RU2295563C1 - Bacterium strain lactococcus lactis subcpecies lactis b-8558 useful in dairy product preparation and method for production of starter culture of strain lactococcus lactis subcpecies lactis b-8558 - Google Patents

Abstract

FIELD: biotechnology, in particular dairy industry with high rheologic and quality characteristics.

SUBSTANCE: invention relates to new natural strain of lactic acid bacteria capable of exopolysaccharide synthesis in amount sufficient to improve rheologic product characteristics without introducing of food supplement and to increase functional properties of products producing with said strain. Starter culture of claimed strain is produced by fermentation under condition of semi-continuous deep culturing on broth containing milky whey, concentrated hydrolyzed milk, KH₂PO₄, Na₂HPO₄, MgSO₄, sodium citrate, and distilled water. Fermentation is carried out at 22-32°C, constant pH 6.0-7.0 up to accumulation of living cell amount of 8.4-9.5 lg CFU/cm³. Biomass is separated, resuspended in protective medium, wherein as cryoprotector defatted milk with increased mass part of dry matter (up to 15 %) or defatted milk blended with gelatin in equal ratio is used; frozen and lyophilized. After drying biomass in bottles is sealed or pre-packed in sealed packing materials.

EFFECT: starter culture with maximal activity; products with improved quality and prolonged storage time.

2 cl, 2 dwg, 3 ex

Description

The invention relates to biotechnology, in particular, the dairy industry and can be used in the production of dairy products with high rheological and quality indicators.

Dairy products play an important role in the diet of various population groups. Dairy products produced by domestic manufacturers meet the established requirements in our country, however, enterprises encounter certain difficulties in obtaining products with stable quality and safety indicators.

To improve the rheological characteristics and increase the shelf life of products in the food industry, polysaccharides of vegetable (natural and modified) and microbiological origin are used in the form of food additives that require special permission for their use. Sometimes only the use of a polysaccharide composition allows a positive result. Thus, manufacturers are faced with many problems and challenges before choosing a food supplement, the use of which should imply the occurrence of minimal changes in the technological chain [1].

At present, Russia has adopted new GOSTs relating to the production of national products in which food additives are not allowed. An alternative to these food additives and a promising direction is the use of natural biologically useful substances with functional properties, which would at the same time contribute to the production of products with the desired properties, stable during storage. One of the factors influencing the course of the biotransformation of the feedstock during the production of fermented dairy products is the starter culture. In recent years, studies have been conducted abroad on the use in the production of dairy products of lactic acid bacteria synthesizing exopolysaccharides (EPS), the main advantage of which is the use of in situ (ie, in situ), which greatly simplifies the tasks of manufacturers [2]. In our country, only recently appeared the first publications on bifidobacteria producing EPS [3]. So, many of the above difficulties in the use of stabilizer polysaccharides can be resolved using lactic acid bacteria producing EPS.

The ability of some strains of lactic acid bacteria to produce EPS is used in the dairy industry abroad to improve the rheological characteristics of sour-milk and other dairy products. Due to the high water-binding ability, the presence of EPS reduces the amount of released serum during production and in the finished product, as well as during storage [4]. EPS, being water-binding agents, reduce the amount of free moisture in the product, thereby increasing the shelf life of the product [5].

Foreign sources of information report on the functional role of polysaccharides synthesized by lactic acid bacteria, in particular, that they exhibit antiulcer and immunomodulatory activity in the human body [6, 7, 8].

Strains of the genera Sphingomonas, Pseudomonas, and Agrobacterium that synthesize EPS are known abroad [9, 10]. But these microorganisms do not have food status, i.e. cannot be used in the production of dairy products.

The strains of Streptococcus thermophilus and Lactobacillus sake, which synthesize exopolysaccharides used in the manufacture of products, are patented abroad. The patents describe morphology, growth optimum, the ability to ferment sugars, and the ability to synthesize exopolymers. It was indicated that the strains and their isolated polysaccharides can be used for the preparation of dietary compositions [11, 12].

