Fermentation method for obtaining a lactulose-containing postbiotic product

Abstract

The invention relates to biotechnology. Proposed is a fermentation method for obtaining a lactulose-containing postbiotic product which includes: preparing a lactulose-containing raw material; cultivating lactase producers in two stages, wherein yeast and lactic acid bacteria are cultivated separately during the first stage under aerobic conditions, the yeast being cultivated at 25-35°С for 8-24 hours under vigorous agitation and the lactic acid bacteria being cultivated at 37-45°С for 8-24 hours without agitation, and the two are cultivated jointly during the second stage under anaerobic conditions at 25-40°С for 12-36 hours; extracting enzymes by autolysis at 40-60°С for 18-30 hours; biosynthesizing lactulose at 30-60°С for 0.1-14 hours using the obtained unpurified enzyme preparation, which is introduced into the lactose-containing raw material to which fructose is added at a lactose:fructose molar concentration ratio of from 1:1 to 1:2; then concentrating the fermented mixture by condensation and / or drying. The invention provides an increased yield of target product from secondary raw milk material within a single cycle.

Description

[0001] ENZYMATIC METHOD OF OBTAINING

[0002] LACTULOSE-CONTAINING A-POSTBIOTIC PRODUCT

[0003] Field of technology to which the invention relates

[0004] The invention relates to food biotechnology and the dairy industry and can be used in the production of a lactulose-containing postbiotic product from secondary milk raw materials using an enzymatic method.

[0005] State of the art

[0006] Lactulose is a prebiotic that is widely used in medicine and the food industry. Lactulose production is based on the isomerization of lactose in alkaline media at high temperatures. An alternative is enzymatic methods, which are considered more environmentally friendly and allow processes to be carried out under milder conditions. Another advantage of lactulose biosynthesis is the possibility of using secondary dairy raw materials as sources of lactose.

[0007] Currently, there are two main ways of enzymatic conversion of lactose into lactulose - isomerization (direct) and transgalactosylation (with intermediate hydrolysis). For the first, cellulose-2-epimerases are used, which do not yet have a safety status, they are not produced on an industrial scale, and the by-product of the reaction is epilactose. The use of cellobiose epimerases allows achieving high yields of lactulose, but genetic engineering and mutagenesis methods are used to obtain these enzymes, which calls into question their safety (Lactulose biotechnology: modern achievements, problems and prospects / S. A. Ryabtseva, A. G. Khramtsov, M. A. Shpak, A. D. Lodygin, G. S. Anisimov, S. N. Sazanova, Yu. A. Tabakova / / Food production technology and engineering. 2023. Vol. 53. No. 1. Pp. 97-122. (In English). blips : / / dgj ; org / 10.21603 / 2074 -94 J, 4 -2023 L- 2419) .

[0008] A method for producing lactulose using whole-cell catalysis is known (patent CN104004699A, published 2014-08-27), according to which recombinant Escherichia coli BL 21 (DE3) is used as a producing bacterial strain to obtain cellobiose epimerase, lactose is used as an inducing agent for enzyme cultivation instead of isopropyl-beta-O-sulfogalactoside (IPTG), and the precipitate obtained by centrifugation is permeabilized with ethyl alcohol and freeze-dried in a vacuum so that they serve as a cellular biocatalyst for the direct conversion of lactose to lactulose. 1-100 ml of the centrifuge sediment of the fermented liquid is suspended in 1 l of ethanol 1-90% (preferably 40%) (v / v) at 4-30°C, stirring for 5-120 min. The powder is obtained by vacuum lyophilization, used to obtain lactulose as a cell catalyst. Lactose is mixed with a buffer liquid to 50-800 g / l, pH is adjusted to 6.0-9.5.The cellular biocatalyst is added to the substrate solution, the conversion conditions are: catalyst 1-100 U / ml, reaction temperature 40-95°C, reaction time 1-24 hours. The disadvantages of the method include the use of genetically modified microorganisms and an enzyme with unproven safety, additional stages of cell centrifugation, their permeabilization (to increase the permeability of cell walls for the enzyme, with the introduction of large quantities of ethyl alcohol) and lyophilic drying.

