CN104293689A - Lactobacillus strain with high adhesive power, and application thereof - Google Patents
Lactobacillus strain with high adhesive power, and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
Abstract
The invention relates to field of feeding probiotics, and particularly relates to a lactobacillus strain with high adhesive power, and an application thereof. The lactobacillus has a good adhesion function for suppressing pathogenic bacteria. The lactobacillus strain with high adhesive power is preserved in China General Microbiological Culture Collection Center (Institute of Microbiology Chinese Academy of Sciences, No.3 Yard No.1, West Beichen Road, Chaoyang District, Beijing 100101, China) on December 9th, 2013, with a preservation number of CGMCC No. 8542. A strain number is ZJ617; and a classification name is lactobacillus reuteri. The lactobacillus strain (ZJ617) with high adhesive power is a strain providing efficiently high adhesion power mode for studying an adhesion mechanism of probiotics or lactobacillus and has important practical significance for developing novel microecological preparations, applying microorganism feed additives, enhancing yoghourt quality, and improving microorganism steady state of human and animals.
Description
Technical field
The present invention relates to feeding field of probiotic bacteria, particularly plant height adhesive power lactic bacilli strains and an application thereof.
Background technology
At the mankind, the animal and plant of nature existence, no matter have a large amount of microorganism participation activities in body surface or body.Microorganism and host have respective Physiology and biochemistry system, but both also exist the relation of mutually promoting and mutually restricting.The normal physiological biochemical parameter of host and pathological parameter are all both interactional results.The enteron aisle of humans and animals is a microenvironment with species diversity, wherein has hundreds of kind microorganism species, and the interaction between different microorganisms flora forms enteric microorganism stable state.Normal people and animal body intestinal microflora composition relative constancy, and intestinal microflora imbalance in animal body can be caused when body is in external environment change or accepts stress reaction.
Probiotic bacterium (Probiotics) is enteric microorganism important composition, plays important prebiotic effect in enteron aisle.Probiotic bacterium is colonizated in humans and animals enteron aisle, reproductive system, can produce the general name of the active beneficial microorganism (bacterium or yeast) of definite health efficacy.Common probiotic bacterium comprises lactobacillus (Lactobacillus species), bifidus bacillus (Bifidobacterium species), suis (Streptococcus species) etc.The mechanism of probiotic bacterium performance function comprises to be improved body's immunity, intervene pathogenic micro-organism thus prevents organism infection, reparation body microorganism species stable state etc.
The vital movement of lactobacillus and humans and animals is closely bound up, is requisite important beneficial flora in humans and animals body, has important physiological function, desirable health care and medical effect.In different environments, it is useful for changing intestinal microflora by introducing lactobacillus, have been reported in much research from the nutrition of this method and treatment benefit, this benefit comprises pathogenic micro-organism in the utilization of the element that has additional nutrients, the minimizing of lactose intolerance, intestinal tract infections control and enteron aisle and suppresses.Be exactly that probiotic bacterium must can tolerate gastral strong acid and high cholate environment to one of selection major criterion of probiotic bacterium in the world, and there is good adhesive capacity competence exertion prebiotic effect.According to research reports, rhamnose lactic acid bacteria Lactobacillus rhamnosus GG (LGG) can Colonization on baby's small intestine, effectively can shorten the diarrhoea time of infants with rotavirus, having simultaneously and have good tolerance to gastral strong acid and high cholate environment, is at present by internationally recognized safe probiotic strain.
In order to play the promoter action of lactobacillus to body health, it must keep certain quantity in the gastrointestinal tract, must have the tolerance certain to the temperature in gi tract, pH, cholate and metal simultaneously, thus prevent from being got rid of fast.The adhesive capacity of lactobacillus to mucomembranous surface can be it provides competitive edge, and by adhering to intestinal epithelial cell attachment sites with pathogenic bacteria competitiveness thus to suppress the growth of pathogenic bacteria to be expanded numerous, this point balances extremely important for maintaining archenteric flora.Lactobacillus acido-philus strain blocks the combination of pathogenic bacteria and cytolemma target acceptor by the adhesion occupy-place of thalline itself; enterohemorrhagic Escherichia coli, Salmonella typhimurium can be suppressed the adhesion of intestinal epithelial cell and invasion and attack, thus play the effect of protection enteron aisle.Obviously, be that lactobacillus plays the prerequisite of prebiotic effect in the long-term field planting of digestive tube to the adhesive capacity of surface epithelial cell attachment sites.
