CN102304489B - Lactobacillus reuteri strain and application thereof - Google Patents

Abstract

The invention discloses a lactobacillus reuteri strain and application thereof. The lactobacillus reuteri strain is lactobacillus reuteri RI021 preserved in China General Microbiological Culture Collection Center on March 11, 2011, and the accession serial number in the collection center is CGMCC (China General Microbiological Culture Collection Center) No. 4650. The lactobacillus reuteri strain and a lactobacillus reuteri preparation thereof are mainly used as feed additives for animals and can be used for regulating the microeubiosis in animal intestines, thus achieving the effect of enhancing the non-specific immune function so as to prevent diseases, simultaneously providing nutritional factors, promoting the digestion and absorption of nutrients, promoting the growth of the animals and improving the feed conversion rate.

Description

One strain lactobacillus reuteri and application thereof

Technical field

The present invention relates to strain lactobacillus reuteri and an application thereof.

Background technology

When in feed, the interpolation microbiotic is made major contribution to intensive animal husbandry development, residual etc. in the generation of the side effect of its generation such as autogenous infection and superinfection, resistance, the destruction of normal intestinal flora and livestock product and environment brings safely serious threat to aquaculture, animal and human food prods, and the novel green safe additive of therefore researching and developing substitute antibiotics is the important directions of present feed advance.

Lactobacillus reuteri (Lactobacillus reuteri) is that in a kind of enteron aisle that inhabits humans and animals, the obligate heterolactic fermentation bacterium with enteron aisle epidermis adhesive capacity is also one of population important in milk-acid bacteria.U.S. food and drug control association (FDA) classified lactobacillus reuteri as safe microbial strains in 1989, and were widely used.China Ministry of Health ratified the probiotic bacterium kind that lactobacillus reuteri can be used for protective foods in 2003.Present domestic lactobacillus reuteri is mainly used in mankind's sour milk food, and report comparatively small amt, more rarely has report and be used for the animal field, major cause may be that this bacterium prepares from the isolation identification to the product and the application of animal in producing do not have systematic research, thereby has limited the application of this bacterial classification in the animal field.Therefore, according to the demand of present livestock industry, develop a kind of lactobacillus reuteri preparation, have vast potential for future development.

Summary of the invention

The purpose of this invention is to provide a strain lactobacillus reuteri (Lactobacillus reuteri) RI021 and application thereof.

Lactobacillus reuteri provided by the present invention (Lactobacillus reuteri) RI021 is numbered CGMCC No.4650 registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.This lactobacillus reuteri strong stress resistance, anti-miscellaneous bacteria ability be strong, have probiotic properties.

Lactobacillus reuteri (Lactobacillus reuteri) RI021 has following biological characteristics:

(1) survival rate after pH2.0 processes 4 hours is 72%;

(2) survival rate after heating 15min is 31.7% in 75 ℃ of water-baths;

The survival rate of (3) placing after 1 month under room temperature is 85.6%;

(4) be seeded in broth culture in 37 ℃, 5%CO ₂Incubator in to cultivate 22 hours growth concentrations the highest, near 10 ¹⁰Cfu/ml (Fig. 1).

Another object of the present invention is to provide a kind of lactobacillus reuteri preparation, and said preparation can substitute existing feeding antibiotic, and has disease-resistant, growth promotion, improves intestinal health, improves the effect of immunizing power.

Lactobacillus reuteri preparation provided by the present invention, its activeconstituents are described lactobacillus reuteri (Lactobacillus reuteri) RI021.

Described preparation has following at least a purposes:

1) product of microecological balance in animal intestine is regulated in preparation;

2) preparation strengthens the product of non-specific immunity;

3) improve the animal-feed transformation efficiency;

4) promote animal to digestion and the absorption of nutritive substance;

5) preparation reduces the product of animal diarrhea disease percentage;

6) preparation improves the product of breeding performonce fo animals.

Above-mentioned lactobacillus reuteri (Lactobacillus reuteri) RI021 or preparation can be used for preparing the intestinal bacteria inhibitor.

Intestinal bacteria in described intestinal bacteria inhibitor specifically can be intestinal bacteria K88, e. coli k99 or intestinal bacteria 987P.

Described raising breeding performonce fo animals can be the production performance that improves weanling pig, is embodied as to improve piglet average daily gain and/or the average daily ingestion amount of piglet.

Described reduction animal diarrhea disease percentage specifically can be presented as the coli-infection grice diarrhoea rate that reduces; Described reduction diarrhea rate specifically can be presented as and reduce coli-infection grice diarrhoea index.

Described preparation specifically can be comprised of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and carrier; Described carrier specifically can be at least a in powdered rice hulls, skim-milk, maltodextrin, wheat bran.

Described carrier can pass through micronization processes.Through after micronization processes, 90% diameter of particle is less than 100 μ m, and median size is less than 50 μ m, and described median size is 50% diameter of particle.

Described preparation specifically can be following A)-any in F):

A) the lactobacillus reuteri preparation A that is comprised of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and described carrier, in described lactobacillus reuteri preparation A, proportioning both is the 8 described carriers of gram: 10 ¹⁰The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1;

B) the lactobacillus reuteri preparation B that is comprised of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and described carrier, in described lactobacillus reuteri preparation B, proportioning both is the 0.5 described carrier of gram: 10 ⁷The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1;

C) the lactobacillus reuteri formulation C that is comprised of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and described carrier, in described lactobacillus reuteri formulation C, proportioning both is the 0.5 described carrier of gram: 10 ⁸The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 1: 1: 1;

D) the lactobacillus reuteri preparation D that is comprised of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and described carrier, in described lactobacillus reuteri preparation D, proportioning both is the 0.4 described carrier of gram: 10 ⁹The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 1: 1: 1: 1;

E) the lactobacillus reuteri preparation E that is comprised of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and described carrier, in described lactobacillus reuteri preparation E, proportioning both is the 0.4 described carrier of gram: 10 ⁷The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Described carrier is comprised of skim-milk and wheat bran, and the mass ratio of described skim-milk and wheat bran is 1: 1;

F) the lactobacillus reuteri preparation F that is comprised of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and described carrier, in described lactobacillus reuteri preparation F, proportioning both is the 0.4 described carrier of gram: 10 ¹⁰The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk and wheat bran is 1: 1: 1.

Lactobacillus reuteri of the present invention (Lactobacillus reuteri) RI021 or described preparation can be used as additive for the preparation of animal-feed, and animal wherein includes but not limited to the various animals such as pig, ox, sheep, chicken.This feed has and the similar function of antibiotic feed, but the side effect of antibiotic-free feed.

In described animal-feed, specifically can contain 10 in the described animal-feed of every gram ⁷Described lactobacillus reuteri (Lactobacillus reuteri) RI021 that CFU is above.

Lactobacillus reuteri of the present invention and lactobacillus reuteri preparation thereof are mainly as the additive of animal-feed, microbiotic in alternative existing animal diets, regulate microecological balance in animal intestine, thereby have the prophylactic effect of the non-specific immune function of enhancing, nutritional factor can also be provided simultaneously, promote the nutraceutical production performance of digesting and assimilating, reducing diarrhoea, promotion growth of animal and improve food conversion ratio, improve weanling pig and growing-finishing pig.

Lactobacillus reuteri of the present invention and lactobacillus reuteri preparation thereof play a role in health care to control animal digestive system disease, can stimulate its gastrointestinal development to growing animal simultaneously, so it is applied in the effect that to play disease-resistant growth-promoting in feed as fodder additives.Simultaneously, lactobacillus reuteri of the present invention and lactobacillus reuteri preparation thereof have no drug resistance residual in animal product with medicine, can not produce potential harm to the mankind's health, be a kind of promising green feed additives.

Describe the present invention in detail below in conjunction with specific embodiment, described embodiment is used for understanding rather than restriction the present invention.

The preservation explanation

Strain name: lactobacillus reuteri

Latin name: Lactobacillus reuteri

Strain number: RI021

Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center

Preservation mechanism is called for short: CGMCC

Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City

Preservation date: on March 11st, 2011

The preservation center numbering of registering on the books: CGMCC No.4650

Description of drawings

Fig. 1 is the growth curve of lactobacillus reuteri (Lactobacillus reuteri) RI021.

Fig. 2 is that the plate trench method is measured the Bacterium lacticum culture to colibacillary inhibition.

Embodiment

The experimental technique that uses in following embodiment is ordinary method if no special instructions.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.

Lactobacillus reuteri Lactobacillus reuteri RI021 of the present invention, the Hungates that can first be inoculated in Rogosa SL agar rolls in the pipe substratum and cultivates, and the inoculum that obtains is inoculated in Rogosa SL meat soup anaerobic culture medium and cultivates.

