CN101974463B - Lactobacillus reuteri and composite viable bacteria preparation thereof - Google Patents
Lactobacillus reuteri and composite viable bacteria preparation thereof Download PDFInfo
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Abstract
The invention provides Lactobacillus reuteri L-HD 5 with the preservation number of CGMCC No.4050 and a composite viable bacteria preparation thereof. The viable bacteria preparation of the invention serves as a feed additive, can obviously improve daily gain of weaned pigs and feed efficiency after being used for feeding, lowers piglet diarrhea rate and improves economic benefit. The invention has popularization and application prospect and provides certain theoretical and practical basis for searching an antibiotic succedaneum.
Description
Technical field
The present invention relates to belong to the probiotics technical field, specifically relate to a kind of lactobacillus reuteri and compound active bacteria formulation thereof.
Background technology
Compound active bacteria formulation is a kind of as probiotics, has been widely used in livestock industry and has cultured.It mainly is made up of two or more different probiotic bacterium Pseudomonas.At present, the bacterial classification of using usually mainly is lactobacillus spp, bifidus bacillus, streptococcus faecium, genus bacillus and yeast etc.The probiotic bacterium combination that will have different biological functions; The compound active bacteria formulation of development through biological antagonist effect, biology take by force oxygen with and reach work such as enhancing immunity in order to keep the gi tract homeostasis; And probiotic bacterium can be in animal body growth metabolism; The various digestive ferments that produce can obviously be strengthened body to the digesting and assimilating of various nutritive ingredients in the feed, and improve the utilization of feed.
At present; The compound active bacteria formulation that is applied to pig is produced in the bacterial strain, and major part is a non-pig source probiotic bacterium, because the generation of microbial population of animal intestinal tract is the result of long-term evolution; A kind of probiotics that is suitable for certain type of animal not necessarily is suitable for another kind of animal, thereby causes effect to be a greater impact.
Lactobacillus reuteri (Lactobacillus reuteri) is the GI bacterium of being everlasting of healthy piglet; The foreign study report shows; Lactobacillus reuteri has stronger tolerance to hydrochloric acid in gastric juice and bile, the through gi tract that can maintain vigour, and can also secrete special antimicrobial substance Reuterin; Effectively suppress the growth of rotavirus and other enteropahtogenic microganisms, thereby prevent and treat diarrhoea.This bacterium is widely used in feeding micro-ecological preparation by Chinese scholars, has good market outlook.
Bifidus bacillus is one of topmost physiological bacterium in the humans and animals enteron aisle, has constituted microflora with other physiological bacterium member, and constitutes a microecosystem with the host.Bifidus bacillus gets into behind the enteron aisle rapidly in the intestines mucosa field planting, and combines to form microbial film, field planting and the invasion of prevention pathogenic bacterium with intestinal epithelial cell; Simultaneously, produce a large amount of lactic acid and acetate, improve environment in the intestines, keep normal intestinal peristalsis, impel pathogenic bacterium to excrete; The directly growth of pathogenic bacterium such as inhibition or antagonism dysentery bacterium, Corynebacterium diphtheriae, Pseudomonas aeruginosa, pathogenic colon bacillus, staphylococcus.Bifidus bacillus can be removed the poisonous and harmful meta-bolites that pathogenic bacterium discharge, and reduces the intracellular toxin source, suppresses the pathogenic flora growth, reduces level of endotoxin in the blood, and the adjustment microecological balance is prevented and treated endotoxemia.Bifidus bacillus also can be synthesized multivitamin (like VITMAIN B1, B2, B6, B12, nicotinic acid, folic acid etc.) and enzyme; Make the enteron aisle acidifying, help the absorption of iron, calcium and vitamins D, prevent the generation of iron-deficiency anaemia and vitamin D deficiency; Can replenish multiple amino acids and trace element.Can activate the cytophagous phagocytic activity of body, the anti pathologic immunity function of enhancing body.Because bifidus bacillus has many-sided beneficial natural disposition such as the microecological balance of keeping, biological antagonist, immunomodulatory, nutrition can; Be widely used in fields such as microorganism feed addictive, medical treatment, health care, food at present, received investigator and human consumer's favor at home and abroad.
