CN104388325A - Feeding saccharomyces boulardii and applications thereof - Google Patents
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Abstract
The invention discloses feeding saccharomyces boulardii and applications thereof. Saccharomyces boulardii is screened out of soil. Through in vitro experimental detection, saccharomyces boulardii has good capacity of resisting salt, gastric acid, artificial intestinal juice and heat. The strain has been sent to the China Center for Type Culture Collection (CCTCC) to be collected, with a collection number of CCTCCNO:M2014211. A high-density fermentation product can be obtained by inoculating the strain to bran under the conditions that the ratio of the material to water is 1 to 2, the additive amount of glucoamylase is 200U/g, the additive amount of soluble starch is 2.4%, the additive amount of ammonium chloride is 0.9%, the fermentation temperature is 30 DEG C and the fermentation period is 60 hours. Through piglet experiments, saccharomyces boulardii can smoothly pass through the gastric and intestinal environments and conduce to giving play to the physiological activity functions in the intestinal tracts, maintaining intestinal microecological balance of animals, improving intestinal health, preventing constipation, controlling bacterial diarrhea and promoting growth of livestock and poultry.
Description
Technical field
The invention belongs to microorganism field, be specifically related to strain feeding cloth Laplace yeast strain and an application thereof.
Background technology
Cloth Laplace yeast by finding in Indonesia's lichee and being separated, after be confirmed as subspecies of yeast saccharomyces cerevisiae.Research shows that cloth Laplace yeast does not have pathogenicity bo, well can grow under 37 DEG C of high temperature, and has unique biological activity.Within 1962, play cloth Laplace yeast active bacteria formulation and be applied to treatment mankind diarrhoea, this yeast not only can degrade pathogenic bacterium produce toxin and in and bacteriotoxin, can also intestinal mucosa be stimulated thus strengthen immunologic function, simultaneously the balance of micro-ecological environment in regulating intestinal canal, stops the invasion and attack of pathogenic bacteria.
Along with developing rapidly of aquaculture, the restriction of feeding antibiotic uses, and ensures that animal productiong security system becomes particularly crucial to the research of green feed additive.Cloth Laplace yeast has due to it unique effect alleviating symptom of diarrhea, and 1993 beginnings for improving monogastric animal nutrition and health, also show good effect as microorganism feed addictive in animal cultivation.This yeast itself also has the various nutritive ingredient of rich in protein VITAMIN and the factor in addition, and for animal supplements various nutrition and somatomedin, promote its healthy growth, its development prospect is very wide.
The present invention is separated and obtains the cloth Laplace yeast SH94 that a strain has excellent prebiotic performance from Hua Zhong Agriculture University's orchard soil, and establishing with wheat bran is the high-density solid fermentation process of main matrix.Animal feeding trials shows, this microbial inoculum can improve efficiency of feed utilization, reduces diarrhea of weaned piglets rate, the potentiality having industrialized developing He apply.
Summary of the invention
The object of this invention is to provide a strain feeding cloth Laplace yeast (Saccharomyces boulardii), this bacterial strain was delivered in China typical culture collection center preservation on 05 19th, 2014, Classification And Nomenclature: cloth Laplace yeast (Saccharomycesboulardii) SH94, preserving number: CCTCC NO:M 2014211, address: Wuhan, China Wuhan University.
Present invention also offers a kind of method that high-density solid fermentation produces cloth Laplace yeast SH94, method is simple, easy.
Another object of the present invention there are provided cloth Laplace yeast (Saccharomyces boulardii) SH94 and is preparing the application in fodder additives.Feeding piglet experimental result display cloth Laplace yeast effectively can reduce feedstuff-meat ratio and significantly reduce diarrhea rate, promotes piglet growth.
In order to achieve the above object, the present invention takes following technical measures:
One strain feeding cloth Laplace yeast (Saccharomyces boulardii), screening obtains by the following method:
1) rhizosphere soil is gathered from Hua Zhong Agriculture University's orchard rhizosphere, by muddy for soil sample 30 DEG C of aerobic enrichment culture in peptone glucose (YPD) liquid nutrient medium containing 50 μ g/mL/mL penbritins, flat board is coated with after suitable dilution, according to the single bacterium colony (white of colonial morphology picking products of typical yeast bacterium, size 1-2mm, moistening protuberance, has ester fragrance), go out yeast by microscopy scalping.
2) checked order and the analysis of microsatellite locus YLR177W distinguished sequence by Physiology and biochemistry, 26S rDNA, finishing screen chooses strain cloth Laplace yeast (Saccharomyces boulardii) SH94.This bacterial strain was delivered in China typical culture collection center preservation on 05 19th, 2014, deposit number: CCTCC NO:M 2014211, address: Wuhan, China Wuhan University.
