CN106148249A - A kind of lactic acid bacteria microbial inoculum being applicable to grass silage and application thereof - Google Patents
A kind of lactic acid bacteria microbial inoculum being applicable to grass silage and application thereof Download PDFInfo
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Abstract
The invention provides a kind of lactic acid bacteria microbial inoculum being applicable to grass silage, described lactic acid bacteria microbial inoculum is made up of Lactobacillus plantarum lyophilized powder and Lactobacillus buchneri lyophilized powder, described Lactobacillus plantarum and Lactobacillus buchneri all isolateds from rye grass straw, Lactobacillus plantarum is homofermentative lactic bacteria, Lactobacillus buchneri is heterofermentation lactobacillus, two strain bacterial strains are preserved in China typical culture collection center on May 25th, 2016, the bacterial strain of described Lactobacillus plantarum is L.plantarumZRR, preserving number is CCTCC No:M2016281, the bacterial strain of described Lactobacillus buchneri is L.buchneriBR2, preserving number is CCTCC No:M2016280.The lactic acid bacteria of the present invention has the advantages such as acceleration grass silage ripe, raising grass silage quality, raising aerobic stability, can be widely applied to grass silage feedstuff preparation field.
Description
Technical field
The invention discloses a kind of lactic acid bacteria microbial inoculum being applicable to grass silage and application thereof, belong to the green grass or young crops in feed stripped
Storage feed processing technology field.
Background technology
Ensiling is the propagation by lactic acid bacteria, and the fermentation substrate in raw material changes into the acidic materials such as lactic acid, maintains acid
Property anaerobic environment, is beneficial to a kind of storage method that silo crop preserves for a long time.Ensilage color is yellowish green, the sour perfume (or spice) of abnormal smells from the patient, soft
Soft succulence, good palatability, extensively apply in Animal husbandry production, especially in ruminant is raised, it has also become important high-quality
Roughage is originated.Compared with becoming Radix Glycyrrhizae with herbage dry in the sun, grass silage can be greatly shortened from gathering in the time preserved, it is to avoid
The forage grass loss that adverse weather causes, makes the nutritional labeling of green forage be preserved to greatest extent.
Lactic acid bacteria is the microoganism additives for ensiling the most promoted, its Main Function be regulation ensiling material in micro-
Biotic component, rapidly becomes dominant microflora, and Competitive assays harmful microorganism grows, and the sugar that rapid conversion is limited drops significantly
Low ensiling pH value, thus it is effectively improved the quality of ensilage, there is bigger potentiality and vast potential for future development.At present, to green grass or young crops
The development of storage lactic acid bacteria is concentrated mainly on the single cultures such as Lactobacillus plantarum.Patent documentation " a kind of Lactobacillus plantarum and warm
Application (CN2016100121 45.2) in season type grass silage " provide isolated from native grass ensilage
One lactobacillus plantarum.But the later stage that ensiling is behind early stage, mid-term and Kaifeng, the microorganism composition of Silage, physics and chemistry
Character and local environment all can occur a series of change.Single lactic acid bacteria product cannot meet the ensiling requirement of change.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) produces more lactic acid, improves silage fermentation quality, but suppresses aerobic rotten ability universal
Poor.Heterofermentative lactic bacteria utilizes WSC to produce more acetic acid, it is suppressed that aerobic bacteria, yeast and mycete and ensiling are raised
Expect aerobic rotten.Patent documentation " a kind of microbe additive (CN 201410677476.9) for preparing ensilage " carries
Having supplied a kind of novel plant lactobacillus ZJY-6 and the microbe additive comprising this bacterium, microbe additive also comprises Bu Shi breast
Any in bacillus, lactobacillus casei, lactobacillus thermophilus, enterococcus, Pediococcus pentosaceus, Bacillus pumilus, pediococcus acidilactici
A kind of.Patent documentation " prepares the method (CN20141063295 of Caulis Sacchari sinensis tail ensilage with Lactobacillus buchneri and Lactobacillus plantarum
3.X) " disclose the method combining ensiling Caulis Sacchari sinensis tail with the microbial inoculum of Lactobacillus buchneri and Lactobacillus plantarum.But these two parts specially
In profit document, Lactobacillus plantarum, Lactobacillus buchneri one are isolatable from silagecorn, and one is purchased from strain storehouse.Microorganism fungus kind
In resource, each strain has the subspecies (bacterial strain) that up to a hundred upper performances the most thousands of are different.According to microorganism, habitat is fitted
Answering property and specificity principle, separating lactic acid bacterium from herbage material, lactic acid bacteria is reapplied in herbage, lactic acid bacteria can obtain more rapidly
Field planting in grass silage, forms dominant microflora.Therefore separating lactic acid bacterium from herbage, selects the lactic acid bacteria bacterium having complementary functions
Strain, the lactic acid bacteria microbial inoculum that preparation herbage is exclusive, is the important guarantee improving grass silage feed quality.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, its object is to provide a kind of lactic acid bacteria microbial inoculum being applicable to grass silage
And application.
