CN102304489A - Lactobacillus reuteri strain and application thereof - Google Patents
Lactobacillus reuteri strain and application thereof Download PDFInfo
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- CN102304489A CN102304489A CN201110285722A CN201110285722A CN102304489A CN 102304489 A CN102304489 A CN 102304489A CN 201110285722 A CN201110285722 A CN 201110285722A CN 201110285722 A CN201110285722 A CN 201110285722A CN 102304489 A CN102304489 A CN 102304489A
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- lactobacillus reuteri
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Fodder In General (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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
Technical field
The present invention relates to a strain lactobacillus reuteri and an application thereof.
Background technology
When the interpolation microbiotic is made major contribution to the intensification animal husbandry development in the feed; Residual etc. in the destruction of the spinoff of its generation such as autogenous infection and superinfection, chemical sproof generation, normal intestinal flora and livestock product and the environment brings serious threat to aquaculture, animal and human food prods's safety, therefore the research and development important directions that to substitute antibiotic novel green safe additive be present feed advance.
Lactobacillus reuteri (Lactobacillus reuteri) is that the obligate heterolactic fermentation bacterium with enteron aisle epidermis adhesive capacity also is one of population important in the milk-acid bacteria in a kind of enteron aisle that inhabits humans and animals.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 the human sour milk food; And report comparatively small amt; More rarely has report and be used for the animal field; Major cause possibly be this bacterium from the isolation identification to the product prepn and the application of animal in producing do not have systematic research, thereby limited of the application of this bacterial classification in the animal field.Therefore,, develop a kind of lactobacillus reuteri preparation, have vast potential for future development according to the demand of present livestock industry.
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 common micro-organisms center.This lactobacillus reuteri strong stress resistance, anti-assorted bacterium ability be strong, have probiotic properties.
Lactobacillus reuteri (Lactobacillus reuteri) RI021 has following biological characteristics:
(1) survival rate after pH2.0 handles 4 hours is 72%;
(2) survival rate behind the heating 15min is 31.7% in 75 ℃ of water-baths;
(3) survival rate of room temperature held after 1 month 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 provides a kind of lactobacillus reuteri preparation, and said preparation can substitute existing feeding antibiotic, and has disease-resistant, promotes growth, 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.
Said preparation has following at least a purposes:
1) product of microecological balance in the animal intestine is regulated in preparation;
2) preparation strengthens the product of non-specific immunity;
3) improve the animal-feed transformation efficiency;
4) promote digestion and the absorption of animal to 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 suppressor factor.
Intestinal bacteria in the said intestinal bacteria suppressor factor specifically can be intestinal bacteria K88, e. coli k99 or intestinal bacteria 987P.
Said 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.
Said reduction animal diarrhea disease percentage specifically can be presented as the coli-infection grice diarrhoea rate that reduces; Said reduction diarrhea rate specifically can be presented as and reduce coli-infection grice diarrhoea index.
Said preparation specifically can be made up of said lactobacillus reuteri (Lactobacillus reuteri) RI021 and carrier; Said carrier specifically can be at least a in powdered rice hulls, skim-milk, maltodextrin, the wheat bran.
Said carrier can pass through micronization processes.Through after the micronization processes, 90% diameter of particle is less than 100 μ m, and median size is less than 50 μ m, and said median size is 50% diameter of particle.
Said preparation specifically can be following A)-in F) any:
A) the lactobacillus reuteri preparation A that is made up of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation A, the proportioning of the two is the said carriers of 8 grams: 10
10The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1;
B) the lactobacillus reuteri preparation B that is made up of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation B, the proportioning of the two is the said carrier of 0.5 gram: 10
7The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1;
C) the lactobacillus reuteri formulation C of being made up of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and said carrier, in the said lactobacillus reuteri formulation C, the proportioning of the two is the said carrier of 0.5 gram: 10
8The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 1: 1: 1;
D) the lactobacillus reuteri preparation D that is made up of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation D, the proportioning of the two is the said carrier of 0.4 gram: 10
9The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 1: 1: 1: 1;
E) the lactobacillus reuteri preparation E that is made up of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation E, the proportioning of the two is the said carrier of 0.4 gram: 10
7The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Said carrier is made up of skim-milk and wheat bran, and the mass ratio of said skim-milk and wheat bran is 1: 1;
F) the lactobacillus reuteri preparation F that is made up of described lactobacillus reuteri (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation F, the proportioning of the two is the said carrier of 0.4 gram: 10
10The described lactobacillus reuteri of cfu (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk and wheat bran is 1: 1: 1.
Lactobacillus reuteri according to the invention (Lactobacillus reuteri) RI021 or said preparation can be used as additive and are used to prepare animal-feed, and animal wherein includes but not limited to various animals such as pig, ox, sheep, chicken.This feed has and the similar function of antibiotic feed, but the spinoff of antibiotic-free feed.
In the said animal-feed, specifically can contain 10 in the said animal-feed of every gram
7Said 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 the alternative existing animal daily ration; Regulate microecological balance in the 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 gi tract to grow to growing animal simultaneously, so it is applied in the effect that can play disease-resistant growth-promoting in the 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 human beings'health, are a kind of promising green feed additives.
Describe the present invention in detail below in conjunction with specific embodiment, said 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 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 measures the probiotic lactobacillus culture to colibacillary inhibition for the plate trench method.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Lactobacillus reuteri Lactobacillus reuteri RI021 of the present invention; The Hungates that can be inoculated in Rogosa SL agar earlier rolls in the pipe substratum and cultivates, and the inoculum that obtains is inoculated in the Rogosa SL meat soup anaerobic culture medium and cultivates.
Wherein said nutrient agar is for being fit to any substratum that lactobacillus reuteri is cultivated, and which kind of substratum those skilled in the art know is fit to the lactobacillus reuteri cultivation.Specifically can select for use 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 said suitable lactobacillus reuteri is cultivated can be the substratum of any suitable lactobacillus reuteri cultivation of prior art, and this is known by those skilled in the art, specifically can be: Tryptones 1-20g, Carnis Bovis seu Bubali cream 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 SULPHATE HEPTAHYDRATE 99.5 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, zero(ppm) water 100-2500mL; Be preferably Tryptones 5-15g, Carnis Bovis seu Bubali cream 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 SULPHATE HEPTAHYDRATE 99.5 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, zero(ppm) water 500-2000mL; Tryptones 10g more preferably, Carnis Bovis seu Bubali cream 10g, yeast soak powder 5g, glucose 10g; Pectinose 5g, sucrose 5g, sodium-acetate 15g, Sodium Citrate 2g; Potassium primary phosphate 6g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.58g, four water manganous sulfate 0.25g, ferrous sulfate 0.03g; Tween-80 1mL, agar 13g, zero(ppm) water 1000mL.
