CN101671634A - Rhamnose lactobacillus M8, rhamnose lactobacillus SLP and preparation method thereof - Google Patents
Rhamnose lactobacillus M8, rhamnose lactobacillus SLP and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a strain of rhamnose lactobacillus M8 and a strain of rhamnose lactobacillus SLP prepared from the same. The preparation method of the rhamnose lactobacillus SLP comprises thefollowing steps: taking the rhamnose lactobacillus M8 as a host, guiding lactobacillus surface protein genes into the host by an electrotransformation method, and incubating to successively express the lactobacillus surface protein genes in the host. The adhesive capability of the rhamnose lactobacillus SLP is greatly strengthened in the intestinal canal of a human body so as to efficiently strengthen the positive effect of the rhamnose lactobacillus in the alimentary canal of the human body.
Description
Technical field
The present invention relates to a kind of lactobacillus rhamnosus bacterial strain, also relate to a kind of engineering bacteria of lactobacillus rhamnosus and preparation method thereof.
Background technology
At present, utilize S-layer proteins (being S-layer albumen) this characteristic of field planting in human intestinal, existing abroad part Study and report, some has been applied to clinical treatment, such as the serious diarrhoea of the caused baby of helicobacter pylori infection etc., its effect highly significant.Yet the research of China aspect probiotic bacterium field planting cultivation is made slow progress always, and only the field planting ability to pathogenic micro-organism had relevant research aspect medical science.In recent years, obtain domestic scholars and popular affirming for the application of functional probiotic bacterium on food, also emerge in an endless stream at the functional foodstuff aspect this field, how to improve the positive effect of these functional foodstuffs in human body alimentary canal, just cause the concern of domestic scholars gradually.
Milk-acid bacteria is a kind of human intravital probiotic bacterium that is present in, and is that the fermenting carbohydrate primary product is the general name that a class of lactic acid does not have gemma, gram-positive bacterium, and it is gained the name because of carbohydrate being fermented into lactic acid; The milk-acid bacteria of the overwhelming majority all is requisite and a flora with important physiological function in the human body, it extensively is present in the enteron aisle of human body, can help digest, help the health of human body intestines and internal organs of the body, so milk-acid bacteria often is regarded as heath food and is added within the sour milk.Lactobacillus rhamnosus is a kind of in the milk-acid bacteria, and it can directly enter enteron aisle by hydrochloric acid in gastric juice, cholate smoothly, supports good digestive function, can set up enteron aisle advantage probiotic bacterium group fast.Lactobacillus rhamnosus has the effect of improvement to the acute gastroenteritis that rotavirus causes, can prevent and shorten the child and suffer from diarrhoea the time, regulates human intestinal, has the effect of the gastrointestinal health of improvement.Lactobacillus rhamnosus has been applied to the nutrient food of capsule, lozenge and powdery type.Lactobacillus rhamnosus also can be added in the food of functional demands such as sour milk, milk powder and lactobacillus drink.Yet,, thereby limited its application in functional foodstuff because that the lactobacillus rhamnosus enteron aisle sticks ability is relatively poor.
Therefore the milk-acid bacteria S-layer proteins can not digested by various digestive ferment in enteron aisle, and the biliary effect of ability, can consider to contain that the proteic milk-acid bacteria of S-layer is developed to probiotic bacterium or as the carrier of antigen-antibody.The more important thing is that existing big quantity research and experiment are all verified, S-layer albumen can also be used for the field planting of probiotic bacterium (especially milk-acid bacteria) at enteron aisle, can improve the adhesive capacity of probiotic bacterium in enteron aisle greatly.Yet, at present knownly contain the proteic milk-acid bacteria of S-layer and few, that includes among the GENBANK also is no more than 20 kinds, and the research of proteic function of S-layer that produces for these milk-acid bacterias and mechanism is very not thorough yet.Because the ability of the sticking power of these milk-acid bacterias differs, therefore seriously restricted the application of milk-acid bacteria in functional foodstuff.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, one strain lactobacillus rhamnosus (Lactobacillusrhamnosus) bacterial strain is provided, and provide by this bacterial strain by a kind of simple, strain lactobacillus rhamnosus SLP that low cost method prepares, this lactobacillus rhamnosus SLP ability of sticking in human intestinal strengthens greatly, can the positive effect of efficient hardening lactobacillus rhamnosus in human body alimentary canal.
