The goal of the study is to evaluate how the consumption of plant-based protein supplements compares to animal-based protein supplements in terms of their effects on metabolic state and body composition in individuals who engage in physical activity.

The increasing awareness of the health advantages of plant-based foods and the rising number of health-conscious consumers are driving the demand for plant-based supplements. Furthermore, there is a tendency for consumers to favor vegetable proteins over animal proteins.

The effect of total protein intake and its various sources on gains in lean body mass and strength in response to resistance training has been extensively studied. However, most research has focused on comparing soy protein preparations to animal-derived preparations, primarily whey protein and casein. Studies suggest that whey protein, which has a higher leucine content, may stimulate muscle protein synthesis more effectively than soy and casein. It is unclear to what extent the type of protein supplement affects strength and lean body mass in studies lasting six weeks or longer. Pea and rice proteins may be suitable replacements for highly allergenic soy protein. Consequently, there is a need for research to assess the impact of these products on human metabolic state and overall health and well-being.

Dietary supplements are commonly used foodstuffs. For a long time, supplements were used mainly by people practicing sports professionally. An increasing number of supplements are dedicated to people for whom sport comes down to health-promoting physical activity. At the same time, cultural, ethical, and environmental trends encourage society to reduce animal products and use plant-based alternatives. The size of the global dietary supplement market was valued at USD 151.9 billion in 2021. It is projected to grow at an average annual rate (CAGR) of 8.9% between 2022 and 2030. Forecasts assume that consumer demand for protein supplements and amino acid products, such as creatine, tyrosine, and citrulline, will guarantee the segment of proteins and amino acids the highest share in CAGR at the level of 13.4%.

Such a dynamic trend brings with it several questions that pose new challenges to the world of science. Can protein products of plant origin compete with the preparations based on proteins of animal origin, which have been present for many years? What effects can the investigators expect from systematic supplementation of vegetable proteins? Which of the vegetable proteins is the best substitute for proteins of animal origin, and does supplementation with vegetable proteins carry no additional risks? In the study, the investigators want to compare four protein preparations: whey protein, pea protein, rice protein, and a preparation composed of pea and rice protein mixed in the right proportion to increase its biological value by complementing the limiting amounts of amino acids. Pea and rice proteins may be a promising replacement for highly allergenic proteins such as soy protein. Therefore, there is a need to research the assessment of the impact of these products on the metabolic state of humans in terms of health and quality of life.

Another research aspect regarding the consumption of proteins of various origins is the assessment of their impact on the intestinal microbiota profile. In recent years, the number of scientific publications on the role of intestinal microbiota in shaping human health has been increasing. It is now known that the intestinal microbiota is not only involved in the processes related to the digestion and absorption of nutrients. Bacteria included in the intestinal microbiota perform various functions, and their proper quantitative and qualitative structure supports the homeostasis of the whole organism, shaping immunity, metabolism, and the synthesis of many chemical compounds.

Research hypotheses:

1. Protein preparations of vegetable origin (rice protein isolate, pea protein isolate) when covering the demand for leucine (protein from food + preparation) will not show significant differences in body composition compared to a protein preparation of animal origin (WPC).
2. The combination of the intervention with the preparations (pea and rice proteins), due to the reduction in the amount of limiting amino acids, will have a more favorable effect on the total protein utilization than the separate intervention with the preparation of rice protein and pea protein.
3. Increased protein supply will help reduce muscle mass loss during intense physical exercise.
4. A diet enriched with a protein preparation affects the biochemical parameters of the blood and the microbiota profile.

2. Importance of the project Resistance exercise is widely regarded as a key strategy in developing muscle mass, strength, and endurance. They are also an effective tool in limiting the progression of sarcopenia. Research shows that resistance training alone may not be enough. Stimulation of protein synthesis by resistance exercise also depends on protein supply . Protein intake greater than the RDA is beneficial in maintaining lean mass during periods of intense exercise or controlled weight loss.

The biological value of protein goes beyond its amino acid composition and digestibility. In healthy individuals, the slow release of amino acids into the portal vein and then into the systemic circulation in response to bolus protein intake improves nitrogen retention and reduces urea production. In addition, the protein is a source of essential amino acids (EAAs), of which branched-chain amino acids (BCAAs) are associated with the stimulation of skeletal muscle protein synthesis. Studies show that whey proteins achieve better nitrogen retention than soy or casein, which is probably due to the high content of essential amino acids (especially leucine). However, leucine consumption above 3.5g per day does not seem to provide additional benefits.