The adhesivity of current research display probiotic bacterium both domestic and external and lactobacillus has strain specificity, and the type strain of high-adhesiveness energy is less, still lacks and adheres to relevant protection and Mechanism Study data.The adhesion studying bacterium is in vivo difficult, and the assessment carried out in in-vitro simulated system has been generally acknowledged method.
Summary of the invention
The invention provides a strain and be separated the lactobacillus with high adhesive power obtained from intestine of young pigs, it has good suppression pathogenic bacteria adhesive function.
The technical solution adopted for the present invention to solve the technical problems is:
One plant height adhesive power lactic bacilli strains, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preservation date on December 9th, 2013, deposit number CGMCC No.8542, strain number is ZJ617, and Classification And Nomenclature is Lactobacillus reuteri.
The present inventor obtains a plant height adhesive power lactic bacilli strains from the weanling pig enteron aisle separation screening of 28 ages in days, purifying.The biological property of this bacterial strain is as follows:
Morphological feature: form obvious bacterium colony on MRS substratum, diameter is between 0.1 ~ 2.0mm, and circular, oyster white, neat in edge, opaque, surface wettability is smooth, non-pigment.Examine under a microscope, strain cell is thin rod shape, not raw spore.Belong to gram-positive microorganism, do not move.
Cultural characteristic: optimum growth temperature 30 ~ 37 DEG C, amphimicrobian, during aerobic, growth is slow, and the initial pH of the most suitable growth is 5.7.Be grown in MRS substratum, medium component: peptone 10.0g/L, beef powder 8.0g/L, yeast powder 4.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.2g, manganous sulfate 0.04g/L, tween 80 1.0g/L.
Lactobacillus Lactobacillus reuteri ZJ617 provided by the invention, there is high adhesive power, there is the tolerance good to heat, acid, cholate and metal (copper and zinc) simultaneously, prove that this bacterial strain has good probiotic properties, can be used to develop probiotic products.Lactobacillus ZJ617 adheres to the competition of intestinal cell to pathogenic bacteria (as intestinal bacteria and Salmonellas), repulsion, expeling suppression aspect have significant effect, can be used to develop the microbial inoculum suppressing pathogenic bacteria.Can at research probiotic bacterium and the adherence mechanism of lactobacillus, and the aspect such as the research and development of probiotics is as type strain.The probiotic properties that this lactobacillus ZJ617 possesses because of it, will have broad application prospects, have very high social benefit, economic benefit and ecological benefits.
A kind of milk-product comprise as described in high adhesive power lactobacillus.
A kind of described application of high adhesive power lactic bacilli strains in fermentative production Yoghourt.
A kind of probiotic feed additive comprises described high adhesive power lactobacillus.The lactobacillus ZJ617 with high adhesive power can suppress pathogenic bacterium effect in animal intestinal, improves animal immunizing power.
As preferably, described probiotic feed additive also comprises carrier, and described high adhesive power lactobacillus and the mass ratio of carrier are 2-3:15-30.Probiotic microorganism fodder additives provided by the invention, in aquatic feeds, its addition is the 0.5-2% of feed gross weight; For in full price pig feed, its addition is the 2-5% of feed gross weight.The present invention can improve growth of animal performance, increases efficiency of feed utilization, reduces feed cost, increases animal ingestion amount, improves immunity function, reduces animal intestinal mortality, reaches the object improving meat quality.
As preferably, described carrier is any one or two or more mixtures in montmorillonite, oyster shell whiting, sweet potato starch and W-Gum.Montmorillonite, oyster shell whiting, sweet potato starch, W-Gum are the nutrition sources required for proliferation of probiotics, improve probiotics survival rate, the probiotics growth and breeding of stimulation of host own, and play protection and adhewsive action to thalline itself.
Concrete, the invention provides a kind of formula, lactobacillus ZJ617 mixes with the mass ratio of carrier by 3:16, and carrier is the mixture of montmorillonite, oyster shell whiting, sweet potato starch and W-Gum, and mass ratio is 1:1:1-2:1-2.Described lactobacillus ZJ617 and carrier, at 300 ~ 350 turns/min, stir 10 ~ 20min, mix, obtained probiotic microorganism fodder additives.
