Lactobacillus acidophilus and-91, used to prepare dairy products, lactobacillus acidophilus h-91, used to prepare dairy products, yeast for the preparation of fermented milk products and the drug contributing to the adaptation of an organism to adverse external influences

 

(57) Abstract:

The invention relates to biotechnology, can be used in the food and dairy industry, medicine, agriculture. Lactobacillus acidophilus And-91, deposited in the collection of the Institute SHMB D-75 from 16.07.1997, and the strain Lactobacillus acidophilus N-91, deposited in the collection of the Institute SHMB D-76 dated 16.07.1997, together form a symbiotic system, characterized citrinum synergistic relationships type, used for making dairy products. Ferment on the basis of them contains a mixture of Lactobacillus acidophilus And-91 and H-91 in the ratio of microorganisms from 4 : 1 to 1 : 4. The drug Vilaflor" contributing to the adaptation of an organism to adverse external influences, on the basis of acidophilic bacteria contains a mixture of Lactobacillus acidophilus A-9l and N-91 in the ratio of microorganisms from 4 : 1 to 1 : 4 when the contents of living cells from 1 to 10 billion cells per 1 g of the drug. The drug showed high efficiency for a number of diseases, as well as for adaptation to adverse environmental conditions. 4 C. and 1 C.p. f-crystals, 2 tab.

The invention relates to the field of biotechnology and strains for the production of fermented milk drinks, their technology can be used in the food and dairy industry, medicine, agriculture and related fields.

Known bacterial preparations for medical purposes, such as colibacterin, laktobakterin, bifidobakterii (Guide to vaccine and serum case. Ed. by Acad. AMS, SSSU I. N. Burgasov, Moscow, "Medicine", 1978) contains one or two strains of microorganisms with the necessary medical and biological characteristics.

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

Known use as adaptogenes ginseng, Siberian ginseng, sea buckthorn, Catnip, mummy, propolis and drugs from other natural sources (C. I. Mashanov, A. A. Pokrovsky. Aromatic plants, M., Agropromizdat, 1991; I. S. Sokolova and other wild and cultivated plants, M , Medicine, 1990).

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

Currently, widespread fermented milk drinks, combining the availability of raw materials with relative safety and efficacy.

In particular, shotonline of pure races lactic acid Streptococcus, Bulgarian, acidophilus and cheese sticks (A., Khramtsov. Waste-free technology in the dairy industry, M., Agropromizdat, 1989, S. 210).

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

To improve the quality of preparations based on milk whey using bacterial additives (yeast), improving mainly nutritional and taste characteristics of drugs.

Known as starter cultures the use of a mixture in the ratio 1:1 crops Str. cremons and Str. diacetitaetis (Ed. St. USSR N 581923, CL A 23 C 9/12).

The lack of leaven is low biological value because of the low antagonistic activity of these microorganisms in relation to extraneous microflora.

Known more effective ferment based on lactic acid bacteria, containing equal parts Saceharomyces cerevisiae, Lactobacillus fermenti and Lactobacillus helveticus (Ed. St. USSR N 1329745, class. And 23 With 9/00).

The lack of leaven is the poor compatibility of the microorganisms that causes the need to use several temperature regimes, low adaptive properties when introduced into the whey

About the and for enhancing the adaptive capacity of the organism under adverse environmental conditions, and used for the preparation of a strain of microorganism and sour leaven (RF Patent N 2064268, 1996, class. And 23 With 9/12). The drink is obtained on the basis of Lactobacillus acidophilus (L. Acidophilus) strain 20T with the addition of microorganisms Str. thermophilus strain 24C, Lactobacillus casei strain 2, Lactobacillus helveticus strain 305-5. To obtain a beverage using a bacterial starter culture containing the specified culture in the ratio of 20-35 wt.%, 20-35 wt.%, 20-35 wt.% and 5-15 wt.%

The disadvantage of the prototype is a complex production technology, to the mutual inhibition of different strains of bacterial cultures.

The challenge faced by the researchers, was the creation of such strains, which, while maintaining the effectiveness of the prototype, would be in a more stable equilibrium and would be similar in nature, which significantly simplify the preparatory stage of the process of obtaining the drink, making possible the manufacture of drink specialists in the household, and could also have an independent meaning as a concentrate useful for bowel when dysbiosis and when the effects of other negative factors.

