CN106148249B - It is a kind of suitable for the lactic acid bacteria agent of grass silage and its application - Google Patents
It is a kind of suitable for the lactic acid bacteria agent of grass silage and its application Download PDFInfo
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- CN106148249B CN106148249B CN201610818343.8A CN201610818343A CN106148249B CN 106148249 B CN106148249 B CN 106148249B CN 201610818343 A CN201610818343 A CN 201610818343A CN 106148249 B CN106148249 B CN 106148249B
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Abstract
The present invention provides a kind of lactic acid bacteria agents suitable for grass silage, the lactic acid bacteria agent is made of lactobacillus plantarum freeze-dried powder and lactobacillus buchneri freeze-dried powder, the lactobacillus plantarum and lactobacillus buchneri is isolated from rye grass stalk, lactobacillus plantarum is homofermentative lactic bacteria, lactobacillus buchneri is heterofermentation lactobacillus, two plants of bacterial strains are preserved in China typical culture collection center on May 25th, 2016, the bacterial strain of the lactobacillus plantarum is L.plantarumZRR, deposit number is CCTCC No:M2016281, the bacterial strain of the lactobacillus buchneri is L.buchneriBR2, deposit number is CCTCC No:M2016280.Lactic acid bacteria of the invention has many advantages, such as to accelerate grass silage maturation, improves grass silage quality, improves aerobic stability, can be widely applied to grass silage feed preparation field.
Description
Technical field
The invention discloses a kind of suitable for the lactic acid bacteria agent of grass silage and its application, belongs to the blueness in feed stripped
Store feed processing technology.
Background technique
Ensiling is the proliferation by lactic acid bacteria, and the fermentation substrate in raw material is converted to the acidic materials such as lactic acid, maintains acid
Property anaerobic environment, in favor of a kind of storage method of silo crop long-term preservation.Ensilage color is yellowish green, smell sour, soft
Soft succulence, palatability are good, are widely applied in Animal husbandry production, especially in ruminant raising, it has also become important is high-quality
Roughage source.Compared with herbage dries into hay, the time from harvesting to storage can be greatly shortened in grass silage, avoided
The loss of forage grass caused by adverse weather, saves the nutritional ingredient of green forage to greatest extent.
Lactic acid bacteria is the microoganism additives for ensiling promoted in recent years, and main function is micro- in adjusting ensiling material
Biotic component, rapidly becomes dominant microflora, the growth of Competitive assays harmful microorganism, and the limited sugar of rapid conversion drops significantly
Low ensiling pH value has larger potentiality and vast potential for future development to effectively improve the quality of ensilage.Currently, to blueness
The development of storage lactic acid bacteria is concentrated mainly on the single cultures such as lactobacillus plantarum.A kind of patent document " lactobacillus plantarum and its warm
Application (CN2016100121 45.2) in season type grass silage " provide it is isolated from native grass ensilage
One lactobacillus plantarum.But ensiling is from the later period behind early period, mid-term and Kaifeng, the microorganism group of Silage at, it is physical and chemical
A series of variation can all occur for property and local environment.Single lactic acid bacteria product is unable to satisfy the ensiling requirement of variation.Separately
On the one hand, lactic acid bacteria is divided into homofermentative lactic bacteria and heterofermentative lactic bacteria by fermented type.Homofermentative lactic bacteria utilizes
Water soluble carbohydrates (WSC) generate more lactic acid, improve silage fermentation quality, but inhibit aerobic rotten ability universal
It is poor.Heterofermentative lactic bacteria generates more acetic acid using WSC, it is suppressed that aerobic bacteria, yeast and mould and ensiling are raised
Expect aerobic rotten.Patent document " a kind of microbe additive (CN 201410677476.9) for being used to prepare ensilage " mentions
A kind of novel plant lactobacillus ZJY-6 and the microbe additive comprising the bacterium are supplied, microbe additive also includes Bu Shi cream
It is bacillus, Lactobacillus casei, lactobacillus thermophilus, enterococcus, Pediococcus pentosaceus, bacillus pumilus, any in Pediococcus acidilactici
It is a kind of.Patent document is " with the method (CN20141063295 of lactobacillus buchneri and lactobacillus plantarum preparation sheath of sugarcane ensilage
It 3.X) " discloses and is combined with the microbial inoculum of lactobacillus buchneri and lactobacillus plantarum come the method for ensiling sheath of sugarcane.But this two parts special
In sharp document, lactobacillus plantarum, lactobacillus buchneri one be isolated from silage corn, one is purchased from strain storehouse.In microorganism fungus kind
In resource, subspecies (bacterial strain) that each strain has up to a hundred or even thousands of upper performances different.Habitat is fitted according to microorganism
Answering property and specific principle, the separating lactic acid bacterium from herbage material reapply lactic acid bacteria in herbage, and lactic acid bacteria rapider can obtain
It is colonized in grass silage, forms dominant microflora.Therefore the separating lactic acid bacterium from herbage, selects the lactic acid bacteria bacterium having complementary functions
Strain, the exclusive lactic acid bacteria agent of preparation herbage are the important guarantees for improving grass silage feed quality.
