CN101974463A - Lactobacillus reuteri and composite viable bacteria preparation thereof - Google Patents

Abstract

The invention provides Lactobacillus reuteri L-HD5 and its composite live bacteria preparation, and its preservation number is CGMCC No.4050. As a feed additive, the living bacteria preparation of the invention can significantly increase the average daily weight gain and feed efficiency of weaned piglets after feeding, reduce the diarrhea rate of piglets, and improve economic benefits. It has a prospect of popularization and application, and provides a certain theoretical and practical basis for seeking alternatives to antibiotics.

Description

Lactobacillus reuteri and its compound live bacteria preparation

technical field

The invention relates to the technical field of microecological preparations, in particular to a Lactobacillus reuteri and a composite live bacteria preparation thereof.

Background technique

As a kind of microecological preparation, compound live bacteria preparation has been widely used in animal husbandry. It mainly consists of two or more different genera of probiotic bacteria. At present, the commonly used strains are mainly Lactobacillus, Bifidobacterium, Streptococcus faecalis, Bacillus and yeast. Combining probiotics with different biological functions, the compound live bacterial preparation developed can maintain the balance of the gastrointestinal environment through biological antagonism, biological oxygen capture and immune enhancement, and the probiotics can grow and metabolize in the animal body. The various digestive enzymes produced can significantly strengthen the body's digestion and absorption of various nutrients in the feed and improve the utilization of the feed.

At present, most of the production strains of compound living bacteria preparations used in pigs are non-pig-derived probiotics. Since the formation of animal intestinal flora is the result of long-term evolution, a microecological preparation suitable for a certain type of animal may not be Suitable for another type of animal, resulting in a greater impact on the application effect.

Lactobacillus reuteri is a common bacterium in the gastrointestinal tract of healthy piglets. Foreign research reports have shown that Lactobacillus reuteri has strong tolerance to gastric acid and bile, and can maintain vitality directly to the gastrointestinal tract. And it can also secrete special antibacterial substance Reuterin, which can effectively inhibit the growth of rotavirus and other intestinal pathogenic microorganisms, so as to prevent and treat diarrhea. The bacterium has been widely used in feed microecological preparations by scholars at home and abroad, and has a good market prospect.

Bifidobacterium is one of the most important physiological bacteria in the intestinal tract of humans and animals. It forms a microbial community with other physiological bacterial members and forms a microecosystem with the host. Bifidobacteria quickly colonize the intestinal mucosa after entering the intestinal tract, and combine with intestinal mucosal epithelial cells to form a microbial film to prevent the colonization and invasion of pathogenic bacteria; at the same time, a large amount of lactic acid and acetic acid are produced to improve the intestinal environment and maintain normal Intestinal peristalsis promotes the excretion of pathogenic bacteria; directly inhibits or antagonizes the growth of Shigella, Salmonella typhi, Pseudomonas aeruginosa, pathogenic Escherichia coli, Staphylococcus and other pathogenic bacteria. Bifidobacteria can remove the toxic and harmful metabolites released by pathogenic bacteria, reduce the source of endotoxin, inhibit the growth of pathogenic bacteria, reduce the level of endotoxin in the blood, adjust the micro-ecological balance, and prevent and treat endotoxemia. Bifidobacteria can also synthesize a variety of vitamins (such as vitamin B1, B2, B6, B12, niacin, folic acid, etc.) and biological enzymes; acidify the intestinal tract, which is beneficial to the absorption of iron, calcium and vitamin D, and prevent iron deficiency Occurrence of anemia and vitamin D deficiency; can supplement a variety of amino acids and trace elements. It can activate the phagocytic ability of the body's phagocytic cells and enhance the body's anti-disease immune function. Bifidobacteria have been widely used in microbial feed additives, medical care, health care, food and other fields because of their beneficial properties in maintaining micro-ecological balance, biological antagonism, immune regulation, and nutrition. favored by consumers.

In addition, domestic and foreign scholars have found that Bacillus natto in the genus Bacillus, because it can produce spores, has the characteristics of acid resistance, alkali resistance, high temperature resistance and extrusion resistance, and is stable in the granulation process and gastric acid environment. It can maintain a high degree of stability and is one of the strains that are more resistant to the environment and can directly reach the small intestine among various probiotics. In addition, Bacillus natto can also produce a variety of proteases, glucoamylases, lipases, and amylases to degrade some complex carbohydrates in plant feeds, thereby improving feed conversion rates, regulating intestinal flora, and enhancing animal health. cellular immune effects.