[0009] The second possible route for the enzymatic production of lactulose is based on the transgalactosylation of lactose in the presence of fructose, for which 0-galactosidases (lactases) are most often used, which have an international safety status (GRAS) and are available on the market, with a well-studied mechanism of action. The main producers of 0-galactosidases for the production of lactulose are the yeast Kluyveromyces lactis or the mold Aspergillus oryzae. Lactulose is mainly synthesized using commercial (purified, concentrated) β-galactosidase preparations, which significantly increases the cost of the finished product (Lactulose biotechnology: modern achievements, problems and prospects / S. A. Ryabtseva, A. G. Khramtsov, M. A. Shpak, A. D. Lodygin, G. S. Anisimov, S. N. Sazanova, Yu. A. Tabakova / / Food production technology and engineering. 2023. Vol. 53. No. 1. Pp. 97-122. (In English).

[0010] Other microorganisms can also be a source of lactase; it is also possible to use partially purified or unpurified enzymes obtained in a single cycle of lactulose production. An enzymatic method for obtaining lactulose is known (patent CN104805152A, published on 12.07.2015). The method begins with obtaining lactase using a mutant strain of Arthrobacter. Cultivation is carried out in a medium containing: a carbon source (glucose 1%, lactose 2% or glycerol 0.2%), one or more nitrogen sources (yeast extract 0.5-1.4% and / or peptone 0.5-1% or corn extract 2.5%), an inorganic salt (calcium chloride, Zn 2+ , magnesium sulfate, potassium hydrogen phosphate, one or more primary potassium phosphates or iron chloride, their concentration is 12-28 mmol / l. Strain cultivation conditions

[0011] Arthrobacter. temperature 25-30°C, duration 0.8-1.5 hours with constant stirring at a speed of 200 rpm. Then the resulting solution is centrifuged for 10-15 minutes at a speed of 5000-6000 rpm. The sediment is dissolved in a buffer with phosphoric acid of the same volume (10 mmol / l, pH 7.0). Then the cells are destroyed by ultrasound and the solution is centrifuged again for 10-15 minutes at 10000-12000 rpm. The supernatant, which is an enzyme preparation of lactase, is collected. For the synthesis of lactulose, a substrate is used consisting of: phosphoric acid buffer solution pH 5.5-8.0, lactose 300 g / l and fructose 250 g / l. Additionally, the substrate may contain 10 mmol / l Zn 2+and 4-6 mmol / l of ferric chloride. The enzyme preparation of lactase is added in the amount of 600 units / l, the solution is kept at 20°C for 1.5 hours. For the purpose of additional purification, anhydrous alcohol is added to the resulting mixture, bringing its final concentration to 65%, centrifuged for 10 minutes at 12,000 rpm, the supernatant is concentrated at 65°C and the amount of lactulose is determined. Disadvantages: use of a mutant strain and complex expensive nutrient media for its cultivation, repeated centrifugation to separate cells, use of large volumes of ethyl alcohol to isolate lactulose.

[0012] The use of crude enzymes reduces the cost of finished products. Moreover, fermented mixtures can contain a variety of metabolic products and components of enzyme-producing microorganism cells that are beneficial to human health, and can be used to obtain postbiotic products, which are considered very promising (Rozhkova I.V., Leonova V.A., Begunova A.V. Postbiotics as Potential Components of Fermented Milk Products with Functional Properties / / Dairy Industry. 2022. No. 3. Pp. 16-18.) Although this new term has appeared recently and its definition is being clarified, many scientists believe that the concept of postbiotics includes inactivated microorganisms that have a beneficial effect on the health of the host. (Salminen, S.; Collado, M.S.; Endo, A.; et al. The International Scientific Association of Probiotics and Prebiotics (I SAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol. 2021, 18, 649-667.).In recent years, patents have appeared on methods for producing postbiotic products.