First goal of the invention of the present invention is to provide a kind of lactic acid bacteria microbial inoculum being applicable to grass silage, described lactic acid
Bacteria agent is made up of Lactobacillus plantarum lyophilized powder and Lactobacillus buchneri lyophilized powder, and described Lactobacillus plantarum and Lactobacillus buchneri are equal
Isolated from rye grass straw, Lactobacillus plantarum is homofermentative lactic bacteria, and Lactobacillus buchneri is heterofermentation lactobacillus,
Two strain bacterial strains are preserved in China typical culture collection center on May 25th, 2016, and the bacterial strain of described Lactobacillus plantarum is
L.plantarumZRR, preserving number is CCTCC No:M2016281, and the bacterial strain of described Lactobacillus buchneri is L.buchneriBR2,
Preserving number is CCTCC No:M2016280；
In described lactic acid bacteria microbial inoculum, the weight ratio of Lactobacillus plantarum lyophilized powder and Lactobacillus buchneri lyophilized powder is 2:1, plants
Viable count in thing lactobacillus lyophilized powder reaches 8 × 109Cfu/g, the viable count in Lactobacillus buchneri lyophilized powder reaches 4 ×
109Cfu/g, the viable count of lactic acid bacteria microbial inoculum reaches 1.2 × 1010cfu/g；
Described lactic acid bacteria microbial inoculum is 2.4 × 10 at the addition of grass silage feedstuff5cfu/g。
The described lactic acid bacteria microbial inoculum be applicable to grass silage, the 16S rDNA gene order of described Lactobacillus plantarum is such as
Shown in SEQ ID No.1, the 16S rDNA gene order of described Lactobacillus buchneri is as shown in SEQ ID No.2.
The Lactobacillus plantarum (L.plantarum ZRR) of the present invention be screen from herbage straw have product acid speed
Degree is fast, energy for growth acid proof lactic acid bacteria strong, good；This bacterial strain has been preserved in China typical culture collection center, preservation
Address: No. 299 Wuhan Universitys of Wuchang District, Wuhan City, Hubei Province Bayi Road in the school, Classification And Nomenclature: Lactobacillus plantarum
Lactobacillus plantarum, preserving number: CCTCC No:M2016281, preservation date is: on May 25th, 2016.
The bacterium colony of described Lactobacillus plantarum (Lactobacillus plantarum) ZRR is creamy white, neat in edge；Carefully
Born of the same parents' microscopic morphology is rod-short, without spore, Gram-positive；Catalase is negative, oxidase negative；There is stronger growth
And product acid activity, the culture medium that initial ph value is 3.0-8.0 can grow, under the conditions of 10 DEG C, present faint growth, 15
Under the conditions of DEG C 40 DEG C, upgrowth situation is good, and under conditions of no more than 6.5%NaCl, upgrowth situation is good.
The Lactobacillus buchneri (L.buchneriBR2) of the present invention be screen from herbage straw have product acetic acid,
Good acid proof lactic acid bacteria；This 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 And Nomenclature: Lactobacillus buchneri Lactobacillus buchneri, preservation
Number: CCTCC No:M2016280, preservation date is: on May 25th, 2016.
