RU2608871C1 - Lactobacillus paracasei 1 bacterial strain used for probiotic preparation production - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to microbiology and biotechnology. Strain Lactobacillus paracasei 1 has high antagonistic activity, high level of acid formation, improved adhesive properties, resistance to some antibiotic preparations and high rate of biomass accumulation. Strain Lactobacillus paracasei is deposited in Russian National Collection of Industrial Microorganisms under registration number VKPM V-11821 and can be used in production of probiotics.

EFFECT: invention allows to normalize piglets intestinal microflora and enhance livestock livability.

1 cl, 1 dwg, 7 tbl, 6 ex

Description

The invention relates to the field of microbiology and biotechnology and is a new bacterial strain of Lactobacillus paracasei 1, which can be used in the microbiological industry and in the production of probiotics.

The bacterial strain Bacillus licheniformis VKPM B-2414D is known from the prior art [Korotchenya I.N., Koburneev I.V., Ginzburg A.S., Luhvich K.A. The bacterial strain Bacillus licheniformis VKM used to produce a probiotic feed additive intended for the production of high-quality feeds that increase productivity and reduce the risk of gastrointestinal diseases of animals, birds and fish. Patent RU 2398872, publ. 09/10/2010, bull. No. 25, stated 2008149890/13, 12/18/2008]. It has a high survival rate due to the formation of spore forms of bacteria, which makes it possible to store it in liquid form at positive temperatures for one year. The disadvantage of this strain is that it is isolated from the soil and is atypical for the microflora of the gastrointestinal tract of animals, therefore, it is rapidly eliminated and has a short probiotic effect.

A known bacterial strain Lactobacillus amylovorus BT - 24/88 VKPM B-6253 isolated from the contents of the cecum of a milk pig [Tarakanov B.V. The bacterial strain LACTOBACILLUS AMYLOVORUS, used to produce the probiotic lactoamilovorin. Patent RU No. 2054478, publ. 02.20.1996, declared 5063804/13, 10/01/1992] used for the preparation of the veterinary probiotic preparation "Lactoamilovorin". This strain is characterized by a high rate of biomass formation (biological concentration for 14-16 hours of cultivation reaches (3.0 ± 1.5) ⋅10 ⁹ CFU / ml), a high level of acid formation. The disadvantages of this strain is the low adhesive activity and the level of antagonistic activity against different types of conditionally pathogenic enterobacteria (see Tables 1 and 2). In addition, the use of strains of Bacillus licheniformis VKM B-2414D and Lactobacillus amylovorus BT - 24/88 VKPM B-6253 is limited to veterinary practice. As part of probiotic preparations intended to normalize human microflora, they are not used.

A known strain of Lactobacillus plantarum 8P-A3. This strain is part of the probiotic preparation "Lactobacterin" [http://www.gastroscan.ru/handbook/145/1878]. It has a high adhesive activity against human red blood cells. The disadvantage of this strain is the low level of acid formation, low rate of increase in biomass: the biological concentration of lactobacilli after 54 hours of cultivation reaches (3.0 ± 0.6) ⋅10 ⁹ CFU / ml. [Dynamics of the content of lactobacilli, microbial metabolites and antibacterial activity of the growing culture of Lactobacillus plantarum 8P-A3 / Chicherin I.Yu., Pogorelsky IP, Lundovskikh IA, Malov AA, Shabalina MR, Darmov I. AT. / Journal of Infectology. - 2013. - No. 3. - S. 50-55]. In addition, the levels of antagonistic and adhesive activities of this strain are low.

The objective of the invention is the expansion of the range of bacterial strains with pronounced probiotic properties, with high antagonistic activity, high levels of acid formation, increased adhesive properties, resistance to a number of antibiotic drugs and a high rate of biomass accumulation.

The implementation of the invention

Strain Lactobacillus paracasei 1 isolated from the intestinal contents of a healthy milk pig. This strain has a high antagonistic activity against opportunistic microflora, pronounced acid formation, natural resistance to a number of antimicrobial agents. A high level of adhesive activity both in relation to human red blood cells and in relation to pig red blood cells opens up prospects for the use of this strain as a part of biological products in both medical and veterinary practice. Lactobacillus paracasei 1 actively propagates in liquid and solid nutrient media, has good survival with preservation of probiotic properties during long-term storage in the form of a suspension. The strain Lactobacillus paracasei 1 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) FSUE State Research Institute of Genetics and Breeding of Industrial Microorganisms (FSUE GosNIIgenetika) under registration number VKPM B-11821 in 2014.

