CN107475147A - A kind of Lactobacillus plantarum LP1 and its application in feed addictive is prepared - Google Patents
A kind of Lactobacillus plantarum LP1 and its application in feed addictive is prepared Download PDFInfo
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- CN107475147A CN107475147A CN201710645393.5A CN201710645393A CN107475147A CN 107475147 A CN107475147 A CN 107475147A CN 201710645393 A CN201710645393 A CN 201710645393A CN 107475147 A CN107475147 A CN 107475147A
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- lactobacillus plantarum
- alfalfa
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- alfalfa silage
- clover
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- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 79
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 79
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 76
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 claims abstract description 49
- 241000219823 Medicago Species 0.000 claims abstract description 41
- 239000004460 silage Substances 0.000 claims abstract description 28
- 241000219793 Trifolium Species 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 27
- 239000002068 microbial inoculum Substances 0.000 claims description 26
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000006041 probiotic Substances 0.000 claims description 7
- 235000018291 probiotics Nutrition 0.000 claims description 7
- 239000003223 protective agent Substances 0.000 claims description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 30
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 26
- 239000004310 lactic acid Substances 0.000 abstract description 15
- 235000014655 lactic acid Nutrition 0.000 abstract description 15
- 238000000855 fermentation Methods 0.000 abstract description 13
- 230000004151 fermentation Effects 0.000 abstract description 13
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 13
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000017854 proteolysis Effects 0.000 abstract description 4
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 abstract description 3
- 230000001681 protective effect Effects 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 19
- 241000894006 Bacteria Species 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 16
- 239000012530 fluid Substances 0.000 description 14
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 11
- 239000008267 milk Substances 0.000 description 11
- 235000013336 milk Nutrition 0.000 description 11
- 210000004080 milk Anatomy 0.000 description 11
- 235000015097 nutrients Nutrition 0.000 description 11
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 10
- 240000004658 Medicago sativa Species 0.000 description 9
- 239000000284 extract Substances 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- 238000009630 liquid culture Methods 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 235000011054 acetic acid Nutrition 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 235000019260 propionic acid Nutrition 0.000 description 5
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000005238 degreasing Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- -1 LA) Chemical compound 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000004459 forage Substances 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Substances OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 229910000906 Bronze Inorganic materials 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 1
- 241001112695 Clostridiales Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 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 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 235000010624 Medicago sativa Nutrition 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000010974 bronze Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- KUNSUQLRTQLHQQ-UHFFFAOYSA-N copper tin Chemical compound [Cu].[Sn] KUNSUQLRTQLHQQ-UHFFFAOYSA-N 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 150000002469 indenes Chemical class 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 239000001393 triammonium citrate Substances 0.000 description 1
- 235000011046 triammonium citrate Nutrition 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Polymers & Plastics (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Animal Husbandry (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Sustainable Development (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of Lactobacillus plantarum LP1 and its application in feed addictive is prepared.Lactobacillus plantarum (Lactobacillus plantarum) LP1 that the present invention protects, its deposit number are CGMCC No.10473.Present invention protection applications of Lactobacillus plantarum (Lactobacillus plantarum) LP1 in alfalfa silage is prepared.Lactobacillus plantarum LP1 can be used as addictive for alfalfa silage, significantly improve lactic acid output, significantly reduce pH value, effectively improve Alfalfa Silage Fermentation Quality；And nonprotein nitrogen and ammonia nitrogen content can be reduced, suppress protein degradation in alfalfa ensilage, effectively improve the feeding value of alfalfa silage；Lactobacillus plantarum LP1 has the advantages that fermentation efficiency is high, cost is cheap simultaneously, can be applied in the production of green environment protective biological feed.
Description
Technical field
The present invention relates to forage feed processing and storage technical field, and in particular to a kind of Lactobacillus plantarum LP1 and its is making
Application in standby feed addictive.
