was seen between animal protein intake and mortality. Unlike our findings, one meta-analysis found that each reduction of three servings of processed meat in a week was associated with a small reduction in the risk of overall cancer mortality over a lifetime. 72 In addition, fish consumption was associated with a lower risk of all cause mortality among high consumers than among those with the lowest intake. 73 Thus lack of a significant association between animal protein intake and mortality in our meta-analysis could be due to combining protein from different animal sources, including poultry, eggs, and dairy foods. Also, the discrepant associations of animal meat and animal protein intake. Here, we compared our findings with a previous meta-analysis 72 on animal meat. In that meta-analysis the exposure variable was meat as a food group, whereas our exposure variable was protein as a nutrient. Animal meat contains fat, sodium, iron, and B vitamins in addition to protein so that those nutrients could affect the risk of mortality differently, whereas animal protein is protein only from animal sources. Therefore, findings for animal meat and animal protein could be different. with mortality explained by the fat content of meat. Some studies investigating the association of animal protein intake and mortality have controlled their analysis for fat intake. 12 15 16 18 58 62 64 In addition, different methods used in the processing and cooking of meats might provide further explanation for the discrepancy.

In the interpretation of our findings, it must be considered that humans do not consume single macronutrients, such as proteins. Dietary intake of other nutrients and biologically active factors in foods containing protein could also account for the association between protein intake and mortality. In addition, when the contribution of a single nutrient is assessed as a disease risk, the interaction between nutrients in the gut should be taken into account. Some studies included in this meta-analysis had controlled for the confounding effects of other macronutrients (fat or carbohydrates). 7 12 14 15 16 17 18 46 57 58 62 64 When we confined the analysis to studies that had made these adjustments, the inverse association of plant protein with all cause and cardiovascular disease mortality changed little, whereas the inverse association between intake of total protein and all cause mortality became non-significant. Therefore, dietary fat intake is not likely to account for the protective association between plant protein intake and mortality. Consumption of animal and plant proteins could be a marker of broader dietary intake patterns—or even of social class, an important independent predictor of many health outcomes. Our findings must be interpreted in this context, and future investigations should consider whether intake of animal and plant proteins is a marker of overall dietary patterns or of social class.

In this study, intake of plant protein was inversely associated with mortality from all causes and cardiovascular disease. The same finding was also seen for intake of total protein and all cause mortality. Given that plant protein is part of total protein, the observed inverse association for intake of total protein seems to be related to its plant protein component. The mechanisms through which plant proteins could affect human health are not well known. Whereas consumption of animal protein was associated with increased concentrations of insulin-like growth factor 1, dietary intake of plant proteins was not associated with raised levels. 74 75 Increased levels of insulin-like growth factor 1 have been linked to an increased risk of age related diseases, such as cancers. 76 77 In addition, dietary plant proteins were associated with favourable changes in blood pressure, waist circumference, body weight, and body composition, which might help to lower the risk of several chronic diseases, including cardiovascular disease and type 2 diabetes. 78 Intake of animal protein, independent of body weight, was associated with hypercholesterolaemia, whereas consumption of plant proteins was associated with low levels of plasma cholesterol. 79 80 81

Bacterial fermentation of plant proteins in the gut could help to lower the production of potentially toxic and carcinogenic metabolites, such as ammonia, amines, phenols, tryptophan metabolites, and sulfides. 82 Bioactive peptides derived from plant proteins could also have beneficial health promoting properties. These proteins and peptides have antioxidative, anti-inflammatory, antihypertensive, and antimicrobial activities. 83 84 85 Anorectic peptides have been shown to exert their antiobesogenic activity through decreasing food intake. 86 Earlier studies have indicated that bioactive peptides could reduce blood cholesterol levels. 87 Furthermore, the different associations between animal or plant proteins and mortality risk could be due to the differences in amino acid composition. Plant proteins contain lower amounts of lysine and histidine amino acids than animal proteins; high intake of these amino acids has been shown to increase secretion of lipoproteins containing apo B. 88 Therefore, intake of plant proteins could be associated with protection against cardiovascular diseases through this mechanism. In addition to amino acids, plant proteins are rich in non-essential amino acids such as arginine and pyruvate precursors, which in turn can lead to upregulation of glucagon and downregulation of insulin secretion. 89 The action of glucagon on hepatocytes is mediated through increased cyclic adenosine monophosphate concentrations, which downregulate the synthesis of required enzymes for de novo lipogenesis and upregulate the low density lipoprotein receptors and production of insulin-like growth factor 1 antagonist. 89

Strengths and weaknesses of this study

This meta-analysis has several strengths. Firstly, the large number of participants and deaths included allowed us to quantitatively assess the association of protein intake and risk of mortality, thus making it more powerful than any single study. Secondly, a dose-response analysis was conducted to evaluate the linear and non-linear associations. Thirdly, because all the studies included were prospective, the influence of recall and selection bias is negligible. In addition, we considered subtypes of total protein intake, including animal and plant proteins. These data provide a comprehensive insight into the association between intake of dietary protein and risk of mortality based on the current evidence.

This study has some limitations, most of which are common to observational studies and meta-analyses. Residual or unmeasured confounding factors could have affected the magnitude of the association between protein intake and mortality. Although most studies had controlled for potential confounders, some did not take into account dietary consumption of other nutrients and others did not consider total energy intake and BMI as covariates. Lack of controlling for other nutrients, such as amount and type of dietary fat, which is present in most food sources of protein, could affect the independent association of protein intake with mortality. In addition, some studies in this review did not report sufficient information to be included in the dose-response meta-analysis. Also, different methods for dietary assessment, including food frequency questionnaires, dietary recall, and records, were used in the included cohorts, and the units of protein intake varied in different studies. Measurement errors in dietary assessment are inevitable and would have tended to underestimate the associations with protein intake. In addition, our conclusions about animal protein intake could have less generalisability to low or middle income economics, in which diets are carbohydrate-rich and consumption of animal sources is low.

Conclusions, policy implications, and future research

We found that high intake of total proteins was associated with a lower risk of mortality from all causes. Intake of plant protein was also associated with a lower risk of mortality from all causes and cardiovascular diseases, which is consistent with its beneficial effects on cardiometabolic risk factors, including blood lipid and lipoprotein profiles, blood pressure, and glycaemic regulation. These findings have important public health implications as intake of plant protein can be increased relatively easily by replacing animal protein and could have a large effect on longevity. Also, an additional 3% of energy from plant proteins a day was associated with a 5% lower risk of death from all causes. Our findings therefore strongly support the existing dietary recommendations to increase consumption of plant proteins in the general population. Extrapolation of these findings to the worldwide population should be done cautiously because most studies included in the meta-analysis are from Western nations and few studies have been reported from other countries. Therefore, further studies are required. Additional studies should also focus on the mechanisms through which dietary protein affect mortality.

What is already known on this topic

Consumption of high protein diets has been suggested to control body weight and improve cardiometabolic abnormalities

Regular consumption of red meat and high intake of animal proteins have been linked to several health problems

Data on the association between different types of proteins and mortality are conflicting

What this study adds

High intake of total protein is associated with a lower risk of mortality from all causes

Intake of plant protein is associated with a lower risk of mortality from all causes and from cardiovascular diseases, and an additional 3% of energy from plant proteins a day is associated with a 5% lower risk of death from all causes

These findings support current dietary recommendations to increase consumption of plant proteins in the general population

Contributors: OS and SN contributed to the literature search, data extraction, and data analysis. AE contributed to study conception, manuscript drafting, and data analysis. WCW contributed to study conception, manuscript drafting, data analysis, and approving the final manuscript. All authors acknowledge full responsibility for the analyses and interpretation of the report. AE is the guarantor. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.

Funding: The meta-analysis was funded by the Research Council of School of Nutritional Sciences and Dietetics of Tehran University of Medical Sciences, Tehran, Iran (grant No 46459). The funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication.

Competing interests: All authors have completed the ICMJE uniform disclosure form at http://www.icmje.org/coi_disclosure.pdf and declare: support from the Research Council of School of Nutritional Sciences and Dietetics of Tehran University of Medical Sciences, Tehran, Iran for the submitted work; no financial relationships with any organisation that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work.

Ethical approval: Not required.

Data sharing: No additional data available.

The corresponding author affirms that the manuscript is an honest, accurate, and transparent account of the study being reported. No important aspects of the study have been omitted and any discrepancies from the study as planned have been disclosed.

Dissemination to participants and related patient and public communities: No patients were involved in setting the research question or the outcome measures, nor were they involved in the design and implementation of the study. We plan to disseminate these findings to participants in our annual newsletter and to the general public in a press release.

This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/ .

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Dietary protein intake and human health

Affiliation.

• 1 Departments of Animal Science and Medical Physiology and Faculty of Nutrition, Texas A&M University, College Station, Texas 77843, USA. [email protected].
• PMID: 26797090
• DOI: 10.1039/c5fo01530h

A protein consists of amino acids (AA) linked by peptide bonds. Dietary protein is hydrolyzed by proteases and peptidases to generate AA, dipeptides, and tripeptides in the lumen of the gastrointestinal tract. These digestion products are utilized by bacteria in the small intestine or absorbed into enterocytes. AA that are not degraded by the small intestine enter the portal vein for protein synthesis in skeletal muscle and other tissues. AA are also used for cell-specific production of low-molecular-weight metabolites with enormous physiological importance. Thus, protein undernutrition results in stunting, anemia, physical weakness, edema, vascular dysfunction, and impaired immunity. Based on short-term nitrogen balance studies, the Recommended Dietary Allowance of protein for a healthy adult with minimal physical activity is currently 0.8 g protein per kg body weight (BW) per day. To meet the functional needs such as promoting skeletal-muscle protein accretion and physical strength, dietary intake of 1.0, 1.3, and 1.6 g protein per kg BW per day is recommended for individuals with minimal, moderate, and intense physical activity, respectively. Long-term consumption of protein at 2 g per kg BW per day is safe for healthy adults, and the tolerable upper limit is 3.5 g per kg BW per day for well-adapted subjects. Chronic high protein intake (>2 g per kg BW per day for adults) may result in digestive, renal, and vascular abnormalities and should be avoided. The quantity and quality of protein are the determinants of its nutritional values. Therefore, adequate consumption of high-quality proteins from animal products (e.g., lean meat and milk) is essential for optimal growth, development, and health of humans.