For solving the problems of the technologies described above, basic ideas of the present invention screen from different separation source exactly and are rich in the proteic lactobacillus strain of S-layer, again S-layer albumen is purified and quantitative analysis, and S-layer protein coding gene be cloned into not contain in the proteic beneficial natural disposition milk-acid bacteria of S-layer (for example lactobacillus rhamnosus) it is efficiently expressed.For this reason, the present invention at first provides a strain lactobacillus rhamnosus M8, it is characterized in that: described lactobacillus rhamnosus M8 is stored in China Committee for Culture Collection of Microorganisms common micro-organisms center and preserving number is the bacterial strain of CGMCC No.3002.This bacterial strain is to separate to obtain from fermented meat, obtains by the plate streaking separation and purification, is accredited as lactobacillus rhamnosus through 16S rRNA.The depositary institution of this bacterial strain is the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, the depositary institution address is positioned at the Institute of Microorganism, Academia Sinica of Datun Road, Chaoyang District, Beijing City, preservation date is on April 7th, 2009, and this bacterial strain is named as lactobacillus rhamnosus M8 (Lr.M8).
The above-mentioned lactobacillus rhamnosus M8 that provides can be used for further preparing engineering bacteria of the present invention---lactobacillus rhamnosus SLP, and described lactobacillus rhamnosus SLP expresses the engineering bacteria that milk-acid bacteria S-layer proteins gene is arranged in the described lactobacillus rhamnosus CGMCC of claim 1 No.3002.Among the above-mentioned lactobacillus rhamnosus SLP, described milk-acid bacteria S-layer albumen preferably is meant lactobacterium helveticus (Lactobacillus helveticus) S-layer proteins, this lactobacterium helveticus strains separation is from daily edible pickles, obtain by the plate streaking separation and purification, be accredited as lactobacterium helveticus through 16S rRNA.Through our experiment repeatedly, discovery selects for use preferred lactobacterium helveticus S-layer albumen can stably import among the above-mentioned lactobacillus rhamnosus M8 of the present invention, and can stably obtain lactobacillus rhamnosus SLP of the present invention, and the lactobacterium helveticus S-layer albumen that the present invention selects for use is to the host bacterial nontoxicity, and its genetic resources also can be obtained simply, easily.
Among the above-mentioned lactobacillus rhamnosus SLP, described lactobacterium helveticus S-layer proteins accounts for 30%～60% of described lactobacillus rhamnosus SLP tropina.Existing lactobacillus rhamnosus does not secrete S-layer albumen, but the S-layer protein secretion of lactobacillus rhamnosus SLP provided by the invention increase greatly than the sugared Bacterium lacticum of wild-type sandlwood, compares the lactobacterium helveticus secretory volume and improves a lot yet.
The present invention also provides the preparation method of a kind of above-mentioned lactobacillus rhamnosus SLP, may further comprise the steps: with lactobacillus rhamnosus CGMCC No.3002 is the host, S-layer protein gene imported among this host by electric method for transformation, through described S-layer protein gene successful expression in this host behind the incubation, obtain lactobacillus rhamnosus SLP.
Among the preparation method of above-mentioned lactobacillus rhamnosus SLP, described S-layer protein gene is a pair of primer of stencil design with lactobacterium helveticus S-layer proteins gene preferably, and by obtaining behind the pcr amplification, the full-length gene order of described primer is as follows:
slp5F(5′-3′)CG?
ATGAAGAAAAATTTAAG BamH?I
slp5R(5′-3′)GG?
TTATTCAAAGTTAGCAA Kpn?I。
Among the preparation method of above-mentioned lactobacillus rhamnosus SLP, described pcr amplification reaction condition optimization is: 94 ℃～95 ℃ pre-sex change 3min～5min; 95 ℃ of sex change 1min; 60 ℃～50 ℃ annealing 30s～1min; 72 ℃ are extended 2min; 10 circulations; 95 ℃ of sex change 1min; 50 ℃ of annealing 1min; 72 ℃ are extended 2min; 25 circulations; 72 ℃ are prolonged 7min.
Among the preparation method of above-mentioned lactobacillus rhamnosus SLP, by our experiment and to subsequent detection result's contrast, the operational condition that described electricity transforms preferably includes: voltage 1500V～1800V, the electric shock time is 3ms～8ms.The specified operational procedure that electricity transforms can directly carry out according to the working specification of electricity conversion instrument.
Among the preparation method of above-mentioned lactobacillus rhamnosus SLP, the temperature during described incubation is preferably about 37 ℃, and the incubation time is preferably about 3h.
