RU2677668C2 - Lactobacillus helveticus d75 and d76 strains and preparation therewith - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: group of inventions relates to biotechnology. Proposed strain Lactobacillus helveticus D75, deposited in the collection of the ARRIAM under number RCAM04483, and the strain Lactobacillus helveticus D76, deposited in the collection of the ARRIAM under the number RCAM04484, intended for use as part of probiotic preparations. Probiotic preparation intended for the preparation of a fermented milk product and containing bacterial cells of the strain Lactobacillus helveticus D75 and Lactobacillus helveticus D76 in a ratio from 1:1 to 1:3 is also proposed.EFFECT: group of inventions ensures the preservation of the properties of fermented milk drink during storage for 20 days.4 cl, 3 ex, 4 tbl

Description

The invention relates to the field of biotechnology, namely to probiotic preparations and bacterial strains used to obtain them. The invention is intended for use in medicine, food and dairy industries, agriculture and related fields.

Nowadays, probiotic preparations are widely known, which are understood to mean compositions containing a functional food ingredient in the form of non-pathogenic and non-toxicogenic microorganisms useful for humans, which, when used systematically in the form of drugs or as part of food products, have a beneficial effect on the human body as a result of normalization composition and (or) increase the biological activity of normal intestinal microflora (GOST R 52349-2005 "Food products. Food products fu tional. Terms and definitions ".

In particular, probiotics in the form of fermented milk drinks are widely used. So, the Health fermented milk drink is obtained on the basis of pasteurized whey fermented with sourdough made from pure races of lactic streptococcus, Bulgarian, acidophilus and cheese sticks (A.G. Khramtsov. Waste-free technology in the dairy industry, M., Agropromizdat, 1989, p. 210). The disadvantage of the drink is the low ability of the strains to adapt in the host enteric media and, as a result, a weakly expressed therapeutic effect.

Known whey drink "Elixir", used in practice as a drug to increase the adaptive capacity of the body under adverse environmental conditions, and strains of microorganisms Lactobacillus acidophilus (strain 20T), Streptococcus thermophilus (strain 24C), Lactobacillus casei (used in its preparation) strain 2), Lactobacillus helveticus (strain 305-5). To obtain a drink using sourdough containing these strains in a ratio of 20-35 wt. %, 20-35 wt. %, 20-35 wt. % and 5-15 wt. % (RU 2064268, 1996). The disadvantage of the drink is the complex technology for producing yeast and the presence of antagonistic relationships between production strains.

The closest in technical essence to the claimed group of the invention is previously developed with the participation of the authors of the present invention, the drug "VITAFLOR" ^® (RU 2160992, 2000). The freeze-dried preparation contains 1-5 billion / g of viable cells of 2 strains of Lactobacillus acidophilus A-91 and H-91 (when depositing strains No. 75 and No. 76, respectively), 0.5-2.5 wt. % gelatin, 5-20 wt. % sucrose, 3-5 wt. % skim milk, 2-8 wt. % water and the remains of the culture fluid. To obtain a fermented milk product, the drug is introduced into milk or the milk mixture and kept at elevated temperature for 16-18 hours.

The product and the fermented milk product have established themselves as effective agents for the prevention and treatment of intestinal infections, as well as promising immunostimulants and adaptogens (RU 2342174, 2008; RU 2487543, 2013; RU 2419443, 2011; RU 2371190, 2009; RU 342174, 2008; RU 2278682 , 2006). Given the complete safety of the drug, its use in the form of a probiotic or probiotic food product (probiotic food) in extreme conditions (space flight, prolonged swimming, other stressful conditions) is promising.

The method of obtaining the drug involves the cultivation of bacteria Lactobacillus acidophilus in a hydrolyzate-milk nutrient medium containing 0.3-0.5 wt. % calcium carbonate, the introduction of a protective medium containing gelatin, sucrose, skim milk, freezing at a temperature of minus 25-30 ° C, freeze-drying at a temperature not exceeding minus 30 ° C and a pressure of 13-15 Pa for 45-48 hours. To obtain a fermented milk drink, the drug is dissolved in water, poured into milk and incubated for 12-18 hours at a temperature of 35-45 ° C.

