CN103333829A - Preparation method of active components of high-moisture alfalfa ensiling bacterial agent - Google Patents
Preparation method of active components of high-moisture alfalfa ensiling bacterial agent Download PDFInfo
- Publication number
- CN103333829A CN103333829A CN2013102660497A CN201310266049A CN103333829A CN 103333829 A CN103333829 A CN 103333829A CN 2013102660497 A CN2013102660497 A CN 2013102660497A CN 201310266049 A CN201310266049 A CN 201310266049A CN 103333829 A CN103333829 A CN 103333829A
- Authority
- CN
- China
- Prior art keywords
- milk
- acid bacteria
- mrs
- bacterial strain
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 72
- 241000219823 Medicago Species 0.000 title claims abstract description 39
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 238000012360 testing method Methods 0.000 claims abstract description 28
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 26
- 241000196324 Embryophyta Species 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 244000199866 Lactobacillus casei Species 0.000 claims abstract description 19
- 235000013958 Lactobacillus casei Nutrition 0.000 claims abstract description 19
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 19
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 19
- 229940017800 lactobacillus casei Drugs 0.000 claims abstract description 19
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 19
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 9
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 7
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 166
- 241000894006 Bacteria Species 0.000 claims description 144
- 235000014655 lactic acid Nutrition 0.000 claims description 83
- 239000007788 liquid Substances 0.000 claims description 79
- 235000015097 nutrients Nutrition 0.000 claims description 61
- 239000007787 solid Substances 0.000 claims description 38
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 28
- 241000186660 Lactobacillus Species 0.000 claims description 27
- 229940039696 lactobacillus Drugs 0.000 claims description 27
- 229920001817 Agar Polymers 0.000 claims description 20
- 239000008272 agar Substances 0.000 claims description 20
- 239000000022 bacteriostatic agent Substances 0.000 claims description 20
- 230000003385 bacteriostatic effect Effects 0.000 claims description 20
- 239000002068 microbial inoculum Substances 0.000 claims description 18
- 230000004913 activation Effects 0.000 claims description 16
- 239000002054 inoculum Substances 0.000 claims description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 15
- 239000008103 glucose Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 15
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 14
- 230000003068 static effect Effects 0.000 claims description 13
- 102000016938 Catalase Human genes 0.000 claims description 12
- 108010053835 Catalase Proteins 0.000 claims description 12
- 125000006850 spacer group Chemical group 0.000 claims description 12
- 230000003213 activating effect Effects 0.000 claims description 11
- 238000001816 cooling Methods 0.000 claims description 11
- 244000005700 microbiome Species 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 239000011248 coating agent Substances 0.000 claims description 9
- 238000000576 coating method Methods 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- 229930003448 Vitamin K Natural products 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 235000019168 vitamin K Nutrition 0.000 claims description 8
- 239000011712 vitamin K Substances 0.000 claims description 8
- 150000003721 vitamin K derivatives Chemical class 0.000 claims description 8
- 229940046010 vitamin k Drugs 0.000 claims description 8
- 239000004310 lactic acid Substances 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 claims description 6
- 241000237502 Ostreidae Species 0.000 claims description 6
- 230000008901 benefit Effects 0.000 claims description 6
- 230000003115 biocidal effect Effects 0.000 claims description 6
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 claims description 6
- CUXQLKLUPGTTKL-UHFFFAOYSA-M microcosmic salt Chemical compound [NH4+].[Na+].OP([O-])([O-])=O CUXQLKLUPGTTKL-UHFFFAOYSA-M 0.000 claims description 6
- 235000020636 oyster Nutrition 0.000 claims description 6
- 239000011734 sodium Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 230000004083 survival effect Effects 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 229930182566 Gentamicin Natural products 0.000 claims description 3
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 239000012984 antibiotic solution Substances 0.000 claims description 3
- 210000000481 breast Anatomy 0.000 claims description 3
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 claims description 3
- 229960002588 cefradine Drugs 0.000 claims description 3
- RDLPVSKMFDYCOR-UEKVPHQBSA-N cephradine Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CCC=CC1 RDLPVSKMFDYCOR-UEKVPHQBSA-N 0.000 claims description 3
- 229960003405 ciprofloxacin Drugs 0.000 claims description 3
- 238000009792 diffusion process Methods 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 229960003276 erythromycin Drugs 0.000 claims description 3
- 238000011156 evaluation Methods 0.000 claims description 3
- 229960002518 gentamicin Drugs 0.000 claims description 3
- 235000011187 glycerol Nutrition 0.000 claims description 3
- 150000002314 glycerols Chemical class 0.000 claims description 3
- 230000005764 inhibitory process Effects 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 230000002062 proliferating effect Effects 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000013207 serial dilution Methods 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 150000004684 trihydrates Chemical class 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 16
- 235000013305 food Nutrition 0.000 abstract description 4
- 206010052804 Drug tolerance Diseases 0.000 abstract 1
- 238000003501 co-culture Methods 0.000 abstract 1
- 230000001629 suppression Effects 0.000 abstract 1
- 241000219793 Trifolium Species 0.000 description 27
- 239000000654 additive Substances 0.000 description 15
- 230000000996 additive effect Effects 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 238000004737 colorimetric analysis Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 150000001720 carbohydrates Chemical class 0.000 description 6
- 235000014633 carbohydrates Nutrition 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 235000019750 Crude protein Nutrition 0.000 description 4
- 239000004459 forage Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- MYKOKMFESWKQRX-UHFFFAOYSA-N 10h-anthracen-9-one;sulfuric acid Chemical compound OS(O)(=O)=O.C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 MYKOKMFESWKQRX-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000001408 fungistatic effect Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000002829 antibacterial sensitivity test Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Abstract
The invention discloses a preparation method of active components of a high-moisture alfalfa ensiling bacterial agent. The active components include lactobacillusplantarum WN5 and lactobacilluscasei SN3. The preparation method comprises the following steps of: 1, separating and purifying bacterial strains; 2, performing a yeast suppression test; 3, performing the drug sensitive test on the bacterial strains; 4, performing the co-culture test of the bacterial strains; and 5, identifying the bacterial strains. When the bacterial agent prepared from the active components prepared by the five steps is used for ensiling high-moisture alfalfa, the water content of the high-moisture alfalfa is up to 81.7 percent, the ensiling time reaches 240 days, the stem leaves of the alfalfa after ensiling are completely stored on the plant, the water content is little changed compared with that of the alfalfa before ensiling, and the protein content is increased; and meanwhile, the antibacterial effect of the ensiling agent is improved, and the hidden danger that the drug tolerance enters a food chain by virtue of the ensiled alfalfa to be spread is reduced.
Description
Technical field
The present invention relates to a kind of preparation method of microbe additive, relate in particular to a kind of preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents.
Background technology
Along with the continuous development of aquaculture and riseing of provision price, develop domestic demand best in quality, that the inexpensive forage feed of safety has become the transformation aquaculture style of economic increase.Clover is the forage grass of phytophagous animal high-quality, its main products is hay, but this production is drenched with rain often, fallen leaves, expensive drying plant and the puzzlement that consumes problems such as a large amount of energy, alfalfa ensilage not only can be avoided above puzzlement, also can reduce nutrient loss to keep the nutritive property of green forage.