The interest of the clinical and consumer market is increasingly focused on the use of protein supplements as dietary ingredients to maintain or increase skeletal muscle mass. Recent studies suggest that the consumption of plant-derived proteins (soy and wheat proteins) causes a weaker reaction of muscle protein synthesis compared to animal-derived proteins. The weaker anabolic properties of plant proteins can be attributed to lower digestibility and a greater synthesis of urea from plant-derived amino acids compared to animal-derived proteins. The latter may be related to plants' relative lack of certain essential amino acids. In addition, most plant proteins are relatively low in leucine, which may further reduce their anabolic properties. The literature data show that proteins of plant origin can support the reduction of excessive body weight and lower the level of lipids in the blood. They can also positively affect kidney and liver function. Research to date has focused mainly on plant proteins derived from soy and wheat, therefore there is a need to thoroughly investigate the effects of plant protein preparations other than soy and wheat.

Selection of protein preparations and justification

The investigators selected 4 protein preparations for the study:

1. - whey protein concentrate (WPC) with a protein content of 80%
2. - rice protein in the form of an isolate (RPI) with a protein content of 80%
3. - pea proteins in the form of an isolate (PPI) with a protein content of 80%
4. - a preparation combining RPI and PPI in a 1 to 1 ratio (MIX) with a protein content of 80, created for the study

Description of the proteins selected for the study:

Whey proteins are distinguished by fast digestion and high content of essential amino acids (EAA), which are necessary to stimulate skeletal muscle protein synthesis. In terms of amino acid composition, whey is similar to muscle proteins. Whey has the highest level of BCAAs. It is the most commonly used protein supplement among athletes and people practicing physical activity. It plays an important role in protein synthesis, increasing lean muscle mass and in the metabolism of carbohydrates that provide energy during exercise, thus improving athletic performance.

Rice proteins - until recently, rice flour was mainly processed by industry to collect starch residues, and proteins used for animal feed. Nowadays, rice proteins are considered by producers as valuable raw materials. The protein content of rice can vary depending on genotype, climate, and cultivation practice, but is typically 7-10% of the weight of the grain. Rice protein is becoming an increasingly popular alternative to animal proteins. It contains all essential amino acids and has hypoallergenic properties. Thanks to these properties, rice protein becomes a food ingredient with a wide range of applications in infant formulas, foodstuffs, and dietary supplements for athletes. The Protein Digestibility Corrected Amino Acid Index (PDCAAS) for rice bran protein is 0.90 and for soy protein is 0.95. These are very similar results and only slightly worse compared to whey 1.00. In addition, brown rice concentrate contains 36% EAA and 18% BCAA. Therefore, rice protein derived from brano r endosperm is a potential replacement for many animal products.

Pea proteins have a well-balanced amino acid profile with a high lysine content and achieve a PDCAAS of 0.89. Relatively low calorific value and the fact that pea proteins do not contain gluten and are a vegan product make them gain more and more recipients. They are fairly well tolerated by most people and easily digestible compared to some other sources of plant protein such as soy or beans. Pea proteins are widely used as an ingredient in food products such as protein drinks, protein powders, protein bars, cookies, and other processed foods. They can also be used as an ingredient in baked goods, soups, sauces, and salads. Studies show that pea proteins can support the process of muscle mass gain in people performing resistance training. Thanks to its availability, low cost, nutritional value, and health benefits, pea protein can be used as a new and effective alternative to soy or animal proteins in functional foods.

Various strategies can be used to improve the anabolic properties of plant proteins, these may include: enrichment of plant protein sources with amino acids, i.e. methionine, lysine, and/or leucine, consumption of more plant protein sources, consumption of multiple sources of protein in to provide a more balanced amino acid profile. Milk and vegetable protein blends achieve more balanced amino acid profiles with higher scores compared to single-component proteins. However, the effectiveness of such nutritional strategies in postprandial muscle protein synthesis requires further research.

Growing awareness of the health benefits of plant-based foods and the growing number of health-conscious consumers are key drivers of demand for plant-based supplements. Consumers are increasingly switching from animal proteins to vegetable proteins. This will likely shift consumers' predisposition towards plant-derived supplements.

Much attention has been devoted to determining the effect of total protein intake and its various sources on gains in lean body mass and strength in response to resistance training. Most studies have focused on the comparison of soy protein preparations with animal-derived preparations based mainly on whey protein and casein. Studies indicate that whey protein, probably related to the higher content of leucine, better stimulates muscle protein synthesis. This process occurs to a greater extent than in the case of proteins such as soy and casein. In 2013, a study was conducted that compared the effects of rice and whey protein on body weight composition. A study conducted for 8 weeks on 24 young men systematically performing strength training showed that there were no differences between rice protein and whey protein when covering the demand for leucine. In 2019, research results were published describing the effect of whey and pea protein supplementation on physiological adaptations after an 8-week intensive functional training. They did not show a significant difference for such parameters as body weight or body fat content after the 8-week intervention. Pea and rice proteins may be a promising replacement for highly allergenic proteins such as soy protein.

Inclusion Criteria:

• Healthy men aged 18-40, practicing systematic physical activity at least twice a week.

Exclusion Criteria:

• food allergies to milk, pea and rice proteins.