The strain can be used for the preparation of liquid and dry forms of probiotic, as well as for the preparation of a biological product in the form of a culture fluid freed from microorganisms. When fed to piglets during weaning, it increases the safety and weight gain of piglets, inhibits the hemolytic forms of bacteria, pathogenic staphylococci, pathogenic and conditionally pathogenic enterobacteria in the gastrointestinal tract, restores the qualitative and quantitative composition of microflora.

The proposed bacterial strain is characterized by the following features.

Cultural and morphological features

On a dense nutrient medium, MRS, after two days of incubation, forms colonies with a diameter of 1-2 mm, opaque, white, shiny, round in shape with a convex hilly surface. The edge of the colony is uneven. Microscopic examination revealed gram-positive rods, straight and rounded. In a liquid nutrient medium, MRS-4 forms diffuse growth, and forms a loose precipitate at the bottom of the tube.

Physiological and biochemical characteristics

Fixed, indisputable facultative anaerobic, catalase-negative, has amylolytic activity. Optimum cultivation conditions: temperature (37 ± 2) ° С, microaerophilic conditions with a carbon dioxide content of 5%. It is able to grow at temperatures of (15 ± 2) ° C and (45 ± 2) ° C, ferments carbohydrates: glucose, starch, fructose, dextrose, mannose, inulin, esculin.

The strain is non-pathogenic, does not have virulence and toxicity.

The culture is stored in a semi-liquid MRS medium at a temperature of 6-8 ° C and in a freeze-dried state.

The strain is resistant to streptomycin at a concentration of more than 50 μg / ml, gentamicin (more than 20 μg / ml) and kanamycin (more than 25 μg / ml).

The invention is illustrated by the following examples.

Example 1. The selection of the strain of Lactobacillus paracasei 1 VKPM B-11821

The strain Lactobacillus paracasei 1 VKPM B-11821 was isolated from the intestinal contents of a milk pig in the Kirov region in 2012 as follows:

1.0 g of feces was introduced into test tubes with 9 ml of saline, ten-fold dilutions were prepared, and 3, 4, and 5 dilutions were plated on plates with a solid nutrient medium MRS of the following composition (g / l): casein hydrolyzate - 10.0; peptone - 10.0; yeast extract - 5.0; glucose - 20.0; three-sodium sodium acetate (CH ₃ COONa⋅H ₂ O) - 5.0; ammonium citrate disubstituted (C ₆ H ₉ O ₇ H (NH ₄ ) ₂ ) - 2.0; potassium phosphate disubstituted (K ₂ NRA ₄ ) - 2.0; magnesium sulphate heptahydrate (MgSO ₄ ⋅ 7H ₂ O) - 0.02; four-water manganese sulfate (MnSO ₄ ⋅H ₂ O) - 0.05; tween 80-1.0; agar - 15.0, distilled water up to 1 l; pH of the medium is 7.0-7.2. The cultivation was carried out at a temperature of 37 ° C under microaerophilic conditions with a carbon dioxide content in the atmosphere of 5% for 24 hours. At the end of cultivation, the appearance of white opaque colonies with a diameter of 1-2 ml was observed on the surface of the agar.

A pure bacterial culture was obtained from an accumulative culture by sieving according to the Drigalski method under similar cultivation conditions. According to phenotypic, cultural-morphological, biochemical characteristics in accordance with [Dworkin, M. Procaryotes. Third edition / M. Dworkin., S. Falkow, E. Rossenberg, K.-H. Schleifer, E. Stackebrandt. - 2001. - Vol. 1] and according to the analysis of the nucleotide sequences of 16S rRNA identified as a strain of Lactobacillus paracasei 1.

Example 2. The study of the adhesiveness of the strain

The adhesive properties of lactobacilli were evaluated using the photocolorimetric rapid method. [Oborin, V.A. Bacteriofixing activity of red blood cells against vaccine strains of the causative agents of plague, anthrax, tularemia, brucellosis and the rationale for its role in the pathogenesis of these diseases: dis. ... Dr. honey. Sciences: 14.03.03 / Oborin V.A. - Saratov, 2011 .-- 261 s: ill.]. The substrate for the adhesion of lactobacilli was human erythrocytes 0 (I) Rh + blood groups and pig red blood cells. Lactobacilli were grown for 24 hours on a dense nutrient medium MRS at a temperature of (37 ± 1) ° C. The grown cultures were washed off with phosphate-buffered saline (pH 7.2-7.3 units) and suspensions were prepared with an optical density of 1.0. 1 ml of suspension of thrice washed red blood cells in phosphate buffer with a concentration of 10 ⁹ cells / ml and 2.5 ml of a suspension of the studied cultures of lactobacilli was added to the tubes. Controls were samples containing 2.5 ml of a suspension of microbial cells and 1 ml of phosphate-buffered saline (control sample No. 1); 1 ml of a suspension of red blood cells and 2.5 ml of phosphate-buffered saline (control sample No. 2). The experimental and control samples were incubated at a temperature of (37 ± 1) ° С for 30 min. To precipitate red blood cells, the samples were centrifuged at a speed of 1000 rpm for 2 min, then the supernatant was taken in a volume of 2 ml and its optical density was measured. In each series of experiments, five independent determinations were performed.