In addition; Discover that through Chinese scholars the bacillus natto in the bacillus is because it can produce gemma; Thereby characteristics such as acidproof, alkaline-resisting, high temperature resistant and anti-extruding are arranged; In pelletization and gastric acid environment, all can keep the stability of height, be in the middle of the various probiotic bacteriums, better and one of bacterial classification of small intestine that can go directly to the environment tolerance.In addition, bacillus natto can also generate multiple protein enzyme, saccharifying enzyme, lypase, glycase, some complicated glucide in the degrading plant forage, thus the transformation efficiency of raising feed is regulated intestinal microflora, strengthens the cell immunoreceptor of animal.
Summary of the invention
The purpose of this invention is to provide a kind of lactobacillus reuteri and by the compound active bacteria formulation of its preparation.
Another object of the present invention provides the application of said lactobacillus reuteri and composite fungus agent thereof.
Lactobacillus reuteri of the present invention (Lactobacillus reuteri) L-HD5 separates from the chitling road; This bacterial strain has been preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on July 30th, 2010; Postcode 100101); It abbreviates CGMCC as, and deposit number is CGMCC No.4050.
The present invention also provides the microbial inoculum that contains lactobacillus reuteri (Lactobacillus reuteri) L-HD5.
Said microbial inoculum also comprises bifidus bacillus and bacillus natto.
Preferably, said microbial inoculum contains by weight:
Lactobacillus reuteri L-HD5 1-10 part
Bifidus bacillus 1-10 part
Bacillus natto 1-10 part.
More preferably, said microbial inoculum contains by weight:
1 part of lactobacillus reuteri L-HD5
1 part of bifidus bacillus
1 part of bacillus natto.
The present invention also provides the fodder additives that contains lactobacillus reuteri (Lactobacillus reuteri) L-HD5 or above-mentioned microbial inoculum.
The present invention further provides the method for the above-mentioned microbial inoculum of preparation; Comprise the steps: lactobacillus reuteri L-HD5, bifidus bacillus and bacillus natto are carried out following operation respectively: carry out liquid submerged fermentation and cultivate; Collect bacterium mud after the centrifugal treating; Through Vacuum Freezing & Drying Technology preparation and collection bacterium powder, at last that three kinds of bacterium powder are composite again.
Wherein, the fermentation culture conditions of said lactobacillus reuteri L-HD5 and bifidus bacillus is: 37 ℃ of temperature, and pH6.0～6.5, static cultivation, liquid amount 60%, inoculum size 3%, incubation time 16～24h, viable count is greater than 10
8CFU/mL.
The fermentation culture conditions of said bacillus natto is: 37 ℃ of temperature, and pH6.0, mixing speed 180rpm, liquid amount 20%, inoculum size 3%, incubation time 24～36h, viable count is greater than 10
8CFU/mL, the gemma rate of formation reaches more than 50%.
The present invention also provides lactobacillus reuteri (Lactobacillus reuteri) L-HD5 or the application of above-mentioned microbial inoculum in fodder additives.
Compound active bacteria formulation of the present invention can significantly improve equal day weight gain of weanling pig head and feed efficiency as fodder additives after raising, reduce the grice diarrhoea rate, increases economic efficiency.Have popularizing application prospect, and certain theory and practical basis are provided for seeking the microbiotic substitute.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Embodiment 1: the preparation of lactobacillus reuteri active bacteria formulation
Actication of culture
(Beijing Luqiao Technology Co., Ltd. produces in the MRS solid medium with lactobacillus reuteri freeze-drying lactobacillus (Lactobacillus reuteri L-HD5) streak inoculation; Lot number is: CM188) surface; 37 ℃ of anaerobism are cultivated 16～24h; Grow bacterium colony until the solid culture primary surface, microscopy is observed thalli morphology and purity.