The disadvantage of the drug is the low stability of the fermented milk drink during storage (no more than 5 days), which manifests itself in a rapid increase in acidity and degree of syneresis due to the properties of the production strains of the drug.

The problem solved by the authors was to increase the stability of the fermented milk drink.

The technical result was achieved by changing the strain formula of the drug, namely, using the strains Lactobacillus helveticus D75 and Lactobacillus helveticus D76 obtained as a result of targeted selection of the strains included in the Vitaflor preparation in the cell ratio from 1: 1 to 1: 3 (optimally 1: 2) ..

The strains of Lactobacillus helveticus D75 and D76 were characterized genetically and culturally morphologically.

The original strains were isolated from the intestines of a healthy child in 1991 in St. Petersburg. They were wild types. Phenotypic identification of wild types was carried out in 1995 by O. Kandler & Nor-bert 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, Vol. 2. The Willians & Wilkins Co., Baltimore. According to the results of phenotypic identification, the strains were assigned to Lactobacillus acidophilus.

Phenotypic identification of breeding types was carried out in 2012 according to the Bergey's Manual of Systematic Bacteriology (Volume 3: The Firmicutes) Editors: Vos, P., Garrity, G., Jones, D., Krieg, NR, Ludwig, W., Rainey, FA, Schleifer, K.-H., Whitman, W. (Eds.), 2009. After complete sequencing of the genomes, the strains were presented in NCBI as Lactobacillus helveticus D75 [GenBank: CP020029.1] and Lactobacillus helveticus D76 [GenBank: CP016827 .one].

The strain Lactobacillus helveticus D75 was deposited in connection with the patent procedure under registration number RCAM04483 in the Departmental collection of beneficial microorganisms for agricultural purposes (VKSM).

Strains are gram-positive long thin sticks with rounded edges, single or in chains of 2-3 cells.

The type of shell structure is firmmouth. They do not form a dispute. Stationary, devoid of flagella and pili. Cell sizes vary depending on cultivation conditions, and especially on the growth phase.

Cultural and morphological features of the strain.

When grown in MPC-1 medium for 48 hours at a temperature of 37 ± 1 ° C, forms a weak turbidity and sediment; in the semi-liquid medium MPC-2 - hanging colonies in the form of “cloves”.

In the thickness of the dense medium, MPC-4 forms whitish colonies of complex configuration with a diameter of 2-4 mm. On the surface of the medium, MRS-4 forms rounded, often irregularly shaped, slightly convex colorless (eventually light beige) colonies with a diameter of 1.8-2.2 mm. The edge of the colonies is wavy, the surface is rough, the structure is folded-granular, and the texture is soft, oily. Colonies consist of tightly laid bundles of cells connected by bridges surrounded by a thin surface film.

Stroke growth is moderate, distinct.

The pigment diffusing into the nutrient medium is absent.

Known physiological and biochemical properties of the strain.

Optional anaerobic, catalase negative, cytochrome oxide-negative. It does not reduce nitrates, it does not dilute gelatin, esculin does not hydrolyze, it does not produce hydrogen sulfide and indole. It does not have hemolytic activity.

Obligatory saccharolytic. Ferments lactose, mannose, galactose, trehalose, glucose, fructose. Weakly ferment sucrose. It does not ferment melicitosis, ribose, rhamnose, xylose, raffinose, cellobiose, maltose, arabinose, sorbitol, melibiosis, mannitol. The final pH value in MPC-1 medium on fermentable sugars is 4.1-4.2.

The type of fermentation is homoenzymatic. The end product of metabolism is DL-lactate.

Milk with litmus restores without gas formation, intensively restores milk with methylene blue.

With growth in milk forms a homogeneous mucous clot. The maximum titratable acidity when grown in milk is at least 230 ° T, in MPC-1 at least 370 ° T.