Therefore, a lot of technology disclose the method for alfalfa ensilage, but because the nutritional character of clover self and colony characteristics cause the effect of alfalfa ensilage in existing disclosed technology or the method undesirable, subject matter is: one, during a part of technical requirements ensiling, need the water content that clover is wilted or airing to 65% is following, the clover in rainy season that the requirement of this water content will cause facing results can't carry out ensiling; Two, clover self is with a large amount of yeast and a spot of milk-acid bacterias, clover needs to use fast, the antibacterial strong lactic bacteria additive of breeding in ensilage, but the alfalfa ensilage product that add agent of lactic acid bacteria will enter food chain when feeding animals, reduce the risk of antibiotics resistance, need to estimate the antibiotics resistance of agent of lactic acid bacteria, namely agent of lactic acid bacteria is to antibiotic sensitivity test.
Feature when the problem that exists with lactic bacteria additive in view of above-mentioned alfalfa ensilage and alfalfa ensilage, prior art can not be finished simultaneously the high moisture content clover is carried out ensiling, improves the fungistatic effect of ensiling agent simultaneously and reduces resistance to enter food chain by the ensiling clover and the problem of the hidden danger propagated.
Summary of the invention
For solving deficiency of the prior art, the object of the invention is to provide a kind of preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents.
The present invention is for achieving the above object, and the technique means that adopts is: a kind of preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents, its activeconstituents be plant lactobacillus (
Lactobacillus plantarum) WN5 and lactobacterium casei SN3 (
Lactobacillus casei).
The preparation method of above-mentioned activeconstituents is:
One, the separation of bacterial strain and purifying: collect the leach liquor of mud, the ratio of 1:10 joins mixing in the sterilized water by volume, and serial dilution is coated on each dilution bacterium liquid 100 microlitres respectively in the MRS solid medium, and 37 ℃ of anaerobism are cultivated 48h; Single bacterium colony that the picking transparent circle is bigger, line obtains pure single bacterium colony 3 times continuously on the MRS solid medium; Whether picking list bacterium colony observes aerogenesis in the MRS liquid nutrient medium that does not add calcium carbonate, the aerogenesis bacterial strain is heterofermentative lactic bacteria, and the anaerogen strain is homofermentative lactic bacteria; Isolate homotype and the heterofermentative lactic bacteria strain of 175 purifying altogether; These bacterial strains insert in the MRS liquid nutrient medium, and 37 ℃ of anaerobism are cultivated 20h, and get 700 microlitre bacterium liquid wherein and add 300 microlitre sterile glycerols, mixing then ,-20 ℃ are frozen;
Two, press down the yeast test: takes out the milk-acid bacteria that preserves under-20 ℃ of conditions, get on the 5 microlitres injection MRS solid medium and be coated with, under the condition of 37 ℃ of temperature, cultivate 12～14h; With the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate 16～18h, the milk-acid bacteria that obtains activating; Inoculum size by 1% does not contain the milk-acid bacteria access that activates in the 250ml triangular flask of calcium carbonate MRS liquid nutrient medium, and liquid amount is 100ml, cultivates 16～18h, at this moment, contains 0.993 * 10 at least in every milliliter of MRS liquid nutrient medium for 37 ℃
10The cfu milk-acid bacteria; 5000 leave heart 5min, collect supernatant liquor, obtain bacteriostatics one; Inoculum size by 1% is equipped with the milk-acid bacteria access of activation in the 250 ml triangular flasks of MRS liquid nutrient medium, and liquid amount is 100 ml; Cultivate 16～18h, at this moment, contain 1.0 * 10 at least in every milliliter of MRS liquid nutrient medium for 37 ℃
10The cfu milk-acid bacteria; 5000 leave heart 5min, collect supernatant liquor, obtain bacteriostatics two; With yeast
Saccharomyces cerevisiae28 ℃ of static cultivation 48h obtain the yeast nutrient solution in the YPD liquid nutrient medium; Double-deck agar plate punch method is measured the bacteriostasis of above-mentioned bacteriostatics one, and double-deck agar plate punch method is measured the bacteriostasis of above-mentioned bacteriostatics two;
Three, the drug sensitive test of bacterial strain: the microbiotic filter paper of preparation different concns, cultivate milk-acid bacteria, adopt the filter paper diffusion process to measure the drug susceptibility of milk-acid bacteria, obtain 6 strains to antibiotic sensitive and the bacterial strain good to the yeast inhibition;
Four, the co-cultivation of bacterial strain test: take out 6 strains of lactic acid bacteria of preserving under-20 ℃ of conditions and activate, the 6 strains breast bacterium liquid of activation is pressed cfu than the mixed of 1:1, obtain carrying out the seed liquor of bacterial strain co-cultivation, inoculum size by 1%, mixed above-mentioned seed liquor is inoculated in MRS liquid nutrient medium not calciferous, 37 ℃ of static cultivations, surveyed bacterial concentration and medium pH value at interval in 1 day, METHOD FOR CONTINUOUS DETERMINATION 7 days, the combination table of bacterial strain WN5 and SN3 reveal best proliferative advantage and survival advantage;
Five, the evaluation of bacterial strain: bacterial strain WN5 and SN3 that the above-mentioned steps screening is obtained carry out biological characteristics and 16S-23S rDNA transcribed spacer Sequence Identification respectively.
Further, the biological characteristics of bacterial strain is identified and is referred in the step 5, bacterial strain WN5 is gram-positive microorganism, and that bacterium colony oyster white, bacterium colony smooth surface, cell are is shaft-like, 10～45 ℃ of growth temperatures, 32～38 ℃ of optimum temperutures, catalase feminine gender, catalase feminine gender, the suitableeest carbon source glucose, the suitableeest inorganic microcosmic salt K
2HPO
4Bacterial strain SN3 is gram-positive microorganism, and bacterium colony oyster white, bacterium colony smooth surface, cell are rod-short, 10～45 ℃ of growth temperatures, 32～38 ℃ of optimum temperutures, catalase feminine gender, catalase feminine gender, the suitableeest carbon source glucose, the suitableeest inorganic microcosmic salt Na
2HPO
4
Further, the Sequence Identification of 16S-23S rDNA transcribed spacer refers in the step 5, nucleotide sequence and the plant lactobacillus WCFS1(AL935263 of the 16S-23S rDNA transcribed spacer of bacterial strain WN5) homology the highest, its homology is 98%; Nucleotide sequence and the lactobacterium casei W56(HE970764 of the 16S-23S rDNA transcribed spacer of bacterial strain SN3) homology the highest, its homology is 97%.
Further, double-deck agar plate punch method is measured and is referred in the step 2, and the MRS solid medium of 20ml is injected the culture dish of diameter 25cm, cooling 1h; Get 6mL yeast nutrient solution and be added to 100ml and be cooled in 45 ℃ the YPD semisolid medium, shake up, and be poured on rapidly on the bottom MRS substratum; On bottom MRS substratum, at interval 3 lml pipettor Tip heads that quilt is cut short are placed at the equidistance place; The height that this quilt is cut short the Tip head is 1cm, and diameter is 0.9cm; The YPD semisolid medium that cooling is injected, the time is 1h, at this moment, makes double-deck substratum; Take out the Tip head, make for the hole of placing bacteriostatics; Get the above-mentioned milk-acid bacteria bacteriostatics one of same concentrations, the volume number is 150 microlitres, adds wherein in two holes; Remain one in contrast, add the sterilization Russia water of 150 microlitres; Cultivate 48h for 37 ℃, measure and record milk-acid bacteria to saccharomycetic antibacterial circle diameter; Component and content that described YPD liquid nutrient medium is every liter are: yeast powder 10 grams, peptone 20 grams, glucose 20 grams; Component and content that described YPD semisolid medium is every liter are: yeast powder 10 grams, peptone 20 grams, glucose 20 grams, 0.7% agar powder.
