RU2112387C1 - Method of production of dry bacterial concentrate for lactic acid dairy product - Google Patents

Abstract

FIELD: dairy industry, microbiology. SUBSTANCE: method involves preparing the strain Lactobacillus acidophilus Ep317/402 by its passage (3-4 times) from liquid cultural medium on solid nutrient medium, control over absence of bacterial contamination, strain culturing, biomass mixing with protective medium, freezing and drying. EFFECT: enhanced quality, prolonged storage time of bacterial concentrate.

Description

 The invention relates to microbiology.

A known method of preparing dry bacterial starter culture for the production of dairy products, including the cultivation of acidophilus bacteria in milk with a solids content of 14 - 16% to 3.7 - 4.0 • 10 billion living cells in 1 ml of liquid starter culture, freezing for 10 - 12 hours at a temperature of minus 40 - 50 ^o C [1].

The closest technical solution to the proposed invention is a method for the production of dry bacterial concentrate for the production of fermented milk product, including the preparation of a strain of Lactobacillus acidophilus Ep 317/402, cultivation on a nutrient medium containing milk whey in an amount of 250 ml, potato extract - 1.6 ml, manganese sulfate - 0.16 g, sodium citrate 6.0 g, tap water up to 1 l, pH 6.0, mixing the resulting biomass with a protective medium containing skim milk 8% dry matter in quantity e 300 mL of sodium citrate - 18 g, sucrose - 35 g of gelatin - 35 g, tap water to 1 liter. The resulting mixture is poured into glass bottles under sterile conditions, then the preparation is frozen and dried. Freezing is carried out at minus 34 ^o C for 12 hours, drying for 48 hours. The final drying temperature is +35 ^o C. The total drying time is 64 hours [2].

 The disadvantage of this method is the shorter shelf life of the concentrate and its insufficient quality.

 The technical result consists in improving the quality and lengthening the shelf life of the bacterial concentrate.

 The essence of the invention lies in the preparation of a seed strain of Lactobacillus acidophilus Ep 317/402 by 3-4 passages from a liquid culture medium onto a solid nutrient medium, selection of typical colonies, followed by control of the absence of bacterial contamination. Then, the obtained seed strain is cultivated on a nutrient medium with a pH of 5.6 - 5.7, which contains milk whey - 170 ml, potato extract diluted with water 1: 6 - 1.4 ml, skim milk - 10 ml, manganese sulfate - 0.165 g, sodium acetate - 6 g and distilled water to 1 liter. The resulting biomass is mixed with a protective medium containing sucrose, gelatin, a milk component and monitored for the absence of contamination of extraneous microflora by plating on MPA (meat-peptone agar) with 9% sodium chloride and Saburo medium. To the resulting biomass add a 15% solution of sodium bicarbonate in an amount of 1.1 l.

 Then the resulting microbial mass is poured into 5 ml vials and subjected to freezing, drying, i.e. freeze-dried for 64 - 66 hours. The pH in the finished product is 6.5 - 7.5.

 The developed scheme for the preparation of the seed strain guarantees the use in production of highly active standard strain corresponding to its passport for all items, which increases the quality of the concentrate.

 The proposed method allows to maintain greater stability and survival of bacteria in the drug.

The activity of the bacterial concentrate is at least 10 ⁷ microbial cells in 1 dose.

 The shelf life of the concentrate is at least 2 years.

 Example.

 A seed strain of Lactobacillus acidophilus Ep 317/402 is prepared, which consists of the following steps.

 1. Sowing the production strain of Lactobacillus acidophilus Ep 317/402 obtained from Armenia in milk in a liquid cultivation medium used for production at the enterprise (0.5 ml in two tubes with 4.5 ml of culture medium. Cultivation medium consists of milk whey - 170 ml of potato extract diluted with water 1: 6 - 1.4 ml, skim milk - 10 ml, manganese sulfate - 0.165 g, sodium acetate - 6 g and distilled water to 1 L. Cultivation of crops is carried out for 12 - 16 h

 2. Reseeding with a loop (0.025 ml) of microbial suspension from each tube by stroke on a dense medium - agar with hydrolyzed milk in Petri dishes. Cultivation of crops 16 - 18 hours. On the surface of the agar at the end of sowing, individual colonies of microorganisms are streaked to study them.