Publication types

• Research Support, Non-U.S. Gov't
• Amino Acids / analysis
• Amino Acids / metabolism
• Dietary Proteins / analysis
• Dietary Proteins / metabolism*
• Growth and Development
• Malnutrition / prevention & control
• Muscle Weakness / prevention & control
• Nutritive Value
• Recommended Dietary Allowances
• Amino Acids
• Dietary Proteins

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Home > Books > Grain and Seed Proteins Functionality

Advances in Food Development with Plant-Based Proteins from Seed Sources

Submitted: 02 September 2020 Reviewed: 28 January 2021 Published: 30 June 2021

DOI: 10.5772/intechopen.96273

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Grain and Seed Proteins Functionality

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Increased awareness on the effects of food on human health and the environment has compelled the need to look for alternative food sources. This resulted in the steady increase in demand for plant-based protein foods as opposed to animal food sources on the premises of significant health benefits, environment-friendly sustainable production systems and moral ethics. This trend has also been reflected in recently reviewed national food guides. Research on plant-based food systems primarily aims to understand the nutritional and functional roles of dietary proteins sourced from crop seeds. Recent scientific advances in this field explore the use innovative technologies in the research and commercial applications of seed proteins. The objective of this paper is to review and summarize key research efforts and recent advances on the utility of seed-sourced proteins in the food product development applications. Important topics covered in the review are: exploration of sources of dietary protein seeds, the status of seed dietary protein research for nutrition and health, and the deployment of new and innovative technologies for developing dietary seed proteins. The topics draw on research and publications on the availability, functionality, quality, genetics, and innovative technologies to develop value-added products from dietary plant-based proteins. The review will fill knowledge gaps in the utilization of emerging plant-based protein food systems in relation to nutritional and health benefits, process technologies and promoting food system sustainability.

• dietary proteins
• grain sources
• essential amino acids
• protein bio-availability
• bioactive peptides
• protein functionality
• plant protein genetics

Author Information

Isaac o. daniel *.

• Excel Agrology – SeedTech. Inc., Canada

Mulualem T. Kassa

• BioTEI Inc., University of Manitoba, Canada

*Address all correspondence to: [email protected]

1. Introduction

Proteins are in the class of biological macromolecules which are necessary for virtually all activities in living organisms as they engage in complex interactions among themselves and other macromolecules like polysaccharides and nucleic acids to drive cellular functions. In this sense, protein intake from food sources plays essential biological roles in the diets of humans and livestock. Among the three macronutrients (carbohydrates, fats, and proteins), protein insufficiency and deficiency in diets has been found to cause more anomalies to human health and wellbeing [ 1 , 2 ]. Food-derived health issues constitutes the new threat to global food security and human health. The Food and Agriculture Organization (FAO) of the United Nations estimated that about 15% of the world’s population is chronically hungry due to nutritional inadequacy [ 3 ]. Gosh et al. [ 4 ] estimated that about 1 billion people face nutritional insecurity, suffering from myriads of nutrient deficiencies and poor health because of insufficient protein intake.

Until recently, dietary proteins have been sourced primarily from animal products including meat, eggs, dairy, and blood. However, the production of dietary proteins from animal food sources is raising adverse ecological footprint concerns. In addition, there is a need to double the present global food production by 2050 [ 5 ]. Meeting this challenge in environmentally sustainable ways compel the search for alternative protein sources. The body of literature that quantifies sustainability of animal-based versus plant-based agroecosystem models is growing and most of them found better sustainability in plant-based protein food system [ 6 ]. For example, Eshel et al. [ 7 ] estimated that by replacing meat proteins with plant alternatives, the US could save 35–50% of the Greenhouse Gas (GHG) emission. Besides this, the cultural practices in animal protein production systems are known to depleting non-renewable resources like phosphorous. Continuing the current rate of phosphorous consumption required in animal production operations was estimated to potentially depleting the limited reserves of the world’s phosphorus within 50–100 years [ 8 , 9 ]. Hence, besides health challenges, findings in the environment frontier warrants further research on plant-based protein alternatives.

The plant-based dietary protein supply is being sustained by the grain commodity markets. Grains constitute important ingredients of the diets of livestock and humans. Generally, grains are botanically the seeds of cereals, pseudo-cereals, and legumes commodity crops [ 10 ]. Most of the commercially available plant protein foods in the industry are made from ingredients containing crops of each of these classes of grains. A visualized analysis of FAO’s [ 11 ] food production datasets in the last decade showed steady growth in the value of food ingredients used in the plant-based protein industry using the pseudo-cereals, legume and cereal crops groups ( Figure 1 ). This data suggests that the availability of grain commodities in commercial quantities enable the market to meet the raw material demand for production of plant protein products.

Value of major grain commodities used as ingredients for producing plant-based protein foods over the last decade. Data adapted from FAOSTAT [ 11 ].

The dominance of cereal crop production value does not necessarily interpret to growth over the years. The steady growth in the value of legumes over the last decade indicates value addition of these crops due to the shifts in the consumption of plant dietary protein sources. Over the years, growing concerns over the health implications of gluten diets common in wheat and other cereal crops compels the need to diversify the sources of plant-based proteins. For example, an analysis of the grains production dataset of cereals against pulses over the decade shows that global cereals production trails behind that of leguminous pulses ( Figure 2 ), depicting the shift to gluten-free diets and the revolution of consumption of high protein crops. The consumption pattern also depicts the research investment in diversifying the sources of plant-based foods with protein composition that are suitable for the production of gluten-free foods. Moreover, concerted research efforts tend to focus on enhanced health benefits [ 12 , 13 ]. Along these trends comes the growing knowledge in grain processing for plant-based protein diets, with ripple effects on research-intensive regulatory policies [ 14 , 15 ].

Gross production value of cereals and pulses grain crops over the last decade. Data from FAOSTAT [ 11 ].

The aim of this chapter was to review recent studies on food development based on dietary protein from grain sources. The review seeks to consolidate the state of knowledge in the actively growing field of plant-based proteins that has elicited numerous publications, innovations and technologies in the last few years. In this review, we probed PubMed and associated libraries along with other sources of compelling information or datasets like FAO and WHO etc. The keywords for the calls in PubMed contained “plant-based seed proteins”, covering 2010 to 2020. We probed four research themes - crop source exploration and diversification, health and functional food development, product improvement through processing for functionality, and crop genetics ( Table 4 ).

2. Exploration of dietary protein sources

In this section, we shall explore the scope of crop exploitation for the production of seed dietary proteins vis-a-vis the development of value-added products in the food industry. It should be noted that while the authors of this chapter recognize the broad diversity of seed protein sources in the plant kingdom, the main focus of this chapter is plant protein sources from the grains, which invariably constitutes the dominant input of the plant-based protein food industry.

2.1 Comparative sources of dietary proteins

Recently, the evaluation of protein quality shifted from raw weight or caloric estimates of food dietary content to estimates of nutrient value in foods. The emphasis of dietary protein quality now tends to be based on the bioavailability of individual nutrients measured in terms of true digestibility of amino acids, namely, the essential amino acids (EAA) content retained after digestion [ 16 , 17 ]. EAAs are the amino acids that humans and experimental animal models do not produce in sufficient amounts de-novo , and so they must be acquired from food sources. There are nine EAAs namely; leucine, isoleucine, valine, lysine, threonine, tryptophan, methionine, phenylalanine and histidine. Fürst et al. [ 18 ] introduced the concept of conditionally indispensable amino acids in terms of adequacy especially in relation to disease conditions, thus extending the list of EAAs to include arginine, cysteine, glutamine, proline, and tyrosine.

Many studies that evaluated animal or vegetal foods for dietary proteins established that plant-based proteins have unbalanced EAA nutritional value when compared with animal-based sources [ 18 , 19 ]. Growing evidences from research are however showing that the EAA content of some seed-sourced proteins are quite comparable to those of animal sources. Table 1 shows data from a recent review of studies that compared amino acid profiles of selected high-protein seeds from cereals (wheat), legumes (soybeans), and a pseudo-cereal (quinoa) with animal food products like whey protein, casein, diary, and beef [ 19 ]. The EAA content is considerably comparable between both food sources. Though the findings have generated ambiguity in comparing protein dietary sources, some answers to this puzzle are coming from the accuracy of measurements of protein food quality in terms of the metrics of digestibility and bio-availability of their EAAs.

Essential amino acid scores (EAA) of selected animal-and plant-based protein sources. (data adapted from Gorissen and Witard [ 19 ].

Scores were calculated based on EAA recommendations for a healthy human adult [ 20 ].

The measurement of protein quality in terms of digestibility and bioavailability of EAAs was revised in the early 1990s to 2012 from Protein Digestibility Corrected Amino Acid Score (PDCAAS) to Digestible Indispensable Amino Acid Score (DIAAS) [ 21 , 22 ]. PDCAAS was dropped because of concerns in the capacity to accurately evaluate protein content in terms of digestibility. Firstly, PDCAAS truncates the scores at 1.00, missing out on proteins with higher digestibility values than 1.00. Secondly, its values likely overestimate protein quality since the method uses fecal analysis to obtain protein digestibility. It misses data on nitrogen disappearance in the large intestine, which is not as a result of protein digestion and absorption, but rather to microbial degradation. On the other hand, DIAAS is considered a superior measure of protein quality because it is calculated using ileal digestibility, and the values are not truncated at 1.0 [ 23 ].

DIAAS is an active area of research in the study of grain-based dietary proteins [ 24 , 25 ]. However, evidences from previous studies that compare grain-based dietary proteins to animal proteins typically indicate that animal proteins have higher digestibility scores compared to plant proteins in the human gut [ 26 , 27 , 28 , 29 ]. One of the studies on plant-based dietary proteins compared digestibility values for four animal proteins and four plant proteins in pig guts instead of rats [ 29 ]. The researchers found that the DIAAS of most of the indispensable amino acids from animal sources like whey protein isolates, whey protein concentrate, and milk protein concentrate were significantly greater ( P  < 0·05) than for pea protein concentrate, soya protein isolate, soya flour and wheat. DIAAS evaluation open new research vistas on the true quality of seed proteins.

