RU2170023C2 - Strain lactobacillus acidophilus a-91 used for preparing fermented-milk products, strain lactobacillus acidophilus h-91 used for preparing fermented-milk products, ferment for preparing fermented-milk products and preparation promoting body adaptation to unfavorable environment effects - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: strain Lactobacillus acidophilus A-91 deposited in collection of VNII SKHMB N D-75 from 16.07.1997 and strain Lactobacillus acidophilus H-91 deposited in collection VNII SKHMB N D-76 from 16.07.1997 form in common symbiotic system characterizing synthrophic type of relationship that is used for preparing fermented-milk products. Ferment based on indicated system has a mixture of strains Lactobacillus A-91 and H-91 taken in the ratio of microorganisms from 4:1 to 1:4. Preparation "Vitaflor" promoting body adaptation to unfavorable environment effects based on acidophilic bacteria has a mixture of strains Lactobacillus acidophilus A-91 and H-91 taken in the ratio of microorganisms from 4: 1 to 1:4 in the content of living cells from 1 to 10 billion cells/1 g of preparation. Preparation showed high effectiveness in some diseases and for body adaptation to unfavorable environment conditions. Invention can be used in food and dairy industry, medicine, agriculture. EFFECT: strains indicated above, valuable medicinal properties, enhanced effectiveness of preparation. 5 cl, 2 tbl

Description

 The invention relates to the field of biotechnology, and in particular to strains for the production of sour-milk drinks, their production technology at home or industrial conditions and products with medicinal properties based on them. The invention can be used in food and dairy industries, medicine, agriculture and related fields.

 Known bacterial drugs for medical use, such as colibacterin, lactebacterin, bifidobacterin (a Guide to vaccine and serum. Edited by Acad. AMS SSSU I. N. Burgasova, Moscow, "Medicine", 1978) contains one or two strains of microorganisms having necessary biomedical characteristics.

 The disadvantage of these drugs is their low therapeutic activity and the inability to use for fermentation of milk.

 It is known to use ginseng, eleutherococcus, sea buckthorn, catnip, mummy, propolis and preparations from other natural sources as adaptagens (V.I. Mashanov, A.A. Pokrovsky. Spicy aromatic plants, M., Agropromizdat, 1991; I.S. . Sokolova and other wild-growing and cultivated plants, M., Medicine, 1990).

 The disadvantage of these drugs is the limited natural resources, non-standard raw materials, the presence of side effects.

 Currently, fermented milk drinks are widely used, combining the availability of raw materials with relative safety and effectiveness.

 In particular, the Health drink is known, obtained on the basis of pasteurized whey, fermented sourdough, prepared from pure races of lactic streptococcus, Bulgarian, acidophilus and cheese sticks (A. G. Khramtsov. Non-waste technology in the dairy industry, M., Agropromizdat, 1989, p. 210).

 The disadvantage of the drink is the low adaptive efficiency and lack of therapeutic effect.

 To improve the quality of preparations based on whey, bacterial additives (starter cultures) are used that mainly improve the nutritional and taste characteristics of the preparations.

 It is known as starter culture to use a mixture in a ratio of 1: 1 cultures of Str. cremons and Str. diacetitaetis (Aut. St. USSR N 581923, class A 23 C 9/12).

 The disadvantage of the starter culture is its low biological value due to the low antagonistic activity of these microorganisms in relation to extraneous microflora.

 A more effective starter culture based on lactic acid bacteria is known, containing equal parts of Saceharomyces cerevisiae, Lactobacillus fermenti and Lactobacillus helveticus (Ed. St. USSR N 1329745, class A 23 C 9/00).

A disadvantage of the starter culture is the poor compatibility of microorganisms, which necessitates the use of several temperature conditions when using them, low adaptive properties when introduced into milk whey
The prototype of the claimed group of inventions is the drink "Elixir", used in practice as a drug to increase the adaptive capabilities of the body under adverse environmental conditions, and the strain of microorganisms and fermented milk sourdough used in its preparation (RF Patent N 2064268, 1996, class A 23 C 9/12). The drink is obtained using Lactobacillus acidophilus (L. Acidophilus) strain 20T with the addition of Str. thermophilus strain 24C, Lactobacillus casei strain 2, Lactobacillus helveticus strain 305-5. To obtain a drink using bacterial starter culture containing these cultures in a ratio of 20-35 wt.%, 20-35 wt.%, 20-35 wt.% And 5-15 wt.%
The disadvantage of the prototype is the complex technology of obtaining, mutual inhibition of strains of various bacterial cultures.

