CN107034157A - A kind of Lactobacillus plantarum and its application - Google Patents
A kind of Lactobacillus plantarum and its application Download PDFInfo
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- CN107034157A CN107034157A CN201710278393.6A CN201710278393A CN107034157A CN 107034157 A CN107034157 A CN 107034157A CN 201710278393 A CN201710278393 A CN 201710278393A CN 107034157 A CN107034157 A CN 107034157A
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- lactobacillus plantarum
- ensilage
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- feed mulberry
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- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 67
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 66
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 66
- 240000000249 Morus alba Species 0.000 claims abstract description 43
- 235000008708 Morus alba Nutrition 0.000 claims abstract description 43
- 239000004460 silage Substances 0.000 claims abstract description 14
- 239000000654 additive Substances 0.000 claims abstract description 10
- 230000000996 additive effect Effects 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 241000196324 Embryophyta Species 0.000 claims description 8
- 241000186660 Lactobacillus Species 0.000 claims description 8
- 229940039696 lactobacillus Drugs 0.000 claims description 8
- 238000005520 cutting process Methods 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 239000003674 animal food additive Substances 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 15
- 238000000855 fermentation Methods 0.000 abstract description 12
- 230000004151 fermentation Effects 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 7
- 235000016709 nutrition Nutrition 0.000 abstract description 4
- 238000012545 processing Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 230000035764 nutrition Effects 0.000 abstract description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 16
- 239000004310 lactic acid Substances 0.000 description 8
- 235000014655 lactic acid Nutrition 0.000 description 8
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- 239000008272 agar Substances 0.000 description 4
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- OBMBUODDCOAJQP-UHFFFAOYSA-N 2-chloro-4-phenylquinoline Chemical compound C=12C=CC=CC2=NC(Cl)=CC=1C1=CC=CC=C1 OBMBUODDCOAJQP-UHFFFAOYSA-N 0.000 description 2
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 235000019750 Crude protein Nutrition 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000235342 Saccharomycetes Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
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- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- 229960002163 hydrogen peroxide Drugs 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
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- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 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 2
- 238000012795 verification Methods 0.000 description 2
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- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 241000282849 Ruminantia Species 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
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 238000004380 ashing Methods 0.000 description 1
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- 241000909929 bacterium ZS-1 Species 0.000 description 1
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- 229920005610 lignin Polymers 0.000 description 1
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- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 239000002304 perfume Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- NESLWCLHZZISNB-UHFFFAOYSA-M sodium phenolate Chemical compound [Na+].[O-]C1=CC=CC=C1 NESLWCLHZZISNB-UHFFFAOYSA-M 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
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- 235000011046 triammonium citrate Nutrition 0.000 description 1
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Classifications
-
- 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
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Fodder In General (AREA)
Abstract
The invention discloses a kind of Lactobacillus plantarum for belonging to ensilage processing technique field and its application.Lactobacillus plantarum (Lactobacillus plantarum) ZS1 of the present invention, preserving number is CGMCC No.13460, available for ensilage is prepared, especially prepares feed mulberry ensilage.Lactobacillus plantarum (Lactobacillus plantarum) ZS1 of the present invention can quickly reduce the pH value of ensilage, and accelerated fermentation processes improve ensiling speed；Feed mulberry Silage Quality is effectively improved, feed mulberry ensilage miscellaneous bacteria quantity is reduced, can preferably preserve feed mulberry nutrition, the problem of feed mulberry fermentation quality is poor is overcome, can widely use.With the present invention Lactobacillus plantarum (Lactobacillus plantarum) ZS1 prepare additive silage effect than commercially available lactic bacteria additive more preferably, and cost it is low, safely, be easy to utilize.
Description
Technical field
The invention belongs to ensilage processing technique field, and in particular to a kind of Lactobacillus plantarum and its application.