Background technology
Alfalfa (Medicago sativa L.) abbreviation clover, it is that most wide leguminous forage, its albumen are cultivated by China
Content is high, and nutritive value is high, is known as the laudatory title of " King of Pasture ".A kind of important form of the Alfalfa Silage as alfalfa products, no
Loss caused by solving the processing of China's alfalfa hay and drenching with rain is only capable of, and the diversity of clover grass product can be increased, is extended
The utilization of green succulent feed.However, clover water-soluble carbohydrate content is low, there is stronger buffer capacity to be unfavorable for green grass or young crops
Store lactic fermentation.
Although the water content of alfalfa ensilage raw material is reduced by wilting can suppress clostridial fermentation, improve fermentation quality,
It is, in North China area and Huang-Huai-Hai, clover harvest season more rainfalls, it is difficult to which it is half-dried to make clover by field wilting
Ensiling.Alfalfa ensilage fermentation plant at initial stage attachment lactic acid bacteria and clostridium and deposit, when ensiling feed moisture content is higher than 60%, ring
When border temperature is more than 37 DEG C, it is easy to clostridium corruption occurs, produces substantial amounts of butyric acid and ammoniacal nitrogen, pH value is made from reducing
Feeding value is reduced into a large amount of protein degradations, and is unfavorable for preserving for a long time.And added in alfalfa ensilage using lactic acid bacteria
Agent, particularly homo-fermentative Lactobacillus plantarum, lactobacillus-fermented can be promoted initial stage in ensiling, improve lactic acid output, fast prompt drop
Low ph value, be advantageous to ensiling and store and suppress protein degradation, improve the feeding value of alfalfa silage.
The content of the invention
It is an object of the invention to provide a kind of Lactobacillus plantarum LP1 and its application in feed addictive is prepared.
Lactobacillus plantarum (Lactobacillus plantarum) LP1 provided by the invention, on 01 30th, 2015
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC；Address：Chaoyang District, Beijing City
The institute 3 of North Star West Road 1, Institute of Microorganism, Academia Sinica；Postcode：100101), deposit number CGMCC
No.10473.Lactobacillus plantarum (Lactobacillus plantarum) LP1 is referred to as Lactobacillus plantarum LP1.
Applications of the present invention protection Lactobacillus plantarum LP1 in alfalfa silage is prepared.
The present invention also protects a kind of microbial inoculum, and Lactobacillus plantarum LP1 and probiotics protective agent are mixed to get.
The microbial inoculum is concretely obtained Lactobacillus plantarum LP1 and probiotics protective agent are freeze-dried mixed.
In every gram of microbial inoculum, the content of the Lactobacillus plantarum LP1 is more than 1 × 1010CFU。
Contain 1 × 10 in every gram of microbial inoculum12CFU Lactobacillus plantarums LP1.
The present invention also protects a kind of preparation method of microbial inoculum, comprises the following steps：Lactobacillus plantarum LP1 and probiotics are protected
Protect agent mixing.
Methods described is concretely：Freeze, obtain described after Lactobacillus plantarum LP1 thalline are mixed with probiotics protective agent
Microbial inoculum.
The preparation method of the Lactobacillus plantarum LP1 thalline is concretely：Lactobacillus plantarum LP1 is seeded to MRS liquid
Cultivate in culture medium, centrifuge cultivating system at the end of culture, collect precipitation (Lactobacillus plantarum LP1 thalline).
In methods described, the OD of cultivating system at the end of culture260nm=4.
In methods described, the initial OD of cultivating system after inoculation260nm=1.8.
In methods described, condition of culture is 37 DEG C, 250rpm shaken cultivations.
In every gram of microbial inoculum, the content of the Lactobacillus plantarum LP1 is more than 1 × 1010CFU。
Contain 1 × 10 in every gram of microbial inoculum12CFU Lactobacillus plantarums LP1.
The concretely defatted milk of probiotics protective agent described in any of the above.
Solid-state milk powder is concretely dissolved in what water obtained by the defatted milk by 10% mass volume ratio (w/v).