Liquid nutrient medium wherein is for being fit to any substratum that lactobacillus reuteri is cultivated, and those skilled in the art also know this liquid nutrient medium, wherein preferred meat soup anaerobic culture medium; Meat soup anaerobic culture medium described here is interpreted as on the basis of ordinary broth anaerobic culture medium, the substratum of suitably revising, and the substratum of said suitable modification can be the substratum of any suitable lactobacillus reuteri cultivation of prior art; This is known by those skilled in the art, preferred Rogosa SL meat soup anaerobic culture medium, and wherein the prescription 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 SULPHATE HEPTAHYDRATE 99.5 0.1-1g, four water manganous sulfate 0.1-1g; Ferrous sulfate 0.01-0.05g, tween-80 0.5-1.5mL, zero(ppm) 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 SULPHATE HEPTAHYDRATE 99.5 0.58g; Four water manganous sulfate 0.25g, ferrous sulfate 0.03g, tween-80 1mL, zero(ppm) 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 the pipe and cultivates, and preferably in incubator, under 37 ℃, cultivate 15-72 hour; Lactobacillus reuteri enlarged culturing wherein preferably in Rogosa SL meat soup anaerobic culture medium, was cultivated 15-80 hour down for 37 ℃.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.The carbon N/P ratio is 5～100: 3～12 in the substratum of wherein said one time fermentation, Secondary Fermentation or three fermentations: 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 the 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 cooperate by same any on the market conventional animal feed at present, and animal-feed protection domain promptly according to the invention is not limited by the conventional animal feed kind that cooperates.
Yet conventional animal feed wherein can be preferably daily ration, further is preferably pig and uses daily ration with daily ration, ruminating animal with daily ration and aquatic animal with daily ration, chicken.For different animal, the prescription of its daily ration has nothing in common with each other, and still, disclosed any daily ration can be as animal-feed of the present invention in the prior art, and its prescription all is the common practise in this field.
Culture medium prescription used among the following embodiment is following:
1, Rogosa SL broth culture
Tryptones 10g, Carnis Bovis seu Bubali cream 10g, yeast soak powder 5g, glucose 10g, pectinose 5g; Sucrose 5g, sodium-acetate 15g, Sodium Citrate 2g, potassium primary phosphate 6g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.58g; Four water manganous sulfate 0.25g, ferrous sulfate 0.03g, tween-80 1mL is settled to 1L with zero(ppm) water.
2, Rogosa SL nutrient agar
Add agar 13g in the 1L Rogosa SL broth culture.
3, MRS broth culture
Peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast soak powder 5g, potassium hydrogenphosphate 2g, citric acid two ammonium 2g, glucose 20g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.58g, four water manganous sulfate 0.25g, sodium-acetate 5g is settled to 1L with zero(ppm) water.
4, MRS nutrient agar
Add agar 14g in the 1L MRS broth culture.
5, glucose nutrient broth
Peptone 10g, Carnis Bovis seu Bubali cream 5g, sodium-chlor 5g, glucose 2.5g is settled to 1L with zero(ppm) 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 healthy piglet
2(stomach, duodenum, jejunum, ileum, caecum and colon), in Sterilization cup, the chyme with on the SPSS flushing mucous membrane is equipped with the mucous membrane immersion in the beaker of 15mL HEPES damping fluid, behind tweezers waggle mucous membrane 5min, obtains supernatant.
1.2 the separation and Culture of bacterial strain
Inject the Hungates that Rogosa SL nutrient agar is housed with asepsis injector absorption 0.2mL supernatant and roll pipe, make supernatant be evenly distributed on agar surface in the pipe with light rolling of have gentle hands.Put into 5%CO
2Incubator; Cultivate after the 72h for 37 ℃; In aseptic technique in the Biohazard Safety Equipment bacterium colony of white needle point size is transferred to the syringe needle picking in the aseptic Rogosa broth culture of anaerobism and to be cultivated, observe meat soup and whether become muddy, have 4 ℃ of refrigerators of muddy placement to preserve subsequent use.
1.3 gramstaining
Draw a small amount of Rogosa SL broth culture with asepsis injector, drop on the 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: decolouring 30s 95% ethanol=3: 7), washing; Drip husky yellow staining fluid and redye 1min, washing is waited to do, and on ordinary optical microscope, observes, 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 the about 0.2mL injection of culture Rogosa SL nutrient agar inclined-plane, 5%CO are housed
2Incubator is cultivated 24h for 37 ℃, grow bacterium colony after, 3% superoxol is added drop-wise on the bacterium colony, be negative if there is bubble to produce explanation, be positive if there is bubble to produce explanation.Culture through anaerobism on the Rogosa SL is cultivated can tentatively be thought lactobacillus (Lactobacillus) through Gram-positive and catalase test feminine gender.
2 results and discussion
This test utilizes from healthy piglet is gastrointestinal tract mucous that the complete selective medium of Rogosa SL filters out 6790 strain probiotic lactobacillus after persalt is proofreaied and correct.In the HEPES damping fluid, shake 5min with piglet is gastrointestinal tract mucous; Draw the 0.2mL supernatant with asepsis injector and pack into and contain rolling of Rogosa SL nutrient agar and cultivate 24h in the pipe, choose white colony again and in the aseptic Rogosa SL broth culture of anaerobism, cultivate.Through gramstaining and catalase test, prove that probiotic lactobacillus is the GI dominant microflora of healthy piglet, explain through the gauged Rogosa SL of hydrochloric acid nutrient agar to be fit to separate gastrointestinal tract mucous probiotic lactobacillus.
The screening of bacterial classification is and loaded down with trivial details process.The seed selection process of most of probiotic lactobacillus is to separate probiotic bacterium from the digestive tube epithelium, so just can obtain to have well to grow the bacterial classification of effect surely.