The strain is characterized by a high level and a wide spectrum of antagonistic activity against museum reference cultures and clinical isolates of pathogenic microflora (S. flexneri, S. sonnei, P. vulgaris, P. mirabilis, E. coli, S. aureus, C. albicans, etc. .d.).

Temperature optimum: (37 ± 1) ° C. Grows at 45 ° C. Does not grow at 15 ° C. The optimal pH of the medium is 5.8 ± 0.2.

Cultivation conditions:

The composition of the nutrient medium (g / l).

MPC-1 (liquid): Peptonized milk (Peptonized milk, Himedia) - 40.0; casein enzymatic hydrolyzate - 10.0; yeast extract (BBL) - 8.0; glucose - 20.0; ammonium citrate - 2.0; fused sodium acetate - 5.0; L-cystine hydrochloride - 0.1; K ₂ NRA ₄ - 2.0; Mg ₂ SO ₄ - 0.2; MnSO ₄ - 0.02; tween-80 - 1.0 ml.

pH of the medium is 6.4.

MPC-2 (semi-liquid): The composition is identical to the composition of MPC-1, agar 0.75.

MPC-4 (dense): The composition is identical to the composition of MPC-1, agar - 20.

Temperature and duration of cultivation: (37 ± 1) ° C. In the environment of MPC-1 - 18-24 hours, in the media MPC-2 and MPC-4 - 48 hours.

Storage conditions

In sublimated form in sealed ampoules at a temperature of 6 ± 2 ° C. Duration of storage - 5 years or more. The protective environment during drying is sucrose 10%, gelatin 1%, pH 7.0 ± 0.2.

With periodic reseeding (no more than 5 transfers from the ampoule), the storage duration is 1 month at a temperature of 6 ± 2 ° C.

The results of genotypic identification of strain D75

Sequencing Technology: PacBio

Sequencing Method: SMRT PacBio RS II

Build Type: De novo assembly

Software: SMRT Portal v.2_3_0

Build Method: HGAP (Precise Build)

Read Library Feature:

1) average read length = 14321 bp .;

2) N50 reads = 23981 bp;

3) number of reads = 92790;

4) the total number of bases = 1328881154;

Read genome coverage: x422.69

Level of completion: completed genome

Genome characterization: contig = 1

Genome ID in the Genome Database (NCBI): CP020029.1

Genome ID in the BioSample Database (NCBI): SAMN05417795

Genome length = 2053066 bp

The number of genes = 2092

The number of pseudogenes = 317

The number of protein coding genes = 1693

The number of rRNA genes = 5, 5, 5 (5S, 16S, 23S)

The number of tRNA genes = 64

Other RNA genes = 3

G / C ratio = 37%

The strain Lactobacillus helveticus D76 was deposited in connection with the patent procedure under registration number RCAM04484 in the Departmental collection of beneficial microorganisms for agricultural purposes (VKSM). Classification: Kingdom - Bacteria; type - Firmicutes; class - Bacilli; squad - Lactobacillales; family - Lactobacillaceae; genus - Lactobacillus; species - Lactobacillus helveticus; strain - D76.

Strains are gram-positive long thin sticks with rounded edges, single or in chains of 2-3 cells.

The type of shell structure is firmmouth. They do not form a dispute. Stationary, devoid of flagella and pili. Cell sizes vary depending on cultivation conditions, and especially on the growth phase.

Cultural and morphological features of the strain.

Gram-positive long thin sticks with rounded edges, single or in chains of 2-3 cells.

The type of shell structure is firmmouth. The dispute does not form. Fixed, devoid of flagella and pili. Cell sizes vary depending on cultivation conditions, and especially on the growth phase. The average cell length is 4.0 μm.

When grown in MPC-1 medium for 48 hours at a temperature of 37 ± 1 ° C, it forms a transparent supernatant and precipitate; in semi-liquid medium MPC-2

- hanging, rounded colonies.

