RU2260042C2 - Strain bifidobacterium bifidum for production of bacterium preparation, bioactive food supplement, ferment, fermented milk product, fermented and non-fermented foodstuff, hygienic and cosmetic products - Google Patents

Abstract

FIELD: biotechnology, food processing and medicine industry , in particular production of bacterium preparations.

SUBSTANCE: Strain Bifidobacterium bifidum is obtained by selection without using of genetic modification methods and is capable to utilize of inulin and cellobiose. Strain accumulates biomass on plant-origin substrates and artificial broth for short period with high bifidobacterium concentration. Strain has acid-forming properties and represents antagonist of pathogenic and saprogenic microflora. Bacterium preparations, bioactive food supplements, ferments, fermented milk products, fermented and non-fermented foodstuffs, hygienic and cosmetic products containing the same have probiotic effect and provide normalization of microbiocenose in human organism, including gastrointestinal and urogenital tracts, skin integument and mucilaginous.

EFFECT: new strain Bifidobacterium bifidum with probiotic and acid-forming properties.

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Description

The invention relates to biotechnology, food and medical industry and can be used in the production of bacterial preparations, biologically active food additives, fermented and non-fermented foods, hygiene and cosmetics.

A known strain of Bifidobacterium bifidum 791, which is the closest analogue to the claimed strain.

This strain was isolated from the intestines of a healthy infant, deposited in the Center for Medical Research “VNIIgenetika” under No. 3300 and is characterized by the following features.

1. Cultural and morphological characteristics.

Gram-positive rod-shaped cells with bifurcations at the ends, motionless, in a smear form clusters in the form of Chinese characters.

In liquid nutrient media (hepatic, hydrolyzate-dairy, casein-yeast) supplemented with 0.75-1% agar, they grow throughout the entire volume except the aerobiosis zone, accumulating biomass by 20-24 hours. Separate colonies are in the form of "comets".

2. Physiological and biochemical characteristics.

Obligate anaerobic. Ferments lactose with the formation of acetic and lactic acids. It does not ferment arabinose, xylose, mannose, cellobiose, inulin, mannitol, sorbitol, salicin.

Fermenting milk at 16-18 hours with the formation of a clot of tender consistency, pH 4.3-4.5.

Acid-forming activity: on the hepatic environment: by 24 hours - 80-90 ° T, by 72 - 90-100 ° T; on a GM environment: up to 100 ° T and 200 ° T, respectively.

3. Antagonistic properties.

The strain exhibits against Shigella Sonne, Flexner, enteropathogenic Escherichia coli, staphylococci.

The disadvantage of this strain is that it can absorb only lactose from carbohydrates. As a result, when it enters the human body, its viability, stability and colonizing activity during the passage of the digestive tract are insufficient.

A known strain of Bifidobacterium bifidum 791 BAG is used to obtain a liquid concentrate of bifidobacteria, sour-milk therapeutic and dietary products and dietary supplements (RF Patent 2165454 from 2001.04.20).

The objective of the invention is to obtain a strain of Bifidobacterium bifidum 791-IN, which provides the utilization of a wider range of carbohydrates, has higher biological activity and resistance to external influences, as a result of which it is more technologically advanced: it accumulates biomass on nutrient substrates from natural plant materials and artificial nutrient media and ferment milk in a shorter time and with a large yield of active biomass.

To obtain a strain of Bifidobacterium bifidum 791-IN, a selection of the strain Bifidobacterium bifidum 791 was performed. Selection was carried out without using genetic modification methods by repeated passages through a synthetic nutrient medium of the following composition:

per 1 liter of distilled water:

Epidermate-0 10 g inulin 10 g Cysteine 0.5 g Sodium Chloride 2.5 g Magnesium Sulfuric Acid 0.5 g Sodium Acetic Acid 0.3 g Agar agar 0.75 g

After the above components were dissolved in the medium, the pH was adjusted to 7.2-7.4, poured into vials and sterilized at 0.5 atm for 30 minutes.

