RU2260041C2 - Method for probiotic production, two strains streptococcus salivarius subsp thermophilus used for probiotic production - Google Patents

Abstract

FIELD: biotechnology, in particular food processing, dairy and medicine industry.

SUBSTANCE: claimed method includes broth preparation, sterilization and cooling. In prepared broth probiotic strain together with two non-lysogenic strains of thermophylic streptococcus in ratio 4:1 and 3:2, respectively, are cultivated. Mass of each probiotic strain Streptococcus salivarius subsp. Thermophilus is built up separately from one another. Then strains are mixed in equal amounts, pre-packed, dried and packed.

EFFECT: increased assortment of probiotic agents with improved properties.

4 cl, 2 tbl, 5 ex

Description

The invention relates to biotechnology, in particular the dairy industry, the food and medical industries, and can be used to obtain probiotics intended for direct consumption or as a biologically active food supplement, to normalize the beneficial microflora of the human gastrointestinal tract.

The advantages of probiotics over fermented milk products with probiotic properties are that they can be consumed with various foods in both ordinary and autonomous conditions; they are convenient for transportation and can be delivered to the most remote corners of the country and at the same time probiotics contribute not only to the restoration of autoflora, but also to a number of vital functions of the human body.

Probiotic cultures have a positive effect on human health [1, 2]. Nevertheless, to enhance the in vivo effect of probiotics, scientists propose using special substances that activate the action of probiotic cultures in the human body [3] or compositions that include microorganisms and their combinations with prebiotics or oligosaccharides [4, 5, 6].

A known method of producing a fermented milk product, including the normalization of milk, the introduction of "Lactusan", a vitamin and mineral supplement, pasteurization, homogenization, cooling, fermentation and fermentation with complex fermentation, cooling, bottling and packaging of the product. This method only applies to obtaining a certain composition of the fermented milk product, which has a short shelf life and is feasible in regions where there is a sufficient amount of raw materials, in particular milk [7].

A known method of obtaining the probiotic "Simbiter-2" and a method of production using fermented milk product [8]. A method for producing a probiotic involves co-culturing in a milk medium bifidobacteria of various species, acetic acid bacteria and propionic acid bacteria, which are mixed with a previously prepared consortium of lactic acid bacteria in a ratio of 4: 1. Describes a method of producing a fermented milk product with a probiotic "Simbiter-2" based on milk or buttermilk, or a mixture of milk with buttermilk, or a mixture of milk with milk whey or a mixture of buttermilk with whey. The disadvantage of the method of obtaining probiotic is that when co-culturing different types of bifidobacteria that differ in the dynamics of development, it is not always possible to guarantee a probiotic of stable quality, where all the types of bifidobacteria used will be present in sufficient quantities. The use of this probiotic is limited only by its use for the production of sour-milk products of a certain composition and having a short shelf life.

Closest to the invention in terms of essential features is a method for producing a dry bacterial preparation "Bifacid" [9], which includes preparing a nutrient medium, heat treating it, cooling to incubation temperature, separate cultivation of specific strains of acidophilic bacteria and bifidobacteria, mixing them in equal proportions, freeze-drying. The disadvantage of this method of obtaining a dry bacterial preparation is the use of culture media that are most favorable for the accumulation of cells of specific strains of acidophilic bacteria and bifidobacteria and do not contain ingredients that enhance the probiotic effect of the drug in the human body.