[0013] Patent CN115606805 (2023-01-17 Postbiotic composition and application thereof in oral health) describes the production of a postbiotic composition that includes the following components in parts by weight: 2-25 parts of inactivated Lactobacillus cnspaius. 5-50 parts of yeast beta-glucan and 1-10 parts of honeysuckle flower powder. The product is intended for oral care, as it can effectively inhibit unwanted microorganisms. The disadvantage is the complexity of the composition, including yeast beta-glucan (immunomodulator) as a separate component, which increases the cost of the finished product.

[0014] In patent RU2658777 (2018-06-22 Method for producing a postbiotic product), a hydrolysate-milk medium is prepared based on a hydrolysate of a protein component obtained by enzymatic hydrolysis, including skim cow's milk and soybean extract in a ratio of 9:1. Stabilizing additives and microorganism growth factors are introduced into the hydrolysate, the resulting hydrolysate-milk medium is sterilized, daily cultures of microorganisms of the genus Lactobacillus and the genus Bifidobacterium or the genus Lactobacillus and the genus Streptococcus are separately introduced into it in equal proportions and cultivated. The resulting mixture is destroyed by heating at 56-80°C for 45-90 minutes or by freezing at temperatures of -5-(-10)°C, kept at this temperature for 10-24 hours, followed by slow defrosting at room temperature or by introducing organic acids into the microbial suspension, the resulting mixture is kept for 24 hours at room temperature and the finished product is stored at a temperature of 2-30°C.The disadvantage of this method is the use of complex and expensive nutrient media, the absence of yeast-based postbiotics and lactulose.

[0015] The closest in technical essence to the proposed method are the methods described in patent EP 3542641 and patent RU 2622078.

[0016] Patent EP 3542641 (Lactulose-containing product and method for producing a lactulose-containing product, published 25.09.2019) describes a method for producing a lactulose-containing product, comprising the following stages: a) obtaining a lactose-containing substrate, b) treating the lactose-containing substrate with β-galactosidase and / or β-glucosidase in the presence of fructose to obtain a substrate containing lactulose; c) inactivating β-galactosidase and / or β-glucosidase by heating; and d) obtaining a lactulose-containing retentate by nanofiltration and / or diafiltration of the lactulose-containing substrate. Permeates obtained by ultrafiltration, preferably of milk, whey, a milk derivative or a whey derivative, are used as raw materials. The permeate is heated (pasteurized and / or sterilized).To carry out step b), 0-galactosidase from Aspergillus niger, Aspergillus oryzae, Kluyveromyces lactis, Bacillus licheniformis or Bifidobacterium bifidum and / or 0-glucosidase from Aspergillus oryzae or Pyrococcus furiosus are used. The process is carried out at a temperature of from 2°C to 42°C, in particular from 5°C to 15°C, preferably from 5°C to 7°C, in a pH range of from 4.0 to 7.0, in particular from 4.0 to 6.5, preferably from 4.5 to 6.5, with an enzymatic activity, in particular normalized enzymatic activity, of from 0.1 nkat to 20 nkat, in particular from 1 nkat to 10 nkat, preferably 2 nkat, per ml of lactose-containing substrate and for a period of from 12 to 72 hours, in particular from 24 to 60 hours, preferably from 40 to 56 hours. Step c) is carried out at a temperature of from 90°C to 150°C, in particular from 100°C to 130°C, preferably from 120°C to 130°C. Nanofiltration is carried out at a transmembrane pressure difference of 10 to 50 bar, in particular 10 to 40 bar, preferably 20 to 40 bar.Before dnafltration, the transmembrane pressure drop is reduced, in particular, from 30 bar to 20 bar. Disadvantages of the method: the use of expensive commercial (purified) enzymes, a long and expensive process of lactulose isolation.