Any substratum that wherein said nutrient agar is cultivated for being fit to lactobacillus reuteri, which kind of substratum those skilled in the art know is fit to the lactobacillus reuteri cultivation.Specifically can select the Hungates of Rogosa SL agar to roll the pipe substratum, the Hungates of Rogosa SL agar described here rolls the pipe substratum and is interpreted as on the basis of the complete selective medium of Rogosa SL, the substratum that the suitable lactobacillus reuteri of suitably revising is cultivated.the substratum that described suitable lactobacillus reuteri is cultivated can be the substratum of any suitable lactobacillus reuteri cultivation of prior art, this dawn known to those skilled in the art, specifically can be: Tryptones 1-20g, extractum carnis 1-20g, yeast soaks powder 1-15g, glucose 1-20g, pectinose 1-15g, sucrose 1-15g, sodium-acetate 5-25g, Sodium Citrate 1-10g, potassium primary phosphate 1-15g, magnesium sulfate heptahydrate 0.1-1.5g, four water manganous sulfate 0.1-1.5g, ferrous sulfate 0.01-0.10g, tween-80 0.1-2.0mL, agar 5-20g, distilled water 100-2500mL, be preferably Tryptones 5-15g, extractum carnis 5-15g, yeast soak powder 3-10g, glucose 5-15g, pectinose 3-10g, sucrose 3-10g, sodium-acetate 10-20g, Sodium Citrate 1-5g, potassium primary phosphate 1-10g, magnesium sulfate heptahydrate 0.1-1g, four water manganous sulfate 0.1-1g, ferrous sulfate 0.01-0.05g, tween-80 0.5-1.5mL, agar 10-15g, distilled water 500-2000mL, Tryptones 10g more preferably, extractum carnis 10g, yeast soak powder 5g, glucose 10g, pectinose 5g, sucrose 5g, sodium-acetate 15g, Sodium Citrate 2g, potassium primary phosphate 6g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, ferrous sulfate 0.03g, tween-80 1mL, agar 13g, distilled water 1000mL.

any substratum that liquid nutrient medium is wherein cultivated for being fit to lactobacillus reuteri, those skilled in the art also know this liquid nutrient medium, preferred meat soup anaerobic culture medium wherein, meat soup anaerobic culture medium described here is interpreted as on the basis of ordinary broth anaerobic culture medium, the substratum of suitably revising, the substratum of described suitable modification can be the substratum of any suitable lactobacillus reuteri cultivation of prior art, this dawn known to those skilled in the art, preferred Rogosa SL meat soup anaerobic culture medium, wherein the formula of substratum is: Tryptones 5-15g, yeast soaks powder 3-10g, glucose 5-15g, pectinose 3-10g, sucrose 3-10g, sodium-acetate 10-20g, Sodium Citrate 1-5g, potassium primary phosphate 1-10g, magnesium sulfate heptahydrate 0.1-1g, four water manganous sulfate 0.1-1g, ferrous sulfate 0.01-0.05g, tween-80 0.5-1.5mL, distilled water 500-2000mL, be preferably Tryptones 10g, yeast soaks powder 5g, glucose 10g, pectinose 5g, sucrose 5g, sodium-acetate 15g, Sodium Citrate 2g, potassium primary phosphate 6g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, ferrous sulfate 0.03g, tween-80 1mL, distilled water 1000mL.

Use above-mentioned substratum, those skilled in the art generally know concrete culturing process, and the Hungates that is preferably above-mentioned Rogosa SL nutrient agar here rolls in pipe and cultivates, and preferably in incubator, under 37 ℃, cultivate 15-72 hour; Lactobacillus reuteri enlarged culturing was wherein cultivated 15-80 hour under 37 ℃ preferably in Rogosa SL meat soup anaerobic culture medium.The pH value of above-mentioned substratum can be 6-7.

Described lactobacillus reuteri CGMCC No.4650 strain liquid is the strain liquid through one time fermentation, Secondary Fermentation or three fermentation gained.In the substratum of wherein said one time fermentation, Secondary Fermentation or three fermentations, the carbon N/P ratio is 5～100: 3～12: 0.5～5; Preferred 100: 10: 3.

Described lactobacillus reuteri preparation media is the substratum of any suitable making lactobacillus reuteri preparation, and its medium component is preferably: molasses, sucrose, glucose, three's weight ratio are 20-60 part: 10-70 part: 10-40 part.

Described lactobacillus reuteri preparation media is the substratum of any suitable making lactobacillus reuteri preparation, and its medium component also comprises peptone on the basis of above-mentioned substratum, and the weight ratio of itself and molasses is 10-30 part: 10-60 part.

Described lactobacillus reuteri preparation media, substratum for any suitable making lactobacillus reuteri preparation, its medium component also comprises phosphoric acid and/or soluble phosphate on the basis of above-mentioned substratum, the weight ratio of itself and molasses is 10-30 part: 10-60 part.

Described aftertreatment be with lactobacillus reuteri bacterium liquid through once, secondary, three fermentations expand numerously, and zymocyte liquid is added in phosphate buffered saline buffer, spraying drying gets final product, the pH of described phosphate buffered saline buffer is 6.5～6.7.

Animal-feed of the present invention comprises: conventional animal feed and lactobacillus reuteri preparation of the present invention.Wherein the content of lactobacillus reuteri preparation is at least 0.001%, is preferably 0.001-3%, more preferably 0.01%-2%, most preferably 0.05%-0.2%.Lactobacillus reuteri preparation of the present invention can with at present on the market any conventional animal feed coordinate, namely animal-feed protection domain of the present invention for the cooperation conventional animal feed kind do not limited.

Yet conventional animal feed wherein can be preferably daily ration, more preferably pig with daily ration, chicken with daily ration, ruminating animal with daily ration and aquatic animal daily ration.For different animals, the formula of its daily ration is different, and still, in prior art, disclosed any daily ration can be as animal-feed of the present invention, and its formula is all the common practise in this field.

Culture medium prescription used in following embodiment is as follows:

1, Rogosa SL broth culture

Tryptones 10g, extractum carnis 10g, yeast soak powder 5g, glucose 10g, pectinose 5g, sucrose 5g, sodium-acetate 15g, Sodium Citrate 2g, potassium primary phosphate 6g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, ferrous sulfate 0.03g, tween-80 1mL is settled to 1L with distilled water.

2, Rogosa SL nutrient agar

Add agar 13g in 1L Rogosa SL broth culture.

3, MRS broth culture

Peptone 10g, extractum carnis 10g, yeast soak powder 5g, dipotassium hydrogen phosphate 2g, citric acid two ammonium 2g, glucose 20g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, sodium-acetate 5g is settled to 1L with distilled water.

4, MRS nutrient agar

Add agar 14g in 1L MRS broth culture.

5, glucose nutrient broth

Peptone 10g, extractum carnis 5g, sodium-chlor 5g, glucose 2.5g is settled to 1L with distilled water.

The isolation identification of embodiment 1, lactobacillus reuteri (Lactobacillus reuteri) RI021

Lactobacillus reuteri of the present invention extraction from pig is gastrointestinal tract mucous, separation, screening, purifying obtain.

(1) isolation and purification of lactobacillus reuteri

1.1 test materials

Get the gastrointestinal tract mucous 5cm of sodium selenite ²(Stomach duodenum, jejunum, ileum, caecum and colon), in Sterilization cup, with the chyme on stroke-physiological saline solution flushing mucous membrane, mucous membrane is immersed be equipped with in the beaker of 15mL HEPES damping fluid, after tweezers waggle mucous membrane 5min, obtain supernatant liquor.

1.2 the separation and Culture of bacterial strain

Inject with asepsis injector absorption 0.2mL supernatant liquor the Hungates that Rogosa SL nutrient agar is housed and roll pipe, make supernatant liquor be evenly distributed on agar surface in pipe with light rolling of have gentle hands.Put into 5%CO ₂Incubator, after 37 ℃ of cultivation 72h, in aseptic technique in Biohazard Safety Equipment, the bacterium colony of white needle point size is transferred to the syringe needle picking in the aseptic Rogosa broth culture of anaerobism and cultivated, observe meat soup and whether become muddy, have 4 ℃ of refrigerator storages of muddy placement standby.

1.3 gramstaining

Draw a small amount of Rogosa SL broth culture with asepsis injector, drop on slide glass, oven dry is fixing gently on spirit lamp flame.Drip violet staining liquid, dye 1min, washing; Drip the Gram's iodine solution mordant dyeing, effect 1min, washing; Drip acetone ethanol mixed solution (acetone: 95% ethanol=3: 7) decolouring 30s, washing; Drip husky yellow staining fluid and redye 1min, washing is waited to do, and observes on ordinary optical microscope, and thalline takes on a red color negative, purple positive.Be the consistent bacillus of Gram-positive form, further carry out catalase test.

1.4 catalase test

Do Rogosa SL slant medium, get culture approximately 0.2mL inject and Rogosa SL nutrient agar inclined-plane to be housed, 5%CO ₂Incubator is cultivated 24h for 37 ℃, after growing bacterium colony, 3% superoxol is added drop-wise on bacterium colony, and be negative if there is no the Bubble formation explanation, be positive if the Bubble formation explanation is arranged.Can tentatively think lactobacillus (Lactobacillus) by the culture that anaerobism on Rogosa SL is cultivated through Gram-positive and catalase test feminine gender.

