Reduced Allergenicity of Foods of Plant Nature by the Method of Enzymatic Hydrolysis

Introduction

Feeding relates to life-sustaining functions, so the intolerance to any food ingredients by the body raises sharply the question about the need for the development of new or modification of known socially significant products[1]. It is necessary to develop food technology, including cooking during which in the food manufacturing process toxic substances would be destroyed or transformed into harmless [2]. Some prospects for feeding optimization are opened by the improvement of the food protein composition  by ensuring it [the last one] with the relevant peptides, amino acids. Peptide hydrolysates can be recommended for feeding weakened organisms, during short-term diets, at high physical loads, with a food intolerance, or intolerance.[3] In recent years the number of people affected by allergic diseases caused by food products, for example, consumption of bakery and cereal products that are rich in high molecular weight proteins significantly increased. Despite the fact that the consumption of bread in the world in general and in Russia in particular is reduced, up to the present time, one of the most important products in the food basket, has always been and remains bread. The level of consumption of bread in Russia as well as in the European countries is 80-95 kg per capita per year [4]. Spare proteins of cereals have strong allergenic properties. For wheat they are gliadin and glutenin – the main components of gluten,the shares/content of which amount40-45 and 35-40 % of the total protein respectively, for rye – scalini and glutenin, the content of which is almost equal to 30-50% of total protein, corn – Seine 50-55 and glutamine 30-45% of the total protein.[5] The molecular weight of components in the spectrum of gliadins (from 10 to 20 fractions) varies from 27 to 75 kDa, for glutenin the molecular weight from 41 to 44 kDa is characteristic [6]. The profile and distribution of fractions of Zein in corn flour is represented by polypeptides of α-Zein (24 kDa and 26 kDa); γ-Zein (22 kDa); the β-Zein (17 kDa ), and δ-Zein, respectively [7]. The most effective method of reducing the toxicity of protein crops is its biocatalytic modification. In the process of hydrolysis by proteolytic enzymes break down protein into peptide fragments having a much lower sensitizing activity [8]. In addition, enzymatic hydrolysates retain all the amino acids contained in raw materials, including essential amino acids.

The purpose of this study was to investigate the influence of enzyme preparations of proteolytic action on the degree of degradation of the protein crops for the production of dietary products with reduced allergenicity.

Materials and Methods

The object of this study was wheat, rye and corn flour. As enzyme systems (FS) the complex of fungal proteases (FS-1), obtained by culturing filamentous fungus Aspergillusniger, was used; (FS-2) is a complex of proteases synthesized by the micromycete A. oryzae. Proteolytic activity was determined by the degree of hydrolysis of hemoglobin [9] . The content of amine nitrogen -by the copper method in the absence of ammonium salts [10]. Total protein content was determined by the Kjeldahl method on automatic [11].

The process of hydrolysis of protein substances of raw grain was carried out by FS proteolytic activities in selected earlier selected conditions: within 2 h at 50°C With рН5,5 [8], the concentration of flour suspension – 30%. To determine the degree of proteolysis of the protein composition of the feedstock and fermentalistic the method of electrophoresis in polyacrylamide gel was used[12].

The composition of the total proteins in cereals was determined by the composition of the individual fractions, the classification of which is based on the principle of solubility [11]. Protein fractions: albumins, globulins, and glutelin that prolamins were extracted by successive processing of flour with water, 5-10% sodium chloride solution, 60-80% aqueous solution of alcohol and of 0,1-0,2% solution of sodium hydroxide. Albumins were fractionated from globulins with the help of ammonium sulfate. The globulins were precipitated at 50%, and albumin at 100% saturation solution of ammonium sulphate. Liquid and solid fractions were separated in a centrifuge (3000 rpm). The content of total and free amino acids were determined by amino acid analyzer HPLC “KNAUER” (Germany).

Results

Protein and amino acid composition of grain raw materials and products of the enzymatic hydrolysis has been researched. Crops differed in protein content. The highest protein content was observed in wheat (21%) and rye (18.2 %), the least one- in corn (13.1 %),with rye proteins containing 46.7% of the essential amino acids having a higher biological value (table. 1).

Table 1: The content of essential amino acids in grain raw materials.

Amino acid (AA)	Content of essential amino acids, mg/g  of flour
	Corn	Rye	Wheat
Threonine	0,454	0,489	0,691
Valine	0,523	0,608	0,835
Methionine	0,224	0,205	0,283
Isoleucine	0,478	0,419	0,742
Leucine	1,595	1,036	1,750
Phenylalanine	0,868	0,944	1,406
Lysine	0,436	0,672	0,743
Tryptophan	2,200	5,644	4,263
The total number of essential amino acids	6,778	10,017	10,713
The total number of amino acids,of them irreplaceable,% of the total number of AA	16,33841,5	21,46746,7	24,86843,1

Characterization of the protein composition of the investigated samples of wheat, rye and corn on solubility is presented in figure 1. The highest albumin content was noted in the composition of proteins of corn flour (15,5%); globulins –  that of globulins – in rye flour(24,5%), glutelins – in wheat (38.6 %).

Figure 1: Components composition was of protein in crops. Click here to View figure

 

The study of the fractional composition of proteins of cereals, selected for the degradation of the allergenic proteins of grain raw materials was carried out using the method of electrophoresis in page. Analysis of the fractional composition of the investigated cereal crops showed the presence of protein fractions in the range from 10 to 97 kDa. At the same time raw wheat was noted to have a higher content of high molecular weight proteins, including glyadenovo and glutaminova fractions in the range from 35 to 44 kDa. In rye and corn raw material the molecular weight of protein fractions was significantly lower. For corn it is typical to have the polypeptides of a molecular weight of 50 kDa, and for rye – to 60 kDa (Fig. 2).