SH94 bacterial strain specifically has typical cloth Laplace yeast physiological and biochemical property: the bacterium colony that this bacterial strain is formed in the upper breeding of peptone agar glucose solid medium (YPD) is circular, large and moistening, is creamy white, surface elevation, smooth non-wrinkled, edge clear, growth is rapidly; Microscopy showed cell individuality is comparatively large, and in subsphaeroidal, polygon budding, can produce thecaspore.Physiological and biochemical property: glucose fermentation, semi-lactosi, maltose, sucrose, raffinose fermentation 1/3, does not assimilate nitrate, not decomposing urea, produces ester, resistance to 10% ethanol.
Molecular biology identification result display cloth Laplace yeast SH94 at microsatellite locus YLR177W distinguished sequence is: CTTAAACAACAGCTCCCAAAAATACTATCCACAGAAACAACAGCAGCAGCAGCAGC AGCAGCAGCAGCAACAACAAAGCATCTTTGACCCGGGAAGAAGATCTTCCTATATT TCTGATGCGCTGATTCAT
(CAG) in microsatellite locus YLR177W 9 appears in cloth Laplace yeast.Above-mentioned sequence size is the number of 130bp, CAG is 9, proves that this yeast is cloth Laplace yeast.Salt resistance ability experimental result shows, and cloth Laplace yeast SH94 upgrowth situation when NaCl massfraction is 11% is good, can also grow, can not grow when NaCl massfraction is 14% when NaCl massfraction is 13%.Resistance to simulated gastric fluid and intestinal juice capacity experimental result display cloth Laplace yeast SH94 process 2.5 hours viable counts in the synthetic gastric juice of pH2.0 to be reduced less than 10%, in the synthetic intestinal juice of pH6.8, then processes 2.5 hours viable counts without considerable change.Temperature capacity experimental result display cloth Laplace yeast SH94 is at 40 DEG C of constant temperature water bath process 6h, and survival rate can reach 60%, and at 70 DEG C of water bath processing 2min and after 80 DEG C of water bath processing 30s, the survival rate of bacterium liquid can maintain more than 60%.
A kind of cloth Laplace yeast high-density solid fermentation method, its step is as follows:
With transfering loop picking cloth Laplace yeast inclined-plane seed, inoculation YPD seed culture medium, cultivates 48h, OD value for 30 DEG C and reaches 2.0-5.0.
The seed liquor of cultivating is inoculated solid fermentation substratum by the inoculum size of 5% (V/W), and cultivate 60h for 30 DEG C, then adding 1.5% trehalose is protective material, and 45 DEG C of cryodryings are pulverized, and obtain feeding cloth Laplace yeast agent.
Solid fermentation culture medium prescription: wheat bran 1000g, material-water ratio 1:2, adds saccharifying enzyme 200U/g, Zulkovsky starch 2.4% (w/w), ammonium chloride 0.9% (w/w) by wheat bran weight.
Described material-water ratio is the mass ratio of wheat bran and water.
The application of cloth Laplace yeast agent SH94 in livestock and poultry cultivation, its application process is:
By a kind of cloth Laplace yeast agent separately or with other microbiobacterial agent compounds, add in animal and fowl fodder or drinking-water in 0.01% ~ 0.3% (w/w) ratio, the security in detoxification of cloth Laplace yeast is good, and the different steps of producing at livestock and poultry cultivation is fed and be may be used for improving intestinal health, prevents constipation, control bacterial diarrhea, promote intestinal absorption thus improve production performance.
Compared with prior art, the present invention has the following advantages:
The invention provides strain cloth Laplace yeast (Saccharomyces boulardii) SH94.This bacterial strain salt tolerant, resistant to gastric juice, resistance to intestinal juice ability by force, can be widely applied in livestock culture industry, can pass through gastroenteric environment smoothly, in enteron aisle, play physiological activity, can maintain animal intestinal micro-ecology balance, promote growth of animals or poultry.
The method of cloth Laplace yeast (Saccharomyces boulardii) SH94 high-density solid state fermentation provided by the invention, compared with liquid state fermentation, equipment is simple, technical difficulty is low, with low cost; Compared with common yeast active bacteria formulation, the microbial inoculum that system of the present invention is joined is matrix with wheat bran, and raw material is easy to get, and the product nutrient after fermentation enriches, and viable bacteria rate is higher.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further details.Embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.In following embodiment, method therefor is ordinary method if no special instructions.