The closest analogue of the invention is patent WO 03102204; C 12 P 19/04; A 23 C 9/123; Novel Streptococcus thermophilus strains producing stable high-molecular-mass exopolysaccharides. De Vuyst L., Vaningelgem F., published December 11, 2003, patenting a new strain of Streptococcus thermophilus producing soluble high molecular weight exopolysaccharides. The strain is donated under the number LMP P-21524, refers to cultures with functional properties due to the production of high molecular weight exopolysaccharides. Culture, as a producer of EPS, or pure EPS are used in food products to give the product functional properties, improve rheological properties, and also as a water-binding agent. The molecular weight, structure, and composition of heteropolysaccharides are characterized. A nutrient medium was selected for the cultivation of the strain — milk with a lactalbumin hydrolyzate.

Thus, an analysis of the above data indicates that at the present stage, it is promising for the food industry of our country to obtain and use in the production process of fermented milk products domestic strains of microorganisms with the ability to synthesize exopolymers classified as GRAS (widely recognized as safe), to which include lactic acid bacteria [13].

Known methods for producing EPS strains with increased synthesis of EPS using genetic manipulation [14, 15, 16, 17] and by changing the cultivation conditions for microorganisms of the genus Sphingomonas and Streptococcus [9, 18]. An analysis of the available data allows us to conclude that the conditions for the production of EPS for each strain are individual and therefore, in each case, it is necessary to identify rational cultivation parameters to ensure the maximum EPS activity of bacteria.

In contrast to existing patents, an EPS-active strain of lactic acid bacteria belonging to another taxonomic group, namely, Lactococcus lactis subspecies lactis, is first proposed. The proposed strain of lactic acid bacteria Lactococcus lactis subspecies lactis LLN-E2 is deposited in the All-Russian collection of industrial microorganisms under the number VKPM B-8558, belongs to the mesophilic and can be used as an import-substituting strain as a producer of soluble exopolysaccharides in the production of dairy products.

The new strain of Lactococcus lactis subspecies lactis VKPM B-8558 is characterized by cultural-morphological, genetic, physiological and biochemical characteristics and technological properties.

The strain Lactococcus lactis subspecies lactis VKPM B-8558, according to the passport, isolated from an enriched culture based on self-fermented cottage cheese obtained from cow's milk. The strain Lactococcus lactis subspecies lactis VKPM B-8558 is gram-positive, facultatively anaerobic, non-spore-forming, motionless, catalase-negative cocci. Cells are located singly, in pairs and in chains of different lengths. When grown in liquid hydrolyzed milk, it grows over the entire height of the volume, in liquid whey medium it forms a biomass sediment, and on sterile skim milk it forms a milk clot uniform throughout the column. On the surface of solid nutrient media (hydrolyzed milk with agar, M02 and MRS) forms oval and round colonies located both on the surface and in the depth of the cultivation medium.

The content of HC in DNA is 37.5 mol%, which confirms its taxonomic affiliation with the genus Lactococcus. The strain of Lactococcus lactis subspecies lactis VKPM B-8558 has two plasmids (figure 1). The stability of preserving the properties of this strain was studied, as a result of which it can be concluded that the strain stably retains its properties.

The optimum development temperature is (26-34) ° С, the pH of the nutrient media is 7.2 ± 0.2; does not form catalase; does not thin the gelatin. It mainly produces L (+) - isomer of lactic acid; resistant to 0.4% phenol and 2% NaCl.

The fermentation activity of sterile milk with a 5% strain is 7.0-8.0 hours, the energy of acid formation after 7.5 hours of cultivation in milk is (60-67) ° T. The number of cells in sterile skim milk averages 2 × 10 ⁸ -5 × 10 ⁸ in 1 cm ³ . The water-retaining ability of the clot formed in milk is (3.5-4.5) cm ³ . The limit of acid formation in sterile skim milk is (94 ± 5.0) ° T. When it develops in milk of normal composition, it forms a viscous clot, which has a mucous consistency and has a clean sour-milk taste without extraneous smacks and odors. The enzyme produces β-galactosidase: the index of lactose-protecting activity is 0.63.

A distinctive feature of Lactococcus lactis subspecies lactis VKPM B-8558 is its natural ability to produce a sufficiently high amount of soluble exopolysaccharides under various cultivation conditions (0.075-0.105 g / 100 cm ³ ), and this property is stably preserved.

For the strain Lactococcus lactis subspecies lactis VKPM B-8558, an individual genetic characterization was obtained using DNA fingerprinting technology with further analysis of amplification product kits, which allows industrial use to protect copyright (Fig. 2).