(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 stomach probiotic lactobacillus and the intestines probiotic lactobacillus of having cultivated 24h respectively, and inoculum size 1% is observed behind 37 ℃ of cultivation 24h.If the muddiness that substratum becomes explains that this bacterial strain is acidproof.Carry out gradient dilution to 10 from inoculation beginning and 8h with asepsis injector taking-up 1mL
-5After, to get the 0.3mL diluent and go up evenly coating at MRS agar (pH5.2), plate is placed on 37 ℃, 5%CO
2In the incubator, cultivate 24h, calculate survival rate.Microscopy is seen whether microbiological contamination then.The initial separation sample retention of the lactobacterium strain that can grow is subsequent use 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 behind 28 ℃ of cultivation 24h.If the muddiness that substratum becomes is explained this bacterial strain bile tolerance.Microscopy is seen whether microbiological contamination then.The initial separation sample retention of the lactobacterium strain that can grow is subsequent use 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 behind 28 ℃ of cultivation 12-72h.If the muddiness that substratum becomes is explained the anti-high-copper of this bacterial strain.Microscopy is checked whether microbiological contamination then.The initial separation sample retention of the lactobacterium strain that can grow is subsequent use 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 behind 28 ℃ of cultivation 12-72h.If the muddiness that substratum becomes is explained the anti-high zinc of this bacterial strain.Microscopy is seen whether microbiological contamination then.The initial separation sample retention of the lactobacterium strain that can grow is subsequent use at-80 ℃ of refrigerators.
2. result and discussion
2.1 acid resistance seed selection
Through the acid resistance seed selection, in the 6790 strain probiotic lactobacillus, have 4753 strain probiotic lactobacillus can be in the Rogosa of pH2.0 SL broth culture growth and breeding, mortality is 30%.This explains that GI most of probiotic lactobacillus acid resistance is better, has 70% probiotic lactobacillus in the gi tract of piglet, to breed.Probiotic lactobacillus is generally through growing at digestive tube epithelium generation probiotic properties surely.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 through 4753 strain probiotic lactobacillus of acid resistance seed selection, have 856 strain probiotic lactobacillus can be in the malt extract medium that contains cholate 0.3% growth and breeding, mortality is 82%.Research shows, cholate is a more adverse factors of the ratio hydrochloric acid in gastric juice that in animal gastrointestinal tract, runs into of intestines probiotic lactobacillus.Chou and Weimer discover that be effective with cholate selectivity domestication probiotic lactobacillus to the tolerance of cholate; The ability force rate parental generation probiotic lactobacillus of the probiotic lactobacillus bile tolerance that domestication obtains afterwards through the several generations cholate is strong; Probiotic lactobacillus produces tolerance to the cholate performance easily; 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 through 856 strain probiotic lactobacillus of bile tolerance performance seed selection, have 257 strain probiotic lactobacillus can be in the Rogosa of 250mg/kg cupric sulfate pentahydrate SL broth culture growth and breeding, mortality is 70%.Prove that this probiotic lactobacillus 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 through 257 strain probiotic lactobacillus of anti-high-copper performance seed selection, have 77 strain probiotic lactobacillus 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 to suffer 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 also is necessary.
3. brief summary
Obtaining 77 strains through this test, to derive from piglet gastrointestinal tract mucous, and environment in the digestive tube is had the lactobacterium strain of certain resistivity, and this lays the foundation for later seed selection work.
(3) the probiotic properties seed selection of lactobacillus reuteri
1.1 the probiotic lactobacillus meta-bolites 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.
Probiotic lactobacillus: 77 strains are acidproof, the probiotic lactobacillus of bile tolerance, anti-high-copper and Nai Gao zinc.
Substratum: Mai Kangkai (MacConkay) substratum (available from the Beijing Tiantan Biological Products Co.ltd).
In the Hungates pipe, add the about 15mL of MRS broth culture, carbonating is processed aseptic anaerobism MRS meat soup.Inoculate respectively in each pipe that 77 strains that are separated in the healthy piglet stomach are acidproof, the probiotic lactobacillus of bile tolerance, anti-high-copper and Nai Gao zinc, probiotic lactobacillus is cultivated the back through 18h and is inoculated in the meat soup by 1%, after cultivating 24h in 37 ℃ of incubators, it is subsequent use to put 4 ℃ of refrigerators preservations.
Suppress coli test: make the Mai Kangkai substratum, behind 121 ℃ of autoclaving 15min, asepticly topple over plate after the heating for dissolving.In the line of plate bottom plate is divided into three parallel zones with marking pen, each zone is with loop-carrier streak inoculation intestinal bacteria nutrient solution (4.0 * 10
8CFU/mL) in media surface, dig one then in the place of the about 3cm of anomaly ware center line and widely be the ditch of 0.5cm, ditch is vertical with the inoculation line, and is with the hot loop-carrier benefit end, as shown in Figure 2.Ditch is filled up (can not overflow) with different probiotic lactobacillus cultures respectively, after putting 37 ℃ of common incubators and cultivating 18h, observe the position that the intestinal bacteria bacterium colony occurs, colibacillary bacterium colony be red, measures and leaves the nearest intestinal bacteria bacterium colony of ditch and the distance of ditch.Each probiotic lactobacillus is done two parallel appearance, with the MV ecbatic.
1.2 probiotic lactobacillus and the seed selection of intestinal bacteria mixed culture restraining effect
Intestinal bacteria (K88, K99 and 987P) source is saved together with 1.1.
Probiotic lactobacillus: 65 strain intestines meta-bolitess have stronger inhibiting probiotic lactobacillus to intestinal bacteria.
Colibacillary detection substratum: Mai Kangkai (MacConkay) substratum and 1.1 together.
Probiotic lactobacillus is inoculated in the aseptic MRS meat soup of anaerobism, behind 37 ℃ of cultivation 24h, puts 4 ℃ of refrigerators and preserves subsequent use.
1.2.1 the different concns probiotic lactobacillus is to colibacillary inhibition
Select a strain probiotic lactobacillus at random, get 5mL culture (8.2 * 10
9CFU/mL) respectively with 5,10, the 15mL nutrient broth mixes, and processes 3 nutrient broths that contain different probiotic lactobacillus culture concentration, the inoculation intestinal bacteria, inoculum size is 5% of a nutrient solution, puts 37 ℃, 5%CO
2 Behind incubator cultivation 4,8 and the 18h, take out nutrient solution with asepsis injector respectively, nutrient solution becomes 10 through gradient dilution
-2～10
-5After, each extent of dilution is done 4 parallel appearance, gets the 0.3mL diluent; Evenly coat on the maconkey agar flat board with " L " rod; Place 37 ℃, common incubator is cultivated 18h, gets colony count 50～150 plate count; With the MV ecbatic, calculate colibacillary quantity in every milliliter of nutrient solution.