Embodiment 1:
One strain cloth Laplace yeast SH94, screening obtains by the following method:
Strains separation and Physiology and biochemistry are identified:
Rhizosphere soil is gathered from Hua Zhong Agriculture University's orchard rhizosphere, by muddy for soil sample 30 DEG C of aerobic enrichment culture in peptone glucose (YPD) liquid nutrient medium containing 50 μ g/mL penbritins, flat board is coated with after suitable dilution, according to the single bacterium colony (white of colonial morphology picking products of typical yeast bacterium, size 1-2mm, moistening protuberance, there is ester fragrance), yeast is gone out by microscopy scalping, finishing screen selects a strain feeding cloth Laplace yeast (Saccharomyces boulardii), this bacterial strain was delivered in China typical culture collection center preservation on 05 19th, 2014, Classification And Nomenclature: cloth Laplace yeast (Saccharomyces boulardii) SH94, preserving number: CCTCC NO:M 2014211, address: Wuhan, China Wuhan University.
SH94 bacterial strain bacterium colony and thalli morphology: this bacterial strain is circular, large and moistening at the upper bacterium colony formed of breeding of peptone agar glucose solid medium (YPD), is creamy white, surface elevation is smooth non-wrinkled, edge clear, and growth is rapidly; Microscopy showed cell individuality is comparatively large, and in subsphaeroidal, polygon budding, can produce thecaspore.
Physiological and biochemical property: glucose fermentation, semi-lactosi, maltose, sucrose, raffinose fermentation 1/3, does not assimilate nitrate, not decomposing urea, produces ester, resistance to 10% ethanol.
Peptone agar glucose liquid nutrient medium (YPD): glucose 20g, Tryptones 20g, yeast powder 10g, water 1000mL, adjusts pH 7.0,115 DEG C of sterilizing 20min.
Peptone agar glucose solid medium (YPD): glucose 20g, Tryptones 20g, yeast powder 10g, agar 18g, water 1000mL, adjust pH 7.0,115 DEG C of sterilizing 20min.
The molecular biology identification of cloth Laplace yeast SH94
Be inoculated in YPD liquid nutrient medium by the bacterial classification SH94 that 80% glycerine in laboratory is preserved, 34h cultivated by 30 DEG C of shaking tables, is separated after rejuvenation for subsequent use through plate streaking.
Picking bacterial strain SH94 mono-bacterium colony one ring, is inoculated in 30 DEG C of shaking tables in YPD liquid nutrient medium and cultivates 34h.Extract test kit (tiangen) with yeast genes and extract DNA, and by the DNA concentration of micro-spectrophotometer Detection and Extraction.With the cerevisiae dna extracted for template, Boul3 (5'-CTTAAACAACAGCTCCCAAA-3') and Boul4 (5'-ATGAATCAGCGCATCAGA AAT-3') is primer, and PCR system is: ddH
2o 28 μ l, 5 × PCR buffer10 μ l, dNTP (10mmol/L) 5 μ l, Boul32 μ l, Boul42 μ l, Taq enzyme 1 μ l, template 2 μ l, PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 53 DEG C of annealing 20s, 72 DEG C extend 20s, total elongation 72 DEG C of 5min, 32 circulations.After reaction terminates, carry out 1.5% agarose gel electrophoresis detection to pcr amplification product, result obtains through amplification the specific band that size is about 100bp.Reclaim and this object band of purifying with axygen purification kit.Then the PMD-18 carrier of Mei Lian Takara company, enzyme disjunctor is: system PCR primer 1.0 μ l, PMD-18T carrier 0.5 μ l, ddH2O 3.5 μ l, Buffer5.0 μ l, and 22 DEG C connect 2h.Enzyme connects product conversion bacillus coli DH 5 alpha competent cell, converted product coating Amp/LB agar plate, cultivate 14h for 37 DEG C, to select in 5 transformants to the 1.5mL centrifuge tube that substratum is housed 37 DEG C, 250r/min shaking culture 8h, carry out bacterium liquid PCR with primer M13R (CAGGAAACAGCTATGACC) and M13F (TGTAAAACGACGGCCAGT) to verify, reaction system is: ddH
2o 16 μ l, 5 × PCR buffer2 μ l, dNTP (10mmol/L) 0.5 μ l, M13F 0.4 μ l, M13R 0.4 μ l, Taq enzyme 0.2 μ l, template 0.5 μ l, reaction conditions is: denaturation 94 DEG C of 5min, sex change 94 DEG C of 30s, anneal 53 DEG C of 25s, extend 72 DEG C of 25s, total elongation 72 DEG C of 5min, 32 circulations.After reaction terminates, carry out 1.5% agarose gel electrophoresis detection to pcr amplification product, bacterium liquid object stripe size being about the correspondence of 200bp is got 0.4ml and is transferred to Li Fei Bioisystech Co., Ltd (Shanghai) to carry out determined dna sequence (sequencing primer is M13R).