Further, the microbiotic filter paper of preparation different concns refers in the step 3, Streptomycin sulphate, Ciprofloxacin and gentamicin all are configured to three concentration gradients of 80 μ g/ml, 320 μ g/ml and 1280 μ g/ml, amoxycilline Trihydrate bp, erythromycin and Cephradine all are configured to three concentration gradients of 0.625 μ g/ml, 20 μ g/ml and 80 μ g/ml, prepare the circular filter paper sheet that diameter is 0.6cm with filter paper, they are put into above-mentioned antibiotic solution, obtain the microbiotic filter paper of different concns;
Further, cultivating milk-acid bacteria in the described step 3 refers to, take out the milk-acid bacteria that preserves under-20 ℃ of conditions, getting 5 microlitres injects on the MRS solid medium, coating, under the condition of 37 ℃ of temperature, cultivate 12～14h, with the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate the milk-acid bacteria that 16～18h obtains activating, the milk-acid bacteria of activation is equipped with in the 250ml triangular flask of MRS liquid nutrient medium by 1% inoculum size access, liquid amount is 100ml, cultivates 16～18h, obtains lactobacillus suspension for 37 ℃, at this moment, contain 0.993 * 10 at least in every milliliter of MRS liquid nutrient medium
10The cfu milk-acid bacteria, the bacterial concentration that dilutes every milliliter of milk-acid bacteria with sterilized water is 1.5 * 10
8Cfu.
Further, the activation of milk-acid bacteria refers in the step 4, and the MRS solid medium that does not add calcium carbonate of 20ml is injected the culture dish of diameter 25cm, cooling 1h, and every ml concn of drawing 100 microlitres is 1.5 * 10
8The lactobacillus suspension of cfu is in substratum central authorities coated plate, get 4 filter papers, these 4 filter papers are respectively 3 microbiotic of the same race but the different microbiotic filter paper of concentration, the 4th filter paper be for containing antibiotic contrast, treat that agar surface absorbs milk-acid bacteria after, paste at interval evenly moderate above-mentioned 4 filter papers in the agar surface of each culture dish, after leaving standstill 5 minutes, the upset plate, 37 ℃ of static cultivation 24h measure and record microbiotic to the antibacterial circle diameter of milk-acid bacteria.
Further, component and content that the MRS liquid nutrient medium that uses in the above-mentioned steps is every liter are: peptone 10 g/L, yeast powder 5 g/L, glucose 10 g/L, sucrose 15 g/L, extractum carnis 10 g/L, diammonium hydrogen citrate 2 g/L, sodium acetate 5 g/L, Sodium.alpha.-ketopropionate 0.18 g/L, L-light cystinic acid hydrochloride 0.05 g/L, protoheme 0.5 g/L, vitamin K
22 g/L, calcium carbonate 10 g/L, K
2HPO
42 g/L, MgSO
47H
2O 0.58 g/L, MnSO
4H
2O 0.25 g/L, Tween-80 1ml/L, the pH value is 6.4,121 ℃ of sterilizations 15 minutes; The component that the MRS solid medium is every liter and content are: add mass percent 1.5% agar powder in the MRS liquid nutrient medium, the pH value is 6.4,121 ℃ of sterilizations 15 minutes.
Beneficial effect of the present invention is: the microbial inoculum that the activeconstituentss of making by above-mentioned five steps are made is during to the high-moisture alfalfa ensilage, high-moisture clover moisture content is up to 81.7%, the ensiling time reaches 240 days, the cauline leaf of clover after the ensiling is complete to be stored on the plant, differ seldom before water content and the ensiling, and protein content also improves to some extent; Improve the fungistatic effect of ensiling agent simultaneously and reduce resistance to enter food chain by the ensiling clover and the hidden danger propagated.
Embodiment
Among the following embodiment, if no special instructions, be ordinary method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels; Described percentage composition or concentration are mass percent if no special instructions.
Substratum used among the following embodiment is as follows: the component that the MRS liquid nutrient medium is every liter and content are: peptone 10 g/L, yeast powder 5 g/L, glucose 10 g/L, sucrose 15 g/L, extractum carnis 10 g/L, diammonium hydrogen citrate 2 g/L, sodium acetate 5 g/L, Sodium.alpha.-ketopropionate 0.18 g/L, L-light cystinic acid hydrochloride 0.05 g/L, protoheme 0.5 g/L, vitamin K
22 g/L, calcium carbonate 10 g/L, K
2HPO
42 g/L, MgSO
47H
2O 0.58 g/L, MnSO
4H
2O 0.25 g/L, Tween-80 1ml/L, the pH value is 6.4,121 ℃ of sterilizations 15 minutes; The component that the MRS solid medium is every liter and content are: add 1.5% agar powder (mass percent) in the above-mentioned MRS liquid nutrient medium, the pH value is 6.4,121 ℃ of sterilizations 15 minutes.