[0017] Patent RU 2622078 (Cl, IPC C12N 9 / 14. Method for producing a combined enzyme preparation of beta-galactosidases / Ryabtseva S.A., Skripnyuk A.A., Kotova A.A., Khramtsov A.G., Rodnaya A.B., Lodygin A.D., Martak A.A. / / prior. 12.01.2016, published. 9.06.2017, bulletin No. 16) describes a method for producing a combined enzyme preparation of beta-galactosidases, including the preparation of lactose-containing raw materials, culturing microorganisms, extracting enzymes, purifying them by ultrafiltration and concentrating them, characterized in that lactose-containing raw materials with a mass fraction of lactose of 3-15% are simultaneously cultured with lactose-fermenting yeast and thermophilic lactic acid bacteria. bacteria at a temperature of (30±2)°C for 7-16 hours, and enzymes are extracted by autolysis at a temperature of (50±5)°C for 6-12 hours.The disadvantage is the slow development of thermophilic lactic acid bacteria at temperatures optimal for yeast (30±2)°C, insufficient accumulation of metabolic products of lactase producers, and, as a consequence, low efficiency of autolysis and activity of the resulting enzyme preparation, especially with respect to transgalactosylation for the biosynthesis of lactulose.

[0018] Implementation of the invention

[0019] The technical result of the proposed method consists in obtaining an active combined lactase preparation for the biosynthesis of lactulose and the production of a lactulose-containing product - a postbiotic from secondary milk raw materials within a single cycle. This technical result is achieved due to a two-stage cultivation of lactase producers (lactose-fermenting yeast and lactic acid bacteria), their effective autolysis and biosynthesis of lactulose using the obtained crude enzyme preparation, inactivation and concentration of the obtained fermented mixture.

[0020] An enzymatic method for obtaining a lactulose-containing postbiotic product is proposed, which includes the preparation of lactose-containing raw materials, the cultivation of lactose-fermenting yeast and thermophilic lactic acid bacteria, the extraction of lactases, the biosynthesis of lactulose using the obtained lactases, the inactivation of cells and enzymes by heating, the concentration of the fermented mixture by thickening and / or drying, characterized in that a two-stage cultivation of lactase producers is used: at the first stage, separate cultivation under aerobic conditions, yeast at a temperature of (25-35) °C for (8-24) hours with intensive stirring, lactic acid bacteria at a temperature of (37-35) °C for (8-24) hours without stirring; at the second stage, the reaction is carried out under anaerobic conditions at a temperature of (25-MO) °C for (12-36) hours, and the enzymes are extracted by autolysis at a temperature of (4(H60) °C for (18-H30) hours., and the biosynthesis of lactulose is carried out using the obtained crude enzyme preparation at a temperature of (30-H5O) °C for (0.1-44) h.

[0021] The method is as follows.

[0022] Secondary milk raw materials (including cheese and / or curd whey, ultrafiltration (UF) permeates of skim milk and whey) containing lactose, nitrogenous and mineral substances necessary for the development of lactose-fermenting yeast and lactic acid microorganisms are used as raw materials and a nutrient medium for cultivating microorganisms. When using milk whey, as a rule, it is purified from residual milk fat and casein dust in special separators. To destroy undesirable microorganisms that can affect the cultivation of lactase producers and the quality of the finished product, pasteurization or sterilization of raw materials is carried out under the modes accepted in the industry. For example, high-temperature pasteurization modes at (85 ^ -87) ° C for 3-5 sec can be used. Lactose-containing raw materials can be natural or thickened, with a mass fraction of lactose in the initial solution from 3 to 15%, which is indicated in patent RU 2622078.The processing modes and the composition of the milk raw material desired for cultivation are known and are not distinctive features of the invention.

[0023] The prepared secondary milk raw material is used for a two-stage cultivation of lactase producers, which are safe lactose-fermenting yeasts and thermophilic lactic acid bacteria used in the food industry and biotechnology, which have the ability to produce lactases, preferably the yeast Kluyveromyces marxianus or Kluyveromyces lactis, and thermophilic lactic acid microorganisms Lactobacillus acidophilus or Streptococcus thermophilus.