Summary of the invention
In view of the deficiencies of the prior art, its purpose is to provide a kind of lactic acid bacteria agents suitable for grass silage by the present invention
And its application.
First goal of the invention of the invention is to provide a kind of lactic acid bacteria agent suitable for grass silage, the lactic acid
Bacteria agent is made of lactobacillus plantarum freeze-dried powder and lactobacillus buchneri freeze-dried powder, and the lactobacillus plantarum and lactobacillus buchneri are equal
Isolated from rye grass stalk, lactobacillus plantarum is homofermentative lactic bacteria, and lactobacillus buchneri is heterofermentation lactobacillus,
Two plants of bacterial strains are preserved in China typical culture collection center on May 25th, 2016, and the bacterial strain of the lactobacillus plantarum is
L.plantarumZRR, deposit number are CCTCC No:M2016281, and the bacterial strain of the lactobacillus buchneri is L.buchneriBR2,
Deposit number is CCTCC No:M2016280；
In the lactic acid bacteria agent, the weight ratio of lactobacillus plantarum freeze-dried powder and lactobacillus buchneri freeze-dried powder is 2:1, is planted
Viable count in object lactobacillus freeze-dried powder reaches 8 × 109Cfu/g, the viable count in lactobacillus buchneri freeze-dried powder reaches 4 ×
109The viable count of cfu/g, lactic acid bacteria agent reach 1.2 × 1010cfu/g；
The lactic acid bacteria agent is 2.4 × 10 in the additive amount of grass silage feed5cfu/g。
The lactic acid bacteria agent suitable for grass silage, the 16S rDNA gene order of the lactobacillus plantarum is such as
Shown in SEQ ID No.1, the 16S rDNA gene order of the lactobacillus buchneri is as shown in SEQ ID No.2.
Lactobacillus plantarum (L.plantarum ZRR) of the invention is screened from herbage stalk with production acid speed
Degree is fast, growth ability is strong, good acid proof lactic acid bacteria；The bacterial strain has been preserved in China typical culture collection center, preservation
Address: No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road in the school, classification naming: lactobacillus plantarum
Lactobacillus plantarum, deposit number: CCTCC No:M2016281, preservation date are as follows: on May 25th, 2016.
The bacterium colony of lactobacillus plantarum (Lactobacillus plantarum) ZRR is creamy white, neat in edge；Carefully
Born of the same parents' microscopic morphology is rod-short, no gemma, Gram-positive；Catalase is negative, oxidase negative；With stronger growth
And product acid activity, it can be grown on the culture medium that initial ph value is 3.0-8.0, faint growth be presented under the conditions of 10 DEG C, 15
Upgrowth situation is good under the conditions of DEG C -40 DEG C, and upgrowth situation is good under conditions of being not more than 6.5%NaCl.
Lactobacillus buchneri (L.buchneriBR2) of the invention be screened from herbage stalk have produce acetic acid,
Good acid proof lactic acid bacteria；The bacterial strain has been preserved in China typical culture collection center, preservation address: Wuhan City, Hubei Province
No. 299 Wuhan Universitys of Wuchang District Bayi Road in the school, classification naming: lactobacillus buchneri Lactobacillus buchneri, preservation
Number: CCTCC No:M2016280, preservation date are as follows: on May 25th, 2016.