Figure 2: The electrophoretogram was of native protein crops (M 1–marker 10-250 kDa; 1–wheat; 2–corn; 3–rye). Click here to View figure

 

In the production of bread and bakery products, cereal grains as the primary ingredient flour derived from wheat, rye or their mixture, and corn is used. If to qualify the crops for the degree of sensitizing activity of proteins, then we can talk about this trend: wheat – rye – corn. [13, 14]. With the goal of creating a product of low toxicity of dietary protein, it is necessary to reduce the length of the protein chain with the formation of peptides. Studies in this area are an important link in the solution of the fundamental problem of ensuring a living body with a complete protein food [15, 16].

For the purpose of selecting dosage of complex proteolytic systems the concentration of the latter was varied from 1 to 7 units of PS/g of flour. The criterion for evaluation was the level of formation  of the amine nitrogen as a result of hydrolysis of proteins in the studied samples of wheat, rye and corn flour.

The results of the research demonstrated that the concentration of the drug  6 units PS/g of flour for FS-1, is optimal from both economic and hydrolytic sides of the process, whereas for FS – 2 the flow dose was lower and amounted to 4 units PS/g   of flour  (Fig.3). It is important to note that as a result of exposure of  FS – 1 the level of accumulation of amino nitrogen in the grain   fermentalistic reached 97.2-101, 1 mg%. for FS – 2 – respectively 116,1-130.6 signatures mg%. Feature of the catalytic properties of proteolytic enzymes is their specificity in relation to the type of peptide bond and the mechanism of their action. Therefore, even in terms of the use of complex enzyme preparations with different specificity, the process of formation of free amino acids is limited, and the main products of enzymatic hydrolysis are the peptides with different molecular weight.

Figure 3: Effect of enzyme preparations of proteolytic action on the process of hydrolysis of proteins of rye, wheat and corn . FS-1, isolated from Aspergillusniger; b. FS-2,isolated from Aspergillusoryzae; Click here to View figure

 

The analysis of amino acids as a result of exposure of enzyme preparations was carried out on concentrated suspensions (30%)of cereals (tabl.2). Amino acids obviously do not cause allergic reactions. The results of the research showed that the impact of FS -2 features a deeper degree of hydrolysis of proteins for all the crops. The accumulation of free amino acids occurred more intensively in the suspension of rye, and the level of free amino acids when using the dosage of FS- 2 was 10% of the total, while the figure for FS-1 was respectively of 7.6.

Table 2: The contents of free amino acids in  fermentalistic grain suspensions depending on the enzyme complex.

Amino acid (AA)	Contents of AA, mg/g of flour.
	Corn		Rye		Wheat
	FS- 1	FS- 2	FS- 1	FS- 2	FS -1	FS- 2
Threonine	0,102	0,116	0,262	0,279	0,129	0,142
Valine	–	0,023	0,031	0,045	0,030	0,031
Methionine	–	–	–	–	–	–
Isoleucine	–	–	0,038	0,036	0,027	0,049
Leucine	0,020	0,036	0,035	0,058	0,029	0,042
Phenylalanine	–	0,015	0,011	0,047	–	0,021
Lysine	0,038	0,040	0,032	0,039	0,012	0,024
Tryptophan	0,302	0,429	0,309	0,637	0,349	0,551
The total number of available A A/ AA is indispensable;in% of the total numberof AA/AA irreplaceable	1,248/0,4627,6/2,9	1,469/0,6599,0/4,1	1,739/0,7188,1/3,3	2,151/1,14110,0/5,3	1,433/0,5765,8/2,3	1,908/0,8607,8/3,5

For a more detailed study of the possibility of liberation from of proteins having allergenic activity, in the manufacture of products using such cereals as wheat, rye and corn was to study the composition of polypeptides in  fermentalistic plant, obtained by the action of the investigated proteolytic enzymes has been thoroughly studied. (Fig. 4-6).

Figure 4: The composition of the polypeptides in fermentalistic wheat (M – marker 10-250 kDa, 1 – processing of wheat, FS-1, 2 – processing of wheat FS-2 ).  Click here to View figure

 

Figure 5: The composition of polypeptides in fermentalistic corn (M – marker 10-250 kDa, 1 – processing of maize, FS-1, 2 – processing of corn FS-2) Click here to View figure

 

Figure 6: The composition of polypeptides in fermentalistic rye (M – marker 10-250 kDa, 1 – processing of rye, FS-1, 2 – processing of rye FS-2). Click here to View figure

 

Processing suspensions of grain raw materials by enzyme preparations of proteolytic action showed that the best results were obtained using the FS – 2. In this case, there was a significant decrease of molecular weight of polypeptides 35 of 40 all the samples of fermentalistic . The results of the use of FS – 1 are less effective , molecular weight of a significant fraction of the proteins remained at the level between 45 and 75 kDa.

Conclusion

It is shown now that pre-treatment of grain raw materials by enzyme preparations of FS-2, obtained by culturing filamentous fungus Aspergillusoryzae, allows to get rid of allergenic proteins in cereals such as wheat, rye and corn, and provides the production of the modified products of cereal crops with reduced sensitizing activity and is enriched with essential amino acids.

Acknowledgement

The research was supported by grant of the Russian Science Foundation № 16-16-00104.

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