Contents of the invention

The object of the present invention is to provide a kind of Lactobacillus reuteri and the compound living bacteria preparation prepared therefrom.

Another object of the present invention is to provide the application of the Lactobacillus reuteri and its composite bacterial agent.

Lactobacillus reuteri (Lactobacillus reuteri) L-HD5 of the present invention is isolated from pig intestines, and this bacterial strain has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee (Address: Chaoyang District, Beijing) on July 30, 2010. No. 3, No. 1 Yard, Beichen West Road, Zip Code 100101), its abbreviation is CGMCC, and the preservation number is CGMCC No.4050.

The present invention also provides a bacterial agent containing Lactobacillus reuteri L-HD5.

The bacterial agent also includes bifidobacteria and bacillus natto.

Preferably, the bacterial agent contains by weight:

Lactobacillus reuteri L-HD5 1-10 copies

Bifidobacterium 1-10 copies

Bacillus natto 1-10 parts.

More preferably, the bacterial agent contains by weight:

Lactobacillus reuteri L-HD5 1 part

Bifidobacterium 1 part

Bacillus natto 1 part.

The present invention also provides a feed additive containing Lactobacillus reuteri L-HD5 or the above bacteria agent.

The present invention further provides a method for preparing the above microbial agent, comprising the following steps: performing the following operations on Lactobacillus reuteri L-HD5, Bifidobacterium and Bacillus natto respectively: carrying out liquid submerged fermentation culture, and collecting after centrifugation bacteria slime, and then prepare and collect the bacteria powder by vacuum freeze-drying technology, and finally compound the three kinds of bacteria powder.

Wherein, the fermentation culture conditions of Lactobacillus reuteri L-HD5 and Bifidobacteria are: temperature 37°C, pH 6.0-6.5, static culture, liquid filling volume 60%, inoculum volume 3%, culture time 16-6. After 24 hours, the number of viable bacteria was greater than 10 ⁸ CFU/mL.

The fermentation and cultivation conditions of Bacillus natto are as follows: temperature 37°C, pH 6.0, stirring speed 180rpm, liquid filling volume 20%, inoculum volume 3%, culture time 24-36h, and the number of viable bacteria is greater than 10 ⁸ CFU/mL , The sporulation rate reaches more than 50%.

The present invention also provides the application of Lactobacillus reuteri L-HD5 or the above bacteria agent in feed additives.

As a feed additive, the compound living bacteria preparation of the invention can significantly increase the average daily head weight gain and feed efficiency of weaned piglets after feeding, reduce the diarrhea rate of piglets, and improve economic benefits. It has a prospect of popularization and application, and provides a certain theoretical and practical basis for seeking alternatives to antibiotics.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Embodiment 1: Preparation of Lactobacillus reuteri live bacteria preparation

Activation of strains

Streak inoculation of Lactobacillus reuteri L-HD5 on the surface of MRS solid medium (produced by Beijing Land Bridge Technology Co., Ltd., batch number: CM188), and anaerobic culture at 37°C for 16-24h, Until bacterial colonies grow on the surface of the solid medium, observe the morphology and purity of the bacteria by microscopic examination.

Preparation of seed solution

Pick the colonies on the surface of the MRS solid medium, inoculate them into a test tube containing 5 mL of seed medium, and incubate anaerobically at ³⁷ °C for 16-24 hours ^. CFU/mL) inoculum amount was inoculated into a 250mL Erlenmeyer flask containing 150mL seed medium, anaerobically cultured at 37°C for 16-24h, until the number of viable bacteria reached 10 ⁸ CFU/mL, it was used as a seed solution for later use.

The composition of the seed medium is as follows: peptone 10g, beef extract 15g, yeast powder 5g, glucose 15g, diammonium citrate 2g, sodium acetate 5g, Tween 801mL, K ₂ HPO ₄ 2g, MgSO ₄ 7H ₂ O 0.5g, MnSO ₄ ·4H ₂ O 0.2g, add distilled water to 1000mL.

liquid submerged fermentation culture

A 5L vertical fermenter was selected for liquid fermentation culture. Among them, the inoculum amount of Lactobacillus reuteri seed solution is 3%, and the filling volume is 60%. After anaerobic culture at 37°C for 20 hours, measure the number of viable bacteria per milliliter of the bacterial solution until the number of viable bacteria reaches 10 ⁹ CFU /mL, put the tank to end the fermentation culture.

