RU2207019C1 - Biologically active food additive and a method of preparation thereof - Google Patents

Abstract

FIELD: food industry. SUBSTANCE: preparation of additive involves addition of adjuvants and daily culture of microorganisms to hydrolyzate medium based on protein component enzymatic hydrolysis product. Enzymatic hydrolysis involves mixing vegetable- or animal-origin raw material with acetic acid followed by boiling, filtration, and dilution. More specifically, hydrolysis is carried out in two stages: the first in acid medium with pepsin and the second in alkali medium with 5-10 g/l of pancreatin. Yield of hydrolyzate is at least 82 vol %, and content of amino acids in hydrolyzate 2.2-2.5 g/l. Hydrolyzate is further stabilized with a number of adjuvants in amounts at least 0.59 vol %, including, g/l: agar-agar 0.8-2.0, sodium chloride 2.0-5.0, peptone 2.5-15.0, cystine or cysteine hydrochloride 0.15-0.5, ascorbic acid 0.3-0.5, and lactulose or lactose 0.15-15.0. Daily culture of microorganisms is selected from species Bifidumbacterium, Lactobacillus, and Streptococcus. Culturing of microorganisms on hydrolyzate medium affords biologically active food additive. EFFECT: increased yield of hydrolyzate with elevated yield of amino acids and increased storage time of additive. 8 cl, 1 tbl, 9 ex

Description

 The invention relates to the food industry, as well as to the production of wellness products and cosmetic preparations using biologically active additives (BAA) that increase the biological value of products and preparations. Supplements can also be used on their own or as a starter for the preparation of wellness products and cosmetic preparations.

 A hydrolyzate usually means an enzymatic hydrolysis product, and a hydrolyzate medium means a hydrolyzate enriched in stabilizing additives. Thus, dietary supplement consists of a hydrolyzate medium and microorganisms grown in it.

 Known dietary supplement for producing adapted fermented milk products containing bifidobacteria, including hydrolyzate-milk environment (GM medium) and 3-5 vol.% Bacteria cultures [Instructions for the preparation of fermented milk bifidumbacterin in dairy kitchens. M., 1988].

The method of its preparation includes the following stages:
- preparation of a GM environment based on a skim milk hydrolyzate and its sterilization;
- the introduction of bacteria into a sterile GM-environment and growing at a temperature of 37.5-38 ^o C for 24 hours in test tubes;
- reseeding of bacterial cultures from tubes to vials in the amount of 3-5% of the volume.

 A disadvantage of the known dietary supplement and the method for its preparation is a short shelf life of the initial titer and activity of microbial cells (hereinafter “shelf life") - 1-2 days.

 There is a method of producing starter culture for a fermented milk product [RF Patent N 2169472], in which vegetable raw materials, soy milk, are used as a protein component, and pepsin is used as an enzyme for hydrolysis. The disadvantage of this method is a narrow range of possibilities for the choice of raw materials, as well as a short shelf life.

 The closest technical solution to the claimed is a dietary supplement, containing a GM environment and daily cultures of microorganisms - bifidobacteria, and / or lactobacilli, and / or thermophilic lactic streptococci - in an amount of 1-5% by volume of the medium. [RF Patent 2052253]. The method for its preparation involves the preparation of a hydrolyzate medium, including the production of a hydrolyzate of skimmed cow's milk - skim milk (boiling skim milk, cooling, adjusting the pH, enzymatic hydrolysis), mixing it with glacial acetic acid; boiling; filtering dilution with distilled water in a certain ratio; the introduction of stabilizing additives; sterilization of the prepared GM medium, the introduction of a daily culture of microorganisms into it and their cultivation. In this case, enzymatic hydrolysis is carried out using pancreatin with a concentration of 1 g / l in a slightly alkaline environment.

 The disadvantage of dietary supplements is a low shelf life for use - 10-20 days.

 The technical task of the invention is to increase the efficiency of the method, namely, increasing the yield of a hydrolyzate with a high content of amino acids, as well as improving the performance of dietary supplements, namely increasing the shelf life.