Embodiment 1
1, the separation of bacterial strain and purifying:
The leach liquor of collecting near the mud Qingdao triumph bridge ratio of 1:10 by volume joins that mixing carries out serial dilution in the sterilized water, and each dilution bacterium liquid 100 microlitres are coated on respectively in the MRS solid medium, and 37 ℃ of anaerobism are cultivated 48h; Single bacterium colony that the picking transparent circle is bigger is rule continuously on the MRS solid medium and is obtained pure single bacterium colony 3 times; Whether picking list bacterium colony observes aerogenesis in the MRS liquid nutrient medium that does not add calcium carbonate, the aerogenesis bacterial strain is heterofermentative lactic bacteria, and the anaerogen strain is homofermentative lactic bacteria; Isolate homotype and the heterofermentative lactic bacteria strain of 175 purifying altogether; These bacterial strains insert in the MRS liquid nutrient medium, and 37 ℃ of anaerobism are cultivated 20h, and get 700 microlitre bacterium liquid wherein and add 300 microlitre sterile glycerols, mixing then, it is frozen to put into-80 ℃ of refrigerators;
2, press down the yeast test:
The activation of milk-acid bacteria: take out the milk-acid bacteria that preserves under-20 ℃ of conditions, get 5 microlitres and inject on the MRS solid medium, coating is cultivated 12～14h under the condition of 37 ℃ of temperature; With the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate the milk-acid bacteria that 16～18h obtains activating; Inoculum size by 1% does not contain the milk-acid bacteria access that activates in the 250ml triangular flask of calcium carbonate MRS liquid nutrient medium, and liquid amount is 100ml, cultivates 16～18h, at this moment, contains 0.993 * 10 at least in every milliliter of MRS liquid nutrient medium for 37 ℃
10The cfu milk-acid bacteria; 5000 leave heart 5min, collect supernatant liquor, obtain bacteriostatics one; Inoculum size by 1% is equipped with the milk-acid bacteria access of activation in the 250 ml triangular flasks of MRS liquid nutrient medium, and liquid amount is 100 ml; Cultivate 16～18h, at this moment, contain 1.0 * 10 at least in every milliliter of MRS liquid nutrient medium for 37 ℃
10The cfu milk-acid bacteria; 5000 leave heart 5min, collect supernatant liquor, obtain bacteriostatics two;
Saccharomycetic liquid culture: with yeast
Saccharomyces cerevisiae28 ℃ of static cultivation 48h in the YPD liquid nutrient medium;
Double-deck agar plate punch method is measured the bacteriostasis of above-mentioned bacteriostatics one: the MRS solid medium of 20ml is injected the culture dish of diameter 25cm, cooling 1h; Get 6mL yeast nutrient solution and be added to 100ml and be cooled in 45 ℃ the YPD semisolid medium, shake up, and be poured on rapidly on the bottom MRS substratum; On bottom MRS substratum, at interval 3 lml pipettor Tip heads that quilt is cut short are placed at the equidistance place; The height that this quilt is cut short the Tip head is 1cm, and diameter is 0.9cm; The YPD semisolid medium that cooling is injected, the time is 1h, at this moment, makes double-deck substratum; Take out the Tip head, make for the hole of placing bacteriostatics; Get the above-mentioned milk-acid bacteria bacteriostatics one of same concentrations, the volume number is 150 microlitres, adds wherein in two holes; Remain one in contrast, add the sterilization Russia water of 150 microlitres; Cultivate 48h for 37 ℃, measure and record milk-acid bacteria to saccharomycetic antibacterial circle diameter;
Double-deck agar plate punch method is measured the bacteriostasis of above-mentioned bacteriostatics two: the MRS solid medium of 20ml is injected the culture dish of diameter 25cm, cooling 1h; Get 6mL yeast nutrient solution and be added to 100ml and be cooled in 45 ℃ the YPD semisolid medium, shake up, and be poured on rapidly on the bottom MRS substratum; On bottom MRS substratum, at interval the Tip head of the lml pipettor that 3 quilts cut short is placed at the equidistance place; The height that this quilt is cut short the Tip head is 1cm, and diameter is 0.9cm; The YPD semisolid medium 1h that cooling is injected makes double-deck substratum; At this moment, take out the Tip head, make for the hole of placing bacteriostatics; Get the above-mentioned milk-acid bacteria bacteriostatics 2 of same concentrations, the volume number is 150 microlitres, adds wherein in two holes; Remain one in contrast, add the water of 150 microlitres; Cultivate 48h for 37 ℃, measure and record milk-acid bacteria to saccharomycetic antibacterial circle diameter;
In the above-mentioned saccharomycetic liquid nutrient medium, component and content that described YPD liquid nutrient medium is every liter are: yeast powder 10 grams, peptone 20 grams, glucose 20 grams;
Above-mentioned yeast nutrient solution is added to 100ml and is cooled in 45 ℃ the YPD semisolid medium, and component and content that described YPD semisolid medium is every liter are: female powder 10 grams, peptone 20 grams, glucose 20 grams, 0.7% agar powder;
3, the drug sensitive test of bacterial strain:
The preparation of microbiotic filter paper concentration: three concentration gradients that Streptomycin sulphate, Ciprofloxacin and gentamicin all are configured to 80 μ g/ml, 320 μ g/ml and 1280 μ g/ml; Amoxycilline Trihydrate bp, erythromycin and Cephradine all are configured to three concentration gradients of 0.625 μ g/ml, 20 μ g/ml and 80 μ g/ml; F4-1 filter paper with Sangon Biotech (Shanghai) Co., Ltd. prepares the circular filter paper sheet that diameter is 0.6cm, and they are put into above-mentioned antibiotic solution, obtains the microbiotic filter paper of different concns;
The cultivation of milk-acid bacteria: take out the milk-acid bacteria that preserves under-20 ℃ of conditions, get 5 microlitres and inject on the MRS solid medium, coating is cultivated 12～14h under the condition of 37 ℃ of temperature; With the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate the milk-acid bacteria that 16～18h obtains activating; The milk-acid bacteria of activation is equipped with in the 250ml triangular flask of MRS liquid nutrient medium by 1% inoculum size access, liquid amount is 100ml, cultivates 16～18h for 37 ℃, obtains and will be used for the lactobacillus suspension of following test, at this moment, contain 0.993 * 10 at least in every milliliter of MRS liquid nutrient medium
10The cfu milk-acid bacteria; The bacterial concentration that dilutes every ml milk-acid bacteria with sterilized water is 1.5 * 10
8Cfu.
The filter paper diffusion process is measured the drug susceptibility of above-mentioned milk-acid bacteria: the MRS solid medium that does not add calcium carbonate of 20ml is injected the culture dish of diameter 25cm, cooling 1h; Every ml concentration of drawing 100 microlitres is 1.5 * 10
8The lactobacillus suspension of cfu is in substratum central authorities, coated plate; Get 4 filter papers, these 4 filter papers are respectively 3 microbiotic of the same race but the different microbiotic filter paper of concentration, and the 4th filter paper be not for containing antibiotic contrast; After treating that agar surface absorbs milk-acid bacteria, paste even moderate above-mentioned 4 filter papers in interval in the agar surface of each culture dish; After leaving standstill 5 minutes, the upset plate, 37 ℃ of static cultivation 24h measure and record microbiotic to the antibacterial circle diameter of milk-acid bacteria.
The result obtains 6 strains to antibiotic sensitive and the bacterial strain good to the yeast inhibition.
4, the co-cultivation of bacterial strain test.
The activation of milk-acid bacteria: take out above-mentioned 6 strains of lactic acid bacteria of preserving under-20 ℃ of conditions, get 5 microlitres and inject on the MRS solid medium, coating is cultivated 12～14h under the condition of 37 ℃ of temperature; With the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate the milk-acid bacteria that 16～18h obtains activating; At this moment, contain 0.893 * 10 at least in every mlMRS liquid nutrient medium
10～1.231 x 10
10The cfu milk-acid bacteria.
The co-cultivation of milk-acid bacteria: the 6 strains breast bacterium liquid of above-mentioned cultivation is mixed the seed liquor that obtains carrying out the bacterial strain co-cultivation in the cfu ratio for the ratio of 1:1, inoculum size by 1%, mixed above-mentioned seed liquor is inoculated in MRS liquid nutrient medium not calciferous, 37 ℃ of static cultivations, surveyed bacterial concentration and medium pH value, METHOD FOR CONTINUOUS DETERMINATION 7 days at interval in 1 day.
The combination table of bacterial strain WN5 and SN3 reveals best proliferative advantage and survival advantage, and in the time of the 1st day, every ml bacterial concentration is at least 0.993 * 10
10Cfu, pH value are 4.5; In the time of the 2nd day, every ml bacterial concentration is at least 0.907 * 10
10Cfu, the pH value is 3.5; In the time of the 3rd day, every ml bacterial concentration is at least 0.902 * 10
10Cfu, the pH value is 4.0; In the time of the 4th day, every ml bacterial concentration is at least 0.839 * 10
10Cfu, the pH value is 4.5; In the time of the 5th day, every ml bacterial concentration is at least 0.827 * 10
10Cfu, the pH value is 4.5; In the time of the 6th day, every ml bacterial concentration is at least 0.832 * 10
10Cfu, the pH value is 4.5; In the time of the 6th day, every ml bacterial concentration is at least 0.842 * 10
10Cfu, the pH value is 4.5; The co-cultivation of this explanation bacterial strain has promoted thalli growth, has significantly improved cell density and bacterial strain survival ability.