Background technology
Increase and industrial expansion with population, China's animal husbandry are developed rapidly, and people and animals strive grain problem increasingly
Sharply, the development of animal husbandry is directly affected.Therefore, scientifically and rationally exploitation Resourse of Forage Plant is the material base developed animal husbandry
Plinth, is to solve a not enough important channel of Forage Germplasm Resources.China scientific research personnel cultivates newest resistance strain-feed mulberry,
Because its is nutritious, palatability is good, easy to digest, with higher protein and calcium content, with the potential ecological value and feeding
Material value, DEVELOPMENT PROSPECT is very wide.Ensilage color is yellowish green, the sour perfume of smell, soft and succulency, palatability are good, in animal husbandry
Extensive use in production, especially in ruminant is raised, it has also become indispensable basal feed.But forage plant is because various
Reason causes Silage Quality bad, influences the palatability of domestic animal.According to the characteristics of feed mulberry, by verification experimental verification, finally think
The lactic acid bacterium number that growth is fast, production acid is high is lowly the reason for feed mulberry Silage Quality is low.
At present, it is to be directed to corn silage, alfalfa ensilage or other forage crops mostly using wide commodity microbial inoculum, will
It applies the effect in feed mulberry ensiling not good.
The content of the invention
The invention aims to overcome in ensilage preparation process in the prior art, lactobacter growth is slow, it is low to produce acid
And cause the problem of feed mulberry Silage Quality is low, propose that one kind can quickly reduce pH value, accelerated fermentation processes effectively improve feeding
Expect the Lactobacillus plantarum of mulberry Silage Quality.
Therefore, the concrete technical scheme of the present invention is as follows：
A kind of Lactobacillus plantarum (Lactobacillus plantarum), bacterial strain is ZS1, and preserving number is CGMCC
No.13460。
Lactobacillus plantarum (Lactobacillus plantarum) ZS1, from feed mulberry raw material.
The 16S rDNA sequences such as SEQ ID No.1 of Lactobacillus plantarum (Lactobacillus plantarum) ZS1
It is shown.
The biological characteristics of Lactobacillus plantarum (Lactobacillus plantarum) ZS1 is：Gram's staining sun
Property bacillus, glucose homofermentation, acid resistance is strong (can be with normal growth in pH3.0), growth rate height (MRS medium cultures
24 hours, OD620nm>2.00), acid production speed is fast (MRS medium cultures 24 hours, pH can drop to less than 4.0), gives birth to wide temperature range
(5-50 DEG C can normal growth), the high-yield lactic acid under the conditions of pure culture and ensiling.
A kind of active component of silage additive is Lactobacillus plantarum (Lactobacillus plantarum) ZS1.
The preparation method of the silage additive, comprises the following steps：By described Lactobacillus plantarum
(Lactobacillus plantarum) ZS1 is cultivated on MRS culture mediums, obtains silage additive.
Applications of described Lactobacillus plantarum (Lactobacillus plantarum) ZS1 in ensilage preparation.
In above-mentioned application, the ensilage is feed mulberry ensilage.
A kind of preparation method of ensilage, comprises the following steps：
1) will feed cutting be fermented, be well mixed；
2) to step 1) the middle Lactobacillus plantarum (Lactobacillus plantarum) added described in claim 1
ZS1；
3) by step 2) in feed vacuum sealing, storage, tunning is ensilage.
Step 1) in treat fermented feed be feed mulberry.
Step 1) in feed cutting length to be fermented be 1~2cm.
Step 2) in every kilogram treat fermented feed add Lactobacillus plantarum (Lactobacillus plantarum) ZS11.0
×105~1.0 × 106It is individual.
Step 3) in holding conditions be：15~45 DEG C of temperature, 30~60d of time.
Beneficial effects of the present invention are：
1st, Lactobacillus plantarum (Lactobacillus plantarum) ZS1 of the invention can be used for preparing ensilage, especially
It is to prepare feed mulberry ensilage.
2nd, Lactobacillus plantarum (Lactobacillus plantarum) ZS1 of the invention can quickly reduce ensilage
PH value, accelerated fermentation processes, improve ensiling speed；Feed mulberry Silage Quality is effectively improved, reduction feed mulberry ensilage is miscellaneous
Bacterium number amount, can preferably preserve feed mulberry nutrition, overcome the problem of feed mulberry fermentation quality is poor, can widely use.
3rd, imitated with Lactobacillus plantarum (Lactobacillus plantarum) ZS1 of present invention additive ensilings prepared
Fruit than commercially available lactic bacteria additive more preferably, and cost it is low, safely, be easy to utilize.