The preparation of seed liquor
Picking MRS solid medium surface colony is inoculated in the test tube that the 5mL seed culture medium is housed, and 37 ℃ of anaerobism are cultivated 16～24h, treat that viable count reaches 10
8CFU/mL is by 2% (about 1 * 10
6CFU/mL) inoculum size is inoculated in the 250mL Erlenmeyer flask that the 150mL seed culture medium is housed, and 37 ℃ of anaerobism are cultivated 16～24h, treat that viable count reaches 10
8CFU/mL, subsequent use as seed liquor.
Seed culture medium is formed as follows: peptone 10g, Carnis Bovis seu Bubali cream 15g, yeast powder 5g, glucose 15g, dibasic ammonium citrate 2g, sodium acetate 5g, tween 80 1mL, K
2HPO
42g, MgSO
47H
2O 0.5g, MnSO
44H
2O 0.2g, zero(ppm) water adds to 1000mL.
Liquid submerged fermentation is cultivated
Select for use the 5L vertical digester to carry out liquid fermentation and culture.Wherein, the inoculum size of lactobacillus reuteri seed liquor is 3%, and liquid amount is after 60%, 37 ℃ of anaerobism is cultivated 20h, to measure every milliliter of viable count in the bacterium liquid, treats that viable count reaches 10
9CFU/mL is put a jar end fermentation culture.
Fermention medium is formed as follows: soy peptone 50g, and glucose 10g, yeast powder 17g, oligose 3g, zero(ppm) water adds to 1000mL, and pH 6.0～6.5.
The preparation of freeze-dried vaccine powder
Through centrifugal, concentrated, the production sequences such as adding lyophilized vaccine, vacuum lyophilization and the check of bacterium powder viable count of fermented liquid, finally prepare the powdery active bacteria formulation.
Wherein, the composition of lyophilized vaccine is following: sucrose 15g, and glycerine 3g, sorbyl alcohol 15g, skim-milk 15g, zero(ppm) water adds to 250mL; Bacterium mud (thalline after centrifugal the concentrating) is 1: 125 (g/mL) with the adding proportion of lyophilized vaccine, and the frozen-dried protective rate reaches more than 70%.
Finished product detection
Get the powdery active bacteria formulation 1g that has prepared, measure the viable count in every gram bacterium powder by GB GB/T 4789.35-2003 method, and the viable count in the bacterium powder is greater than 10
9CFU/g.
Embodiment 2: the preparation of bifidobacteria viable bacteria preparation
Actication of culture
With freeze-dried bifidobacteria kind (bifidobacteria infantis; Available from China Committee for Culture Collection of Microorganisms common micro-organisms center; Numbering: CGMCC No.1.2202) streak inoculation is in MRS solid culture primary surface; 37 ℃ of anaerobism are cultivated 16～24h, grow bacterium colony until the solid culture primary surface, and microscopy is observed thalli morphology and purity.
The preparation of seed liquor
Picking MRS solid medium surface colony is inoculated in the test tube that the 5mL seed culture medium is housed, and 37 ℃ of anaerobism are cultivated 16～24h, treat that viable count reaches 10
8CFU/mL is by 2% (about 1 * 10
6CFU/mL) inoculum size is inoculated in the 250mL Erlenmeyer flask that the 150mL seed culture medium is housed, and 37 ℃ of anaerobism are cultivated 16h～24h, treat that viable count reaches 10
8CFU/mL, subsequent use as seed liquor.
Seed culture medium is formed as follows: peptone 10g, Carnis Bovis seu Bubali cream 15g, yeast powder 5g, glucose 15g, dibasic ammonium citrate 2g, sodium acetate 5g, tween 80 1mL, K
2HPO
42g, MgSO
47H
2O 0.5g, MnSO
44H
2O 0.2g, zero(ppm) water adds to 1000mL.