The bacterium colony of described Lactobacillus buchneri (Lactobacillus buchneri) BR2 is canescence, and thickness, edge are whole
Together；Cell microscopic morphology is shaft-like in being, without spore, Gram-positive；Catalase is negative, oxidase negative；Have stronger
Growth and product acid activity, the culture medium that initial ph value is 3.0 7.5 can grow, under the conditions of 10 DEG C, present faint life
Long, under the conditions of 15 DEG C 40 DEG C, upgrowth situation is good, and under conditions of no more than 6.5%NaCl, upgrowth situation is good.
Second goal of the invention of the present invention is to provide the application in preparation grass silage feedstuff of the above-mentioned lactic acid bacteria microbial inoculum.
Described grass silage feed producing method is as follows:
Raw material prepares: after herbage cradles, it is ensured that cleaning, nothing are gone rotten rotten, and are cut to 2-3cm.
Bacterial strain activates: L.plantarumZRR, L.buchneriBR2 of freezen protective are inoculated in MRS liquid training respectively
Support in base, at temperature 37 DEG C, cultivate 18-24h, such Secondary Culture obtain for 2-3 time described activation L.plantarumZRR,
L.buchneriBR2 strain；Described MRS fluid medium composition is as follows: 10g peptone, 5g Carnis Bovis seu Bubali cream, 4g yeast leaching powder,
20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate
Add 1000mL distilled water, regulate pH to 6.5,121 DEG C of sterilizing 15min.
The preparation of mycopowder: the strain inoculation fermentation respectively of above-mentioned activation, lyophilization, pulverizing, adjuvant are mixed and freeze
Dry bacterium powder, the lyophilized powder of L.plantarumZRR, L.buchneriBR2 mixes according to the part by weight of 2:1, makes lactic acid bacteria microbial inoculum
The viable count of lyophilized powder reaches 1.2 × 1010cfu/g。
The modulation of grass silage: moisture during herbage raw material ensiling controls at 65%-70%, weighs 200g herbage former
Material loads in vacuum packaging plastic bag, by the mycopowder of above-mentioned preparation with 2.4 × 105The inoculum concentration of cfu/g is inoculated in raw material, adopts
After being vacuum-packed with vacuum packing machine, it is placed in storeroom and ferments.
The condition of described fermentation is: fermentation temperature is 15-25 DEG C, and fermentation time is 30-60d.The quality inspection of ensiling herbage
Survey: after fermenting-ripening, take out part ensiling herbage and carry out microorganism composition and fermentation quality analysis.
The lactic acid bacteria microbial inoculum that the present invention provides has a following good effect:
(1) according to microorganism to the adaptability in habitat and specificity principle, from herbage, raising grass silage product are filtered out
Exclusive bacterium L.plantarumZRR, L.buchneriBR2 of matter.(2) present invention is through creative work, finds lactic acid bacteria microbial inoculum
The best proportioning of effect is Lactobacillus plantarum and Lactobacillus buchneri, and its weight ratio is 2:1, and lactic acid bacteria microbial inoculum is at grass silage feedstuff
Addition be 2.4 × 105cfu/g.(3) compared with existing single lactic acid bacteria, inoculation L.plantarumZRR,
The lactic acid bacteria microbial inoculum of L.buchneriBR2, can quickly become dominant microflora at earlier fermentation, reduces pH value；The content of lactic acid
Significantly improving, ammonia nitrogen content declines, and is conducive to improving the palatability of ensilage, improves the fermentation quality of grass silage；
Significantly improve acetic acid content, reduce the content of ethanol, suppress the growth of the fungus such as mycete, yeast in ensilage after opening bag, carry
High aerobic stability, thus be conducive to extending the pot-life of ensilage.
Lactic acid bacteria microbial inoculum the most of the present invention has acceleration grass silage maturation, improves grass silage quality, raising
The advantages such as aerobic stability, can be widely applied to grass silage feedstuff preparation field.
