RU2115724C1 - Strain of bacterium streptococcus lactis - a producer of nisin - Google Patents

Abstract

FIELD: microbiology, biotechnology. SUBSTANCE: new strain of Streptococcus lactis VKPLM B-7430 - a producer of food polypeptide preserving agent nisin. New strain provides high rate of biosynthesis of nisin at operated periodic regime of culturing (activity is 11 500-12 500 IU/ml). Strain shows the increased capability to ferment different carbohydrates and alcohols. EFFECT: new strain indicated above, increased yield of the end product.

Description

 The invention relates to biotechnology and can be used in the food, dairy, microbiological and canning industry as a polypeptide preservative nisin. Traditional methods of preserving food products through sterilization are accompanied by the destruction of valuable components of the products used. Reducing the loss of food components by the addition of organic acids usually changes the taste of the food product. As an alternative, natural food preservatives are considered. The producers of nisin are strains of lactic acid bacteria Streptococcus lactis. Nisin inhibits the development of gram-positive microorganisms: streptococci, staphylococci, sarcinomas, spore-forming bacteria, including thermophilic microflora. The biological mechanism of nisin is associated with impaired permeability of bacterial cytoplasmic membranes. Violation of the membrane potential initiates the formation of pores through which bacteriocin molecules pass. Nisin inhibits the germination of spores in a number of bacillary forms of bacteria, which in turn allows to reduce the time of heat treatment of food during canning and prevents the destruction of vitamins and change the taste of human food.

 Known mutant strain of Streptococcus lactis VKPM B-4591 - producer of the antibiotic nisin (USSR author's certificate N 1687616, class C 12 P 21/00, C 12 N 1/20, BI N 40, 1991).

 The antibiotic activity of this recombinant strain on a mineral medium with glucose or maltose is 4000 - 4200 IU / ml with a fermentation time of 8 - 9.5 hours.The disadvantage of the known mutant strain is the relatively low activity of the final target product - nisin and its insufficient stability under production conditions. Known strain of Streptococcus lactis VKPM B-6793 - producer of nisin bacteriocin (RF patent N 2061042, class C 12 N 1/20, C 12 P 1/04, C 12 R 1: 4, BI N 15, 1996). The disadvantages of this known strain are the relatively low activity of nisin - 4800 IU / ml and a smaller spectrum of fermentation of sugars and alcohols.

The closest in technical solution and production indicators is a strain of Streptococcus lactis VKPM B-6795 - producer of bacteriocin nisin (RF patent N 2059716, class C 12 N 1/20, C 12 P 1/04, BI N 13, 1996), which provides activity of 5600 IU / ml, productivity of 2520 IU / cm ³ / h at a flow rate of the medium D = 0.45 h ^-1 . The disadvantages of its use in production are the smaller spectrum of carbohydrate fermentation and the relatively lower activity of nisin synthesis in hydrolyzed milk.

 The objective of the invention is to obtain a new strain-producer of nisin, providing a high yield of the target product and actively fermenting a wider range of different carbohydrates and alcohols.

 The technical result consists in obtaining the final target product - the polypeptide preservative nisin with a concentration two times higher - 11500 - 12500 IU / ml and in fermentation, in contrast to the prototype strain Str. lactis VKPM B-6795, arabinose, rhamnose, sorbitol, glycerol, dulcite.

 The strain Streptococcus lactis 47-28 is stored in the All-Russian collection of industrial microorganisms of the Institute of GosNIIgenetika under the number VKPM B-7430.

Several active strains of Streptococcus lactis were selected from the Museum of Lactic Acid VNIIMS (Uglich) in a periodic aseptic process and tested in a continuous process with an increasing flow rate of the medium (D = 0.3 - 0.5 h ^-1 ) in the Biostat apparatus . The second stage of selection was the selection of active, large colonies when seeding samples from a batch and continuous process in an apparatus on meat-peptone agar and an agarized milk hydrolyzate, followed by cultivation in rocking flasks of the active isolate of the strain Streptococcus lactis VKPM B-7430 in an optimal liquid nutrient medium and the determination of nizinobrazuyuschego ability by diffusion into agar with the test organism Bacillus coaqueans.