The invention has the beneficial effects as follows: the lactic bacilli strains ZJ617 with high adhesive capacity, for research probiotic bacterium or the adherence mechanism of lactobacillus provide effective high adhesive power type strain, simultaneously at development of new probiotics, using microbe fodder additives, strengthen Quality of Yoghourt, improve humans and animals microorganism stable state all there is important practice significance.
Accompanying drawing explanation
Fig. 1 is the microscopy picture of lactobacillus;
Fig. 2 is separated to obtain lactobacillus enteron aisle source distribution schematic diagram;
Fig. 3 is the biochemical identification partial results of lactobacillus;
Fig. 4 is the full length fragment amplification electrophoretogram of lactobacillus 16S rDNA;
Fig. 5 is the evolutionary tree collection of illustrative plates being separated acquisition 85 strain lactobacillus 16SrDNA;
Fig. 6 is the microscopy figure of high adhered strains ZJ617 and low adhered strains ZJ610;
Fig. 7 is that lactobacillus is to colibacillary Competitive assays result figure (CFU/well × 10
4);
Fig. 8 is that lactobacillus is to colibacillary repulsion result figure (CFU/well × 10
4);
Fig. 9 is that lactobacillus suppresses result figure (CFU/well × 10 to colibacillary removal
4);
Figure 10 is competitive inhibitory effect (CFU/well × 10 of lactobacillus to Salmonellas
4);
Figure 11 is repulsion restraining effect (CFU/well × 10 of lactobacillus to Salmonellas
4);
Figure 12 is lactobacillus and goes surface protein lactobacillus to expeling restraining effect (CFU/well × 10 of Salmonellas
4).
Embodiment
For further setting forth the present invention for the technique means reaching predetermined goal of the invention and take and effect, below in conjunction with accompanying drawing and preferred embodiment, the effect adhering to the competition of intestinal cell according to the lactobacillus ZJ617 with high adhesive power provided by the invention to pathogenic bacteria, repulsion, expeling suppress is described, is described in detail as follows.
In the present invention, if not refer in particular to, all parts, per-cent are weight unit, and the equipment adopted and raw material etc. all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the ordinary method of this area.
Embodiment 1:
The present invention obtains mucus and chyme from the weanling pig enteron aisle of 28 ages in days, rear coated plate Anaerobic culturel, single clone carries out purifying and cultivates the pure bacterium colony of acquisition, obtain lactobacillus 85 strain lactobacillus (part bacterial strain is as Fig. 1), wherein Jejunal chyme 36 strain, ileum chyme 3 strain, jejunum mucus 18 strain and ileum mucus 28 strain (as Fig. 2).
The pure bacterium obtained carries out methylene blue dyeing, microscope is utilized to go out milk-acid bacteria according to form preliminary screening, rear use 23 kinds of sugar-fermenting biochemical tubes confirm the kind of lactobacillus, and biochemical tube is lactose, maltose, N.F,USP MANNITOL, sucrose, pectinose, wood sugar, trehalose, raffinose, fructose, rhamnosyl, semi-lactosi, cellobiose, melibiose, melizitose, seminose, sorbyl alcohol, arabitol, salicin, Vitamin C2, gluconate, arginine hydrolysis, D-ribose, amygdaloside biochemical tube (partial results is as Fig. 3) respectively.By biochemical identification, confirm that bacterium of obtaining is lactobacillus according to color reaction.
Extract the genomic dna of 85 strain lactobacilluss, by forward primer and reverse primer, the total length to the 16srDNA of lactobacillus increases (Fig. 4), and checks order.
The nucleotide sequence of forward primer is selected from the one in SEQ ID No.1-SEQ ID No.4, i.e. 5 '-CACGGATCCAGTTTGAT (C/T) (A/C) TGGCTCAG-3 '; The nucleotide sequence of reverse primer is selected from SEQ ID No.5 or SEQ ID No.6, i.e. 5 '-GTGAAGCTTAGGG (C/T) TACCTTGTTACGACTT-3 '.
Lactobacillus 16S rDNA full length fragment amplification, length is about 1500bp, identical with expected results.The gene order recorded in sequence BLAST software and GenBank (EF088327) and RDP database carries out similarity analysis, and in Clustal X (1.8) routine package, carry out multiple sequence with 16S rRNA gene order close in GenBank and mate and arrange (Multiple alignments) and analyze, through finding that the lactobacillus that we obtain is mainly three kinds with Genbank comparison, Yue Shi lactobacillus (Lactobacillus johnsonill strain) 1 strain, saliva lactobacillus (Lactobacillus salivarius strains) 54 strains, lactobacillus reuteri (Lactobacillus reuteri strains) 30 strains.