The primary seed of the strain Bifidobacterium bifidum 791 in a volume of 5% was added to the above medium and cultivated at 37 ° C for 24 hours. Then, 30 passages of the culture grown on this medium were conducted through an environment of the same composition, after each of which clones were selected according to the signs of maximum biomass accumulation during the cultivation cycle. If at the first passages a microbial yield of not more than 10 ⁴ -10 ⁵ in 1 ml was obtained, then at the 25th passage the number of CFU of bifidobacteria reached 10 ⁹ and remained at that level during subsequent passages. This is due to the increased production of inulinase as a result of prolonged passage on a medium containing inulin.

As a result of the selection, the strain Bifidobacterium bifidum 791-IN was obtained, which was deposited in the State collection of microorganisms - representatives of the normal human microflora under No. 162.

The inventive strain can be used in the production of bacterial preparations in various forms, including capsules, suppositories, ointments; biologically active food additives, starter cultures, fermented and non-fermented foods, including dairy products, juices, drinks, kvass, etc. Also, the strain Bifidobacterium bifidum 791-IN can be used in the production of hygienic and cosmetic products, for example, creams, gels, nourishing masks.

The technical result of the claimed invention is the production of bacterial preparations, biologically active food additives, starter cultures, fermented and non-fermented foods, hygienic and cosmetic products, which contain the Bifidobacterium bifidum 791-IN strain as an active principle of probiotic orientation, which provides for the utilization of a wider range of carbohydrates and therefore more technological than the prototype, has a higher biological activity and resistance to external activities, which provides a more active colonization of the intestine and the restoration of resident microflora, and will also expand the arsenal of technical means of therapeutic and prophylactic orientation. The inventive strain has the following properties.

Cultural and morphological: gram-positive homogeneous rods, uniformly stained with methylene blue, located mainly in chains.

Physiological and biochemical: ferments lactose, inulin, cellobiose with the formation of acetic and lactic acids and traces of formic and propionic acids.

Arabinose, xylose, mannose, mannitol, sorbitol, salicin does not ferment. In all culture media accepted in production, at least lg9 microbial number grows with accumulation of biomass of bifidobacteria in 16-18 hours. The maximum biomass accumulation in this case exceeds the analogue by 0.5-1 log. The activity of acid formation in this case also exceeds those of the prototype. When cultured on a medium with inulin, the biomass yield exceeds the prototype by 4 orders of magnitude.

The data are presented in table 1.

Table 1
The number of CFU in 1 ml and the active acidity in ° T after 16-18 hours of cultivation Culture medium B. Bifidum 791 B. Bifidum 791-IN Hepatic (Blaurocca) 2 × 10 ⁸ 60 ° T 6 × 10 ⁸ 60 ° T Hydrolyzed milk 5 × 10 ⁸ 100 ° T 8 × 10 ⁹ 140 ° T Milk 1 × 10 ⁸ 70 ° T 5 × 10 ⁸ 90 ° T Epidermal with inulin 1 × 10 ⁵ 40 ° T 1 × 10 ⁹ 100 ° T

Antagonistic properties of the claimed strain are manifested in relation to Shigella Sonnet, Flexner, Salmonella, enteropathogenic Escherichia coli, Staphylococcus aureus, Proteus and are more pronounced than in the prototype (table 2).

table 2
Test Crop Growth Delay Zone strain S.sonnei S.flexnery E.coli S.aureus Pr.vulgaris B. bifidum 791 16.1 15.3 15.1 14.5 17.3 B. bifidum 791-IN 22.5 29.0 18.3 25.0 19.6

Thus, from the above data it is seen that the new strain of Bifidobacterium bifidum 791-IN is highly technological, actively increases the biomass of bifidobacteria on production media, while its productivity is on average 0.5 lg superior to the prototype; unlike the prototype, it utilizes inulin and cellobiose and grows on substrates containing these carbohydrates; differs in higher activity of acid formation than the prototype. This increases the functional value of products and preparations prepared on the basis of such a strain. When a culture of such a strain enters the human body, a more extensive set of plant substrates is utilized, as a result of which colonization of the intestinal mucous membranes, displacement of pathogenic and putrefactive microflora are more active, and resident microflora is more effectively restored.

