RU2477313C1 - STRAIN Enterococcus hirae, USED TO MAKE CULTURED MILK FOODS - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain Enterococcus hirae BC - 37 having high antagonistic activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number VKPMV-10090 and may be used, for instance, in production of such cultured milk foods as kefir, cottage cheese or ryazhenka.

EFFECT: invention makes it possible to produce a strain having high antagonistic activity and high speed of milk turning sour.

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Description

The invention relates to biotechnology and the dairy industry and is a new strain of mesophilic streptococcus, intended for the production of dairy products and bacterial preparations.

Known strains of Streptococcus citrovorus, Streptococcus paracitrovorus, Streptococcus diacetilactis, with high resistance to growth inhibitors, the ability to stably form a viscous milk clot, good compatibility with other strains of lactobacilli and pronounced physiological and biochemical properties.

The disadvantage of these strains is the lower marginal acidity and rate of fermentation of milk.

Known strain Enterococcus durans RSH (VKPM B-8717) patent No. 2273662. Since 1982, E. durans has been used in the dairy industry as a component of starter cultures, as well as a probiotic in medicine (Rocha JM, Malcata FX, On the microbiological profile of traditional Portuguese sourdough. J. Food Prot. 62 (12): 1416-29 , 1999).

It is also shown that E. durans in a number of indicators (resistance to bile and acid shock, in vitro cholesterol assimilation) is superior to known probiotic microorganisms (Dora IA Pereira and Glenn R., Gibson Cholesterol Assimilation by Lactic Acid Bacteria and Bifidobacteria Isolated from the Humman Gut // Applied and Environmental Microbiology, vol. 68, No. 9, p. 489-4693, 2002).

The disadvantage of this strain is the lower marginal acidity and rate of fermentation of milk.

The objective of the invention is the creation of a dairy product using lactobacilli on accessible nutrient media with high antagonistic activity.

The problem is solved by using nutrient media of dense and liquid consistency: sterile skim milk, hydrolyzed milk (pH 6.8-7.0).

The local strain Enterococcus hirae VKPM B-10090 isolated in vivo from the microflora of kefir fungus.

The identification of the strain was carried out according to the method described in the book of L. A. Bannikov. "Breeding of lactic acid bacteria and their use in the dairy industry." - M., "Food Industry", 1975.

The strain is deposited in the All-Russian Collection of Industrial Microorganisms and has a number VKPM B-10090.

The local strain Enterococcus hirae is isolated in vivo from the surface of an alfalfa plant (flower).

The identification of the strain was carried out according to the method described in the book of L. Bannikova. (1975). The strain Enterococcus hirae was deposited in VKPM under the number B-10090.

The strain Enterococcus hirae VKPM B-10090 has the following characteristics:

1. Cultural and morphological:

- optional anaerobic;

- gram-positive cocci;

- diameter 1.0 ± 0.02 μm;

- motionless;

- located in pairs or in short chains;

- do not form endospores.

On the surface of the agar medium (hydrolyzed milk) after 24 hours at a temperature of 37 ° C form:

- round colonies,

- size 2.2 mm (average);

- the nature of the contour of the edge is even;

- relief is convex;

- surface - smooth, shiny;

- White colour;

- the structure is homogeneous;

- consistency - pasty.

The study of the cultural or morphological properties of the selected strain of lactic acid microorganisms was carried out on a dense nutrient medium - agarized milk.

2. Physiological and biochemical:

- milk fermentation rate - 9 hours;

- the limit of acid formation is 201 ° T;

- the strain ferments glucose, galactose, lactose, maltose, dextrin;

- grows in milk at a temperature of 40-45 ° C;

- grows in hydrolyzed milk containing NaCl (2%, 3%, 4%, 6.5%);

- grows in meat-peptone broth (at pH 9.2; 9.6);

- grows in hydrolyzed milk with bile content (20%, 30%, 40%);

- grows in milk containing methylene blue (0.0005%, 0.005%, 0.01%, 0.1%);

- withstands heating for 30 minutes at a temperature of 55 ° C, 60 ° C.

