STUDY OF PROTEINS OF SOME AMARANTH VARIETIES OF THE AMARANTHACEAE FAMILY

ABSTRACT

The study of proteins in the aerial parts of local amaranth varieties grown in Uzbekistan is of practical importance, since the chemical composition of introduced plants often changes depending on climatic conditions. The prospects for the aerial parts of amaranth as raw materials for complex processing as a source of products and additives for the food industry are determined by their rich chemical composition and biological value. In particular, methods for preparing amaranth seeds for pressing and selecting optimal conditions, analysis of proteins in the aerial parts of amaranth, and a method for complex processing of amaranth cake by a chemical method with the isolation of protein isolate contribute to the production of various products for functional nutrition. To accomplish the assigned tasks, the work used well-known modern chemical and physicochemical methods for analyzing seeds, seed proteins and amaranth meal.

АННОТАЦИЯ

Вопросы изучения белков надземных частей местных сортов амаранта выращиваемых в Узбекистане иметь практическое значение, поскольку у интродуцированных растений часто изменяется химический состав в зависимости от климатических условий. Перспектива надземной части амаранта как сырья для комплексной переработки в качестве источника получения продуктов и добавок для пищевой промышленности определяются их богатым химическим составом и биологической ценностью. В частности, способы подготовки семян амаранта к прессованию и выбрать оптимальные условия, анализ белков надземных частей амаранта, способ комплексной переработки жмыха амаранта химическим методом с выделением белкового изолята способствует для производства разных товаров для функционального питание. Для выполнения поставленных задач в работе использовались известные современные химические и физико-химические методы анализа семян, белков семян и шрота амаранта.

Keywords: amaranth, cake, meal, protein, functional food.

Ключевые слова: амарант, жмых, шрот, белок, функциональное питание.

Relevance of the work. In recent years, the agriculture of Uzbekistan has been enriched, along with economically valuable crops, with new species of introduced plants. In the process of introducing new plants, new varieties appear, depending on climatic conditions, such as the length of sunny days, the fertility of crop lands, good water supply, etc.

According to modern requirements for the production and processing of agricultural products, each economically valuable crop is subjected to complex processing in order to more fully use resources, using non-waste technologies. Integrated processing of renewable plant resources and the creation of virtually waste-free technologies contribute to solving the global environmental problem. The rational use of natural resources allows us to expand the range of products for food, pharmacological and technical purposes and create additional sources of secondary raw materials.

Purpose of research. The prospects for the aerial parts of amaranth as raw materials for complex processing as a source of products and additives for the food industry are determined by their rich chemical composition and biological value. The development of a method for the complex processing of cake using a chemical method with the isolation of protein isolates, the determination of optimal conditions for the isolation of proteins from the aerial parts of local amaranth varieties contributes to the production of various products for functional nutrition. During the research, the physicochemical characteristics of seeds of local amaranth varieties were studied; grains and their protein content; The chemical method of processing the cake was refined.

During the research, the physicochemical characteristics of seeds of local amaranth varieties were studied; their protein content; The chemical method of processing the cake with the isolation of the protein fraction was refined.

Knowledge of the problem. Amaranth is a promising reproducible plant raw material that attracts the attention of researchers and agricultural practitioners due to its high protein content with a balanced set of essential amino acids, high yield, high content of vitamins and mineral salts. The fairly high content of flavonoids, protein, pectin, and carotenoids in some types of amaranth suggests its use for obtaining a number of biologically active compounds that can be used in medical practice and in organizing functional nutrition. The introduction and use of amaranth can be considered as a way to solve complex problems in the production of feed, food and medicines.

Despite the large number of works on the problems of research and use of amaranth, a number of issues require additional study. Thus, in the works of many researchers, the special importance of amaranth is primarily determined by its nutritional advantages and does not pay due attention to research on the use of amaranth seeds as a raw material for increasing the nutritional value of food products, the problems of complex processing of amaranth seeds and the development of modern non-traditional industrial technologies.

Amaranth is an exceptional source of high-quality protein, containing high amounts of lysine, methionine and cysteine ​​combined with a finely balanced composition of amino acids [1]. Amaranth proteins are highly digestible by the human body. It has been established that amaranth protein reduces the level of cholesterol in the blood plasma during hypercholesterolemia by 27%, and when mixed with casein – by 48%. The authors of the work expressed the opinion that amaranth protein affects the metabolism of endogenous cholesterol [2].

In conditions of increasing stratification of society, deterioration of the nutritional structure of the bulk of the population, an increase in the supply of products using plant proteins to the market could, to some extent, improve the existing situation. Integrated processing of renewable plant resources and the creation of virtually waste-free technologies contribute to solving the global environmental problem. The rational use of natural resources allows us to expand the range of products for food, pharmacological and technical purposes and create additional sources of secondary raw materials.