 Separately grown colonies are examined in a binocular magnifying glass or under a small magnification of a microscope in order to select colonies typical of a strain - by their size, configuration, color, etc., and mark them for further study. From the number of selected colonies, smears are made on glass slides, fixed, stained according to Gram and microscopic.

 If smears contain microbes with typical shapes and sizes for the strain, these colonies are subject to further reseeding and study.

 3. Reseeding the selected colonies (5 colonies from a Petri dish) in 2 bottles with 50 ml of culture medium in each. Cultivation of crops 12 - 16 hours

 Purpose: adaptation of the strain to a new culturing medium for it: obtaining a homogeneous microbial suspension of the strain in an amount sufficient to further obtain the desired number of inoculum strain and to study all its properties listed above.

 Samples are taken from the microbial suspension grown in the bottles for a comprehensive study of the strain.

 4. Reseeding of 5 ml of microbial suspension from vials on the surface of agar with hydrolyzed milk in mattresses. Cultivation of crops 16 - 18 hours

 Purpose: growing the microbial mass of the strain to obtain the required number of inoculum strain.

 5. Wash away the grown microbial culture from the surface of the agar in the matrices. For this purpose, 40 ml of sterile protective drying medium (sucrose-gelatin-milk medium) is added to each mattress.

 Purpose: obtaining a microbial suspension of the strain for the preparation of the required number of seed strain.

 From the washed microbial suspension, smears are prepared separately from each mattress, which are microscopic to control the absence of extraneous microflora. In the absence of such, the washed-off microbial suspension is drained from all mattresses into one sterile flask. The contents of the flask are thoroughly mixed on a Shuttel apparatus, a microbial suspension sample is taken for microscopy and determination of the concentration of lactobacilli (the number of live lactobacilli in 1 ml of suspension).

 6. Spill microbial suspension in a drying medium of 2 ml into bottles and its lyophilization.

 Purpose: obtaining a guaranteed homogeneous, refined, stable inoculum strain that allows you to get standard uterine cultures for the production of all series of products.

 Since the lyophilized strain is controlled only 2 times a year, greater material savings are achieved for its control and reduced labor costs.

 Then get the biomass of acidophilic bacteria.

 To obtain the biomass of acidophilic bacteria, the obtained inoculum strain Lactobacillus acidophilus Ep 317/402 is seeded in a bottle with sterile milk in an amount of 150 ml (culture of the 1st passage), then the obtained culture of the 1st passage is transferred to 10 bottles of 300 ml each with sterile milk (receiving culture of the 2nd passage). The culture of the 2nd passage (uterine culture) is reseeded in 5 bottles with a cultivation medium, which is used in an amount of 34 l, followed by mixing of the cultivation medium with a protective drying medium in an amount of 34 l.

After mixing with a protective medium, a check is made for the absence of contamination of extraneous microflora by inoculation on MPA (meat peptone agar) with 9% sodium chloride and Saburo medium, as well as to determine the number of viable lactobacilli in 1 ml. To the resulting biomass add a 15% solution of sodium bicarbonate in an amount of 1.1 l. Then, the resulting microbial mass is poured into 5 ml vials and subjected to freezing, drying, i.e., freeze-drying for 64 hours. The final temperature of the concentrate at the end of drying is not higher than 30 ^o C. The pH in the finished product is 7.0.

Claims (1)

 A method of producing a dry bacterial concentrate for a fermented milk product, comprising cultivating a strain of Lactobacillus acidophilus Ep 317/402 on a nutrient medium containing dairy whey, potato extract, manganese sulfate and water, mixing the resulting biomass with a protective medium, freezing and drying, characterized in that before by culturing the strain, it is prepared by 3–4 passages from a liquid cultivation medium to a solid nutrient medium, selection of typical colonies, followed by control of the absence bacterial contamination, and the cultivation of the strain is carried out on a medium with a pH of 5.6 - 5.7, which additionally contains skim milk in an amount of 9 - 11 ml and sodium acetate in an amount of 5.9 - 6.1 g, while the pH of the concentrate is 6.5 - 7.5.4