 The challenge facing the researchers was the creation of such strains that, while maintaining the effectiveness of the prototype, would be in more stable equilibrium and would be close in nature, which would greatly simplify the preparatory stages of the process of obtaining a drink, making it possible to make a drink by non-specialists in the household, and could also have independent significance as a concentrate useful for the intestines with dysbiosis and other negative factors.

 This problem is solved by the creation of strains of L. acidophllus A-91 and H-91, fermented milk sourdough containing these strains, and a drug based on it.

 The strain Lactobacillus acidophllus A-91 was isolated from the intestines of a healthy child in 1991 in Leningrad, is a wild type and was identified by the identifier: O. Kandlor & N. Weiss 1986., Regular, Nonsporing Grampositive Rods // In; P.H Sneath, N. S. Mair, M. E. Sharpe & J.G. Holt (ed.) Bergey, s manual of Systematic bacteriology, v. 2, The Willians & Wilkin & Co., Baltumore.

 The strain was deposited in the collection of the All-Russian Research Institute of Agricultural Medicine No. 75 of 07.16.1997.

The strain is characterized by the following features
Cultural and morphological features
When grown on a base medium for identification of lactobacilli (N 1) for 48 hours at a temperature of 38-40 ^o C, uniform turbidity forms in the broth, in a semi-liquid medium - hanging, drop-like colonies, in the thickness of the agar - whitish colonies of complex configuration up to 1.5 -2.0 mm in diameter. On the surface of a solid agar medium, roundish, slightly convex colorless (over time light yellow) colonies are formed with a diameter of 1.8-2.2 mm. The edge of the colonies is wavy-lobed, the surface is rough, the structure is folded and granular, the texture is soft, oily. Stroke growth is moderate, distinct. The pigment diffusing into the nutrient medium is absent.

Cell morphology
Gram-positive long thin sticks with rounded edges, single or in chains of several cells. They do not form a dispute. Stationary, devoid of flagella and pili. The type of shell structure is firmmouth. Cell sizes vary depending on culture conditions - and especially - on the growth phase. The average cell length is 2.86 μm.

Physiological and biochemical characteristics of the strain
Optional anaerobic, catalase negative, cytochrome oxidase negative. It does not reduce nitrates, it does not dilute gelatin, esculin weakly hydrolyzes, and does not produce hydrogen sulfide and indole. O / F - test on a modified Hugh-Leifson medium is positive. Obligatory saccharolytic.

Saccharolytic activity
Ferments lactose, mannose, galactose, trehalose, glucose, fructose. Weakly ferment sucrose. It does not ferment melicitosis, ribose, rhamnose, xylose, raffinose, cellobiose, maltose, arabinose, sorbitol, melibiosis, mannitol. The final pH value on fermentable sugars is 4.1-4.3 in the base medium N 1. The type of fermentation is homoenzymatic (the final metabolic product is lactate). Milk cures with the formation of a mucous, loose clot, the maximum titratable acidity of which is 250 T, milk with litmus restores without gas formation, intensively restores milk with methylene blue.

The temperature optimum is 38-40 ^o C. It grows at 45 ^o C. It does not grow at a temperature of 15 ^o C. The optimal pH value is 5.6-6.0. Antibiotic sensitivity
The strain is characterized by high sensitivity to chloramphenicol, oxacillin, carbenicillin, ampicillin, novobiocin, erythromycin, moderate sensitivity to ristomycin, monomycin, gentamicin, kanamycin, neomycin. Resistant to polymyxin and 5-NOC.

Adhesive properties
Highly adhesive strain. The adhesion index on transplantable cell cultures of HEP-2 is 53.3 / 3.0 (in the numerator is the percentage of adhered cells of HEP-2, in the denominator is the average number of bacteria on the cell of HEP-2.

Method, conditions and composition of media for storage of strain
The strain is stored in sterile skim milk at 4 ^o with a periodic margin of 1 time in 20 days or in freeze-dried form in sealed ampoules (shelf life of 2 years or more). Protective medium when dried - sucrose 10%, gelatin 1%, at pH 7.0 +0.2.