Among the preparation method of above-mentioned lactobacillus rhamnosus SLP, in the described incubation process and preparation finish the back and the lactobacillus rhamnosus SLP that makes is transformed the back all use in the culturing process and transform the back substratum, substratum comprises the component of following massfraction after this conversion: bone collagen polypeptide 0.5%～1.5%, yeast extract paste 0.5%, glucose 0.5%, KH
2PO
40.3%, tween 80 0.5%, Tomato juice 5%, sodium acetate 0.5% and Trisodium Citrate 0.2%; Culture temperature is 37 ℃, initial pH value of medium 6.0, rotating speed 150r/min, incubation time 48 hours.This conversion back substratum is that we prepare voluntarily at the characteristic of lactobacillus rhamnosus, and its culture effect is better.
Lactobacillus rhamnosus SLP after adopting polyacrylamide gel electrophoresis to above-mentioned cultivation detects, and the result shows a large amount of S-layer albumen and obtains expressing.
Compared with prior art, the invention has the advantages that: by transgenic technology, on lactobacillus rhamnosus, the field planting that makes milk-acid bacteria S-layer albumen be successfully applied to lactobacillus rhamnosus is cultivated with milk-acid bacteria S-layer albumen successful expression.Produce the lactobacillus rhamnosus SLP of preparation with the inventive method, its surface specific is expressed a large amount of S-layer albumen, and its field planting time in enteron aisle is prolonged greatly.In with the goods of this lactobacillus rhamnosus SLP as starter; because lactobacillus rhamnosus SLP has obtained relevant characterization of adsorption; help the field planting in the enteron aisle in human body of this probiotic bacterium body; make probiotic bacterium occupy the enteron aisle surface; repel erosion and the attack of pathogenic bacteria, the protection HUMAN HEALTH to the human intestinal superficial cell.Utilize expression to have the proteic lactobacillus rhamnosus SLP of S-layer to make functional foodstuff and not only can promote the growth of enteron aisle beneficial microorganism, improve HUMAN HEALTH, and can obtain the good social economic benefit.In addition, the preparation method of lactobacillus rhamnosus SLP provided by the invention is simple, can reduce production costs, and improves tiring of lactobacillus rhamnosus SLP.
A kind of lactobacillus rhamnosus bacterial strain (Lactobacillus rhamnosus M8), its depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC), preserving number is CGMCC No.3002, and preservation date is on April 7th, 2009.
Description of drawings
Fig. 1 is the electrophorogram that utilizes the polyacrylamide gel electrophoresis technology that the present embodiment result is detected, swimming lane 1 expression albumen Marker among the figure, bacterium liquid before swimming lane 2 expressions are induced, back bacterium liquid is induced in swimming lane 3 expressions, supernatant liquor after the swimming lane 4 expression supersound process, swimming lane 5 expression supersound process postprecipitations, swimming lane 6 expression GHCl handle supernatant liquor, swimming lane 7 expression GHCl handle precipitation, swimming lane 8 expression wild-type lactobacterium helveticus S-layer proteins.
Embodiment
Embodiment:
A kind of preferred implementation of the present invention is as follows.
Step 1, the following raw material of preparation and equipment:
Step 1.1 is prepared substratum
Step 1.1.1 prepares fresh MRS agar plate and MRS liquid nutrient medium, is mainly used in separation, actication of culture and the dilution of single bacterium colony.
Step 1.1.2 prepares to transform the back substratum, and its component comprises (mass percent): bone collagen polypeptide 0.5%～1.5%, yeast extract paste 0.5%, glucose 0.5%, KH
2PO
40.3%, tween 80 0.5%, Tomato juice 5%, sodium acetate 0.5% and Trisodium Citrate 0.2%; The pH value is 6.0.
Step 1.2 is prepared damping fluid: 0.5mol/L sucrose, 7mmol/L KH
2PO
4And K
2HPO
4, 1mmol/L MgCl
2, pH7.4.
Step 1.3 is prepared electric converting apparatus: high-voltage pulse electric shock conversion instrument.
Step 1.4 is obtained the SLP protein gene
Step 1.4.1 designs primer: with lactobacterium helveticus (ATCC 15009) S-layer proteins gene (the Genebank accession number: AJ388559) be template design primer, amplification S-layer proteins full-length gene, as follows:
slp5F(5′-3′)CG?
ATGAAGAAAAATTTAAG BamH?I
slp5R(5′-3′)GG?
TTATTCAAAGTTAGCAA Kpn?I
Bolded section is a restriction enzyme site.