This problem is solved by creating strains of L. acidophllus a-91 and H-91, dairy starter culture containing these strains, etc is in, Leningrad, is a wild type and was identified by the determinant: 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 is deposited in the collections of the Institute SHMB N D 75 dated 16.07.1997.

The strain is characterized by the following signs

Cultural morphological traits

When grown in a basic environment for the identification of lactobacilli (N 1) for 48 hours at a temperature of 38-40oC in broth forms a uniform suspension in semi-solid medium - hanging, drop the colony, in the thickness of the agar - whitish colonies complex configuration to 1.5-2.0 mm in diameter. On the surface of solid agar medium are formed rounded, slightly convex, colorless (pale yellow) colonies with a diameter of 1,8 - 2,2 mm of the edge of the colonies - wavy-lobed, surface - roughened structure - fold-grained, consistency - soft, oil. The growth of the stroke is moderate, beadlike. The pigment diffusing into the nutrient medium, is missing.

The morphology of the cells

Gram long thin sticks with rounded ends, single or in chains of several cells. Spores do not form. NEPAD the t culturing conditions - and especially from the phase of growth. The average length of the cells is 2.86 microns.

Physiological and biochemical characteristics of the strain

Optional gone anaerobic, catalytically, zithromaxadministering. Nitrates are not reducyruet, gelatino not thins, esculin hydralizine poorly, hydrogen sulfide and indole is not produced. O/F - test on the modified environment Hugh Leifson positive. Obligate Charolais.

Caroliina activity

Ferments lactose, mannose, galactose, trehalose, glucose, fructose. Poorly fermented sucrose. Not ferments melezitose, ribose, rhamnose, xylose, raffinose, cellobiose, maltose, arabinose, sorbitol, melibiose, mannitol. The final pH value on formatiruem sugars 4,1-4,3 in the base environment N 1. The type of fermentation - homofermentative (the end product of metabolism is lactate). Milk storageset with the formation of mucous, loose bunch, maximum citirama acidity which is 250 Tons, milk with litmus paper restores without the formation of gas, intensively restores milk with methylene blue.

Temperature optimum 38-40oC. Grows at 45oC. does Not grow at a temperature of 15oC. Optimal SNK levomycetin, oxacillin, carbenicillin, ampicillin, novobiocin, erythromycin, average sensitivity to ristomycin, monomitsina, gentamycin, kanamycin, neomycin. Resistant to polymyxin and 5-NOK.

Adhesive properties

High adhesive strain. The index of the adhesive on transplantable cell cultures NER-2 is 53,3/3,0 (in the numerator is the percentage of adhered cells NER-2, in the denominator - the average number of bacteria on the cell NER-2.

The method, conditions and medium composition for the storage of strain

Strain keep of sterile non-fatty milk at 4owith periodic preponderance of 1 every 20 days or in lyophilized form in a sealed ampoule (age 2 years and over) Protective environment at drying - sucrose 10%, gelatin 1%, at pH 7,0+0,2.

The method, conditions and medium composition for growth of strain

The strain is grown under 38-40oin the basic environment N 1 or planting environment N 2.

The composition of the basic environment N 1 (s), g 1 l:

enzymatic hydrolysate of casein - 10

yeast extract - 5

meat extract - 10

glucose - 20

ammonium citrate, disubstituted - 2

sodium acetate - 5

MgSO47H2O - 0,5

MgSO44H2O - the Yat 15 g of agar.

The sowing medium N 2, g/l:

Acid hydrolysate of casein - 10

yeast extract - 5

glucose - 10

Na2HPO4H2O - 1,75

KCl - 3

sodium acetate - 10

MgSO47H2O - 0,2

MgSO44H2O - 0,016

the pH was adjusted to 6.7 and sterilized for 15 minutes at 121oC.

The strain Lactobacillus acidophilus N-91 was isolated from the intestine of a healthy child in 1991, Leningrad, is a wild type. Identified by the determinant: 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 is deposited in the collections of the Institute SHMB N D 76 dated 16.07.1997.

The strain is characterized by the following signs

Cultural morphological traits

When grown in a basic environment for the identification of lactobacilli (N 1) for 48 hours at a temperature of 38-40oC in broth forms a uniform suspension in semi-solid medium - hanging, drop the colony, in the thickness of the agar - whitish colonies complex configuration to 2-4 mm in diameter. On the surface of solid agar medium are formed rounded, slightly convex, colorless (pale yellow) colony diameter of 1.5-2.0 mm Edge Colo the t bar moderate, showing clearly. The pigment diffusing into the nutrient medium, is missing.