5, the evaluation of bacterial strain
Milk-acid bacteria WN5 and SN3 that the above-mentioned steps screening is obtained carry out biological characteristics and 16S-23S rDNA transcribed spacer Sequence Identification respectively.
The biological characteristics of bacterial strain: that bacterial strain WN5 is that gram-positive microorganism, bacterium colony oyster white, bacterium colony smooth surface, cell are is shaft-like, 10 ℃～45 ℃ of growth temperatures, 32 ℃～38 ℃ of optimum temperutures, catalase feminine gender, catalase feminine gender, the suitableeest carbon source glucose, the suitableeest inorganic microcosmic salt K
2HPO
4; Bacterial strain SN3 is that gram-positive microorganism, bacterium colony oyster white, bacterium colony smooth surface, cell are rod-short, 10 ℃～45 ℃ of growth temperatures, 32 ℃～38 ℃ of optimum temperutures, catalase feminine gender, catalase feminine gender, the suitableeest carbon source glucose, the suitableeest inorganic microcosmic salt Na
2HPO
4
The sequence of 16S-23S rDNA transcribed spacer: nucleotide sequence and the plant lactobacillus WCFS1(AL935263 of the 16S-23S rDNA transcribed spacer of bacterial strain WN5) homology is the highest, and its homology is 98%; Nucleotide sequence and the lactobacterium casei W56(HE970764 of the 16S-23S rDNA transcribed spacer of bacterial strain SN3) homology the highest, its homology is 97%.
Based on above feature, the bacterial strain WN5 that above-mentioned steps 1 screening is obtained be accredited as plant lactobacillus (
Lactobacillus plantarum), the bacterial strain SN3 that above-mentioned steps 1 screening is obtained be accredited as lactobacterium casei (
Lactobacillus casei).Above-mentioned three strain bacterium are all in No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 04 12nd, 2013, plant lactobacillus (
Lactobacillus plantarum) preserving number of WN5 is CGMCC NO. 7469, lactobacterium casei (
Lactobacillus casei) preserving number of SN3 is CGMCC NO. 7470.
Embodiment 2
Protoheme and vitamin K
2The raising plant lactobacillus (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) the propagation density of SN3 CGMCC NO. 7470.
The activation of milk-acid bacteria: take out the plant lactobacillus that 1 screening of the above-mentioned example preserved under-20 ℃ of conditions obtains (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) SN3 CGMCC NO. 7470, to get 5 microlitres respectively and inject on the MRS solid medium, coating is cultivated 12～14h under the condition of 37 ℃ of temperature; With the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate the milk-acid bacteria that 16～18h obtains activating, at this moment, contain 0.993 * 10 at least in every mlMRS liquid nutrient medium
10The cfu milk-acid bacteria.
Plant lactobacillus (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) co-cultivation of SN3 CGMCC NO. 7470: with two kinds of Bacterium lacticum liquid of above-mentioned cultivation in cfu than mixing the seed liquor that obtains carrying out the bacterial strain co-cultivation for the ratio of 1:1, inoculum size by 10% is inoculated in mixed above-mentioned seed liquor inoculating lactic acid bacterium respectively MRS liquid nutrient medium not calciferous, does not contain calcium carbonate and vitamin K
2The MRS liquid nutrient medium and do not contain calcium carbonate, protoheme and vitamin K
2The MRS liquid nutrient medium in, 37 ℃ of static cultivations are measured substratum respectively at the cell density of 16h. 3 repetition are established in test, and the mean value of 3 repetitions is end value. every ml cell density of three kinds of substratum is respectively 1.007 * 10
10Cfu, 0.994 * 10
10Cfu and 0.999 * 10
10Cfu. the result shows protoheme and vitamin K
2The raising plant lactobacillus (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) the propagation density of SN3 CGMCC NO. 7470.
Embodiment 3
Protoheme and vitamin K
2The raising plant lactobacillus (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) anti-hunger and the acidproof ability of SN3 CGMCC NO. 7470.
The activation of milk-acid bacteria: take out the plant lactobacillus that 1 screening of the above-mentioned example preserved under-20 ℃ of conditions obtains (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) SN3 CGMCC NO. 7470, simultaneously, take out the above-mentioned example of preserving under-20 ℃ of conditions 1 and screen other two strains bacterium ML_2 and the YN3_4 that obtains; Get 5 microlitres respectively and inject on the MRS solid medium, coating is cultivated 12～14h under the condition of 37 ℃ of temperature; With the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate the milk-acid bacteria that 16～18h obtains activating, at this moment, contain 0.993 * 10 at least in every mlMRS liquid nutrient medium
10The cfu milk-acid bacteria.
The co-cultivation of bacterial strain: with the plant lactobacillus of above-mentioned cultivation (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) SN3 CGMCC NO. 7470 bacterium liquid in cfu than mixing the seed liquor 1 that obtains the bacterial strain co-cultivation for the ratio of 1:1, and the bacterium liquid of other two strains bacterium ML_2 and YN3_4 mixes the seed liquor 2 that obtains the bacterial strain co-cultivation in the cfu ratio for the ratio of 1:1, inoculum size by 10%, respectively mixed above-mentioned seed liquor is connect 1 and seed liquor 2 be inoculated in MRS liquid nutrient medium not calciferous, 37 ℃ of static cultivations, measure respectively and cultivated 1 day, 2 days, 3 days, 4 days, 5 days, cell density when 6 days and 7 days. seed liquor 1 is after cultivating 1 day (24h), and pH is 3.5; Seed liquor 1 is after cultivating 1 day (24h), and pH is that 3 repetitions are established in the 3.8. test, and the mean value of 3 repetitions is as shown in table 1.
The cell density of the different seed liquor of table 1 under different incubation times
The result shows seed liquor 1, namely plant lactobacillus (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) the co-cultivation bacterium liquid of SN3 CGMCC NO. 7470 improved cell density, strengthened the survival ability of bacterial strain under acidic conditions, strengthened the survival ability of bacterial strain under the nutritive substance depletion conditions.
Embodiment 4
Utilize plant lactobacillus (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) SN3 CGMCC NO. 7470 makes the microbiobacterial agent of high-moisture alfalfa ensilages and the application in the high-moisture alfalfa ensilage.
The activation of milk-acid bacteria: take out the plant lactobacillus that 1 screening of the above-mentioned example preserved under-20 ℃ of conditions obtains (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) SN3 CGMCC NO. 7470, to get 5 microlitres respectively and inject on the MRS solid medium, coating is cultivated 12～14h under the condition of 37 ℃ of temperature; With the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate the milk-acid bacteria that 16～18h obtains activating, at this moment, contain 0.993 * 10 at least in every mlMRS liquid nutrient medium
10The cfu milk-acid bacteria.