In the thickness of the dense medium, MPC-4 forms whitish colonies of complex configuration with a diameter of 2-4 mm. On the surface of the medium, MRS-4 forms rounded, often irregularly shaped, slightly convex colorless (eventually light beige) colonies with a diameter of 1.5-2.0 mm. The edge of the colonies is wavy, the surface is rough, the structure is folded-granular, and the texture is soft, oily. Colonies consist of tightly laid bundles of cells connected by bridges surrounded by a thin surface film.

Stroke growth is moderate, distinct.

The pigment diffusing into the nutrient medium is absent.

Known physiological and biochemical properties of the strain.

Optional anaerobic, catalase-negative, cytochrome oxidase-negative. It does not reduce nitrates, it does not dilute gelatin, esculin does not hydrolyze, it does not produce hydrogen sulfide and indole. It does not have hemolytic activity.

Obligatory saccharolytic. Ferments lactose, mannose, galactose, trehalose, glucose, fructose. Weakly ferment sucrose. It does not ferment melicitosis, ribose, rhamnose, xylose, raffinose, cellobiose, maltose, arabinose, sorbitol, melibiosis, mannitol. The final pH in MPC-1 fermented sugars is 4.2-4.3.

The type of fermentation is homoenzymatic. The end product of metabolism is DL-lactate.

Milk with litmus restores without gas formation, intensively restores milk with methylene blue.

When grown in milk, it forms a homogeneous loose clot. The maximum titratable acidity when grown in milk is at least 220 ° T, in MPC-1 - at least 330 ° T.

The strain is characterized by a high level and a wide spectrum of antagonistic activity against museum reference cultures and clinical isolates of pathogenic microflora (S. flexneri, S. sonnei, P. vulgaris, P. mirabilis, E. coli, S. aureus, C. albicans, etc. .d.).

Temperature optimum: (37 ± 1) ° C. Grows at 45 ° C. Does not grow at 15 ° C. The optimal pH of the medium is 5.8 ± 02.

Cultivation conditions:

The composition of the nutrient medium (g / l).

MPC-1 (liquid): Peptonized milk (Peptonized milk, Himedia) - 40.0; casein enzymatic hydrolyzate - 10.0; yeast extract (BBL) - 8.0; glucose - 20.0; ammonium citrate - 2.0; fused sodium acetate - 5.0; L-cystine hydrochloride - 0.1; K ₂ NRA ₄ - 2.0; Mg ₂ SO ₄ - 0.2; MnSO ₄ - 0.02; tween-80 - 1.0 ml. pH of the medium is 6.4.

MPC-2 (semi-liquid): The composition is identical to the composition of MPC-1, agar 0.75.

MPC-4 (dense): The composition is identical to the composition of MPC-1, agar - 20.

Temperature and duration of cultivation: (37 ± 1) ° C. In the environment of MPC-1 - 18-24 hours, in the media MPC-2 and MPC-4 - 48 hours.

Storage conditions

In sublimated form in sealed ampoules at a temperature of 6 ± 2 ° C. Duration of storage - 5 years or more. The protective environment during drying is sucrose 10%, gelatin 1%, pH 7.0 ± 0.2.

With periodic reseeding (no more than 5 transfers from the ampoule), the storage duration is 1 month at a temperature of 6 ± 2 ° C.

The results of genotypic identification of strain D76

Sequencing Technology: PacBio

Sequencing Method: SMRT PacBio RS II

Build Type: De novo assembly

Software: SMRT Portal v.2_3_0

Build Method: HGAP (Quick Build)

Read Library Feature:

1) average read length = 14125 bp .;

2) N50 reads = 18990 bp .;

3) number of reads = 77334;

4) total number of bases = 1092367582

Read Genome Cover Level: x375

Level of completion: completed genome

Genome characterization: contig = 1

Genome ID in the Genome Database (NCBI): CP016827.1

Genome ID in the BioSample Database (NCBI): SAMN05417796

Genome length = 2058319 bp

The number of genes = 2097

The number of pseudogenes = 321

The number of protein coding genes = 1694

The number of rRNA genes = 5, 5, 5 (5S, 16S, 23 S)