The prospects for amaranth seeds as raw materials for complex processing as a source of products and additives for the food industry are determined by their rich chemical composition and biological value. Below are examples of methods and technologies for complex processing of grain and amaranth phytomass.

Researchers have proposed a method for obtaining oil, protein and starch products from amaranth grain germs [3]. The method includes grinding amaranth seeds, dispersing the crushed mixture to obtain a starch fraction and a mixture of germs and shells of amaranth seeds. Next, cold pressing of the mixture of germs and shells of amaranth seeds is carried out at a temperature not exceeding 40°C. The resulting oil is settled and submitted for filtration. The cold-pressed cake is mixed with oil sludge and subjected to a second pressing, releasing the oil and cake in the form of germ flakes as the protein fraction. The resulting oil retains the most valuable component, squalene, in a biologically active state, and the protein fraction (germ flakes) can be used in food production.

Methods have been developed for complex processing of amaranth seeds to produce new types of food products and additives of high nutritional value: high-protein flour, flour fractions enriched with oil and carbohydrates. One of the methods proposed in [4] is the method of dry separation of crushed seeds, as a result of which two fractions are obtained. The protein-lipid fraction contains up to 38% protein, up to 18% oil and is prepared for the extraction of high-quality amaranth oil. The granulometric composition and structure of the protein-lipid fraction have a positive effect on the drainage properties of the material and improve the efficiency of oil extraction with a more complete transition of squalene and other biologically valuable components into it. After oil extraction, cakes and meals can be processed into protein flour or amaranth concentrates and protein isolates. The second fraction is a carbohydrate concentrate with a low content of protein, fiber and oil. This fraction is suitable for use in various areas of industrial production (starch, bakery, confectionery, perfume, pharmaceutical, etc.).

Another method of complex processing of amaranth seeds proposed in [4] is a biotechnological method, including the hydrolytic effect of complex enzyme preparations on amaranth flour. As a result, a semi-finished product (raw material) is obtained, which can be used in the production of a wide range of energy drinks, fermented milk and other health care and mass nutrition products. The insoluble residue after hydrolysis (protein-lipid concentrate) with an oil content of up to 27.5% is the raw material for extracting amaranth oil from it with the subsequent production of amaranth protein products. Due to its qualitative and quantitative composition, the insoluble residue can also be of interest in the production of a wide range of food products.

Russian scientists, using a pilot plant under conditions of mechanoacoustic influence, have developed a highly effective, environmentally friendly innovative technology for isolating practically valuable substances from the above-ground part (phytomass) of amaranth. The technology makes it possible to intensify the process and ensure a high yield of final products using soft food acids: citric, succinic, etc. Based on this technology, a method has been developed for producing effective feed additives with high nutritional value and increased digestibility, enriched with biologically active compounds. The influence of additions of amaranth phytomass and its pulp on the process of methanogenesis when using sewage sludge as a raw material was studied; At the same time, a stimulating effect of amaranth-based additives was discovered [5]. Using this technology, a wide range of valuable compounds are extracted in a single technological chain by sequential extraction with 70% ethanol, distilled water, organic acid solution and soda solution: rutin, which has P-vitamin activity, food coloring amaranthine of betacyanin nature, high-quality, balanced in amino acid composition protein. The advantages of the technology are low cost and environmental friendliness. During its development, a new approach was used, which makes it possible, under conditions of mechanoacoustic influence, to intensify the hydrolysis-extraction process, to achieve environmentally friendly conditions under which the high biological activity of the target products is maintained. At the same time, it becomes possible to replace the mineral acids used in traditional methods of pectin production with organic ones: citric, succinic, lactic. It is possible to use whey as an extractant, which allows the use of intermediate products of amaranth processing as highly effective feed additives with high nutritional value and increased digestibility, as well as solving the environmental problem associated with the formation of large amounts of wastewater that must be disposed of using traditional methods of pectin production. It has been shown that amaranth pulp is an effective co-substrate for productive methane fermentation and biogas production.

Amaranth seeds are small, lenticular in shape. The seed mass is characterized by significant variation in quality within one species, namely, the size of the seeds, their weight, degree of ripening, and humidity differ, which is associated with the unevenness of seed ripening not only on one plant, but also in one inflorescence [6]. The seeds are covered with a shell that can be easily removed when crushed. During crushing, the embryo and seed coat (bran fraction), rich in protein and lipids, can be separated from the endosperm, which consists mainly of starch. The structure of the amaranth seed allows, when crushed or crushed, to obtain two fractions - bran, represented by the germ and surface tissues of the seed, enriched with oil and protein, and a fraction consisting mainly of endosperm and enriched with starch [7].