Numbering ZJ is adopted to name bacterial strain.The Neighbor-Joining method constructing system in MAGA3.1 routine package is adopted to set (Fig. 5).
The present invention selects to be used for follow-up lactobacillus 40 strain carrying out CCL188 Caco-2 cell adhesion experiment.
Set up Caco-2 cell culture system, Growth of Cells in containing 5% foetal calf serum DMEM/F12 substratum in, 5%CO
237 DEG C of cultivations.Cell is inoculated in 6 orifice plate culture plates (1 × 10
6individual/hole), culturing cell abundance reaches 80%, rinses 3 times with phosphoric acid buffer PBS, adds not containing the DMEM/F12 substratum of foetal calf serum, in 5%CO
2cultivate under 37 DEG C of conditions, construct lactobacillus in analogue body and adhere to the optimal adhesion model of Caco-2 cell.Respectively the analysis of Caco-2 cell ability is carried out to 40 strain lactobacilluss of above-mentioned selection.Found that, wherein lactobacillus ZJ617 has high adhesive capacity, is 12.35CFU/Caco-2, is significantly higher than other bacterial strains, as low adhered strains ZJ610.High adhered strains ZJ617 and low adhered strains ZJ610 is shown in Fig. 6, and statistics is in table 1.
Table 1. lactobacillus adheres to quantity
To selected, there is high adhesive power bacterial strain ZJU617 and control strain LGG lactobacillus, carry out heat-resisting, bile tolerance, acidproof, CuSO respectively
4and ZnSO
4analyzing, calculating colony number or bacterium liquid OD value by cultivating certain hour.Bacterial strain tolerance analysis shows: high adhesive power lactobacillus ZJ617 and the LGG obtained compares, and all has good tolerance generally to heat, acid, cholate and metal (copper and zinc).
Table 2. lactobacillus is to heat, acid, cholate and metal (copper and zinc) tolerance
1, high adhered strains ZJ617 is to the competitive inhibition of the adhesion intestinal cell of E. coli K88
Above-mentioned high adhesive power bacterial strain ZJ617, selects low adhesive power bacterial strain ZJ610 for contrast simultaneously, selects University of Georgia of the rhamnose lactic acid bacteria LGG(U.S. to give) be standard control.Carry out lactobacillus and E. coli K88 to adhere to and compete, i.e. lactobacillus (1.0 × 10
6and intestinal bacteria (1.0 × 10 CFU)
6cFU) add containing Caco-2 cell (1.0 × 10 simultaneously
5cell) culture hole.Cultivate 2h at 37 DEG C, remove non-adherent bacteria.The aseptic PBS of Caco-2 cell cleans.Adherent bacteria with 0.05% Triton X-100 wash-out, elutriant is inoculated into culture medium culturing and counting after gradient dilution, analyze lactobacillus adhere to competition Microecological effects.
Lactobacillus the results are shown in Figure 7 to intestinal bacteria Competitive assays, and synthesis result is in table 3.The high-adhesiveness lactobacillus ZJ617 that separation obtains and lactobacillus LGG is under race condition, E. coli adhesion intestinal cell quantity significantly declines (P<0.05), and result shows that obtained lactobacillus ZJ617 has good restraining effect to intestinal bacteria.
Table 3. lactobacillus competition bacillus coli K88 adheres to the ability of Caco-2 cell
2, high adhered strains ZJ617 suppresses the repellency that E. coli K88 adheres to intestinal cell
Above-mentioned high adhesive power bacterial strain ZJ617, selects low adhesive power bacterial strain ZJ610 for contrast simultaneously, selects University of Georgia of the rhamnose lactic acid bacteria LGG(U.S. to give) be standard control.Carry out lactobacillus and E. coli K88 to adhere to and repel, i.e. lactobacillus (1.0 × 10
6cFU) add containing Caco-2 cell (1.0 × 10
5cell) culture hole.Cultivate 1h at 37 DEG C, do not stick the aseptic PBS eccysis of lactobacillus.Add intestinal bacteria (1.0 × 10
6cFU), 1h is cultivated at 37 DEG C.Adherent bacteria with 0.05% Triton X-100 wash-out, elutriant is inoculated into culture medium culturing and counting after gradient dilution, analyze lactobacillus adhere to repel Microecological effects.
Lactobacillus repels suppression to intestinal bacteria and the results are shown in Figure 8, and synthesis result is in table 4.The high-adhesiveness lactobacillus ZJ617 that separation obtains and lactobacillus LGG is under rejection condition, E. coli adhesion intestinal cell quantity significantly declines (P<0.05), and result shows that obtained lactobacillus ZJ617 has good restraining effect to intestinal bacteria.
Table 4. lactobacillus repels the ability that bacillus coli K88 adheres to Caco-2 cell
3, high adhered strains ZJ617 suppresses the expeling that E. coli K88 adheres to intestinal cell
Above-mentioned high adhesive power bacterial strain ZJ617, selects low adhesive power bacterial strain ZJ610 for contrast simultaneously, selects University of Georgia of the rhamnose lactic acid bacteria LGG(U.S. to give) be standard control.Carry out lactobacillus and E. coli K88 ad-hesion removal rate suppresses, i.e. intestinal bacteria (1.0 × 10
6cFU) add containing Caco-2 cell (1.0 × 10
5cell) culture hole.1h is cultivated, the aseptic PBS eccysis of non-adherent bacteria at 37 DEG C.Add lactobacillus (1.0 × 10
6cFU), 1h is cultivated at 37 DEG C.Adherent bacteria with 0.05% Triton X-100 wash-out, elutriant is inoculated into culture medium culturing and counting after gradient dilution, analyzes the Microecological effects that lactobacillus ad-hesion removal rate suppresses.
Lactobacillus suppresses to the results are shown in Figure 9 to the intestinal bacteria property removed, and synthesis result is in table 5.The high-adhesiveness lactobacillus ZJ617 that separation obtains and lactobacillus LGG is under removal property rejection condition, E. coli adhesion intestinal cell quantity significantly declines (P<0.05), and result shows that obtained lactobacillus ZJ617 has good restraining effect to intestinal bacteria.
The table 5. lactobacillus property removed suppresses bacillus coli K88 to adhere to the ability of Caco-2 cell
4, high adhered strains ZJ617 is to the competitive inhibition of the adhesion intestinal cell of Salmonellas S.enteritidis50335
Above-mentioned high adhesive power bacterial strain ZJ617, selects low adhesive power bacterial strain ZJ610 for contrast simultaneously, selects University of Georgia of the rhamnose lactic acid bacteria LGG(U.S. to give) be standard control.Carry out lactobacillus and Salmonellas S.enteritidis50335 to adhere to and compete, i.e. lactobacillus (1.0 × 10
6and Salmonellas (1.0 × 10 CFU)
6cFU) add containing Caco-2 cell (1.0 × 10 simultaneously
5cell) culture hole.Cultivate 2h at 37 DEG C, remove non-adherent bacteria.The aseptic PBS of Caco-2 cell cleans.Adherent bacteria with 0.05% Triton X-100 wash-out, elutriant is inoculated into culture medium culturing and counting after gradient dilution, analyze lactobacillus adhere to competition Microecological effects.
Lactobacillus the results are shown in Figure 10 to Salmonellas Competitive assays, and synthesis result is in table 6.The high-adhesiveness lactobacillus ZJ617 that separation obtains and lactobacillus LGG is under race condition, Salmonellas adheres to intestinal cell quantity and significantly declines (P<0.05), and result shows that obtained lactobacillus ZJ617 has good restraining effect to Salmonellas.
Table 6. lactobacillus competition Salmonellas 50335 adheres to the ability of Caco-2 cell
5, high adhered strains ZJ617 suppresses the repellency that Salmonellas S.enteritidis50335 adheres to intestinal cell
Above-mentioned high adhesive power bacterial strain ZJ617, selects low adhesive power bacterial strain ZJ610 for contrast simultaneously, selects University of Georgia of the rhamnose lactic acid bacteria LGG(U.S. to give) be standard control.Carry out lactobacillus and Salmonellas S.enteritidis50335 to adhere to and repel, i.e. lactobacillus (1.0 × 10
6cFU) add containing Caco-2 cell (1.0 × 10
5cell) culture hole.Cultivate 1h at 37 DEG C, do not stick the aseptic PBS eccysis of lactobacillus.Add Salmonellas (1.0 × 10
6cFU), 1h is cultivated at 37 DEG C.Adherent bacteria with 0.05% Triton X-100 wash-out, elutriant is inoculated into culture medium culturing and counting after gradient dilution, analyze lactobacillus adhere to repel Microecological effects.
Lactobacillus repels suppression to Salmonellas and the results are shown in Figure 11, and synthesis result is in table 7.The high-adhesiveness lactobacillus ZJ617 that separation obtains and lactobacillus LGG is under rejection condition, Salmonellas adheres to intestinal cell quantity and significantly declines (P<0.05), and result shows that obtained lactobacillus ZJ617 has good restraining effect to Salmonellas.
Table 7. lactobacillus repels the ability that Salmonellas 50335 adheres to Caco-2 cell
6, high adhered strains ZJ617 suppresses the expeling that Salmonellas S.enteritidis50335 adheres to intestinal cell
Above-mentioned high adhesive power bacterial strain ZJ617, selects low adhesive power bacterial strain ZJ610 for contrast simultaneously, selects University of Georgia of the rhamnose lactic acid bacteria LGG(U.S. to give) be standard control.Carry out lactobacillus and Salmonellas S.enteritidis50335 ad-hesion removal rate suppresses, i.e. Salmonellas (1.0 × 10
6cFU) add containing Caco-2 cell (1.0 × 10
5cell) culture hole.1h is cultivated, the aseptic PBS eccysis of non-adherent bacteria at 37 DEG C.Add lactobacillus (1.0 × 10
6cFU), 1h is cultivated at 37 DEG C.Adherent bacteria with 0.05% Triton X-100 wash-out, elutriant is inoculated into culture medium culturing and counting after gradient dilution, analyzes the Microecological effects that lactobacillus ad-hesion removal rate suppresses.
Lactobacillus suppresses to the results are shown in Figure 12 to the Salmonellas property removed, and synthesis result is in table 8.The high-adhesiveness lactobacillus ZJ617 that separation obtains and lactobacillus LGG is under removal property rejection condition, Salmonellas adheres to intestinal cell quantity and significantly declines (P<0.05), and result shows that obtained lactobacillus ZJ617 has good restraining effect to Salmonellas.
The table 8. lactobacillus property removed suppresses Salmonellas 50335 to adhere to the ability of Caco-2 cell
Conclusion: the lactic bacilli strains ZJ617 with high adhesive capacity, for research probiotic bacterium or the adherence mechanism of lactobacillus provide effective high adhesive power type strain, simultaneously at development of new probiotics, using microbe fodder additives, strengthen Quality of Yoghourt, improve humans and animals microorganism stable state all there is important practice significance.
The milk-product of embodiment 2 containing bacterial strain of the present invention
Composition of raw materials (in 1 ton):
Different probiotic bacterium bacterial classifications has different qualities and function more specifically, takes the rear probiotic effects to HUMAN HEALTH generation and is also not quite similar.The present invention is Lactobacterium acidophilum, on the basis of the combination of bifidobacterium lactis and bifidus longum bb three kinds of specific probiotic bacteriums, increase the lactobacillus ZJ617 with high adhesive power, be made into Yoghourt, result through carrying out the detection of relevant clinical medical science and assessment to sour-milk product of the present invention shows, Yoghourt containing described ZJ617 probiotic combinations of the present invention, it has the superior effect comprehensively promoting human intestinal health, this effect is simple with Lactobacterium acidophilum higher than what report in prior art, bifidobacterium lactis and bifidus longum bb are the effect sum combining the Yoghourt made, the Yoghourt of the probiotic combinations containing high adhesive power ZJ617 bacterial strain of the present invention is while having good mouthfeel, unobstructed enteron aisle can be played, improve intestinal microflora micro-ecological environment, promote the effect that nutritive ingredient is digested and assimilated, particularly stomachache is improved in promotion human intestinal health, improve abdominal distension, improve gasteremphraxis, improve stomach heavy, improve poor appetite, improve enteron aisle exhaust and/or improve the aspects such as defecation condition and all there is unusual effect, also can effectively improve micro-ecological environment in body in addition, the viable count of probiotic bacterium in remarkable increase body, reduce pathogenic bacteria as Salmonellas, the viable count of intestinal bacteria etc.
Above-mentioned milk-product formula provided by the invention, actually can carry out regulating other components according to demand, also only can comprise a kind of bacterial classification of high adhesive power lactic bacilli strains provided by the invention.