The solid-state milk powder concretely high protein degreasing high-calcium milk powder, more specifically can be purchased from Inner Mongol Erie industry collection
Limited company of group, article No. are 6907992440071 high protein degreasing high-calcium milk powder.
The present invention also protects application of the microbial inoculum in alfalfa silage is prepared.
The present invention also protects a kind of preparation method of alfalfa silage (method first), comprises the following steps：By the bacterium
Agent is applied on clover, is carried out ensiling, is obtained alfalfa silage.
In methods described first, the clover concretely squaring period alfalfa.
In methods described first, the water content of the clover concretely 55.64%-67.26%.
In methods described first, the water content of the clover concretely 55.64% or 67.26%.
In methods described first, the clover is by pretreatment；The preprocess method is：Clover is shredded to 2-3cm.
In methods described first, the microbial inoculum is applied to clover and gone forward, is comprised the following steps：Microbial inoculum is dissolved using water,
Room temperature activates 25min.The water is distilled water.
In methods described first, the microbial inoculum applied amount is that every gram of clover is inoculated with 0.5-20 × 105CFU Lactobacillus plantarum
LP1.The microbial inoculum applied amount concretely every gram of clover inoculation 8 × 105CFU Lactobacillus plantarum LP1.
In methods described first, the temperature of the ensiling is 20 DEG C -40 DEG C.
Concretely 20 DEG C of the temperature of the ensiling.
Concretely 30 DEG C of the temperature of the ensiling.
Concretely 40 DEG C of the temperature of the ensiling.
The time of the ensiling is 45 days.
The present invention also protects a kind of preparation method of alfalfa silage (method second), comprises the following steps：Will be described
Lactobacillus plantarum LP1 is applied on clover, is carried out ensiling, is obtained alfalfa silage.
In methods described second, the clover concretely squaring period alfalfa.
In methods described second, the water content of the clover concretely 55.64%-67.26%.
In methods described second, the water content of the clover concretely 55.64% or 67.26%.
In methods described second, the clover is by pretreatment；The preprocess method is：Clover is shredded to 2-3cm.
In methods described second, the applied amount of the Lactobacillus plantarum LP1 is inoculated with 0.5-20 × 10 for every gram of clover5CFU's
Lactobacillus plantarum LP1.The microbial inoculum applied amount concretely every gram of clover inoculation 8 × 105CFU Lactobacillus plantarum LP1.
In methods described second, the temperature of the ensiling is 20 DEG C -40 DEG C.
Concretely 20 DEG C of the temperature of the ensiling.
Concretely 30 DEG C of the temperature of the ensiling.
Concretely 40 DEG C of the temperature of the ensiling.
The time of the ensiling is 45 days.
The present invention also protects the alfalfa silage that methods described first or methods described second are prepared.
The invention provides a kind of Lactobacillus plantarum LP1, Lactobacillus plantarum LP1 can be used as addictive for alfalfa silage, significantly
Lactic acid output is improved, pH value is significantly reduced, effectively improves Alfalfa Silage Fermentation Quality；And nonprotein nitrogen can be reduced and ammoniacal nitrogen contains
Amount, suppress protein degradation in alfalfa ensilage, effectively improve the feeding value of alfalfa silage；Lactobacillus plantarum LP1 simultaneously
Have the advantages that fermentation efficiency is high, cost is cheap, can be applied in the production of green environment protective biological feed.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
MRS fluid nutrient mediums are made up of solute and solvent；The solute and its concentration in MRS fluid nutrient mediums are：
Peptone 10.0g/L, powdered beef 5.0g/L, dusty yeast 4.0g/L, glucose 20.0g/L, sodium acetate 5.0g/L, Triammonium citrate
2.0g/L, Tween 80 1mL/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate 0.2g/L, manganese sulfate 0.05g/L；The solvent is water.