2.2 Seed sources of dietary proteins

Figure 3 summarizes the amino-acid content of plant food sources of proteins as compiled by FAO. The visualized summary indicates a linear increase in protein and EAA contents from cereal sources to pulses and oilseed crops. The shift to pulses for grain-based proteins was recognized by the 68th United Nations (UN) General Assembly’s declaration of 2016 as the “International Year of Pulses” (IYP) [ 30 ]. The UN-FAO in their implementation of the declaration recognized 12 types of pulses: dry beans, dry broad beans, dry peas, chickpeas, cowpeas, pigeon peas, lentils, Bambara beans, vetches, lupins and pulses nes (not elsewhere specified – minor pulses that do not fall into one of the other categories) [ 30 ]. It’s known that pulses and oilseed crops like soybeans are leguminous species, which are capable of fixing atmospheric nitrogen in symbiosis with Rhizobium (nitrogen fixing bacteria). The profile of legume proteins is mainly albumin, globulin, prolamins, and glutelin in varying compositions [ 31 ]. In grain pulses, legumin and vicilins a predominant and in soybeans there are mainly glycinin and beta-conglycinin, and 2S albumin, all of which generally belongs to the globulin family of seed storage proteins [ 31 ].

Dietary protein and equivalent essential amino acids (EAA) of cereals and legume sources. Data from FAO [ 11 ].

Data on digestibility and bioavailability of legume proteins in terms of DIAAS is still growing. Much of what is known thus far about DIAAS scores of digestibility of EAAs from plant-based proteins comes from comparison of food proteins in the animal guts [ 26 , 27 , 28 , 29 ]. There are however a number of studies reported on DIAAS of legume grains in the guts of different ages of experimental animals and humans. A recent article reported a study on the true digestibility values (percentage of the total indispensable AA from ileal extracts) of some Chinese pulses. The results of the experiment in humans older than 3 years to adults shows that DIAAS was 88% for kidney bean, 86% for mung bean, 76% for chickpeas, 68% for peas, 64% for adzuki bean and 60% for broad beans [ 32 ]. In another study, Kashyap et al. [ 33 ] used the isotopic method to estimate DIAAS for mung bean and reported that the true mean ileal IAA digestibility of mung bean was 70.9 ± 2.1% after dehulling, demonstrating inconsistencies in methodologies of amino acid digestibility and indicating research gaps and need for elaborate datasets for seed dietary protein measurements to meet the quality challenge in the development of grain-based proteins [ 33 ].

As knowledge is advancing on protein quality evaluation of plant-based food sources, Herreman et al. [ 34 ] recently published a comprehensive review of DIAAS scores for 17 various sources of dietary proteins including some seed sources. The data shows that animal sources of dietary protein have high digestibility of lysine and methionine, comparable only with pea and soybeans, while the cereal sources showed the lowest DIAASS for these EAAs ( Figure 4 ). The higher digestibility estimates of lysine and methionine in potatoes and hemp than cereal seeds and some pulses as shown in in Herreman’s dataset, indicates that there are non-conventional sources of plant dietary proteins besides cereals and grain legumes. There are reports on pseudo-cereals (Amaranth, quinoa, hemp, and chia) as sources of plant-based protein ingredients comparable with animal proteins in human diet because of the special functional properties [ 35 , 36 , 37 ]. Other workers reported the presence of high levels of limiting EAAs i.e. lysine and sulfur containing amino acids (methionine + cystine) in cereal and legume proteins respectively [ 38 , 39 , 40 ]. Mattila et al. [ 41 ] published the nutritional values for seven plant-based dietary protein sources namely: buckwheat, fava bean, flaxseed, hemp seed, lupin, quinoa, and rapeseed. The sheer volume of plant species waiting to be explored as dietary protein sources provide opportunities for more research and reviews, especially on DIAAS, to consolidate the knowledge for scaling these research outcomes.

Digestibility scores (DIAAS) of limiting EAAs (lysine and methionine+cysteine) and DIAAS of 17 dietary protein sources according to the 0.5-to 3-year-old reference pattern score. Data from Herreman et al. [ 34 ].

3. Advances in seed protein development for nutritional and health benefits

Much of the interest in plant-based protein sources are driven by health reasons. Since dietary protein and it’s EAAs provide nitrogen (N), which is required to support basic metabolic processes such as protein synthesis and all other cellular activities, it’s crucial to the health of the living systems. Hence advances in this area of research had been very steady in the last decade. We have reviewed a number of reports on health benefits of various grain-based proteins firstly as nutrient sources and secondly as revolutionary bio-refinery health products.

3.1 Functional foods and nutritional benefits from seed dietary proteins

Health Canada defines functional foods as “ordinary food that has components or ingredients added to give it a specific medical or physiological benefit, other than a purely nutritional effect” [ 42 ].

Because plant-based dietary proteins are not known to provide all the EAAs, Krajcovicova-Kudlackova [ 43 ] identified the risk of lower protein synthesis for vegans due to reduced lysine and indispensable Sulfur EAAs in many single plant-based proteins diets. That is same risk of falling short of the recommended daily allowance (RDA) for to achieve N-balance (i.e., N-loss = N-intake), which is about the efficient use of dietary proteins depending on Metabolic Demand (MD) [ 44 , 45 , 46 ]. This coupled with lower bio-availability of plant-based proteins compared to animal proteins compels the need to augment plant protein foods for limiting EAAs. This is the background for research on producing functional foods with plant-based proteins.

Recent reviews show that research in this area can be rounded up in two main strategies – protein complementation and fortification [ 47 , 48 ]. It’s however noteworthy that both research strategies work with protein/EAA quality evaluation in most of the projects. Protein complementation strategies have been studied in various combinations of blending foods that are deficient in certain EAAs with other ingredients that provides the limiting EAAs. Protein blending strategies can either be plant with plant sources, or plant sources with other protein sources to complement limiting EAAs. Márquez-Mota [ 49 ] found that blending low lysine cereal proteins (corn) with low Sulfur amino acids of legume (soybeans) proteins elicited improved metabolism (mTORC1-signaling pathway and hepatic polyribosome profile). Another published research strategy of plant protein complementation involves blending with protein of animals (casein, whey and diary) with plant-sourced ones (soybeans isolates or concentrates) [ 50 , 51 , 52 ].

Berrazaga et al. [ 49 ] detailed 16 clinical studies over the last ten years that assesses nutritional and anabolic properties of plant-based protein sources in animal models and humans with various MDs involving muscle synthesis. Engelen et al. [ 53 ] reported that fortifying soy proteins with branched-chain amino acids (leucine, isoleucine, and valine) relieved muscle wasting in elderly patients with chronic obstructive pulmonary diseases elderly patients. One research question raised in this area of studies is understanding specific nutritional requirements at individual level in different stages and lifestyles. Hopefully, advances in the field of nutrigenomics will open opportunities to fill this wide knowledge gap.

3.2 Bioactive peptides and nutraceutical activities of seed proteins

Nutraceutical products are isolated or purified from foods and generally sold in medicinal forms or as a pharmaceutical alternative which claims physiological benefits or provide protection against chronic disease [ 54 ]. Bioactive peptides have nutraceutical activities, in the intestine, they get absorbed into the blood circulation and exert systemic physiological effects in target tissues. They are sequences between 2 and 20 amino acids that have been reported to inhibit chronic diseases by playing various roles such as antioxidative, immunomodulatory, antihypertensive, hypo-cholesterolemic, anti-obesity and antimicrobial [ 55 ]. They are inactive when they are part of the parent protein sequence, but become activated upon release by in vivo digestion, in vitro enzymatic hydrolysis/fermentation, and food processing with acid, alkali, or heat [ 56 ].

Plant-based proteins are rich sources of bioactive peptides that have specific physiological and biochemical functions. Literature on bioactive peptides sources from seed proteins with physiological effects and health benefits are enormous [ 57 , 58 ]. Soybeans has been the most exploited seed source of bioactive peptides with nutraceutical activities on more than 40 health conditions as demonstrated by publication on over 100 products [ 59 ]. The field of research into bioactive peptides is very active and the literature resources is vast and diverse, but we have summarized a few common bioactive peptides made from seed proteins in Table 2 according to Karami & Akbari-Adergani [ 60 ].

Seed protein derived bioactive peptides with antioxidant activity. Data adapted from Karami & Akbari-Adergani [ 60 ].

4. Advances in the improvement of seeds for plant-based proteins

Researchers employ different methodologies drawn from different scientific fields towards improving plant-based proteins. The strategies for improving plant-based proteins in literature can be viewed as focused on functional improvement on the front-end and on the back-end is genetic improvement of seed protein quality traits in source crops. Investigations on the two strategies draw on mixtures of scientific methodologies. Most studies on functional improvement investigates physico-chemical and sensory properties of food products made with plant-based protein ingredients [ 72 ], while back-end studies leverage basic crop improvement methodologies that integrate various -omics techniques together with modern plant genetics and breeding. In this section, we will review studies related to the functionality of plant-based protein food products and the genetic improvement strategies of their source crops.

4.1 Seed protein analogs of animal protein foods

Plant protein analogs of animal protein foods are the most popular products in the contemporary plant-based protein gaining markets globally. Analogs are substitutes either used as whole foods of ingredients in producing either meat or dairy alternatives. Meat alternatives strives to resemble meat in appearance, texture and taste when hydrated and cooked [ 73 ], necessitating functionality and sensory research on them. Owusu-Apenten [ 74 ] defined protein functionality in foods as measuring the structure of dietary proteins in the context of their performance in food compositions. Functionality testing for food formulations differ between food types, so that the testing required for meat analogs are different from dairy analogs. While the functionality evaluation for meat products includes rheological properties, chewiness, and sensory values like color and taste [ 75 ], the functional evaluation of dairy analog products is by emulsification, foaming, gelation [ 76 ] besides sensory properties like whiteness and flow.

A review of most of the meat alternative products in the market shows that they are made from plant proteins from wheat, rye, barley, and oats containing gluten (gliadins and glutelin), soybeans containing β-conglycinin protein bodies, legumes (prominently peas) containing glycinin and vicilin proteins; and legumin, oilseeds like Canola containing albumins, globulins, glutelin [ 76 ]. Studies on functional properties of plant proteins of meat analog products are very dynamic because the formulations differ in structural forms such as flour, protein concentrates, protein isolates, and peptides. These structural forms interact with protein contents of the ingredients, hence, research and testing of functional properties in terms of physico-chemical composition and sensory evaluation continues to be an area of active research for meat alternatives [ 77 ]. Excellent and current reviews (up to 2020) provide details of physico-chemical studies of meat alternatives in the market [ 76 , 78 , 79 ]. An active area of research is the investigation of reconstruction techniques of plant protein sources. Shia and Xiong [ 80 ] summarized studies in physico-chemical interactions and the aggregation of plant proteins into particles and anisotropic fibrils to impart meat-like texture; they concluded that thermo-extrusion is the principal re-constructuring technique for meat-like fiber synthesis from plant proteins [ 79 ]. Moreover, some workers are investigating digestibility as regulatory interests seeks more transparency including information on protein bio-availability in commercial meat analog products [ 39 , 78 , 81 ]. Kumar et al. [ 75 ] published an up-to-date review of health implications of proteins in existing meat analog products.