The method, conditions and composition of the media for the propagation of the strain
The strain is grown at 38-40 ^o in the base medium N 1 or inoculum medium N 2.

The composition of the base medium N 1 (MPC), g in 1 l:
casein enzymatic hydrolyzate - 10
yeast extract - 5
meat extract - 10
glucose - 20
ammonium citrate, disubstituted - 2
sodium acetate - 5
MgSO ₄ • 7H ₂ O - 0.5
MgSO ₄ • 4H ₂ O - 0.5
Tween-80 - 1.0
The pH was adjusted to 6.2-6.4 and sterilized for 15 minutes at 121 ^° C. To obtain a solid medium, 15 g of agar was added.

Sowing medium N 2, g / l:
Casein Acid Hydrolyzate - 10
yeast extract - 5
glucose - 10
Na ₂ HPO ₄ • H ₂ O - 1.75
KCl - 3
sodium acetate - 10
MgSO ₄ • 7H ₂ O - 0.2
MgSO ₄ • 4H ₂ O - 0.016
The pH was adjusted to 6.7 and sterilized for 15 minutes at 121 ^o C.

 The strain Lactobacillus acidophilus H-91 was isolated from the intestines of a healthy child in 1991 in the city of Leningrad, is a wild type. Identified by the identifier: O. Kandlor & N. Weiss 1986., Regular, Nonsporing Grampositive Rods // In; P. H Sneath, N. S. Mair, M. E. Sharpe & J.G. Holt (ed.) Bergey, s manual of Systematic bacteriology, v. 2, The Willians & Wilkin & Co., Baltumore.

 The strain was deposited in the collection of the All-Russian Research Institute of Agricultural Medicine N 76 D of July 16, 1997.

The strain is characterized by the following features
Cultural and morphological features
When grown on a base medium for identification of lactobacilli (N 1) for 48 hours at a temperature of 38-40 ^o C, uniform turbidity forms in the broth, in a semi-liquid medium - hanging, drop-like colonies, in the thickness of the agar - whitish colonies of complex configuration up to 2-4 mm in diameter. On the surface of a solid agar medium, roundish, slightly convex colorless (over time light yellow) colonies with a diameter of 1.5-2.0 mm are formed. The edge of the colonies is wavy, the surface is rough, the structure is folded-granular, the texture is soft, oily. Stroke growth is moderate, clearly visible. The pigment diffusing into the nutrient medium is absent.

Cell morphology
Gram-positive long thin sticks with rounded edges, single or in chains of 2-4. They do not form a dispute. Stationary, devoid of flagella and pili. The type of shell structure is firmmouth. Cell sizes vary depending on cultivation conditions - and especially - on the growth phase. The average cell length is 4.0 μm.

Physiological and biochemical characteristics of the strain
Optional anaerobic, catalase-negative, cytochrome oxidase-negative. It does not reduce nitrates, it does not dilute gelatin, esculin does not hydrolyze, and does not produce hydrogen sulfide and indole. The O / F test on the modified Hugh-Leifson medium is positive. Obligatory saccharolytic.

Saccharolytic activity
Ferments lactose, mannose, galactose, trehalose, glucose, fructose. Weakly ferment sucrose. It does not ferment melicitosis, ribose, rhamnose, xylose, raffinose, cellobiose, maltose, arabinose, sorbitol, melibiosis, mannitol. The final pH value on fermentable sugars is 4.1-4.3 in the base medium N 1. The type of fermentation is homoenzymatic (the final metabolic product is lactate).

 Milk chokes with the formation of lactic acid. The nature of the clot is chopped, loose. The maximum titratable acidity is 190-200 T. The duration of milk coagulation with 3% yeast is 6 hours. Milk with litmus restores without the formation of gas, intensively restores milk with methylene blue.

The temperature optimum is 38-40 ^o C. It grows at 45 ^o C. It does not grow at a temperature of 15 ^o C. The optimal pH value is 5.6-6.0.

Antibiotic sensitivity
The strain is characterized by high sensitivity to chloramphenicol, oxacillin, carbenicillin, ampicillin, novobiocin, erythromycin, moderate sensitivity to ristomycin, monomycin, gentamicin, kanamycin, neomycin. Resistant to polymyxin and 5-NOC, nalidixic acid, colimycin.

