RU2453591C1 - Strain lactobacillus rhamnosus used for making lactic acid bacillus containing products - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: strain Lactobacillus rhamnosus LK-25 is recovered from a healthy infant, actively propagates in a nutrient medium with accumulation of an production biomass in a short 12-14-hour period of cultivation time of the high lactic acid bacillus concentration, is able to sour milk. The strain Lactobacillus rhamnosus LK-25 exhibits acid-forming activity, antagonist activity with respect to pathogenic microorganisms and opportunistic pathogens. The strain Lactobacillus rhamnosus LK-25 is deposited in Russian National Collection of Industrial Microorganisms, No. B- 10984. That allows the strain Lactobacillus rhamnosus LK-25 being used in making the products with lactic acid bacilli - bacterial preparations, biologically active food additives, fermented and non-fermented foodstuff, hygienic and cosmetic agents.

EFFECT: normalised microbiocenosis of a human body.

2 tbl, 5 ex

Description

The invention relates to the food, cosmetic, biotechnological and medical industries, in particular, it is used for the preparation of dairy, fermented and non-fermented foods, hygiene and cosmetics, dietary supplements and bacterial preparations.

It is known that the normal obligate microflora of the human body plays an important role for a healthy state of health: it participates in metabolic processes, synthesizes vitamins, utilizes harmful substances, is an antagonist to conditionally pathogenic and pathogenic microflora, and contributes to the formation of the human body's resistance to harmful factors .

Currently, due to adverse environmental conditions, stress, prolonged antibacterial, radiation and chemotherapy, dysbiosis resulting from past diseases, the number of people suffering from disorders of the normal microflora of the body is increasing. In this regard, for the treatment and prevention of these disorders, increasing attention is being paid to both drugs and food products containing normal human microflora, in particular lactobacilli.

The following types of lactobacilli are found in the human intestine:

Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus rhamnosus and others. Known patents for inventions, the basis of which are lactobacilli (patent No. 2259192, Russia, patent No. 2270855, Russia, patent No. 2170023, Russia, patent No. 2413761, Russia, patent No. 2376366, Russia, patent No. 2141521, Russia, patent No. 2108999, Russia, patent No. 78446711, USA, patent No. 7354757, USA, patent No. 6004551, USA, patent No. 6093394, USA, patent No. 637297, Switzerland; patent No. 2134179, Germany; patent No. 0430642, Japan).

The quality of medicinal products and preparations is largely due to the properties of the producer strain - its specific activity and adaptability. A large assortment of biologically active and technologically advanced strains of lactobacilli makes it possible to develop new mono-and complex medical nutrition products and drugs that can provide high colonization resistance of the human intestine, restore its normal microflora and reduce the incidence of various intestinal infections.

The objective of the present invention is to obtain a strain of microorganisms of the species Lactobacillus rhamnosus LK-25 with high biological activity, resistance to environmental conditions, with stabilizing acid formation in the entire temperature range of cell development, antagonistic activity against pathogenic and conditionally pathogenic microflora that ferment milk and multiplies by artificial nutrient media of various compositions.

The strain Lactobacillus rhamnosus LK-25 was obtained from a baby, deposited in VKPM, FSUE GosNIIgenetika, collection No. B-10984.

The inventive strain can be used in the production of fermented milk products, fermented foods, such as kvass, beer, etc., unfermented foods, such as juices, drinks, etc., starter cultures for the preparation of fermented milk products and biologically active food additives. The strain Lactobacillus rhamnosus LK-25 can also be used for the production of hygiene and cosmetics, for example creams, lotions, napkins, soaps, powders, tampons, nourishing masks, gels, women's pads, cosmetic milk, various powders, etc.

In addition, the strain of Lactobacillus rhamnosus LK-25 can be used in the production of bacterial preparations, in the form of suppositories, oral and other similar agents.

The technical result of the claimed invention is to obtain a strain of Lactobacillus rhamnosus LK-25, which can be used as a biologically active substance in dairy, fermented and non-fermented foods, starter cultures, hygiene and cosmetics, dietary supplements and bacterial preparations. Lactobacillus rhamnosus LK-25 has biologically and technologically significant properties, such as acid-forming activity, the ability to increase microbial biomass in various nutrient media. The strain Lactobacillus rhamnosus LK-25 is antagonistically active against pathogenic and opportunistic microorganisms.

