CN105132322A - Lactobacillus plantarum and application thereof - Google Patents
Lactobacillus plantarum and application thereof Download PDFInfo
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- CN105132322A CN105132322A CN201510559899.5A CN201510559899A CN105132322A CN 105132322 A CN105132322 A CN 105132322A CN 201510559899 A CN201510559899 A CN 201510559899A CN 105132322 A CN105132322 A CN 105132322A
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Abstract
The invention discloses lactobacillus plantarum GLM101 and an application thereof in preparing a feed additive. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number CGMCC No. 11156. The lactobacillus plantarum GLM101 has very strong inhibiting ability for escherichia coli, staphylococcus aureus, salmonella typhi, vibrio vulnificus and aeromonas hydrophila, and also has excellent acid-resisting and cholate-resisting abilities. The lactobacillus plantarum GLM101 can be used for adjusting the microecological balance inside the intestines of animals, has the action of enhancing the nonspecific immunity function to prevent diseases, and also can provide trophic factors, promote digestive absorption of nutrients, promote growth of animals, and improve the feed conversion ratio.
Description
Technical field
The present invention relates to strain novel bacterial and an application thereof, be specifically related to the isolation identification of a lactobacillus plantarum GLM101, and the application in fungistat and fodder additives.
Background technology
In cultural technique field, antibiotic use can prevent Animal diseases effectively, reduces mortality of animals, reduces the risk that meat pollutes.But its abuse in animal-feed causes animal food antibiotic remains to exceed standard, animal and human's body is made to there is risk microbiotic being produced to resistance.Complete prohibition food animal was eaten microbiotic feed additive for promoting growth by European Union from 2006, and the risk that the health of microbiotic to animals and humans causes also has caused the attention of agricultural sector of China.In the antibiotic reasonable employment of cultivating link of strengthening management and animal foods, antibiotic remains exceeds standard, and becomes the focus that field of food safety is paid close attention to.
Probiotic bacterium is the normal microflora in animal intestinal, does not have toxic side effect to animal body.It is attached in animal intestinal, produces the material that some suppress growth of pathogenic bacteria on the one hand, in addition in growth metabolism process, can secrete multiple enzyme, promotes animal digesting and assimilating feed, improves feedstuff-meat ratio, promotes animal health growth.Therefore probiotics preparation becomes the focus of domestic and international industry research.
Lactobacillus plantarum belongs to lactobacillus genus, gram-positive microorganism, and optimum growth temperature is 30 ~ 37 DEG C, amphimicrobian, optimal pH about 6.5.The life of plant lactobacillus and the mankind is in close relations, is a kind of milk-acid bacteria be common in cream, meat and many vegetable fermentation goods, by stomach and be colonizated in enteron aisle play beneficial effect.It has material impact to enteric microorganism, no matter at food fermentation, or in fields such as industrial lactic fermentation and fodder additivess, all has a wide range of applications.
Summary of the invention
The object of this invention is to provide a lactobacillus plantarum LactobacillusplantarumGLM101.
Another object of the present invention is to provide above-mentioned plant lactobacillus preparing the application in fodder additives.
The technical solution used in the present invention is:
Bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center by applicant, depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, the bacterial strain that preservation center receives applicant provide on July 23rd, 2015.The preserving number that preservation center gives this culture is CGMCCNo.11156, and the Classification And Nomenclature of suggestion is plant lactobacillus Lactobacillusplantarum, and the bacterial strain in qualification preservation on July 23rd, 2015 is survival.
The invention has the beneficial effects as follows:
The invention provides a lactobacillus plantarum GLM101, it has significant restraining effect to intestinal bacteria, streptococcus aureus, salmonella typhi, Vibrio vulnificus and Aeromonas hydrophila; There is good acid resistance, can survive under the condition of pH2.0; There is bile tolerance ability, can grow under 1 ~ 3 ‰ cholate condition.
The lactobacillus plantarum provided of the present invention can be used for preparing fodder additives, and be applicable to should in the whole process of livestock and poultry, aquaculture.Bacterial strain GLM101 effectively can improve the production performance of child care pig, also can significantly improve the health level of swinery, reduces the use of microbiotic and other drug, saves cost.