Example 1. Obtaining a lyophilized bacterial preparation.

A protective drying medium (sucrose 80% and gelatin 8% in a volume of 10-12%) is added to a suspension of a 22-24-hour culture of bifidobacteria of the strain Bifidobacterium bifidum 791-IN and poured into 5 ml penicillin vials, frozen at -40-45 ° C and freeze-dried from a frozen state in a vacuum dryer. The resulting preparation contains in 1 g of dry biomass at least 10 ⁸ living bifidobacteria cells.

Example 2. The use of strain Bifidobacterium bifidum 791-IN as a starter culture for the preparation of a product of therapeutic and functional nutrition.

A suspension of a 14-16 hour culture of the strain Bifidobacterium bifidum 791-IN is introduced into sterile milk in a volume of 5% by volume of milk, mixed and cultured for 8-10 hours until a clot forms, after which the product is cooled and drained. The result is a therapeutic and prophylactic dairy product with a bifidobacteria content of at least 10 ⁸ in 1 ml.

Example 3. Obtaining a liquid form of a biologically active food supplement.

The culture inoculum of the strain Bifidobacteriun bifidum 791-IN is added in a volume of 5-10% to a fermenter with a nutrient medium of the following composition per 1 liter of product: 5% aqueous Jerusalem artichoke extract, 2% collagen hydrolyzate, 0.5% sodium chloride, 0 , 5% rosehip extract, 1% pectin, 0.1% agar agar and cultured for 6-8 hours at 37.5-38 ° C and sterilely poured into containers. The result is a biologically active food supplement with a content of bifidobacteria of at least 10 ⁸ in 1 ml. The resulting additive has a pronounced immunocorrective effect.

Example 4. Obtaining an encapsulated form of a biologically active food supplement.

To a suspension of a 22-24 hour culture of bifidobacteria of the strain Bifidobacterium bifidum 791-IN, a drying medium in a volume of 20% is added and spray dried. 1 g of finished biomass contains at least 10 ⁹ viable bifidobacteria.

For encapsulation, a mixture is prepared, which in equal parts includes the following ingredients: dried biomass of bifidobacteria, lactulose, pectin, dextrin. To the mixture, based on the resulting mass, add 1% magnesium stearate and 1% ascorbic acid. After thorough mixing, encapsulation is carried out in capsules No. 0 of 0.3 g. One capsule contains at least 10 ⁸ viable bifidobacteria.

Example No. 5. The use of strain Bifidobacterium bifidum 791-IN to obtain hygiene and cosmetics. Dry biomass of bifidobacteria strain with a content of bifidobacteria of at least 10 ⁹ in 1 g is introduced into the composition of the nutrient cream at the rate of 1 g per 100 g of cream.

Example No. 6. The use of strain Bifidobacterium bifidum 791-IN for the preparation of juice.

The culture inoculum of the strain Bifidobacterium bifidum 791-IN in a volume of 5-10% is introduced into a fermenter with a nutrient medium of the following composition per 1 liter of product: 5% aqueous Jerusalem artichoke extract, 2% collagen hydrolyzate, 0.5% sodium chloride, 0 , 5% rosehip extract, 1% pectin, 0.1% agar agar, cultured for 6-8 hours at 37.5-38 ° C and introduced into juice, mixed and poured into containers. The result is a product with a concentration of live bifidobacteria of at least 10 ⁶ / ml.

Example No. 7. The use of strain Bifidobacterium bifidum 791-IN to obtain hygiene products.

The dry biomass of bifidobacteria strain with a bifidobacteria content of at least 10 ⁹ in 1 g is introduced into the composition of the toothpaste at the rate of 1 g per 100 g of paste.

Claims (1)

The strain of Bifidobacterium bifidum 791-IN GKNM GU MNIIEM MH RF them. G.N. Gabričevskogo No. 162, used to obtain bacterial preparations, biologically active food additives, starter cultures, sour-milk, fermented and non-fermented foods, hygiene and cosmetics.