The essence of the proposed technical solution lies in the fact that in the method of producing a probiotic, which involves the use of a nutrient medium based on skim milk with an increased mass fraction of dry solids, sodium citrate and with the addition of ingredients that provide not only biomass with a high number of bacterial cells of probiotic cultures, but and colonization of the used microorganisms in the human intestine. In this case, separate cultivation of different types of probiotic cultures (Bifidobacterium adolescentis, Bif. Longum, Bif. Bifidum, Bif. Infantis, Bif. Breve, Lactobacillus acidophilus, L. casei, L. plantarum, L. bulgaricus and others) is carried out that meet modern requirements for probiotic strains, under rational conditions, but with their joint development with non-isogenic strains of Streptococcus salivarius subsp. thermophilus. As non-lysogenic cultures, special strains of Streptococcus salivarius subsp are used. thermophilus, one of which has a high natural ability to produce the enzyme β-galactosidase (strain Streptococcus salivarius subsp. thermophilus VKPM B-7985), and the other (strain Streptococcus salivarius subsp. thermophilus VKPM B-7984) exopolysaccharides: glucose and. Co-cultivation of probiotic strains with strains of thermophilic lactic streptococcus allows to increase the number of viable cells in probiotic (Table 1), accumulate biologically active substances (antibiotic substances, lactic acid, acetic acid, etc.) and obtain immobilized cells of probiotic strains due to their attachment to polysaccharides.

The use of non-lysogenic strains of thermophilic lactic streptococcus reduces the likelihood of phagolysis in the process of fermentation of a nutrient medium when receiving a probiotic.

The method uses a specially selected strain of Streptococcus salivarius subsp. thermophilus VKPM B-7985, which has a high natural ability to produce the enzyme β-galactosidase.

The ability to produce the enzyme β-galactosidase was evaluated by an individual index of lactobacillus activity. The lactose-protecting activity index (Il) was determined as the ratio of the amount of lactose fermented by a specific strain (L) to the maximum amount of fermented lactose (L _max ) from the sample: Il = L / L _max . To obtain a probiotic, it was recommended to use strains of thermophilic lactic streptococcus with an index of lactobacillus activity not lower than 0.85.

Strains of Streptococcus salivarius subsp. thermophilus VKPM B-7985 and Streptococcus salivarius subsp. thermophilus VKPM B-7984 are characterized by cultural-morphological, genetic, physiological and biochemical characteristics and technological properties.

Strain Streptococcus salivarius subsp. thermophilus VKPM B-7984 is a gram-positive, facultative anaerobic, non-spore-forming, motionless, catalase-negative cocci. Cells are arranged in pairs (diplococci) and in the form of chains of different lengths. When grown in liquid hydrolyzed milk, it grows over the entire height of the volume, and on sterile skim milk it forms a milk clot uniform throughout the entire column of milk. On the surface of solid nutrient media (hydrolyzed milk with agar and sucrose) forms small colonies of a rounded shape with a granular structure, deep colonies have a lenticular shape.

The content of HC in DNA is 38.7 mol%, which confirms its taxonomic belonging to the genus Streptococcus. Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 does not have plasmid DNA, which indicates its ability to stably maintain its characteristic signs and properties.

The optimum development temperature is (37-42) ° C, the pH of the nutrient medium is 7.2 ± 0.2; does not form catalase; ferments carbohydrates: lactose, glucose, sucrose. When fermented, lactose forms L (+) - lactic acid. Does not ferment: maltose, mannitol, salicin, sorbitol, rhamnose, dextrin. It can develop on nutrient media in the range of pH 5.5-8.3; resistant to 20% bile and 2.0% NaCl; unstable to 0.1% methylene blue and alkaline reaction medium (pH 9.6); does not restore litmus milk.

The fermentation activity of sterile milk with a 3% strain is 4.0-6.5 hours, the energy of acid formation after 5 hours of cultivation in milk is (60-65) ° C, the limit of acid formation after 7 days is (100 ± 3.4) ° FROM. The number of cells in sterile skim milk averages 9.1 · 10 ⁸ -2.5 · 10 ⁹ in 1 cm ³ ; the water holding capacity of the clot formed in milk is (3.6 ± 0.4) cm ³ . When it develops in milk of normal composition, it forms a viscous clot, which has a mucous consistency and has a clean sour-milk taste without extraneous smacks and odors. The enzyme produces β-galactosidase: the index of lactobacillus activity is 0.88 (table 2).