It is known that the methods of using each strain with EPS ability are individual, i.e. to different conditions, the strains synthesize different amounts of EPS and, in order to maximize the synthesis of EPS, special cultivation conditions are determined. For this, it is necessary to obtain a EPS strain with maximum activity for the production of polymers for use in the production of dairy products in order to obtain high rheological parameters.

The proposed method differs in the cultivation conditions of the strain Lactococcus lactis subspecies lactis VKPM B-8558 to obtain the maximum amount of EPS, which includes: preparing a nutrient medium (medium composition per 1 l: 230 ÷ 270 ml (90 ÷ 140 g) milk whey (curd or cheese, clarified), 230 ÷ 270 ml concentrated hydrolyzed milk, 1.8 ÷ 2.3 g KH ₂ PO ₄ , 2.2 ÷ 2.8 g Na ₂ HPO ₄ , 0.08 ÷ 0.15 g MgSO ₄ , 5 ÷ 8 g sodium citrate, distilled water (up to 1 l)). Dry whey is restored, deoxidized (25% NH ₄ OH or 40% NaOH) to pH = 6.5–6.7, kept for 20-30 minutes. Then, other components are filtered and introduced, stirred, poured into containers and sterilized at t = 117 ± 1 ° C (0.8 atm) for 40 minutes, fermentation under conditions of semi-continuous deep cultivation at a temperature of 22 ÷ 32 ° C on the prepared nutrient medium for 7.5 ÷ 14 hours at a constant pH value equal to (6.5 ± 0.5), at which a sufficient number of living cells accumulate - (8.4 ÷ 9.5) log CFU in 1 cm ³ and the maximum the number of EPS - 0,075 ÷ 0,105 g / 100 cm ³ . After cultivation, the cell biomass is centrifuged with the culture fluid in a centrifuge with cooling at 2800-3500 rpm for 10-25 minutes. Then the sediment constituting the resulting biomass is resuspended in a protective medium in a ratio of 1: 1 or 1: 2. As cryoprotectants use skim milk with an increased mass fraction of solids (up to 15%) or skim milk mixed with gelatin in the same ratio. Next, freeze the starter culture at a temperature not exceeding minus 30 ± 2 ° C. Drying is carried out by the freeze-drying method until the mass fraction of moisture in dry biomass reaches no more than 4%. After drying, the biomass in the vials is sterilely sealed or packaged in sealed packaging materials. Store dry starter EPS culture at a temperature of minus 18 ± 2 ° C for no more than 6 months.

The technical result from the implementation of the invention is the social effect aimed at improving the population and obtaining a natural product without the inclusion of food additives, and the economic effect obtained by excluding from the production stage of the purchase, quality control, preparation and introduction of food additives. This goal is achieved through the use of a strain of Lactococcus lactis subspecies lactis VKPM B-8558, producing EPS (0.075 ÷ 0.105 g / 100 cm ³ ), which improves the rheological performance of the product without making food additives and enhances the functional properties of the obtained products using this strain of lactic acid bacteria.

Example 1

Preparing a nutrient medium (per 1 l): 250 ml (100 g) whey (curd, clarified), 250 ml concentrated hydrolyzed milk, 2 g KN ₂ PO ₄ , 2.5 g Na ₂ HPO ₄ , 0.12 g MgSO ₄ , 6 g sodium citrate, distilled water (up to 1 liter). Dry whey is restored, deoxidized (25% NH ₄ OH) to pH = 6.5, incubated for 20 minutes. Then, other components are filtered and introduced, stirred, poured into containers and sterilized at t = 117 ° C (0.8 atm) for 40 minutes, then cooled to a temperature of 22 ° C. 50 ml of the activated culture of Lactococcus lactis subspecies lactis VKPM B-8558 are added to the prepared nutrient medium in the fermenter in an amount of 1 l. After that, the process of increasing the bacterial biomass is started, carried out at a temperature of 22 ° C, pH = 7.0 for 14 hours. Then the biomass is centrifuged, mixed with a protective medium (skim milk with gelatin in a ratio of 1: 1), frozen at a temperature of minus 30 ° C and freeze-dried to a mass fraction of moisture in dry biomass of not more than 4%. After drying, the biomass in the vials is sterilely sealed or packaged in sealed packaging materials. Store a dry starter culture at a temperature of minus 18 ± 2 ° C for no more than 6 months.