The microbe additive of high-moisture alfalfa ensilage is made: with the plant lactobacillus of above-mentioned cultivation (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) the bacterium liquid of SN3 CGMCC NO. 7470 in cfu than mixing the seed liquor that obtains carrying out co-cultivation for the ratio of 1:1, inoculum size by 10%, mixed above-mentioned seed liquor is inoculated in the MRS liquid nutrient medium not calciferous, 37 ℃ of static cultivation 14h～20h make that cell density reaches 0.993 * 10 in every ml liquid nutrient medium
10Cfu～0.832 * 10
11Cfu, per kilogram adds 30ml～35ml microbe additive during ensiling.
The application of microbe additive in the high-moisture alfalfa ensilage: the ratio that adds 30ml～35ml microbe additive in high-moisture clover (water content is 79.7%-80.6%) in the per kilogram clover adds the mentioned microorganism additive; Before adding microbiobacterial agent, it is long that the high-moisture clover is cut into 5cm ~ 6cm; After adding microbiobacterial agent, turn microbial inoculum and clover so that they mix; Then, the clover of adding microbial inoculum is encased in the ensiling device; Room temperature is placed; Measure ensiling clover material contained moisture (%), pH value, crude protein (%), water-soluble carbohydrate (%), ammonia-state nitrogen (%), total free aminoacids (%) and reducing sugar (%) in 2 days, 5 days, 9 days, 15 days, 30 days and 90 days at interval respectively.3 repetitions are established in test, and the mean value of 3 repetitions is end value.
The ensiling effect of high-moisture alfalfa ensilage material: open the ensiling device in different time sections, evenly accurately take by weighing 20 g samples; Add 180 mL distilled water, stir, add preservative film; 4 ℃ of following lixiviate 24 h are vat liquor through four layers of gauze and twice filtration of qualitative filter paper gained liquid.Vat liquor is used for measuring alfalfa ensilage material pH value and ammonia-state nitrogen (NH
3-N).The pH value uses thunder magnetic PHS-25 type pH instrument to measure, and ammonia nitrogen content adopts phenol-clorox colorimetric method for determining.Water content is sample weightless weight behind dry 48 h in 65 ℃ of baking ovens, water-soluble carbohydrate adopts anthrone-sulfuric acid colorimetric method for determining, crude protein adopts Kjeldahl determination to measure (FOSS company full-automatic Kjeldahl determination device), reducing sugar adopts 3,5-dinitrosalicylic acid (3,5-Dinitrosalicylic acid, DNS) colorimetric method for determining, total free aminoacids adopt ninhydrin colorimetry to measure.Measurement result is as shown in table 2.
Microbiobacterial agent is to the ensiling effect of high-moisture clover in table 2 example 4
As seen from Table 2, with the clover that microbe additive in the example 4 is handled, 2 days～90 days different ensiling time period, every index of ensiling all reached good effect.The pH value remains on level less than 5 in the different ensiling time, the content of protein, water-soluble carbohydrate and reducing sugar does not all have because the prolongation of ensiling time shows obvious minimizing, and the content of water content, ammonia-state nitrogen and total free aminoacids does not all have because the prolongation of ensiling time shows obvious increase.
Embodiment 5
Utilize plant lactobacillus (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) SN3 CGMCC NO. 7470 makes the microbiobacterial agent of high-moisture alfalfa ensilages and the application in the high-moisture alfalfa ensilage.
The activation of milk-acid bacteria: take out the plant lactobacillus that 1 screening of the above-mentioned example preserved under-20 ℃ of conditions obtains (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) SN3 CGMCC NO. 7470, to get 5 microlitres respectively and inject on the MRS solid medium, coating is cultivated 12～14h under the condition of 37 ℃ of temperature; With the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate the milk-acid bacteria that 16～18h obtains activating, at this moment, contain 0.993 * 10 at least in every mlMRS liquid nutrient medium
10The cfu milk-acid bacteria.
The microbe additive of high-moisture alfalfa ensilage is made: with the plant lactobacillus of above-mentioned cultivation (
Lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
Lactobacillus casei) the bacterium liquid of SN3 CGMCC NO. 7470 in cfu than mixing the seed liquor that obtains carrying out co-cultivation for the ratio of 1:2, inoculum size by 10%, mixed above-mentioned seed liquor is inoculated in the MRS liquid nutrient medium not calciferous, 37 ℃ of static cultivation 14h～20h make that cell density reaches 0.993 * 10 in every ml liquid nutrient medium
10Cfu～0.832 * 10
11Cfu, per kilogram adds 30ml～35ml microbe additive during ensiling..
The application of microbe additive in the high-moisture alfalfa ensilage: the ratio that adds 30ml～35ml microbe additive in high-moisture clover (water content is 79.7%-80.6%) in the per kilogram clover adds the mentioned microorganism additive; Before adding microbiobacterial agent, it is long that the high-moisture clover is cut into 5cm～6cm; After adding microbiobacterial agent, turn microbial inoculum and clover so that they mix; Then, the clover of adding microbial inoculum is encased in the ensiling device; Room temperature is placed; Measure ensiling clover material contained moisture (%), pH value, crude protein (%), water-soluble carbohydrate (%), ammonia-state nitrogen (%), total free aminoacids (%) and reducing sugar (%) in 2 days, 5 days, 9 days, 15 days, 30 days and 90 days at interval respectively.3 repetitions are established in test, and the mean value of 3 repetitions is end value.
The ensiling effect of high-moisture alfalfa ensilage material: open the ensiling device in different time sections, evenly accurately take by weighing 20 g samples; Add 180 mL distilled water, stir, add preservative film; 4 ℃ of following lixiviate 24 h are vat liquor through four layers of gauze and twice filtration of qualitative filter paper gained liquid.Vat liquor is used for measuring alfalfa ensilage material pH value and ammonia-state nitrogen (NH
3-N).The pH value uses thunder magnetic PHS-25 type pH instrument to measure, and ammonia nitrogen content adopts phenol-clorox colorimetric method for determining.Water content is sample weightless weight behind dry 48 h in 65 ℃ of baking ovens, water-soluble carbohydrate adopts anthrone-sulfuric acid colorimetric method for determining, crude protein adopts Kjeldahl determination to measure (FOSS company full-automatic Kjeldahl determination device), reducing sugar adopts 3,5-dinitrosalicylic acid (3,5-Dinitrosalicylic acid, DNS) colorimetric method for determining, total free aminoacids adopt ninhydrin colorimetry to measure.Measurement result is as shown in table 3.
Microbiobacterial agent is to the ensiling effect of high-moisture clover in table 3 example 5
As seen from Table 2, with the clover that microbe additive in the example 5 is handled, 2 days ~ 90 days different ensiling time period, every index of ensiling all reached good effect.The pH value remains on level less than 5 in the different ensiling time, the content of protein, water-soluble carbohydrate and reducing sugar does not all have because the prolongation of ensiling time shows obvious minimizing, and the content of water content, ammonia-state nitrogen and total free aminoacids does not all have because the prolongation of ensiling time shows obvious increase.
Embodiment recited above is described preferred implementation of the present invention; be not that scope of the present invention is limited; design under the spiritual prerequisite not breaking away from the present invention; various distortion and improvement that the common engineering technical personnel in this area make technical solution of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (9)
1. the preparation method of a high-moisture alfalfa ensilage microbial inoculum activeconstituents is characterized in that: activeconstituents be plant lactobacillus (
Lactobacillus plantarum) WN5 and lactobacterium casei SN3 (
Lactobacillus casei).