Embodiment 3
A kind of probiotic microorganism fodder additives comprises described high adhesive power lactobacillus ZJ617, this fodder additives is made up of lactobacillus ZJ617 and carrier, the quality of lactobacillus ZJ617 and carrier is than general control at 2-3:15-30, and carrier is any one or two or more mixtures in montmorillonite, oyster shell whiting, sweet potato starch and W-Gum.
Concrete, the present embodiment provides a kind of formula, and lactobacillus ZJ617 mixes with the mass ratio of carrier by 3:16, and carrier is the mixture of montmorillonite, oyster shell whiting, sweet potato starch and W-Gum, and mass ratio is 1:1:1:2.Described lactobacillus ZJ617 and carrier, at 300 ~ 350 turns/min, stir 10 ~ 20min, mix, obtained probiotic microorganism fodder additives.
Probiotic microorganism fodder additives provided by the invention, in aquatic feeds, its addition is the 0.5-2% of feed gross weight; For in full price pig feed, its addition is the 2-5% of feed gross weight.The present invention can improve growth of animal performance, increases efficiency of feed utilization, reduces feed cost, increases animal ingestion amount, improves immunity function, reduces animal intestinal mortality, reaches the object improving meat quality.
Above-described embodiment is one of the present invention preferably scheme, not does any pro forma restriction to the present invention, also has other variant and remodeling under the prerequisite not exceeding the technical scheme described in claim.
<110> Zhejiang A & F University
<120> mono-plant height adhesive power lactic bacilli strains and application thereof
<130> WHF001
<160> 6
<170> PatentIn version 3.3
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cacggatcca gtttgatcat ggctcag 27
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cacggatcca gtttgattat ggctcag 27
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cacggatcca gtttgatcct ggctcag 27
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cacggatcca gtttgattct ggctcag 27
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gtgaagctta gggctacctt gttacgactt 30
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gtgaagctta gggttacctt gttacgactt 30
Claims (6)
1. a plant height adhesive power lactic bacilli strains, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date on December 9th, 2013, deposit number CGMCC No.8542.
2. a probiotic feed additive comprises high adhesive power lactobacillus as claimed in claim 1.
3. probiotic feed additive according to claim 2, is characterized in that: also comprise carrier, and described high adhesive power lactobacillus and the mass ratio of carrier are 2-3:15-30.
4. probiotic feed additive according to claim 3, is characterized in that: described carrier is any one or two or more mixtures in montmorillonite, oyster shell whiting, sweet potato starch and W-Gum.
5. milk-product comprise high adhesive power lactobacillus as claimed in claim 1.
6. the application of a high adhesive power lactic bacilli strains according to claim 1 in fermentative production Yoghourt.
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|---|---|---|---|
| CN201410043216.6A CN104293689A (en) | 2014-01-29 | 2014-01-29 | Lactobacillus strain with high adhesive power, and application thereof |
Applications Claiming Priority (1)
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| CN201410043216.6A CN104293689A (en) | 2014-01-29 | 2014-01-29 | Lactobacillus strain with high adhesive power, and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105062933A (en) * | 2015-09-11 | 2015-11-18 | 北京博锦元生物科技有限公司 | Lactobacillus reuteri and application thereof |
| CN105219683A (en) * | 2015-11-04 | 2016-01-06 | 广东省农业科学院动物科学研究所 | One strain has L. reuteri strain and the application thereof of prebiotic characteristics |
| CN106036800A (en) * | 2016-06-29 | 2016-10-26 | 晋城市鸿生生物科技有限公司 | Production method of special starch for frying meat balls |
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| CN102586155A (en) * | 2012-03-07 | 2012-07-18 | 江南大学 | Lactobacillus plantarum N13 and use thereof |
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| WENMING ZHANG ET AL.: "Adhesive ability means inhibition activities for lactobacillus against pathogens and S-layer protein plays an important role in adhesion", 《ANAEROBE》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062933A (en) * | 2015-09-11 | 2015-11-18 | 北京博锦元生物科技有限公司 | Lactobacillus reuteri and application thereof |
| CN105219683A (en) * | 2015-11-04 | 2016-01-06 | 广东省农业科学院动物科学研究所 | One strain has L. reuteri strain and the application thereof of prebiotic characteristics |
| CN105219683B (en) * | 2015-11-04 | 2018-03-13 | 广东省农业科学院动物科学研究所 | One plant of L. reuteri strain with prebiotic characteristics and its application |
| CN106036800A (en) * | 2016-06-29 | 2016-10-26 | 晋城市鸿生生物科技有限公司 | Production method of special starch for frying meat balls |
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