Plant lactobacillus of the present invention can be used for regulating microecological balance in animal intestine, have and strengthen non-specific immune function prophylactic effect, nutritional factor can also be provided simultaneously, promote nutraceutically to digest and assimilate, promote growth of animal and improve food conversion ratio.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited thereto.
The separation of embodiment 1 plant lactobacillus GLM101, screening, qualification and preservation
The separation of 1.1 bacterial strains
Get sodium selenite faecal samples, 10,100,1000 times of dilutions are carried out respectively with sterilized water, get 200 μ L and coat MRS solid medium, cultivate 48 hours for 37 DEG C, picking colony, line MRS substratum, after 4 purifying, picking list bacterium colony is cultured to logarithmic phase to MRS meat soup, adds final concentration 20% glycerine, saves backup at-80 DEG C.Check order through gramstaining, catalase test, nitrate reduction test and 16SrDNA.
1.2 strain morphology characterized
Get single bacterium colony of purifying bacterial strain (called after GLM101), be transferred on MRS solid medium (agar), in 37 DEG C of constant incubators, cultivate 24h, 36h and 48h, observe the features such as the size of its bacterium colony, color, edge, projection, slickness, viscosity, transparency respectively.Result shows, and bacterial strain GLM101 forms neat in edge on MRS solid medium, smooth, thickness, surface luster, opaque bacterium colony.Examine under a microscope, for shaft-like.
The physiological and biochemical property of 1.3 bacterial strains
Picking bacterial strain GLM101, at the upper fresh culture thing cultivating 24h of MRS solid medium (agar), carries out Physiology and biochemistry test.Result shows, and GLM101 is Gram-positive bacillus.
1.3.1API20NE identification mark
Utilize French Mei Liai API20NE standard identification systems to measure bacterial strain GLM101 to utilize carbon source and produce sour situation, API20NE test bar is made up of the tubule that 20 contain dry substrate or substratum.
1) API20NEAUX medium component: (NH
4)
2sO
42g, agar 1.5g, inorganic salt basis 82.8mg, amino acid 250mg, VITAMIN and nutrition substrate 35.9mg, phosphoric acid buffer 0.04M adds to 1000ml, and final pH is 7.0 ~ 7.2;
NaCl0.85% substratum: NaCl8.5g, H
2o1000mL.
2) on flat board, cultivate bacterial strain, contain in the substratum of 0.85%NaCl from the pure single bacterium colony of picking the flat board of isolate 1 ~ 4 part to 2ml with inoculating needle, carefully grind well to reach homogeneous bacterial suspension, desired concn is 0.5 Maxwell unit norm turbidity.The ampoul tube opening AUX substratum adds the remaining physiological saline bacteria suspension of 200 μ L (6 ~ 8) to ampoule, carefully mixes, but bubble will have been avoided to produce.
3) the AUX substratum of salt solution bacteria suspension and inoculation adds corresponding test bar tubule respectively,
gLU,
aDHwith
cIREthen cover with mineral oil.
4) cover cultivation box, cultivate observe phenomena after 24 hours in 30 DEG C, result is as shown in table 1.
Table 1. bacterial strain GLM101 biochemical identification result
Note :+: represent that reaction is for positive;-: represent that reaction is for negative;
As can be seen from Table 1, bacterial strain of the present invention can glucose, amygdaloside, pectinose, saltpetre, polychrom, be carbon source to nitro-D-methylgalactose.
1.3.2APIZYM identification mark
Utilize French Mei Liai APIZYM system, can system and study bacterial strain GLM101 enzymatic productivity rapidly, APIZYM test bar is made up of 20 tubules, and its bottom is the upholder that a specialized designs contains enzyme substrates and damping fluid.Step is as follows:
1) choose Fresh bacterial cultures with 2ml sterile distilled water and prepare a bacteria suspension, its turbidity is between McFarlandNo5 and No6.
2) with suction pipe inoculation, in each cup of test bar, 2 samples (65 microlitre) are accessed.