 The problem is solved in that the method of producing dietary supplements for food involves the preparation of a hydrolyzate medium based on a hydrolyzate of a protein component, including enzymatic hydrolysis, mixing with acetic acid, boiling, filtering, diluting, introducing stabilizing additives into the hydrolyzate, sterilizing the resulting hydrolyzate medium, introducing a daily culture of microorganisms , their cultivation. To obtain a hydrolyzate of the protein component, raw materials of plant or animal origin are used. In this case, enzymatic hydrolysis is carried out in two stages, one in an acidic medium using pepsin, and the other in an alkaline medium using pancreatin with a concentration of 5-10 g / l. In this case, the hydrolyzate yield from which the hydrolyzate medium is prepared is at least 82 vol.%, And the amino acid content is 2.2-2.5 g / l. As stabilizing additives, the amount of which is not less than 0.59 vol.%, Agar-agar in the amount of 0.8-2.0, NaCl - 2.0-5.0, lactulose or lactose - 0.15-15 , 0, peptone - 2.5-15.0, cystine or cysteine hydrochloride - 0.15-0.5, ascorbic acid - 0.3-0.5 g / L. Algae extract may be used as a protein component of plant origin. As a protein component for the preparation of the hydrolyzate medium, a mixture of animal and vegetable components in a ratio of 1: 0.1 ÷ 9.0 can be used.

Obtained in this way dietary supplement includes a hydrolyzate medium, prepared on the basis of hydrolyzate and stabilizing additives, and daily cultures of microorganisms Bifidumbacterium, Lactobacillus, Streptococcus in the following ratio, vol.%:
Hydrolyzate with yield - At least 82
and amino acid content - 2.2-2.5 g / l
Microorganism cultures - 1.0-7.0
Stabilizing Additives - At least 0.59
As additives, agar-agar is used in an amount of 0.8-2.0, NaCl - 2.0-5.0, lactulose - 0.15-15.0, peptone - 2.5-15.0, cystine or cysteine hydrochloric acid - 0.15-0.5, ascorbic acid - 0.3-0.5 g / l. An additional effect is provided by the use of the following additives: alfalfa extract - 0.1-2.0 vol.%, Ethyl alcohol - 0.5-10.0 vol.%, Malt extract or extract of Fervital or yeast autolysate - 0.1-2, 0 vol.%, Glycerol - 0.5-10.0 vol.%.

 Hydrolysis using only one enzyme (pancreatin or pepsin, or trypsin, etc.) leads to enzymatic decomposition of only a portion of the proteins and, therefore, makes the nutrient medium for microorganisms less saturated with easily digestible amino acids.

 As a result of the use of two-stage enzymatic hydrolysis using at one stage pancreatin with a concentration of 5-10 g / l, the hydrolyzate yield sharply increases (82-86%) as compared with the analogue and prototype (73-78%), where enzymatic hydrolysis with the use of pancreatin in a slightly alkaline environment, and compared with those methods of enzymatic hydrolysis that are carried out only in an acidic environment using pepsin (RF patent 2169472, hydrolyzate yield - 70%). The indicated regularity is observed both for raw materials of animal origin, and for raw materials of plant origin and their mixtures (see table 1). At the same time, the process of increasing the hydrolyzate yield is accompanied by an increase in its amino acid content to 2.2-2.5 g / l, determined by the number of carboxyl groups by the method of formol titration (the hydrolyzate yield is characterized by the total content of amino acids, peptides, polypeptides, mineral salts).

 Moreover, the concentration of pancreatin below the lower limit does not allow to obtain such a high yield of hydrolyzate, when the concentration of pancreatin above the upper limit is not observed beneficial effect.

 The use of a biphasic substrate (raw materials of animal and plant origin) allows you to prepare a nutrient medium with a more diverse composition of substances necessary for the growth and development of microorganisms.

 The above stabilizing additives have a complex effect on the nutrient medium, being both stabilizers and growth factors, sources of carbon (lactose, lactulose, alcohol, glycerin), nitrogen (cystine or hydrochloric cysteine, hydrolysates), protein and vitamins (peptone, ascorbic acid, extracts of fervital, malt and alfalfa, yeast autolysate).