The morphology of the cells

Gram long thin sticks with rounded ends, single or in chains 2-4. Spores do not form. Motionless, devoid of flagella and pilej. Type of structure shell - Firmicutes. The size of cells varies depending on cultivation conditions and especially from the phase of growth. The average length of the cells is 4.0 μm.

Physiological and biochemical characteristics of the strain

Optional gone anaerobic, catalytically, cytochromoxidase-negative. Nitrates are not reducyruet, gelatino not thins, esculin not hydralizine, hydrogen sulfide and indole is not produced. O/F-test on the modified environment Hugh Leifson positive. Obligate Charolais.

Caroliina activity

Ferments lactose, mannose, galactose, trehalose, glucose, fructose. Poorly fermented sucrose. Not ferments melezitose, ribose, rhamnose, xylose, raffinose, cellobiose, maltose, arabinose, sorbitol, melibiose, mannitol. The final pH value on formatiruem sugars 4,1-4,3 in the base environment N 1. The type of fermentation - homofermentative (the end product of metabolism is Li. Maximum citirama acidity is 190-200 So the duration of the coagulation of milk when you make 3% of the leaven - 6 hours. Milk with litmus paper restores without the formation of gas, intensively restores milk with methylene blue.

Temperature optimum 38-40oC. Grows at 45oC. does Not grow at a temperature of 15oC. the Optimal pH environment of 5.6-6.0.

Sensitivity to antibiotics

The strain is characterized by high sensitivity to levomycetin, oxacillin, carbenicillin, ampicillin, novobiocin, erythromycin, average sensitivity to ristomycin, monomitsina, gentamycin, kanamycin, neomycin. Resistant to polymyxin and 5-NOK, nalidixic acid, colimycin.

Adhesive properties

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

The method, conditions and medium composition for growth of strain

The strain is grown under 38-40oC in the base environment N 1 or planting environment N 2.

The composition of the basic environment N 1 (s), g 1 l:

enzymatic g is nokaly, disubstituted - 2

sodium acetate - 5

MgSO47H2O - 0,5

MgSO44H2O - 0,5

Tween-80 - 1,0

the pH was adjusted to 6.2 - 6.4 and sterilized for 15 minutes at 121oC. To obtain a solid medium is injected 15 g of agar.

The sowing medium N 2, g/l:

acid hydrolysate of casein - 10

yeast extract - 5

glucose - 10

Na2HPO4H2O - 1,75

KCl - 3

sodium acetate - 10

MgSO47H2O - 0,2

MgSO44H2O - 0,016

the pH was adjusted to 6.7 and sterilized for 15 minutes at 121oC.

As can be seen from the above, these strains are very similar among themselves on a wide range of characteristics.

The main difference between strains are adhesive and antagonistic properties (strain a-91 - high adhesive, has a high antagonistic activity, strain H-91 - sredneetazhnye, antagonistic activity is high), belonging to the mucosa (a-91) and nessisity (H-91) races acidophilic Bacillus; the nature of growth in liquid media (a-91 - inoculate independent, N-91 - inoculate dependent)

The overall positive effect of both strains is the ability to form together a sustainable symbiotic system, karakterea on the human body, subjected to negative external factors.

This system exists when the ratio of strains a-91 to H-91 from 1:4 to 4:1, while maintaining these properties, although the best results are achieved when the ratio of microorganisms 2:1, which allowed us to use a mixture of strains in the specified ratio as a starter and the drug 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 establishing a drug on the basis of the obtained bioculture, a study was conducted antagonistic activity of various mixtures of strains in relation to the reference strains, which are used pathogenic and conditionally-pathogenic strains from the collection gisk named after. L. A. Lytvyn recommended for studies of industrial strains of eubiotics for compliance with the requirements of Pharmacopeia articles.

The experiments showed that the resulting symbiotic culture significantly exceeds the efficiency 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 you who enoy environment strains a-91 and H-91 in the ratio from 1:4 to 4:1 at a total content of bacteria in the sourdough 1109-11010cells per gram, which along with the above functions can simultaneously perform the function of the drug, contributing to the adaptation of an organism to adverse external influences. Testing the obtained yeast showed its complete harmlessness oral introduction into the body.