[0024] The first stage of cultivation is carried out separately under aerobic conditions to accumulate the biomass of the producers. The yeast is cultivated at a temperature of (25-35) °C for (8^-24) hours with intensive mixing. At temperatures below 25 °C, the lactase-producing yeast develops very slowly, and above 35 ° the cells begin to die. When culturing for less than 8 hours, sufficient biomass is not accumulated and the required activity of the enzyme preparation is not achieved; after 24 hours of cultivation, the rate of biomass growth decreases, and some cultures show a decrease in the activity of the enzyme preparation. Intensive mixing promotes aeration of the medium, in which the yeast accumulates biomass faster, since a reaction of complete oxidation of carbohydrates occurs with the release of a large amount of energy.Thermophilic lactic acid bacteria grow better at higher temperatures and without oxygen (since they are facultative anaerobes), so they are cultivated separately at a temperature of (37-M5) °C for (8-24) hours without stirring. At temperatures below 37 °C, thermophilic bacteria develop slowly, and above 45 °, the cells begin to die. When cultivated for less than 8 hours, sufficient biomass does not accumulate and the required activity of the enzyme preparation is not achieved; after 24 hours of cultivation, the rate of biomass growth decreases, and some cultures show a decrease in the activity of the enzyme preparation.

[0025] The second stage of cultivation is carried out jointly under anaerobic conditions at a temperature of (25-М0)°С for (12А36) h. Anaerobic conditions are necessary for the process of alcoholic fermentation of yeast, which produces ethyl alcohol, which can promote the permeabilization of yeast cells and lactic acid bacteria, as a result of which the process of lactase release (extraction) from cells is facilitated. During anaerobic fermentation, other metabolic products of lactic acid bacteria and yeast also accumulate, including enzymes, lactic, acetic and other organic acids, vitamins, etc., which can promote the intensification of cell destruction and the production of a more active enzyme preparation, as well as enrich the postbiotic product.

[0026] The enzymes are extracted by autolysis of producer cells in a joint culture at a temperature of (4(H60)°C for (18A30) h. At a temperature below 40°C, cell autolysis proceeds slowly; above 60°C, the activity of the enzyme preparation decreases. With autolysis for less than 18 h, some of the producer cells remain intact, and they can subsequently utilize lactose and lactulose during biosynthesis. With autolysis for more than 30 h, the activity of the enzyme preparation decreases. The enzyme preparation is not purified, due to which its high transglycosylating activity is preserved, and is immediately used for the biosynthesis of lactulose.

[0027] Lactulose biosynthesis is carried out using the obtained crude enzyme preparation, which is introduced into lactose-containing raw materials with the addition of fructose, at a temperature of (3 (H60) ° C for (0.1-H4) h. At temperatures below 30 ° C and above 60 ° C, the rate of lactulose formation decreases sharply. Lactulose begins to be synthesized in the first minutes of the reaction, and its maximum concentration when using some enzyme combinations can be achieved after 6 minutes, after 14 hours of fermentation, a decrease in its concentration is observed, apparently due to the predominance of the hydrolysis reaction. The content of lactose and fructose in the substrate can vary within a wide range of molar concentration ratios, mainly at a ratio of 1:1 to 1:4, which is known from publications (Lactulose biotechnology: modern achievements, problems and prospects / S. A. Ryabtseva, A. G. Khramtsov, M. A. Shpak, A. D. Lodygin, G. S. Anisimov, S. N. Sazanova, Yu. A.Tabakova / / Equipment and technology of food production. 2023. Vol. 53. No. 1. Pp. 97-122. (In English). bs: / / doj.o^A0 1E03 / 2074 -94] D-2023 -1^419) and is not a distinctive feature of the invention.

[0028] The fermented mixture with lactulose is inactivated by heating and concentrated to obtain a lactulose-containing product - a postbiotic with healthy components. The modes of inactivation of cells and enzymes are known (for example, in the above-mentioned patent EP 3542641 at a temperature of 90°C to 150°C), the simplest method is boiling for 3-5 minutes. Due to the fact that the technology uses starter microorganisms with proven safety, no complex purification of the product from toxic products of their metabolism is required. Moreover, the metabolic products and components of inactivated cells of lactose-fermenting yeast (for example, vitamins, antioxidants, glucans, mannans, amino acids, bioactive peptides, etc.) and lactic acid bacteria (organic acids, exopolysaccharides, peptidoglycans, etc.) have health-promoting properties.In this case, inexpensive by-product milk raw materials (whey or permeate) are used both as lactose-containing raw materials for cultivating yeast and lactic acid bacteria, and as a source of lactose for its biotransformation into lactulose, which reduces the cost of the finished product. Concentration of the enzyme mixture is carried out in vacuum evaporators and / or by spray drying under known process conditions.