Biomaterial preservation is proved
Classification And Nomenclature：Lactobacillus plantarum；Strain number：ZS1
Preservation mechanism：China General Microbiological culture presevation administrative center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On December 15th, 2016
Collection is registered on the books numbering：CGMCC No.13460
Embodiment
Following embodiment facilitates a better understanding of the present invention, but is not limited to the present invention.Such as without special in following embodiments
Illustrate, be conventional method.
GFG (business microbial inoculum), purchased from Sichuan Gaofuji Biotechnology Co., Ltd., culture medium is biological from the extensive and profound in meaning star in Beijing
Science and Technology Ltd. (Beijing) buys.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1：Lactobacillus plantarum (Lactobacillus plantarum) ZS1 separation is identified with bacterial strain
1st, the separation of bacterial strain
Using dilution-plate method separating plant lactobacillus：Feed mulberry original 10g is weighed under aseptic condition, be placed in equipped with 90mL without
In the conical flask of bacterium distilled water, shake up, as dilute 10 times of sample suspension, be by 10 times of direct serial dilutions of dilution method afterwards
Can.Appropriate dilution is chosen, takes 0.1mL to be coated on MRS culture medium flat plates, 48h is cultivated in 30 DEG C of insulating boxs, flat board is carried out
Count.According to features such as bacterium colony size, form and colors, picking colonies typical carries out gram stain microscopy and hydrogen peroxide
Enzyme test.The negative bacterial strain of every Gram-positive, catalase test can be primarily determined that as lactobacillus, by its
Line purifying twice, is inoculated into MRS solid slope culture mediums, 4 DEG C of refrigerators and saved backup on MRS culture mediums.
MRS culture mediums：Peptone (Proteose peptone NO.3) 10.0g, powdered beef (Beef extract)
5.0g, dusty yeast (Yeast extract) 4.0g, glucose (Dextrose) 20.0g, tween (Polysorbate 80) 1mL,
Triammonium citrate (Ammonium citrate tribasic) 2.0g, sodium acetate (NaAc) 5.0g, magnesium sulfate (MgSO4·7H2O)
0.2g, manganese sulfate (MnSO4·4H2O) 0.05g, dipotassium hydrogen phosphate (K2HPO4) 2.0g, distilled water (H2O) 1000mL, solid culture
Base adds 15g/L agar (Agar) again, and 121 DEG C, sterilize 20min.
2nd, screening high-yield lactic acid, acidproof and fast growth lactobacillus
The lactobacillus separated from feed mulberry raw material, carry out Gram's staining, glucose aerogenesis, catalase test,
Give birth to temperature (5,10,45,50 DEG C) and fertility pH (3.0,3.5,4.0,4.5,9.0), carbon source through fermentation, the pH value fermented in 24h
And OD620nmDeng bio-chemical characteristics.
Physiological and biochemical test method is as follows：
(1) Gram's staining is with reference to the elegant pearl chief editor's in east《Common bacteria system identification handbook》.
(2) Lactobacillus plantarum growth pH regulations use 2mol/L NaOH and 2mol/L HCl.
(3) glucose aerogenesis is tested：Experimental strain is seeded on MRS culture mediums and in anaerobic culture box culture 48h, chosen
Take single bacterium colony to be inoculated in the test tube containing 5mLMRS fluid nutrient mediums (pH6.5) (Du Shi tubules back-off is in vitro), place
In common 37 DEG C of culture 48h of constant incubator, observe and record result, produce bubble is designated as the positive, and bubble is not produced then
For feminine gender.
(4) Catalase determination is tested：Experimental strain is seeded on MRS culture mediums and in anaerobic culture box culture
48h, picking single bacterium colony, which is coated on, to be added dropwise on the culture dish of 3% (w/w) hydrogenperoxide steam generator, has seen whether bubble generation,
Produce bubble for catalase positive, bubble is not produced is then feminine gender.
(5) the Bacillus acidi lactici biochemical identification suit analysis that sugar fermentation is produced using Beijing road and bridge technical concern Co., Ltd.
For Gram-positive, the experimental strain of negative catalase is set with using the biochemical identification containing 8 kinds of carbon sources, is compareed and is
One Maxwell turbidity opacity tube, test method is carried out according to specification, 37 DEG C of incubated 48h, log.
