Method for processing oats and oat bran

Abstract

FIELD: biotechnology.SUBSTANCE: invention is a method for processing oats and oat bran, including sieve sorting of oats and oat bran, with the removal of mineral and other inclusions, followed by grinding oats and oat bran in a hammer mill to particles of at least 50 microns in size, followed by extraction of lipids from the crushed raw materials using liquid CO2 extraction technology to obtain unrefined oat oil with a yield of 5% of the mass of oats and bran crushed to a fraction of 0.25-0.45 mm at a carbon dioxide pressure of 350 atm, a temperature of 40 to 50 °C, fluid flow rate of 50 g / min and extraction duration of 60 minutes, the meal formed after extraction of crushed oats and oat bran, comprising starch, vegetable protein, fibre, beta-glucan, water-soluble vitamins, is treated with amylolytic enzymes in the following order: heat-resistant alpha amylase at a temperature of 80 to 90 °C with cooling to 55-60 °C with isoamylase, followed by treatment with glucoamylase of the resulting suspension of starch oligomers and the conversion of these oligomers into glucose with the destruction of amylose and amyloic acid starch to glucose; then the resulting suspension of glucose, vegetable protein, fibre, beta-glucan, water-soluble vitamins is heated to a temperature of 85-90 °C, then cooled to a temperature of 50-55 °C and enzymatic hydrolysis of protein to amino acids is carried out using proteolytic enzymes, then from the resulting suspension of glucose, amino acids and beta-glucan, the fibre is separated by centrifugation and the suspension is sterilized in an autoclave at a temperature of 120±1 °C, then cooled to the temperature for culturing lactic acid bacteria in the resulting nutrient medium.EFFECT: obtaining a culture medium for cultivating lactic acid bacteria.1 cl, 1 tbl, 1 ex

Description

[0001] Current trends in the development of cosmetics include the use of lactic acid bacteria metabolites. Lactic acid bacteria are very demanding of their nutrient environment. Their development requires amino acids, B vitamins, and nucleic acid components (purine and pyrimidine bases).

[0002] In this regard, the search for raw materials and processing methods to produce a nutrient medium for cultivating lactic acid bacteria and obtaining metabolic products used in the production of cosmetic media is highly relevant. Moreover, these raw materials should provide the nutrient medium with natural substances that improve the quality of cosmetic products.

[0003] Thus, during the analysis of nutrient media used for culturing lactobacilli, it can be noted that lactobacilli are auxotrophic organisms and therefore are extremely demanding of artificial nutrient media. To stimulate their growth, various additives are added to the nutrient medium: yeast extract, Tween-80, and yeast autolysate, which contains valuable soluble forms of protein and B vitamins. Certain peptides and amino acids (L-arginine hydrochloride, cysteine ​​hydrochloride) have been shown to enhance the growth of lactic acid bacteria. Proteolytic enzymes are used to hydrolyze proteins and convert them into forms (peptides and amino acids) more accessible to lactobacilli.

[0004] In skim milk and ordinary nutrient media, the yield of lactic acid bacteria is 1.0×10 9 -2.0×10 9cells / ml. However, milk cannot be used as a medium for accumulating lactic acid bacteria cells without special pretreatment due to protein coagulation, which greatly complicates cell separation. The use of nutrient media containing tryptone, yeast extract, glucose, or lactose for culturing lactobacilli allows for a high cell yield. However, these media are impractical for the mass production of bacterial concentrate.

[0005] To reduce the cost of bacterial preparations for lactic acid bacteria cell growth, it is proposed to use cheese whey with the addition of various components, including skim milk. However, this requires additional labor for protein extraction and whey clarification. The buffering properties of the medium are crucial for lactic acid bacteria growth. Whey, for example, has a lower buffering capacity than skim milk, which negatively impacts cell accumulation. Buffer salts such as sodium citric acid, phosphoric acid, and acetic acid are known to favorably affect the growth of lactic acid bacteria.