3), after inoculation, 4 hours are cultivated in 37 DEG C of incubators.
4), after cultivating, 1 ZYMA reagent and 1 ZYMB reagent is added.Observe color and to record result as shown in table 2:
Table 2. bacterial strain GLM101 biochemical identification result
Note :+: represent that reaction is for positive;-: represent that reaction is for negative; W: represent that reaction is more weak.
As can be seen from Table 2, bacterial strain GLM101 of the present invention has functions such as producing L-LEU arylamine enzyme, α-amino-isovaleric acid arylamine enzyme, beta galactoside enzyme, alpha-gluconase activity enzyme, β-glucosaccharase, N-acetyl-glucosaminidase.
1.4 the molecular biology identification of bacterial strain
1) utilize sky to follow bacterial genomes extraction test kit (TIANAMPBacteriaDNAKit) to extract the genomic dna of bacterial strain GLM101, step is by described in shop instruction requirement.Utilize bacterial universal primers 27F, 1492R amplification bacterial strain 16SrDNA fragment.
2) primer pair sequence as above is, 1492R:TACCTTGTTACGACTT, 27F:AGAGTTTGATCCTGGCTCAG.
3) result of the 16SrRNA gene sequencing of bacterial strain is as SEQIDNO.1.NCBI finding after comparison, the similarity of itself and type strain Lactobacillusplantarumsubsp.plantarumATCC14917 (T) is reached for 99.93%, the relevant physiological and biochemical index of comprehensive bacterial strain GLM101 and molecular biology identification result, in conjunction with its morphological feature, we are by its called after LactobacillusplantarumGLM101, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in 7 months 23 days year in 2015, its deposit number is CGMCCNO.11156.
The functional screening of 1.5 bacterial strains
1.5.1 acid resisting test.
Early stage is chosen in experiment, and source separation and purification is to lactic bacterium strains in contrast from sodium selenite enteron aisle, plant, cheese, commercial like product etc., through qualifications such as gramstaining, catalase test, nitrate reduction test and 16SrDNA order-checkings, these lactic bacterium strains belong to enterococcus faecalis 4 strain, faecium 2 strain, lactobacillus fermentum 2 strain, lactobacterium casei 2 strain, Lactobacillus pentosus 2 strain, plant lactobacillus 5 strain, lactobacillus reuteri 3 strain, lactobacillus rhamnosus 3 strain, lactobacillus salivarius 2 strain, Lactobacterium acidophilum 1 strain respectively.16SrDNA order-checking compare of analysis the results are shown in following table 3.
Table 3. is correlated with the qualification result of separating obtained lactic bacterium strains
Experimental technique: with HCl regulates liquid substratum to pH2.0.By bacterial classification with the inoculum size of 5% (V/V) inoculate above-mentioned each process respectively, 37 DEG C of quiescent culture.After inoculating initial and inoculation, 120min gets in above-mentioned each process and cultivates bacterium liquid, carries out plate count, thus calculates the survival rate of bacterium, with the bacterium liquid of pH6.0 for contrast.Bacterium liquid viable count × 100% of the bacterium liquid viable count/pH6.0 of survival rate (%)=pH to be measured.Experimental result is in table 4.
Experimental result:
Table 4.GLM101 and the acidproof Experimental comparison results of other isolated strains
From experimental result, the survival rate when bacterial strain be separated is under pH2.0 condition is respectively 1.2% ~ 76.3%, and wherein the survival rate of bacterial strain GLM101 is the highest, reaches 76.3%.
Carry out acid resistance detection to bacterial strain GLM101 further, experimental technique is:
If 4 different pH value process, be 1.5,2.5,3.5,4.5 by HCl regulates liquid substratum to pH value respectively.By bacterial classification with the inoculum size of 5% (V/V) inoculate above-mentioned each process respectively, 37 DEG C of quiescent culture.In inoculation after initial and inoculation 30,60,90,120,150min gets in above-mentioned each process and cultivates bacterium liquid, carry out plate count, thus calculate the survival rate of bacterium, with the bacterium liquid of pH6.0 for contrast.Bacterium liquid viable count × 100% of the bacterium liquid viable count/pH6.0 of survival rate (%)=pH to be measured.Experimental result is in table 5.