2. the preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents according to claim 1, it is characterized in that: the preparation method of described activeconstituents is:
One, the separation of bacterial strain and purifying: collect the leach liquor of mud, the ratio of 1:10 joins mixing in the sterilized water by volume, and serial dilution is coated on each dilution bacterium liquid 100 microlitres respectively in the MRS solid medium, and 37 ℃ of anaerobism are cultivated 48h; Single bacterium colony that the picking transparent circle is bigger, line obtains pure single bacterium colony 3 times continuously on the MRS solid medium; Whether picking list bacterium colony observes aerogenesis in the MRS liquid nutrient medium that does not add calcium carbonate, the aerogenesis bacterial strain is heterofermentative lactic bacteria, and the anaerogen strain is homofermentative lactic bacteria; Isolate homotype and the heterofermentative lactic bacteria strain of 175 purifying altogether; These bacterial strains insert in the MRS liquid nutrient medium, and 37 ℃ of anaerobism are cultivated 20h, and get 700 microlitre bacterium liquid wherein and add 300 microlitre sterile glycerols, mixing then ,-20 ℃ are frozen;
Two, press down the yeast test: takes out the milk-acid bacteria that preserves under-20 ℃ of conditions, get on the 5 microlitres injection MRS solid medium and be coated with, under the condition of 37 ℃ of temperature, cultivate 12～14h; With the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate 16～18h, the milk-acid bacteria that obtains activating; Inoculum size by 1% does not contain the milk-acid bacteria access that activates in the 250ml triangular flask of calcium carbonate MRS liquid nutrient medium, and liquid amount is 100ml, cultivates 16～18h, at this moment, contains 0.993 * 10 at least in every milliliter of MRS liquid nutrient medium for 37 ℃
10The cfu milk-acid bacteria; 5000 leave heart 5min, collect supernatant liquor, obtain bacteriostatics one; Inoculum size by 1% is equipped with the milk-acid bacteria access of activation in the 250 ml triangular flasks of MRS liquid nutrient medium, and liquid amount is 100 ml; Cultivate 16～18h, at this moment, contain 1.0 * 10 at least in every milliliter of MRS liquid nutrient medium for 37 ℃
10The cfu milk-acid bacteria; 5000 leave heart 5min, collect supernatant liquor, obtain bacteriostatics two; With yeast
Saccharomyces cerevisiae28 ℃ of static cultivation 48h obtain the yeast nutrient solution in the YPD liquid nutrient medium; Double-deck agar plate punch method is measured the bacteriostasis of above-mentioned bacteriostatics one, and double-deck agar plate punch method is measured the bacteriostasis of above-mentioned bacteriostatics two;
Three, the drug sensitive test of bacterial strain: the microbiotic filter paper of preparation different concns, cultivate milk-acid bacteria, adopt the filter paper diffusion process to measure the drug susceptibility of milk-acid bacteria, obtain 16 strains to antibiotic sensitive and the bacterial strain good to the yeast inhibition;
Four, the co-cultivation of bacterial strain test: take out 6 strains of lactic acid bacteria of preserving under-20 ℃ of conditions and activate, the 6 strains breast bacterium liquid of activation is pressed cfu than the mixed of 1:1, obtain carrying out the seed liquor of bacterial strain co-cultivation, inoculum size by 1%, mixed above-mentioned seed liquor is inoculated in MRS liquid nutrient medium not calciferous, 37 ℃ of static cultivations, surveyed bacterial concentration and medium pH value at interval in 1 day, METHOD FOR CONTINUOUS DETERMINATION 7 days, the combination table of bacterial strain WN5 and SN3 reveal best proliferative advantage and survival advantage;
Five, the evaluation of bacterial strain: bacterial strain WN5 and SN3 that the above-mentioned steps screening is obtained carry out biological characteristics and 16S-23S rDNA transcribed spacer Sequence Identification respectively.
3. the preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents according to claim 2 is characterized in that: double-deck agar plate punch method is measured and is referred in the step 2, and the MRS solid medium of 20ml is injected the culture dish of diameter 25cm, cooling 1h; Get 6mL yeast nutrient solution and be added to 100ml and be cooled in 45 ℃ the YPD semisolid medium, shake up, and be poured on rapidly on the bottom MRS substratum; On bottom MRS substratum, at interval 3 lml pipettor Tip heads that quilt is cut short are placed at the equidistance place; The height that this quilt is cut short the Tip head is 1cm, and diameter is 0.9cm; The YPD semisolid medium that cooling is injected, the time is 1h, at this moment, makes double-deck substratum; Take out the Tip head, make for the hole of placing bacteriostatics; Get the above-mentioned milk-acid bacteria bacteriostatics one of same concentrations, the volume number is 150 microlitres, adds wherein in two holes; Remain one in contrast, add the sterilization Russia water of 150 microlitres; Cultivate 48h for 37 ℃, measure and record milk-acid bacteria to saccharomycetic antibacterial circle diameter; Component and content that described YPD liquid nutrient medium is every liter are: yeast powder 10 grams, peptone 20 grams, glucose 20 grams; Component and content that described YPD semisolid medium is every liter are: yeast powder 10 grams, peptone 20 grams, glucose 20 grams, 0.7% agar powder.
4. the preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents according to claim 2, it is characterized in that: the microbiotic filter paper of preparation different concns refers in the step 3, with Streptomycin sulphate, Ciprofloxacin and gentamicin all are configured to 80 μ g/ml, three concentration gradients of 320 μ g/ml and 1280 μ g/ml, the amoxycilline Trihydrate bp, erythromycin and Cephradine all are configured to 0.625 μ g/ml, three concentration gradients of 20 μ g/ml and 80 μ g/ml, prepare the circular filter paper sheet that diameter is 0.6cm with filter paper, they are put into above-mentioned antibiotic solution, obtain the microbiotic filter paper of different concns.
5. the preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents according to claim 2, it is characterized in that: cultivate milk-acid bacteria in the described step 3 and refer to, take out the milk-acid bacteria that preserves under-20 ℃ of conditions, getting 5 microlitres injects on the MRS solid medium, coating, under the condition of 37 ℃ of temperature, cultivate 12～14h, with the single bacterium colony on the transfering loop picking MRS solid medium, be inoculated in the 10ml test tube that the 5mlMRS substratum is housed, under the condition of 37 ℃ of temperature, cultivate the milk-acid bacteria that 16～18h obtains activating, the milk-acid bacteria of activation is equipped with in the 250ml triangular flask of MRS liquid nutrient medium by 1% inoculum size access, liquid amount is 100ml, cultivates 16～18h, obtains lactobacillus suspension for 37 ℃, at this moment, contain 0.993 * 10 at least in every milliliter of MRS liquid nutrient medium
10The cfu milk-acid bacteria, the bacterial concentration that dilutes every milliliter of milk-acid bacteria with sterilized water is 1.5 * 10
8Cfu.