Example 2

Prepare culture medium (per 1 liter): 230 ml (90 g), whey (cheese, clarification), 270 ml of concentrated hydrolysed milk, 1.8 g of KH ₂ PO _4, 2.2 g Na ₂ HPO _4, 0.08 g MgSO ₄ , 5 g sodium citrate, distilled water (up to 1 l). Dry whey is restored, deoxidized (40% NaOH solution) to pH = 6.7, incubated for 30 minutes. Then, other components are filtered and introduced, stirred, poured into containers and sterilized at t = 118 ° C (0.8 atm) for 40 minutes, then cooled to a temperature of 27 ° C. 50 ml of the activated culture of Lactococcus lactis subspecies lactis VKPM B-8558 are added to the prepared culture medium in the fermenter in an amount of 1 L. After that, the process of increasing bacterial biomass begins, carried out at a temperature of 27 ° C, pH = 6.5 for 11.5 hours. Then the biomass is centrifuged, mixed with a protective medium (skim milk with gelatin in a ratio of 1: 1), frozen at a temperature of minus 32 ° C, and freeze-dried to a mass fraction of moisture in dry biomass of not more than 4%. After drying, the biomass in the vials is sterilely sealed or packaged in sealed packaging materials. Store a dry starter culture at a temperature of minus 18 ± 2 ° C for no more than 6 months.

Example 3

Preparing a nutrient medium (per 1 liter): 270 ml (140 g) whey (curd, clarified), 230 ml concentrated hydrolyzed milk, 2.3 g KN ₂ PO ₄ , 2.8 g Na ₂ HPO ₄ , 0.15 g MgSO ₄ , 8 g sodium citrate, distilled water (up to 1 l). Dry whey is restored, deoxidized (40% NaOH solution) to pH = 6.6, incubated for 25 minutes. Then, other components are filtered and introduced, stirred, poured into containers and sterilized at t = 116 ° C (0.8 atm) for 40 minutes, then cooled to a temperature of 32 ° C. 50 ml of the activated culture of Lactococcus lactis subspecies lactis VKPM B-8558 are added to the prepared nutrient medium in the fermenter in an amount of 1 l. After that, the process of increasing bacterial biomass is started, carried out at a temperature of 32 ° C, pH = 6.0 for 7.5 hours. Then the biomass is centrifuged, mixed with a protective medium (skim milk with an increased mass fraction of solids (12%)), frozen at a temperature of minus 28 ° C, and freeze-dried to a mass fraction of moisture in dry biomass of not more than 4%. After drying, the biomass in the vials is sterilely sealed or packaged in airtight packaging materials. Store a dry starter culture at a temperature of minus 18 ± 2 ° C for no more than 6 months.

Literature

1. Greshnov A.G., Vzorov A.L., Nikitkov V.A. Nutritional supplements from The Nutra Sweet Kelco Company (UK). // Food industry. - 1997. - No. 11 - p. 68-71.

2. Degeest Bart, Vaningelgem Frederic, Luc De Vuyst Microbial physiology, fermentation kinetics, and process engineering of heteropolysaccharide production by lactic acid bacteria. // International Dairy Journal, 11, 2001, p. 747-757.

3. Novik GI, Astapovich NI, Kübler I., Gamyan A. Characterization of polysaccharides secreted by Bifidobacterium adolescentis 94 BIM. // Microbiology. - 2002. - Volume 71. - No. 2. - p.205-210.

4. Hartley D. L., Denariaz G. The role of lactic acid bacteria in yogurt fermentation. // Int. J. Immunotherapy. - 1993. - 9 (1) - P.3-17.

5. Ricciardi A. and Clementi F. Exopolysaccharides from lactic acid bacteria: structure, production and technological applications. // Ital. Journal Food Sci. - 2000 - Vol. 12. - No. 1. - P.23-45.

6. Stigele F., Sebastien J.F. Vincent, Faber E.J., Newell J.W., Kamerling J.P. and Neeser J.R. Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide. // Molecular Microbiology. - 1999 - Vol. 32. - No. 6. - P.1287-1295.

7. Patent 2002010242 Republic of Korea, Int: C 12 N 1/20; Eur: A 61 K 31/715; Microorganism isolated from Chinese elm (Ulmus sp.) And method for producing immunostimulating exopolysaccharide having anticancer activity using the same microorganism. Yang et al. Publ. 02/04/2002., Http: v3.espacenet.com.