The acidproof experimental result of table 5 bacterial strain GLM101
From experimental result, the bacterial strain be separated pH1.5,2.5,3.5,4.5 time survival rate be respectively 2.1%, 66.9%, 88.0%, 95.1%, illustrate that this bacterium has stronger acid resistance, minimum can tolerable pH range be 1.5 ~ 2.5.
1.5.2 bacteriostatic test
Experimental strain is chosen laboratory and is in earlier stage separated the plant lactobacillus of originating from plant, intestine of young pigs, commercial like product etc., and indicator strain chooses intestinal bacteria (EscherichiacoliATCC8739), streptococcus aureus (StaphylococcusaureusATCC6538), salmonella typhi (SalmonellatyphiCMCC (B) 50071), Vibrio vulnificus (VibriovulnificusATCC27562) and Pseudomonas aeruginosa (PseudomonasaeruginosaATCC9027).Adopt Odontothrips loti, coated respectively by indicator strain on MRS agar plate, every glass adds the lactic acid bacteria culture solution that 0.2mL cultivates 24h, cultivates in constant incubator, observes cultivation results, and measure antibacterial circle diameter size in plate, experimental result is in table 6.
Experimental result:
Table 6. bacterial strain GLM101 bacteriostatic experiment comparing result
Note: experiment repetition 3 times, result is three mean values.
From experimental result, the bacterial strain be separated has good restraining effect to common causative indicator, and wherein bacterial strain GLM101 is good with other originating species Bacterium lacticum to the bacteriostasis of indicator.
In bacteriostatic test, also have detected bacterial strain GLM101 of the present invention to the restraining effect of Pseudomonas aeruginosa, the Odontothrips loti that working method is same as above, experimental result shows, bacterial strain GLM101 group creates the inhibition zone that diameter is 17.5mm, and control group occurs without inhibition zone, the bacterial strain GLM101 further illustrating the present invention's separation has good restraining effect to common causative indicator.
1.5.3 the test of tolerance cholate.
Experimental strain is chosen laboratory and is separated the lactic bacterium strains (table 3) of originating from plant, intestine of young pigs etc. early stage, after strain culturing 16h, collects thalline, rinse 2 times by PBS solution at 4 DEG C of 12000g after centrifugal 4 minutes.Add in the PBS solution containing cholate 0.7% (w/v), after 37 DEG C of cultivation 4h, be diluted to 10
-4mRS is dull and stereotyped in concentration coating, after 37 DEG C of Anaerobic culturel 48h, observes colony growth situation on MRS flat board.
Result shows, and in all assay plate, only GLM101, GLM271, GLM274, GLM280, GLM321, GLM322, GLM344, GLM347 colony number exceedes and grows 1.0*10
8cFU/ml, wherein the survival volume of bacterial strain GLM101 is the highest, reaches 3.8*10
8cFU/ml, concrete outcome is in table 7.
The each bacterial strain of table 7. processes the survival bacterium amount after 4h through cholate 0.7% (w/v)
Note:----represent survival milk-acid bacteria bacterium colony Shuo≤1.0*10
6cFU/ml.
The preparation method of embodiment 2 bacterial strain GLM101 solid fermentation product
1) bacterial classification and seed liquor preparation thereof
Lactic bacterium strains GLM101 is in the activation of MRS agar plate, and picking list colony inoculation, in MRS liquid tube, after 37 DEG C of cultivation 24h, is transferred in MRS liquid nutrient medium with 2% inoculum size, as seed liquor after cultivating 24.
MRS substratum main component is peptone 10.0g, extractum carnis 10.0g, yeast extract paste 5.0g, diammonium hydrogen citrate 2.0g, glucose 20.0g, tween 80 1.0mL, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganous sulfate 0.25g, distilled water 1000mL, pH6.2 ~ 6.6.Optimal pH 6.0, optimum temperuture 37 DEG C, amphimicrobian.