Physiology and biochemistry method is used, the newborn bar of high-yield lactic acid, acidproof and fast growth plant is filtered out from 103 plants of bacterial strains
Bacterium ZS1.As a result show, Lactobacillus plantarum ZS1 is Gram-positive, homo-fermentative bacillus, in 5-50 DEG C and pH3.0-9.0 bars
It can all be grown under part, with stronger acid resistance (table 1), can ferment most of carbohydrates (table 2), pH drops to less than 4.0 after fermentation 24h
And OD620nm>2.00 (tables 3).
The Lactobacillus plantarum ZS1 of table 1 colonial morphology and physio-biochemical characteristics
Note：-, do not grow；+, normal growth.
The Lactobacillus plantarum ZS1 of table 2 sugar source fermenting experiment result
Note：-, do not grow；+, normal growth.
The Lactobacillus plantarum ZS1 of table 3 rate of producing acid and growth rate
3rd, the identification of bacterial strain
The high-yield lactic acid of screening, acidproof and fast growth lactobacillus are seeded in 0.5mLMRS fluid nutrient mediums,
30 DEG C, 180rmp overnight incubations carry out strain idenfication using bacterium solution PCR.
Universal primer of the primer from the 16S rDNA gene magnifications of bacterium：
27F：5’-AGAGTTTGATCCTGGCTCAG-3’
1492R：5’-GGTTACCTTGTTACGACTT-3’
PCR reaction systems (40 μ L)：
Reaction condition：
Amplified production Song Meiji bio tech ltd be sequenced, 16S rDNA sequences as shown in SEQ ID No.1,
Sequence alignment is carried out on NCBI, with reference to the Physiology and biochemistry data of bacterial strain, the final bacterial strain for determining separation belongs to Lactobacillus plantarum
Lactobacillus plantarum, are named as ZS1.
Embodiment 2：The preparation of feed mulberry ensilage
The preparation of feed mulberry ensilage comprises the following steps：
(1) the feed mulberry slightly dried is cut to 2cm or so, be well mixed；
(2) Lactobacillus plantarum (Lactobacillus plantarum) ZS1, every kilogram is added into step (1) feed mulberry
Feed mulberry adds Lactobacillus plantarum (Lactobacillus plantarum) ZS1 about 1.0 × 106It is individual, and with no added (CK),
GFG (business microbial inoculum) is control, each 3 repetitions of processing；
(3) by step (2) feed mulberry load 30cm × 20cm polyethylene bag silo, per it is packed enter 100g, it is close with vacuum
The pumping of envelope machine, sealing, are placed in room temperature storage 60d.
1st, ensilage nutritional quality is determined
The conventional nutrients assay method of feed mulberry ensilage is as follows：
The measure of dry (DM)：Using air dry oven, feed mulberry ensilage is dried under 65 DEG C of constant temperatures
48h, cooling is weighed；Crude protein (CP) is measured using the full-automatic Kjeldahl determination devices of FOSS 8400；Neutral detergent fiber, acid
Property the fiber type analysis systems of washing fiber utilization Ankom 220 be measured, soluble sugar (WSC) content uses Anthrone Sulphuric acid
Method is determined.Acid detergent fiber is digested with 72% sulfuric acid, residue is ashed, the part escaped in ashing i.e. acidic cleaning wood
Quality.
As shown in table 4, though difference is not notable between each nutriment, addition plant is newborn for nutriment after feed mulberry ensiling
Bacillus (Lactobacillus plantarum) ZS1 ensilage, its dry, crude protein and soluble sugar content are higher than
CK and GFG, and neutral detergent fiber, acid detergent fiber and acidic cleaning content of lignin are slightly below and are not added with.Therefore, add
The nutritional quality of Lactobacillus plantarum (Lactobacillus plantarum) ZS1 feed mulberry ensilage is slightly higher.