Sequencing result shows that the fragment sequence that bacterial strain SH94 is amplified by Boul3 and boul4 is:
CTTAAACAACAGCTCCCAAAAATACTATCCACAGAAACAACAGCAGCAGCAGCAGCAGCAGCAGCAGCAACAACAAAGCATCTTTGACCCGGGAAGAAGATCTTCCTATATTTCTGATGCGCTGATTCAT
This fragment is positioned at yeast saccharomyces cerevisiae microsatellite locus YLR177W, above-mentioned Sequentially continuous occurs that (CAG) number of times is 9, and (CAG) 9 in microsatellite locus YLR177W appears in cloth Laplace yeast, determine that bacterial strain SH94 is cloth Laplace yeast thus.
Embodiment 2:
The salt resistance ability analysis of cloth Laplace yeast SH94
Salts solution configures: the NaCl adding different amount in the PA bottle that 5mLYPD liquid nutrient medium is housed, makes massfraction be followed successively by 10%, and 11%, 12%, 13%, 14%, 15%.
By activated good bacterial strain with 1% inoculum size be inoculated in aforesaid liquid, 37 DEG C of shaking table constant temperature culture 34h, observe growth conditions.
Result to be presented at when NaCl massfraction is 11% cloth Laplace yeast SH94 still can well-grown, can also grow when NaCl massfraction is 13%, can not grow when NaCl massfraction is 14%, illustrate that this bacterial strain has good resistance to osmotic pressure ability, can keep active in the digestive tube that osmotic pressure is higher.
Embodiment 3:
The resistant to gastric juice capability analysis of cloth Laplace yeast SH94
The compound method of simulated gastric fluid: be add distilled water in the hydrochloric acid of 0.1kg/L to regulate pH to 2.0 at 16.4mL massfraction.1g stomach en-is added in every 100mL solution, after it fully dissolves, degerming with the millipore filtration of 0.22um.
Get the sub-liquid of cloth Laplace barms growing to the later stage centrifugal, the twice rear Eddy diffusion of the brine with 0.9%, with 10
7the amount of mL-1 is inoculated in simulated gastric fluid, in 37 DEG C of water-baths, hatch cultivation, is coated with YPD flat board measures viable count every 0.5h sampling dilution.
After result display cloth Laplace yeast SH94 stops 2.5h in the gastric juice of pH2.0, viable count still has 90%, shows that this bacterial strain has stronger tolerance to gastric juice.
Embodiment 4:
The capability analysis of resistance to simulated intestinal fluid of cloth Laplace yeast SH94
The compound method of simulated intestinal fluid: get 6.8gKH
2pO
4dissolve in 500mL distilled water, with massfraction 4g/LNaOH damping fluid regulator solution pH value to 6.8, be settled to 1000mL.1g trypsinase is added in every 100mL solution, after it fully dissolves, degerming with the millipore filtration of 0.22um.
Thalline after embodiment 3 being processed (be after gastric juice is cultivated, have the strain solution of 90% viable count) is again centrifugal, and Eddy diffusion again after the brine with 0.9% twice, with 10
7the amount of mL-1 is inoculated in simulated intestinal fluid, in 37 DEG C of water-baths, hatch cultivation, is coated with YPD flat board measures viable count every 0.5h sampling dilution.
After result display cloth Laplace yeast SH94 stops 2.5h in the simulated intestinal fluid of pH6.8, viable count is almost constant, shows that this bacterial strain has stronger tolerance to intestinal juice, can pass through gastric juice and arrive small intestine maintenance activity and play a role, be applicable to the feeding microbial inoculum of preparation.
Embodiment 5:
Cloth Laplace yeast SH94 temperature capacity is analyzed
The temperature and time that cloth Laplace yeast liquid provides according to table 1 is heat-treated, is coated with the dull and stereotyped viable count measured after treatment of different temperature different time with the dilution of YPD substratum.
Table 1 cloth Laplace yeast liquid thermal treatment temp and time
Result display cloth Laplace yeast SH94 is at 40 DEG C of process 6h, and survival rate is to 60%, and at 70 DEG C of process 2min and after 80 DEG C of process 30s, survival rate maintains 60%, illustrates that this bacterial strain has certain temperature capacity.