Diary analogs in the market are mostly milk, cheese and yoghurt products [ 79 , 82 ]. There are current comprehensive reviews of functionality and sensory evaluation of diary products including milk-like foods from crop plant sources. McClements [ 83 ] compared plant-based milks with cow’s milk with fortified plant -based milks. In the review, two methods of formulating plant-based milk from various crop sources; mechanically breaking down certain plant materials to produce a dispersion of oil bodies and other colloidal matter in water, or by forming oil-in-water emulsions by homogenizing plant-based oils and emulsifiers with water. The review highlighted the physico-chemical properties (viscosity and flow index), structural properties (mean particle diameter and separation rate), and sensory evaluations (whiteness) of various formulations of plant-based milks ( Table 3 ). The data presented shows that the plant milk analog composition have comparable values in structure, optical properties, rheology, stability, and digestibility with cow’s milk ( Table 3 ). Martinez-Padilla et al. [ 79 ] also reviewed various crop sources of plant-based milk analogs for protein digestibility and compared them with cow’s milk. Sim et al. [ 82 ] reviewed plant-based yogurts made by the fermentation of grain-based milks, imparting fermented flavors and probiotic cultures and thereby reducing the protein content of yogurts. The researchers addressed these challenges by exploring high-pressure processing (HPP) of plant protein ingredients as an alternative structuring strategy for the improvement of plant-based yogurts.

Physico-chemical properties of milk analogs from plant-based food sources. Data table reproduced from McClements [ 83 ].

Mean particle diameter (D 32 and D 43 ).

Though research in functional properties of these food classes continues, each emerging formulation of analogs raises research questions on functionality and quality in terms of digestibility.

4.2 Leveraging on modern genetics and breeding for seed protein improvement

The genetic improvement of seed proteins began with discovery of corn endosperm carrying the Opaque-2 gene in homozygous recessive state [ 84 ]. This constituted the genetic background for the development of quality protein maize (QPM) parental populations with increased levels of amino acids lysin and tryptophan. Corn varieties with Opaque-2 double recessive mutant gene are noted for up to 94% lysin content with about 90% bio-availability against 62% lysin content in Opaque-2 heterozygous recessive corn populations [ 85 ]. For example, the Provitamin A biofortified corn varieties are created through marker assisted pyramiding strategies of β -Carotene Hydroxylase, Lycopene- ε -Cyclase and Opaque2 genes through backcrosses and selection breeding [ 86 ].

Performing the same feat achieved in corn in other crops was more challenging. Galili and Amir [ 87 ] compiled a review of studies that involved seed protein improvement by genetically manipulating amino acid contents right from the discovery of Opaque-2 up till 2013. The review showed that apart from maize, classical genetics rarely produced commercially viable varieties in other crops, hence the transgenic breeding methods were engaged. To date, only two genetically modified (GM) events have been commercialized in cereal crops to modify AA-traits [ 88 ]. These are the dapA-gene ( Corynebacterium glutamicum ), which increases free-Lys content and the cor-dapA gene, which encodes the enzyme that catalyzes the first reaction in the Lys biosynthetic pathway [ 88 ]. However, the introgression of foreign genes affects the acceptability of GM crops for cultivation due to the possibility of potential toxicity, allergenic effects, genetic drifts to other crops, and environmental hazards.

Within the last decade, alternative techniques have been developed that makes it possible to avoid the introgression of foreign genes and transgenic GM crops including e.g., cisgenesis, intragenesis and genome editing [ 88 , 89 ]. Genome editing techniques include engineered endonucleases/meganucleases (EMNs), zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) [ 90 , 91 , 92 ]. Genome editing has been used in maize and soybeans to target the gene that encodes enzymes that catalyzes the first step in the biosynthesis pathway of some EAAs [ 90 , 92 ]. However, research studies that incorporate these strategies for seed dietary proteins in seeds are still sparse, though there are reviews on the possible application of these technologies for improving other seed quality traits [ 93 ]. There are prospects of generating populations for improving seed proteins without transgenic breeding with these technologies.

5. Future research gaps

With the current global awareness, the development of best possible organoleptic and nutritious qualities of food from sustainable plant proteins to feed the ever-increasing global population will continue despite the enormous knowledge been generated in the last decade ( Table 4 ). The data on the knowledge base confirms the assertions that opportunities exist to overcome technology obstacles and nutrition and safety challenges in further developing the alternative plant-based protein markets from grain crop sources [ 94 ].

References from calls on PubMed and associated libraries with various research themes and call terms including “plant-based seed proteins”. Calls were restricted to each year of 2010 to 2020. Other references were accessed from associated journals within PubMed and associated libraries.

The health products from plant-based proteins are the key selling points for the emerging consumer shift, because it is where significant growth in research and innovations is happening ( Table 4 ). In this case, the discovery of bioactive peptides is a critical research area in the dynamics of peptide sources, sequences, structure, networks, and functionality in relation to specific health issues or even emergencies like the SARS-CoV-2 pandemic [ 95 ]. A call for bioactive peptides in PubMed for COVID generated 723 reference listings in January 2021. Secondly, the standardization of protein bio-availability is also an active area of knowledge generation that falls under the seed protein quality testing for diversification of protein sources. Under methods of production, the evaluation of functionality has become a space for multiplied research activities as the industry continues to innovate formulations. Composite EAA strategies continues to generate new nutraceuticals, which is exposing new knowledge gaps for the standardization of protocols for protein bio-availability measurements (PDCAAS in the US and PER in Europe) to DIAAS for global regulatory compliance with bio-availability measurements [ 96 ]. Thirdly, industry acceptance thrives on organoleptic acceptance, texture and taste of ever-increasing formulations of animal protein plant analogs, thus standardizing sensory evaluation techniques requires continuing research efforts as products are formulated. Lastly, the field of genetics and breeding of plant protein crops is a space where the knowledge gap remains very wide. Being at the base of the value chain for plant protein innovations, genetics promises future gains for the protein production systems. Besides genetic engineering techniques, one prominent approach in the future of advancing plant-based food production systems is the emerging breeding technique that combines the use of artificial intelligence (AI) individual seed selection, cloud-based omics diversity databases and machine learning algorithms to identify and develop situation specific protein varieties in a short time. With the cloud computing support and robust prediction algorithms, the capacity to analyze large genomic and phenotypic datasets enables scientists and breeders to easily associate genomic sequences with beneficial traits. The outlook for the development of dietary protein seeds with these advances promises the possibility of personalized nutrition, the possibility of cost-effective trait development, accelerated breeding cycles, and better management of environmental resources for better nutrition.

Moreover, with the expanding knowledge in plant proteins will come the need for environmental datasets across the value chain from field to the table. Dynamic datasets on environmental footprints will continue to be in demand to settle contentions of the animal protein and the emerging plant protein industries and strike the balance in the industry.

6. Conclusion

The combination of various factors that compels research and innovations in the field of plant-based dietary proteins include the realities of proven nutritional and health benefits and its benefit in promoting ecologically sustainable food production systems. Research efforts in this field have generated a body of knowledge that requires to be updated and consolidated on a steady basis given the fast pace of research activities and volume of scientific publications. This review provides a modest update on the place of seeds (grains) in the development of plant-based protein foods. The review focused on PubMed library and other literature resources to probe the subjects of crop sources of dietary proteins, the state of functional and health benefits from seed-based dietary proteins, functionality manipulations to achieve animal protein analogs, and the state of crop genetics in the improvement of grain-based dietary proteins. The review illuminates the enormity of information and the fast pace of knowledge generation in three key research themes which in turn creates new knowledge gaps that draws from the other research themes. These key knowledge areas are: (1) Continuous generation of health-related functional foods and nutraceuticals from grain-based proteins. The development of bioactive peptides for specific health issues at specific personal physiological conditions will continue to be an active research area with potentials for advancing nutrigenomics sciences in the near future. (2) Plant protein quality research in terms of bioavailability and functionality of the ever-increasing fortification strategies. The pace of identification and formulation of plant protein foods creates knowledge gaps that demands research attention for the harmonization of regulatory policies in the various global jurisdictions for promoting the seed protein innovation markets. (3) At the base of the value chain of plant-based proteins is the genetics and breeding of targeted dietary protein and nutritional traits. The future will see the application of advancing omics tools, databases, and networks to the breeding of new varieties in record time for the emerging plant-based protein food systems.

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IX. Proteins

This chapter provides an introduction and discussion of proteins and amino acids that are important in the nutrition of food-producing animals.

New Terms Amino acid Dipeptide Essential amino acid Nonessential amino acid Peptide bond Polypeptide Protein

Chapter Objectives

• To describe the chemical structure of proteins and amino acids
• To discuss the different classification of protein and amino acids
• To discuss essential and nonessential amino acids

What Are Proteins?

The word proteins was coined by a Dutch chemist G. J. Mulder and originated from the Greek word “ proteios” , meaning first or most important. Proteins are organic compounds made up of different building blocks (basic units) called amino acids joined together by peptide bonds (Figure 9.1). A dipeptide contains one peptide bond and two amino acids, whereas a tripeptide contains three amino acids and two peptide bonds. A peptide with more than ten amino acids is called a polypeptide. Proteins are essentially large polypeptides. The structure of a protein is determined first by the sequence of individual amino acids it has in the polypeptide chain. This is also called the primary structure of the protein.

Protein: Functions

• Body proteins (e.g., muscle, hair, hooves, skin)
• Blood proteins (e.g., albumin, globulin)
• Tissue proteins (e.g., collagen, keratin)
• Enzymes and hormones
• Immune system antibodies and other peptide growth factors

Protein Functions: Proteins are vital for life and are the major structural components of animal tissues (e.g., skin, muscles, wool, feather, tendons, eggs). In addition, proteins are also involved in biochemical (e.g., enzymes), immunological (e.g., immunoglobulins), transportational (e.g., lipoproteins), and other regulatory (e.g., hormones) activities. Proteins can also provide energy when needed. Many of the structures in animal tissue (e.g., muscle) and metabolic reactions (e.g., enzymes, hormones) are catalyzed by proteins. Therefore, protein synthesis is essential for maintaining life process. Provision of adequate dietary protein and amino acids are essential for maintaining growth, health, and productivity in food-producing animals. Intestinal microflora can synthesize proteins from nonprotein sources in ruminant animals.