Liquid submerged fermentation is cultivated
By 3% inoculum size, liquid amount 60% is inoculated in the 5L fermentor tank with bifidus bacillus seed liquid, and pH6.5 after 37 ℃ of anaerobism are cultivated 24h, measures every milliliter of viable count in the bacterium liquid, treats that viable count reaches 10
9CFU/mL is put a jar end fermentation culture.
Fermention medium is formed as follows: soy peptone 50g, and glucose 10g, yeast powder 17g, oligose 3g, zero(ppm) water adds to 1000mL, pH6.5.
The preparation of freeze-dried vaccine powder
Through centrifugal, concentrated, the production sequences such as adding lyophilized vaccine, vacuum lyophilization and the check of bacterium powder viable count of fermented liquid, finally prepare the powdery active bacteria formulation.
Finished product detection
Get the powdery active bacteria formulation 1g that has prepared, measure the viable count in every gram bacterium powder, record viable count in the bacterium powder greater than 10 by GB GB/T 4789.35-2003 method
9CFU/g.
Embodiment 3: the preparation of bacillus natto active bacteria formulation
Actication of culture
With the bacillus natto freeze-drying lactobacillus (available from China Committee for Culture Collection of Microorganisms common micro-organisms center; Numbering: CGMCC No.1.1086) (the extensive and profound in meaning star biotechnology in Beijing Ltd produces in nutrient agar in streak inoculation; Lot number is: 20060808) surface; 37 ℃ of aerobic cultivation 12～24h grow bacterium colony until media surface, and microscopy is observed thalli morphology and purity.
The preparation picking nutrient agar surface colony of seed liquor is inoculated in and the 5mL nutrient broth medium is housed (Beijing Luqiao Technology Co., Ltd. produces, and lot number is: in test tube CM106), cultivate 12～24h, treat that viable count reaches 10 for 37 ℃
8CFU/mL is by 2% (about 1 * 10
6CFU/mL) inoculum size is inoculated in the 250mL Erlenmeyer flask that 150mL nutrient broth liquid nutrient medium is housed, and cultivates 12～24h, treats that viable count reaches 10 for 37 ℃
8More than the CFU/mL, subsequent use as seed liquor.
Liquid submerged fermentation is cultivated
The bacillus natto seed liquor is inoculated in the 5L fermentor tank that fermention medium is housed by 3% inoculum size, pH6.0, mixing speed 180rpm behind 20%, 37 ℃ of aerobic cultivation 24～36h of liquid amount, measures every milliliter of viable count in the bacterium liquid, treats that viable count reaches 10
8More than the CFU/mL, the gemma rate of formation reaches more than 80%, and (skimmer is a vegetables oil, addition 1%, unit: g/ml) to put a jar end fermentation culture.
Fermention medium is formed as follows: peptone 10g, and beef powder 3.0g, NaCl 5.0g, glucose 1.0g, zero(ppm) water adds to 1L, pH6.0.
The preparation of freeze-dried vaccine powder
Through centrifugal, concentrated, the production sequences such as adding lyophilized vaccine, vacuum lyophilization and the check of bacterium powder viable count of fermented liquid, finally prepare the powdery active bacteria formulation.
Finished product detection
Get the powdery active bacteria formulation 1g that has prepared, measure the viable count in every gram bacterium powder, record viable count in the bacterium powder greater than 10 by GB NY-T 1461-2007 method
9CFU/g.
Embodiment 4: the preparation of compound active bacteria formulation
Three kinds of powdery active bacteria formulations that prepared completion are composite in 1: 1: 1 ratio of mass ratio, can obtain compound active bacteria formulation 45g, the active bacteria formulation finished product total viable count that after detection is composite, obtains is greater than 10
10CFU/g.
Effect: composite fungus agent of the present invention adopts the preparation of lyophilize resist technology, guarantees the preservation period of the high viable count of product significantly, and the total viable count of product is not less than 10 in 1 year
7CFU/g.