The bacterium colony of lactobacillus buchneri (Lactobacillus buchneri) BR2 is in canescence, sticky, edge is whole
Together；Cell microscopic morphology be in rod-shaped, no gemma, Gram-positive；Catalase is negative, oxidase negative；With relatively strong
Growth and product acid activity, initial ph value be 3.0-7.5 culture medium on can grow, faint life is presented under the conditions of 10 DEG C
Long, upgrowth situation is good under the conditions of 15 DEG C -40 DEG C, and upgrowth situation is good under conditions of being not more than 6.5%NaCl.
Second goal of the invention of the invention is to provide application of the above-mentioned lactic acid bacteria agent in preparation grass silage feed.
The grass silage feed producing method is as follows:
Raw material preparation: after herbage cradles, guarantee cleaning, go bad without going rotten, and be cut to 2-3cm.
Bacterial strain activation: L.plantarumZRR, L.buchneriBR2 of freezen protective are inoculated in the training of MRS liquid respectively
Support in base, cultivate 18-24h at 37 DEG C of temperature, obtain for such secondary culture 2-3 time described in activation L.plantarumZRR,
L.buchneriBR2 strain；The described MRS fluid nutrient medium composition is as follows: 10g peptone, 5g beef extract, 4g yeast extract,
20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate
1000mL distilled water is added, adjusts pH to 6.5,121 DEG C of sterilizing 15min.
The preparation of bacterium powder: by the strain of above-mentioned activation, inoculation fermentation, freeze-drying, crushing, auxiliary material are mixed and made into jelly respectively
The freeze-dried powder of dry bacterium powder, L.plantarumZRR, L.buchneriBR2 is mixed according to the weight ratio of 2:1, makes lactic acid bacteria agent
The viable count of freeze-dried powder reaches 1.2 × 1010cfu/g。
The modulation of grass silage: it is former to weigh 200g herbage in 65%-70% for moisture content control when herbage raw material ensiling
Material is fitted into vacuum packaging polybag, by the bacterium powder of above-mentioned preparation with 2.4 × 105The inoculum concentration of cfu/g is inoculated into raw material, is adopted
After being vacuum-packed with vacuum packing machine, it is placed in storeroom and ferments.
The condition of the fermentation are as follows: fermentation temperature is 15-25 DEG C, fermentation time 30-60d.The quality of ensiling herbage is examined
It surveys: after fermenting-ripening, taking out part ensiling herbage progress microorganism group and analyzed at fermentation quality.
Lactic acid bacteria agent provided by the invention has following good effects:
(1) adaptability in habitat and specific principle are filtered out from herbage and improves grass silage product according to microorganism
Exclusive bacterium L.plantarumZRR, L.buchneriBR2 of matter.(2) present invention passes through creative work, finds lactic acid bacteria agent
The best proportion of effect is lactobacillus plantarum and lactobacillus buchneri, and weight ratio 2:1, lactic acid bacteria agent is in grass silage feed
Additive amount be 2.4 × 105cfu/g.(3) compared with existing single lactic acid bacteria, inoculation L.plantarumZRR,
The lactic acid bacteria agent of L.buchneriBR2 can quickly become dominant microflora in earlier fermentation, reduce pH value；The content of lactic acid
It significantly improves, ammonia nitrogen content decline is conducive to the palatability for improving ensilage, improves the fermentation quality of grass silage；
Acetic acid content is significantly improved, the content of ethyl alcohol is reduced, the growth for inhibiting the fungies such as mould, yeast in ensilage after bag is opened, mentions
High aerobic stability, to be conducive to extend the pot-life of ensilage.
Therefore lactic acid bacteria agent of the present invention, which has, accelerates grass silage maturation, improves grass silage quality, improves
The advantages that aerobic stability, can be widely applied to grass silage feed preparation field.
Detailed description of the invention
Fig. 1-1 is Different adding amount ensiling napier grass pH dynamic change；
Fig. 1-2 is Different adding amount ensiling napier grass lactic acid bacteria dynamic change；
Fig. 1-3 is the variation of Different adding amount ensiling napier grass dynamic of bacteria；
Fig. 1-4 is Different adding amount ensiling napier grass mould dynamic change；
Fig. 2-1 is different microbial inoculum ensiling rye grass pH dynamic changes；
Fig. 2-2 is different microbial inoculum ensiling rye grass lactic acid bacteria dynamic changes；
Fig. 2-3 is the change of different microbial inoculum ensiling rye grass dynamic of bacteria；
Fig. 2-4 is different microbial inoculum ensiling rye grass mould dynamic changes；
Mould dynamic change when Fig. 3-1 is different microbial inoculum ensiling rye grass ensilings and after aerobic exposure；
Yeast dynamic change when Fig. 3-2 is different microbial inoculum ensiling rye grass ensilings and after aerobic exposure.