The composition of the fermentation medium is as follows: 50g of soybean peptone, 10g of glucose, 17g of yeast powder, 3g of oligosaccharides, distilled water to 1000mL, pH 6.0-6.5.

Preparation of freeze-dried bacterial powder

Through the production procedures such as centrifugation, concentration, addition of freeze-drying protective agent, vacuum freeze-drying, and inspection of the number of live bacteria in the bacteria powder, the powdery live bacteria preparation is finally prepared.

Wherein, the composition of lyoprotectant is as follows: 15g of sucrose, 3g of glycerin, 15g of sorbitol, 15g of skimmed milk powder, and distilled water is added to 250mL; 125 (g/mL), the freeze-drying protection rate reached more than 70%.

Finished product inspection

Take 1 g of the prepared powdery viable bacteria preparation, and measure the number of viable bacteria in each gram of bacteria powder according to the method of national standard GB/T 4789.35-2003, and the number of viable bacteria in the bacteria powder is greater than 10 ⁹ CFU/g.

Embodiment 2: the preparation of Bifidobacterium live bacteria preparation

Activation of strains

Freeze-dried strains of Bifidobacterium infantis (Bifidobacterium infantis, purchased from the General Microbiology Center of China Committee for Culture Collection of Microbial Cultures, No.: CGMCC No. 1.2202) were inoculated on the surface of MRS solid medium by streaking and cultured anaerobically at 37°C for 16 ~24h, until colonies grow on the surface of the solid medium, observe the morphology and purity of the bacteria by microscopic examination.

Preparation of seed solution

Pick the colonies on the surface of the MRS solid medium, inoculate them into a test tube containing 5 mL of seed medium, and incubate anaerobically at ³⁷ °C for 16-24 hours ^. CFU/mL) inoculum amount was inoculated into a 250mL Erlenmeyer flask containing 150mL seed medium, and anaerobically cultured at 37°C for 16h to 24h, until the number of viable bacteria reached 10 ⁸ CFU/mL, and it was used as a seed solution for later use.

The composition of the seed medium is as follows: peptone 10g, beef extract 15g, yeast powder 5g, glucose 15g, diammonium citrate 2g, sodium acetate 5g, Tween 801mL, K ₂ HPO ₄ 2g, MgSO ₄ 7H ₂ O 0.5g, MnSO ₄ ·4H ₂ O 0.2g, add distilled water to 1000mL.

liquid submerged fermentation culture

The bifidobacterium seed liquid is inoculated in a 5L fermenter with an inoculation amount of 3%, and a liquid filling amount of 60%. After anaerobic cultivation at 37°C for 24 hours, the number of viable bacteria in each milliliter of the bacterial liquid is measured. When the number of viable bacteria reaches 10 ⁹ CFU/mL, the fermentation is completed by putting in the tank.

The composition of the fermentation medium is as follows: 50 g of soybean peptone, 10 g of glucose, 17 g of yeast powder, 3 g of oligosaccharides, distilled water added to 1000 mL, pH 6.5.

Preparation of freeze-dried bacterial powder

Through the production procedures such as centrifugation, concentration, addition of freeze-drying protective agent, vacuum freeze-drying, and inspection of the number of live bacteria in the bacteria powder, the powdery live bacteria preparation is finally prepared.

Finished product inspection

Take 1 g of the prepared powdery live bacteria preparation, measure the number of viable bacteria per gram of the bacteria powder according to the method of national standard GB/T 4789.35-2003, and the number of viable bacteria in the bacteria powder is greater than 10 ⁹ CFU/g.

Embodiment 3: the preparation of bacillus natto live bacteria preparation

Activation of strains

The freeze-dried strains of Bacillus natto (purchased from the General Microbiology Center of China Microbiological Culture Collection Management Committee, number: CGMCC No. 1.1086) were streaked and inoculated on nutrient agar medium (produced by Beijing Aoboxing Biotechnology Co., Ltd., batch number For: 20060808) surface, aerobic culture at 37°C for 12 to 24 hours until colonies grow on the surface of the culture medium, and the morphology and purity of the bacteria were observed by microscopic examination.