Nutriment (g kg after the feed mulberry ensiling of table 4-1DM)
Note：Having the representative of same letter tail tag per column data, there was no significant difference, and no same letter then has conspicuousness poor
It is different
2nd, ensilage fermentation quality determination
The fermentation quality analysis method of feed mulberry ensilage is as follows：
Ammonia nitrogen content utilizes phenol-sodium hypochlorite colorimetric method for determining；Organic acidity test uses the efficient liquid of Shimadzu GC-14 types
Chromatography (chromatographic column：KC-811column, Shimadzu, Japan), detector：SPD-M10AVP, mobile phase：3mmol L-1
Perchloric acid, flow velocity 1mLmin-1, 50 DEG C of column temperature, Detection wavelength 210nm, the μ L of sample size 5.
Feed mulberry ensiling after fermentation quality as shown in table 5, compared with CK and GFG, adds Lactobacillus plantarum
(Lactobacillus plantarum) ZS1 can significantly reduce feed mulberry ensiling pH, ammoniacal nitrogen and acetic acid content (P ＜ 0.05),
And significantly improve lactic acid content (P ＜ 0.05), that is, significantly improve feed mulberry Silage Quality.
Feed mulberry ensiling after fermentation quality (the g kg of table 5-1DM)
Note：Having the representative of same letter tail tag per column data, there was no significant difference, and no same letter then has conspicuousness poor
It is different
3rd, ensilage microbiological analysis
Feed mulberry ensilage aseptically takes 10g (to change gloves in sampling process, prevent dirt immediately after opening
Dye), add the physiological saline of 90mL sterilizings, 150rpm vibration 2h, gradient dilution coating.Micro organism quantity, which is determined, uses flat board meter
Number method, lactic acid bacteria MRS solid mediums, saccharomycete rose-bengal solid medium (Rose Bengal Medium
Agar) count, Escherichia coli and mould Yihong methylene blue solid medium (eosin-methylene blue medium
Agar) count.Lactic acid bacteria 30 DEG C of culture 2d of anaerobic box, Escherichia coli, mould 37 DEG C of culture 2d of biochemical cultivation case, saccharomycete
With 28 DEG C of culture 2d of biochemical cultivation case.
Rose-bengal solid medium (g/L)：Peptone 5g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, glucose 10g, chlorine
Mycin 0.1g, rose-bengal 0.033g, agar 18.5g.
Yihong methylene blue solid medium (g/L)：Peptone 5g, beef extract powder 3g, lactose 10g, sodium chloride 5g, eosine
0.4g, methylenum careuleum 0.065g, agar 14g.
Microbiological indicator as shown in table 6, as a result shows addition Lactobacillus plantarum (Lactobacillus after feed mulberry ensiling
Plantarum) ZS1 can significantly reduce feed mulberry ensilage miscellaneous bacteria quantity.
Microbiological indicator (log cfu g after the feed mulberry ensiling of table 6-1FM)
Note：Having the representative of same letter tail tag per column data, there was no significant difference, and no same letter then has conspicuousness poor
It is different.
SEQUENCE LISTING
<110>China Agricultural University
<120>A kind of Lactobacillus plantarum and its application
<130> 2017
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1437
<212> DNA
<213>Lactobacillus plantarum（Lactobacillus plantarum）
<400> 1
tgcagtcgaa cgctttttct ttcaccggag cttgctccac cgaaagaaaa ggagtggcga 60
acgggtgagt aacacgtggg taacctgccc atcagaaggg gataacactt ggaaacaggt 120
gctaataccg tataacaatc gaaaccgcat ggtttcggtt tgaaaggcgc ttttgcgtca 180
ctgatggatg gacccgcggt gcattagcta gttggtgagg taacggctca ccaaggcaac 240
gatgcatagc cgacctgaga gggtgatcgg ccacattggg actgagacac ggcccaaact 300
cctacgggag gcagcagtag ggaatcttcg gcaatggacg aaagtctgac cgagcaacgc 360
cgcgtgagtg aagaaggttt tcggatcgta aaactctgtt gttagagaag aacaaggatg 420
agagtagaat gttcatccct tgacggtatc taaccagaaa gccacggcta actacgtgcc 480
agcagccgcg gtaatacgta ggtggcaagc gttgtccgga tttattgggc gtaaagcgag 540
cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg 600
aaactgggaa acttgagtgc agaagaggag agtggaattc catgtgtagc ggtgaaatgc 660