Protein requirements vary with life stages and are high during phases of fast growth in young animals and during pregnancy and lactation. Like other macronutrients, proteins contain carbon, oxygen, and hydrogen. In addition, proteins also contain nitrogen and sulfur (in some amino acids). It is the nitrogen that makes proteins very unique in animal nutrition with respect to its digestibility, metabolism, and disposal within the animal body.

Classification of Proteins

Proteins can be classified based on their shape; solubility in water, salt, acid, base, or alcohol; or according to the nature of the prosthetic group.

Classification Based on Solubility and Prosthetic Group

• Albumin (water soluble; present as albumen in egg white; in blood circulation, it performs various functions [e.g., as a carrier of lipids])
• Globulin (soluble in dilute neutral solutions; functions as part of the immune system in body defense [e.g., immunoglobulins]
• Keratins (e.g. wool, hair, feather, hooves, horn)
• Collagen (can be converted to gelatin when heated; present in bone, teeth, tendons, and soft connective tissue)
• Lipoproteins (lipid-carrying protein)
• Hemoprotein (proteins with heme units)
• Glycoproteins (proteins with sugar)
• Nucleoprotein (proteins bound to nucleic acid

These proteins have limited nutritional value but are important in biochemical, structural, and other metabolic functions. For example, feather meal is high in protein (keratin) but very low in digestibility and is of limited use in animal nutrition as a feed ingredient. Amino acids in the polypeptide chain in feather meal form disulfide bonds (-S-S-), which twist the polypeptide chain into a specific coiled structure such as helix or sheet. This is called a secondary structure. These bonds account for the tough physical properties of hooves and horns and their low digestibility.

The disruption of secondary structure by heat treatment causes denaturation of the proteins (e.g., egg white coagulation during cooking). Certain antinutritional factors in feed (e.g., trypsin inhibitor in soybean meal) are proteins. Heat processing denatures trypsin inhibitor in soybean meal and can enhance digestibility.

Amino Acids: Amino acids are the building blocks of proteins. There are more than 300 different amino acids known to exist in nature. Out of these, about 20 amino acids are important constituents of animal proteins and are associated with muscles, connective tissues, skin, feathers, horns, blood, enzymes, and hormones. There about 10 amino acids that should be present in the diet of animals because animal tissues cannot synthesize them or cannot make the adequate amount needed for metabolic functions; these are called essential amino acids. A few other amino acids such as citrulline and ornithine do not occur in animal tissues but are involved in cellular metabolic functions.

All amino acids by definition contain at least one amino group (-NH2) and one carboxyl group (–COOH) on the C atom adjacent to the carboxyl group (Figure 9.2). An exception to this is proline (imino acid), which is lacking a free amino group. The general structure of an amino acid is shown below by the amino acid glycine, the simplest of the amino acids (Figure 9.2). The R group (shown in the red circle) in amino acids varies for different amino acids. The R group is the remainder of the molecule or any other group attached to the C atom. In the case of glycine, it is an H group. The amino group (NH2) provides basic properties to the amino acid, and the carboxyl (COOH) group provides acidic properties. Amino acids important in animal nutrition are alpha (α) amino acids, which are carboxylic acids with an amino group on the α-carbon (or the first carbon attached to a functional group). A list of amino acids important in animal nutrition, their essentiality and classification are shown in Table 9.1.

Amino acids can exist in two isomeric forms, the D- and L-isomers. The D- and L-amino acids differ in their configuration of groups around the asymmetric α-carbon. Only L-amino acids are used in protein synthesis, except methionine, where both D- and  L-amino acids can be used by the animal. DL methionine is commonly used as an amino acid supplement in animal feeds.

Essential Amino Acids: Animal body can synthesize some amino acids in sufficient amounts. However, animals cannot synthesize some amino acids, or not in the amount that is needed for body requirements. Such amino acids need to be provided through diet in monogastric animals; these are called essential (indispensable) amino acids. Pigs, dogs, and humans need a total of 10, and chickens and cats need a total of 11 essential amino acids. A list of essential amino acids needed by monogastric animals is shown below. It should be borne in mind that other nonessential amino acids are also physiologically important for metabolic functions in the body and are made from other precursors available through diet (e.g., carbohydrates, nonprotein nitrogenous substances). The need for essential amino acids varies in animals. For example, horses need essential amino acids, whereas ruminant animals (e.g., cattle, sheep, goats) generally do not have a requirement of essential amino acids as they are synthesized by rumen microbes.

List of Essential Amino Acids and Their Common Abbreviations

• Arginine (Arg)
• Histidine (His)
• Lysine (Lys)
• Isoleucine (Ile)
• Leucine (Leu)
• Methionine (Met)
• Phenylalanine (Phe)
• Threonine (Thr)
• Tryptophan (Try)
• Valine (Val)

In addition to these 10 essential amino acids, cats and chickens need the following extra amino acids. Cats need taurine (Tau), and chickens need glycine (Gly).

• Proteins can be found in structural components of the body and are needed for many metabolic functions.
• The presence of Nitrogen makes protein unique.
• More than 300 amino acids are identified. But only 20 amino acids are used to synthesize all proteins.
• Three features of a typical amino acid include a carbon skeleton, a carboxyl group, and an amino group.
• Acidic amino acids contain more carboxyl groups, and basic amino acids contain more amino groups. Neutral amino acids contain an equal number of carboxyl and amino groups.
• Sulfur-containing amino acids are methionine and cysteine. Among these amino acids, methionine is essential because animals cannot synthesize it. Cysteine is not considered essential because if S is available, the body can make it.
• Aromatic amino acids contain a ring structure.
• Imino acids contain an imino instead of an amino group (e.g., proline).
• Essential amino acids are those that cannot be synthesized by the animal body. There are 10 essential amino acids; cats need taurine, and chickens need glycine.
• Amino acids are joined together by peptide bonds. A long chain of amino acids formed this way is called a polypeptide.
• The primary structure of an amino acid is determined by the sequence of individual amino acids in the polypeptide chain. The amino acids in the polypeptide chain form disulfide bonds and hydrogen bonds, which twist the polypeptide chain into a specific coiled structure, such as a helix or a sheet. This is called a secondary structure.  Proteins can be classified based on their shape; solubility in water, salt, acid, base, or alcohol; or according to the nature of prosthetic groups.

Review Questions

• Name the linkage between two amino acids in a protein.
• What are the essential amino acids? Why are they essential?
• Compared to carbohydrates, why are proteins unique?
• Which amino acid is essential to chickens but not humans? How about to cats?
• Name one amino acid from the following groups: acidic, basic, aromatic, and sulfur-containing.
• Differentiate between essential and nonessential amino acids.
• Is proline an amino acid?
• List the 10 essential amino acids for monogastric animals.
• How many peptide bonds are there in a tripeptide?
• Give an example of a globular, fibrous, and conjugated protein.

A Guide to the Principles of Animal Nutrition Copyright © 2019 by Gita Cherian is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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Protein – Which is Best?

Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed.

• Higher protein needs are seen in athletic populations.
• Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein consumed from primarily animal sources.
• With a proper combination of sources, vegetable proteins may provide similar benefits as protein from animal sources.
• Casein protein supplementation may provide the greatest benefit for increases in protein synthesis for a prolonged duration.

Introduction

The protein requirements for athletic populations have been the subject of much scientific debate. Only recently has the notion that both strength/power and endurance athletes require a greater protein consumption than the general population become generally accepted. In addition, high protein diets have also become quite popular in the general population as part of many weight reduction programs. Despite the prevalence of high protein diets in athletic and sedentary populations, information available concerning the type of protein (e.g. animal or vegetable) to consume is limited. The purpose of this paper is to examine and analyze key factors responsible for making appropriate choices on the type of protein to consume in both athletic and general populations.

Role of Protein

Proteins are nitrogen-containing substances that are formed by amino acids. They serve as the major structural component of muscle and other tissues in the body. In addition, they are used to produce hormones, enzymes and hemoglobin. Proteins can also be used as energy; however, they are not the primary choice as an energy source. For proteins to be used by the body they need to be metabolized into their simplest form, amino acids. There have been 20 amino acids identified that are needed for human growth and metabolism. Twelve of these amino acids (eleven in children) are termed nonessential, meaning that they can be synthesized by our body and do not need to be consumed in the diet. The remaining amino acids cannot be synthesized in the body and are described as essential meaning that they need to be consumed in our diets. The absence of any of these amino acids will compromise the ability of tissue to grow, be repaired or be maintained.

Protein and Athletic Performance

The primary role of dietary proteins is for use in the various anabolic processes of the body. As a result, many athletes and coaches are under the belief that high intensity training creates a greater protein requirement. This stems from the notion that if more protein or amino acids were available to the exercising muscle it would enhance protein synthesis. Research has tended to support this hypothesis. Within four weeks of protein supplementation (3.3 versus 1.3 g·kg -1 ·day -1 ) in subjects’ resistance training, significantly greater gains were seen in protein synthesis and body mass in the group of subjects with the greater protein intake (Fern et al., 1991 ). Similarly, Lemon et al. ( 1992 ) also reported a greater protein synthesis in novice resistance trained individuals with protein intakes of 2.62 versus 0.99 g·kg -1 ·day -1 . In studies examining strength-trained individuals, higher protein intakes have generally been shown to have a positive effect on muscle protein synthesis and size gains (Lemon, 1995 ; Walberg et al., 1988 ). Tarnapolsky and colleagues ( 1992 ) have shown that for strength trained individuals to maintain a positive nitrogen balance they need to consume a protein intake equivalent to 1.8 g·kg -1 ·day -1 . This is consistent with other studies showing that protein intakes between 1.4 – 2.4 g·kg -1 ·day -1 will maintain a positive nitrogen balance in resistance trained athletes (Lemon, 1995 ). As a result, recommendations for strength/power athletes’ protein intake are generally suggested to be between 1.4 - 1.8 g·kg -1 ·day -1 .