A distinctive feature of Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 is a natural ability to produce a sufficiently high amount of exopolysaccharides (247-282) mg / dm ³ .

Streptococcus salivarius subsp. thermophilus strain VKPM B-7985 is gram-positive, facultatively anaerobic, non-spore-forming, motionless, catalase-negative cocci. Cells are arranged in pairs (diplococci). When grown in liquid hydrolyzed milk, it grows over the entire height of the volume, and on sterile skim milk it forms a milk clot uniform throughout the column of milk with separation on the surface of the whey. On the surface of solid nutrient media (hydrolyzed milk with agar and sucrose) forms small colonies of a rounded shape with a granular structure, deep colonies have a lenticular shape.

The content of HC in DNA is 39.9 mol%, which confirms its taxonomic belonging to the genus Streptococcus. Streptococcus salivarius subsp. thermophilus strain VKPM B-7985 does not have plasmid DNA, which indicates its ability to stably maintain its characteristic signs and properties. The optimum development temperature is (37-42) ° C, the pH of the nutrient medium is 7.2 ± 0.2; does not form catalase and ferments carbohydrates: lactose, glucose, sucrose. When fermented, lactose forms L (+) - lactic acid. It can develop on nutrient media in the range of pH 6.0-8.3; resistant to 20% bile; unstable to NaCl, to an alkaline reaction of the medium (pH 9.60), to 0.1% methylene blue and does not restore litmus milk.

The fermentation activity of sterile milk with a 3% strain is 4.0-7.0 hours, the energy of acid formation after 5 hours of cultivation in milk is (70-75) ° C, the limit of acid formation after 7 days is (109 ± 3.2) ° FROM. The number of cells in sterile skim milk averages 5.0 · 10 ⁸ -6.0 · 10 ⁸ in 1 cm ³ ; the water holding capacity of the clot formed in milk is (5.0 ± 0.1) cm ³ . When it develops in milk of normal composition, it forms a medium-density clot with a whey compartment, which has a pure sour-milk taste without extraneous smacks and odors. Produces exopolysaccharides (47-62) mg / dm ³ in small quantities. A distinctive feature of Streptococcus salivarius subsp. VKPM strain B-7985 is a natural ability to produce a sufficiently high amount of β-galactosidase enzyme: an individual index of lactose-protecting activity is 1.0 (Table 2).

The technical result from the implementation of the invention is a social effect aimed at improving the population, and which is achieved by increasing the arsenal of probiotic agents with improved properties. This goal is achieved through the joint use of probiotic cultures and specially selected natural strains: Streptococcus salivarius subsp. thermophilus strain VKPM B-7985, with high ability to produce β-galactosidase, and Streptococcus salivarius subsp. thermophilus VKPM B-7984 producing exopolysaccharides, which increases the epithelial adhesion of probiotic cultures and helps to restore autoflora in the recipient's body.

Example 1. In 1 dm ³ skim milk with a mass fraction of solids of 12% and 0.75% sodium citrate make 4 cm ³ syrup "Lactusan", mix and sterilize at a temperature of 112 ° C for 20 minutes, cool to a temperature of 38 ° C and make 100 cm ³ 5% sterile solution of vitamin C and mix. The prepared medium is transferred to a fermenter, into which 40 cm ^{3 of an} activated culture of Bifidobacterium adolescentis, 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985. After that, the process of increasing the biomass of bacteria begins, carried out at a temperature of 37 ° C, before the onset of the stationary phase of the development of the probiotic strain, adjust the pH with a 5% ammonia solution to 6.0. Then, in the case of using biomass in liquid form, it is poured into sterile bottles, hermetically sealed and labeled. To obtain biomass in dry form, it is mixed with a protective medium (sucrose-gelatin), poured into bottles or trays and dried by sublimation. After drying, the bottles are sealed sterilely; the mass dried on the trays, packaged in bags. Store a ready-made liquid probiotic at a temperature of 4 ° C for no more than 30 days, and dry at a temperature of minus 18 ° C for no more than 6 months.