Specific embodiment
The present invention will be further described with reference to the accompanying drawings and examples, and embodiment below facilitates a better understanding of this
Invention, but do not limit the present invention.Experimental method in following embodiments is unless otherwise specified conventional method；Used
Material, reagent etc., are commercially available unless otherwise specified.
1 lactobacillus plantarum of embodiment (Lactobacillus plantarum) ZRR, lactobacillus buchneri
The separation and identification of (Lactobacillus buchneri) BR2
1, it isolates and purifies
It is isolated and purified from rye grass stalk to lactobacillus plantarum ZRR and lactobacillus buchneri BR2, is for homofermentation respectively
Lactic acid bacteria and heterofermentation lactobacillus, specific as follows: it weighs 10g rye grass straw sample and is added in the 90ml distilled water of sterilizing,
1h is shaken using constant-temperature table, then 10 times of gradient dilutions, take 10 respectively-3、10-4With 10-5Times sample diluting liquid is coated on MRS
On solid medium, culture 48h is then taken out, size, form and the color of foundation bacterium colony, the list grown on picking MRS
Bacterium colony carries out catalase experiment and Gram's staining.Negative catalase and Gram-positive person are fixed tentatively and are
Lactic acid bacteria purifies 2 times in continuing scribing line on MRS solid medium.- 80 DEG C are stored in the MRS culture medium of 25% glycerol, bacterial strain
It is respectively designated as ZRR, BR2.
2, Physiology and biochemistry detects
Lactic acid bacteria ZRR can be grown on the culture medium that initial ph value is 3.0-8.0, and faint life is presented under the conditions of 10 DEG C
Long, upgrowth situation is good under the conditions of 15 DEG C -40 DEG C, and upgrowth situation is good under conditions of being not more than 6.5%NaCl.Lactic acid bacteria
BR2 can be grown on the culture medium that initial ph value is 3.0-7.5, faint growth be presented under the conditions of 10 DEG C, in 15 DEG C of -40 DEG C of items
Upgrowth situation is good under part, is resistant to 3.0% salinity.Part physicochemical property is as shown in table 1.Bacterial strain ZRR, BR2 can
The carbon source utilized is as shown in table 2.
3, Molecular Identification
Lactic acid bacteria strains ZRR, BR2 of 48h will be cultivated on solid plate, using colony PCR amplification, carry out 16rRNA gene
Sequence Identification.The 16S rRNA gene magnification universal primer of primer selection bacterium: 27F and 1492R.PCR reaction system is 50 μ l:
Premix Taq25 μ l, 27F and 1492R (being 20 μM) 1 μ l, supplement sterile purified water to 50 μ l directly bacterium colony are added anti-
System is answered, PCR amplification is carried out, obtains PCR product.PCR product is sent into sequencing, result is shown in sequence 1,2 in sequence table.
The physio-biochemical characteristics of table 1 bacterial strain ZRR, BR2
The carbon source through fermentation test result of table 2 bacterial strain ZRR, BR2
Note :+reaction is represented as the positive；- reaction is represented as feminine gender；W represents reaction as weakly positive
By above-mentioned identification, lactic acid bacteria strains ZRR, BR2 have been preserved in Chinese Typical Representative culture on May 25th, 2016
Collection, preservation address: No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road in the school, classification naming: plant cream bar
Bacterium Lactobacillus plantarum, deposit number: CCTCC No:M2016281, classification naming: lactobacillus buchneri
Lactobacillus buchneri, deposit number: CCTCC No:M2016280.
Application of 2 different strains of embodiment in rye grass ensiling
Raw material preparation: after rye grass cradles, guarantee cleaning, go bad without going rotten, and be cut to 2-3cm.