Accompanying drawing explanation
Fig. 1-1 dynamically changes for Different adding amount ensiling grassiness pH；
Fig. 1-2 is that Different adding amount ensiling grassiness lactic acid bacteria dynamically changes；
Fig. 1-3 is the change of Different adding amount ensiling grassiness dynamic of bacteria；
Fig. 1-4 dynamically changes for Different adding amount ensiling grassiness mycete；
Fig. 2-1 dynamically changes for different microbial inoculum ensiling rye grass pH；
Fig. 2-2 dynamically changes for different microbial inoculum ensiling rye grass lactic acid bacterias；
Fig. 2-3 is that different microbial inoculum ensiling rye grass dynamic of bacteria becomes；
Fig. 2-4 dynamically changes for different microbial inoculum ensiling rye grass mycetes；
Mycete when Fig. 3-1 is different microbial inoculum ensiling rye grass ensiling and after aerobic exposure dynamically changes；
Yeast when Fig. 3-2 is different microbial inoculum ensiling rye grass ensiling and after aerobic exposure dynamically changes.
Detailed description of the invention
The present invention will be further described with embodiment below in conjunction with the accompanying drawings, and below example facilitates a better understanding of this
Invention, but do not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method；Used
Material, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1 Lactobacillus plantarum (Lactobacillus plantarum) ZRR, Lactobacillus buchneri
The separation of (Lactobacillus buchneri) BR2 and qualification
1, isolated and purified
From rye grass straw isolated and purified to Lactobacillus plantarum ZRR with Lactobacillus buchneri BR2, be for homofermentation respectively
Lactic acid bacteria and heterofermentation lactobacillus, specific as follows: to weigh in the 90ml distilled water that 10g rye grass straw sample adds sterilizing,
Utilize constant-temperature table to shake 1h, then 10 times of gradient dilutions, take 10 respectively-3、10-4With 10-5Times sample diluting liquid is coated on MRS
On solid medium, cultivate 48h, be then taken out, according to the size of bacterium colony, form and color, the list of growth on picking MRS
Bacterium colony, carries out catalase experiment and Gram’s staining.Negative catalase and Gram-positive person are fixed tentatively and is
Lactic acid bacteria, continues line purification 2 times 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 detection
Lactic acid bacteria ZRR can grow in the culture medium that initial ph value is 3.0-8.0, presents faint life under the conditions of 10 DEG C
Long, under the conditions of 15 DEG C-40 DEG C, upgrowth situation is good, and under conditions of no more than 6.5%NaCl, upgrowth situation is good.Lactic acid bacteria
BR2 can grow in the culture medium that initial ph value is 3.0-7.5, presents faint growth under the conditions of 10 DEG C, at 15 DEG C of-40 DEG C of bars
Under part, upgrowth situation is good, it is possible to the salinity of tolerance 3.0%.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
Lactobacilli strain ZRR, BR2 of 48h will be cultivated on solid plate, use colony PCR amplification, carry out 16rRNA gene
Sequence Identification.Primer selects the 16S rRNA gene amplification universal primer of antibacterial: 27F and 1492R.PCR reaction system is 50 μ l:
Premix Taq25 μ l, 27F and 1492R (being 20 μMs) 1 μ l, bacterium colony, to 50 μ l, is directly added anti-by supplementary sterile purified water
Answer system, carry out PCR amplification, obtain PCR primer.PCR primer is sent to order-checking, and result is in sequence table shown in sequence 1,2.
The physio-biochemical characteristics of table 1 bacterial strain ZRR, BR2
The carbon source through fermentation result of the test of table 2 bacterial strain ZRR, BR2
Note :+represent reaction for the positive；Represent reaction for feminine gender；W represents reaction for the weak positive
Through above-mentioned qualification, lactobacilli strain ZRR, BR2, on May 25th, 2016, have been preserved in Chinese Typical Representative culture
Preservation center, preservation address: No. 299 Wuhan Universitys of Wuchang District, Wuhan City, Hubei Province Bayi Road in the school, Classification And Nomenclature: plant breast bar
Bacterium Lactobacillus plantarum, preserving number: CCTCC No:M2016281, Classification And Nomenclature: Lactobacillus buchneri
Lactobacillus buchneri, preserving number: CCTCC No:M2016280.