8. US patent 5700787, Int: A 61 K 31/715; Eur: A 61 K 39/02; Capsular polysaccharide immunomodulator. Trianabos et al. Publ. 12/23/1997., Http: v3.espacenet.com.

9. US Patent 2004077056, Int: C 08 B 37/00; Eur: C 08 B 37/00; Production of exopolysaccharides unattached to the surface of bacterial cells. Yamazaki M., Armentrout R. et al. Publ. 04/22/2004., Http: v3.espacenet.com.

10. U.S. Patent 4,567,140, Int: C 12 P 39/00; Eur: C 12 P 19/04; Microbial polysaccharides, process for their preparation, microorganisms suitable for this and use of the polysaccharides. Voelskow et al. Publ. 01/28/1986., Http: v3.espacenet.com.

11. U.S. Patent 2004023361, Int: A 61 K 31/715; Eur: C 12 P 19/04; Lactic acid bacteria producing polysaccharide similar to those in human milk and corresponding gene. Pot B., Neeser J.R. and other publ. 02/05/2004., Http: v3.espacenet.com.

12. US Patent 5834043, Int: A 23 C 9/12; Eur: A 21 D 2/18 B; Lactobacillus sake like strains, production and use of their exopolysaccharide. Ledeboer A.M., Vreeker R. et al. Publ. 11/10/1998., Http: v3.espacenet.com.

13. Degeest B., Mozzi F., De Vuyst L. Effect of medium composition and temperature and pH changes on exopolysaccharide yields and stability during Streptococcus thermophilus LY03 fermentations. // International Journal of Food Microbiology 79 (2002) p. 161-174.

14. Patent 5639601 Australia, Int C 12 N 15/31; Eur: C 12 N 15/52; Streptococcus thermophilus operons involved in exopolysaccharide (eps) synthesis. Mengaud J., Rallu F. et al. Publ. 10/30/2001., Http: v3.espacenet.com.

15. Patent 9962316 World Intellectual Property Organization (WIPO), Eur: C 12 N 9/10 D1; Lactic acid bacteria producing exopolysaccharides. Germond J.E. and other publ. 12/09/1999., Http: v3.espacenet.com.

16. Patent 280974 New Zealand, Int C 12 N 15/52; Eur: C 12 N 15/52; Lactic bacterium DNA coding for enzymes involved in exopolysaccharide (EPS) production and its use. Mollet B. and Stingele F. Publ. 08/26/1998., Http: v3.espacenet.com.

17. Patent 2002130709 Russia, C 12 N 9/90; Overproduction of exopolysaccharides by mutants of lactic acid bacteria. Anba J. Agier J. and others Publ. 04/20/2004., Http://www.rupto.ru.

18. Patent 03102204 World Intellectual Property Organization (WIPO), Int C 12 P 19/04; Eur: A 23 C 9/123 H; Novel Streptococcus thermophilus strains producing stable high-molecular-mass exopolysaccharides. De Vuyst L., Vaningelgem F. Publ. 12/11/2003., Http: v3.espacenet.com.

Claims (2)

1. The bacterial strain Lactococcus lactis subspecies lactis VKPM B-8558, used in the production of dairy products. 2. A method of obtaining a starter culture of a strain of Lactococcus lactis subspecies lactis VKPM B-8558, which provides for the preparation of a nutrient medium containing whey - 230 ÷ 270 ml (90 ÷ 140 g), concentrated hydrolyzed milk - 230 ÷ 270 ml, KH ₂ PO ₄ - 1.8 ÷ 2.3 g, Na ₂ HPO ₄ - 2.2 ÷ 2.8 g, MgSO ₄ - 0.0 8 ÷ 0.15 g, sodium citrate - 5 ÷ 8 g, distilled water (up to 1 l ), sterilization, cooling, application of the activated culture of Lactococcus lactis subspecies lactis VKPM B-8558 and fermentation under conditions of semi-continuous deep cultivation at a temperature of 22 ÷ 32 ° C with constantly maintained enii pH 6.0 ÷ 7.0 to accumulation amount of live cells 8,4 ÷ 9,5 lg KOE / cm ³ and a content of exopolysaccharides 0.075 ÷ 0.105 g / 100 cm ^3, separation of the biomass, resuspension in a protective environment, freezing and subsequent sublimation drying.

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