Experimental part. 1. Selection of optimal conditions for preparing amaranth seeds for pressing.

1.1. Determination of physico-chemical characteristics of amaranth seeds.

The physicochemical characteristics of amaranth seeds were determined according to the methods described in [6], the linear dimensions of the seeds, the weight of 1000 seeds and the volumetric mass - according to [8].

1.1.1. Determination of linear dimensions of seeds

1.1.2. Apparatus, materials and reagents

We used an indicator table micrometer TIN-1.

From a sample of 100 g, 500 seeds were selected, their length, width and thickness were measured, and then the average sizes were calculated.

1.2.1. Determination of the mass of 1000 seeds [8].

1.2.2. Apparatus, materials and reagents

Technical scales were used in this work.

1.2.3. Carrying out analysis

A sample of seeds is mixed and distributed in an even layer in the form of a square, divided diagonally into four triangles, and from each two opposite triangles, 1000 seeds are counted, 500 seeds from each triangle, selecting them in a row. The two selected samples are weighed on technical scales with an accuracy of 0.01 g. The difference between the mass of two parallel samples should not exceed 5% of the average mass. The mass of the two samples is summed up.

1.3. Determination of seed volumetric mass

1.3.1. Apparatus, materials and reagents

For the analysis, a laboratory grain purka, a glass beaker with a capacity of 50 g, and an analytical balance were used.

1.3.2. Carrying out analysis

To determine the volumetric mass, the purku was filled with amaranth seeds, the excess seeds were removed, the contents were poured into a glass and a pre-weighed glass and weighed on an analytical balance. The analysis was carried out in duplicate.

1.4. Determining the optimal degree of seed grinding

1.4.1. Apparatus, materials and reagents

The work used analytical balances, a ball mill, a sifting device with a sieve cell diameter of 0.1-10 mm, and a mini-press.

1.4.2 Conducting analysis

For the experiments, amaranth seeds with an oil content of 6.4% were used. 100 g of seeds were ground in a ball mill. The resulting mixture of particles of different sizes was separated sequentially using a sieving device on sieves with two sieves into 3 parts - large, medium, small. Large particles larger than 7 mm in size were separated from the ground seeds by sifting using the first sieve with holes with a diameter of no more than 0.7 mm. Particles less than 7 mm in size fell through the holes of the first sieve onto the second. The residue on the first sieve was removed into a collection container. Along with large particles, large particles of destroyed shells also fell into this collection from the surface of the first sieve. The second sieve had a hole diameter of less than 0.7 mm, but not less than 0.5 mm, where the second separation of the crushed material took place. Small grinding particles fell into the holes of the second sieve, and medium particles were removed from the surface of the second sieve into another collection container. The resulting fractions were pressed on a mini-press and the oil yield was determined.

1.5. Determining the optimal moisture content of amaranth seeds

Determination of the optimal moisture content of amaranth seeds for their subsequent pressing was carried out using the recommended method [8] in duplicate.

1.5.1. Apparatus, materials and reagents

The work used analytical balances, a drying cabinet, and a desiccator.

1.5.2. Carrying out analysis

5 g of crushed and weighed seeds on an analytical balance were transferred to 2 pre-dried and weighed bottles and dried in an oven at 100-105 °C for 4 hours. After the specified time, the bottles with the samples were quickly removed from the cabinet, covered with lids and placed in a desiccator for 10-15 minutes. The cooled and weighed bottles were again placed in the drying oven for 30 minutes, then removed, cooled and weighed. This was repeated until a constant weight was achieved.

A constant weight was considered achieved when the difference between weighings did not exceed 0.001 g.

Seed moisture was calculated using the following formula:

                                                                                                                              (1)

Here P₁ is the mass of seeds before drying, g;

where, P₁ is the weight of seeds in g before drying;

Р₂ - weight of seeds in g after drying;

Р - weight of seeds, in grams.

The average of two parallel determinations was taken as the final result. The discrepancies between parallel determinations did not exceed 0.3%.

To reduce humidity, crushed seeds were dried in a drying cabinet, and to increase moisture, they were moistened.

2. Determination of the total protein content and protein fraction of local amaranth varieties.

Total protein. To extract total protein, amaranth seeds and pods were ground into powder and degreased with acetone, acetone: ether (1:1). To isolate total protein, the acetone-dried powder was extracted with an alkaline solution, centrifuged, precipitated from the protein solution with ammonium sulfate, dialyzed in running water, and lyophilized.

Fractional composition of protein. Determination of the content and fractional composition of protein in amaranth seeds and cake was carried out according to the method of Kalkar and Kjeldahl [9].