2 results and discussion

This test utilization complete selective medium of Rogosa SL after persalt is proofreaied and correct from sodium selenite is gastrointestinal tract mucous filters out 6790 strain Bacterium lacticum.Shake 5min with piglet is gastrointestinal tract mucous in the HEPES damping fluid, draw the 0.2mL supernatant liquor with asepsis injector and pack into and contain rolling of Rogosa SL nutrient agar and cultivate 24h in pipe, then choose white colony and cultivate in the aseptic Rogosa SL broth culture of anaerobism.Through gramstaining and catalase test, prove that Bacterium lacticum is the GI dominant microflora of sodium selenite, illustrate that the Rogosa SL nutrient agar of proofreading and correct through hydrochloric acid is fit to separate gastrointestinal tract mucous Bacterium lacticum.

The screening of bacterial classification is and loaded down with trivial details process.The Breeding Process of most of Bacterium lacticum is to separate probiotic bacterium from the digestive tube epithelium, so just can obtain to have well surely to grow the bacterial classification of effect.

(2) the resistance seed selection of lactobacillus reuteri

1.1 acid resistance seed selection

Preparation pH value is respectively 2.0,2.5,3.0,3.5 Rogosa SL broth culture, and inoculates respectively stomach Bacterium lacticum and the intestines Bacterium lacticum of having cultivated 24h, and inoculum size 1% is observed after 37 ℃ of cultivation 24h.If the muddiness that substratum becomes illustrates that this bacterial strain is acidproof.Carry out gradient dilution to 10 from inoculation beginning and 8h with asepsis injector taking-up 1mL ^-5After, get the 0.3mL diluent in the upper evenly coating of MRS agar (pH5.2), plate is placed on 37 ℃, 5%CO ₂In incubator, cultivate 24h, calculate survival rate.Then microscopy is seen whether microbiological contamination.The initial separation sample retention of the lactobacterium strain that can grow is standby at-80 ℃ of refrigerators.

1.2 bile tolerance performance seed selection

Preparation contains the malt extract medium of pig cholate 0.3%, and the bacterium culture of 24h has been cultivated in inoculation, and inoculum size 1% is observed after 28 ℃ of cultivation 24h.If the muddiness that substratum becomes illustrates this bacterial strain bile tolerance.Then microscopy is seen whether microbiological contamination.The initial separation sample retention of the lactobacterium strain that can grow is standby at-80 ℃ of refrigerators.

1.3 anti-high-copper performance seed selection

Preparation contains the broth culture of 250mg/kg cupric sulfate pentahydrate, and the bacterium culture of 24h has been cultivated in inoculation, and inoculum size 1% is observed after 28 ℃ of cultivation 12-72h.If the muddiness that substratum becomes illustrates the anti-high-copper of this bacterial strain.Then microscopy checks whether microbiological contamination.The initial separation sample retention of the lactobacterium strain that can grow is standby at-80 ℃ of refrigerators.

1.4 anti-high zinc performance seed selection

Preparation contains the meat soup anaerobic culture medium of 1% zinc oxide, and the bacterium culture of 24h has been cultivated in inoculation, and inoculum size 1% is observed after 28 ℃ of cultivation 12-72h.If the muddiness that substratum becomes illustrates the anti-high zinc of this bacterial strain.Then microscopy is seen whether microbiological contamination.The initial separation sample retention of the lactobacterium strain that can grow is standby at-80 ℃ of refrigerators.

2. result and discussion

2.1 acid resistance seed selection

By the acid resistance seed selection, in 6790 strain Bacterium lacticum, have 4753 strain Bacterium lacticum can be in the Rogosa of pH2.0 SL broth culture growth and breeding, mortality is 30%.The GI most of Bacterium lacticum acid resistances of this explanation are better, have 70% Bacterium lacticum to breed in the gi tract of piglet.Bacterium lacticum is generally by surely growing at digestive tube epithelium generation probiotic properties.Therefore, if bacterial classification does not have tolerance to hydrochloric acid in gastric juice, be difficult to the production performance of animal is produced good effect.

2.2 bile tolerance performance seed selection

To carry out the seed selection of bile tolerance performance by 4753 strain Bacterium lacticum of acid resistance seed selection, have 856 strain Bacterium lacticum can be in the malt extract medium that contains cholate 0.3% growth and breeding, mortality is 82%.Studies show that, cholate is the more disadvantageous factor of ratio hydrochloric acid in gastric juice that the intestines Bacterium lacticum runs in animal gastrointestinal tract.Chou and Weimer research finds that taming Bacterium lacticum with the cholate selectivity is effective to the tolerance of cholate, the energy force rate parental generation Bacterium lacticum of the Bacterium lacticum bile tolerance that domestication obtains afterwards through the several generations cholate is strong, Bacterium lacticum easily produces tolerance to the cholate performance, and have certain heredity, the lactobacillus reuteri that selects bile tolerance has great importance aborning.

2.3 anti-high-copper performance seed selection

To carry out the seed selection of anti-high-copper performance by 856 strain Bacterium lacticum of bile tolerance performance seed selection, have 257 strain Bacterium lacticum can be in the Rogosa of 250mg/kg cupric sulfate pentahydrate SL broth culture growth and breeding, mortality is 70%.Prove that this Bacterium lacticum has stronger anti-high copper feature.Because the high-copper daily ration has somatotrophic effect, the lactobacillus reuteri that the daily ration of the commonplace use of China, so selection at present has anti-high-copper is extremely necessary.

2.5 anti-high zinc performance seed selection

To carry out the seed selection of anti-high zinc performance by 257 strain Bacterium lacticum of anti-high-copper performance seed selection, have 77 strain Bacterium lacticum can be in containing the meat soup anaerobic culture medium of 1% zinc oxide growth and breeding, mortality is 70%.High zinc has and prevents from suffering from diarrhoea, promotes the effect of growing, and therefore utilizes zinc oxide to prepare pig starter feed in China, and especially the producer of weanling pig feed is a lot.Therefore the lactobacillus reuteri that selects anti-high zinc is also necessary.

3. brief summary

Derive from piglet by this test acquisition 77 strains gastrointestinal tract mucous, environment in digestive tube is had the lactobacterium strain of certain resistivity, this lays the foundation for later seed selection work.

(3) the probiotic properties seed selection of lactobacillus reuteri

1.1 Lactobacillus metabolite suppresses the seed selection of intestinal bacteria ability

Intestinal bacteria (K88, K99 and 987P) are available from China Veterinary Drugs Supervisory Inst..K88 at the preserving number of Chinese medicine bacterium preservation administrative center is: CMCC44742, K99 at the preserving number of Chinese medicine bacterium preservation administrative center is: CMCC44820,987P at the preserving number of Chinese medicine bacterium preservation administrative center is: CMCC44317.

Bacterium lacticum: 77 strains are acidproof, the Bacterium lacticum of bile tolerance, anti-high-copper and Nai Gao zinc.

Substratum: Mai Kangkai (MacConkay) substratum (available from the Beijing Tiantan Biological Products Co.ltd).

Add approximately 15mL of MRS broth culture in the Hungates pipe, carbonating is made aseptic anaerobism MRS meat soup.Inoculate respectively in each pipe that 77 strains that are separated in the sodium selenite stomach are acidproof, the Bacterium lacticum of bile tolerance, anti-high-copper and Nai Gao zinc, Bacterium lacticum is inoculated in meat soup by 1% after 18h cultivates, and after cultivating 24h in 37 ℃ of incubators, puts 4 ℃ of Refrigerator stores standby.

Suppress coli test: make the Mai Kangkai substratum, after 121 ℃ of autoclaving 15min, asepticly topple over plate after heating for dissolving.In plate bottom line, plate is divided into three parallel zones with marking pen, each zone loop-carrier streak inoculation intestinal bacteria nutrient solution (4.0 * 10 ⁸CFU/mL) in media surface, then approximately dig one and widely be the ditch of 0.5cm in the place of 3cm at anomaly ware center line, ditch is vertical with the inoculation line, with the hot loop-carrier benefit end, as shown in Figure 2.Ditch is filled up (can not overflow) with different Bacterium lacticum cultures respectively, after putting 37 ℃ of cultivation 18h of common incubator, the position of intestinal bacteria bacterium colony appears in observation, and colibacillary bacterium colony is red, measures from the nearest intestinal bacteria bacterium colony of ditch and the distance of ditch.Each Bacterium lacticum is made two Duplicate Samples, represents result with mean value.

1.2 Bacterium lacticum and the seed selection of intestinal bacteria mixed culture restraining effect

Intestinal bacteria (K88, K99 and 987P) source is same with 1.1 joints.

Bacterium lacticum: 65 strain intestines meta-bolitess have stronger inhibiting Bacterium lacticum to intestinal bacteria.

Colibacillary detection substratum: Mai Kangkai (MacConkay) substratum and 1.1 together.