Bacterial strain activation: L.plantarumZRR, L.buchneriBR2 of freezen protective are inoculated in the training of MRS liquid respectively
Support in base, cultivate 18-24h at 37 DEG C of temperature, obtain for such secondary culture 2-3 time described in activation L.plantarumZRR,
L.buchneriBR2 strain；The described MRS fluid nutrient medium composition is as follows: 10g peptone, 5g beef extract, 4g yeast extract,
20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate
1000mL distilled water is added, adjusts pH to 6.5,121 DEG C of sterilizing 15min.
The preparation of bacterium powder: by the strain of above-mentioned activation, inoculation fermentation, freeze-drying, crushing, auxiliary material are mixed and made into jelly respectively
Dry bacterium powder, lactobacillus plantarum L.plantarumZRR (hereinafter referred to as ZRR), lactobacillus buchneri L.buchneri BR2 (following letter
Claim BR2) freeze-dried powder mixed according to different weight ratio, so that the viable count of lactic acid bacteria freeze drying powder is reached 1.2 × 1010cfu/g。
The mixed weight ratio of five groups of different strain groups is respectively:
Test group 1:100% lactobacillus plantarum ZRR；
Test group 2:100% lactobacillus buchneri BR2；
Test group 3:33% lactobacillus plantarum ZRR, 67% lactobacillus buchneri BR2 (1:2)；
Test group 4:50% lactobacillus plantarum ZRR, 50% lactobacillus buchneri BR2 (1:1)；
Test group 5:67% lactobacillus plantarum ZRR, 33% lactobacillus buchneri BR2 (2:1)；
The modulation of rye grass ensiling: moisture content when rye grass raw material ensiling is controlled in 65%-70%, is weighed 200g and is herded
Grassland material is fitted into vacuum packaging polybag, and the bacterium powder of above-mentioned preparation is pressed 0.04 ‰ (i.e. 4.8 × 105Cfu/g inoculum concentration)
It is inoculated into raw material, after being vacuum-packed using vacuum packing machine, is placed in storeroom and ferments.
After 42d fermenting-ripening, ensiling sample is taken to carry out attributional analysis.90ml aqua sterilisa is added in ensiling sample, sufficiently shakes
It swings, appropriate amount of fluid is taken to carry out the measurement of pH and organic acid, and take appropriate ensilage to be placed in 65 DEG C of baking ovens and air-dry to constant weight, survey
Its fixed dry matter.It adds influence of the different strain to rye grass Silage Quality and is shown in Table 3.
As shown in Table 3, test group 1 individually adds ZRR, and lactic acid content highest, pH and ethanol content are minimum, shows screening
Exclusive bacterium ZRR can be colonized in herbage rapidly, and inverted sugar is lactic acid, and inhibiting other bacterium inverted sugars is ethyl alcohol, and rapid acidification mentions
High Silage Quality, but acetic acid content is minimum, is unfavorable for the aerobic stability in fermentation later period.Test group 2 individually adds BR2, acetic acid
Content highest, but lactic acid producing amount is minimum in test group, pH and ethanol content highest.Individually addition ZRR or BR2, is not able to satisfy
Technical requirements of both Silage Quality and aerobic stability.Therefore, two plants of bacterial strains are compounded, different adding proportion groups is screened
It closes.Test group 3,4,5 the result shows that: the lactic acid content of test group 5 is up to 10.31%, ethanol content minimum 0.81%,
And the acetic acid content between tri- groups of BR2 of 100%, 67%, 33% does not have difference.Therefore, test group 5 is 67%ZRR, 33%BR2
The adding proportion of (2:1) combines, and can be quickly lactic acid and acetic acid by the soluble sugar conversion in rye grass, reduce pH, significantly
Ethanol content is reduced, aerobic stability is improved.
Table 3: influence (%/dry matter) of the addition different strain to rye grass Silage Quality
Note: with column data subscript, different letters indicate significant difference (P < 0.05)
Application of the lactic acid bacteria agent of 3 Different adding amount of embodiment in napier grass ensiling
The implementation steps such as raw material preparation, actication of culture, the preparation of bacterium powder, napier grass ensiling modulation are referring to embodiment 2, in bacterium powder
The freeze-dried powder of L.plantarumZRR, L.buchneri BR2 are mixed according to the ratio of 2:1 according to the result of embodiment 2 in preparation
It closes, the viable count of lactic acid bacteria agent freeze-dried powder is made to reach 1.2 × 1010cfu/g.By the bacterium powder of preparation respectively with 0.01 ‰,
0.02 ‰, 0.04 ‰, 0.1 ‰, 0.2 ‰ (i.e. 1.2 × 105、2.4×105、4.8×105、1.2×106、2.4×106cfu/g)
Inoculum concentration be inoculated into napier grass, after being vacuum-packed using vacuum packing machine, be placed in storeroom and ferment.