1.2.2 contain probiotic lactobacillus and colibacillary quantitative relation in the nutrient broth of 1/4 probiotic lactobacillus
Get 5mL probiotic lactobacillus 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), the quantity of probiotic lactobacillus is colibacillary 10 times in the initial mixed solution, and the pH that uses 0.1M NaOH solution to regulate nutrient solution is 7.0, after putting 37 ℃ of incubators and cultivating 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 done 3 parallel appearance, and MRS nutrient agar (pH5.4) is put 37 ℃, 5%CO
2Incubator is cultivated 18h, gets colony count 50～150 plate count, representes the quantity of probiotic lactobacillus with MV, calculates the quantity of probiotic lactobacillus in every milliliter of nutrient solution.Directly get the 1mL nutrient solution through gradient dilution to 10 after cultivating 24h with asepsis injector
-2After, get the 0.3mL diluent and place on the maconkey agar, evenly be coated with " L " rod, each pipe is done 3 parallel appearance, and flat board is placed 37 ℃, and common incubator is cultivated 18h, calculates each dull and stereotyped intestinal bacteria colony count, with the MV ecbatic.
1.3 the seed selection of lactic acid production
Probiotic lactobacillus: the strong probiotic lactobacillus of 63 strain Chinese People's Anti-Japanese Military and Political College enterobacteria abilities.
The MRS broth culture is regulated pH to 5.4 with Glacial acetic acid min. 99.5, mixes the back and puts into the Hungates pipe, and every pipe dress 10mL meat soup fills 1.5%CO while hot
21min seals, and autoclaving is processed the aseptic meat soup of anaerobism, and 63 strain probiotic lactobacillus after the aseptic meat soup of this anaerobism is cultivated 24h, are inoculated in the MRS meat soup by 1% and cultivate 20h respectively.Get the 2mL nutrient solution; The centrifugal 15min of 3500rpm gets supernatant, after 10 times of ultrapure water dilutions; With the lactic acid concn in D-lactate detection test kit (numbering K-DATE, the graceful bio tech ltd of last Hypon) and the Technicon RA 100 biochemical instruments enzymatic assays supernatants.Each bacterial strain is surveyed two parallel appearance, with the MV ecbatic.
2 results
2.1 the probiotic lactobacillus meta-bolites suppresses colibacillary seed selection
The probiotic lactobacillus meta-bolites suppresses colibacillary effect to be index from the nearest intestinal bacteria bacterium colony of ditch and the distance of ditch, and distance is big more, explains that fungistatic effect is good more.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 antimicrobial substance porous in the ditch to cross agar and suppress intestinal bacteria; Along with the increase from the ditch distance, the concentration of the antimicrobial substance in porous past is just more little.The distance of intestinal bacteria bacterium colony and ditch is big more, explains that the bacteriostasis of the contained antimicrobial substance of probiotic lactobacillus meta-bolites is strong more.Can measure the probiotic lactobacillus meta-bolites simultaneously to the colibacillary restraining effect of 3 strains with the plate trench method at same plate, and can only measure the colibacillary restraining effect of a strain at same plate with plate borehole method.Have 65 strain probiotic lactobacillus 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, probiotic lactobacillus 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 probiotic lactobacillus and intestinal bacteria vitro culture
2.2.1 different ratios probiotic lactobacillus RI021 nutrient solution and the quantitative relation of intestinal bacteria under the mixed culture condition.
The result sees table 2.
Contain different ratios probiotic lactobacillus RI021 nutrient solution in table 2 nutrient broth to colibacillary restraining effect (unit: CFU/mL)
2.2.2 probiotic lactobacillus and the intestinal bacteria quantitative relation under the mixed culture condition
Quantitative relation (the unit: CFU/mL) of table 3 probiotic lactobacillus and intestinal bacteria mixed culture
Can find out that from table 2 probiotic lactobacillus of different concns is similar to colibacillary restraining effect during with the intestinal bacteria mixed culture, explaining that the nutrient broth that contains 25% probiotic lactobacillus RI021 is enough to be used for doing suppresses colibacillary test.Probiotic lactobacillus RI021 and intestinal bacteria see table 3 with the quantitative relation of intestinal bacteria mixed culture in the nutrient broth that contains 25% probiotic lactobacillus RI021 culture.Visible from table, RI021 has stronger bacteriostasis relatively to three kinds of serotype intestinal bacteria.Quantitative relation when external test tube is cultivated between different microorganisms can be used as the interactional in vivo a kind of index of reflection mikrobe.
2.3 the acid producing ability of probiotic lactobacillus
Through measuring, the acid producing ability of probiotic lactobacillus 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 the production bacterial classification of probiotic lactobacillus RI021 as probiotic bacterium.This strain bacterium is accredited as lactobacillus reuteri (Lactobacillus acidophilus) through China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), registers 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 the 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, every at preceding 24h at a distance from 1h, after the 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 and is gone up evenly coating at MRS nutrient agar (pH5.2), and each gradient is done 3 parallel appearance, and plate is placed on 37 ℃, 5%CO
2Incubator in, cultivate 24h, the extent of dilution of the colony count 50～150 in the ware of making even is as calculating usefulness, each gradient is done 3 parallel appearance, with the MV ecbatic.Every milliliter of bacterial concentration is represented with logarithmic value.
1.2 acidproof survival rate
The MRS broth culture transfers the pH joint to 5.4 with Glacial acetic acid min. 99.5, again with concentrated hydrochloric acid with pH regulator to 2.0, place Hungates to roll pipe, each pipe dress 20mL meat soup is processed the aseptic meat soup of anaerobism.The back that cools down adds the culture of 1mL lactobacillus reuteri 16h in every pipe, when beginning, 2h, 4h, 6h and 8h take a sample respectively, measures survival rate.After taking out 1mL and carry out gradient dilution with asepsis injector, 10
-4～10
-7Extent of dilution is got the 0.3mL diluent and is gone up evenly coating at MRS nutrient agar (pH5.2), and each gradient is done 3 parallel appearance, and plate is placed on 37 ℃, 5%CO
2Incubator in, cultivate 24h, the plate count of the colony count 50～150 in the ware of making even is with the MV ecbatic.