Bacterium lacticum is inoculated in the aseptic MRS meat soup of anaerobism, after 37 ℃ of cultivation 24h, puts 4 ℃ of Refrigerator stores standby.

1.2.1 the different concns Bacterium lacticum is to colibacillary inhibition

The random strain Bacterium lacticum of selecting is got 5mL culture (8.2 * 10 ⁹CFU/mL) respectively with 5,10, the 15mL nutrient broth mixes, and makes 3 nutrient broths that contain different Bacterium lacticum culture concentration, the inoculation intestinal bacteria, inoculum size is 5% of nutrient solution, puts 37 ℃, 5%CO ₂After incubator cultivation 4,8 and 18h, take out nutrient solution with asepsis injector respectively, nutrient solution becomes 10 through gradient dilution ^-2～10 ^-5After, each extent of dilution is made 4 Duplicate Samples, get the 0.3mL diluent, evenly coat on the maconkey agar flat board with " L " rod, be placed in 37 ℃, common incubator is cultivated 18h, gets colony number the plate count of 50～150, represent result with mean value, calculate colibacillary quantity in every milliliter of nutrient solution.

1.2.2 contain Bacterium lacticum and colibacillary quantitative relation in the nutrient broth of 1/4 Bacterium lacticum

Get 5mL Bacterium lacticum culture (10 ⁸CFU/mL) mix with the 15mL nutrient broth respectively, inoculation 24h culture of Escherichia coli, inoculum size is respectively 10% (10 of nutrient solution ⁷CFU/mL), in initial mixed solution, the quantity of Bacterium lacticum is colibacillary 10 times, and the pH that regulates nutrient solution with 0.1M NaOH solution is 7.0, after putting 37 ℃ of incubators cultivation 24h, takes out nutrient solution with asepsis injector respectively, and nutrient solution is through gradient dilution to 10 ^-5, 10 ^-6, 10 ^-7After, to get the 0.3mL diluent and evenly coat on the MRS nutrient agar with " L " rod, the nutrient solution of each gradient is made 3 Duplicate Samples, and MRS nutrient agar (pH5.4) is put 37 ℃, 5%CO ₂Incubator is cultivated 18h, gets colony number the plate count of 50～150, represents the quantity of Bacterium lacticum with mean value, calculates the quantity of Bacterium lacticum in every milliliter of nutrient solution.Directly get the 1mL nutrient solution through gradient dilution to 10 with asepsis injector after cultivating 24h ^-2After, get the 0.3mL diluent and be placed on maconkey agar, evenly be coated with " L " rod, each pipe is done 3 Duplicate Samples, and flat board is placed in 37 ℃, and common incubator is cultivated 18h, calculates each dull and stereotyped intestinal bacteria colony number, represents result with mean value.

1.3 the seed selection of lactic acid production

Bacterium lacticum: the 63 strong Bacterium lacticum of strain Chinese People's Anti-Japanese Military and Political College's enterobacteria ability.

The MRS broth culture is regulated pH to 5.4 with Glacial acetic acid, puts into the Hungates pipe after mixing, and every pipe dress 10mL meat soup fills 1.5%CO while hot ₂1min, sealing, autoclaving is made the aseptic meat soup of anaerobism, and 63 strain Bacterium lacticum then are inoculated in MRS meat soup by 1% and cultivate 20h respectively after the aseptic meat soup of this anaerobism is cultivated 24h.Get the 2mL nutrient solution, the centrifugal 15min of 3500rpm gets supernatant liquor, after 10 times of ultrapure water dilutions, with the lactic acid concn in D-ALPHA-Hydroxypropionic acid detection kit (numbering K-DATE, the graceful bio tech ltd of upper Hypon) and Technicon RA 100 biochemical instruments enzymatic assays supernatant liquors.Each bacterial strain is surveyed two Duplicate Samples, represents result with mean value.

2 results

2.1 Lactobacillus metabolite suppresses colibacillary seed selection

Lactobacillus metabolite suppresses colibacillary effect take from the distance of the nearest intestinal bacteria bacterium colony of ditch and ditch as index, apart from larger, illustrates that fungistatic effect is better.The plate trench method that this test is adopted is the improvement to the plate borehole method of classics, ultimate principle is to be placed on antibiont mass-energy in ditch to penetrate agar and suppress intestinal bacteria, along with the increase from the ditch distance, the concentration that can permeate antimicrobial substance in the past is just less.The distance of intestinal bacteria bacterium colony and ditch is larger, illustrates that the bacteriostasis of the contained antimicrobial substance of Lactobacillus metabolite is stronger.Can measure simultaneously Lactobacillus metabolite to the 3 colibacillary restraining effect of strain with the plate trench method at same plate, and can only measure a colibacillary restraining effect of strain at same plate with plate borehole method.Have 65 strain Bacterium lacticum to the antibacterial distance of intestinal bacteria K88 and K99 between 1.0～2.7cm, further study the quantitative relation of they and intestinal bacteria mixed culture.Wherein, Bacterium lacticum RI021 is respectively 2.1cm, 2.2cm, 1.9cm to the antibacterial distance of intestinal bacteria K88,987P and K99.

2.2 the quantitative relation of Bacterium lacticum and intestinal bacteria vitro culture

2.2.1 different ratios Bacterium lacticum RI021 nutrient solution and the quantitative relation of intestinal bacteria under the mixed culture condition.

The results are shown in Table 2.

Contain different ratios Bacterium lacticum RI021 nutrient solution in table 2 nutrient broth to colibacillary restraining effect (unit: CFU/mL)

2.2.2 Bacterium lacticum and the intestinal bacteria quantitative relation under the mixed culture condition

Quantitative relation (the unit: CFU/mL) of table 3 Bacterium lacticum and intestinal bacteria mixed culture

As can be seen from Table 2, the Bacterium lacticum of different concns is similar to colibacillary restraining effect during to the intestinal bacteria mixed culture, illustrates that the nutrient broth that contains 25% Bacterium lacticum RI021 is enough to do to suppress colibacillary test.Bacterium lacticum RI021 and intestinal bacteria see Table 3 with the quantitative relation of intestinal bacteria mixed culture in the nutrient broth that contains 25% Bacterium lacticum RI021 culture.As seen from the table, RI021 has relatively strong bacteriostasis to three kinds of serotype intestinal bacteria.Quantitative relation when external test tube is cultivated between different microorganisms can be used as reflection microorganism interactional a kind of index in vivo.

2.3 the acid producing ability of Bacterium lacticum

After measured, the acid producing ability of Bacterium lacticum RI021 is 0.7231g/L, and compares with other bacterial strain of surveying, has higher acid producing ability.

3 brief summaries

According to bacteriostatic test, strain separating position and acid producing ability, filter out Bacterium lacticum RI021 as the production bacterial classification of probiotic bacterium.This strain bacterium is accredited as lactobacillus reuteri (Lactobacillus acidophilus) through China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), register on the books and be numbered CGMCC No.4650 in the preservation center.The RI021 cell is shaft-like, Gram-positive, and other biological characteristics is as shown in table 4.

The biological characteristics of table 4 RI021

This lactobacillus reuteri (Lactobacillus acidophilus) is a kind of of lactobacillus (Lactobacillus).

Lactobacillus reuteri described in following embodiment all refers to lactobacillus reuteri (Lactobacillus acidophilus) RI021 CGMCC No.4650 if no special instructions.

The biological characteristic research of embodiment 2, lactobacillus reuteri (Lactobacillus acidophilus) RI021 CGMCC No.4650

1.1 growth curve

Dress 300mL MRS broth culture in the 500mL Erlenmeyer flask.By 1% inoculum size inoculation lactobacillus reuteri culture, at front 24h every 1h, after 24h the 28th, 32,36,40,44, the 48h sampling, after getting the 1mL nutrient solution and carrying out gradient dilution, 10 ^-2～10 ^-6Extent of dilution is got the 0.3mL diluent in the upper evenly coating of MRS nutrient agar (pH5.2), and each gradient is done 3 Duplicate Samples, and plate is placed on 37 ℃, 5%CO ₂Incubator in, cultivate 24h, the extent of dilution of the colony number 50～150 in the ware of making even is as calculating use, each gradient is done 3 Duplicate Samples, represents result with mean value.Every milliliter of bacterial concentration represents with logarithmic value.

1.2 acidproof survival rate

The MRS broth culture transfers the pH joint to 5.4 with Glacial acetic acid, then with concentrated hydrochloric acid with pH regulator to 2.0, be placed in Hungates and roll pipe, each pipe dress 20mL meat soup is made the aseptic meat soup of anaerobism.Cooling down adds the culture of 1mL lactobacillus reuteri 16h in rear every pipe, when beginning, 2h, 4h, 6h and 8h take a sample respectively, measures survival rate.After carrying out gradient dilution with asepsis injector taking-up 1mL, 10 ^-4～10 ^-7Extent of dilution is got the 0.3mL diluent in the upper evenly coating of MRS nutrient agar (pH5.2), and each gradient is done 3 Duplicate Samples, and plate is placed on 37 ℃, 5%CO ₂Incubator in, cultivate 24h, the plate count of the colony number 50～150 in the ware of making even represents result with mean value.