The microorganism dynamic change of ensiling napier grass detects: respectively in ensiling the 0th, 1,3,5,7,14,21,28,35,42d, taking
Part ensiling napier grass out carries out pH detection and microorganism composition analysis.Microorganism composition analysis includes lactic acid bacteria, mould, bacterium
Counting.Weigh 10g ensilage, 90ml aqua sterilisa be added, fullys shake, then with aqua sterilisa with 10 times of dilution methods to sample into
Row gradient dilution chooses dilution appropriate respectively and is coated on MRS culture medium, potato dextrose agar, nutrition fine jade
It on rouge, is placed in constant incubator and cultivates, lactic acid bacteria, mould, aerobic bacteria are counted respectively.The cream of Different adding amount
The microorganism dynamic result of sour bacteria agent ensiling napier grass is as shown in Fig. 1-1,1-2,1-3,1-4.
By microorganism composition analysis in Fig. 1-1,1-2,1-3,1-4 it is found that the ensiling napier grass of addition lactic acid bacteria and control
Group is compared, and pH is remarkably decreased, and lactic acid bacterium number dramatically increases, and the growth bacterium of bacterium and mould is suppressed completely.With high additive amount
0.04 ‰, 0.1 ‰, 0.2 ‰ (i.e. 1.2 × 105、2.4×105、4.8×105、1.2×106、2.4×106Cfu/g it) compares,
(i.e. the 2.4 × 10 of 0.02 ‰5Cfu/g) few additive, lactic acid bacteria agent can become advantage lactic acid in napier grass ensiling system
Bacterium is acidified ensilage, inhibits the growth of the bacterium, mould in ensilage.
Application of the 4 lactic acid bacteria agent of embodiment in rye grass ensiling.
The implementation steps such as raw material preparation, actication of culture, the preparation of bacterium powder, rye grass ensiling modulation are referring to embodiment 2, in rye
Four test groups are set in careless ensiling modulation, are control group, commodity microbial inoculum, lactobacillus plantarum ZRR, lactic acid bacteria agent, quotient respectively
Savoring microbial inoculum to be is lactic acid bacteria for straw ensilings, product indicias such as herbage, corns commercially.The addition of three kinds of microbial inoculums
Amount is the test result in embodiment 3 --- 0.02 ‰ (i.e. 2.4 × 105cfu/g).Respectively ensiling the 0th, 1,3,5,7,14,
21, part ensiling rye grass is taken out in 28,35,42d, carries out pH detection and microorganism composition analysis.The detection method of microorganism is joined
According to embodiment 3.In addition after 42d fermenting-ripening, ensiling sample is taken to carry out attributional analysis.90ml aqua sterilisa is added in ensiling sample,
It fullys shake, takes appropriate amount of fluid to carry out the measurement of organic acid and ammoniacal nitrogen, and appropriate ensilage is taken to be placed in 65 DEG C of baking oven apoplexy
It does to constant weight, measures its amount of dry matter.After measuring the aerobic exposure of ensiling rye grass simultaneously, the microorganism dynamic of different microbial inoculums becomes
Change.The pH and microorganism dynamic change of different microbial inoculum ensiling rye grasses are as shown in Fig. 2-1,2-2,2-3,2-4.Different microbial inoculum ensilings
Microorganism dynamic change after the aerobic exposure of rye grass is as shown in Fig. 3-1,3-2.