Cultural-morphological and physiological-biochemical properties of the strain Streptococcus lactis VKPM B-7430:
on meat and peptone agar forms small, smooth, shiny whitish colonies with a diameter of 1.5 - 2.0 mm, the edge of the colony is smooth, the profile is elevated;
on an agarized milk hydrolyzate, the colonies are small, white, smooth, shiny with a smooth edge, the profile is elevated, the diameter of the colony is 0.8-1.5 mm.

 Cells: oval cocci 1.05 - 1.5, 1.05 - 2.1, 1.2 - 1.4 microns, mainly forming pairs and chains of 4-6-8 cells.

Bacteria are gram-positive, indisputable, motionless, do not dilute gelatin, nitrates do not restore, catalase-negative. It is fermented from carbohydrates: glucose, fructose, galactose, lactose, sucrose, maltose, arabinose, mannose, rhamnose, xylose; of alcohols - mannitol, sorbitol, glycerin, dulcite. Does not form indole and hydrogen sulfide. Milk quickly coagulates, does not peptone, the duration of coagulation of milk with a 10% inoculum is 14-16 hours, the blood does not hemolize. Optional anaerobic, fermentation type - homoenzymatic, cultivation temperature 20 - 32 ^o C, optimum 30 ^o C, medium pH 6.5 - 6.8, the content of G + C in DNA varies between 33-42 mol%. The strain is phage resistant. The culture is stored in freeze-dried state in ampoules at a temperature of - 4 ± 2 ^o C for at least 12 months and in the form of a milk clot in 12% skim sterile milk with monthly reseeding and storage in a domestic refrigerator at a temperature of 4 - 6 ^o C.

 Information confirming the possibility of carrying out the invention.

Example 1. The strain of Streptococcus lactis VKPM B-7430 is restored as follows: the contents of the ampoule are introduced into a test tube with sterile skimmed 12% milk with the addition of 1% yeast autolysate and incubated at a temperature of 30 ^o C for 24 hours, 3 passages are made of it. From the last passage of the restored culture, an inoculum is prepared in skim milk in test tubes or small cones, 10% of the inoculum is introduced into 750 ml rocking flasks and batch cultivation is carried out periodically at 220 rpm for 14-16 hours. Hydrolyzed medium is used as a nutrient medium milk and whey.

Preparation: skimmed milk powder is diluted with water to a solids content of 30 g / l, add 20 g / l of whey, boil, cool to 45 - 50 ^o C, bring to pH = 7.2 - 7.5 units, make protosubtilin, dissolved in a small amount of warm distilled water at the rate of 1 g / l of medium. The hydrolysis is carried out at a temperature of 45 ^o C for 6 hours. At the end of the hydrolysis, the pH of the medium is adjusted to 7.0 units and sterilized for 30 minutes at 1 at. Nutrient medium can be used for rocking flasks and fermenters. The regulation of the pH of the medium in the flasks is made by adding 10% NaOH solution. The formation activity of the nisin polypeptide preservative by the strain of Streptococcus lactis VKPM B-7430 in the periodic mode in flasks is 10,250 IU / ml.

 Example 2. The cultivation of a strain of Streptococcus lactis VKPM B-7430 was carried out in a periodic aseptic mode in the apparatus "Fermatron" with a working volume of 50 l for 15 hours, with the same parameters and composition of the medium that are indicated in example 1, inoculum obtained in shakers flasks with a volume of 750 ml, in an amount of 5% were introduced sterilely under a torch into the Fermatron apparatus, the pH was adjusted by continuously supplying a 10% NaOH solution with automatic pH stating. As a result, at the end of the logarithmic growth phase, the largest amount of nisin is formed 11500 - 12500 IU / ml. Under the same conditions, the Str.lactis VKPM B-6795 prototype strain showed an activity of 6,000 IU / ml.

 Thus, a more active strain of Str. lactis VKPM B-7430, which provides the yield of the final target product - the food polypeptide preservative nisin is two times higher than the prototype - 11500 - 12500 IU / ml and fermenting a wider range of carbohydrates and alcohols.

Claims (1)

 The bacterial strain Streptococcus lactis VKPM B-7430 is a producer of the food polypeptide preservative nisin.