The calculation formula of survival rate is:
S
Acid=n
x/ n
0
S
AcidFor handle the lactobacillus reuteri survival rate of back different time through pH2.0; n
0Be every milliliter of viable count before the pH2.0 processing; n
xBe every milliliter of viable count behind pH2.0 processing 2,4,6, the 8h.
1.3 heat-resisting survival rate
The MRS broth culture, regulating pH is 6.7, places Hungates to roll pipe, each pipe dress 20mL meat soup is processed 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 behind the 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 taking out 1mL and carry out gradient dilution with asepsis injector, 10
-4～10
-7Extent of dilution is got the 0.3mL diluent and is gone up evenly coating at MRS nutrient agar (pH5.2), and each gradient is done 3 parallel appearance, and plate is placed on 37 ℃, 5%CO
2In the incubator, cultivate 24h, the colony count in the ware of making even is 50～150 plate count, with the MV ecbatic.
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 the heat treated; n
1Be every milliliter of viable count behind the heat treated 15min.
1.4 storage tolerance survival rate
The MRS broth culture, regulating pH is 6.7, places Hungates to roll pipe; Every pipe dress 20mL MRS broth culture, process the aseptic meat soup of anaerobism after, add the culture that the 1mL lactobacillus reuteri is cultivated 16h in every pipe; Be placed in 37 ℃ of incubators behind the 16h, sampling 1mL carries out live bacterial count.The room temperature held is after 1 month, and 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 and is gone up evenly coating at MRS nutrient agar (pH5.2), and each gradient is done 3 parallel appearance, and plate is placed on 37 ℃, 5%CO
2Incubator in, cultivate 24h, get colony count and be 50～150 plate count, with the MV ecbatic.
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
0Be every milliliter of viable count before preserving; 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 the adaptive process of mikrobe to new growing environment lag period; In this course; It is constant or descend that mikrobe shows as quantity, and himself macromole and micromolecular composition are adjusted, and also can produce specific material such as enzyme simultaneously and wait and adapt to new environment.Logarithmic phase be mikrobe to after the 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, because the thalli growth metabolism is to the consumption of nutritive substance and the accumulation of toxic products, the growth and breeding speed of bacterium decline.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 is as shown in Figure 1, and is visible from Fig. 1, and the concentration of lactobacillus reuteri has a declining tendency when beginning, and process 2h is afterwards from 10
3One magnitude rises rapidly, to 22h near 10
10One magnitude; To 40h, the number of bacteria level begins slow decline, 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 that 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 is seen table 5 through the cell concentration that pH2.0 handles different time.
Table 5 pH2.0 handles the influence (unit: CFU/mL) of different time to lactobacillus reuteri viable bacteria concentration
Visible from table 5,2h before pH2.0 handles, the quantity of lactobacillus reuteri almost remains on 100% survival rate, is that 72%, the 6h drops to 34.4% to the 4h decrease in survival rate.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 an ideal comparatively for its anti-restraining effect of crossing hydrochloric acid in gastric juice or killing action, 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
Lactobacillus reuteri RI021 handles 15min survival rate afterwards through 75 ℃ and reaches 31.7%.The pelleting temperature of general pig starter feed is between 70 ℃～85 ℃; After high temperature resistant survival rate low also be probiotic lactobacillus as one of major limitation sexual factor of fodder additives; According to the research of Fuller (1989), probiotic lactobacillus does not have viable bacteria after handling 10min through 75 ℃ basically.See that from heat-resisting survival rate the lactobacillus reuteri of this test seed selection can tolerate the high temperature when granulating, and will have application promise in clinical practice as fodder additives.
2.4 the storage tolerance survival rate of lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650
After one month storage, 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 regulates pH to 6.7 with acetic acid, put 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; Through drying and crushing, the lactobacillus reuteri preparation A that the lactobacillus reuteri preparation that promptly obtains being made up of lactobacillus reuteri and carrier is formed.Among this lactobacillus reuteri preparation A, the proportioning of the two is 8 gram carriers: 10
10The cfu lactobacillus reuteri, said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1.
2, lactobacillus reuteri is inoculated in the Rogosa SL broth culture, regulates pH to 6.7 with acetic acid, put common incubator and cultivate after 18-24 hour for 37 ℃, 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 were carried out micronization processes in advance, and (90% diameter of particle is less than 100 μ m, and median size is less than 50 μ m; Said median size is 50% diameter of particle), the lactobacillus reuteri preparation B that the lactobacillus reuteri preparation that promptly obtains being made up of lactobacillus reuteri and carrier is formed.Among this lactobacillus reuteri preparation B, the proportioning of the two is 0.5 gram carrier: 10
7The cfu lactobacillus reuteri; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1.
3, place fermentor tank to carry out one grade fermemtation with cultivating the lactobacillus reuteri CGMCC No.4650 that obtains in the step 1, cultivate after 18-24 hour for 37 ℃, use the sterilized water gradient dilution, 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.Other adds suitable quantity of water, and regulates pH to 6.7 with acetic acid.The carbon N/P ratio is 100: 5: 0.5 in the substratum.
By weight, will contain 10 parts of strain liquids, 2 parts of the powdered rice hulls of 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 (90% diameter of particle are less than 100 μ m; Median size is less than 50 μ m; Said median size is 50% diameter of particle), the lactobacillus reuteri formulation C that the lactobacillus reuteri preparation that promptly obtains being made up of lactobacillus reuteri and carrier is formed, the proportioning of the two are the said carrier of 0.5 gram: 10
8The cfu lactobacillus reuteri; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 1: 1: 1.
4, place seeding tank to carry out one grade fermemtation with cultivating the lactobacillus reuteri CGMCC No.4650 that obtains in the step 1, place the production jar to carry out second order fermentation then, use the sterilized water gradient dilution, obtaining concentration is (10
7-10
10) strain liquid of cfu/g.Two-stage fermentation jar 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 other adds suitable quantity of water.And with acetic acid adjusting pH to 6.7.The carbon N/P ratio is 100: 10: 3 in the substratum.
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 (90% diameter of particle are less than 100 μ m; Median size is less than 50 μ m; Said median size is 50% diameter of particle), promptly obtain the lactobacillus reuteri preparation D that forms by lactobacillus reuteri and carrier, the proportioning of the two is the said carrier of 0.4 gram: 10
9The cfu lactobacillus reuteri; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 1: 1: 1: 1.