Similarly, to prevent significant losses in lean tissue endurance athletes also appear to require a greater protein consumption (Lemon, 1995 ). Although the goal for endurance athletes is not necessarily to maximize muscle size and strength, loss of lean tissue can have a significant detrimental effect on endurance performance. Therefore, these athletes need to maintain muscle mass to ensure adequate performance. Several studies have determined that protein intake for endurance athletes should be between 1.2 – 1.4 g·kg -1 ·day -1 to ensure a positive nitrogen balance (Freidman and Lemon, 1989 ; Lemon, 1995 ; Meredith et al., 1989 ; Tarnopolsky et al., 1988 ). Evidence is clear that athletes do benefit from increased protein intake. The focus then becomes on what type of protein to take.

Protein Assessment

The composition of various proteins may be so unique that their influence on physiological function in the human body could be quite different. The quality of a protein is vital when considering the nutritional benefits that it can provide. Determining the quality of a protein is determined by assessing its essential amino acid composition, digestibility and bioavailability of amino acids (FAO/WHO, 1990 ). There are several measurement scales and techniques that are used to evaluate the quality of protein.

Protein Rating Scales

Numerous methods exist to determine protein quality. These methods have been identified as protein efficiency ratio, biological value, net protein utilization, and protein digestibility corrected amino acid score.

Protein Efficiency Ratio

The protein efficiency ratio (PER) determines the effectiveness of a protein through the measurement of animal growth. This technique requires feeding rats a test protein and then measuring the weight gain in grams per gram of protein consumed. The computed value is then compared to a standard value of 2.7, which is the standard value of casein protein. Any value that exceeds 2.7 is considered to be an excellent protein source. However, this calculation provides a measure of growth in rats and does not provide a strong correlation to the growth needs of humans.

Biological Value

Biological value measures protein quality by calculating the nitrogen used for tissue formation divided by the nitrogen absorbed from food. This product is multiplied by 100 and expressed as a percentage of nitrogen utilized. The biological value provides a measurement of how efficient the body utilizes protein consumed in the diet. A food with a high value correlates to a high supply of the essential amino acids. Animal sources typically possess a higher biological value than vegetable sources due to the vegetable source’s lack of one or more of the essential amino acids. There are, however, some inherent problems with this rating system. The biological value does not take into consideration several key factors that influence the digestion of protein and interaction with other foods before absorption. The biological value also measures a protein’s maximal potential quality and not its estimate at requirement levels.

Net Protein Utilization

Net protein utilization is similar to the biological value except that it involves a direct measure of retention of absorbed nitrogen. Net protein utilization and biological value both measure the same parameter of nitrogen retention, however, the difference lies in that the biological value is calculated from nitrogen absorbed whereas net protein utilization is from nitrogen ingested.

Protein Digestibility Corrected Amino Acid Score

In 1989, the Food & Agriculture Organization and World Health Organization (FAO/WHO) in a joint position stand stated that protein quality could be determined by expressing the content of the first limiting essential amino acid of the test protein as a percentage of the content of the same amino acid content in a reference pattern of essential amino acids (FAO/WHO, 1990 ). The reference values used were based upon the essential amino acids requirements of preschool-age children. The recommendation of the joint FAO/WHO statement was to take this reference value and correct it for true fecal digestibility of the test protein. The value obtained was referred to as the protein digestibility corrected amino acid score (PDCAAS). This method has been adopted as the preferred method for measurement of the protein value in human nutrition (Schaafsma, 2000 ). Table 1 provides a measure of the quantity of various proteins using these protein rating scales.

Protein quality rankings.

Adapted from: U.S Dairy Export Council, Reference Manual for U.S. Whey Products 2nd Edition, 1999 and Sarwar, 1997 .

Although the PDCAAS is currently the most accepted and widely used method, limitations still exist relating to overestimation in the elderly (likely related to references values based on young individuals), influence of ileal digestibility, and antinutritional factors (Sarwar, 1997 ).

Amino acids that move past the terminal ileum may be an important route for bacterial consumption of amino acids, and any amino acids that reach the colon would not likely be utilized for protein synthesis, even though they do not appear in the feces (Schaarfsma, 2000 ). Thus, to get truly valid measure of fecal digestibility the location at which protein synthesis is determined is important in making a more accurate determination. Thus, ileal digestibility would provide a more accurate measure of digestibility. PDCAAS, however, does not factor ileal digestibility into its equation. This is considered to be one of the shortcomings of the PDCAAS (Schaafsma 2000 ).

Antinutritional factors such as trypsin inhibitors, lectins, and tannins present in certain protein sources such as soybean meal, peas and fava beans have been reported to increase losses of endogenous proteins at the terminal ileum (Salgado et al., 2002 ). These antinutritional factors may cause reduced protein hydrolysis and amino acid absorption. This may also be more effected by age, as the ability of the gut to adapt to dietary nutritional insults may be reduced as part of the aging process (Sarwar, 1997 ).

Protein Sources

Protein is available in a variety of dietary sources. These include foods of animal and plant origins as well as the highly marketed sport supplement industry. In the following section proteins from both vegetable and animal sources, including whey, casein, and soy will be explored. Determining the effectiveness of a protein is accomplished by determining its quality and digestibility. Quality refers to the availability of amino acids that it supplies, and digestibility considers how the protein is best utilized. Typically, all dietary animal protein sources are considered to be complete proteins. That is, a protein that contains all of the essential amino acids. Proteins from vegetable sources are incomplete in that they are generally lacking one or two essential amino acids. Thus, someone who desires to get their protein from vegetable sources (i.e. vegetarian) will need to consume a variety of vegetables, fruits, grains, and legumes to ensure consumption of all essential amino acids. As such, individuals are able to achieve necessary protein requirements without consuming beef, poultry, or dairy. Protein digestibility ratings usually involve measuring how the body can efficiently utilize dietary sources of protein. Typically, vegetable protein sources do not score as high in ratings of biological value, net protein utilization, PDCAAS, and protein efficiency ratio as animal proteins.

Animal Protein

Proteins from animal sources (i.e. eggs, milk, meat, fish and poultry) provide the highest quality rating of food sources. This is primarily due to the ‘completeness’ of proteins from these sources. Although protein from these sources are also associated with high intakes of saturated fats and cholesterol, there have been a number of studies that have demonstrated positive benefits of animal proteins in various population groups (Campbell et al., 1999 ; Godfrey et al., 1996 ; Pannemans et al., 1998 ).

Protein from animal sources during late pregnancy is believed to have an important role in infants born with normal body weights. Godfrey et al. ( 1996 ) examined the nutrition behavior of more than 500 pregnant women to determine the effect of nutritional intake on placental and fetal growth. They reported that a low intake of protein from dairy and meat sources during late pregnancy was associated with low birth weights.

In addition to the benefits from total protein consumption, elderly subjects have also benefited from consuming animal sources of protein. Diets consisting of meat resulted in greater gains in lean body mass compared to subjects on a lactoovovegetarian diet (Campbell et al., 1999 ). High animal protein diets have also been shown to cause a significantly greater net protein synthesis than a high vegetable protein diet (Pannemans et al., 1998 ). This was suggested to be a function of reduced protein breakdown occurring during the high animal protein diet.

There have been a number of health concerns raised concerning the risks associated with protein emanating primarily from animal sources. Primarily, these health risks have focused on cardiovascular disease (due to the high saturated fat and cholesterol consumption), bone health (from bone resorption due to sulfur-containing amino acids associated with animal protein) and other physiological system disease that will be addressed in the section on high protein diets.

Whey is a general term that typically denotes the translucent liquid part of milk that remains following the process (coagulation and curd removal) of cheese manufacturing. From this liquid, whey proteins are separated and purified using various techniques yielding different concentrations of whey proteins. Whey is one of the two major protein groups of bovine milk, accounting for 20% of the milk while casein accounts for the remainder. All of the constituents of whey protein provide high levels of the essential and branched chain amino acids. The bioactivities of these proteins possess many beneficial properties as well. Additionally, whey is also rich in vitamins and minerals. Whey protein is most recognized for its applicability in sports nutrition. Additionally, whey products are also evident in baked goods, salad dressings, emulsifiers, infant formulas, and medical nutritional formulas.

Varieties of Whey Protein

There are three main forms of whey protein that result from various processing techniques used to separate whey protein. They are whey powder, whey concentrate, and whey isolate. Table 2 provides the composition of Whey Proteins.

Composition (%) of whey protein forms.

Adapted from Geiser, 2003 .

Whey Protein Powder

Whey protein powder has many applications throughout the food industry. As an additive it is seen in food products for beef, dairy, bakery, confectionery, and snack products. Whey powder itself has several different varieties including sweet whey, acid whey (seen in salad dressings), demineralized (seen primarily as a food additive including infant formulas), and reduced forms. The demineralized and reduced forms are used in products other than sports supplements.

Whey Protein Concentrate

The processing of whey concentrate removes the water, lactose, ash, and some minerals. In addition, compared to whey isolates whey concentrate typically contains more biologically active components and proteins that make them a very attractive supplement for the athlete.

Whey Protein Isolate (WPI)

Isolates are the purest protein source available. Whey protein isolates contain protein concentrations of 90% or higher. During the processing of whey protein isolate there is a significant removal of fat and lactose. As a result, individuals who are lactose-intolerant can often safely take these products (Geiser, 2003 ). Although the concentration of protein in this form of whey protein is the highest, it often contain proteins that have become denatured due to the manufacturing process. The denaturation of proteins involves breaking down their structure and losing peptide bonds and reducing the effectiveness of the protein.

Whey is a complete protein whose biologically active components provide additional benefits to enhance human function. Whey protein contains an ample supply of the amino acid cysteine. Cysteine appears to enhance glutathione levels, which has been shown to have strong antioxidant properties that can assist the body in combating various diseases (Counous, 2000 ). In addition, whey protein contains a number of other proteins that positively effect immune function such as antimicrobial activity (Ha and Zemel, 2003 ). Whey protein also contains a high concentration of branched chain amino acids (BCAA) that are important for their role in the maintenance of tissue and prevention of catabolic actions during exercise. (MacLean et al., 1994 ).

Casein is the major component of protein found in bovine milk accounting for nearly 70-80% of its total protein and is responsible for the white color of milk. It is the most commonly used milk protein in the industry today. Milk proteins are of significant physiological importance to the body for functions relating to the uptake of nutrients and vitamins and they are a source of biologically active peptides. Similar to whey, casein is a complete protein and also contains the minerals calcium and phosphorous. Casein has a PDCAAS rating of 1.23 (generally reported as a truncated value of 1.0) (Deutz et al. 1998 ).