The calculation formula of survival rate is:

S _Acid=n _x/ n ₀

S _AcidLactobacillus reuteri survival rate for different time after processing through pH2.0; n ₀For pH2.0 processes front every milliliter of viable count; n _xBe every milliliter of viable count after pH2.0 processing 2,4,6,8h.

1.3 heat-resisting survival rate

The MRS broth culture, regulating pH is 6.7, is placed in Hungates and rolls pipe, each pipe dress 20mL meat soup is made the aseptic meat soup of anaerobism.Add the culture after the 1mL lactobacillus reuteri is cultivated 16h in every pipe, be placed in 37 ℃ of incubators after 16h, sampling is carried out live bacterial count and is cultivated, and the Hungates pipe is placed on rapidly in 75 ℃ of water-baths heats 15min simultaneously, and live bacterial count is carried out in sampling.After carrying out gradient dilution with asepsis injector taking-up 1mL, 10 ^-4～10 ^-7Extent of dilution is got the 0.3mL diluent in the upper evenly coating of MRS nutrient agar (pH5.2), and each gradient is done 3 Duplicate Samples, and plate is placed on 37 ℃, 5%CO ₂In incubator, cultivate 24h, the colony number in the ware of making even is 50～150 plate count, represents result with mean value.

The calculation formula of survival rate is:

S _Heat=n ₁/ n ₀

S _HeatFor through the lactobacillus reuteri survival rate after 75 ℃ of processing; n ₀Be every milliliter of viable count before heat treated; n ₁Be every milliliter of viable count after heat treated 15min.

1.4 storage tolerance survival rate

The MRS broth culture, regulating pH is 6.7, is placed in Hungates and rolls pipe, every pipe dress 20mL MRS broth culture after making the aseptic meat soup of anaerobism, adds the culture that the 1mL lactobacillus reuteri is cultivated 16h in every pipe, be placed in 37 ℃ of incubators after 16h, sampling 1mL carries out live bacterial count.Place under room temperature after 1 month, sampling 1mL carries out live bacterial count and cultivates.After method of counting carries out gradient dilution with asepsis injector taking-up 1mL, 10 ^-4～10 ^-7Extent of dilution is got the 0.3mL diluent in the upper evenly coating of MRS nutrient agar (pH5.2), and each gradient is done 3 Duplicate Samples, and plate is placed on 37 ℃, 5%CO ₂Incubator in, cultivate 24h, get colony number and be the plate count of 50～150, represent result with mean value.

The calculation formula of survival rate is:

S _Storage=n ₁/ n ₀

S _StorageBe the lactobacillus reuteri survival rate after preserving through 1 month; n ₀For preserving front every milliliter of viable count; n ₁For preserving every milliliter of viable count after 1 month.

2 results and discussion

2.1 the growth curve of lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650

Growth curve mainly reflects a kind of microbial growth characteristic, and microbial growth generally experiences lag period, logarithmic phase, stationary phase and decline phase four-stage, and this is a kind of typical growth curves model.Be that microorganism is to the adaptive process of new growing environment lag period, in this course, it is constant or descend that microorganism shows as quantity, and himself macromole and micromolecular composition are adjusted, and also can produce specific material such as enzyme etc. simultaneously and adapt to new environment.Logarithmic phase be microorganism to after new environmental adaptation, growth and breeding speed is the stage of geometricprogression, is a fastest stage of quantity growth, shows as the increase of thalline quantity and weight.But arrived the latter stage of logarithmic phase, to the consumption of nutritive substance and the accumulation of toxic products, the growth and breeding speed of bacterium descends due to the thalli growth metabolism.Be the stage that rate of bacterial growth and rate of death tend to balance stationary phase.The decline phase bacterial number obviously descends.Measure growth curve and have vital role for definite suitable fermentation time.

The growth curve of lactobacillus reuteri as shown in Figure 1, as seen from Figure 1, the concentration of lactobacillus reuteri the time has a declining tendency in beginning, through after 2h from 10 ³The individual order of magnitude rises rapidly, to 22h near 10 ¹⁰The individual order of magnitude, to 40h, the order of magnitude of bacterium begins slow decreasing, and major cause is that substratum nutritive substance density loss and harmful meta-bolites increase cause the dead increase of thalline self-dissolving, the thalline quantity of growth and breeding is more more than dead thalline, causes total number of viable to reduce.Can find out from growth curve, be 20～28h after cultivation the best harvesting time of lactobacillus reuteri.

2.2 the acidproof survival rate of lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650

Lactobacillus reuteri sees Table 5 through the cell concentration that pH2.0 processes different time.

Table 5 pH2.0 processes different time to lactobacillus reuteri viable bacteria concentration as influencing factor (unit: CFU/mL)

As seen from Table 5,2h before pH2.0 processes, the quantity of lactobacillus reuteri almost remains on 100% survival rate, drops to 72%, the 6h to the 4h survival rate and drops to 34.4%.This lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650 is that a strain separates the bacterial classification from duodenal mucosa, this survival rate should be comparatively desirable for its anti-restraining effect or killing action of crossing hydrochloric acid in gastric juice, shows that also this bacterial strain can have the viable count of sufficient amount to arrive duodenum.

2.3 the heat-resisting survival rate of lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650

The survival rate of lactobacillus reuteri RI021 after 75 ℃ are processed 15min reaches 31.7%.The pelleting temperature of general pig starter feed is between 70 ℃～85 ℃, after high temperature resistant survival rate low be also Bacterium lacticum as one of major limitation sexual factor of fodder additives, according to the research of Fuller (1989), Bacterium lacticum there is no viable bacteria after processing 10min through 75 ℃.From heat-resisting survival rate, the lactobacillus reuteri of this test seed selection can tolerate the high temperature when granulating, and will have application prospect preferably as fodder additives.

2.4 the storage tolerance survival rate of lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650

After the storage of month, the survival rate of lactobacillus reuteri is 85.6%.

The preparation of embodiment 3, lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650 preparation

1, lactobacillus reuteri is inoculated in the glucose nutrient broth, and with vinegar acid for adjusting pH to 6.7, puts 37 ℃ of degree of common incubator and cultivate after 18-24 hour, use the sterilized water gradient dilution, obtaining concentration is (10 ⁷-10 ¹⁰) strain liquid of cfu/g.

By weight, to contain 0.5 part of mixing of 1 part of 1 part of 1 part of 1 part of strain liquid, powdered rice hulls, skim-milk, maltodextrin, wheat bran of lactobacillus reuteri CGMCC No.4650, drying is pulverized, the lactobacillus reuteri preparation A that the lactobacillus reuteri preparation that namely obtains being comprised of lactobacillus reuteri and carrier forms.In this lactobacillus reuteri preparation A, proportioning both is 8 gram carriers: 10 ¹⁰The cfu lactobacillus reuteri, described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1.

2, lactobacillus reuteri is inoculated in Rogosa SL broth culture, with vinegar acid for adjusting pH to 6.7, puts 37 ℃ of common incubators and cultivate after 18-24 hour, use the sterilized water gradient dilution, obtaining concentration is (10 ⁷-10 ¹⁰) strain liquid of cfu/g.

By weight, to contain 1 part of 1 part of 1 part of 10 parts of strain liquids, powdered rice hulls, skim-milk, maltodextrin and 0.5 part of mixing of wheat bran of lactobacillus reuteri CGMCC No.4650, spraying drying, powdered rice hulls and wheat bran carried out micronization processes in advance, and (90% diameter of particle is less than 100 μ m, median size is less than 50 μ m, described median size is 50% diameter of particle), the lactobacillus reuteri preparation B that the lactobacillus reuteri preparation that namely obtains being comprised of lactobacillus reuteri and carrier forms.In this lactobacillus reuteri preparation B, proportioning both is 0.5 gram carrier: 10 ⁷The cfu lactobacillus reuteri; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1.

3, the lactobacillus reuteri CGMCC No.4650 that cultivation in step 1 is obtained is placed in fermentor tank and carries out one grade fermemtation, cultivates after 18-24 hour for 37 ℃, uses the sterilized water gradient dilution, and obtaining concentration is (10 ⁷-10 ¹⁰) strain liquid of cfu/g.The fermentation tank culture medium component is skim-milk, peptone, sucrose, lactose, glycerine, starch, and six weight ratio is 10 parts: 15 parts: 2 parts: 3 parts: 1 part: 2 parts.Separately add suitable quantity of water, and with vinegar acid for adjusting pH to 6.7.In substratum, the carbon N/P ratio is 100: 5: 0.5.