2) inoculate
With 3%v/v inoculum size the seed liquor of preparation is inoculated into configuration high-density solid medium (containing wheat bran 20%, dregs of beans 24%, cane molasses 2%, gac 2%, calcium carbonate 3%, cycloheptaamylose 4% and water 45%, wherein per-cent is mass percent).Wheat bran 20%, dregs of beans 24%, cane molasses 2%, gac 2%, calcium carbonate 3%, cycloheptaamylose 4% and water 45%
3) ferment
Substratum sealed fermenting 48h at 30 ~ 37 DEG C of bacterial classification will be connected.Then tunning is dried 4.1h at 55 DEG C, obtain bacterial strain GLM101 solid fermentation product.
Get above-mentioned fermentating drying product, gradient dilution, coating MRS substratum, after Anaerobic culturel 48h, calculate colony number, the experimental result viable bacteria content shown in fermentating drying product of the present invention is 3.6*10
10cFU/g.
Embodiment 3 bacterial strain GLM101 solid fermentation product is on the impact of child care pig production performance
3.1 laboratory animal and grouping
Choose 32 age in days weanling pig 216 for experimental subjects, wherein each 108 of experimental group, control group, feed to 77 ages in days.Experiment to end of day April 18 in 2013, went through 45 days phases from 4 days March in 2013.
3.2 experimental design
Control group 1: basal diet does not add bacterial strain GLM101 solid fermentation product;
Control group 2: basal diet adds 0.1% market like product;
Experimental group: add 0.1% (mass percent) bacterial strain GLM101 solid fermentation product in basal diet, experiment detailed design is as shown in table 8.
Table 8. daily ration structure
3.3, results and analysis
(1) fermentation strain impact that child care pig body weight is increased
It is as shown in table 9 that body weight increases situation, experimental group initial weight 8.99kg/ head, the heavy 25.41kg/ head in end, day weight gain 364.89g; Control group initial weight 9.08kg/ head, the heavy 25.14kg/ head in end, day weight gain 356.89g.Experimental group day weight gain than control group 8.00g more than 1, at the many 0.36kg of the equal net gain of experimental session head.
The impact that table 9. fermentation strain increases child care pig body weight
(2) bacterial strain GLM101 is on the impact of child care pig incubation rate
Child care pig incubation rate situation is as shown in table 10, and experimental group chooses 108 piglets, and experimental session eliminates 4, dead 1, livestock on hand 103 at the end of experiment, and incubation rate is 95.37%; Control group chooses 108 weanling pigs, and experimental session eliminates 4, and dead 5, livestock on hand 99 at the end of experiment, incubation rate is 91.67%.Experimental group significantly improves than control group incubation rate.
Table 10 fermentation strain is on the impact of child care pig incubation rate
(3) bacterial strain GLM101 is on the impact of child care pig feedstuff-meat ratio
The feed consumption situation in child care stage is as shown in table 11, and in the child care stage, the head of experimental group all increases weight as 16.42kg, and the equal feed consumption of head is 30.37kg, and feedstuff-meat ratio is 1.85.Experimental group compares remarkable decline than the feedstuff-meat ratio of control group.
Table 11 fermentation strain is on the impact of child care stage feedstuff-meat ratio
3.4, effect discussion and explanation
This experiment show experimental group every child care pig comparatively basal feed control group mean body weight improve 0.36kg, average daily gain improves 8.0g, and dead mortality reduces 3.70%, and feedstuff-meat ratio reduces 0.47.Experiment proves, bacterial strain GLM101 effectively can improve the production performance of child care pig, and is significantly better than the market like product of contrast.The use of bacterial strain GLM101 can significantly improve the health level of swinery in addition, can reduce the use of microbiotic and other drug, can save cost equally.
In sum, plant lactobacillus of the present invention can be used for regulating microecological balance in animal intestine, have and strengthen non-specific immune function prophylactic effect, nutritional factor can also be provided simultaneously, promote nutraceutically to digest and assimilate, promote growth of animal and improve food conversion ratio.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
<110> Guangzhou Glam Biotechnology Co., Ltd.