5, place seeding tank to carry out double fermentation with cultivating the lactobacillus reuteri CGMCC No.4650 that obtains in the step 2, place the production jar to carry out three grade fermemtation then, use the sterilized water gradient dilution, 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 in 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 regulate pH to 6.7 with acetic acid.
By weight; To contain 1 part of mixing of 1 part in 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; Said median size is 50% diameter of particle), promptly obtain the lactobacillus reuteri preparation E that forms by lactobacillus reuteri and carrier, the proportioning of the two is the said carrier of 0.4 gram: 10
7Lactobacillus reuteri; Said carrier is made up of skim-milk and wheat bran, and the mass ratio of said 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 in 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 (90% diameter of particle are less than 100 μ m; Median size is less than 50 μ m; Said median size is 50% diameter of particle), promptly obtain the lactobacillus reuteri preparation F that forms by lactobacillus reuteri and carrier, the proportioning of the two is the said carrier of 0.4 gram: 10
10The cfu lactobacillus reuteri; Said carrier is made up of powdered rice hulls, skim-milk and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk and wheat bran is 1: 1: 1.
Experimental example 4, lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650 preparation are to the influence of piglet production performance
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 component at random 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 is divided into groups
To be divided into be two processing in test, and handling 1 be test group, and basal diet interpolation weight ratio is any among the lactobacillus reuteri preparation A-F among 0.1% the embodiment 3; Handling 2 is control group, and table 6 is seen in each arrangement of handling of basal diet.The composition and the nutritive ingredient of basal diet are seen table 6.
The basal diet of table 6 growth test is formed 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; Vitamin G, 5.22mg; Pantothenic acid, 20.0mg; Nicotinic acid, 26.0mg; Cobalamin, 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 the house remains on 24～27 ℃.Free choice feeding, each hurdle circle are equipped with the duck-beak type water fountain and supply piglet freely to drink water.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, the strict health and epidemic prevention system of carrying out of feeding piglet control measures.
1.5 sample collection and processing
Piglet individual weight, record day weight gain and feed food consumption are weekly claimed in test when beginning, 7d, 14d and 21d.
1.6 data statistics
(SPSS Inc., USA) statistics is handled in the check of the independent sample t of statistical software to test all The data SPSS12.0.
2. result and discussion
2.1 the lactobacillus reuteri preparation to weaned piglet after the influence of growth performance
Table 7 lactobacillus reuteri formulation C is to the influence of weanling pig growth performance
Annotate: the table intermediate value is a mean+SD.
Find out from table 7; Wean back 1wk, 2wks and 3wks, piglet average daily gain (ADG) comparison of test group you can well imagine high 38.5%, 44.1% and 27.4% according to component; Significant difference (P＜0.05); Average daily ingestion amount (ADFI) comparison you can well imagine high 21.3%, 41.8% and 18.6%, significant difference (P＜0.05) according to component).
Carry out same experiment with lactobacillus reuteri preparation A, B, D, E and F and also obtained identical conclusion.
Experimental example 5, lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650 are to the influence of coli-infection grice diarrhoea
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 into 2 groups at random, single cage is raised in metabolic cage, and the room temperature of respiratory chamber is controlled at 25 ℃～27 ℃, 12 piglets of treatment group oral liquid lactobacillus reuteri preparation of drinking water every day, and drinking every day into, the sum of lactobacillus reuteri CGMCC No.4650 is about 10
7CFU, 12 piglet drinking public water supplies of control group are as 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, the colibacillary of three kinds of serotypes is in equal proportions in this mixed solution, a twice-daily, 1d attacks poison back piglet and still freely drinks tap water and search for food altogether, measures and attacks poison back diarrhoea disease time and diarrhoea index, 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 diarrhoea index
The diarrhoea 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 the interior piglet of suffering from diarrhoea of 7d multiply by 7 after attacking poison.
Table 7 diarrhoea standards of grading
1.4 statistical study
SPSS12.0 is adopted in the statistical study of data, and (SPSSInc., USA) statistical study is carried out in the independent sample t-check of software.
2 results and discussion
Test-results is seen table 8, and the test group diarrhea disease percentage descends 64.8% than control group, the diarrhoea index decreased 63.Control group 2d after attacking poison promptly has first piglet to suffer from diarrhoea, and experimental group is just found first grice diarrhoea until attack poison back 4d.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 superior to microbiotic or recombinant vaccine.The lactobacillus reuteri of the lactic acid producing rhzomorph that in the lactobacillus reuteri preparation, contains has a good Chinese People's Anti-Japanese Military and Political College enterobacteria characteristic external, according to have in the Hoefling report newborn piglet diarrhoea 26% since enterotoxigenic Escherichia coli cause.The test group piglet is 7d before attacking poison, the oral always water that contains lactobacillus reuteri, and lactobacillus reuteri exists at gi tract chyme and mucous membrane in a large number, and at specific gi tract microhabitat performance prebiotic effect.Piglet receives intestinal bacteria when attacking poison; Because lactobacillus reuteri can suppress colibacillary growth and breeding; Thereby protected body to avoid the heavy damage that a large amount of breedings of intestinal bacteria cause effectively, reduced the severity of diarrhea disease percentage and diarrhoea, postponed the diarrhoea disease time simultaneously.
Table 8 lactobacillus reuteri preparation E is to the influence of 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.
Experimental example 6, lactobacillus reuteri (Lactobacillus reuteri) RI021 CGMCC No.4650 preparation are to the influence of early-weaned piglets gi tract microorganism species, nutrient digestibility
1 materials and methods
1.1 experimental animal and daily ration are handled
Select 48 28 ± 2d weanling pigs, body weight 7.67 ± 1.09kg is by completely random block design animal experiment; Test divides 2 processing, and each handles 6 repetitions, and 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 handles 3 circle boars, 3 circle sows.Handling 1 is test group, and basal diet adds the lactobacillus reuteri preparation F of 0.1% (mass content) embodiment 3; Handling 2 is control group, and basal diet adds the 50mg/kg carbadox, and table 9 is seen in each arrangement of handling.The duration of test feeding piglet is in fully closed child care piglet house, and the temperature in the house remains on 24～27 ℃.Free choice feeding, each hurdle circle are equipped with the duck-beak type water fountain and supply piglet freely to drink water.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, the strict health and epidemic prevention system of carrying out of feeding piglet control measures.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 the zeyssatite of interpolation 0.5% is as the digestibility of indicator mensuration nutritive substance in 1 all daily rations, and the basal diet composition is seen 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 to measure the microorganism species of content and mucous membrane; The content of getting stomach, duodenum, jejunum epimere, jejunum stage casing, jejunum hypomere, ileum, caecum, the colon section of falling and colon liter section is loaded in the plastics tubing, is placed in-80 ℃ of refrigerators.Testing last three days is that the fresh excreta of just having discharged from anus is collected by unit with the circle, is placed in-20 ℃ of refrigerators, measures the nutrient digestion rate.