Alfalfa extracts liquid culture medium：1kg alfalfas, in chopping plus 5L water, 50 DEG C of water-bath 2h, obtain extract solution；
By extract solution first with four layers of filtered through gauze, then filtered with quantitative filter paper (Hangzhou Special Paper Industry Co., Ltd., the type of nova 201),
Collect filtrate；By 121 DEG C of filtrate autoclaving 15 minutes, then it is dispensed into standby in sterilized test tube.
Defatted milk：Solid-state milk powder is dissolved in distilled water by 10% mass volume ratio (w/v).
Solid-state milk powder：High protein degreasing high-calcium milk powder, Inner Mongolia Yili Industry Group Co., Ltd, article No.：
6907992440071。
Separation, screening and the identification of embodiment 1, Lactobacillus plantarum (Lactobacillus plantarum) LP1
First, the separation screening of bacterial strain
Inner Mongol Huhehaote City sheepskin soak (in sheepskin manufacturing process, the liquid after clear water immersion rawhide) is gathered,
30min is shaken with oscillator, using distilled water 10-1-10-6Gradient dilution, 1mL is taken to connect from each dilution gradient dilution respectively
Kind adds the MRS solid mediums after 10-15ml sterilizes, shaken up, treat its cooled and solidified, stand Anaerobic culturel into culture dish
48h.Picking colony form, size, color and glossiness have the single bacterium colony of significant difference, repeat to rule, and until obtain pure bacterium
Fall.Picking single bacterium colony carries out Gram's staining and catalase test.The bacterium of every Gram-positive, negative catalase
Strain tentatively regards as lactic acid bacteria, then goes out one plant of growth ability with alfalfa extract solution Screening of Media and acid producing ability is high
Lactic acid bacteria, bacterial strain LP1 is named as, its growth ability and acid producing ability are as shown in table 1.
The assay method of strain growth ability and acid producing ability：Strain to be tested is inoculated into 10mL MRS fluid nutrient mediums
In, it is inoculated into after cultivating for 2 generations by the inoculum concentration of 3% (volume ratio) in alfalfa extraction liquid culture medium, 37 DEG C, 250rpm vibrations
Culture.The sample once cultivated is taken out at interval of 12h, is taken 3 times, for the last time to be sampled after culture 36h, all samples are in wavelength
The absorbance of sample is measured under 620nm, and determines the pH value of zymotic fluid.Using the clover of non-inoculating strain extract liquid culture medium as
Control, compare absorbance of the different strains in clover extracts liquid culture medium in shaken cultivation 36h under wavelength 620nm with it is right
Maximum difference according between, and the pH value of zymotic fluid with compare between maximum difference, it is maximum to filter out absorbance rise,
PH value declines maximum lactic acid bacteria strains.
The bacterial strain LP1 of table 1 growth ability and acid producing ability
| \ | Growth abilitya | Acid producing abilityb |
| Bacterial strain LP1 | 1.152 | 2.45 |
Note：A, lactic acid bacteria strains cultivated in clover extracts liquid culture medium in 36h absorbance under wavelength 620nm with
Maximum difference between control；B, lactic acid bacteria strains clover extract liquid culture medium in cultivate 36h in zymotic fluid pH value with it is right
Maximum difference according between.
2nd, bacterial strain LP1 identification
Bacterial strain LP1 biological characteristics：Bacterial strain LP1 cultivates 24h on MRS solid mediums, and strain growth is good, can shape
Into the milky bacterium colony of neat in edge；Bacterial strain Gram's staining is positive, and the cellular morphology under microscope is rod-short, without bud
Spore；Oxidase negative, there is stronger growth and product acid activity in clover extracts liquid culture medium.
The assimilative capacicy of bacterial strain different carbon source is detected according to GB4789.35-2010, its experiment to carbon source through fermentation
As a result it is as shown in table 2.