The product synthesized by the strain is lactic acid.

3. Antibiotic activity:

- the diameter of the zone of inhibition of growth in relation to the representative of the pathogenic microflora Staphylococcus aureus was 23 mm;

- the diameter of the zone of growth inhibition in relation to the representative of opportunistic microflora of Escherichia coli was 20 mm.

Determination of the antibiotic activity of the Enterococcus hirae strain VKPM B-10090 was carried out on solid nutrient medium (MPA) by agar diffusion.

The method, conditions and composition of the media for long-term storage of the strain - lyophilization; at a temperature of 4 ° C; MPG environments Rogosa; milk with chalk; milk with yeast autolysate, glucose, litmus, liver extract and CaCO ₃ ; agarized milk cheese whey.

The method, conditions and composition of the media for the propagation of the strain at a temperature of 30-37 ° C, medium: agarized milk cheese whey; skim milk, unsalted cheese whey with a content of at least 4.0% lactose.

Optimum conditions and composition of the medium for fermentation - temperature - 37 ° C; environments: cow's milk; unsalted cheese whey with a content of at least 4.0% lactose, skimmed cow's milk.

The proposed new strain Enterococcus hirae VKPM B-10090, isolated from the surface of an alfalfa plant (flower), is well adapted to local conditions, easy to cultivate and store, can be used in the production of fermented milk products, is non-zoo and phytopathogenic, does not pose a risk to others reasons.

Due to its pronounced biological and antibiotic properties (the ability to form a viscous milk clot, the diameter of the zone of inhibition of growth of Staphylococcus aureus is 23 mm, Escherichia coli is 20 mm), the strain is of industrial value for the dairy industry.

Example 1. The use of strain Enterococcus hirae VKPM B-10090 in the production of kefir

Milk is weighed, sorted and filtered, subjected to primary processing. It must be fresh, natural and meet the requirements of the standard.

In the production of kefir, milk is cooled to a fermentation temperature of 20-25 ° C. Already pasteurized milk is poured into the fermenter tank and pre-prepared yeast is added - 5-6% of the milk mass. Ferment is also enriched with Enterococcus hirae strain VKPM B-10090. After thorough mixing, the contents of the tank are left alone, ensuring a constant temperature with a water jacket. The milk is fermented to obtain a dense clot with an acidity of 90-100 ° T. The duration of ripening is 10-12 hours at a temperature of at least 20 ° C. Upon reaching the necessary acidity and viscosity of the clot, the circulation of water in the jacket is stopped, ice water with a temperature of 1-2 ° C is supplied to the interstitial space and a stirrer is turned on to stir the clot and more quickly cool it to the ripening temperature. After reaching a clot temperature of 12-16 ° C, the supply of cooling water is stopped and it is left alone for 4-6 hours for the maturation and development of yeast. Then the contents of the tank are further cooled to a temperature of 8-10 ° C to complete ripening.

Example 2. The use of strain Enterococcus hirae VKPM B-10090 in the production of cottage cheese

Curd production in the traditional way. The technological process consists of the following operations: acceptance and preparation, milk separation, normalization, pasteurization, cooling, fermentation and fermentation of normalized milk, cutting the clot, separating whey and pouring the clot, self-pressing and pressing the clot, cooling, packaging, packaging, storage and transportation of cottage cheese .

Dairy raw materials intended for the production of cottage cheese are cleaned on milk separators or filtered through three layers of gauze or other filter cloth. The purified milk is heated to 37 ± 2 ° C and separated on cream separators. In the manufacture of cottage cheese, fat, bold and peasant milk is normalized by fat, taking into account the mass fraction of protein in whole milk, so that a finished product with a given content of fat and moisture is obtained. Skimmed or normalized milk is pasteurized at a temperature of 78 ± 2 ° C with an exposure of 15-20 s in plate or tubular pasteurization-cooling units or in capacitive devices. After pasteurization, the milk is cooled to the fermentation temperature.