Experimental example 1: the application experiment of lactobacillus reuteri L-HD5 freeze-dried powder preparation
Select 36 of the healthy weanling pigs of 35 ages in days at random, maternal paternal line is landrace; 36 piglets are divided into two groups at random, and test group one is a lactobacillus reuteri L-HD5 freeze-dried powder preparation group, the piglet diet of feeding (not containing microbiotic)+lactobacillus reuteri L-HD5 freeze-dried powder preparation group (addition be basal diet 1%); Test group two is a control group, the basal diet of feeding, and test period is 30d, the immunity of piglet is undertaken by pig farm routine immunization program.Final experimental data is following:
Test group one every piglet average daily gain is 0.415Kg, and control group is 0.360Kg, and test group improves day weight gain 7.1% than control group.
Test group one every average daily material consumption of piglet is 0.668Kg, and control group is 0.700Kg, and test group reduces daily material consumption 2.3% than control group.
Test group one diarrhea rate is 12.5%, and control group is 32.2%, and test group reduces diarrhea rate 19.7% than control group.
Animal is fed to test and shows, can improve the weanling pig day weight gain to weanling pig Feeding Pig source lactobacillus reuteri L-HD5 freeze-dried powder preparation, reduces daily material consumption and diarrhea disease percentage.
Experimental example 2
Select 54 of the healthy weanling pigs of 35 ages in days at random, maternal paternal line is landrace; 54 piglets are divided into three groups at random, and test group one is a lactobacillus reuteri L-HD5 freeze-dried powder preparation group, the piglet diet of feeding (not containing microbiotic)+lactobacillus reuteri L-HD5 freeze-dried powder preparation group (addition be basal diet 1%); Test group two is compound active bacteria formulation group, the piglet diet of feeding (not containing microbiotic)+embodiment 4 prepared compound active bacteria formulations (addition be basal diet 1%); Test group three is a control group, the basal diet of feeding, and test period is 30d, the immunity of piglet is undertaken by pig farm routine immunization program.Final testing data is following:
Test group one every piglet average daily gain is 0.415Kg, and test group two every piglet average daily gains are 0.563Kg, and control group is 0.360Kg, and test group one improves day weight gain 7.1% than control group, and test group two improves day weight gain 22% than control group.
Test group one every average daily material consumption of piglet is 0.668Kg; Test group two every average daily material consumptions of piglet are 0.690Kg; Control group is 0.700Kg, and test group one reduces daily material consumption 2.3% than control group, and test group two reduces daily material consumption 0.7% than control group.
Test group one diarrhea rate is 12.5%, and test group two diarrhea rates are 9.6%, and control group is 32.2%, and test group reduces diarrhea rate 19.7% than control group, and test group two reduces diarrhea rate 22.6% than control group.
Application experiment shows, can significantly improve the weanling pig day weight gain to the weanling pig compound active bacteria formulation of feeding, and reduces daily material consumption and diarrhea disease percentage.
The more single lactobacillus reuteri preparation of compound active bacteria formulation benefit comes into force with remarkable.This possibly be because three kinds of probiotic bacteriums in the compound active bacteria formulation have synergistic function.
At first, bacillus natto belongs to aerobic bacteria, in the growth and breeding process, consumes the oxygen in the environment behind the entering enteron aisle, can suppress the growth of aerobic pathogenic bacterium, but help the breeding of anerobess such as lactobacillus reuteri and bifidus bacillus; Secondly, the growth of lactobacillus reuteri and bifidus bacillus has increased the acidity in the enteron aisle again, has more strengthened the inhibition to the pathogenic bacterium growth.So the more single bacteria preparation of compound active bacteria formulation has shown the more significant benefit usefulness that comes into force.
Claims (10)
1. lactobacillus reuteri (Lactobacillus reuteri) L-HD5, its deposit number is CGMCC No.4050.
2. the microbial inoculum that contains the said lactobacillus reuteri of claim 1 (Lactobacillus reuteri) L-HD5.
3. the fodder additives that contains the said lactobacillus reuteri of claim 1 (Lactobacillus reuteri) L-HD5 or the said microbial inoculum of claim 2.