The carbon source through fermentation experimental result of the LP1 bacterial strains of table 2
Note："+" is the positive；"-" is feminine gender
Bacterial strain LP1 16S rDNA sequences are expanded and are sequenced, sequencing result is as shown in the sequence 1 of sequence table.16s
RDNA qualification results show that the similitude of bacterial strain LP1 and the plurality of plants lactobacillus in ncbi database is 99%.
Identified more than, it may be determined that bacterial strain LP1 belongs to Lactobacillus plantarum, therefore is named as plant breast bar again
Bacterium LP1.
3rd, Lactobacillus plantarum LP1 preservation
Lactobacillus plantarum (Lactobacillus plantarum) LP1, it is micro- that China is preserved on 01 30th, 2015
Biological inoculum preservation administration committee common micro-organisms center (abbreviation CGMCC；Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3, Institute of Microorganism, Academia Sinica；Postcode：100101), deposit number CGMCCNo.10473.Lactobacillus plantarum
(Lactobacillus plantarum) LP1 is referred to as Lactobacillus plantarum LP1.
4th, growth measurements of the Lactobacillus plantarum LP1 under different pH, temperature and salinity environmental condition
1st, Lactobacillus plantarum LP1 is inoculated in different initial pH (3,4,4.5,5,6,7 and 8) MRS fluid nutrient mediums,
37 DEG C of quiescent cultures 2 days, after culture terminates, growing state is compared with blank MRS fluid nutrient mediums.As a result it is as shown in table 3.As a result
It has been shown that, Lactobacillus plantarum LP1 be able to can grow in the case where initial ph value is 4.0-8.0 condition of culture.
Growing states of the Lactobacillus plantarum LP1 of table 3 in different initial pH environment
| The initial pH of culture medium | Bacterial strain LP1 |
| 3.0 | + |
| 4.0 | + |
| 4.5 | + |
| 5.0 | + |
| 6.0 | + |
| 7.0 | + |
| 8.0 | + |
Note："+" is the positive；"-" is feminine gender
2nd, Lactobacillus plantarum LP1 is inoculated in MRS fluid nutrient mediums, carries out following division operation：
Group A：4 DEG C of quiescent cultures 14 days；
Group B：10 DEG C of quiescent cultures 2 days；
Group C：20 DEG C of quiescent cultures 2 days；
Group D：30 DEG C of quiescent cultures 2 days；
Group F：40 DEG C of quiescent cultures 2 days；
Group G：50 DEG C of quiescent cultures 7 days；
Group H：60 DEG C of quiescent cultures 7 days.
After culture terminates and blank MRS fluid nutrient mediums compare growing state.As a result it is as shown in table 4.As a result show, plant
Lactobacillus LP1 can not grow at a temperature of 4 DEG C, 50 DEG C and 60 DEG C, and upgrowth situation is good under the conditions of 10 DEG C -40 DEG C.
Growing states of the Lactobacillus plantarum LP1 of table 4 in different initial pH environment
| Cultivation temperature | Bacterial strain LP1 |
| 4℃ | - |
| 10℃ | + |
| 20℃ | + |
| 30℃ | + |
| 40℃ | + |
| 50℃ | - |
| 60℃ | - |
Note："+" is the positive；"-" is feminine gender
3rd, by Lactobacillus plantarum LP1 be inoculated in respectively the MRS fluid nutrient mediums containing 3% (mass percent) sodium chloride and
In MRS fluid nutrient mediums containing 6.5% (mass percent) sodium chloride, 37 DEG C of quiescent cultures 4 days, lactobacter growth shape is observed
Condition.As a result it is as shown in table 5.As a result show, Lactobacillus plantarum LP1 can be containing 3% (mass percent) and 6.5% (quality hundred
Point ratio) grow under conditions of sodium chloride.