By weight, 10 parts of strain liquids, 2 parts of the powdered rice hulls that will contain lactobacillus reuteri CGMCC No.4650,1 part of skim-milk, 1 part of maltodextrin, 1 part of mixing of wheat bran, spraying drying, powdered rice hulls and wheat bran pass through micronization processes in advance, and (90% diameter of particle is less than 100 μ m, median size is less than 50 μ m, described median size is 50% diameter of particle), the lactobacillus reuteri formulation C that the lactobacillus reuteri preparation that namely obtains being comprised of lactobacillus reuteri and carrier forms, proportioning both are the 0.5 described carrier of gram: 10 ⁸The cfu lactobacillus reuteri; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 1: 1: 1.

4, the lactobacillus reuteri CGMCC No.4650 that cultivation in step 1 is obtained is placed in seeding tank and carries out one grade fermemtation, then is placed in the production tank and carries out second order fermentation, uses the sterilized water gradient dilution, and obtaining concentration is (10 ⁷-10 ¹⁰) strain liquid of cfu/g.Two-stage fermentation tank nutrient media components is 12 parts of powdered rice hulls, 10 parts of peptones, 10 parts of skim-milks, 1 part of sucrose, 2 parts of lactose, 1 part of glycerine, 3 parts of starch, mixes, and separately adds suitable quantity of water.And with vinegar acid for adjusting pH to 6.7.In substratum, the carbon N/P ratio is 100: 10: 3.

By weight, to contain 1 part of mixing of 1 part of 1 part of 1 part of 10 parts of strain liquids, powdered rice hulls, skim-milk, maltodextrin, wheat bran of lactobacillus reuteri CGMCC No.4650, spraying drying, powdered rice hulls and wheat bran pass through micronization processes in advance, and (90% diameter of particle is less than 100 μ m, median size is less than 50 μ m, described median size is 50% diameter of particle), namely obtain the lactobacillus reuteri preparation D that formed by lactobacillus reuteri and carrier, proportioning both is the 0.4 described carrier of gram: 10 ⁹The cfu lactobacillus reuteri; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 1: 1: 1: 1.

5, the lactobacillus reuteri CGMCC No.4650 that cultivation in step 2 is obtained is placed in seeding tank and carries out double fermentation, then is placed in the production tank and carries out three grade fermemtation, uses the sterilized water gradient dilution, and obtaining concentration is (10 ⁷-101 ⁰) strain liquid of cfu/g.The fermentation tank culture medium component is 12 parts of powdered rice hulls, 6 parts of peptones, 10 parts of skim-milks, 1 part of sucrose, 1 part, molasses, and 2 parts of lactose, 1 part of glycerine, 3 parts of starch, the carbon N/P ratio is 100: 7: 2.Add entry, and with vinegar acid for adjusting pH to 6.7.

By weight, to contain 1 part of mixing of 1 part, 6 parts of strain liquids, wheat bran, skim-milk of lactobacillus reuteri CGMCC No.4650, spraying drying, wheat bran passes through micronization processes in advance, and (90% diameter of particle is less than 100 μ m, median size is less than 50 μ m, described median size is 50% diameter of particle), namely obtain the lactobacillus reuteri preparation E that formed by lactobacillus reuteri and carrier, proportioning both is the 0.4 described carrier of gram: 10 ⁷Lactobacillus reuteri; Described carrier is comprised of skim-milk and wheat bran, and the mass ratio of described skim-milk and wheat bran is 1: 1.

6, contain lactobacillus reuteri CGMCC No.4650 strain liquid preparation method with step 4.

By weight, to contain 1 part of mixing of 1 part, 2 parts of 10 parts of strain liquids, powdered rice hulls, wheat bran, skim-milk of lactobacillus reuteri CGMCC No.4650, spraying drying, powdered rice hulls and wheat bran pass through micronization processes in advance, and (90% diameter of particle is less than 100 μ m, median size is less than 50 μ m, described median size is 50% diameter of particle), namely obtain the lactobacillus reuteri preparation F that formed by lactobacillus reuteri and carrier, proportioning both is the 0.4 described carrier of gram: 10 ¹⁰The cfu lactobacillus reuteri; Described carrier is comprised of powdered rice hulls, skim-milk and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk and wheat bran is 1: 1: 1.

The impact on weaned piglets of experimental example 4, lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650 preparation

1.1 materials and methods:

Experimental animal

Choose 48 of the DLY ternary hybrid piglets of 28 ± 2d wean, mean body weight 7.56 ± 0.52kg, distinguishing at random component by body weight is 2 treatment group, every group of 6 repetitions, each repeats 4 piglets.

1.2 lactobacillus reuteri preparation

Adopt the lactobacillus reuteri preparation A-F of embodiment 3.

1.3 test grouping

To be divided into be two processing in test, and processing 1 be test group, and basal diet interpolation weight ratio is any in lactobacillus reuteri preparation A-F in 0.1% embodiment 3; Processing 2 is control group, and each arrangement of processing of basal diet sees Table 6.Composition and the nutritive ingredient of basal diet see Table 6.

The basal diet of table 6 growth test forms and trophic level

Annotate: ¹. crude protein, Methionin, methionine(Met), Gelucystine, Threonine, calcium and phosphorus are measured value.

². per kilogram Preblend provides: vitamin A, 11,000IU; Vitamin D3 500,000 I.U/GM, 1503IU; Vitamin-E, 44.1IU; Vitamin K, 4.0mg; Riboflavin, 5.22mg; Pantothenic acid, 20.0mg; Nicotinic acid, 26.0mg; Vitamin B12,0.01mg; Manganese, 35.0mg; Iron, 100.0mg; Zinc, 90.0mg; Copper, 16.5mg; Iodine, 0.30mg; Selenium, 0.30mg.

1.4 feeding and management

Test in national feed Engineering Technical Research Centre respiratory chamber and carry out.21 days trial periods.The duration of test feeding piglet is in fully closed child care piglet house, and the temperature in house remains on 24～27 ℃.Free choice feeding, each hurdle circle are equipped with Duckbill-type drinking trough and freely drink water for piglet.1% Preblend autogamy of basal diet does not contain any microbiotic.The immunity of piglet is undertaken by the immune programme for children of pig routine veterinary transmissible disease, feeding piglet control measures strict implement health and epidemic prevention system.

1.5 sample collection and processing

Test claims the piglet individual weight when beginning, 7d, 14d and 21d, record day weight gain and feed food consumption weekly.

1.6 data statistics

Test the independent sample t check processing statistics of all the data SPSS12.0 (SPSS Inc., USA) statistical software.

2. result and discussion

2.1 the lactobacillus reuteri preparation on weaned piglet after the impact of growth performance

The impact of table 7 lactobacillus reuteri formulation C on Growth Performance of Weaning Piglets

Annotate: the table intermediate value is mean+SD.

As seen from Table 7,1wk, 2wks and 3wks after wean, the piglet average daily gain (ADG) of test group be you can well imagine high by 38.5% than the contrast component, 44.1% and 27.4%, significant difference (P＜0.05), average daily ingestion amount (ADFI) you can well imagine high 21.3%, 41.8% and 18.6%, significant difference (P＜0.05) than the contrast component).

Carry out same experiment with lactobacillus reuteri preparation A, B, D, E and F and also obtained identical conclusion.

The impact on the coli-infection grice diarrhoea of experimental example 5, lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650

1. materials and methods

The lactobacillus reuteri preparation E of embodiment 3.

1.1 experimental animal and feeding and management

Select the healthy DLY ternary hybrid piglet of 24 28 ± 2d wean, body weight is 7.66 ± 1.00kg.Be divided at random 2 groups, single cage is raised in metabolic cage, and the room temperature of respiratory chamber is controlled at 25 ℃～27 ℃, and 12 oral liquid lactobacillus reuteri preparations of piglet drinking-water every day for the treatment of group are drunk into the sum of lactobacillus reuteri CGMCC No.4650 be about 10 every day ⁷CFU, 12 piglet drinking public water supplies of control group in contrast.(total count is 10 with the mixed-culture medium of 20mL e. coli k99, K88 and 987P (source with embodiment 1) to test 8d ⁸CFU) oral challenge, in this mixed solution, the colibacillary of three kinds of serotypes is in equal proportions, a twice-daily, 1d, attack malicious rear piglet and still freely drink tap water and search for food altogether, measures and attack rear disease time and the Scours index of suffering from diarrhoea of poison, trial period 14d.

1.2 test daily ration

The test piglet basal diet of searching for food does not contain any microbiotic, prepares with reference to the requirement that NRC (1998) recommends.The table 6 of embodiment 4 is seen in the basal diet configuration.

1.3 Scours index

Scours index is that the diarrhoea head multiply by corresponding diarrhoea score, and the diarrhoea score is calculated according to the diarrhoea standards of grading of table 7.Diarrhea rate is that the inferior piglet number divided by every day of the total head of piglet of attacking diarrhoea in the rear 7d of poison multiply by 7.

Table 7 diarrhoea standards of grading

1.4 statistical study

Statistical study is carried out in the independent sample t of statistical study employing SPSS12.0 (SPSSInc., the USA) software of data-check.