Preparation of seed solution Pick colonies on the surface of nutrient agar medium, inoculate them into a test tube containing 5 mL of nutrient broth medium (produced by Beijing Land Bridge Technology Co., Ltd., batch number: CM106), and incubate at 37°C for 12-24 hours. When the number of bacteria reaches 10 ⁸ CFU/mL, inoculate 2% (about 1×10 ⁶ CFU/mL) inoculum into a 250 mL Erlenmeyer flask containing 150 mL of nutrient broth liquid medium, and incubate at 37°C for 12 to 24 hours. When the number of viable bacteria reaches more than 10 ⁸ CFU/mL, it is used as a seed solution for later use.

liquid submerged fermentation culture

The Bacillus natto seed solution was inoculated in a 5L fermenter equipped with a fermentation medium with a 3% inoculation amount, pH 6.0, a stirring speed of 180 rpm, a liquid filling volume of 20%, and aerobic cultivation at 37°C for 24 to 36 hours. Measure the number of viable bacteria per milliliter of bacterial liquid, wait until the number of viable bacteria reaches more than 10 ⁸ CFU/mL, and the spore formation rate reaches more than 80%, put the tank to finish the fermentation culture (the defoaming agent is vegetable oil, the addition amount is 1%, unit: g/ml).

The composition of the fermentation medium is as follows: peptone 10g, beef powder 3.0g, NaCl 5.0g, glucose 1.0g, distilled water to 1L, pH 6.0.

Preparation of freeze-dried bacterial powder

Through the production procedures such as centrifugation, concentration, addition of freeze-drying protective agent, vacuum freeze-drying, and inspection of the number of live bacteria in the bacteria powder, the powdery live bacteria preparation is finally prepared.

Finished product inspection

Take 1 g of the prepared powdery live bacteria preparation, and measure the number of viable bacteria per gram of the bacteria powder according to the national standard NY-T 1461-2007 method, and the measured number of viable bacteria in the bacteria powder is greater than 10 ⁹ CFU/g.

Embodiment 4: the preparation of composite living bacteria preparation

Compound the three prepared powdery live bacteria preparations according to the mass ratio of 1:1:1 to obtain 45g of the compound live bacteria preparation ^. CFU/g.

Effect: The composite microbial agent of the present invention is prepared by freeze-drying protection technology, which greatly guarantees the shelf life of the product with a high viable count, and the total viable count of the product within one year is not less than 10 ⁷ CFU/g.

Experimental example 1: Application experiment of Lactobacillus reuteri L-HD5 freeze-dried powder preparation

Randomly select 36 healthy weaned piglets at the age of 35 days, all of which are Landrace pigs; the 36 piglets were randomly divided into two groups, the first test group was the Lactobacillus reuteri L-HD5 freeze-dried powder preparation group, and the piglets were fed daily Grain (without antibiotics)+Lactobacillus reuteri L-HD5 freeze-dried powder preparation group (addition amount is 1% of the basal diet); the second test group is the control group, fed with the basal diet, and the test time is 30d. The immunization of piglets was carried out according to the routine immunization procedures of the pig farm. The final experimental data is as follows:

The average daily gain of each piglet in the test group 1 was 0.415Kg, and that of the control group was 0.360Kg, and the daily gain of the test group was 7.1% higher than that of the control group.

The average daily feed consumption per piglet in the test group 1 was 0.668Kg, and that in the control group was 0.700Kg. Compared with the control group, the daily feed consumption in the test group was reduced by 2.3%.

The diarrhea rate of the test group 1 was 12.5%, and that of the control group was 32.2%. Compared with the control group, the rate of diarrhea in the test group was reduced by 19.7%.

Animal feeding experiments showed that feeding pig-derived Lactobacillus reuteri L-HD5 freeze-dried powder preparation to weaned piglets can increase the daily weight gain of weaned piglets, reduce daily feed consumption and the incidence of diarrhea.

Experimental example 2

Randomly select 54 healthy weaned piglets at the age of 35 days, all of which are landrace pigs; the 54 piglets are randomly divided into three groups, the first test group is the Lactobacillus reuteri L-HD5 freeze-dried powder preparation group, and the piglets are fed every day. grain (without antibiotics)+Lactobacillus reuteri L-HD5 freeze-dried powder preparation group (the addition amount was 1% of the basic diet); the second experimental group was the compound live bacteria preparation group, and the piglets were fed with the diet (without Antibiotic)+compound live bacteria preparation (addition amount is 1% of basal ration) made in embodiment 4; Test group three is control group, feeds basal ration, and test time is 30d, and the immunity of piglet is according to pig farm Routine immunization procedures were performed. The final test data is as follows:

The average daily gain of each piglet in the test group 1 was 0.415Kg, the average daily gain of each piglet in the test group 2 was 0.563Kg, and that of the control group was 0.360Kg. Compared with the control group, the test group 1 increased the daily gain by 7.1%, and the test group 2 Compared with the control group, the daily weight gain was increased by 22%.

The average daily feed consumption per piglet in test group 1 was 0.668Kg, the average daily feed consumption per piglet in test group 2 was 0.690Kg, and that in the control group was 0.700Kg. Compared with the control group, the daily feed consumption in test group 1 was reduced by 2.3%. Compared with the control group, the experimental group 2 reduced the daily feed consumption by 0.7%.

The diarrhea rate of the first test group was 12.5%, that of the second test group was 9.6%, and that of the control group was 32.2%.

Application experiments show that feeding compound live bacteria preparations to weaned piglets can significantly increase the daily weight gain of weaned piglets, reduce daily feed consumption and the incidence of diarrhea.

The probiotic effect of compound live bacteria preparation is more significant than single Lactobacillus reuteri preparation. This may be due to the synergistic effect of the three probiotics in the compound live bacteria preparation.

First of all, Bacillus natto belongs to aerobic bacteria. After entering the intestinal tract, it consumes oxygen in the environment during the growth and reproduction process, which can inhibit the growth of aerobic pathogenic bacteria, but helps Lactobacillus reuteri and Bifidobacteria, etc. The reproduction of anaerobic bacteria; secondly, the growth of Lactobacillus reuteri and bifidobacteria increases the acidity in the intestinal tract, which further strengthens the inhibition of the growth of pathogenic bacteria. Therefore, the compound live bacteria preparation showed a more significant probiotic effect than the single bacteria preparation.

Claims (10)

1. Lactobacillus reuteri (Lactobacillus reuteri) L-HD5, its preservation number is CGMCC No.4050. 2. the bacterial agent that contains the bacterial strain described in claim 1. 3. bacterial agent as claimed in claim 2, it also comprises bifidobacteria, bacillus natto and lyoprotectant, wherein the component of said lyoprotectant is: sucrose 10g-15g, glycerol 3g-5g , sorbitol 10g-15g, skim milk powder 10g-15g, add distilled water to 250mL; add 125ml of lyoprotectant to each gram of the sludge composed of Lactobacillus reuteri L-HD5, Bifidobacterium and Bacillus natto. 4. bacterial agent as claimed in claim 3, is characterized in that, described microbial agent contains by weight: Lactobacillus reuteri L-HD5 1-10 servings Bifidobacterium 1-10 copies Bacillus natto 1-10 parts. 5. bacterial agent as claimed in claim 4, is characterized in that, described microbial agent contains by weight: Lactobacillus reuteri L-HD5 1 part Bifidobacterium 1 part Bacillus natto 1 part. 6. A feed additive containing the strain according to claim 1 or the microbial agent according to any one of claims 2-5. 7. The method for preparing the bacterial agent described in any one of claims 4 to 6, comprising the steps of: carrying out the following operations respectively with Lactobacillus reuteri L-HD5, Bifidobacterium and Bacillus natto: carrying out liquid deep layer Fermentation, centrifugation to collect the bacteria sludge, and then prepare and collect the bacteria powder by vacuum freeze-drying technology, and finally compound the three bacteria powders. 8. The method according to claim 7, characterized in that the fermentation and culture conditions of the Lactobacillus reuteri L-HD5 and Bifidobacteria are: temperature 37°C, pH 6.0-6.5, static culture, liquid filling The dosage is 60%, the inoculum size is 3%, the culture time is 16-24 hours, and the number of viable bacteria is greater than 10 ⁸ CFU/mL. 9. The method according to claim 7, wherein the fermentation and cultivation conditions of the Bacillus natto are: temperature 37°C, pH 6.0, stirring speed 180rpm, liquid loading 20%, inoculum 3%, The culture time is 24-36 hours, the number of viable bacteria is greater than 10 ⁸ CFU/mL, and the sporulation rate reaches over 50%. 10. The application of the bacterial strain according to claim 1 or the microbial agent according to any one of claims 2 to 5 in feed additives.