Growing states of the Lactobacillus plantarum LP1 of table 5 in salt environment
Note："+" is the positive；"-" is feminine gender
Embodiment 2, Lactobacillus plantarum (Lactobacillus plantarum) LP1 prepare alfalfa silage
First, prepared by bacterium powder
Lactobacillus plantarum LP1 is inoculated in 10ml MRS fluid nutrient mediums, 37 DEG C, 250rpm shaken cultivations, cultivated for 2 generations
Afterwards (bacterium solution initial OD in 60ml MRS fluid nutrient mediums is inoculated into by the inoculum concentration of 3% (volume ratio)260nm=1.8), 37 DEG C,
250rpm shaken cultivations 18-24h (the final OD of bacterium solution260nm=4), cultivating system is centrifuged, bacterial sediment is collected, by bacterial sediment
Defatted milk after being sterilized with 10ml freezes after mixing, and obtains solid-state microbial inoculum, the Lactobacillus plantarum concentration in solid-state microbial inoculum for 1 ×
1012CFU g-1(1 × 10 need to be more than10CFU g-1)。
2nd, prepared by alfalfa ensilage
Clover：Last batch of squaring period alfalfa cutting occasion of Hebei Zhuozhou plantation is on November 06th, 2016, is cut
2-3cm is broken to, is 55.64% and 67.26% by wilting in various degree to water content.
1st, the solid-state microbial inoculum for taking step 1 to prepare, dissolved using 5ml distilled water, room temperature activation 25min, obtain liquid microbial inoculum
(Lactobacillus plantarum LP1 content is 2 × 10 in 5ml liquid microbial inoculums9CFU)。
The 2nd, the 5ml liquid microbial inoculum that step 1 obtains all is sprayed on 2500g clovers to (water content is 55.64% He
67.26%), every gram of clover inoculation 8 × 105CFU Lactobacillus plantarum LP1.Control group (CK) sprays 5ml sterilized waters.Using poly-
Vinyl bag (180 × 260mm) vacuumizes sealing and makes alfalfa ensilage, every bag of about 250g, then stores respectively to 20,30 and 40 DEG C
Under insulating box in, ensiling 45 days.
3rd, alfalfa ensilage effect detection
1st, every bag of alfalfa ensilage sample is opened respectively, weighs the ensilage 20g fully mixed, adds 180mL distilled water, is used
Mixer blends 1min, is successively filtered with 4 layers of gauze and qualitative filter paper (Hangzhou Special Paper Industry Co., Ltd., nova 102), collects
Filtrate.
Following Indexs measure is carried out to filtrate：PH value, lactic acid (lactic acid, LA), acetic acid (acetic acid,
AA), propionic acid (propionic acid, PA), butyric acid (butyrate acid, BA), ammoniacal nitrogen (ammonia nitrogen,
) and Free Amino Nitrogen (amino acid nitrogen, AA-N) AN.
PH value is determined using thunder magnetic PHS-3C types pH meter.
Lactic acid, acetic acid, propionic acid and butyric acid use high-efficient liquid phase chromatogram technique analysis.Instrument：Shimadzu 20A high performance liquid chromatography
Instrument；Detector：Shimadzu SPD-M20A detectors；Chromatographic column：ShodexRSpak KC-811 chromatographic columns (8mm × 300mm)；Flowing
Phase：3mmol·L-1Perchloric acid (top pure grade)；Detection wavelength：210nm；Flow velocity：1mL·min-1；Sample size：5μL；Column temperature 50
℃.Filtrate to be measured is with upper machine testing after 0.45 μm of water system membrane filtration.
Lactic acid, acetic acid, propionic acid and butyric acid standard items (Geel, Belgium) are purchased from the limited public affairs of Beijing lark prestige chemical technology
Department.
The appearance time of lactate standard product is 8.1min；Go out peak position under the same conditions within ± 0.2min, can be with
Regard as same substance.
The appearance time of acetic acid standard items is 9.6min；Go out peak position under the same conditions within ± 0.2min, can be with
Regard as same substance.
The appearance time of propionic acid standard items is 11.2min；Go out peak position under the same conditions within ± 0.2min, can be with
Regard as same substance.
The appearance time of butyric acid standard items is 13.8min；Go out peak position under the same conditions within ± 0.2min, can be with
Regard as same substance.
