## 4 Discussion  
The human body does not store amino acids in the same way that it does fatty acids or carbohydrates. As a result, it is essential to ensure that the daily intake of amino acids needed for protein synthesis and other specific metabolic functions is sufficient. The concentration of amino acids in the blood remains relatively stable, which means that inadequate dietary protein intake can increase muscle protein breakdown, while excessive protein intake can lead to the breakdown of proteins for energy [69]. In the United States, the population's average recommended dietary allowance for protein is 0.8 grams of protein per kilogram of body weight per day [49]. In the European Union, based on nitrogen balance data, the European Food Safety Authority (EFSA) has established an average requirement of 0.66 grams of protein per kilogram of body weight per day for healthy adults, regardless of gender. Therefore, the recommendation for daily protein intake has been set at 0.83 grams of protein per kilogram of body weight per day [70], which corresponds to around 10-12% of total energy intake (E%). However, these recommendations may vary at the national level. For example, the Spanish recommen  
Figure 2: (**A**) Assessment of cytotoxicity using MTT; (**B**). Assessment of cytotoxicity using LDH assay. S1—_Pleurotus_ spp. flour; S2—pea powder; S3—almond powder; S4—yeast powder; SS—spirulina powder; CN = Control—untreated NCTC cells with culture media; ***, ***, *** = \(p\) value \(<\) 0.05; ns = statistically non-significant. dation is 0.93-1.2 g of protein per kilogram of body weight per day [71] and the Finnish recommendation is 1.1-1.3 grams of protein per kilogram of body weight per day [72]. The protein content of raw materials is strongly influenced by the environment (pedo-climatic conditions, soil chemistry, pollution), variety and processing grade, which justifies the variability between the studies published so far [73; 74; 75; 76; 17]. Results obtained by our study indicate a high percentage of protein for the pea powder (\(77.96\pm 0.70\%\)) and Spirulina powder (\(64.79\pm 2.20\%\)); out of the tested ingredients, these are the most balanced in terms of fat and carbohydrate percentages, and their correlation with energy values. _Pleurotus_ spp. has a low fat content, which is in accordance with the literature data. The lipid fraction of mushrooms generally comprises representative compounds from all lipid classes (free fatty acids, mono-, di- and triglycerides, sterols, sterol esters and phospholipids). Pea and yeast powders had a low fat content but with a high energy value due to a high protein content in the case of pea powder, and carbohydrates for yeast powder. According to [PERSON] et al. (2012) [77], it was found that the relatively high protein intake highlights the success of the so-called \"low-carbohydrate\" diet, which is usually high in protein. Reduced carbohydrate intake had no effect on decreasing body weight and fat mass during energy restriction, while increasing daily absolute protein promoted body weight loss while reducing fat mass during the weight loss phase. Even though the highest energy value was obtained for almond powder due to the higher fat content and yeast powder for the high carbohydrate content, the preferred raw materials are pea and spirulina powder due to the total protein content. According to [PERSON] et. al., 2004 [78], it was shown that the ninhydrin test for the quantification of amino groups in protein structure was more difficult than the analysis of free amino acids. The method of quantifying amino acids by Cd-ninhydrin has proven to be more effective in highlighting \(\alpha\)-amino acids. In this context, a higher content of free amino acids for _Pleurotus_ spp. flour was shown, while the soluble protein content was 6.15 mg/g. The administration of nutritional sources with a higher content of free amino acids are more indicated due to the absorption rate and assimilation efficiency higher than proteins [79].