Adhesive properties
Relatively low adhesive strain. The adhesiveness index on transplantable cell cultures of HEP-2 is 6.5 / 1.5 (in the numerator is the percentage of adhered cells of HEP-2, in the denominator is the average number of bacteria on the cell of HEP-2).

The method, conditions and composition of the media for the propagation of the strain
The strain is grown at 38-40 ^o C in the base medium N 1 or inoculum medium N 2.

The composition of the base medium N 1 (MPC), g in 1 l:
casein enzymatic hydrolyzate - 10
yeast extract - 5
meat extract - 10
glucose - 20
ammonium citrate, disubstituted - 2
sodium acetate - 5
MgSO ₄ • 7H ₂ O - 0.5
MgSO ₄ • 4H ₂ O - 0.5
Tween-80 - 1.0
The pH was adjusted to 6.2-6.4 and sterilized for 15 minutes at 121 ^° C. To obtain a solid medium, 15 g of agar was added.

Sowing medium N 2, g / l:
casein acid hydrolyzate - 10
yeast extract - 5
glucose - 10
Na ₂ HPO ₄ • H ₂ O - 1.75
KCl - 3
sodium acetate - 10
MgSO ₄ • 7H ₂ O - 0.2
MgSO ₄ • 4H ₂ O - 0.016
The pH was adjusted to 6.7 and sterilized for 15 minutes at 121 ^o C.

 As can be seen from the foregoing, these strains are very similar to each other in a wide range of characteristics.

The main difference between the strains are adhesive and antagonistic properties (strain A-91 is highly adhesive, has medium antagonistic activity, strain H-91 is moderately adhesive, antagonistic activity is high), belonging to the mucosa (A-91) and non-mucous (H-91) races of acidophilus sticks; growth pattern in liquid media (A-91 - independent inoculum, H-91 - dependent inoculum)
The common positive effect of both strains is the ability to form a jointly stable symbiotic system, characterized by a syntrophic and synergistic type of relationship and capable of exerting a normalizing effect on the human body exposed to negative external factors.

 The specified system exists at a ratio of strains A-91 to H-91 from 1: 4 to 4: 1, while maintaining these properties, although better results are achieved with a ratio of microorganisms of 2: 1, which allowed the mixture of strains in the specified ratio to be used as starter and preparation based on it.

 The main comparative properties of the strains and their mixtures in the ratio of microorganisms 2: 1 are shown in table 1.

 To assess the possibility of creating a drug based on the obtained bioculture, a study was conducted of the antagonistic activity of various mixtures of strains in relation to reference strains, for which pathogenic and conditionally pathogenic strains from the GISK collection were used. L.A. Tarasovich recommended for the study of industrial strains of eubiotics for compliance with the requirements of pharmacopeia articles.

 The experiments showed that the resulting symbiotic culture significantly exceeds the effectiveness of individual microorganisms and causes complete inhibition of growth of E. coli 0157, Sh. Flexneri 331, Sh. Flexneri 170, Sh. Sonnae 5063, Pr. Vulgaris 177, St. aureus 209 (table 2).

As mentioned above, an acidophilic starter culture was created on the basis of a symbiotic mixture, containing, along with the remains of the nutrient medium, strains A-91 and H-91 in a ratio of 1: 4 to 4: 1 with a total content of microorganisms in the leaven of 1 • 10 ⁹ -1 • 10 ¹⁰ cells per gram, which, along with the above functions, can simultaneously perform the functions of a drug that helps the body adapt to adverse external influences. Tests of the obtained starter culture showed its complete harmlessness when administered orally into the body.

 For its receipt, the Center for State Sanitary and Epidemiological Supervision MSCh-24 of St. Petersburg issued a hygienic certificate N 7-97 of 02/10/97. The obtained yeast can be used to obtain a milk drink in liquid form or subjected to lyophilization and pressing for the purpose of further use in the form of tablets.

The resulting tablets have a white or yellowish color with a characteristic sour-milk smell and taste.
When dissolved in water, the tablet forms a homogeneous suspension. When diluted with a 0.85% solution of sodium chloride, the pH of the solution is 45-55. The acidification activity of the starter culture is not less than 200 T.

Ferment contains at least 5 billion living cells per g and can retain its properties for a year when stored in a dry, dark place at 2-8 ^o C. At the same time, by the end of this period, in 1 g of ferment, the content of viable bacteria should be at least 3 billion cells.