AN uses phenol-hypochlorous acid colorimetric method for determining.
AA-N uses the bronze medal colorimetric method for determining of indenes three.
2nd, remaining ensilage dries 48h at 65 DEG C, after crushed 40 mesh sieves with plant pulverizer, carries out following index inspection
Survey：Dry (dry matter, DM), total nitrogen (crude protein, TN), nonprotein nitrogen (non-protein
nitrogen,NPN)。
DM is determined using oven drying method.
NPN uses Kjeldahl nitrogen determination after trichloroacetic acid precipitation.
Peptide nitrogen content calculates according to formula：Peptide-N=NPN-(AA-N+AN).
As a result as shown in table 6 and table 7.
Influences of the Lactobacillus plantarum LP1 to different moisture content Alfalfa Silage Fermentation Quality under 6 different reserve temperatures of table
Influences of the Lactobacillus plantarum LP1 to different moisture content alfalfa ensilage nitrogen component under 7 different reserve temperatures of table
In summary, Lactobacillus plantarum LP1 can be used as addictive for alfalfa silage, significantly improve lactic acid output, significantly reduce
PH value, effectively improve Alfalfa Silage Fermentation Quality；And nonprotein nitrogen and ammonia nitrogen content can be reduced, suppress albumen in alfalfa ensilage
Matter is degraded, and effectively improves the feeding value of alfalfa silage；Lactobacillus plantarum LP1 has fermentation efficiency height, cost low simultaneously
The advantages that honest and clean, it can be applied in the production of green environment protective biological feed.
<110>China Agricultural University
<120>A kind of Lactobacillus plantarum LP1 and its application in feed addictive is prepared
<160> 1
<210> 1
<211> 1457
<212> DNA
<213>Lactobacillus plantarum（Lactobacillus plantarum）
<400> 1
gcttgctata atgcagtcga cgaactctgg tattgattgg tgcttgcatc atgatttaca 60
tttgagtgag tggcgaactg gtgagtaaca cgtgggaaac ctgcccagaa gcgggggata 120
acacctggaa acagatgcta ataccgcata acaacttgga ccgcatggtc cgagtttgaa 180
agatggcttc ggctatcact tttggatggt cccgcggcgt attagctaga tggtggggta 240
acggctcacc atggcaatga tacgtagccg acctgagagg gtaatcggcc acattgggac 300
tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac aatggacgaa 360
agtctgatgg agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa actctgttgt 420
taaagaagaa catatctgag agtaactgtt caggtattga cggtatttaa ccagaaagcc 480
acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt 540
attgggcgta aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc ttcggctcaa 600
ccgaagaagt gcatcggaaa ctgggaaact tgagtgcaga agaggacagt ggaactccat 660
gtgtagcggt gaaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctgg 720
tctgtaactg acgctgaggc tcgaaagtat gggtagcaaa caggattaga taccctggta 780
gtccataccg taaacgatga atgctaagtg ttggagggtt tccgcccttc agtgctgcag 840
ctaacgcatt aagcattccg cctggggagt acggccgcaa ggctgaaact caaaggaatt 900
gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct 960
taccaggtct tgacatacta tgcaaatcta agagattaga cgttcccttc ggggacatgg 1020
atacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080
acgagcgcaa cccttattat cagttgccag cattaagttg ggcactctgg tgagactgcc 1140
ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg 1200
ctacacacgt gctacaatgg atggtacaac gagttgcgaa ctcgcgagag taagctaatc 1260
tcttaaagcc attctcagtt cggattgtag gctgcaactc gcctacatga agtcggaatc 1320
gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380
ccgtcacacc atgagagttt gtaacaccca aagtcggtgg ggtaaccttt taggaaccag 1440
ccgcctaagg tgaaccc 1457
Claims (8)
1. applications of Lactobacillus plantarum (Lactobacillus plantarum) LP1 in alfalfa silage is prepared.