 The introduction of these additives into the semifinished product (base) of the nutrient medium — a hydrolyzate with a high yield and amino acid content, obtained by two-stage bienzymatic hydrolysis, in an amount predominantly greater than conventional, allows to increase the viability of microorganisms and, without compromising the performance properties of dietary supplements, increase their shelf life to 35 and more than a day.

 When the content of stabilizing additives in an amount less than the lower boundary of the intervals, a significant increase in shelf life is not observed. When the content of stabilizing additives in quantities greater than the upper boundary of the intervals, there is no beneficial effect.

 Bacterial cultures of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidumbacterium longum, Bifidumbacterium bifidum, Bifidumbacterium infantis, Bifidumbacterium adolescentis can be used as seed. Streptococcus thermophilus, Streptococcus mesophilus. Streptococcus cremoris, Streptococcus lactis, etc.

Sowing crops is first carried out in test tubes, which are maintained at 37.5-38 ^o With during the day. Then, from a test tube with pronounced growth, the bacterial culture is subcultured into vials with a sterile hydrolyzate medium prepared by the indicated method.

 The use of these concentrations of agar-agar (0.8-2.0 g / l) higher compared to the prototype (0.75 g / l) not only provides the medium with a semi-liquid consistency, contributing to the uniform distribution of microorganisms throughout the volume of the medium, but also has stabilizing effect on the vitality of organisms.

 The use of two-stage enzymatic hydrolysis, as is known, contributes to a deeper decomposition of broad-spectrum proteins to the level of amino acids, however, there is no direct proportionality and even monotonicity in the laws associated with the conditions of hydrolysis and the final characteristics of the hydrolyzate. This feature of biochemical processes is emphasized by many authors, including L.Ya. Telishevskaya [Protein hydrolysates. M., 2000, p. 117-125], indicating a huge discrepancy between the calculated and actual data. So even when using three-stage (three-stage) hydrolysis and immobilized enzymes, the hydrolyzate yield does not exceed 70% in the absence of monotonicity in the accumulation of amine nitrogen. The hydrolyzate yield of 50% in the two-stage hydrolysis with pepsin and pancreatin is achieved according to the calculation in 1.6 hours, and in fact - in 67 hours. Optimization of such a multi-factor non-monotonic process is extremely difficult. So the above-described method of producing dietary supplements with a very high shelf life without losing the original properties of the content of microbial cells was found by numerous experimental studies. In this case, dietary supplement with the above properties does not work if it is obtained from a hydrolyzate with a lower yield and amino acid content and non-compliance with the conditions of the method.

 Optimization of the composition of dietary supplements is also difficult due to the lack of proportionality and monotony in the influence of the composition of dietary supplements on its performance. Otherwise, the task of obtaining dietary supplements with an infinitely long shelf life and with an infinitely large content of microbial cells would be solved by increasing the number and quantity of stabilizing additives.

 Thus, the stated proposals are particular solutions to the problem when the general solution is known with a positive effect, the possibility of which does not follow from the disclosure of the content of the general solution.

In the process of preparing the hydrolyzate medium, two-stage enzymatic hydrolysis is carried out as follows. They take the protein component of an animal (for example, skimmed cow's milk - reverse) or vegetable (for example, soy drink or algae extract) origin or a mixture thereof. The pH of the resulting mixture was adjusted to 1.5-2.0 with a concentrated solution of hydrochloric acid. Then make pepsin at the rate of 10.0 g / l and carry out thermostating of the mixture 37-38 ^o C for 5 hours. In the process of hydrolysis, the pH shifts to the alkaline side by 0.2-0.4. After hydrolysis with pepsin in an acidic medium, the pH is adjusted with a 20% sodium hydroxide solution to a pH of 7.8-8.0. Next, pancreatin is added at a rate of 5-10 g / l and the mixture is thermostated at 37-38 ^o C for 3 hours, setting the pH shifting to the acid side every 30-40 minutes to a value of 7.5-7.7. The resulting hydrolyzate is boiled for 20-30 minutes, filtered, diluted with water and stabilizing additives are added. After which the medium is sterilized and, after cooling, daily cultures of microorganisms are introduced in a predetermined quantity and they are cultured for 16 hours at t = 37-38 ^o C.