6. the preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents according to claim 2, it is characterized in that: the activation of milk-acid bacteria refers in the step 4, the MRS solid medium that does not add calcium carbonate of 20ml is injected the culture dish of diameter 25cm, cooling 1h, every ml concn of drawing 100 microlitres is 1.5 * 10
8The lactobacillus suspension of cfu is in substratum central authorities coated plate, get 4 filter papers, these 4 filter papers are respectively 3 microbiotic of the same race but the different microbiotic filter paper of concentration, the 4th filter paper be for containing antibiotic contrast, treat that agar surface absorbs milk-acid bacteria after, paste at interval evenly moderate above-mentioned 4 filter papers in the agar surface of each culture dish, after leaving standstill 5 minutes, the upset plate, 37 ℃ of static cultivation 24h measure and record microbiotic to the antibacterial circle diameter of milk-acid bacteria.
7. according to the preparation method of claim 2 or 3 or 4 or 5 or 6 described high-moisture alfalfa ensilage microbial inoculum activeconstituentss, it is characterized in that: the component that the MRS liquid nutrient medium is every liter and content are: peptone 10 g/L, yeast powder 5 g/L, glucose 10 g/L, sucrose 15 g/L, extractum carnis 10 g/L, diammonium hydrogen citrate 2 g/L, sodium acetate 5 g/L, Sodium.alpha.-ketopropionate 0.18 g/L, L-light cystinic acid hydrochloride 0.05 g/L, protoheme 0.5 g/L, vitamin K
22 g/L, calcium carbonate 10 g/L, K
2HPO
42 g/L, MgSO
47H
2O 0.58 g/L, MnSO
4H
2O 0.25 g/L, Tween-80 1ml/L, the pH value is 6.4,121 ℃ of sterilizations 15 minutes; The component that the MRS solid medium is every liter and content are: add mass percent 1.5% agar powder in the MRS liquid nutrient medium, the pH value is 6.4,121 ℃ of sterilizations 15 minutes.
8. the preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents according to claim 2, it is characterized in that: the Sequence Identification of 16S-23S rDNA transcribed spacer refers in the step 5, nucleotide sequence and the plant lactobacillus WCFS1(AL935263 of the 16S-23S rDNA transcribed spacer of bacterial strain WN5) homology the highest, its homology is 98%; Nucleotide sequence and the lactobacterium casei W56(HE970764 of the 16S-23S rDNA transcribed spacer of bacterial strain SN3) homology the highest, its homology is 97%.
9. the preparation method of high-moisture alfalfa ensilage microbial inoculum activeconstituents according to claim 2, it is characterized in that: the biological characteristics of bacterial strain is identified and is referred in the step 5, bacterial strain WN5 is gram-positive microorganism, and that bacterium colony oyster white, bacterium colony smooth surface, cell are is shaft-like, 10～45 ℃ of growth temperatures, 32～38 ℃ of optimum temperutures, catalase feminine gender, catalase feminine gender, the suitableeest carbon source glucose, the suitableeest inorganic microcosmic salt K
2HPO
4Bacterial strain SN3 is gram-positive microorganism, and bacterium colony oyster white, bacterium colony smooth surface, cell are rod-short, 10～45 ℃ of growth temperatures, 32～38 ℃ of optimum temperutures, catalase feminine gender, catalase feminine gender, the suitableeest carbon source glucose, the suitableeest inorganic microcosmic salt Na
2HPO
4
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310266049.7A CN103333829B (en) | 2013-06-28 | 2013-06-28 | A kind of high-moisture alfalfa ensilage microbial inoculum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310266049.7A CN103333829B (en) | 2013-06-28 | 2013-06-28 | A kind of high-moisture alfalfa ensilage microbial inoculum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103333829A true CN103333829A (en) | 2013-10-02 |
| CN103333829B CN103333829B (en) | 2016-05-18 |
Family
ID=49242034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310266049.7A Active CN103333829B (en) | 2013-06-28 | 2013-06-28 | A kind of high-moisture alfalfa ensilage microbial inoculum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103333829B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1738541A (en) * | 2003-01-14 | 2006-02-22 | 味之素株式会社 | Silage additive and a process for preparing silage using it |
| CN101735964A (en) * | 2008-11-18 | 2010-06-16 | 中国农业大学 | Microbial inoculum for preparing feed and application thereof |
-
2013
- 2013-06-28 CN CN201310266049.7A patent/CN103333829B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1738541A (en) * | 2003-01-14 | 2006-02-22 | 味之素株式会社 | Silage additive and a process for preparing silage using it |
| CN101735964A (en) * | 2008-11-18 | 2010-06-16 | 中国农业大学 | Microbial inoculum for preparing feed and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 于颖等: "异型乳酸菌2种常用鉴定方法的比较", 《食品与发酵工业》, vol. 37, no. 1, 31 December 2011 (2011-12-31), pages 102 - 105 * |
| 张慧杰等: "青贮饲料中乳酸菌的分离鉴定及优良菌株筛选", 《草地学报》, vol. 19, no. 1, 31 January 2011 (2011-01-31), pages 137 - 141 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103333829B (en) | 2016-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102640885B (en) | Applications of Lactobacillus plantarum in complete-strain Triticumspp silage | |
| CN102907572A (en) | Banana silage microbial additive and preparation method thereof | |
| CN105948838B (en) | A kind of compound lactobacillus microbial manure and its preparation and application | |
| CN108998393A (en) | One bacillus subtilis and its preparation have the Pu'er tea method for adjusting function of intestinal canal | |
| CN102864093B (en) | Lactobacillus plantarum suitable for silage straw and use method thereof | |
| CN104365995A (en) | Microbial additive for preparing silage | |
| CN101735964A (en) | Microbial inoculum for preparing feed and application thereof | |
| CN107828681A (en) | Wrap up in bag ensiling mixed lactic bacteria preparation in Alpine-arctic Pastoral | |
| CN106635911A (en) | Composite strain fermentation agent and preparation method of silage | |
| CN107988117A (en) | A kind of compound probiotic decomposing agent and preparation method and application | |
| CN102994421B (en) | Lactic acid bacteria suitable for ensiling oat and applications thereof | |
| CN107603923A (en) | 3 strains of lactic acid bacteria of Alpine-arctic Pastoral ensiling | |
| CN101225364A (en) | Discovery of forage grass attached lactobacillus plantarum and enterococcus mundtii and use thereof | |
| JP2021035952A (en) | Hay preservative and methods for preservation of hay | |
| CN107653203A (en) | A kind of Lactobacillus rhamnosus and its application in feed addictive is prepared | |
| CN107022509A (en) | A kind of separation method of lactic bacteria composition and its application | |
| CN102178126B (en) | Straw micro-stage additive and preparation method thereof | |
| CN102640886B (en) | Applications of Lactobacillus plantarum in Brassica oleracea outer wrapper leaf silage | |
| CN104431646A (en) | High water content alfalfa silage bacterial agent active component preparation method | |
| CN104543602B (en) | A kind of switchgrass ensiling processing method | |
| CN103333829B (en) | A kind of high-moisture alfalfa ensilage microbial inoculum | |
| CN103315195B (en) | High-moisture alfalfa silage inoculants bacteria | |
| CN103749958B (en) | A kind of silage composition and application thereof containing wheat stalk and alta fascue | |
| CN109943505A (en) | A kind of compound probiotic microbial inoculum for harmless treatment excrement slag bed padding | |
| CN102450525A (en) | Leguminous forage silage complex bacteria additive |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |