Protein digestibility is typically defined as the rate at which ingested protein is hydrolyzed to amino acids, dipeptides, and tripeptides suitable for absorption⁷. In vivo methods using animals or humans, while providing the most accurate results, are time-consuming and costly. Therefore, in vitro digestion models offer a valuable alternative to animal and human models, enabling rapid screening of food components⁸. Static in vitro digestion involves in vitro oral, gastric, and/or small intestinal digestion experiments performed under constant physicochemical conditions in each simulated phase⁹. These digestion models are widely used to study the structural changes, digestibility, and release of food components under simulated gastrointestinal conditions^8,10.

In this study, simple, economical, and reproducible in vitro digestion models based on human physiology were developed to investigate the digestibility of protein powders under simulated gastrointestinal conditions. The amino acid bioavailability of a protein source is best conceptualized as the amount and diversity of amino acids digested and absorbed into the bloodstream after ingestion¹¹. However, data on the digestibility of commercially available powdered protein sources commonly used in sports nutrition, especially comparative analyses under standardized in vitro conditions, are lacking in the literature. The need to integrate information on protein consumption is critical for both practical and future research. The Dumas method involves incinerating a sample of defined mass at high temperature in the presence of oxygen; the gases produced are then reduced with copper and dried while trapping CO2, and finally nitrogen is quantified using a universal detector. The Dumas method for quantitative nitrogen determination is an alternative to classical methods for analyzing the protein content of foods by measuring the molecular weight of an unknown gas¹².

This study was conducted to determine the protein content, label compliance, and digestibility rates of various standard protein powders commercially available to athletes as dietary supplements using the Dumas method in a simulated in vitro environment.

Study Design and Sample Collection: Eighteen samples (n=18) were used to determine protein and digestibility levels by purchasing commercially available athlete protein powders, including three each of whey, bovine milk casein, soy, egg white, beef protein, and pea. The origin and brand categorization of the products are given in Table 1. Bovine milk casein sodium salt (Sigma C3400) was used as a standard.

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Table 1: Information on protein powders

Gastric Digestion Simulation: The protein powder samples were weighed to yield 5.0 g each. HCl (0.2 N, 34.0 mL) was added to each flask containing the sample. The mixture was shaken for 5 minutes, then its pH was adjusted to 1.9 with 1.0 N NaOH, and the mixture was placed in a water bath at 37°C. At the same time, 2.08 g of pepsin was added to a flask and dissolved in 80 mL of distilled water. The fresh pepsin solution prepared for each flask was added to the mixture, whose pH was adjusted to 1.9, and the mixture was kept for 30 minutes with occasional stirring. At the end of the period, 0.2 N Na2HPO4 2H2O buffer was added to the mixture, and the pH was adjusted to 7.5 to terminate pepsin enzyme activity.

Small Intestine Digestion Simulation: In this stage of the study, 16.00 g of pancreatin was put into a conical flask and dissolved in 80 mL of distilled water, and 1.0 mL of fresh pancreatin solution was added to each conical flask (0.200 g of pancreatin for each conical flask) after the stomach digestion stage was completed. It was kept for 150 minutes, stirring occasionally.

Dumas Method: After the digestion time with both enzymes, the mixture was removed from the water bath. Two mL of the mixture was taken, 1.25 mL of saturated TCA was added to stop the digestion and precipitate the undigested proteins, and the mixture was made homogeneous by shaking. From this mixture, after the digestion was complete and the mixture was thoroughly shaken to make it homogeneous, 0.2 mL was taken serially with a micropipette, and the nitrogen content was measured using the Dumas method. The values were used to determine the amount of nitrogen before centrifugation.

To determine the nitrogen amount, analysis was performed on the supernatant obtained by centrifuging the mixture after stopping small intestine digestion for 20 minutes at 12500 rpm. The nitrogen amount determination from the mix before centrifugation was used to determine the total protein amount of the sample, and the nitrogen amount determination from the supernatant after centrifugation was used to determine the total protein amount digested proportionally^13-15. The nitrogen factor used in calculating protein content was 6.38 for whey, 6.36 for casein, 5.70 for soy, and 6.25 for egg white, meat, and pea powders. The protein content was obtained by multiplying the nitrogen content by the nitrogen factor. The digestion rate was calculated with the following formula:

% Digestibility Rate = (Supernatant protein amount / Pre-centrifugation mixture protein amount) x 100

Statistical Analysis: Three measurements were taken from each protein powder sample, resulting in a total of nine replications for each protein. Nine replications were obtained for the standard. The normality distribution of the determined observation values was measured using the Shapiro-Wilk normality test. Homogeneity of variance was checked using the Bartlett test. Statistical significance between digestibility, Nitrogen Amount Before Centrifugation (mg), and Nitrogen Amount After Centrifugation (mg) values among protein powders was determined using analysis of variance and the Tukey multiple comparison test in case of significance. All statistical analyses were performed using the R statistical software (www.r-project.org/). The significance level was determined as p<0.05.

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Table 2: Protein contents of samples

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Figure 1: Digestion rates of protein powder bars

The digestion rates for each sample are shown in Table 3. Table 4 also shows statistical data. According to the findings and statistical evaluations, the highest digestibility rate among the protein powder groups was meat powder protein (66.21%). Casein powder digestibility ranked next (60.89%), followed by standard bovine milk casein (56.75%), whey protein powder (54.50%), pea protein powder (53.66%), and soy protein powder (43.76%). In comparison, the protein powder with the lowest digestibility rate was egg white protein (36.87%). The difference between the groups was found to be statistically significant for the samples analyzed (p<0.05). In the Turkish Food Codex Sports Food Communiqué, it was emphasized that protein with a minimum net protein utilization of 70% (from egg, milk, and meat) offered as sports foods should be more than 70% in dry matter. In our study, all of the powders complied with the Turkish Food Codex Sports Nutrition Communiqué².

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Table 3: Digestion rate of protein powders

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Table 4: One-way ANOVA data according to the R statistical program

This study determined the protein content and in vitro digestion rates of diverse types of protein powders commonly used as dietary supplements by athletes using the Dumas method. It was noteworthy that egg white protein powder digestibility in a simulated in vitro environment, determined by the Dumas method, was relatively low. The amino acid composition of egg proteins has long led to their acceptance as a reference protein for humans¹⁸. Egg white powders are subjected to thermal processing, dry heating, or spray drying. Although heat-treated egg protein is known to be more digestible than raw egg protein, ovomucoid does not react with human trypsin and is also relatively heat stable^19,20. Ovomucoid is quantitatively the most critical trypsin inhibitor. The underrepresentation of ovomucoid-derived peptides may be explained by the known resistance of ovomucoid to digestion even after heat treatment¹⁸. Therefore, the low digestibility level of egg white powder protein may be due to the high heat resistance of ovomucoid. The high digestibility of meat powder proteins may be due to the high particle size and high bioaccessibility of the spray-dried powder, as calculated by digestibility factors²¹.

Almeida et al.²² investigated the in vitro digestibility of commercially available whey proteins from the United States and Brazil. In vitro protein digestibility experiments revealed significantly higher digestibility for whey and standard caseinate powder than the current study's findings. Soy powder digestibility, however, is more comparable to that of other protein sources. The lower digestibility rates of the standard caseinate and soy powder used in our study may be due to the three-hour digestion time we limited compared to other studies and the method used. The low digestibility of soy protein compared to other protein sources is consistent with the findings of Pires et al.²³. Pires et al. ²³ explained the low digestibility of plant proteins by the formation of protein complexes with anti-nutritional factors in their structures, thereby reducing their digestibility. Mokrane et al. ²⁴ subjected the seeds of seven Algerian sorghum plants to in vitro protein digestion tests, using the Dumas method for protein determination, and reported that the low digestibility, below 50%, was due to the resistance of kafirin protein, the main proteins in sorghum, to peptidase enzyme due to its rich disulfide cross-links. Sindayikengera and Xia²⁵ found significantly higher digestibility levels for whey protein concentrate and sodium caseinate, which contain protein, compared to the current study. This may be due to the Kjeldahl method being used for protein analysis after in vitro digestion with a commercial complex prepared from Bacillus enzymes.

Amino acid scores are an important parameter in determining the digestibility of protein powders. It is also worth noting that the most widely accepted methods for assessing protein quality are the protein digestibility-corrected amino acid score (PDCAAS) and the digestible indispensable amino acid score (DIAAS) ²⁶. The low or high protein digestibility of foods significantly affects the PDCAAS and DIAAS value^7,27. Therefore, this study is promising to fill this gap in the literature, as future studies plan to determine the amino acid score in addition to the digestibility of protein powders.

Consequently, this study compared the protein content and digestibility rates of commercially available animal and plant protein sources using the Dumas method in a simulated environment. This study also addressed label compliance of commercial protein powder sources. Accurate food labeling allows consumers to know exactly what ingredients a product contains and helps them make more informed health and nutrition choices. Regulations stipulate that compliance is mandatory, but they may not always provide a process for resolving non-compliance issues. This study highlights discrepancies in label information for commercially available protein powders and will guide consumer preferences. Furthermore, it will serve as a guide for future studies on determining amino acid scores in protein powders or assessing digestibility beyond digestibility in peptidomic studies using mass spectrometry.

Acknowledgment
We would like to thank the Proofreading & Editing Office of the Dean for Research at Erciyes University for copyediting and proofreading service for this manuscript.

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