Microorganism dynamic when Fig. 2-1,2-2,2-3,2-4 are different microbial inoculum ensiling rye grass ensilings and after aerobic exposure becomes
Change, respectively control group, commodity microbial inoculum, lactobacillus plantarum ZRR and lactic acid bacteria agent group.Commodity microbial inoculum, lactobacillus plantarum ZRR and
Lactic acid bacteria agent group can quickly produce acid, reduce rapidly pH, inhibit bacteria and mold growth, but commodity microbial inoculum is in ensiling later period cream
Sour bacterium is in reduction trend, and lactic acid bacteria can stablize survival in the processing group of lactic acid bacteria agent.Table 4 is the different microbial inoculums of addition to rye grass
The influence of Silage Quality.Compared with the control group, commodity microbial inoculum, lactobacillus plantarum ZRR and lactic acid bacteria agent can significantly improve blueness
The lactic acid content of feed is store, significantly reduces the content of the organic acids such as ammoniacal nitrogen and ethyl alcohol, propionic acid, butyric acid, wherein lactobacillus plantarum
ZRR and the lactic acid content of lactic acid bacteria agent group are above commodity microbial inoculum group.The acetic acid content highest of lactic acid bacteria agent group simultaneously is shown
It writes and is higher than commodity microbial inoculum, lactobacillus plantarum ZRR group.Fig. 3-1,3-2 are micro- after the different aerobic exposures of microbial inoculum ensiling rye grass
Biodynamic variation.After aerobic exposure, since lactic acid bacteria agent group produces the presence of acetic acid and a large amount of lactic acid bacterias, can effectively it inhibit
The growth of mould, yeast improves aerobic stability.
Table 4 adds influence (%/dry matter) of the different microbial inoculums to rye grass Silage Quality
Note: with column data subscript, different letters indicate significant difference (P < 0.05)
Therefore, by Fig. 2-1,2-2,2-3,2-4, Fig. 3-1,3-2 and table 4 the result shows that, with control group, commodity bacterium
Agent, lactobacillus plantarum ZRR are compared, and by adding lactic acid bacteria agent of the present invention in ensiling raw material, can effectively improve institute
The total content of lactic acid in ensilage is obtained, the content of ammoniacal nitrogen, propionic acid, butyric acid is reduced, to be conducive to improve ensilage
Palatability improves nutritive value；The content of ethyl alcohol is significantly reduced, acetic acid content is improved, inhibits mould, yeast etc. in ensilage
The growth of fungi improves its aerobic stability, to be conducive to extend the pot-life of ensilage, achieves unexpected
Technical effect.
<110>OrganizationName: Jiangsu Province Agriculture Science Institute
Application Project
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<120>Title: a kind of suitable for the lactic acid bacteria agent of grass silage and its application
<130> AppFileReference :
<140> CurrentAppNumber :
<141> CurrentFilingDate : ____-__-__
Sequence
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<213> OrganismName : Lactobacillus plantarum
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cagtgctgca gctaacgcat taagcattcc gcctggggag tacggccgca aggctgaaac 900
tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagctac 960
gcgaagaacc ttaccaggtc ttgacatact atgcaaatct aagagattag acgttccctt 1020
cggggacatg gatacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1080
taagtcccgc aacgagcgca acccttatta tcagttgcca gcattaagtt gggcactctg 1140
gtgagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1200
tatgacctgg gctacacacg tgctacaatg gatggtacaa cgagttgcga actcgcgaga 1260
gtaagctaat ctcttaaagc cattctcagt tcggattgta ggctgcaact cgcctacatg 1320
aagtcggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt 1380
gtacacaccg cccgtcacac catgagagtt tgtaacaccc aaagtcggtg gggtaacctt 1440
ctaggaactc agccgctaga gtgtaacaga agtccggg 1478
<212> Type : DNA
<211> Length : 1478
SequenceName : 1
SequenceDescription :
Sequence
--------
<213> OrganismName : Lactobacillus buchneri
<400> PreSequenceString :
cttgcacttg aaagatttaa cattgagacg agtggcgaac tggtgagtaa cacgtgggta 60
acctgccctt gaagtagggg ataacacttg gaaacaggtg ctaataccgt ataacaacca 120
aaaccacctg gttttggttt aaaagacggc ttcggctgtc actttaggat ggacccgcgg 180
cgtattagct tgttggtaag gtaacggcct accaaggcga tgatacgtag ccgacctgag 240
