DETERMINATION AND QUANTITATIVE ANALYSIS OF FREE AMINO ACIDS IN THE MUSHROOM Agaricus bisporus

ABSTRACT

In this study, high-performance liquid chromatography (HPLC) was employed to analyze the composition of free amino acids in Agaricus bisporus. The analysis identified a total of 20 amino acids, of which 17 were quantitatively determined. Among them, histidine, methionine, and threonine were detected in notably high concentrations, highlighting their significant biological roles. These findings characterize Agaricus bisporus as a food source of considerable biological value and suggest its potential as a promising raw material not only for dietary and nutritional applications but also for the development of nutraceutical products.

АННОТАЦИЯ

В данном исследовании для анализа состава свободных аминокислот в Agaricus bisporus использовался метод высокоэффективной жидкостной хроматографии (ВЭЖХ). В результате анализа было идентифицировано 20 аминокислот, из которых 17 были количественно определены. Наиболее высокие концентрации показали гистидин, метионин и треонин, что подчеркивает их важное биологическое значение. Полученные данные позволяют охарактеризовать Agaricus bisporus как пищевой источник с высокой биологической ценностью, а также рассматривают его как перспективное сырьё не только для диетического и лечебного питания, но и для создания нутрацевтических препаратов.

Keywords: Agaricus bisporus; amino acids; extraction; high-performance liquid chromatography (HPLC); biological value.

Ключевые слова: Agaricus bisporus; аминокислоты; экстракция; высокоэффективная жидкостная хроматография (ВЭЖХ); биологическая ценность.

Introduction.

In modern life, conditions such as depression and stress are increasingly prevalent among humans. One of the primary causes of these conditions can be attributed to hormonal imbalances in the human body. This can also be explained by an insufficient intake of essential nutraceutical compounds in the diet. Amino acids, which play a crucial role in hormone synthesis, are of significant importance. Amino acids participate in nearly all physiological processes in the body and perform vital functions. As we know, the main structural component of body cells is protein, which is composed of amino acid units. Amino acids primarily enter the body through dietary sources. Foods rich in amino acids include fish, eggs, nuts, meat, legumes, and dairy products. Edible mushrooms can also be included in this list. Among edible mushrooms, Agaricus bisporus is the most widely cultivated and utilized species as a dietary source. These mushrooms are valuable not only due to their high amino acid content but also because they contain numerous bioactive compounds, peptides, polysaccharides, and other molecules with antimicrobial, anticancer, and antioxidant properties [2]. Although the literature highlights the high protein content of A. bisporus, practical studies have sometimes shown lower levels. This can be explained by the presence of non-protein nitrogenous compounds in their composition. Analytical studies have revealed that mushrooms contain free amino acids along with urea, which accounts for the lower-than-expected protein levels [1]. A. bisporus grows naturally in the wild and is also cultivated commercially. The differences in composition and taste between these two strains are attributed to variations in substrate and compost composition. Metabolites differ significantly between these strains [5–6]. Widely consumed worldwide, this mushroom can be preserved and consumed not only in its fresh form but also through canning, freezing, salting, and even as dried powder. Freezing does not significantly affect its nutritional value, protein components, or taste [3]. Compared to preserved Pleurotus ostreatus, A. bisporus has been shown to have higher nitrogen and protein content [4–8].

Material and methods

Sample preparation and extraction. Samples of Agaricus bisporus were cultivated at the experimental field of the Microbiology Laboratory of Karshi State University. The fruiting bodies were extracted using ethanol as the solvent. The resulting extract was kept under cold conditions, leading to the formation of a precipitate. This precipitate was separated and subsequently subjected to amino acid analysis.

Isolation of free amino acids. Proteins and peptides were precipitated from the aqueous extract in centrifuge tubes. For this purpose, 1 ml of the extract was treated with 20% TCA solution. After 10 minutes of incubation, the precipitate was separated by centrifugation at 8000 rpm for 15 minutes. A 0.1 ml portion of the supernatant was collected and lyophilized. The hydrolysate was evaporated, and the dry residue was dissolved in a triethylamine–acetonitrile–water mixture (1:7:1) and dried again. To ensure complete acid neutralization, this step was repeated twice. Phenylthiocarbamyl (PTC) derivatives of amino acids were obtained by reacting with phenylisothiocyanate according to the Steven method.

HPLC conditions. The amino acid derivatives were analyzed by high-performance liquid chromatography (HPLC). An Agilent Technologies 1200 chromatograph equipped with a DAD detector and a 75 × 4.6 mm Discovery HS C18 column was used. Solvent A consisted of 0.14 M CH₃COONa + 0.05% TEA (pH 6.4), and Solvent B was CH₃CN. The flow rate was maintained at 1.2 ml/min, with absorbance measured at 269 nm. The gradient program was as follows: 1–6% B/0–2.5 min; 6–30% B/2.51–40 min; 30–60% B/40.1–45 min; 60% B/45.1–50 min; and 60–0% B/50.1–55 min.

Results and discussion

20 amino acids were identified from Agaricus bisporus samples, the quantitative values of 17 of which are presented in the following table:

Table 1.

 Amino acids in Agaricus bisporus

The results of the study demonstrated that Agaricus bisporus contains a wide range of essential amino acids. Histidine (7.77 mg/g) was detected at a notably high concentration, highlighting its importance as a biologically active compound. Similarly, the elevated levels of threonine and methionine further support the characterization of A. bisporus as a dietary product that promotes protein metabolism and supports various metabolic processes. These amino acids are of particular importance for growth, immune system activity, and tissue regeneration. Interestingly, tryptophan and glutamine were not detected in the analyzed samples. This absence could be explained by potential losses during technological procedures, such as extraction or lyophilization, or by the intrinsic instability of these amino acids under experimental conditions. Therefore, optimizing extraction parameters and employing complementary analytical approaches in future studies would be valuable for improving the detection of these compounds. Overall, the findings confirm that A. bisporus represents not only a nutritionally rich food source with high biological value but also a promising raw material for pharmaceutical and nutraceutical applications.

Conclusion

The findings of this study revealed that Agaricus bisporus contains 17 free amino acids with a total concentration of 26.18 mg/g. These results highlight the mushroom as a food source of high biological value. In particular, the abundance of histidine and methionine underlines its potential as a promising raw material for the development of biologically active supplements. The diverse amino acid composition of A. bisporus enhances its applications in nutrition, dietary practices, and the pharmaceutical field. In practical terms, this mushroom can be utilized as a natural source of proteins and essential amino acids to produce nutraceuticals, functional foods, dietary additives, and health-promoting products. Moreover, A. bisporus holds notable economic significance: as a fast-growing and high-yielding cultivated mushroom, it serves as a cost-effective and profitable raw material for agriculture and the food industry, offering both nutritional benefits and economic returns.

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