Example 1. Freeze-dried monostrain activated in skim milk containing growth factors, for 16-18 hours at a temperature of 37-40 ^o C (the initial value of inoculation is 10 ⁶ CFU / ml). Next, strains A-91 and H-91 were grown at 38-40 ^o C for 12 hours on seed medium N 2.

The pH was adjusted to 6.7 and sterilized for 15 minutes at 121 ^o C, after which the culture fluids were mixed, achieving a ratio of cells of strains A-91 to H-91 close to 2: 1 with a total content of living cells in the mixture, depending on the task from 1 to 5 billion. A drug with a lower cell content is lyophilized to obtain a powder containing about 10 billion cells per g.

If it is necessary to obtain a liquid starter culture, pour 10 ml of boiled and cooled to room temperature water into a container containing 5 g of powder, shake it and pour into 0.5 l of boiled and cooled to 30-40 ^o C milk. Fermented milk is put for 8-12 hours in a warm place. The resulting sourdough is a well-formed clot resembling sour cream and contains 5-8 billion cells per ml. Sourdough should be stored in the refrigerator. Can be used within 20 days.

To obtain a fermented milk drink on its basis, milk is boiled, cooled to a temperature of 30-40 ^o C and add to it the ferment obtained in the above manner at the rate of 1 tablespoon per 0.5 l of milk. Fermented milk is put in a warm place at 30-40 ^o C for 8-12 hours. The resulting fermented milk product is ready for use.

 For dietary purposes and for prevention, the obtained fermented milk product can be consumed throughout the day. When used for medicinal purposes, it is advisable to use it on an empty stomach in the morning and evening before bedtime. The recommended single dose in this case for adults is 1 glass, for children - from 1 tablespoon to 1 glass, depending on age.

 Example 2. A dry preparation containing acidophilic starter culture obtained in the conditions of Example 1 was tested at the St. Petersburg Medical Academy of Postgraduate Education on the basis of the gastroenterological and gynecological departments. Under observation were 31 people (20 people with chronic enterocolitis, 5 people with hypermotor dyskinesia of the colon, 6 people with impaired normal vaginal microflora). The drug was given 2 tablets 2 times a day in the morning and in the evening for 3-5 days. The results obtained showed in all patients an improvement in the psychoemotional state, a decrease in asthenic phenomena, feelings of bloating and rumbling in the abdomen, normalization of stool, and a decrease in pain.

 Example 3. On the basis of the Military Medical Academy, tests were carried out on the possibility of using the drug to combat motion sickness and to increase the body's adaptation to high altitude conditions (rise from sea level to a height of 2500 m and back within 2 hours in a pressure chamber).

 Tests conducted on a group of 20 volunteers showed that the number of motion sickness under conditions that mimic a 5-point storm with a preliminary - 15-20 minutes before the start of the test - taking 2 tablets of the drug obtained according to the method of example 1, decreased from 60 to 25% .

 When using a pressure chamber, the average time to normalize the main parameters of the cardiovascular (CVS) and immune (IS) system with the preliminary intake of 4 tablets of the drug was reduced by CVS from 36 to 27 hours, by IP - from 48 to 32 hours, which makes the drug promising for further study in this direction.

Claims (5)

 1. The bacterial strain Lactobacillus acidophilus A-91 of the All-Russian Research Institute of Agricultural D-75 D-75 symbiont of the strain Lactobacillus acidophilus H-91 used for the preparation of fermented milk products.  2. The bacterial strain Lactobacillus acidophilus H-91 of the All-Russian Research Institute of Agricultural D-76 D-76 is a symbiont of the strain Lactobacillus acidophilus A-91 used for the preparation of fermented milk products.  3. Ferment for the preparation of fermented milk products based on bacteria Lactobacillus acidophilus, characterized in that it contains a mixture of strains of Lactobacillus acidophilus A-91 and H-91 in the ratio of microorganisms from 4: 1 to 1: 4.  4. Fermentation according to claim 3, characterized in that the ratio of strains A-91 to H-91 is 1: 1.  5. A drug that helps the body adapt to adverse external influences based on acidophilic bacteria, characterized in that it contains a mixture of strains of Lactobacillus acidophilus A-91 and H-91 in the ratio of microorganisms from 4: 1 to 1: 4 as acidophilic bacteria living cells from 1 to 10 billion cells per 1 g of the drug.

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