agggtaatcg gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta 300
gggaatcttc cacaatggac gaaagtctga tggagcaacg ccgcgtgagt gatgaagggt 360
ttcggctcgt aaaactctgt tgttggagaa gaacaggtgt cagagtaact gttgacatct 420
tgacggtatc caaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 480
ggtggcaagc gttgtccgga tttattgggc gtaaagcgag cgcaggcggt tttttaggtc 540
tgatgtgaaa gccttcggct taaccggaga agtgcatcgg aaaccgggag acttgagtgc 600
agaagaggac agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac 660
cagtggcgaa ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag catgggtagc 720
gaacaggatt agataccctg gtagtccatg ccgtaaacga tgagtgctaa gtgttggagg 780
gtttccgccc ttcagtgctg cagctaacgc attaagcact ccgcctgggg agtacgaccg 840
caaggttgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 900
attcgatgct acgcgaagaa ccttaccagg tcttgacatc ttctgccaac ctaagagatt 960
aggcgttccc ttcggggaca gaatgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1020
gagatgttgg gttaagtccc gcaacgagcg caacccttat tgttagttgc cagcattcag 1080
ttgggcactc tagcaagact gccggtgaca aaccggagga aggtggggat gacgtcaaat 1140
catcatgccc cttatgacct gggctacaca cgtgctacaa tggacggtac aacgagtcgc 1200
gaaaccgcga ggtcaagcta atctcttaaa gccgttctca gttcggattg taggctgcaa 1260
ctcgcctaca tgaagttgga atcgctagta atcgtggatc agcatgccac ggtgaatacg 1320
ttcccgggcc ttgtacacac cgcccgtcac a 1351
<212> Type : DNA
<211> Length : 1351
SequenceName : 2
SequenceDescription :
Claims (4)
1. a kind of lactic acid bacteria agent suitable for grass silage, which is characterized in that the lactic acid bacteria agent is by lactobacillus plantarum
Freeze-dried powder and lactobacillus buchneri freeze-dried powder composition, the lactobacillus plantarum and lactobacillus buchneri are separated from rye grass stalk
It obtains, lactobacillus plantarum is homofermentative lactic bacteria, and lactobacillus buchneri is heterofermentation lactobacillus, and two plants of bacterial strains were in 2016 5
The moon is preserved in China typical culture collection center on the 25th, and the bacterial strain of the lactobacillus plantarum isL.plantarumZRR, preservation
It number is CCTCC No:M2016281, the bacterial strain of the lactobacillus buchneri isL.buchneriBR2, deposit number are CCTCC No:
M2016280；
In the lactic acid bacteria agent, the weight ratio of lactobacillus plantarum freeze-dried powder and lactobacillus buchneri freeze-dried powder is 2:1, plant cream
Viable count in bacillus freeze-dried powder is 8 × 109Cfu/g, the viable count in lactobacillus buchneri freeze-dried powder are 4 × 109Cfu/g, lactic acid
The viable count of bacteria agent is 1.2 × 1010cfu/g。
2. the lactic acid bacteria agent according to claim 1 suitable for grass silage, it is characterised in that: the lactobacillus plantarum
16S rDNA gene order as shown in SEQ ID No.1, the 16S rDNA gene order such as SEQ ID of the lactobacillus buchneri
Shown in No .2.
3. the application as described in claim 1 suitable for the lactic acid bacteria agent of grass silage preparation grass silage feed.
4. application according to claim 3, which is characterized in that the grass silage feed producing method is as follows:
1) raw material preparation: after herbage cradles, guarantee cleaning, go bad without going rotten, and be cut to 2-3cm；
2) bacterial strain activates: by freezen protectiveL.plantarumZRR、L.buchneriBR2 is inoculated in MRS Liquid Culture respectively
In base, 18-24h is cultivated at 37 DEG C of temperature, obtains activated spawn such secondary culture 2-3 times；
3) preparation of bacterium powder: by the strain of above-mentioned activation, inoculation fermentation, freeze-drying, crushing, auxiliary material are mixed and made into freeze-drying respectively
Bacterium powder,L.plantarumZRR、L.buchneriThe freeze-dried powder of BR2 is mixed according to the weight ratio of 2:1, freezes lactic acid bacteria agent
The viable count of dry powder reaches 1.2 × 1010cfu/g；
4) modulation of grass silage: moisture content control when herbage raw material ensiling in 65%-70%, by the bacterium powder of above-mentioned preparation with
2.4×105The inoculum concentration of cfu/g is inoculated into raw material, and ensiling herbage is made through everfermentation.
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| CN107354115A (en) * | 2017-09-04 | 2017-11-17 | 本农国际有限公司 | Lactobacillus plantarum for ensilage fermentation |
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