Example 2. In 2 dm ³ skim milk with a mass fraction of solids of 13% and 0.75% sodium citrate make 10 cm ³ syrup "Lactusan", mix and sterilize at a temperature of 112 ° C for 20 minutes, cool to a temperature of 38 ° C and make 200 cm ³ 5% sterile solution of vitamin C and mix. The prepared medium is transferred in equal amounts to two fermenters, in one of which 40 cm ^{3 of the} activated culture of Bifidobacterium bifidum, 5 cm ^{3 of} Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the other, 30 cm ^{3 of} Lactobacillus acidophilus, 10 cm ^{3 of} Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 10 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985. After that, the process of increasing the biomass of bacteria begins, carried out at a temperature of 37 ° C, before the onset of the stationary phase of the development of the probiotic strain, adjust the pH with a 5% ammonia solution to 6.0. Then the finished biomass from the first fermenter is introduced into the finished biomass in the second fermenter, receiving a polyprobiotic, mixed, poured into sterile bottles, hermetically sealed and labeled. To obtain biomass in dry form, it is mixed with a protective medium (sucrose-gelatin), poured into bottles or trays and dried by sublimation. After drying, the bottles are sealed sterilely; the mass dried on the trays, packaged in bags. Store a ready-made liquid probiotic at a temperature of 4 ° C for no more than 30 days, and dry at a temperature of minus 18 ° C for no more than 6 months.

Example 3. In 3 dm ³ skim milk with a mass fraction of solids of 14% and 0.75% sodium citrate make 15 cm ³ syrup "Lactusan", mix and sterilize at a temperature of 112 ° C for 20 min, cool to a temperature of 38 ° C and make 300 cm ³ 5% sterile solution of vitamin C and mix. The prepared medium is transferred in equal amounts to three fermenters, in one of which 30 cm ^{3 of the} activated culture of Lactobacillus acidophilus, 10 cm ^{3 of} Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 10 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the second - 40 cm ³ Lactobacillus casei, 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the third - 40 cm ³ Lactobacillus plantarum, 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985. After that, the process of increasing the biomass of bacteria begins, carried out at a temperature of 37 ° C, before the onset of the stationary phase of the development of the probiotic strain, adjust the pH with a 5% ammonia solution to 6.0. Then, the finished biomass from the first fermenter is mixed under sterile conditions with the finished biomass from the second and third fermenters in equal amounts, receiving a polyprobiotic, mixed, poured into sterile bottles, hermetically sealed and labeled. To obtain biomass in dry form, it is mixed with a protective medium (sucrose-gelatin), poured into bottles or trays and dried by sublimation. After drying, the bottles are sealed sterilely; the mass dried on the trays, packaged in bags. Store a ready-made liquid probiotic at a temperature of 4 ° C for no more than 30 days, and dry at a temperature of minus 18 ° C for no more than 6 months.

Example 4. In 4 dm ³ skim milk with a mass fraction of solids of 14% and 0.75% sodium citrate make 20 cm ³ syrup "Lactusan", mix and sterilize at a temperature of 112 ° C for 20 minutes, cool to a temperature of 38 ° C and make 400 cm ³ 5% sterile solution of vitamin C and mix. The prepared medium is transferred in equal amounts to four fermenters, in one of which 30 cm ^{3 of the} activated culture of Lactobacillus delbrueckii subsp. bulgaricus, 10 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 10 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the second, 40 cm ^{3 of} Bifidobacterium adolescentis, 5 cm ^{3 of} Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the third - 40 cm ³ Lactobacillus casei, 5 cm ³ Str. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the fourth - 40 cm ³ Bifidobacterium breve, 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985. After that, the process of increasing the biomass of bacteria begins, carried out at a temperature of 37 ° C, before the onset of the stationary phase of the development of the probiotic strain, adjust the pH with a 5% ammonia solution to 6.0. Then, the prepared biomass from the first fermenter is mixed under sterile conditions with the prepared biomass from the second, third and fourth fermenters in equal amounts, receiving a polyprobiotic, mixed, poured into sterile bottles, hermetically sealed and labeled. To obtain biomass in dry form, it is mixed with a protective medium (sucrose-gelatin), poured into bottles or trays and dried by sublimation. After drying, the bottles are sealed sterilely; the mass dried on the trays, packaged in bags. Store a ready-made liquid probiotic at a temperature of 4 ° C for no more than 30 days, and dry at a temperature of minus 18 ° C for no more than 6 months.