Embodiment 6:
High-density solid fermentation produces the method for cloth Laplace yeast agent, and step is as follows:
With transfering loop picking solution cloth Laplace yeast inclined-plane seed, inoculation YPD liquid nutrient medium, cultivates 48h, OD for 30 DEG C
600nmvalue reaches 3.0, obtains seed liquor.
The seed liquor of cultivating is seeded to solid fermentation substratum by the inoculum size of 5% (v/m), leavening temperature 30 DEG C, fermentation period 60h, and viable count can reach 45.9 hundred million CFU/g siccatives.
The formula of above-described solid fermentation substratum is: wheat bran charge amount 30g, material-water ratio 1:2 (w/w), adds saccharifying enzyme 200U/g, Zulkovsky starch 2.4% (w/w), ammonium chloride 0.9% (w/w) by wheat bran weight.
Utilize tray (long 320mm, wide 240mm, dark 45mm) to amplify 10 times of fermentation culture cloth Laplace yeast, wheat bran charge amount is 300g, and after adopting above-mentioned technique to ferment, cloth Laplace yeast viable count can reach 32.3 hundred million CFU/g siccatives.
Adding 1.5% trehalose is protective material, and 45 DEG C of cryodryings are pulverized, and survival rate is more than 80%.
Embodiment 7:
Cloth Laplace yeast SH94 feeds and raises weanling pig effect assessment
Carry out as fodder additives (addition is in basal diet per ton) weanling pig effect analysis of feeding by cloth Laplace yeast agent (viable count 2,000,000,000/g), method is: select the weanling pig 225 that 35 age in days body weight are close, through checking different not remarkable (± 0.10kg) (P>0.05) of each process mesosome method of double differences.Piglet random number is divided into 5 groups, often organizes 3 repetition hurdles, 15, every hurdle (male and female is random).5 groups are respectively:
Blank group: basal diet;
Microbiotic group: basal diet per ton+microbiotic amoxycilline Trihydrate bp (50g);
Low dose group: add low dosage microbial inoculum (500g) in basal diet per ton;
Middle dosage group: dosage microbial inoculum (1000g) in adding in basal diet per ton;
High dose group: add high dosage microbial inoculum (2000g) in basal diet per ton.
Confined swine housing is raised, and temperature controls in 28 (± 3) DEG C.Test pig free choice feeding, drinking-water.Immunization is carried out by pig farm conventional procedure.Trial period is 28d.
Shown in result table 2, cloth Laplace yeast agent can improve efficiency of feed utilization (feedstuff-meat ratio reduces by 0.16), reduces diarrhea of weaned piglets rate (diarrhea rate reduces by 59.5%), can play and replace antibiotic effect.
Table 2 weanling pig is fed cloth Laplace yeast results
SEQUENCE LISTING
<110> Hua Zhong Agriculture University
<120> mono-strain feeding cloth Laplace yeast and application thereof
<130> mono-strain feeding cloth Laplace yeast and application thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 130
<212> DNA
<213> cloth Laplace yeast
<400> 1
cttaaacaac agctcccaaa aatactatcc acagaaacaa cagcagcagc agcagcagca 60
gcagcagcaa caacaaagca tctttgaccc gggaagaaga tcttcctata tttctgatgc 120
gctgattcat 130
Claims (7)
1. the cloth Laplace yeast be separated, is characterized in that: cloth Laplace yeast (
saccharomyces boulardii) SH94, preserving number: CCTCC NO:M 2014211.
2. the high-density solid fermentation method of cloth Laplace yeast according to claim 1, step comprises the inoculum size inoculation solid fermentation substratum of seed liquor by 5%V/W, and cultivate 60 h for 30 DEG C, then adding 1.5% trehalose is protective material, 45 DEG C of cryodryings are pulverized, and obtain feeding cloth Laplace yeast agent;
Described solid fermentation culture medium prescription: wheat bran 1000g, material-water ratio 1:2, adds saccharifying enzyme 200U/g, Zulkovsky starch 2.4%w/w, ammonium chloride 0.9%w/w by wheat bran weight.
3. described in claim 1, the application in animal feedstuff additive prepared by cloth Laplace yeast.
4. application according to claim 3, is characterized in that: described animal is piglet.
5. described in claim 1, the application in anti-diarrheal prepared by cloth Laplace yeast.
6. cloth Laplace yeast according to claim 1 is reducing the application in the low feedstuff-meat ratio of piglet.
7. cloth Laplace yeast according to claim 1 is promoting the application in piglet growth.
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