1.3 microbiological analysis
The aseptic gi tract chyme 1g that takes by weighing different sites is dissolved in the SPSS of 99mL, in the bottle of SPSS, places 5～8 little granulated glass spherees.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 in rolling the pipe substratum accordingly, is carried out lactobacillus spp, bifidus bacillus and anaerobic bacteria culture.10
-2～10
-5Each extent of dilution is got the 0.3mL diluent carries out intestinal bacteria, enterobacteria and aerobic bacteria in corresponding plate culture medium counting.Mucous membrane microbioassay method is with reference to the method for Rojas and Conway (1996), the aseptic 1cm that gets
2Gastrointestinal mucosa to there not being the visible chyme, blots lip-deep water with aseptic filter paper with aseptic PBS solution flushing mucous membrane; With aseptic nipper tissue block is immersed the aseptic HEPES damping fluid of 10mL then; And constantly 5min is washed in vibration, and then carries out 10 times of dilutions step by step, up to 10
-3At each extent of dilution, get the 0.3mL diluent in corresponding flat board or roll the pipe substratum in.Mikrobe 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; Potassium hydrogenphosphate, 2g; Agar, 13g; Yihong-Y, 0.4g; Methylene blue, 0.065g; Zero(ppm) water, 1000mL; PH, 7.1), lactobacillus spp is with the 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 SULPHATE HEPTAHYDRATE 99.5,0.58g; Four water manganous sulfates, 0.25g; Ferrous sulfate, 0.03g; Tween-80,1g; Agar, 13g; 0.1% resazurin, 1mL; Zero(ppm) water, 1000mL; Regulate pH to 5.2 with acetic acid).Bifidus bacillus is with bifidus bacillus substratum (BM) (glucose, 20g; Trypticase, 20g; Yeast soaks powder, 10g; Peptone, 10g; Tomato juice, 333mL; Tween-80,2g; Zero(ppm) water, 1000mL; PH 6.6, lime carbonate, 10g; Halfcystine, 0.5g; 0.1% resazurin, 1mL).Aerobic bacteria and anerobes are with aerobic bacteria and anaerobic bacteria culture base (junket peptone, 15g; Glucose, 5g; The L-Gelucystine, 0.5g; Sodium thioglycollate, 0.5g; Yeast soaks powder, 5g; Sodium-chlor, 2.5g; 0.1% resazurin, 1mL; Agar, 13g, zero(ppm) water, 1000mL; PH7.1).Anerobes, lactobacillus spp and bifidus bacillus substratum 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 behind the cultivation 48h.Each extent of dilution is made three flat boards or is rolled pipe, with the flat board of 50～150 bacterium colonies or the extent of dilution work counting usefulness of rolling pipe.
1.4 chemical analysis takes out ight soil from refrigerator, after to be unit with the circle with ight soil mix, get and be positioned over about 300g in the aluminium box, the aluminium box is placed on forced air drying 96h in 65 ℃ the baking oven.After daily ration and faecal samples are pulverized, cross 40 mesh sieves.The chyme sample carries out lyophilize, is used to measure amino acid whose daily ration sample and chyme and pulverizes 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 appearance of PARR1281 (PARR Instrument Corp., the U.S.) to measure.Amino acid (removing sulfur-containing amino acid and tryptophane) is measured with automatic analyzer for amino acids (L-8800 of Hitachi, Japan) behind 110 ℃ of following 6mol/L hydrochloric acid hydrolysis 24h by standard GB/T18246-2000 (2001); Sulfur-containing amino acid (comprising methionine(Met) and halfcystine) according to standard GB/T15399-1994 (1996) 110 ℃ down with peroxyformic acid (1mL hydrogen peroxide+9mL formic acid) oxidation 16h after, use hydrochloric acid hydrolysis method mensuration; Tryptophane uses HPLC (Tianjin, island LC-10A, Japan) to measure behind 110 ℃ of following 4mol/L sodium hydroxide hydrolysis 24h according to standard GB/T18246-2000 (2001).The method of (1974) such as employing McCarthy is measured the salt acid insoluble ash.Promptly get about 10g sample, boil 30min with 100mL 4mol/L HCl after, with no ash content filter paper filtering, wash until anacidity, then more than 650 ℃ of following ashing 6h with boiling water.
1.5 digestibility calculation formula
Calculate the all-digestive tract digestibility of amino acid ileal digestibility or nutrient by following formula:
D＝100-(C1×P2)÷(C2×P1)×100
Wherein, D=amino acid ileal digestibility or nutrient all-digestive tract digestibility (%),
C1=cattle salt acid insoluble ash to be measured content (%),
Nutrient content in P2=chyme or the ight soil (%),
Salt acid insoluble ash content (%) in C2=chyme or the ight soil,
P1=feed nutrient content to be measured (%).
1.6 data statistics
(SPSS Inc., USA) statistics is handled in the check of the independent sample t of statistical software to test all The data SPSS9.0for Windows.
2 results and discussion
2.1 lactobacillus reuteri preparation F is to the influence of weanling pig digestive tube chyme microorganism species
Can be found out that by table 10 enterobacteria quantity raises (P＜0.01) at jejunum, caecum and the colon utmost point significantly, significantly raise at ileum (P＜0.05), and significantly reduce (P＜0.01) at duodenum does not have significant difference between two groups in stomach.Lactobacillus spp quantity all raises (P＜0.01) than the carbadox group utmost point at stomach, duodenum, jejunum and ileum significantly, and (P＜0.05) significantly raises at caecum.Bifidus bacillus quantity extremely significantly raises (P＜0.01) at stomach, jejunum, ileum and caecum, and (P＜0.05) significantly raises at duodenum and colon.The anerobes sum raises (P＜0.01) at stomach, duodenum, jejunum and the ileum utmost point significantly, in caecum and then significantly rising (P＜0.05) of colon.Some research reports show that probiotic lactobacillus can reduce enterobacteria and the intestinal bacteria quantity in weanling pig enteron aisle and the ight soil, and aspect the inhibition growth in piglets, these two kinds of bacterium play an important role.This result of experiment shows that lactobacillus reuteri preparation F can improve the quantity of lactobacillus spp, bifidus bacillus, anerobes, intestinal bacteria, enterobacteria and aerobic bacteria in the chyme of the most positions of digestive tube.