 The following are examples of the implementation of the method of preparing dietary supplements of various compositions. Comparison of the properties of the hydrolyzate and dietary supplement according to the claimed composition and method with the prototype (example 1) and a well-known source - RF patent 2169472 (example 2) are shown in the table.

Example 3. Prepare a hydrolyzate medium in the following way. 5 l of the protein component is treated - skim milk - by boiling for 30 minutes, after cooling, bienzymatic hydrolysis is carried out in two stages by the above method. The hydrolyzate yield and amino acid content are determined. Next, the pH was adjusted to 4.5 with glacial acetic acid (if necessary, storage), the hydrolyzate was boiled for 15 minutes and filtered. Then the hydrolyzate is diluted with purified water in a ratio of 1: 1, additives are added: 20.0 g of NaCl, 8.0 g of agar-agar, 25.0 g of peptone, 1.5 g of lactulose, 1.5 g of hydrochloric acid cystine, 5.0 g of ascorbic acid. The resulting hydrolyzate medium is sterilized for 45 minutes at a pressure of 0.5 atm with steam preheating for 30 minutes. The sterile hydrolyzate medium is cooled and the daily culture of microorganisms is introduced. Incubated at 37 ^o C for 6-20 hours.

 No additional stabilizing additives. As a culture of microorganisms - Bifidumbacterium longum in an amount of 500 ml (4.8% by volume of the medium).

The prepared dietary supplement is a cloudy brown liquid with an acidic taste and a sour-milk smell. The bacteria content is not less than 10 ⁹ microbial cells per ml. The shape of the cells is classic. After storage at 4 ^o C for 35 days, dietary supplement has not lost its original properties. Extraneous microflora is absent.

 Example 4. Carried out analogously to example 3, but the sequence of steps in bienzymatic hydrolysis is reversed. After sterilization of the medium, NaCl - 20.0 g, agar-agar - 10.0 g, peptone - 60.0 g, hydrochloric cysteine - 3.0 g, ascorbic acid - 3.0 g, lactulose - 60 are introduced as a stabilizing additive , 0 g. Culture of microorganisms - Streptococcus mesophilus - 3 vol. % Additional additives - alfalfa extract - 0.3 vol.%, Malt extract - 1.5 vol.%.

 Example 5. Carried out analogously to example 3, but as a protein component for the hydrolyzate take soybean drink. After stabilizing the medium, NaCl - 20.0 g, agar-agar - 10.0 g, peptone - 60.0 g, hydrochloric cystine - 3.0 g, ascorbic acid - 4.0 g, lactulose - 60 are introduced as stabilizing additives , 0 g, the concentration of the culture of Streptococcus thermophilus - 4 vol. % An additional additive is alfalfa extract 2 vol.%.

 Example 6. Carried out analogously to example 3, but as a protein component using a mixture of skim milk and soybean drink in a ratio of 1: 1. Soya drink is prepared according to a known recipe at a concentration of soya powder in water during the preparation of the drink, equal to 75 g / L. After stabilizing the medium, NaCl - 50.0 g, agar-agar - 20.0 g, peptone - 150.0 g, hydrochloric acid cystine - 2.0 g, ascorbic acid - 5.0 g, lactulose - 100, are introduced as stabilizing additives. 0 g, the concentration of the culture of Bifidumbacterium bifidum - 7 vol.%. Additional additives: yeast autolysate - 1 vol.% And ethyl alcohol - 1 vol. %

 Example 7. Carried out analogously to example 3, but as a protein component using an extract of algae with a concentration of 10 g / L. After stabilizing the medium, NaCl - 20.0 g, agar-agar - 15.0 g, peptone - 40.0 g, hydrochloric acid cysteine - 3.0 g, ascorbic acid - 3.0 g, lactulose - 40 are introduced as stabilizing additives , 0 g. An additional additive - ethyl alcohol - 10 vol.%, The culture of microorganisms Lactobacillus plantarum - 2 vol.%.