Step 1.4.2 carries out pcr amplification reaction by following reaction scheme:
10×Buffer 5μL
dNTPs(10mM) 1μL
primer(20uM) 1μL
Template (100ng/ μ L) 1 μ L
Taq?DNA?polymerase 1μL
It is 50 μ L that ultrapure water adds to total amount
95 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min; 60 ℃～50 ℃ annealing 1min; 72 ℃ are extended 2min; 10 circulations; 95 ℃ of sex change 1min; 50 ℃ of annealing 1min; 72 ℃ are extended 2min; 25 circulations; 72 ℃ are extended 7min.
Detect PCR result by agarose gel electrophoresis, order-checking is finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, determines to obtain lactobacterium helveticus S-layer proteins gene (S-layer albumen full-length gene) behind the pcr amplification.
Above-mentioned gained S-layer albumen full-length gene is connected with plasmid pQE30 obtains plasmid DNA, order-checking is confirmed to make up errorless back and is imported in the e. coli jm109, detects expression, waits until this plasmid DNA standby after confirming to express.
Step 2, utilize above-mentioned raw materials and equipment to prepare lactobacillus rhamnosus SLP of the present invention
The single bacterium colony of step 2.1 a lactobacillus rhamnosus CGMCC of picking No.3002 from the above-mentioned MRS agar plate is inoculated in the above-mentioned MRS liquid nutrient medium 37 ℃ of overnight incubation.
The cultivation of step 2.2 competent cell
Overnight culture in the step 2.1 is carried out serial dilution 10
2～10
6Doubly, be inoculated in the above-mentioned MRS liquid nutrient medium that contains 2% glycine and preheating, 37 ℃ of standing over night are cultured to logarithmic phase makes OD600 reach 0.8～1.Again this overnight culture of 5mL being inoculated in 100mL contains in the above-mentioned MRS liquid nutrient medium of 2% glycine and preheating, 37 ℃ leave standstill and are cultured to the early stage OD600 of logarithm and reach 0.2～0.3, add 10 μ g sodium ampicillins (Ampicillin), 37 ℃ are continued to leave standstill and are cultured to OD600 and reach 0.4～0.5.
Step 2.3 centrifuge washing and cell are resuspended
The nutrient solution that obtains in the step 2.2 is transferred in the centrifuge tube, at room temperature the centrifugal 10min of 6000 * g; Abandon supernatant, use and twice of the isopyknic above-mentioned damping fluid washed cell of this nutrient solution.Use the above-mentioned damping fluid re-suspended cell of this nutrient solution volume 1/100 again, place and obtain the competent cell suspension on ice.
The competent cell suspension that step 2.4 is drawn the above-mentioned preparation of 100 μ L in the ice-cold micro-aseptic centrifuge tube of 0.5mL, ice bath 5min.Add 4 μ L～5 μ L above-mentioned plasmid DNA for preparing to be transformed (DNA concentration is 100ng/ μ L), ice bath 5min.The mixed solution of competent cell suspension and plasmid DNA is added in the ice-cold 2mm electric shock conversion cup, touch the electric shock cup and be positioned at electric shock glass bottom, and must not produce foam to guarantee mixed solution.Dry electric shock tank outer water of condensation and fog, the cup that will shock by electricity is put into the electric shock instrument.
Step 2.5 high-voltage pulse electric shock transforms
Regulate the electric shock instrument, making electricimpulse is 25 μ F, voltage 1.7kV, resistance 200 Ω, electric shock time 5ms.
The recovery of step 2.6 transformant
After electricimpulse finishes, from electric shock tank, take out the electric shock cup fast, add after the conversion of 5mL precooling in the substratum; Be transferred to behind the mixing under 37 ℃ of temperature and carry out incubation, leave standstill incubation 3h, make microbiotic (Ampicillin) marker gene of lactobacillus rhamnosus recovery and expression plasmid coding.
Step 2.7 transformant is cultivated and screening
Respectively power taking hit after the conversion cell stoste and with the electric shock transformant liquid 100 μ L that transform after the substratum dilution of back, be layered on after the conversion that contains 100 μ g Ampicillin in the culture medium flat plate, 37 ℃ of anaerobic conditions are inverted down and are cultivated; Occur bacterium colony in 24h～72h, tentatively being defined as is a kind of engineering bacteria bacterium colony of lactobacillus rhamnosus.
Step 3, analyzing and testing result
Step 3.1 abduction delivering
Single bacterium colony on picking above-mentioned steps 2.7 middle plateforms is confirmed to make up through BamHI and KpnI double digestion and order-checking and is carried out abduction delivering after errorless.