The application in rye grass ensiling of embodiment 2 different strains
Raw material prepares: after rye grass cradles, it is ensured that cleaning, nothing are gone rotten rotten, and are cut to 2-3cm.
Bacterial strain activates: L.plantarumZRR, L.buchneriBR2 of freezen protective are inoculated in MRS liquid training respectively
Support in base, at temperature 37 DEG C, cultivate 18-24h, such Secondary Culture obtain for 2-3 time described activation L.plantarumZRR,
L.buchneriBR2 strain；Described MRS fluid medium composition is as follows: 10g peptone, 5g Carnis Bovis seu Bubali cream, 4g yeast leaching powder,
20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate
Add 1000mL distilled water, regulate pH to 6.5,121 DEG C of sterilizing 15min.
The preparation of mycopowder: the strain inoculation fermentation respectively of above-mentioned activation, lyophilization, pulverizing, adjuvant are mixed and freeze
Dry bacterium powder, Lactobacillus plantarum L.plantarumZRR (hereinafter referred to as ZRR), Lactobacillus buchneri L.buchneri BR2 (letter below
Claim BR2) lyophilized powder according to different part by weight mixing, make the viable count of lactic acid bacteria freeze drying powder reach 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 during rye grass raw material ensiling controls at 65%-70%, weighs 200g and herds
Grassland material loads in vacuum packaging plastic bag, by the mycopowder of above-mentioned preparation by 0.04 ‰ (i.e. 4.8 × 105Cfu/g) inoculum concentration
It is inoculated in raw material, after using vacuum packing machine vacuum packaging, is placed in storeroom and ferments.
After 42d fermenting-ripening, take ensiling sample and carry out attributional analysis.Ensiling sample adds 90ml aquesterilisa, fully shakes
Swing, take appropriate amount of fluid and carry out the mensuration of pH and organic acid, and take appropriate ensilage and be placed in 65 DEG C of baking ovens and air-dry to constant weight, survey
Its dry fixed.Add different strain and the impact of rye grass Silage Quality is shown in Table 3.
As shown in Table 3, test group 1 individually adds ZRR, and lactic acid content is the highest, and pH and ethanol content are minimum, shows screening
Exclusive bacterium ZRR energy field planting in herbage rapidly, Nulomoline is lactic acid, and suppressing other Bacterial Transformation sugar is ethanol, and rapid acidification carries
High Silage Quality, but acetic acid content is minimum, is unfavorable for the aerobic stability in fermentation later stage.Test group 2 individually adds BR2, acetic acid
Content is the highest, but lactic acid producing amount is minimum in test group, and pH and ethanol content are the highest.Individually add ZRR or BR2, it is impossible to meet
Technology requirement of both Silage Quality and aerobic stability.Therefore, compounding two strain bacterial strains, screen different adding proportion groups
Close.Test group 3,4, the result of 5 shows: the lactic acid content of test group 5 is up to 10.31%, ethanol content minimum 0.81%,
And 100%, 67%, 33% BR2 tri-groups between acetic acid content there is no difference.Therefore, test group 5 i.e. 67%ZRR, 33%BR2
(2:1) adding proportion combination, can be quickly lactic acid and acetic acid by the soluble sugar conversion in rye grass, reduction pH, significantly
Reduce ethanol content, improve aerobic stability.
Table 3: add the different strain impact (%/dry) on rye grass Silage Quality
Note: with column data subscript difference letter representation significant difference (P < 0.05)
The application in grassiness ensiling of the lactic acid bacteria microbial inoculum of embodiment 3 Different adding amount
Implement step prepared by raw material preparation, actication of culture, mycopowder, grassiness ensiling modulation etc. with reference to embodiment 2, at mycopowder
In preparation, the lyophilized powder of L.plantarumZRR, L.buchneri BR2 is mixed by the result according to embodiment 2 according to the ratio of 2:1
Close, make the viable count of lactic acid bacteria microbial inoculum lyophilized powder reach 1.2 × 1010cfu/g.By the mycopowder 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 in grassiness, use vacuum packing machine vacuum packaging after, be placed in storeroom and ferment.