2 results and discussion

Test-results sees Table 8, and the test group diarrhea disease percentage descends 64.8% than control group, and Scours index has descended 63.Control group 2d after attacking poison namely has first piglet diarrhea, and experimental group is just found first grice diarrhoea until attack the rear 4d of poison.In China, many experimental studies have proved the diarrhoea that probiotic bacterium can effectively prevent and treat sucking piglets and weanling pig, and some effect is better than microbiotic or recombinant vaccine.The lactobacillus reuteri of the lactic acid producing rhzomorph that contains in the lactobacillus reuteri preparation has a good Chinese People's Anti-Japanese Military and Political College enterobacteria characteristic external, according to having 26% because enterotoxigenic Escherichia coli causes in Hoefling report newborn piglet diarrhoea.The test group piglet is 7d before attacking poison, the oral water that contains lactobacillus reuteri always, and lactobacillus reuteri exists in a large number at gi tract chyme and mucous membrane, and brings into play prebiotic effect in specific gi tract microhabitats.When piglet is subject to challenge with E.coli; because lactobacillus reuteri can suppress colibacillary growth and breeding; thereby effectively protected body to avoid the grievous injury that the intestinal bacteria amount reproduction causes, reduced the severity of diarrhea disease percentage and diarrhoea, postponed simultaneously the diarrhoea disease time.

The impact of table 8 lactobacillus reuteri preparation E on the coli-infection diarrhea of weaned piglets

Carry out same experiment with other five kinds of lactobacillus reuteri preparations of embodiment 3 and also obtained identical conclusion.

The impact on early-weaned piglets gi tract microorganism species, nutrient digestibility of experimental example 6, lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650 preparation

1 materials and methods

1.1 experimental animal and daily ration are processed

Select 48 28 ± 2d weanling pigs, body weight 7.67 ± 1.09kg, by complete randomized block design animal experiment, test minute 2 processing, each processes 6 repetitions, each repeats 4 piglets, high-rise online flat supporting, the hurdle circle is of a size of 2.0 * 2.0m, and every circle is a repetition, each processes 3 circle boars, 3 circle sows.Processing 1 is test group, and basal diet adds the lactobacillus reuteri preparation F of 0.1% (mass content) embodiment 3; Processing 2 is control group, and basal diet adds the 50mg/kg carbadox, and each arrangement of processing sees Table 9.The duration of test feeding piglet is in fully closed child care piglet house, and the temperature in house remains on 24～27 ℃.Free choice feeding, each hurdle circle are equipped with Duckbill-type drinking trough and freely drink water for piglet.1% Preblend autogamy of basal diet does not contain any microbiotic.The immunity of piglet is undertaken by the immune programme for children of pig routine veterinary transmissible disease, feeding piglet control measures strict implement health and epidemic prevention system.Trial period 21d claims the piglet individual weight when on-test, 7d, 14d and 21d, record day weight gain and feed food consumption weekly.In the end in 1 all daily rations, the diatomite of interpolation 0.5% is measured the digestibility of nutritive substance as indicator, and the basal diet composition sees Table 6.

The arrangement of table 9 experimental animal

1.2 sample collection and processing

Each repeats to get a piglet to test 21d, fetches the intestines chyme after butchering and measures amino acid digestibility.Asepsis is got stomach, each 5cm of duodenum, jejunum, ileum, caecum and colon is long, after the ligation of two ends, be placed in-80 ℃ of refrigerators, be used for measuring the microorganism species of content and mucous membrane, get the content that Stomach duodenum, jejunum epimere, jejunum stage casing, jejunum hypomere, ileum, caecum, the colon section of falling and colon rise section and be loaded in plastics tubing, be placed in-80 ℃ of refrigerators.Test and collected the fresh excreta of just having discharged from anus take circle as unit in last three days, be placed in-20 ℃ of refrigerators, measure the nutrient digestion rate.

1.3 microbiological analysis

The aseptic gi tract chyme 1g that takes different sites is dissolved in the stroke-physiological saline solution of 99mL, places 5～8 little granulated glass spherees in the bottle of stroke-physiological saline solution.And then carry out 10 times of dilutions step by step, until 10 ^-7, 10 ^-5～10 ^-7Each extent of dilution is got the 0.3mL diluent and carry out lactobacillus, bifidus bacillus and anaerobic bacteria culture in being rolled accordingly the pipe substratum.10 ^-2～10 ^-5Each extent of dilution is got the 0.3mL diluent and carry out the counting of intestinal bacteria, enterobacteria and aerobic bacteria in corresponding plate culture medium.Mucous membrane microbioassay method is with reference to the method for Rojas and Conway (1996), the aseptic 1cm that gets ²Gastrointestinal mucosa rinses mucous membrane to without the visible chyme with aseptic PBS solution, blots lip-deep water with aseptic filter paper, then with aseptic nipper, tissue block is immersed the aseptic HEPES damping fluid of 10mL, and constantly 5min is rinsed in vibration, and then carries out 10 times of dilutions step by step, until 10 ^-3At each extent of dilution, get the 0.3mL diluent in corresponding flat board or roll in the pipe substratum.Microorganism is cultivated with selective medium, and the making of substratum is with reference to the method for Chen Tianshou (1995), and intestinal bacteria and enterobacteria substratum are with Yihong methylene blue (EMB) substratum (peptone, 10g; Lactose, 10g; Dipotassium hydrogen phosphate, 2g; Agar, 13g; Yihong-Y, 0.4g; Methylene blue, 0.065g; Distilled water, 1000mL; PH, 7.1), the lactobacillus complete selective medium of Rogosa SL (Tryptones, 10g; Yeast soaks powder, 5g; Glucose, 10g; Pectinose, 5g; Sucrose, 5g; Sodium-acetate, 15g; The citric acid ammonium, 2g; Potassium primary phosphate, 6g; Magnesium sulfate heptahydrate, 0.58g; Four water manganous sulfates, 0.25g; Ferrous sulfate, 0.03g; Tween-80,1g; Agar, 13g; 0.1% resazurin, 1mL; Distilled water, 1000mL; With vinegar acid for adjusting pH to 5.2).Bifidus bacillus Medium of Bifidobacterium (BM) (glucose, 20g; Trypticase, 20g; Yeast soaks powder, 10g; Peptone, 10g; Tomato juice, 333mL; Tween-80,2g; Distilled water, 1000mL; PH 6.6, calcium carbonate, 10g; Halfcystine, 0.5g; 0.1% resazurin, 1mL).Aerobic bacteria and anerobe aerobic bacteria and anaerobic bacteria culture base (junket peptone, 15g; Glucose, 5g; CYSTINE, 0.5g; Sodium thioglycollate, 0.5g; Yeast soaks powder, 5g; Sodium-chlor, 2.5g; 0.1% resazurin, 1mL; Agar, 13g, distilled water, 1000mL; PH7.1).Anerobe, lactobacillus and Medium of Bifidobacterium are made into Hungates and roll pipe, and aerobic bacteria, intestinal bacteria and enterobacteria are cultivated with dull and stereotyped.Hungates pipe and substratum plate are placed on 37 ℃ of incubators, count after cultivation 48h.Each extent of dilution is made three flat boards or is rolled pipe, is used as to count with the dull and stereotyped of 50～150 bacterium colonies or the extent of dilution that rolls pipe.

1.4 chemical analysis takes out ight soil from refrigerator, after take circle as unit, ight soil being mixed, get the 300g left and right and be positioned in the aluminium box, the aluminium box is placed on forced air drying 96h in the baking oven of 65 ℃.After daily ration and faecal samples are pulverized, cross 40 mesh sieves.The chyme sample carries out lyophilize, is used for measuring amino acid whose daily ration sample and chyme was pulverized 60 mesh sieves.Thick moisture, crude protein, coarse ash, calcium and the total phosphorus of daily ration and excrement measured with reference to State Standard of the People's Republic of China GB/T6435-1986 (2001), GB/T 6432-1994 (2001), GB/T 6438-1992 (2001), GB/T 6436-1992 (2001) and GB/T 6437-1992 (2001) respectively.Wherein, crude protein adopts the KJELTEC1035 fully-automatic analyzer to measure.

Energy adopts the full-automatic energometry instrument of PARR1281 (PARR Instrument Corp., the U.S.) to measure.Amino acid (except sulfur-containing amino acid and tryptophane) is measured with automatic analyzer for amino acids (L-8800 of Hitachi, Japan) after 110 ℃ of lower 6mol/L hydrochloric acid hydrolysis 24h by standard GB/T/T18246-2000 (2001); Sulfur-containing amino acid (comprising methionine(Met) and halfcystine) after using peroxyformic acid (1mL hydrogen peroxide+9mL formic acid) oxidation 16h under 110 ℃, uses the hydrochloric acid hydrolysis method to measure according to standard GB/T/T15399-1994 (1996); Tryptophane uses high pressure liquid chromatography (Shimadzu LC-10A, Japan) to measure after 110 ℃ of lower 4mol/L sodium hydroxide hydrolysis 24h according to standard GB/T/T18246-2000 (2001).The method of (1974) such as employing McCarthy is measured ash insoluble in hydrochloric acid.Namely get approximately 10g sample, after boiling 30min with 100mL 4mol/L HCl, use without the ash content filter paper filtering, rinse until anacidity with boiling water, then more than 650 ℃ of lower ashing 6h.