Table 10 lactobacillus reuteri preparation F is to the influence (unit: 1g CFU/ gram, weight in wet base) of weanling pig digestive tube chyme microorganism species
Annotate: the table intermediate value is a mean+SD.
2.2 lactobacillus reuteri preparation F is to the influence of nutrient digestion rate
Can find out 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 all-digestive tract apparent digestibilities such as energy, dry-matter, organism and calcium and amino acid terminal ileum apparent digestibility all do not had remarkably influenced (P＞0.05).Phytate phosphorus is the important obstruction material that influences absorption of nutrient ingredients in the feed, and lactic acid can be alleviated this inhibition, improves the utilization ratio of phosphorus.Lactobacillus reuteri has the ability (result sees embodiment 1) of higher generation lactic acid in this preparation when in-vitro screening.The result shows that the lactobacillus reuteri preparation is also little to the influence of the nutrient digestion rate of weanling pig.
Table 11 lactobacillus reuteri preparation F is to the influence (unit: %) of weanling pig daily ration nutrient apparent digestibility
Annotate: the table intermediate value is a mean+SD.
Experimental example 7, different strain liquid carrier adsorb prepared preparation long term storage stability at room temperature
1, experimental technique
Lactobacillus reuteri CGMCC No.4650 is inoculated in the glucose nutrient broth, and regulates pH to 6.7 with acetic acid, put 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.The proportioning of carrier and lactobacillus reuteri CGMCC No.4650 is 0.5 gram carrier in the preparation that is made into carrier: 10
9Cfu lactobacillus reuteri (Lactobacillus reuteri) RI021.
Be made into and measure spawn activity (concentration through bacterial classification in the preparation embodies) behind the preparation at once, placed for 1,6,12,18 week respectively under the normal temperature then after, measure spawn activity more respectively; Calculate and place the per-cent that the back spawn activity accounts for the preceding spawn activity of placement.
2, experimental result
As shown in table 12, explain that preparation of the present invention is more stable in carrier.
Table 12 different strain liquid carrier adsorbs the Journal of Sex Research steady in a long-term of prepared preparation
Claims (10)
1. lactobacillus reuteri (Lactobacillus reuteri) RI021 is numbered CGMCC No.4650 registering on the books of China Committee for Culture Collection of Microorganisms common micro-organisms center.
2. lactobacillus reuteri preparation, it is characterized in that: the activeconstituents of said preparation is the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021.
3. preparation according to claim 2 is characterized in that: said preparation has following at least a purposes:
1) product of microecological balance in the 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 digestion and the absorption of animal to nutritive substance;
5) preparation reduces the product of animal diarrhea disease percentage;
6) preparation improves the product of breeding performonce fo animals.
4. according to claim 2 or 3 described preparations, it is characterized in that: said preparation is made up of the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and carrier; Said carrier is at least a in powdered rice hulls, skim-milk, maltodextrin and the wheat bran.
5. preparation according to claim 4 is characterized in that: in the said preparation, the proportioning of the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and said carrier is: (10
7-10
10) cfu: (0.4-8) gram carrier;
Said preparation specifically can be A)-in F) any one:
A) the lactobacillus reuteri preparation A that is made up of the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation A, the proportioning of the two is the said carriers of 8 grams: 10
10The described lactobacillus reuteri of cfu claim 1 (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1;
B) the lactobacillus reuteri preparation B that is made up of the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation B, the proportioning of the two is the said carrier of 0.5 gram: 10
7The described lactobacillus reuteri of cfu claim 1 (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 2: 2: 1;
C) the lactobacillus reuteri formulation C of being made up of the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and said carrier, in the said lactobacillus reuteri formulation C, the proportioning of the two is the said carrier of 0.5 gram: 10
8The described lactobacillus reuteri of cfu claim 1 (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 2: 1: 1: 1;
D) the lactobacillus reuteri preparation D that is made up of the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation D, the proportioning of the two is the said carrier of 0.4 gram: 10
9The described lactobacillus reuteri of cfu claim 1 (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk, maltodextrin and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk, maltodextrin and wheat bran is 1: 1: 1: 1;
E) the lactobacillus reuteri preparation E that is made up of the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation E, the proportioning of the two is the said carrier of 0.4 gram: 10
7The described lactobacillus reuteri of cfu claim 1 (Lactobacillus reuteri) RI021; Said carrier is made up of skim-milk and wheat bran, and the mass ratio of said skim-milk and wheat bran is 1: 1;
F) the lactobacillus reuteri preparation F that is made up of the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and said carrier, among the said lactobacillus reuteri preparation F, the proportioning of the two is the said carrier of 0.4 gram: 10
10The described lactobacillus reuteri of cfu claim 1 (Lactobacillus reuteri) RI021; Said carrier is made up of powdered rice hulls, skim-milk and wheat bran, and the mass ratio of said powdered rice hulls, skim-milk and wheat bran is 1: 1: 1.
6. the animal-feed that contains arbitrary said preparation among the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and the claim 2-5.
7. animal-feed according to claim 6 is characterized in that: in the said animal-feed, contain 10 in the said animal-feed of every gram
7The described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 that cfu is above.
8. the following at least a purposes of arbitrary said preparation among the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 and the claim 2-5:
1) product of microecological balance in the 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 digestion and the absorption of animal to nutritive substance;
5) preparation reduces the product of animal diarrhea disease percentage;
6) preparation improves the product of breeding performonce fo animals.
9. the application of arbitrary said preparation in preparation intestinal bacteria suppressor factor among the described lactobacillus reuteri of claim 1 (Lactobacillus reuteri) RI021 or the claim 2-5.
10. application according to claim 9, the intestinal bacteria in the said intestinal bacteria suppressor factor are intestinal bacteria K88, e. coli k99 or intestinal bacteria 987P.
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| CN2011102857222A CN102304489B (en) | 2011-09-23 | 2011-09-23 | Lactobacillus reuteri strain and application thereof |
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