Cultural and morphological properties of the strain.

Cells are gr + medium-length sticks with rounded ends, can be folded into long chains, ferment milk. On the surface of dense nutrient media form round colonies with smooth edges. Separately grown colonies on a semi-liquid nutrient medium have an elongated shape.

Physico-biochemical properties of the strain.

Indisputable, motionless facultative anaerobic. The temperature of cultivation varies in a wide range of 34-40 degrees Celsius. It does not form catalase, it does not dilute gelatin, it does not form hydrogen sulfide, it does not restore nitrates. Synthesizes L (+) - lactic acid without the formation of gas, acidifying the growth medium to a pH of 4.2-3.6. It ferments rhamnose, arabinose, sorbitol, beckons, lactose, maltose, sucrose, does not ferment raffinose, melibiosis.

The strain fermented milk by 12-14 hours of cultivation.

The strain Lactobacillus rhamnosus LK-25 has antagonistic activity against pathogenic and conditionally pathogenic microflora (Table 1).

Table 1. Antagonistic activity of the strain L. rhamnosus LK-25 Strain Zone of growth inhibition of test cultures mm / M ± m Sh. sonnei Sh. flexneri E. coli S. aureus Pr. vulgaris L. rhamnosus LK-25 25 ± 1.4 20 ± 1.6 18 ± 2.4 20 ± 1.2 22 ± 0.8

The strain actively increases the microbial mass during growth on hydrolyzate-milk medium (GM), Bifidum medium (BP), milk (M), producing acids (Table 2).

table 2 The accumulation of microbial mass (CFU / ml lg 10) and the activity of acid formation (in degrees Turner) of the strain Lactobacillus rhamnosus LK-25 in the dynamics of development NUTRITION CULTIVATION TIME
BIOLOGICAL ACTIVITY 12 h 24 h 48 h CFU ° T CFU ° T CFU ° T GM 8.2 96 9,4 110 7.6 172 Bf 8.0 84 9.2 98 7.4 126 M 8.2 78 8.6 108 8.4 136

The strain maximally accumulates microbial mass by 12-14 hours of cultivation, reaching in 1 ml of at least log 8-9 microbial cells, producing acids.

Thus, the proposed strain of Lactobacillus rhamnosus LK-25 is technologically advanced, because it multiplies in various nutrient media with the accumulation of production biomass with a high concentration of lactobacilli; possesses acid-forming activity, as well as antagonistic activity in relation to pathogenic and conditionally pathogenic microorganisms.

Sour-milk, fermented, non-fermented food products, hygienic, cosmetics, biologically active additives and bacterial preparations containing the Lactobacillus rhamnosus LK-25 strain can be used in the food, medical, biotechnological and cosmetic industries.

The ability of the strain Lactobacillus rhamnosus LK-25 to increase biomass in various nutrient media, acid-forming activity, antagonistic activity against pathogenic and conditionally pathogenic microflora, as well as other properties useful to the human body, characteristic of the species Lactobacillus acidophilus, make it possible to use the strain in the composition various corrective agents for therapeutic purposes.

Example 1. The use of strain Lactobacillus rhamnosus LK-25 in the composition of biologically active additives.

1 passage: a lyophilized strain of Lactobacillus rhamnosus LK-25 in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by ten-fold dilutions to 10 ^-9 . Incubated at a temperature of 38-40 degrees Celsius until the formation of visible growth in the last dilutions.

2 passage: a grown culture of 10 ^-7-9 dilutions in a volume of up to 10% is introduced into the nutrient medium and cultivated at a temperature of 38-40 degrees Celsius for 12-14 hours until growth with a visible aerobiosis zone is formed.

3 passage: the resulting biomass in an amount of up to 10% of the volume is introduced into the accumulation medium and cultivated for 12-14 hours at 38-40 degrees Celsius to obtain biomass with a content of live lactobacilli of at least 10 ⁸ / ml.

The result is a biologically active additive, which is a liquid concentrate with a high, not less than 10 ⁸ / ml, content of lactobacilli.

Example 2. The use of strain Lactobacillus rhamnosus LK-25 for the preparation of fermented and non-fermented foods.