4. the method for preparing the said microbial inoculum of claim 2; Comprise the steps: lactobacillus reuteri L-HD5, bifidus bacillus and bacillus natto are carried out following operation respectively: carry out liquid submerged fermentation and cultivate; Collect bacterium mud after the centrifugal treating; Through Vacuum Freezing & Drying Technology preparation and collection bacterium powder, at last that three kinds of bacterium powder are composite again.
5. method according to claim 4 is characterized in that, said lactobacillus reuteri L-HD5, bifidus bacillus and three kinds of bacterium powder of bacillus natto are according to composite in 1: 1: 1 ratio of mass ratio.
6. method according to claim 4 is characterized in that, vacuum lyophilization is to add the 125ml lyophilized vaccine respectively in the bacterium mud of every gram lactobacillus reuteri L-HD5, bifidus bacillus and bacillus natto; Its 250mL of described lyophilized vaccine is through taking by weighing sucrose 15g, glycerine 3g, and sorbyl alcohol 15g, skim-milk 15g puts into volumetric flask, and adding distil water obtains to 250mL.
7. method according to claim 4 is characterized in that, the fermentation culture conditions of said lactobacillus reuteri L-HD5 and bifidus bacillus is: 37 ℃ of temperature; PH6.0～6.5, static cultivation, liquid amount 60%; Inoculum size 3%, incubation time 16～24h, viable count is greater than 10
8CFU/mL.
8. method according to claim 4 is characterized in that, the fermentation culture conditions of said bacillus natto is: 37 ℃ of temperature, and pH6.0, mixing speed 180rpm, liquid amount 20%, inoculum size 3%, incubation time 24～36h, viable count is greater than 10
8CFU/mL, the gemma rate of formation reaches more than 50%.
9. any product that said method makes of claims 4-8.
10. the said lactobacillus reuteri of claim 1 (Lactobacillus reuteri) L-HD5 or the application of the said microbial inoculum of claim 2 in fodder additives.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102304489B (en) * | 2011-09-23 | 2013-06-12 | 北京龙科方舟生物工程技术有限公司 | Lactobacillus reuteri strain and application thereof |
| WO2015170159A1 (en) * | 2014-05-05 | 2015-11-12 | Giovanni Mogna | A composition for use in treating or preventing viral or bacterial infections in a subject undergoing anti-tumor chemotherapy, leukemia treatment or aids therapy comprising l. reuteri ler03 and/or l. salivarius ls06. |
| CN107047933B (en) * | 2016-08-30 | 2019-11-12 | 安徽省农业科学院畜牧兽医研究所 | A kind of preparation method and application of solid-state lactobacillus reuteri probiotics |
| CN106359904A (en) * | 2016-11-25 | 2017-02-01 | 广东省农业科学院动物科学研究所 | Application of lactobacillus reuteri in replacement of feed antibiotic |
| CN107475117A (en) * | 2017-07-03 | 2017-12-15 | 桐乡恒基生物股份有限公司 | A kind of freeze drying protectant for lactobacillus reuteri and lyophilized store method |
| CN108102976B (en) * | 2018-02-05 | 2020-10-27 | 富乐顿生物工程科技（北京）有限公司 | Lactobacillus reuteri SS23-27 and application thereof in preparation of pure probiotic yogurt |
| CN110269175A (en) * | 2019-06-05 | 2019-09-24 | 嘉兴浙华质检技术服务有限公司 | A kind of spraying micro-ecology preparation and preparation method thereof for feed anticorrosion |
| CN115141766A (en) * | 2022-05-26 | 2022-10-04 | 广州朴成生物科技有限公司 | Application of composite microecological preparation in preparation of preparation for repairing intestinal tract injury caused by weaning stress of piglets |
| CN115141765A (en) * | 2022-05-26 | 2022-10-04 | 广州朴成生物科技有限公司 | Composite microecological preparation for repairing intestinal tract injury caused by piglet weaning stress and preparation method thereof |
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