[0029] The method is confirmed by examples.

[0030] Example 1. Cheese whey, purified from fat and casein dust, is pasteurized at (85 ^ -87) °C for 3-5 sec. The whey is then cooled and used for culturing lactase producers and for the biosynthesis of lactulose in industrial fermenters. In the first stage, Kluyveromyces lactis yeast is cultivated under aerobic conditions at a temperature of (25-H30) °C for (22 ^ -24) h with intensive stirring, lactic acid bacteria Lactobacillus acidophilus are cultivated under aerobic conditions at a temperature of (37-H0) °C for (22 ^ -24) h without stirring. The obtained suspensions of yeast and lactic acid bacteria are combined and a second stage of joint cultivation is carried out under anaerobic conditions at a temperature of (25-N30)°C for (12-N4) h. After this, lactases are extracted from the cells of microorganisms by autolysis at a temperature of (40-N5)°C for (28-N30) h.After autolysis, the resulting enzyme preparation is added to pasteurized and cooled cheese whey with fructose dissolved in it at a molar ratio of lactose:fructose of 1:1, and lactulose biosynthesis is carried out at a temperature of (30-40)°C for (12-14) hours, after which the mixture is inactivated by boiling for several minutes, cooled and sent for vacuum concentration, which is carried out at a temperature of 65°C. Concentration is carried out to a mass fraction of dry matter (40-45)%. A condensed postbiotic product is obtained, containing about 5% lactulose, as well as metabolites and components of inactivated yeast cells and lactic acid bacteria.

[0031] Example 2. Dry UV permeate of skim milk is dissolved in hot water at (90^-95)°C based on achieving a mass fraction of lactose in the solution of 15%. The permeate is then cooled and used for culturing lactase producers and for the biosynthesis of lactulose in industrial fermenters. In the first stage, the yeast Kluyveromyces marxianus is cultivated under aerobic conditions at a temperature of (30-105) °C for (8-10) hours with intensive stirring. Lactic acid bacteria Streptococcus thermophilus are cultivated under aerobic conditions at a temperature of (4(1045) °C for (8-10) hours without stirring. The resulting suspensions of yeast and lactic acid bacteria are combined and the second stage of cultivation is carried out - jointly under anaerobic conditions at a temperature of (35-100) °C for (34-36) hours. After this, lactases are extracted from the cells of microorganisms by autolysis at a temperature of (55-100) °C for (18-100) hours.After completion of autolysis, the resulting enzyme preparation is added to the permeate with fructose dissolved in it at a molar ratio of lactose:fructose of 1:2 and lactulose biosynthesis is carried out at a temperature of (55-100)°C for (0.1-10.5) hours, after which the mixture is inactivated by boiling for several minutes, cooled and sent for spray drying. A dry product is obtained - a postbiotic with a mass fraction of dry matter of 95%, containing about 15% lactulose, as well as metabolites and components of inactivated yeast cells and lactic acid bacteria.

Claims

Invention formula An enzymatic method for obtaining a lactulose-containing postbiotic product, including the preparation of lactose-containing raw materials, a two-stage cultivation of lactase producers: lactose-fermenting yeast and thermophilic lactic acid bacteria, lactase extraction, lactulose biosynthesis using the obtained lactases, inactivation of cells and enzymes by heating, concentration of the fermented mixture by thickening and / or drying, wherein at the first stage of cultivation separate cultivation is carried out under aerobic conditions: yeast at a temperature of 25-35°C for 8-24 hours with intensive stirring, lactic acid bacteria at a temperature of 37-45°C for 8-24 hours without stirring; at the second stage - joint - under anaerobic conditions at a temperature of 25-40°C for 12-36 hours;the extraction of enzymes is carried out by autolysis at a temperature of 40-60°C for 18-30 hours, and the biosynthesis of lactulose is carried out using the obtained crude enzyme preparation, which is introduced into lactose-containing raw materials with the addition of fructose in a ratio of molar concentrations of lactose: fructose from 1:1 to 1:2, at a temperature of 30-60°C for 0.1-14 hours;

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