The number of tRNA genes = 64

Other RNA genes = 3

G / C ratio = 37%

Common marker genetic regions for strains D76 and D75 that distinguish these strains from other bacteria

1) The gene BCM45_RS08730 (BCM44_RS01540), encoding a protein of the EpsG superfamily, is located on chromosome D76 from 1725915 bp up to 1727033 bp (from 291350 bp to 292468 bp on D75 chromosome). Included in a block of 3 genes (the target gene is located between two adjacent genes encoding hypothetical proteins belonging to the glycosyltransferase group). To the left of the genetic block are glycosyl transferase genes, exopolysaccharide synthesis genes, and a multi-resistant MFS transporter. On the right is transposase.

2) Gene BCM45_RS08735 (BCM44_RS01545), encoding a protein of the glycosyltransferase family, located on chromosome D76 from 1727041 bp up to 1728057 bp (from 292476 bp to 293492 bp on D75 chromosome).

3) Gene BCM45_RS10040 (BCM44_RS02850), encoding a hypothetical protein located on chromosome D76 from 1990879 bp up to 1991445 bp (from 556314 bp to 556880 bp on D75 chromosome). The translation product of the target gene is partially similar to the glycosyltransferase family of proteins. The gene is located next to the resolvase gene (on the right in a straight chain) and two genes of hypothetical proteins according to the scheme: transposase - hypothetical protein - hypothetical protein - hypothetical protein - resolvase.

4) The BCM45_RS00535 gene (BCM44_RS03680), encoding the ATP-binding protein of the ABC transporter, is located on the D76 chromosome from 98906 bp up to 100492 bp (from 721280 bp to 722866 bp on D75 chromosome).

The nucleotide sequences of these genes for strain D75 are shown in table 1, for strain D76 in table 2.

The areas of difference between strains D75 and D76 are shown in table 3.

Example 1. The method of obtaining the drug

Sublimated monostrains are activated in skim milk containing growth factors for 16-18 hours at a temperature of 37 ± 1 ° C (initial inoculation is 10 ⁶ CFU / ml). Then, strains D75 and D76 were grown at 37 ± 1 ° C for 12 hours in MPC-1 medium.

Activated strains contribute to skim milk in a ratio of 1: 1 (sample 1); 1: 2 (sample 2): and 1: 3 (sample 3) and cultured for 18 hours at 37 ± 1 ° C. After that, the pH of the culture fluid is adjusted to 6.7 ± 0.2, a protective drying medium is introduced, poured into containers and sublimated.

The finished product is a porous mass containing at the end of the shelf life of at least 1 billion cells in 1 gram.

Example 2. A method of producing a fermented milk drink

10 ml of boiled and cooled to room temperature water are poured into a container containing 0.25 g of the preparation of Example 1, the resulting suspension is shaken and the suspension obtained is transferred to 0.5 l of boiled and cooled to 37 ± 1 ° C milk. Fermented milk is put for 12-16 hours in a warm place.

The finished drink is a well-formed, slightly viscous clot of creamy consistency and contains 0.5-1 billion cells in 1 ml.

Claims (4)

1. The strain Lactobacillus helveticus D75, deposited in the collection of the Federal State Budgetary Institution VNIISHMB under the number RCAM04483, intended for use in probiotic preparations. 2. The strain Lactobacillus helveticus D76, deposited in the collection of the Federal State Budgetary Institution VNIISKhMB under the number RCAM04484, intended for use in probiotic preparations. 3. A probiotic preparation intended for the preparation of a fermented milk product, characterized in that it contains cells of bacterial strains according to paragraphs. 1 and 2 in a ratio of 1: 1 to 1: 3. 4. The probiotic preparation according to claim 3, characterized in that it contains cells of bacterial strains according to claims. 1 and 2 in a ratio of 1: 2.

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