Colony inoculation after step 3.1.1 picking transforms in the substratum, 37 ℃, leaves standstill overnight incubation after 4mL contains the conversion of 100 μ g/mL Ampicillin;
The bacterium liquid that step 3.1.2 gets after the 2.5mL incubated overnight is inoculated in 2 * 50mL conversion back substratum (containing 100 μ g/mLAmpicillin), and 37 ℃, 300rpm is cultured to OD600 and reaches 0.5～0.7;
Before inducing, takes out step 3.1.3 1mL bacterium liquid (noninduced control), and centrifugal, precipitation is resuspended among 50 μ L1 * SDS-PAGE loading buffer, preserve the standby preceding bacterium liquid that obtains inducing for-20 ℃;
Step 3.1.4 adds sec.-propyl-β-D thiogalactoside (IPTG) to final concentration 1mM;
Step 3.1.5 continues to cultivate 4h～5h, gets the bacterium liquid after 1mL cultivates, and is centrifugal, precipitation is resuspended among 100 μ L1 * SDS-PAGE loading buffer again, preserves the standby back bacterium liquid that obtains inducing for-20 ℃;
Step 3.1.6 collects thalline with the centrifugal 10min of 4000 * g.
Step 3.2 supersound process
Step 3.2.1 is resuspended in the thalline of the collection among the step 3.1.6 in the 5mL sterilized water;
Step 3.2.2 adds a certain amount of N,O-Diacetylmuramidase, and to make final concentration be 1mg/mL, places 30min on ice;
Step 3.2.3200W～6 10s of 300W ultrasonication, each 10s at interval, entire operation is all carried out on ice;
Step 3.2.4 is centrifugal 20min～30min under 4 ℃, 10000 * g condition, carefully pours out supernatant (crude extract A, soluble protein), places to preserve on ice to obtain supernatant liquor after the supersound process;
Step 3.2.5 is resuspended in the 5mL sterilized water with the supersound process postprecipitation, is the suspension (crude extractB, insoluble protein) of insoluble substance.
Slightly carrying of step 3.3 S-layer proteins
The thalline of collecting among the step 3.1.6 is resuspended among the 3mL 5M GHCl room temperature treatment 30min.The centrifugal 10min of 13000 * g makes solubilized protein and bacterial cell surface isolation.GHCl is handled supernatant liquor to be changed in the new vial (sealing with dialysis membrane), or change in the dialysis tubing, the dialysis 24h under 4 ℃ with 5L distilled water or 50mM Tris-HCl damping fluid, about therebetween 12h changes water once, and gained is exactly the S-layer proteins crude extract in the dialysis tubing.
Step 3.4 utilizes the polyacrylamide gel electrophoresis technology to analyze
Utilize the polyacrylamide gel electrophoresis technology to bacterium liquid before inducing, induce that supernatant liquor after back bacterium liquid, the supersound process, supersound process postprecipitation, GHCl handle supernatant liquor, GHCl handles precipitation and analyzes, and analytical results and albumen Marker and wild-type lactobacterium helveticus S-layer proteins compared, the result is as shown in Figure 1.
As seen from Figure 1, the molecular weight of wild-type lactobacterium helveticus S-layer proteins (seeing swimming lane 8) is 48kDa, protein expression also occurred on swimming lane 3,4 and 6 the corresponding position in Fig. 1, and has found no protein expression in swimming lane 5 corresponding positions, illustrate that foreign gene has obtained expression, justacrine is outside born of the same parents.Swimming lane 2 and 3 shows that S-layer albumen obtains on the lactobacillus rhamnosus surface expressing; The S-layer albumen that swimming lane 4 and 5 explanations are expressed is soluble proteins, but not inclusion body; Swimming lane 6 and 7 explanation S-layer albumen have correctly been expressed the lactobacillus rhamnosus surface, but not are secreted in the liquid phase.From Fig. 1 we as can be seen, among the lactobacillus rhamnosus SLP of present embodiment preparation, the expression amount of lactobacterium helveticus S-layer proteins accounts for 50% of lactobacillus rhamnosus SLP tropina.