[0006] Casein-yeast medium contains an expensive imported ingredient, cysteine, which significantly increases its cost. Furthermore, this medium, like the MRS medium specially developed for the cultivation and isolation of lactobacilli and other lactic acid bacteria, contains yeast autolysate at a rate of 0.65 liters per 1.0 liter of medium. Yeast autolysate is not a technologically advanced component; it has the consistency of a suspension, requiring additional expensive equipment to separate the solid and liquid phases. A comparative analysis of known nutrient media suitable for culturing lactobacilli shows that its success depends on the quality and composition of the medium, determined by both the type of protein base and the specific stimulating components.

[0007] Glucose, lactose, and sucrose are recommended as carbohydrate sources for the preparation of nutrient media. The development of lactic acid bacteria is significantly influenced by various microelements present in the medium. Therefore, sodium chloride, sodium acetate, diammonium citrate, sodium citrate, and sources of manganese, magnesium, phosphorus, and iron are used. This fact requires a careful and targeted approach to the selection of raw materials rich in the above-mentioned components.

[0008] It has been established that animal-based raw materials, namely dry enzymatic peptone from casein, meat extract, skim milk hydrolysate, and liver extract, are used as components for cultivating lactobacilli in all of the above-mentioned environments. However, in some countries, the use of animal-based components in the production of immunobiological products and vaccines is restricted due to the prevalence of bovine spongiform encephalopathy in cattle, as well as the fact that animal products may contain antibiotics, nitrates, and chemicals, which negatively impact the cultivation of microorganisms. Therefore, the use of lactic acid bacteria metabolites in cosmetics is unacceptable.

[0009] An analysis of scientific and technical information and patent sources reveals a trend toward decreasing use of animal-based raw materials in the production of lactic acid bacteria-based products, with their replacement by plant-based raw materials. The use of plant components may allow for the improvement of existing and the development of new, effective nutrient media for lactic acid bacteria. The author believes that the potential of such media is also linked to a comparative cost assessment of plants and various animal-based products when used as the basis or component of nutrient media for cultivating lactobacilli. However, the list of plant-based nutrient media suitable for growing lactobacilli is limited.Given the high demand for such media, associated with the intensification of the production of cosmetics, improving their quality and safety for the human body, the problem of developing new and improving existing nutrient media for the cultivation of lactobacilli remains relevant.

[0010] In this regard, the aim of the author's research was to develop a new nutrient medium based on plant raw materials, which should, first of all, meet the nutritional needs of lactobacilli, and should also not be expensive.

[0011] The following developments are known from the studied level of technology.

[0012] A method for cultivating Leuconostoc mesenteroides VKPM B-9280 bacteria is known, patent 2346037 (RU). This cultivation method is implemented using a nutrient medium containing 175 g / L of sucrose, which is inoculated with Leuconostoc mesenteroides VKPM B-9280 bacteria at 8% of the nutrient medium volume and cultivated without aeration for 12 hours at a temperature of 27°C and a pH of 7.0. The invention allows for increased biomass yield. The nutrient medium for cultivating lactic acid bacteria is applicable in the food industry for the production of dietary and diabetic products, as well as in medicine.

[0013] However, in addition to sucrose, it is necessary to add bacterial growth stimulants to the nutrient medium, which leads to increased production costs.

[0014] A method for cultivating lactic acid bacteria using disintegrated biomass of Chlorella vulgaris microalgae as a growth stimulator is also known (patent 2824891 (RU)). This method involves cultivating lactic acid bacteria of the genus Bacillus coagulans for 4-5 days at a temperature of 50°C, an initial pH of 6.5-7.5, and a stirring rate of 70-80 rpm in a nutrient medium containing beet molasses. However, molasses contains numerous substances that can end up in the final product—a cosmetic. Disadvantages of this method for producing lactic acid also include the need for a step of recycling the accumulated microalgae biomass after wastewater treatment, as well as the low concentration of stimulating substances, since they are external metabolites.