Apparatus, materials and reagents

The work used a magnetic stirrer, refrigerated centrifuge, spectrophotometer SF-46, FEC, pH meter, analytical and technical balances, freeze-drying device, distiller, dialysis paper, granulated alkali KOH and NaOH, Na₂HPO₄, KH₂PO₄, ammonium sulfate, sodium chloride, tris-hydroxymethylaminomethane, ninhydrin, diethyl ether, acetone.

Conducting the experiment

Amaranth seeds and cake were ground to a powder and degreased with acetone and a mixture of acetone: ether (1:1). Isolation of total protein was carried out from the resulting acetone powder using alkaline extraction, centrifugation, precipitation with ammonium sulfate, dialysis in running water and lyophilization of the protein solution.

Determination of the fractional composition of seed protein

Acid hydrolysis of the protein was carried out with 5,7 N hydrochloric acid under vacuum conditions at a temperature of 110 °C for 24 hours. The hydrolyzate was evaporated and the sum of amino acids was obtained. The composition of amino acids was analyzed by TLC on silfol plates in the system butanol-acetic acid-pyridine-water (15:3:10:12), the developer was 1% ninhydrin in acetone. To identify the components, we used a mixture of essential standard amino acids: lysine, valine, methionine, isoleucine, leucine, threonine, tyrosine, phenylalanine, histidine, arginine. It was found that the total protein of amaranth seeds contains all the essential amino acids and is enriched with lysine, threonine, and phenylalanine.

3. Complex processing of amaranth cake with the isolation of protein isolate.

Conducting the experiment

At stage I, water-soluble carbohydrates were isolated from the cake sample. The squalene fraction was isolated from the wet residue by treating it with an alcohol solution of KOH (stage II). After removing the alkaline solution, the residue was analyzed for the content of alkali-soluble proteins.

Results and discussion.

Determination of the physicochemical characteristics of amaranth seeds showed that the linear dimensions of the seeds have the following average dimensions: length - 0.6-0.8 mm, width 0.5-0.6 mm, thickness 0.3-0.5 mm. The mass of 1000 pieces of amaranth seeds is 0.875 g. According to the analysis results, the volumetric mass of amaranth seeds was 837 g/l.

According to the pressing results, the optimal grinding size for amaranth seeds is a particle size of 0.6-0.7 mm (Table 1).

Table 1.

Yield of press oil from fractions of ground seeds with different particle sizes

As a result of determining the moisture content of the raw materials and the influence of the parameter on the yield of press oil, it was proven that the oil yield is higher when the seed moisture content is 10-11% (Table 2).

Table 2.

Yield of press oil from fractions with optimal particle size depending on the moisture content of the raw material

When determining the total protein content and protein fraction of local amaranth varieties, the results showed that in all studied amaranth varieties it was proven that the total protein content in the bran was higher than in their seeds. This is the basis for the high efficiency of obtaining food products with high nutritional value for livestock and functional nutrition for everyday life from amaranth flour. According to the results, 22.5% of total protein was found in the seeds, and 27.7% in the cake. The total protein content in seeds and meal of local amaranth varieties is given below (Table 3).

Table 3.

Total protein of seeds of local amaranth varieties, in%

When determining the fractional composition of seed protein, the total protein was fractionated and glutelin and albumin fractions were isolated, the content of which was 68% and 46%, respectively (Table 4).

Table 4.

General protein-fractional composition of local variety amaranth seeds, in %

From the table above it can be seen that in all studied amaranth varieties the amount of the glutelin fraction turned out to be significantly higher than the albumin fraction. This provides an excellent basis for creating new food supplements rich in amino acids beneficial for human health based on these amaranth varieties.

In order to refine the method of complex processing of amaranth cake, experiments were carried out to isolate first water-soluble polysaccharides from it, then from the wet residue - the squalene fraction, and from the remaining cake - protein isolate. The results are presented in Table 5.

Table 5.

The influence of difficult processing conditions of amaranth variety Marhamat on the yield of protein isolate

From the table data it is clear that it is advisable to carry out sequential processing of the cake under the conditions of experiment 2, at which ~3.2% alkali-soluble proteins (protein isolate).

Conclusions. Thus, a qualitative and quantitative characteristic of the protein complex of amaranth seed cake has been given and, as a result of the research, it has been established that the optimal particle size of crushed seeds is 0.6-0.7 mm; the optimal moisture content of raw materials before pressing is 10-11%; Amaranth cake contains 27.7% of total protein. Using a chemical method, a fraction containing squalene and a 3.2% alkali-soluble protein isolate were successively obtained from the cake. 22.6% protein was found in the meal.

The data obtained can be used in the process of complex processing of amaranth plant raw materials for the production of functional nutrition products for the prevention of various diseases, baby food products, teas and porridges in everyday nutrition.

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