Sourdough is prepared on pure cultures of mesophilic lactic streptococci, including the culture Enterococcus hirae VKPM B-10090. Before adding to the milk, the surface layer of the starter culture is carefully removed with a clean, disinfected bucket and removed. Then the leaven is mixed to a homogeneous consistency with a clean whorl (when prepared in starter tubes) or a stirrer and poured into the prepared milk in an amount of 5% of the total mass. Duration of fermentation of milk 6 hours

An aqueous solution of calcium chloride (mass fraction of calcium chloride 30-40%) is added to milk after fermentation: 400 g per 1000 kg of fermented milk. The preparation and preparation of a solution of calcium chloride is carried out in accordance with the Instructions for technological control at dairy enterprises. After adding a salt solution to fermented milk, a 1% solution of rennet is introduced at the rate of 1 g of the preparation with an activity of 100,000 ME per 1000 kg of milk.

Rennet powder or pepsin is introduced into milk in the form of a 1% aqueous solution prepared in boiled water and cooled to 36 ± 3 ° C. The enzyme solution is introduced into milk with constant stirring. After 10-15 minutes after making the enzyme solution, stirring is completed and milk is left at rest until a dense clot with an acidity of 61 ± 5 ° T for cottage cheese of 9% and 18% fat content, 65 ± 5 ° T for peasant and 71 ± 5 ° T for low-fat cottage cheese.

To process the clot, use manual lyres, in which a thin stainless steel wire serves as knives. With such wire knives, the clot is cut into cubes measuring 2 × 2 × 2 cm. The clot is first cut along the length of the bath into horizontal layers, and then into vertical and vertical lengths. After this treatment, the clot is left for 40-60 minutes to separate serum and increase acidity. The separated serum is drained from the bath. The clot after draining the whey is poured into coarse calico or dacron bags measuring 40 × 80 cm. The bags are filled about 70%, which is 7-9 kg of cottage cheese. Then the bags are tied and stacked one on top of another in a self-pressing bath, a press trolley or a UPT unit for pressing and cooling the curd.

In order to accelerate the separation of serum, as well as in case of poor serum excretion, the clot is heated by supplying steam or hot water to the interstitial space of the curd bath. For uniform heating, the upper layers of the clot are moved with a wooden or metal plate (shovel) from one wall of the bath to another. The clot is heated to 40 ± 2 ° C for 30-40 minutes for cottage cheese of 9% and 18% fat content, 35 ± 2 ° C for 20-40 minutes for peasant and 36 ± 2 ° C for 15- 20 minutes for low-fat cottage cheese.

Self-pressing of cottage cheese lasts at least 1 hour. Pressing continues until cottage cheese with a mass fraction of moisture is provided for by the regulatory documentation. For cottage cheese of 18% fat, it is 65%; 9% fat - 73; peasant - 74.5; dining room - 76; low-fat - 80; for diet fruit 11% fat - 64, 9% fat - 66, 4% fat - 77 and nonfat - 79% moisture. When developing low-fat cottage cheese, dehydration of the clot can be carried out on a cottage cheese separator. After separation and pressing, the curd is cooled. Cottage cheese is cooled in bags located on the shelves of the refrigerator or in carts or tubs. Cottage cheese is cooled to a temperature of 12 ± 2 ° C and sent for packaging and labeling.

Example 3. The use of strain Enterococcus hirae VKPM B-10090 in the production of fermented baked milk

Normalized milk with a fat content of 4% is subjected to heat treatment at 100 ° C without exposure, cooled to 38 ° C, add fermentation for fermented baked milk in an amount of 2%, activated biomass of bifidobacteria in milk in an amount of 0.1% and Enterococcus hirae strain VKPM B-10090 on sterile milk in an amount of 0.5%. Fermentation is carried out at a temperature of 37 ° C until titratable acidity of 65 ° T is achieved. Next, the product is slowly cooled to 6 ° C, after which the bottling is carried out.

Claims (1)

The strain Enterococcus hirae VKPM B-10090 used in the production of dairy products.