[0015] A method for cultivating lactic acid bacteria based on the Enterococcus faecium B-2240 D strain is known (Patent for Invention of the Russian Federation No. 2205216, IPC C12N 1 / 20, C12R 7 / 56, C12N 1 / 20, C12R 1 / 01, 2000). This method involves fermenting a monoculture of Enterococcus faecium bacteria on a nutrient medium prepared using waste and secondary resources from the processing industries, separating the culture fluid into solid and liquid phases, and isolating the target products. The invention allows for obtaining the product with a yield of up to 98%.

[0016] The disadvantages of this invention include the presence of a stage of separation of the culture fluid into solid and liquid phases, as well as the use of a protein-carbohydrate mixture (namely, grain and potato waste, milk whey, etc.) as a nutrient medium, which limits the use of these carbon sources in the production of cosmetics.

[0017] A known method for producing lactic acid based on microbiological synthesis (Patent for Invention of the Russian Federation No. 2306340, IPC C12P 7 / 56, C12N 1 / 20, C12R 1 / 225, 2005). Lactic acid is obtained by fermenting a sugar-containing medium with lactic acid bacteria of the Lactobacillus delbrueckii strain VKPM B-8744 at a culture age of 48-72 hours in the presence of malt sprouts in an amount of 12-15 g / l, treated with water acidified with lactic acid to pH 4.0-5.0 and the enzyme preparation xyloglucanophoetidine in an amount corresponding to 200-600 cd / l xylanase activity. Chalk is used as a neutralizing agent in the proposed method in an amount of 60% of the total amount of the mixture used. A disadvantage of this method is the use of large quantities of granulated sugar (380-420 g / l) to prepare the nutrient medium, which increases the cost of producing the finished product. However, the pH is maintained at 4.0.

[0018] A method for producing lactic acid is known (RU Patent for Invention No. 1243354, IPC C12P 7 / 56, 2002). According to the proposed method, lactic acid is obtained using a lactic acid-producing strain of Lactobacillus delbriickii bacteria in a sugar-containing nutrient medium. Neutralization is carried out using calcium carbonate under heating, followed by purification and isolation. Product quality is improved and the process is accelerated by the addition of active silicic acid in an amount of 60-80 mg / L, followed by removal of the resulting precipitate in a centrifugal force field with a separation factor of 3500-4500. The supernatant liquid is magnetized in a field with a magnetic induction of 0.01-0.15 T before isolation of the target product. The proposed method for producing lactic acid provides a calcium lactate yield of 1460.0 g / l after 50 hours of fermentation, which leads to a total process productivity of 2.92 g / (l-h) and a lactic acid yield of 93.9%.

[0019] The disadvantages of this invention include the relatively low yield of lactic acid and the use of expensive components of the nutrient medium (carbon and nitrogen sources).

[0020] A method is known that involves adding pectin to the nutrient medium at a rate of 5-6 g / l (0.5-0.6%), which ensures the accumulation of microorganism biomass and imparts prebiotic properties to the fermented milk product (RU Patent No. 2023396).

[0021] A solution is also known under patent No. 2819883 (RU), namely a microbial composition including lactic acid bacteria Lactobacillus parabuchneri VKM B-3553D, Lactobacillus plantarum VKM B-3552D, Lactobacillus acidophilus VKM B-3563D, Enterococcus faecium BKMB-3551D, yeast Brettanomyces bruxellensis BKM Y-3064D and microscopic fungi Penicillium chrysogenum BKM F-4934D or Penicillium chrysogenum BKM F-4935D, used for processing organic waste of animal and plant origin. The invention provides an expansion of the arsenal of means with an increased rate of decomposition of organic remains of animal and plant origin and the suppression of phytopathogenic microorganisms. However, the use of metabolic products of bacteria cultivated on such cheap food sources is unacceptable.

[0022] The closest technical solution involves using a nutrient medium for cultivating lactobacilli to produce a probiotic supplement. The nutrient medium contains beet and corn molasses, on which Lactobacillus agilis microorganisms are cultivated with a titer of at least 1.0 x 10 5 CFU / ml. The use of sucrose-containing molasses in nutrient media for cultivating lactic acid bacteria with the potential to use their metabolic products in cosmetics will require thorough purification of associated organic and mineral substances. Even such expensive purification will not eliminate the possibility of their contamination of the finished cosmetic product.