Example 5. In 5 dm ³ skim milk with a mass fraction of solids of 12% and 0.75% sodium citrate make 20 cm ³ syrup "Lactusan", mix and sterilize at a temperature of 112 ° C for 20 minutes, cool to a temperature of 38 ° C and add 500 cm ³ 5% sterile solution of vitamin C and mix. The prepared medium is transferred in equal amounts to five fermenters, in one of which 30 cm ^{3 of the} activated culture of Lactobacillus acidophilus, 10 cm ^{3 of} Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 10 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the second, 30 cm ^{3 of} Lactobacillus delbmeckii subsp. bulgaricus, 10 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 10 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the third - 40 cm ³ Lactobacillus plantarum, 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the fourth - Bifidobacterium bifidum, 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985; in the fifth - Bifidobacterium infantis, 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7984 and 5 cm ³ Streptococcus salivarius subsp. thermophilus strain VKPM B-7985. After that, the process of increasing the biomass of bacteria begins, carried out at a temperature of 37 ° C, before the onset of the stationary phase of the development of the probiotic strain, adjust the pH with a 5% ammonia solution to 6.0. Then, the prepared biomass from the first fermenter is mixed under sterile conditions with the prepared biomass from the second, third, fourth and fifth fermenters in equal amounts, receiving a polyprobiotic, mixed, poured into sterile bottles, hermetically sealed and labeled. To obtain biomass in dry form, it is mixed with a protective medium (sucrose-gelatin), poured into bottles or trays and dried by sublimation. After drying, the bottles are sealed sterilely; the mass dried on the trays, packaged in bags. Store a ready-made liquid probiotic at a temperature of 4 ° C for no more than 30 days, and dry at a temperature of minus 18 ° C for no more than 6 months.

Literature

1. US Patent 5,589,168, A 23 C 009/12, C 12 R 001/46 Probiotic / Alien; William D .; Linggood; Margaret A. - Claim 09/02/1994; publ. 12/31/1996.

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Claims (4)

1. A method of obtaining a probiotic, including the preparation of a nutrient medium containing skim milk with a given dry matter content, sodium citrate - 0.75 wt.%, Sterilization, cooling, separate cultivation of the probiotic strain - lactobacilli and bifidobacteria with the accumulation of biomass, packaging and freeze-drying , packaging, characterized in that the cultivation of lactobacilli and / or bifidobacteria is carried out in conjunction with strains of Streptococcus salivarius subsp. thermophilus VKPM B-7984 and Streptococcus salivarius subsp. thermophilus B-7985 in a ratio of 4: 1 or 3: 2, respectively, and mixing the accumulated biomass in equal quantities, while the Lactusan syrup is additionally added to the nutrient medium in an amount of 0.3-0.6 wt.%, and after cooling the medium vitamin C is added to it in an amount of 0.4-0.6 wt.%. 2. The method according to claim 1, characterized in that the nutrient medium contains skim milk with a solids content of 12-14 wt.%. 3. The strain of Streptococcus salivarius subsp. thermophilus VKPM B-7984 used to obtain a probiotic. 4. The strain of Streptococcus salivarius subsp. thermophilus VKPM B-7985, used to obtain a probiotic.