On the receipt by the Center of state sanitary and epidemiological supervision Department-24-Petersburg issued a health certificate N 7-97 from 10.02.97. The resulting yeast can be used to produce dairy beverage in liquid form or subjected to freeze-drying and pressing for further use in the form of tablets.

The obtained tablets are white or yellowish in color with a characteristic sour smell and taste

When dissolved in water pill forms a homogeneous suspension. When breeding of 0.85% sodium chloride solution, the pH of the solution is 45-55. The activity of acid generating ferment - not less than 200 Tons

Sourdough contains at least 5 billion live cells per g and can retain their properties during the year when stored in a dry dark place at 2-8oC. At the end of this period, 1 g yeast content of viable bacteria must be at least 3 billion cells.

The use of the owls at a temperature of 37-40oC (the initial value of planting is 106CFU/ml). Further strains a-91 and H-91 were grown at 38-40oC within 12 hours at the seed environment N 2.

the pH was adjusted to 6.7 and sterilized for 15 minutes at 121oC, after which the culture fluid is mixed, achieving a ratio of cells of strains a-91 to H-91 close to 2: 1 when the total content of living cells in the mixture depending on the task, from 1 to 5 billion Drug with a lower content of cells lyophilizer, receiving powder containing about 10 billion cells / g

If necessary, obtain a liquid starter culture into a container containing 5 g of powder pour 10 ml of boiled and cooled to room temperature water, shake and pour 0.5 liters of boiling and cooled to 30-40oC milk. Fermented milk set for 8-12 hours in a warm place. Received leaven is a well-formed clot, reminiscent of sour cream and contains 5-8 billion cells per ml Yeast should be stored in the refrigerator. Can be used for 20 days.

To obtain on the basis of a fermented milk drink milk is boiled, cooled to a temperature of 30-40oC and make obtained in this way ferment for 1 dining room lcny product is ready for use.

Dietary purposes and for the prevention of the obtained milk product may be consumed throughout the day. When used in therapeutic purposes, it is advisable to drink on an empty stomach in the morning and at night before bedtime. The recommended single dose in this case for adults - 1 Cup, for children from 1 tablespoon to 1 Cup depending on age.

Example 2. The dry product containing acidophilus yeast obtained in the conditions of example 1, was tested in St. Petersburg medical Academy of post-diploma education on the basis of gastro - enterological and gynecology departments. Under supervision there were 31 people (20 people with chronic enterocolitis, 5 people with hypermotor dyskinesia colon, 6 people with disorders of the normal microflora of the vagina). The drug was given 2 tablets 2 times a day morning and evening in 3-5 days. The results showed all patients improved emotional state, reducing asthenic phenomena, feelings of bloating and rumbling in the abdomen, normalization of stool, the reduction of pain.

Example 3. At the Military medical Academy were tested for possible use of the drug to Bo the s 2500 m and back within 2 hours in a pressure chamber).

Tests conducted on a group of 20 volunteers showed that the number okazyvayuschihsya under conditions simulating a 5-point storm when the preliminary - for 15 - 20 min before test - taking 2 tablets of a preparation obtained by the method of example 1, was reduced from 60 to 25%.

When using hyperbaric chambers average time normalization of the main parameters of the cardiovascular (CVS) and immune (IP) system in the preliminary reception 4 tablets of the drug decreased by CCC from 36 to 27 hours, IP - from 48 to 32 hours, which makes the drug promising for further development in this direction.

1. The bacterial strain Lactobacillus acidophilus A-91 Institute SHMB D-75 symbiont strain Lactobacillus acidophilus N-91, used to prepare dairy products.

2. The bacterial strain Lactobacillus acidophilus N-91 Institute SHMB D-76 symbiont strain Lactobacillus acidophilus a-91, used to prepare dairy products.

3. Leaven for the preparation of fermented milk products based on bacteria Lactobacillus acidophilus, characterized in that it contains a mixture of Lactobacillus acidophilus And-91 and H-91 in the ratio of microorganisms from 4 : 1 to 1 : 4.

4. Ferment under item 3, characterized in that the ratio of strains a-91 to H-91 Zastavna the basis of acidophilic bacteria, characterized in that as acidophilus bacteria it contains a mixture of Lactobacillus acidophilus And-91 and H-91 in the ratio of microorganisms from 4 : 1 to 1 : 4 when the contents of living cells from 1 to 10 billion cells per 1 g of the drug.

 

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