1 passage: a lyophilized strain of Lactobacillus rhamnosus LK-25 in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by ten-fold dilution to 10 ^-9 . Incubated at 38-40 degrees Celsius until the formation of visible growth in the last dilutions.

2nd passage: a grown culture of 10 ^-7-9 dilutions in a volume of up to 10% is introduced into the nutrient medium and cultivated at a temperature of 38-40 degrees Celsius for 12-14 hours until a dense growth is formed.

3 passage: the resulting biomass in an amount of up to 10% of the volume is introduced into the accumulation medium and cultivated for 12-14 hours at 38-40 degrees Celsius to obtain biomass with a content of live lactobacilli of at least 10 ⁸ / ml.

Getting a fermented or non-fermented food product: the grown biomass is introduced into food raw materials (milk, juices, beer, kvass, etc.) in an amount up to 10% of the volume, mixed and poured into containers. The result is a product with a concentration of live lactobacilli at least 10 ⁶ / ml

Example 3. The use of strain Lactobacillus rhamnosus LK-25 for the preparation of fermented milk product.

1 passage: lyophilized strain is introduced into the culture medium and titrated by ten-fold dilutions to 10 ^-9 . Incubated at 38-40 degrees Celsius until the formation of visible growth in the last dilutions. Or dry lyophilized biomass of lactobacilli is introduced in an amount of 1-2 doses in a test tube with 30 ml of culture medium. Cultivate at a temperature of 38-40 degrees Celsius to obtain biomass containing live lactobacilli at least 10 ⁷ / ml.

2nd passage: the grown culture of the strain Lactobacillus rhamnosus LK-25 from the last dilutions or from a test tube with grown biomass is introduced into the accumulation medium with a volume of up to 10% and cultivated at a temperature of 38-40 degrees Celsius until a biomass with a content of lactobacilli of at least 10 ⁸ / ml is obtained .

3rd passage: the resulting biomass of the strain Lactobacillus rhamnosus LK-25 in a volume of up to 10% is introduced into sterile milk, cultivated until a clot forms (fermented milk product). The number of passages No. 2 can be increased depending on the volume of production.

Example 4. The use of strain Lactobacillus rhamnosus LK-25 to obtain bacterial preparations.

The culture of the strain of the 1st or 2nd passage (Example 1) is introduced into the nutrient medium, cultivated at a temperature of 38-40 degrees Celsius until the biomass of lactobacilli with the content of living cells of at least 10 ⁸ in 1 ml is obtained. Protective drying media (sucrose-gelatin medium, skim milk) are added to the resulting biomass. The resulting suspension is poured into ampoules, vials and other containers, frozen at a temperature of minus 35-45 degrees Celsius and lyophilized from a frozen state in a vacuum dryer. The resulting preparation contains at least 10 ⁷ living cells of lactobacilli. This content of lactobacilli is consistent with scientific and technical documentation for a similar drug with lactobacilli - "Lactobacterin" and is sufficient to use the resulting funds as a drug - eubiotics.

Example 5. The use of strain Lactobacillus rhamnosus LK-25 to obtain hygiene and cosmetics.

The culture of the strain of the 1st or 2nd passage (Example 1) is introduced into the nutrient medium, cultivated at a temperature of 38 ± 1 ° C until the biomass of lactobacilli with the content of living cells of at least 10 ⁸ in 1 ml is obtained. The resulting biomass of lactobacilli of the strain Lactobacillus rhamnosus LK-25 is introduced in an amount of not more than 1% in the composition of soap or cream.

The clinical efficacy of Lactobacillus rhamnosus was studied as part of the Normoflorin liquid dietary supplement on the basis of the Scientific Center for Children's Health of the Russian Academy of Medical Sciences (NCHZ RAMS). Under observation were 46 children aged 3 to 12 months with a diagnosis of atopic dermatitis of mild to moderate degree. "Normoflorin" was received for 30 days in doses corresponding to the Instructions for Supplementation. The control group of children "Normoflorin" did not receive. As a result of the studies conducted against the background of the use of a dietary supplement with Lactobacillus rhamnosus, a significant improvement in digestion processes, relief of manifestations of atopic dermatitis, and improvement of skin structure were noted in comparison with the control. Good tolerance was noted. Adverse events were not recorded.

Claims (1)

The strain of Lactobacillus rhamnosus VKPM B-10984 used to obtain products containing lactobacilli.