1.5 digestibility calculation formula

Calculate as follows the digestibility of amino acid ileal digestibility or nutrient:

D＝100-(C1×P2)÷(C2×P1)×100

Wherein, D=amino acid ileal digestibility or nutrient digestibility (%),

C1=cattle salt acid insoluble ash to be measured content (%),

Nutrient content in P2=chyme or ight soil (%),

Ash insoluble in hydrochloric acid content (%) in C2=chyme or ight soil,

P1=feed nutrient content to be measured (%).

1.6 data statistics

Test the independent sample t check of all the data SPSS9.0for Windows (SPSS Inc., USA) statistical software and process statistics.

2 results and discussion

2.1 the impact of lactobacillus reuteri preparation F on weanling pig digestive tube chyme microorganism species

As can be seen from Table 10, enterobacteria quantity is in jejunum, caecum and the colon utmost point raise significantly (P＜0.01), significantly raise at ileum (P＜0.05), and significantly reduce (P＜0.01) at duodenum, there is no significant difference between two groups, stomach.Lactobacillus quantity all raises (P＜0.01) than the carbadox group utmost point significantly at Stomach duodenum, jejunum and ileum, and (P＜0.05) significantly raises at caecum.Bifidobacteria extremely significantly raises (P＜0.01) at stomach, jejunum, ileum and caecum, and (P＜0.05) significantly raises at duodenum and colon.The anerobe sum raises (P＜0.01) significantly at Stomach duodenum, jejunum and the ileum utmost point, in caecum and significantly rising (P＜0.05) of colon.Some research reports show, Bacterium lacticum can reduce enterobacteria and the intestinal bacteria quantity in weanling pig enteron aisle and ight soil, and aspect the growth that suppresses piglet, these two kinds of bacterium play an important role.The result of this experiment shows, lactobacillus reuteri preparation F can improve the quantity of lactobacillus, bifidus bacillus, anerobe, intestinal bacteria, enterobacteria and aerobic bacteria in the chyme of digestive tube most positions.

The impact (unit: 1g CFU/ gram, weight in wet base) of table 10 lactobacillus reuteri preparation F on weanling pig digestive tube chyme microorganism species

Annotate: the table intermediate value is mean+SD.

2.2 the impact of lactobacillus reuteri preparation F on the nutrient digestion rate

As can be seen from Table 11, compare with carbadox, lactobacillus reuteri preparation F has significantly improved the all-digestive tract apparent digestibility (P＜0.05) of crude protein and total phosphorus in the daily ration, but the all-digestive tract apparent digestibilities such as energy, dry-matter, organism and calcium and ileal apparent amino acids digestibility all had no significant effect (P＞0.05).Phytate phosphorus is the important obstruction material that affects absorption of nutrient ingredients in feed, and lactic acid can be alleviated this inhibition, improves the utilization ratio of phosphorus.In this preparation, lactobacillus reuteri has the ability (the results are shown in embodiment 1) than the acid of high yield lactogenesis when in-vitro screening.Result shows, the lactobacillus reuteri preparation is on the impact of the nutrient digestion rate of weanling pig and little.

Impact (the unit: %) of table 11 lactobacillus reuteri preparation F on weanling pig daily ration Nutrient Apparent Digestibility

Annotate: the table intermediate value is mean+SD.

Experimental example 7, the prepared preparation of different strain liquid carrier adsorption long term storage stability at room temperature

1, experimental technique

Lactobacillus reuteri CGMCC No.4650 is inoculated in the glucose nutrient broth, and with vinegar acid for adjusting pH to 6.7, puts common incubator 37 degree and cultivate after 18-24 hour, use the sterilized water gradient dilution, obtaining concentration is 10 ¹⁰The strain liquid of cfu/g.

The strain liquid carrier is respectively powdered rice hulls, wheat bran, skim-milk, maltodextrin and molasses.In the preparation that is made into carrier, the proportioning of carrier and lactobacillus reuteri CGMCC No.4650 is 0.5 gram carrier: 10 ⁹Cfu lactobacillus reuteri (Lactobacillus reuteri) RI021.

Measure at once spawn activity (concentration by bacterial classification in preparation embodies) after being made into preparation, after then placing respectively for 1,6,12,18 week under normal temperature, then measure respectively spawn activity; Calculate and place the per-cent that rear spawn activity accounts for the front spawn activity of placement.

2, experimental result

As shown in table 12, illustrate that preparation of the present invention is more stable in carrier.

The Journal of Sex Research steady in a long-term of the prepared preparation of table 12 different strain liquid carrier adsorption

Claims (10)

1. lactobacillus reuteri (Lactobacillusreuteri) RI021 is numbered CGMCC No.4650 registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.

2. lactobacillus reuteri preparation, it is characterized in that: described preparation is comprised of lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021 and carrier; Described carrier is powdered rice hulls, skim-milk, maltodextrin and wheat bran;

The proportioning of lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021 and described carrier is: (10 ⁷-10 ¹⁰) cfu:(0.4-8) the gram carrier.

3. preparation according to claim 2, it is characterized in that: described preparation has following at least a purposes:

1) product of microecological balance in animal intestine is regulated in preparation;

2) preparation strengthens the product of animal non-specific immunity;

3) improve the animal-feed transformation efficiency;

4) promote animal to digestion and the absorption of nutritive substance;

5) preparation reduces the product of animal diarrhea disease percentage;

6) preparation improves the product of breeding performonce fo animals;

Described animal is pig.

4. according to claim 2 or 3 described preparations, it is characterized in that: described preparation specifically can be A)-any one in D):

A) the lactobacillus reuteri preparation A that is comprised of lactobacillus reuteri claimed in claim 1 (Lactobacillusreuteri) RI021 and described carrier, in described lactobacillus reuteri preparation A, proportioning both is the 8 described carriers of gram: 10 ¹⁰Cfu lactobacillus reuteri claimed in claim 1 (Lactobacillusreuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2:2:2:1;

B) the lactobacillus reuteri preparation B that is comprised of lactobacillus reuteri claimed in claim 1 (Lactobacillusreuteri) RI021 and described carrier, in described lactobacillus reuteri preparation B, proportioning both is the 0.5 described carrier of gram: 10 ⁷Cfu lactobacillus reuteri claimed in claim 1 (Lactobacillusreuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2:2:2:1;

C) the lactobacillus reuteri formulation C that is comprised of lactobacillus reuteri claimed in claim 1 (Lactobacillusreuteri) RI021 and described carrier, in described lactobacillus reuteri formulation C, proportioning both is the 0.5 described carrier of gram: 10 ⁸Cfu lactobacillus reuteri claimed in claim 1 (Lactobacillusreuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2:1:1:1;

D) the lactobacillus reuteri preparation D that is comprised of lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021 and described carrier, in described lactobacillus reuteri preparation D, proportioning both is the 0.4 described carrier of gram: 10 ⁹Cfu lactobacillus reuteri claimed in claim 1 (Lactobacillusreuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk, maltodextrin and wheat bran is 1:1:1:1.

5. a lactobacillus reuteri preparation, be following E) or F):

E) the lactobacillus reuteri preparation E that is comprised of lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021 and carrier, in described lactobacillus reuteri preparation E, proportioning both is the 0.4 described carrier of gram: 10 ⁷Cfu lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021; Described carrier is comprised of skim-milk and wheat bran, and the mass ratio of described skim-milk and wheat bran is 1:1;

F) the lactobacillus reuteri preparation F that is comprised of lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021 and carrier, in described lactobacillus reuteri preparation F, proportioning both is the 0.4 described carrier of gram: 10 ¹⁰Cfu lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021; Described carrier is comprised of powdered rice hulls, skim-milk and wheat bran, and the mass ratio of described powdered rice hulls, skim-milk and wheat bran is 1:1:1.

6. the animal-feed that contains arbitrary described preparation in lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021 or claim 2-4; Described animal is pig.

7. animal-feed according to claim 6, is characterized in that: in described animal-feed, contain 10 in the described animal-feed of every gram ⁷Lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021 that cfu is above.

8. the following at least a purposes of arbitrary described preparation in lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021 or claim 2-5:

1) product of microecological balance in animal intestine is regulated in preparation;

2) preparation strengthens the product of animal non-specific immunity;

3) improve the animal-feed transformation efficiency;

4) promote animal to digestion and the absorption of nutritive substance;

5) preparation reduces the product of animal diarrhea disease percentage;

6) preparation improves the product of breeding performonce fo animals;

Described animal is pig.

9. the application of arbitrary described preparation in preparation intestinal bacteria inhibitor in lactobacillus reuteri claimed in claim 1 (Lactobacillus reuteri) RI021 or claim 2-5.

10. application according to claim 9, the intestinal bacteria in described intestinal bacteria inhibitor are intestinal bacteria K88, e. coli k99 or intestinal bacteria 987P.

Images