Casein exists in milk in the form of a micelle, which is a large colloidal particle. An attractive property of the casein micelle is its ability to form a gel or clot in the stomach. The ability to form this clot makes it very efficient in nutrient supply. The clot is able to provide a sustained slow release of amino acids into the blood stream, sometimes lasting for several hours (Boirie et al. 1997 ). This provides better nitrogen retention and utilization by the body.

Bovine Colostrum

Bovine colostrum is the “pre” milk liquid secreted by female mammals the first few days following birth. This nutrient-dense fluid is important for the newborn for its ability to provide immunities and assist in the growth of developing tissues in the initial stages of life. Evidence exists that bovine colostrum contains growth factors that stimulate cellular growth and DNA synthesis (Kishikawa et al., 1996 ), and as might be expected with such properties, it makes for interesting choice as a potential sports supplement.

Although bovine colostrum is not typically thought of as a food supplement, the use by strength/power athletes of this protein supplement as an ergogenic aid has become common. Oral supplementation of bovine colostrum has been demonstrated to significantly elevate insulin-like-growth factor 1 (IGF-1) (Mero et al., 1997 ) and enhance lean tissue accruement (Antonio et al., 2001 ; Brinkworth et al., 2004 ). However, the results on athletic performance improvement are less conclusive. Mero and colleagues ( 1997 ) reported no changes in vertical jump performance following 2-weeks of supplementation, and Brinkworth and colleagues ( 2004 ) saw no significant differences in strength following 8-weeks of training and supplementation in both trained and untrained subjects. In contrast, following 8-weeks of supplementation significant improvements in sprint performance were seen in elite hockey players (Hofman et al., 2002 ). Further research concerning bovine colostrum supplementation is still warranted.

Vegetable Protein

Vegetable proteins, when combined to provide for all of the essential amino acids, provide an excellent source for protein considering that they will likely result in a reduction in the intake of saturated fat and cholesterol. Popular sources include legumes, nuts and soy. Aside from these products, vegetable protein can also be found in a fibrous form called textured vegetable protein (TVP). TVP is produced from soy flour in which proteins are isolated. TVP is mainly a meat alternative and functions as a meat analog in vegetarian hot dogs, hamburgers, chicken patties, etc. It is also a low-calorie and low-fat source of vegetable protein. Vegetable sources of protein also provide numerous other nutrients such as phytochemicals and fiber that are also highly regarded in the diet diet.

Soy is the most widely used vegetable protein source. The soybean, from the legume family, was first chronicled in China in the year 2838 B.C. and was considered to be as valuable as wheat, barley, and rice as a nutritional staple. Soy’s popularity spanned several other countries, but did not gain notoriety for its nutritional value in The United States until the 1920s. The American population consumes a relatively low intake of soy protein (5g·day -1 ) compared to Asian countries (Hasler, 2002 ). Although cultural differences may be partly responsible, the low protein quality rating from the PER scale may also have influenced protein consumption tendencies. However, when the more accurate PDCAAS scale is used, soy protein was reported to be equivalent to animal protein with a score of 1.0, the highest possible rating (Hasler, 2002 ). Soy’s quality makes it a very attractive alternative for those seeking non-animal sources of protein in their diet and those who are lactose intolerant. Soy is a complete protein with a high concentration of BCAA’s. There have been many reported benefits related to soy proteins relating to health and performance (including reducing plasma lipid profiles, increasing LDL-cholesterol oxidation and reducing blood pressure), however further research still needs to be performed on these claims.

Soy Protein Types

The soybean can be separated into three distinct categories; flour, concentrates, and isolates. Soy flour can be further divided into natural or full-fat (contains natural oils), defatted (oils removed), and lecithinated (lecithin added) forms (Hasler, 2002 ). Of the three different categories of soy protein products, soy flour is the least refined form. It is commonly found in baked goods. Another product of soy flour is called textured soy flour . This is primarily used for processing as a meat extender. See Table 3 for protein composition of soy flour, concentrates, and isolates.

Protein composition of soy protein forms.

Soy concentrate was developed in the late 1960s and early 1970s and is made from defatted soybeans. While retaining most of the bean’s protein content, concentrates do not contain as much soluble carbohydrates as flour, making it more palatable. Soy concentrate has a high digestibility and is found in nutrition bars, cereals, and yogurts.

Isolates are the most refined soy protein product containing the greatest concentration of protein, but unlike flour and concentrates, contain no dietary fiber. Isolates originated around the 1950s in The United States. They are very digestible and easily introduced into foods such as sports drinks and health beverages as well as infant formulas.

Nutritional Benefits

For centuries, soy has been part of a human diet. Epidemiologists were most likely the first to recognize soy’s benefits to overall health when considering populations with a high intake of soy. These populations shared lower incidences in certain cancers, decreased cardiac conditions, and improvements in menopausal symptoms and osteoporosis in women (Hasler, 2002 ). Based upon a multitude of studies examining the health benefits of soy protein the American Heart Association issued a statement that recommended soy protein foods in a diet low in saturated fat and cholesterol to promote heart health (Erdman, 2000 ). The health benefits associated with soy protein are related to the physiologically active components that are part of soy, such as protease inhibitors, phytosterols, saponins, and isoflavones (Potter, 2000 ). These components have been noted to demonstrate lipid-lowering effects, increase LDL-cholesterol oxidation, and have beneficial effects on lowering blood pressure.

Isoflavones

Of the many active components in soy products, isoflavones have been given considerably more attention than others. Isoflavones are thought to be beneficial for cardiovascular health, possibly by lowering LDL concentrations (Crouse et al., 1999 ) increasing LDL oxidation (Tikkanen et al., 1998 ) and improving vessel elasticity (Nestel et al., 1999 ). However, these studies have not met without conflicting results and further research is still warranted concerning the benefits of isoflavones.

Soy Benefits for Women

An additional focus of studies investigating soy supplementation has been on women’s health issues. It has been hypothesized that considering that isoflavones are considered phytoestrogens (exhibit estrogen- like effects and bind to estrogen receptors) they compete for estrogen receptor sites in breast tissue with endogenous estrogen, potentially reducing the risk for breast cancer risk (Wu et al. 1998 ). Still, the association between soy intake and breast cancer risk remains inconclusive. However, other studies have demonstrated positive effects of soy protein supplementation on maintaining bone mineral content (Ho et al., 2003 ) and reducing the severity of menopausal symptoms (Murkies et al., 1995 ).

High Protein Diets

Increased protein intakes and supplementation have generally been focused on athletic populations. However, over the past few years high protein diets have become a method used by the general population to enhance weight reduction. The low-carbohydrate, high protein, high fat diet promoted by Atkins may be the most popular diet used today for weight loss in the United States (Johnston et al., 2004 ). The basis behind this diet is that protein is associated with feelings of satiety and voluntary reductions in caloric consumption (Araya et al., 2000 ; Eisenstein et al., 2002 ). A recent study has shown that the Atkins diet can produce greater weight reduction at 3 and 6 months than a low-fat, high carbohydrate diet based upon U.S. dietary guidelines (Foster et al., 2003 ). However, potential health concerns have arisen concerning the safety of high protein diets. In 2001, the American Heart Association published a statement on dietary protein and weight reduction and suggested that individuals following such a diet may be at potential risk for metabolic, cardiac, renal, bone and liver diseases (St. Jeor et al., 2001 ).

Protein Intake and Metabolic Disease Risk

One of the major concerns for individuals on high protein, low carbohydrate diets is the potential for the development of metabolic ketosis. As carbohydrate stores are reduced the body relies more upon fat as its primary energy source. The greater amount of free fatty acids that are utilized by the liver for energy will result in a greater production and release of ketone bodies in the circulation. This will increase the risk for metabolic acidosis and can potentially lead to a coma and death. A recent multi-site clinical study (Foster et al., 2003 ) examined the effects of low-carbohydrate, high protein diets and reported significant elevation in ketone bodies during the first three months of the study. However, as the study duration continued the percentage of subjects with positive urinary ketone concentrations became reduced, and by six months urinary ketones were not present in any of the subjects.

Dietary Protein and Cardiovascular Disease Risk

High protein diets have also been suggested to have negative effects on blood lipid profiles and blood pressure, causing an increase risk for cardiovascular disease. This is primarily due to the higher fat intakes associated with these diets. However, this has not been proven in any scientifically controlled studies. Hu et al., ( 1999 ) have reported an inverse relationship between dietary protein (animal and vegetable) and risk of cardiovascular disease in women, and Jenkins and colleagues ( 2001 ) reported a decrease in lipid profiles in individuals consuming a high protein diet. Furthermore, protein intake has been shown to often have a negative relationship with blood pressure (Obarzanek et al., 1996 ). Thus, the concern for elevated risk for cardiovascular disease from high protein diets appears to be without merit. Likely, the reduced body weight associated with this type of diet is facilitating these changes.

In strength/power athletes who consume high protein diets, a major concern was the amount of food being consumed that was high in saturated fats. However, through better awareness and nutritional education many of these athletes are able to obtain their protein from sources that minimizes the amount of fat consumed. For instance, removing the skin from chicken breast, consuming fish and lean beef, and egg whites. In addition, many protein supplements are available that contain little to no fat. It should be acknowledged though that if elevated protein does come primarily from meats, dairy products and eggs, without regard to fat intake, there likely would be an increase in the consumption of saturated fat and cholesterol.

Dietary Protein and Renal Function

The major concern associated with renal function was the role that the kidneys have in nitrogen excretion and the potential for a high protein diet to over-stress the kidneys. In healthy individuals there does not appear to be any adverse effects of a high protein diet. In a study on bodybuilders consuming a high protein (2.8 g·kg -1 ) diet no negative changes were seen in any kidney function tests (Poortsman and Dellalieux, 2000 ). However, in individuals with existing kidney disease it is recommended that they limit their protein intake to approximately half of the normal RDA level for daily protein intake (0.8 g·kg -1 ·day -1 ). Lowering protein intake is thought to reduce the progression of renal disease by decreasing hyperfiltration (Brenner et al., 1996 ).