gtagatatat ggaggaacac cagtggcgaa ggcggctctc tggtctgtaa ctgacgctga 720
ggctcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga 780
tgagtgctaa gtgttggagg gtttccgccc ttcagtgctg cagctaacgc attaagcact 840
ccgcctgggg agtacgaccg caaggttgaa actcaaagga attgacgggg gcccgcacaa 900
gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc 960
ctttgaccac tctagagata gagcttcccc ttcgggggca aagtgacagg tggtgcatgg 1020
ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat 1080
tgttagttgc catcattcag ttgggcactc tagcgagact gccggtgaca aaccggagga 1140
aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa 1200
tgggaagtac aacgagtcgc gaagtcgcga ggctaagcta atctcttaaa gcttctctca 1260
gttcggattg taggctgcaa ctcgcctaca tgaagccgga atcgctagta atcgcggatc 1320
agcacgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag 1380
tttgtaacac ccgaagtcgg tgaggtaacc tttggagcca gccgcctaag tgatgat 1437
Claims (10)
1. a kind of Lactobacillus plantarum (Lactobacillus plantarum), bacterial strain is ZS1, and preserving number is CGMCC
No.13460。
2. a kind of silage additive, it is characterised in that the active component of the additive is the plant described in claim 1
Lactobacillus (Lactobacillus plantarum) ZS1.
3. the preparation method of the silage additive described in claim 2, it is characterised in that comprise the following steps：By right
It is required that Lactobacillus plantarum (Lactobacillus plantarum) ZS1 described in 1 is cultivated on MRS culture mediums, ensiling feeding is obtained
Feed additives.
4. Lactobacillus plantarum (Lactobacillus plantarum) ZS1 described in claim 1 is in ensilage preparation
Using.
5. application according to claim 4, it is characterised in that the ensilage is feed mulberry ensilage.
6. a kind of preparation method of ensilage, it is characterised in that comprise the following steps：
1) will feed cutting be fermented, be well mixed；
2) to step 1) middle Lactobacillus plantarum (Lactobacillus plantarum) ZS1 added described in claim 1；
3) by step 2) in feed vacuum sealing, storage, tunning is ensilage.
7. preparation method according to claim 6, it is characterised in that step 1) in treat that fermented feed is feed mulberry.
8. preparation method according to claim 6, it is characterised in that step 1) in feed cutting length to be fermented be 1~
2cm。
9. preparation method according to claim 6, it is characterised in that step 2) in every kilogram treat that fermented feed adds plant
Lactobacillus (Lactobacillus plantarum) ZS1 1.0 × 105~1.0 × 106It is individual.
10. preparation method according to claim 6, it is characterised in that step 3) in holding conditions be：Temperature 15~45
DEG C, 30~60d of time.
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| CN113943667A (en) * | 2020-07-15 | 2022-01-18 | 中国农业大学 | Lactobacillus plantarum separated from camel rumen and application of lactobacillus plantarum in silage |
| CN114058531A (en) * | 2021-03-16 | 2022-02-18 | 中国农业大学 | Bacteriocin-producing lactobacillus plantarum and compound application thereof in silage |
| CN114107085A (en) * | 2021-09-14 | 2022-03-01 | 内蒙古农业大学 | Lactobacillus plantarum ING8 and application thereof |
| CN114231440A (en) * | 2021-11-24 | 2022-03-25 | 内蒙古农业大学 | Lactobacillus plantarum ING1 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108265016A (en) * | 2018-01-29 | 2018-07-10 | 四川农业大学 | A kind of lactic bacteria additive and its application, preparation method |
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| CN114058531A (en) * | 2021-03-16 | 2022-02-18 | 中国农业大学 | Bacteriocin-producing lactobacillus plantarum and compound application thereof in silage |
| CN114058531B (en) * | 2021-03-16 | 2023-05-30 | 中国农业大学 | Bacteriocin-producing lactobacillus plantarum and compound application thereof in silage |
| CN114107085A (en) * | 2021-09-14 | 2022-03-01 | 内蒙古农业大学 | Lactobacillus plantarum ING8 and application thereof |
| CN114231440A (en) * | 2021-11-24 | 2022-03-25 | 内蒙古农业大学 | Lactobacillus plantarum ING1 and application thereof |
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