 Example 8. Carried out analogously to example 3, but as a protein component using a mixture of skim milk and soybean drink in a ratio of 1: 0.1. As a stabilizing additive after sterilization of the medium is introduced NaCl - 20.0 g, agar-agar - 15.0 g , peptone - 60.0 g, hydrochloric cystine - 3.0 g, ascorbic acid - 4.0 g, lactulose - 60.0 g. As a culture of microorganisms Streptococcus thermophilus - 3 vol.%. Additional additives - Fervital extract - 2 vol.%, Glycerin - 10 vol.%.

 Example 9. Carried out analogously to example 3, but as a protein component using a mixture of skim milk and soybean drink in a ratio of 1: 9.0. After stabilizing the medium, NaCl - 50.0 g, agar-agar - 20.0 g, peptone - 30.0 g, hydrochloric acid cystine - 5.0 g, ascorbic acid - 50.0 g, lactulose - 100 are introduced as stabilizing additives , 0 g. As a culture of microorganisms Lactobacillus acidophilus - 1 vol.%. An additional additive - alfalfa extract - 0.75 vol.%.

These tables clearly demonstrate an increase in the shelf life of dietary supplements of the proposed composition prepared by the above method compared to the prototype, while the important operational properties (titer of lactic acid bacteria equal to 10 ⁸ -10 ¹⁰ microbial cells per milliliter, the classical form of microbial cells) is maintained at the same level as in a freshly prepared product. In addition, the efficiency of the production method, i.e. degree of use of the protein component. The hydrolyzate yield reaches 82-86%.

Claims (6)

1. A method of producing dietary supplements for food, comprising preparing a hydrolyzate medium based on a hydrolyzate of a protein component, including enzymatic hydrolysis, adding acetic acid, boiling, filtering, diluting, introducing stabilizing additives into the hydrolyzate, sterilizing the obtained hydrolyzate medium, introducing a 24-hour culture of microorganisms, and culturing them , characterized in that to obtain a hydrolyzate of the protein component using raw materials of animal and / or vegetable origin, with enzymatic g the hydrolysis is carried out one step in an acidic environment using pepsin, the other in an alkaline medium using pancreatin with a concentration of 5-10 g / l, from the obtained hydrolyzate with a yield of at least 82% and an amino acid content of 2.2-2.5 g / l prepare the hydrolyzate medium with the introduction of stabilizing additives in the hydrolyzate in an amount of not less than 0.59 vol.%, including at a concentration of g / l:
Agar-agar - 0.8-2.0
Sodium Chloride - 2.0-5.0
Peptone - 2.5-15.0
Cystine or cysteine hydrochloride - 0.15-0.5
Ascorbic acid - 0.3-0.5
Lactulose or Lactose - 0.15-15
2. The method according to claim 1, characterized in that the algae extract is used as a protein component of plant origin.  3. The method according to claim 1, characterized in that as a protein component for the preparation of the hydrolyzate medium, a mixture of animal and vegetable components in a ratio of 1: 0.1-9.0 is used. 4. A biologically active food supplement (BAA) obtained by the method of claim 1, comprising a hydrolyzate medium prepared on the basis of a hydrolyzate and stabilizing additives, and daily cultures of microorganisms Bifidumbacterium, Lactobacillus, Streptococcus, wherein the additive contains components in the following ratio, vol .%:
Hydrolyzate with yield - At least 82
and amino acid content - 2.2-2.5 g / l
Microorganism cultures - 1.0-7.0
Stabilizing Additives - At least 0.59
including at a concentration of g / l:
Agar-agar - 0.8-2.0
Sodium Chloride - 2.0-5.0
Peptone - 2.5-15.0
Cystine or cysteine hydrochloride - 0.15-0.5
Ascorbic acid - 0.3-0.5
Lactulose or lactose - 0.15-15.0
5. Supplements according to claim 4, characterized in that as a stabilizing additive, alfalfa extract is used in an amount of 0.1-2.0 vol.%.  6. Supplements according to claim 4, characterized in that ethyl alcohol in the amount of 0.5-10.0% by volume is used as a stabilizing additive.  7. Supplements according to claim 4, characterized in that as a stabilizing additive, malt extract, or Fervital extract, or yeast autolysate in an amount of 0.1-2.0 vol.% Are used.  8. The dietary supplement according to claim 4, characterized in that glycerol is used as a stabilizing additive in an amount of 0.5-10.0% by volume.

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