＜110〉Agricultural University Of Hunan
＜120〉lactobacillus rhamnosus M8, lactobacillus rhamnosus SLP and preparation method thereof
<160>2
<210>1
<211>25bp
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>
cggga?tccat?gaaga?aaaat?ttaag 25
<210>2
<211>25bp
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>
ggggt?acctt?attca?aagtt?agcaa 25
Claims (10)
1, a strain lactobacillus rhamnosus M8 is characterized in that: described lactobacillus rhamnosus (Lactobacillus rhamnosus) M8 is stored in China Committee for Culture Collection of Microorganisms common micro-organisms center and preserving number is the bacterial strain of CGMCC No.3002.
2, a strain lactobacillus rhamnosus SLP is characterized in that: described lactobacillus rhamnosus SLP expresses the engineering bacteria that milk-acid bacteria S-layer proteins gene is arranged in the described lactobacillus rhamnosus M8 of claim 1.
3, lactobacillus rhamnosus SLP according to claim 2 is characterized in that: described milk-acid bacteria S-layer proteins is meant lactobacterium helveticus (Lactobacillus helveticus) S-layer proteins.
4, lactobacillus rhamnosus SLP according to claim 3 is characterized in that: described lactobacterium helveticus S-layer proteins accounts for 30%～60% of described lactobacillus rhamnosus SLP tropina.
5, the preparation method of a kind of lactobacillus rhamnosus SLP as claimed in claim 2, may further comprise the steps: described lactobacillus rhamnosus M8 is the host with claim 1, milk-acid bacteria S-layer proteins gene is imported among this host by electric method for transformation, through described milk-acid bacteria S-layer proteins gene successful expression in this host behind the incubation, obtain lactobacillus rhamnosus SLP.
6, the preparation method of lactobacillus rhamnosus SLP according to claim 5, it is characterized in that: described milk-acid bacteria S-layer proteins gene is to be template design primer with lactobacterium helveticus S-layer proteins gene, by obtaining behind the pcr amplification, the full-length gene order of described primer is as follows:
slp5F(5′-3′)CG?GGATCC?ATGAAGAAAAATTTAAG BamH?I
slp5R(5′-3′)GG?GGTACC?TTATTCAAAGTTAGCAA Kpn?I。
7, the preparation method of lactobacillus rhamnosus SLP according to claim 6 is characterized in that: the condition of described pcr amplification reaction is: 94 ℃～95 ℃ pre-sex change 3min～5min; 95 ℃ of sex change 1min; 60 ℃～50 ℃ annealing 30s～1min; 72 ℃ are extended 2min; 10 circulations; 95 ℃ of sex change 1min; 50 ℃ of annealing 1min; 72 ℃ are extended 2min; 25 circulations; 72 ℃ are prolonged 7min.
According to the preparation method of claim 5 or 6 or 7 described lactobacillus rhamnosus SLP, it is characterized in that 8, the operational condition that described electricity transforms comprises: voltage 1500V～1800V, the electric shock time is 3ms～8ms.
9, according to the preparation method of claim 5 or 6 or 7 described lactobacillus rhamnosus SLP, it is characterized in that: the temperature during described incubation is 37 ℃, the time 3h of incubation.
10, according to the preparation method of claim 5 or 6 or 7 described lactobacillus rhamnosus SLP, it is characterized in that substratum comprises the component of following massfraction after the conversion of using in the described incubation process: bone collagen polypeptide 0.5%～1.5%, yeast extract paste 0.5%, glucose 0.5%, KH
2PO
40.3%, tween 80 0.5%, Tomato juice 5%, sodium acetate 0.5% and Trisodium Citrate 0.2%.
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| CN105053524A (en) * | 2015-07-14 | 2015-11-18 | 湖南农业大学 | Rapeseed dreg microbial fermentation feed and preparation method and application thereof |
| CN105394600A (en) * | 2015-11-24 | 2016-03-16 | 湖南农业大学 | Fermented fat and preparation method thereof |
| CN105454988A (en) * | 2015-11-24 | 2016-04-06 | 湖南农业大学 | Flavor beef pie and preparation method thereof |
| CN105920049A (en) * | 2016-06-02 | 2016-09-07 | 扬州大学 | Application of lactobacillus rhamnosus grx19 in regulation of intestinal floras |
| CN106520598A (en) * | 2016-10-25 | 2017-03-22 | 天益健康科学研究院（镇江）有限公司 | Lactobacillus rhamnosus and application |
| CN107217022A (en) * | 2017-07-18 | 2017-09-29 | 邓禹 | One plant of Lactobacillus rhamnosus and its application |
| CN109182166A (en) * | 2018-08-27 | 2019-01-11 | 南昌大学 | One plant has effects that the Lactobacillus rhamnosus of relief of constipation and its application |
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