<120> mono-lactobacillus plantarum and application thereof
<130>
<160>1
<170>PatentInversion3.5
<210>1
<211>1415
<212>DNA
<213> plant lactobacillus LactobacillusplantarumGLM101
<400>1
gcgtgctatacatgcaagtcgacgaactctggtattgattggtgcttgcatcatgattta60
catttgagtgagtggcgaactggtgagtaacacgtgggaaacctgcccagaagcggggga120
taacacctggaaacagatgctaataccgcataacaacttggaccgcatggtccgagcttg180
aaagatggcttcggctatcacttttggatggtcccgcggcgtattagctagatggtgggg240
taacggctcaccatggcaatgatacgtagccgacctgagagggtaatcggccacattggg300
actgagacacggcccaaactcctacgggaggcagcagtagggaatcttccacaatggacg360
aaagtctgatggagcaacgccgcgtgagtgaagaagggtttcggctcgtaaaactctgtt420
gttaaagaagaacatatctgagagtaactgttcaggtattgacggtatttaaccagaaag480
ccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggat540
ttattgggcgtaaagcgagcgcaggcggttttttaagtctgatgtgaaagccttcggctc600
aaccgaagaagtgcatcggaaactgggaaacttgagtgcagaagaggacagtggaactcc660
atgtgtagcggtgaaatgcgtagatatatggaagaacaccagtggcgaaggcggctgtct720
ggtctgtaactgacgctgaggctcgaaagtatgggtagcaaacaggattagataccctgg780
tagtccataccgtaaacgatgaatgctaagtgttggagggtttccgcccttcagtgctgc840
agctaacgcattaagcattccgcctggggagtacggccgcaaggctgaaactcaaaggaa900
ttgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagctacgcgaagaac960
cttaccaggtcttgacatactatgcaaatctaagagattagacgttcccttcggggacat1020
ggatacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccg1080
caacgagcgcaacccttattatcagttgccagcattaagttgggcactctggtgagactg1140
ccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctg1200
ggctacacacgtgctacaatggatggtacaacgagttgcgaactcgcgagagtaagctaa1260
tctcttaaagccattctcagttcggattgtaggctgcaactcgcctacatgaagtcggaa1320
tcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacacc1380
gcccgtcacaccatgagagtttgtaacacccaaag1415
Claims (7)
1. plant lactobacillus, its Classification And Nomenclature is
lactobacillusplantarumgLM101, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, and deposit number is CGMCC
no.11156.
2. plant lactobacillus described in claim 1
lactobacillusplantarumthe application of GLM101 in antibacterial.
3. plant lactobacillus described in claim 1
lactobacillusplantarumgLM101 is preparing the application in fodder additives.
4. prepare the method for plant lactobacillus solid fermentation product for one kind, it is characterized in that: the method be by plant lactobacillus bacterial classification according to claim 1 by 2 ~ 4% inoculum size be inoculated in high-density solid-state fermentation culture medium, sealed fermenting under the condition of 30 ~ 37 DEG C, again by tunning in 45 ~ 60 DEG C of oven dry;
The formula of described high-density solid-state fermentation culture medium is wheat bran 15% ~ 25%, dregs of beans 19% ~ 29%, cane molasses 1% ~ 3%, gac 1% ~ 3%, calcium carbonate 2% ~ 4%, cycloheptaamylose 2% ~ 6% and water 40% ~ 50%, and wherein percentage sign is mass percent.
5. a kind of method preparing plant lactobacillus solid fermentation product according to claim 4, it is characterized in that: the formula of described high-density solid-state fermentation culture medium is wheat bran 20%, dregs of beans 24%, cane molasses 2%, gac 2%, calcium carbonate 3%, cycloheptaamylose 4% and water 45%, wherein percentage sign is mass percent.
6. a plant lactobacillus solid fermentation product, is characterized in that: its preparation method is method according to claim 4.
7. a kind of plant lactobacillus solid fermentation product according to claim 6 is preparing the application in fodder additives.
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