2. a kind of microbial inoculum, it is to mix Lactobacillus plantarum (Lactobacillus plantarum) LP1 with probiotics protective agent
Arrive.
3. microbial inoculum as claimed in claim 2, it is characterised in that：Contain 1 × 10 in every gram of microbial inoculum12CFU Lactobacillus plantarums
(Lactobacillus plantarum)LP1。
4. a kind of preparation method of microbial inoculum, comprises the following steps：By Lactobacillus plantarum (Lactobacillus plantarum)
LP1 mixes with probiotics protective agent.
5. application of the microbial inoculum described in Claims 2 or 3 in alfalfa silage is prepared.
6. a kind of preparation method of alfalfa silage, comprises the following steps：Microbial inoculum described in Claims 2 or 3 is applied to
On clover, ensiling is carried out, obtains alfalfa silage.
7. a kind of preparation method of alfalfa silage, comprises the following steps：By Lactobacillus plantarum (Lactobacillus
Plantarum) LP1 is applied on clover, is carried out ensiling, is obtained alfalfa silage.
8. the alfalfa silage that the method described in claim 6 or 7 is prepared.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108060104A (en) * | 2018-01-29 | 2018-05-22 | 四川农业大学 | A kind of lactobacillus plantarum and its application, screening calibration method |
| CN112746044A (en) * | 2021-01-28 | 2021-05-04 | 郑州大学 | Lactobacillus plantarum mutant strain and application thereof in alfalfa silage |
| CN113142396A (en) * | 2021-06-11 | 2021-07-23 | 中国农业大学 | Microbial inoculum for preparing leguminous forage silage |
| CN113355265A (en) * | 2021-06-30 | 2021-09-07 | 广东温氏大华农生物科技有限公司 | Lactobacillus plantarum and application thereof |
| CN114836477A (en) * | 2022-06-14 | 2022-08-02 | 江苏省农业科学院 | Chicken liver fermentation process method |
| CN116716228A (en) * | 2023-07-24 | 2023-09-08 | 中国农业大学 | Lactobacillus plantarum EL6 with flavonoid content improving function and application thereof |
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| CN108060104A (en) * | 2018-01-29 | 2018-05-22 | 四川农业大学 | A kind of lactobacillus plantarum and its application, screening calibration method |
| CN108060104B (en) * | 2018-01-29 | 2019-01-29 | 四川农业大学 | A kind of lactobacillus plantarum and its application, screening calibration method |
| CN112746044A (en) * | 2021-01-28 | 2021-05-04 | 郑州大学 | Lactobacillus plantarum mutant strain and application thereof in alfalfa silage |
| CN112746044B (en) * | 2021-01-28 | 2022-04-19 | 郑州大学 | Lactobacillus plantarum mutant strain and application thereof in alfalfa silage |
| CN113142396A (en) * | 2021-06-11 | 2021-07-23 | 中国农业大学 | Microbial inoculum for preparing leguminous forage silage |
| CN113355265A (en) * | 2021-06-30 | 2021-09-07 | 广东温氏大华农生物科技有限公司 | Lactobacillus plantarum and application thereof |
| CN113355265B (en) * | 2021-06-30 | 2022-11-08 | 广东温氏大华农生物科技有限公司 | Lactobacillus plantarum and application thereof |
| CN114836477A (en) * | 2022-06-14 | 2022-08-02 | 江苏省农业科学院 | Chicken liver fermentation process method |
| CN114836477B (en) * | 2022-06-14 | 2023-08-01 | 江苏省农业科学院 | Chicken liver fermentation process |
| CN116716228A (en) * | 2023-07-24 | 2023-09-08 | 中国农业大学 | Lactobacillus plantarum EL6 with flavonoid content improving function and application thereof |
| CN116716228B (en) * | 2023-07-24 | 2024-04-19 | 中国农业大学 | Lactobacillus plantarum EL6 with flavonoid content improving function and application thereof |
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