[0023] The proposed technical solution is a method for cultivating lactic acid bacteria based on oats and oat bran for the preparation of nutrient media used for cosmetic purposes.

[0024] Essence of the invention

[0025] Oats and bran contain proteins, starch, fiber, lipids, vitamins, and minerals. Oats and bran also contain beta-glucan, which has beneficial effects on the human body.

[0026] A combination of mechanical and biocatalytic processing methods for oats and oat bran will convert the natural substances of oats and oat bran (proteins, starch, fiber, lipids, vitamins, and minerals) into carbon sources (glucose, amino acids), growth stimulants, and minerals accessible to lactic acid bacteria. This will ensure the synthesis of metabolic products by lactic acid bacteria, which are important substances for the production of cosmetics. Furthermore, the proposed oat and bran processing method will preserve beta-glucan in its natural state (unchanged). The bacterial metabolic products and beta-glucan will thus be incorporated into multifunctional cosmetics.

[0027] Furthermore, the proposed method for processing oats and bran involves the preliminary extraction of lipids, allowing for more efficient use of proteins, starch, vitamins, growth stimulants, and minerals in the production of a nutrient medium for cultivating lactic acid bacteria. The extracted lipids can be used to produce certain cosmetics. It is noted that the centrate, containing glucose, amino acids, beta-glucan, and dissolved vitamins, is a complete nutrient medium for cultivating lactic acid bacteria. The content of reducing substances in the nutrient medium is regulated by dilution with drinking water and is determined to be optimal for lactic acid bacteria culture and can be 6-7%.

[0028] The proposed method for processing oats and bran enables the production of feed from the resulting secondary resources, ensuring comprehensive utilization of raw materials and reducing the cost of producing a nutrient medium for cultivating lactic acid bacteria. The resulting substance has a beneficial effect on human skin and can be used in cosmetics and the food industry.

[0029] Disclosure and implementation of the claimed method

[0030] The method for obtaining and processing a nutrient medium from oats and oat bran for using the resulting substances in the food and cosmetic industries includes sieve sorting of oats and oat bran, with the removal of mineral and other foreign inclusions, followed by grinding in a hammer mill or other apparatus of oats and oat bran to particles no less than 50 microns in size, followed by extraction of lipids from the crushed raw materials using liquid CO2 extraction technology, where for the complete extraction of beta-glucan and fat-soluble vitamins, treatment with supercritical carbon dioxide at elevated pressures and temperatures is used, with the production of high-quality unrefined oat oil with a yield of 5% of the mass of oats and bran crushed to a fraction of 0.25-0.45 mm at a carbon dioxide pressure of 350 atm, a temperature of 40 to 50 ° C, fluid flow rate of 50 g / min and extraction duration of 60 minutes,at the same time, fat-soluble vitamins are extracted from the crushed raw materials along with lipids, and the product (meal) formed after the extraction of crushed oats and oat bran, containing starch, vegetable protein, fiber, beta-glucan, water-soluble vitamins, is processed with amylolytic enzymes to convert the starch in its composition into glucose in the following order: with heat-resistant alpha amylase at a temperature of 80 to 90°C with cooling to 55-60°C with isoamylase, followed by processing the resulting suspension of oligomers from starch with glucoamylase and the conversion of these oligomers into glucose with the destruction of amylose and amylopectin of starch to glucose; then the resulting suspension of glucose, vegetable protein, fiber, beta-glucan, and water-soluble vitamins is heated to a temperature of 85-90°C to inactivate the amylolytic enzymes introduced for starch hydrolysis, then cooled to a temperature of 50-55°C and enzymatic hydrolysis of the protein to amino acids is carried out,using proteolytic enzymes to convert beta-glucan from a solid to a dissolved state, then from the resulting suspension of glucose, amino acids and beta-glucan, the fiber is separated by centrifugation, where the separated fiber with accompanying residual non-hydrolyzed fragments of starch and protein is a complex of substances that is used as a feed additive; then the nutrient medium is sterilized in an autoclave at a temperature of (120 ± 1) ° C, then cooled to the cultivation temperature of the used lactic acid bacteria culture, where for mesophilic bacterial species, the optimal growth temperature is from 25 to 32 ° C, a minimum of 10 ° C, and for thermophilic bacterial species, the optimal growth temperature is from 38 to 45 ° C, and a minimum of 20 to 22 ° C, while the optimal acidity of the nutrient medium is maintained - 6.5 ± 0.2 pH,Next, the seed material is added to the nutrient medium in an amount of 5-10% of the volume of the nutrient medium; then the bacteria are cultivated, which is carried out under aerobic conditions, in which the nutrient medium is saturated with sterile air at a flow rate of 0.5 to 1.5 m, 3 sterile air per 1 m3 3 nutrient medium per minute with continuous stirring of the nutrient medium and maintaining the cultivation temperature, with the duration of cultivation until the complete depletion of glucose, reaching a content of 10 in the nutrient medium 9CFU / ml of culture fluid, while beta-glucan and some amino acids remain in the nutrient medium; then, the enzymes are extracted from the bacteria by autolysis at 40-60°C for 18-30 hours with continuous stirring of the nutrient medium, as a result of which the intracellular products of bacterial metabolism are converted into a culture fluid with the formation of a suspension consisting of biologically active substances formed during the processing of oats and oat bran, and synthesized by lactic acid bacteria, containing monomers and oligopolymers, beta-glucan, vitamins, flavonoids, polysaccharides, organic acids, amino acids, stigmasterol, steroid saponins, bacteriocins, macro- and microelements, then from the suspension of the above biologically active substances by centrifugation (from 10 to 14 thousand revolutions per minute, temperature 5-15°C and duration of 15-20 minutes) the cell walls of lactic acid bacteria containing biopolymers, beta-glucan,products of bacterial metabolism in a dissolved state: amino acids, vitamins and other above-mentioned biologically active substances, including those formed during the processing of oats and oat bran.

[0031] The technical result is the production of a biological substance in the form of a combination of substances - biopolymers, beta-glucan from oats, bacterial metabolic products, and other biologically active substances with the possibility of their further concentration and stable production in liquid and dry form in apparatuses under vacuum, for use in food and cosmetic products.

[0032] Example of application of the proposed technical solution

[0033] Cultivation of leuconostoc mesenteroides bacteria on fermentolysate.

[0034] Leuconostoc Mesenteroides is a heterofermentative lactic acid bacterium whose growth requires available carbon sources and protein nitrogen. The nutrient medium obtained from oats and oat bran using the proposed method for culturing microorganisms contains reducing substances (a carbon source) and protein nitrogen, which favorably affects microbial development, biomass growth, and the synthesis of biologically active substances.

[0035] According to the proposed technical solution, a nutrient medium was prepared from oats and oat bran with a RV concentration of 0.5 to 4%. To produce lactic acid bacteria biomass, a submerged cultivation method was used in a bioreactor with a stirrer and heating. Process control:

[0036] pH 6.8, stirrer speed 60 rpm, temperature 37±1°C,

[0037] Medium volume 3000 ml.

[0038] The volume of the applied seed crop is 10%.

[0039] Gas saturation level 1 min-1 (1 liter of air per minute).

[0040] rO 2 - 20% (percentage of dissolved oxygen).

[0041] Cultivation was carried out until glucose was completely depleted and the number of CFU / ml of culture liquid and the biomass yield were determined.

[0042] The cultivation duration was 8 hours.

[0043] After completion of cultivation, bacterial cells were lysed at a temperature of 80±1°C for 5 hours.

[0044] Then the suspension was separated by centrifugation into lactic acid bacteria cell wall biopolymers and oat beta-glucan and dissolved metabolic products synthesized by lactic acid bacteria.

[0045] As can be seen from the results presented in Table 1 (below), the CFU / ml content and biomass yield increase with increasing RS content in the oat nutrient medium. The biomass also increases in the content of exopolysaccharides synthesized by lactic acid bacteria and in the content of oat beta-glucan.

[0046]

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