The following criteria were used to assess the severity of the adhesive properties of lactobacilli in relation to human and porcine erythrocytes: with an adhesion index of more than 40% - a high level of adhesive activity, with an adhesion index of 15 to 40% - an average level of adhesive activity, with an adhesion index of 5 to 15% - low level of adhesive activity, with an adhesion index of less than 5%, the degree of adhesion of bacteria was considered zero.

The adhesive properties of lactobacilli in relation to human and pig red blood cells were evaluated by the formula:

where PA is the indicator of adhesion,

D _k1 - the optical density of the supernatant in the 1st control sample,

D _K2 - the optical density of the supernatant in the 2nd control sample,

D _op - the optical density of the supernatant in the experimental sample.

The average values of the results are presented in table 1.

Analyzing the data of table 1, we can conclude that the adhesive activity of the strain L. paracasei 1 both in relation to human red blood cells and in relation to pig red blood cells is high, the strain L. plantarum 8P-A3 has high adhesive activity only in relation to red blood cells person. The adhesion level of L. amylovorus BT - 24/88 to pig erythrocytes is high, but 8% (in terms of adhesion) lower than that of the claimed strain.

The adhesive activity of bacteria is an important factor in the process of bacteria colonizing the mucous membranes of the body, in particular, the mucosa of the gastrointestinal tract. A high level of adhesive activity provides a long-term persistence of lactobacilli in the gastrointestinal tract, therefore, a more pronounced inhibitory effect on opportunistic microflora due to antagonistic activity, favorable conditions are also created for the preferential development of beneficial microflora. A high level of adhesive activity both in relation to human red blood cells and in relation to pig red blood cells opens up prospects for the use of this strain as a part of biological products in both medical and veterinary practice.

Example 3. Antagonistic properties of the strain

Evaluation of the antagonistic properties of lactobacillus strains was carried out in vitro using a two-layer method according to Frederick. [Fridericq P. Actions antibiotiques reciproques chez les Enterobacteriacees Revue Belge de Patologie et de Medicine Experimentale, 19, Supp. 4 1-107 (1948)].

The essence of this technique is as follows: 0.01 ml daily lactobacillus broth culture is applied to a cup with a solid nutrient medium MRS. Crops are incubated for 24 hours at a temperature (36 ± 2 ° C) with a CO ₂ content of 5% in the gas mixture. For lysis of bacteria in colonies, filter paper was placed on the lid of an inverted Petri dish and 1 ml of chloroform was poured and exposed for 30 minutes. Then the cups are aired for 3-5 minutes. Indicator test strains, for which antagonistic activity is determined, are grown in meat-peptone broth during the day, then 0.5 ml is added to 2.5 ml of molten agar melted and cooled to (56 ± 2) ° С, quickly mixed and poured on the surface of nutrient agar with colonies of the studied strains. Tilting the cup from side to side evenly distribute the semi-liquid agar on the surface. Then the crops are incubated for 24 hours at a temperature of (36 ± 2) ° С. Around the colonies of lactobacilli should appear zones of inhibition of growth of test strains.

As test strains, we used a set of cultures of different types of enterobacteria isolated from the intestinal contents of weaned piglets, which are very slow in growth. As a control test strain, E. coli strain C 600 was used, which is a universal indicator strain and is used to evaluate the antagonistic properties of different types of microorganisms. Table 2 presents data on the level of antagonistic activity of strains of L. paracasei 1, L. plantarum 8P-A3, L. amylovorus BT - 24/88.

Analyzing the data of Table 2, we can conclude that the most pronounced antagonistic properties among the studied have the strain L. Paracasei 1, on average exceeding the level of antagonistic activity of the strains (in diameter of the zone of growth inhibition) L. plantarum 8P-A3, by 5- 40%, L. amylovorus BT-24/88 by 5-25%.

Example 4. The study of the level of acid formation

It is known that the antagonistic properties of lactobacilli are largely due to acid-forming activity. In this regard, it seemed appropriate to study this property in lactobacilli of the studied strains according to the Turner method (see Table 3).

To identify the ability to acid formation, Lactobacillus paracasei 1 VKPM B-11821 was inoculated into vials with 25 ml of MRS liquid nutrient medium and incubated at a temperature of (37 ± 1) ° С for 48 hours. At the end of the incubation, the acid-forming activity of the lactobacilli was determined by the Turner titrometric method. Each sample of the culture fluid with a volume of 10.0 cm ^{3 was} titrated with 0.1 M NaOH solution. In this case, the pH of the NaOH solution was controlled potentiometrically, its value should be (8.5 ± 0.1) units.

The calculations were performed according to the formula:

where a is the volume of a 0.1 M solution of NaOH, which went to the titration, ml;

K - correction to the titer of NaOH;

T ₀ - Turner degree, a conditional value expressed in the amount of alkali used in the titration of 1.0 ml of the test suspension.

Example 5. The use of strain Lactobacillus paracasei 1 for the preparation of liquid, dry and high-level (exempted from microorganisms of the culture fluid) forms of the probiotic preparation

To prepare a liquid form of a probiotic preparation, L. paracasei 1 culture frozen in a 10% glycerol solution (biological concentration is (1.2 ± 0.4) ⋅ ^{10 10} CFU / ml) obtained by deep cultivation in a LiFlus bioreactor was used as seed GX (Bio-Tron Inc, Korea), followed by concentration by centrifugation. For the cultivation of lactobacilli used liquid nutrient medium MRS on cabbage broth, pH (7.0 ± 0.5) units. Lactobacilli were cultured at a temperature of (37 ± 1) ° С with aeration (4 L / min) with a stirrer rotation speed of 60 rpm. As a carbon source, glucose was used, which was introduced into the nutrient medium during its preparation to a final concentration of 2%. The growing process was carried out for 16 hours, sampling was carried out every three hours in order to determine the biological concentration and the absence of extraneous microflora. PH adjustment of the culture fluid during the cultivation of lactobacilli was not performed.

The strain Lactobacillus paracasei 1 has a high rate of biomass accumulation. When studying the biomass accumulation rate at the initial inoculum dose of the strain (0.9 ± 0.1) ⋅10 ⁸ CFU / ml, samples were taken every three hours and the biological concentration of bacteria in the culture fluid was determined. The determination results are presented in table 4.

A graphical representation of the growth dynamics of lactobacilli during cultivation in a bioreactor is presented in the Figure.

At the end of the deep cultivation process, samples of the native culture were taken and the concentration of hydrogen ions in the liquid biological product was determined. The biological concentration of lactobacilli was not less than 6⋅10 ⁹ CFU / ml (see Table 5). To ensure good survival of lactobacilli during storage for up to 1 month at a temperature of (6 ± 2) ° C, sucrose was added to the culture fluid to a final concentration of 10%.

The use of strain Lactobacillus paracasei 1 for the preparation of a dry form of a probiotic preparation

To obtain a dry form of the biological product used culture fluid obtained as in the above example. To concentrate the bacterial mass, an Avanti JE centrifuge (Beckman Coulter, USA) was used with a centrifugation mode of 3000 rpm for 8 min at 25 ° C. The supernatant was decanted, after which a protective lyophilization medium containing 20% sucrose and 2% gelatin was added to the resulting lactobacillus suspension in a ratio of 1 volume of protective medium to 1 volume of microbial culture concentrate. At the next stage, lyophilization was carried out using conventional technology using a freezone 12 freeze dryer (Labconco, USA). In 1 g of the obtained dry preparation contained not less than 19-10 ¹⁰ billion live microbial cells (see Table 5).

Preparation of a centrate of culture fluid Lactobacillus paracasei 1

The centrate is a secondary product obtained by the production of a dry form of a probiotic preparation. It contains metabolic products, nutrient components and lysed bacteria. To obtain a centrate of the culture fluid Lactobacillus paracasei 1 used a supernatant obtained after concentration of bacteria by centrifugation.

Example 6. Growth-promoting and therapeutic properties of the drug based on Lactobacillus paracasei 1.

The growth-promoting and therapeutic properties of the biological product were tested on weaned piglets at the age of (25 ± 4) days immediately after weaning from the sow. During this period, due to a change in the diet of food and stress, they are most susceptible to various infections, especially to the gastrointestinal.

In the experiment used three forms of biological product obtained as described in example 3: liquid, dry and centrate of the culture fluid. In total, 120 piglets were involved in the experiment, of which 4 groups of 25-30 animals were formed: 3 experimental and 1 control. Each group of animals was placed in a separate section. The preparations were drunk once a day for two cycles of 10 days each. The interval between cycles was 10 days.

During the first cycle, the first experimental group of piglets was dosed with a liquid preparation in the amount of 3-4 ml per head and 8-10 ml during the second feeding cycle; the second group received a dry form of the drug in an amount of 0.2-0.3 g per head during the first cycle of taking the biological product and 0.4-0.6 g per second cycle of administration; the third group of piglets received a microbial culture centrate in the amount of 10-15 ml per head during the first cycle of taking the drug and in the amount of 20-25 ml during the second cycle, the fourth group of piglets - control - did not receive a probiotic preparation. The diet and conditions of piglets were the same in all sections. Conducted daily clinical observation and registration of live weight of piglets with a frequency of once a week in each group. The survival of piglets was also evaluated (see Table 6) and a qualitative and quantitative species composition of the microflora of intestinal contents was analyzed (see Table 7).

Analyzing the data of table 6, we can conclude that the studied forms of the biological product based on the strain L. paracasei 1 increase the average daily gain of piglets by 7.6-20.3% compared with the control groups, the average value of live weight by 2.1- 10.5%, and safety by 2.7-10%. According to the results of daily clinical observation, it was noted that the incidence and severity of the course of diseases in the experimental groups is lower compared to the control.

The microflora of feces of piglets was investigated during the entire period of drinking probiotic preparations. The population composition of bacteria of the Escherichia coli group, lactobacilli, bifidumbacteria, staphylococci, hemolytic forms of bacteria, conditionally pathogenic and pathogenic enterobacteria, and yeast-like bacteria was determined. The results of microbiological studies are presented in table 7.

The data in table 7 show that immediately after weaning piglets from the sow and the first cycle of probiotic therapy, a decrease in the content of E. coli with normal enzymatic activity was observed in all groups of piglets. In the experimental groups, by the end of probiotic therapy, the content of lactose-positive Escherichia coli increased: in the first and second groups - almost to the initial value, in the third experimental group it also increased, but remained an order of magnitude lower than normal. In the control group, the content of lactose-positive Escherichia coli after weaning decreased and remained below the norm by almost 3 orders of magnitude.

The content of lactobacilli and bifidobacteria in the first and second experimental groups returned to the end of probiotic therapy. In the third experimental group, the content of lactobacilli and bifidobacteria by the end of probiotic therapy also increased, but remained below the norm by almost an order of magnitude. In the control group, the content of lactobacilli after weaning of piglets decreased and remained below the norm by more than two orders of magnitude. The content of bifidobacteria in the control group remained almost an order of magnitude lower than normal.

The content of non-pathogenic staphylococci in the intestinal contents of piglets of the first and second experimental groups remained at the initial level, in the third experimental and control group it increased towards the end of probiotic therapy.

The content of enterobacteria with weakly expressed enzymatic activity in the control group of piglets was at the level of (6.95 ± 2.04) lg CFU / g throughout the experiment. In the experimental groups, before taking the probiotic, the content of enterobacteria with weakly expressed enzymatic activity was at the level of (4.50 ± 2.54) lg CFU / ml, and at the end of the experiment, pathogenic enterobacteria and enteropathogenic and lactose-negative E. coli were not seeded.

The content of hemolytic forms of bacteria in the intestines of weaned piglets of the control group was - (8.1 ± 1.6)% at the beginning of the experiment, in the first, second and third experimental group: (7.3 ± 0.83)%, (7, 29 ± 0.56)% and (7.0 ± 0.16)%, respectively. By the end of the experiment, hemolytic forms of bacteria were found only in piglets of the control group (6.15 ± 1.91%).

The content of enterococci in the feces of piglets of all groups was at the level of (7.21 ± 1.19) log CFU / ml and did not significantly change throughout the course of probiotic therapy. The content of citrate-reducing microorganisms {Pseudomonas aeruginosa) before taking the probiotic in all groups was (4.49 ± 1.07) lg CFU / ml, after taking the probiotic from the intestinal contents of the piglets of the experimental groups, this category of microorganisms was not sown, the concentration in the control group was (4 , 58 ± 0.71) log CFU / ml.

Thus, the strain of Lactobacillus paracasei 1 has a pronounced antagonistic activity against enteropathogenic microorganisms, a high level of acid formation, high adhesive activity against human erythrocytes and pig red blood cells, normalizes the intestinal microflora of piglets, has a growth-promoting effect, increases the safety of the livestock.

Claims (1)

The bacterial strain Lactobacillus paracasei 1 VKPM B-11821, intended for use in probiotic preparations of veterinary and medical purposes.

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