Dietary Protein and Bone

High protein diets are associated with an increase in calcium excretion. This is apparently due to a consumption of animal protein, which is higher in sulfur-based amino acids than vegetable proteins (Remer and Manz, 1994 ; Barzel and Massey, 1998 ). Sulfur-based amino acids are thought to be the primary cause of calciuria (calcium loss). The mechanism behind this is likely related to the increase in acid secretion due to the elevated protein consumption. If the kidneys are unable to buffer the high endogenous acid levels, other physiological systems will need to compensate, such as bone. Bone acts as a reservoir of alkali, and as a result calcium is liberated from bone to buffer high acidic levels and restore acid-base balance. The calcium released by bone is accomplished through osteoclast-mediated bone resorption (Arnett and Spowage, 1996 ). Bone resorption (loss or removal of bone) will cause a decline in bone mineral content and bone mass (Barzel, 1976 ), increasing the risk for bone fracture and osteoporosis.

The effect of the type of protein consumed on bone resorption has been examined in a number of studies. Sellmeyer and colleagues ( 2001 ) examined the effects of various animal-to- vegetable protein ratio intakes in elderly women (> 65 y). They showed that the women consuming the highest animal to vegetable protein ratio had nearly a 4-fold greater risk of hip fractures compared with women consuming a lower animal to vegetable protein ratio. Interestingly, they did not report any significant association between the animal to vegetable protein ratio and bone mineral density. Similar results were shown by Feskanich et al ( 1996 ), but in a younger female population (age range = 35 – 59 mean 46). In contrast, other studies examining older female populations have shown that elevated animal protein will increase bone mineral density, while increases in vegetable protein will have a lowering effect on bone mineral density (Munger et al., 1999 ; Promislow et al., 2002 ). Munger and colleagues ( 1999 ) also reported a 69% lower risk of hip fracture as animal protein intake increased in a large (32,000) postmenopausal population. Other large epidemiological studies have also confirmed elevated bone density following high protein diets in both elderly men and women (Dawson-Hughes et al., 2002 ; Hannan et al., 2000 ). Hannon and colleagues ( 2000 ) demonstrated that animal protein intake in an older population, several times greater than the RDA requirement, results in a bone density accruement and significant decrease in fracture risk. Dawson-Hughes et al ( 2002 ), not only showed that animal protein will not increase urinary calcium excretion, but was also associated with higher levels of IGF-I and lower concentrations of the bone resorption marker N-telopeptide.

These conflicting results have contributed to the confusion regarding protein intake and bone. It is likely that other factors play an important role in further understanding the influence that dietary proteins have on bone loss or gain. For instance, the intake of calcium may have an essential function in maintaining bone. A higher calcium intake results in more absorbed calcium and may offset the losses induced by dietary protein and reduce the adverse effect of the endogenous acidosis on bone resorption (Dawson-Hughes, 2003 ). Furthermore, it is commonly assumed that animal proteins have a higher content of sulfur-containing amino acids per g of protein. However, examination of Table 4 shows that this may not entirely correct. If protein came from wheat sources it would have a mEq of 0.69 per g of protein, while protein from milk contains 0.55 mEq per g of protein. Thus, some plant proteins may have a greater potential to produce more mEq of sulfuric acid per g of protein than some animal proteins (Massey, 2003 ). Finally, bone resorption may be related to the presence or absence of a vitamin D receptor allele. In subjects that had this specific allele a significant elevation in bone resorption markers were present in the urine following 4-weeks of protein supplementation, while in subjects without this specific allele had no increase in N-telopeptide (Harrington et al., 2004 ). The effect of protein on bone health is still unclear, but it does appear to be prudent to monitor the amount of animal protein in the diet for susceptible individuals. This may be more pronounced in individuals that may have a genetic endowment for this. However, if animal protein consumption is modified by other nutrients (e.g. calcium) the effects on bone health may be lessened.

Potential acid as sulfate from sulfur-containing amino acids.

Adapted from Massey, 2003 .

Protein Intake and Liver Disease Risk

The American Heart Association has suggested that high protein diets may have detrimental effects on liver function (St. Jeor et al., 2001 ). This is primarily the result of a concern that the liver will be stressed through metabolizing the greater protein intakes. However, there is no scientific evidence to support this contention. Jorda and colleagues ( 1988 ) did show that high protein intakes in rats produce morphological changes in liver mitochondria. However, they also suggested that these changes were not pathological, but represented a positive hepatocyte adaptation to a metabolic stress.

Protein is important for the liver not only in promoting tissue repair, but to provide lipotropic agents such as methionine and choline for the conversion of fats to lipoprotein for removal form the liver (Navder and Leiber, 2003a ). The importance of high protein diets has also been acknowledged for individuals with liver disease and who are alcoholics. High protein diets may offset the elevated protein catabolism seen with liver disease (Navder and Leiber, 2003b ), while a high protein diet has been shown to improve hepatic function in individuals suffering from alcoholic liver disease (Mendellhall et al., 1993 ).

Comparisons between Different Protein Sources on Human Performance

Earlier discussions on protein supplementation and athletic performance have shown positive effects from proteins of various sources. However, only limited research is available on comparisons between various protein sources and changes in human performance. Recently, there have been a number of comparisons between bovine colostrum and whey protein. The primary reason for this comparison is the use by these investigators of whey protein as the placebo group in many of the studies examining bovine colostrum (Antonio et al., 2001 ; Brinkworth et al., 2004 ; Brinkworth and Buckley, 2002 ; Coombes et al., 2002 ; Hofman et al., 2002 ). The reason being that whey protein is similar in taste and texture as bovine colostrum protein.

Studies performed in non-elite athletes have been inconclusive concerning the benefits of bovine colostrum compared to whey protein. Several studies have demonstrated greater gains in lean body mass in individuals supplementing with bovine colostrum than whey, but no changes in endurance or strength performance (Antonio et al., 2001 ; Brinkworth et al., 2004 ). However, when performance was measured following prolonged exercise (time to complete 2.8 kJ·kg -1 of work following a 2-hour ride) supplement dosages of 20 g·day -1 and 60 g·day -1 were shown to significantly improve time trial performance in competitive cyclists (Coombes et al., 2002 ). These results may be related to an improved buffering capacity following colostrum supplementation. Brinkworth and colleagues ( 2004 ) reported that although no performance changes were seen in rowing performance, the elite rowers that were studied did demonstrate an improved buffering capacity following 9-weeks of supplementation with 60 g·day -1 of bovine colostrum when compared to supplementing with whey protein. The improved buffering capacity subsequent to colostrum supplementation may have also influenced the results reported by Hofman et al., ( 2002 ). In that study elite field hockey players supplemented with either 60 g·day -1 of either colostrum or whey protein for 8-weeks. A significantly greater improvement was seen in repeated sprint performance in the group supplementing with colostrum compared to the group supplementing with whey protein. However, a recent study has suggested that the improved buffering system seen following colostrum supplementation is not related to an improved plasma buffering system, and that any improved buffering capacity occurs within the tissue (Brinkworth et al., 2004 ).

In a comparison between casein and whey protein supplementation, Boirie and colleagues ( 1997 ) showed that a 30-g feeding of casein versus whey had significantly different effects on postprandial protein gain. They showed that following whey protein ingestion the plasma appearance of amino acids is fast, high and transient. In contrast, casein is absorbed more slowly producing a much less dramatic rise in plasma amino acid concentrations. Whey protein ingestion stimulated protein synthesis by 68%, while casein ingestion stimulated protein synthesis by 31%. When the investigators compared postprandial leucine balance after 7-hours post ingestion, casein consumption resulted in a significantly higher leucine balance, whereas no change from baseline was seen 7-hours following whey consumption. These results suggest that whey protein stimulates a rapid synthesis of protein, but a large part of this protein is oxidized (used as fuel), while casein may result in a greater protein accretion over a longer duration of time. A subsequent study showed that repeated ingestions of whey protein (an equal amount of protein but consumed over a prolonged period of time [4 hours] compared to a single ingestion) produced a greater net leucine oxidation than either a single meal of casein or whey (Dangin et al., 2001). Interestingly, both casein and whey are complete proteins but their amino acid composition is different. Glutamine and leucine have important roles in muscle protein metabolism, yet casein contains 11.6 and 8.9 g of these amino acids, respectively while whey contains 21.9 and 11.1 g of these amino acids, respectively. Thus, the digestion rate of the protein may be more important than the amino acid composition of the protein.

In a study examining the effects of casein and whey on body composition and strength measures, 12 weeks of supplementation on overweight police officers showed significantly greater strength and lean tissue accruement in the subjects ingesting casein compared to whey (Demling and DeSanti, 2000 ). Protein supplementation provided a relative protein consumption of 1.5 g·kg·day -1 . Subjects supplemented twice per day approximately 8–10 hours apart.

Only one study known has compared colostrum, whey and casein supplementation (Fry et al., 2003 ). Following 12-weeks of supplementation the authors reported no significant differences in lean body mass, strength or power performances between the groups. However, the results of this study should be examined with care. The subjects were comprised of both males and females who were resistance training for recreational purposes. In addition, the subject number for each group ranged from 4–6 subjects per group. With a heterogeneous subject population and a low subject number, the statistical power of this study was quite low. However, the authors did analyze effect sizes to account for the low statistical power. This analysis though did not change any of the observations. Clearly, further research is needed in comparisons of various types of protein on performance improvements. However, it is likely that a combination of different proteins from various sources may provide optimal benefits for performance.

Conclusions

It does appear that protein from animal sources is an important source of protein for humans from infancy until mature adulthood. However, the potential health concerns associated with a diet of protein consumed primarily from animal sources should be acknowledged. With a proper combination of sources, vegetable proteins may provide similar benefits as protein from animal sources. Maintenance of lean body mass though may become a concern. However, interesting data does exist concerning health benefits associated with soy protein consumption.

In athletes supplementing their diets with additional protein, casein has been shown to provide the greatest benefit for increases in protein synthesis for a prolonged duration. However, whey protein has a greater initial benefit for protein synthesis. These differences are related to their rates of absorption. It is likely a combination of the two could be beneficial, or smaller but more frequent ingestion of whey protein could prove to be of more value. Considering the paucity of research examining various sources of protein in sport supplementation studies, further research appears warranted on examining the benefits of these various protein sources.

Biographies

Jay R. HOFFMAN

Department of Health and Exercise Science. The College of New Jersey

Research interests

Endocrinology of sports performance and nutritional supplementation.

E-mail: ude.jnct@jnamffoh

Michael J. FALVO

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