The dynamic change-detection of microorganism of ensiling grassiness: respectively ensiling the 0th, 1,3,5,7,14,21,28,35,42d, take
Go out part ensiling grassiness, carry out pH detection and microorganism composition analysis.Microorganism composition analysis includes lactic acid bacteria, mycete, antibacterial
Counting.Weigh 10g ensilage, add 90ml aquesterilisa, fully shake, then with 10 times of dilution methods, sample is entered with aquesterilisa
Row gradient dilution, chooses suitable diluent respectively and coats MRS culture medium, potato dextrose agar, nutrition fine jade
On fat, it is placed in constant incubator cultivating, respectively lactic acid bacteria, mycete, aerobic bacteria is counted.The breast of Different adding amount
The microorganism dynamic result of acid bacteria agent ensiling grassiness 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, add ensiling grassiness and the comparison of lactic acid bacteria
Group is compared, and pH is remarkably decreased, and lactic acid bacterium number dramatically increases, and the growth bacterium of antibacterial and mycete is the most suppressed.With high addition
0.04 ‰, 0.1 ‰, 0.2 ‰ (i.e. 1.2 × 105、2.4×105、4.8×105、1.2×106、2.4×106Cfu/g) compare,
(i.e. the 2.4 × 10 of 0.02 ‰5Cfu/g) few additive, lactic acid bacteria microbial inoculum can become advantage lactic acid in grassiness ensiling system
Bacterium, is acidified ensilage, the antibacterial in suppression ensilage, the growth of mycete.
The application in rye grass ensiling of the embodiment 4 lactic acid bacteria microbial inoculum.
Implement step prepared by raw material preparation, actication of culture, mycopowder, rye grass ensiling modulation etc. with reference to embodiment 2, in rye (Secale cereale L.)
Grass ensiling modulation arranges four test group, is matched group respectively, commodity microbial inoculum, Lactobacillus plantarum ZRR, lactic acid bacteria microbial inoculum, business
Savoring microbial inoculum is commercially, and for the straw ensiling such as herbage, Semen Maydis, product indicia is lactic acid bacteria.The interpolation of three kinds of microbial inoculums
Amount is the result of the test 0.02 ‰ (i.e. 2.4 × 10 in embodiment 35cfu/g).Respectively ensiling the 0th, 1,3,5,7,14,
21,28,35,42d, take out part ensiling rye grass, carry out pH detection and microorganism composition analysis.The detection method ginseng of microorganism
According to embodiment 3.Additionally after 42d fermenting-ripening, take ensiling sample and carry out attributional analysis.Ensiling sample adds 90ml aquesterilisa,
Fully concussion, takes appropriate amount of fluid and carries out the mensuration of organic acid and ammoniacal nitrogen, and takes appropriate ensilage and be placed in 65 DEG C of baking oven apoplexy
Do to constant weight, measure its amount of dry matter.After measuring the exposure of ensiling rye grass aerobic, the microorganism of different microbial inoculums dynamically becomes simultaneously
Change.PH and the microorganism of different microbial inoculum ensiling rye grasses dynamically change as shown in Fig. 2-1,2-2,2-3,2-4.Different microbial inoculum ensilings
Rye grass aerobic expose after microorganism dynamically change as shown in Fig. 3-1,3-2.
Microorganism when Fig. 2-1,2-2,2-3,2-4 are different microbial inoculum ensiling rye grass ensiling and after aerobic exposure dynamically becomes
Change, respectively matched group, commodity microbial inoculum, Lactobacillus plantarum ZRR and lactic acid bacteria microbial inoculum group.Commodity microbial inoculum, Lactobacillus plantarum ZRR and
Lactic acid bacteria microbial inoculum group all can quickly produce acid, reduces rapidly pH, suppression antibacterial and fungus growth, but commodity microbial inoculum is at ensiling later stage breast
Acid bacterium is in reducing trend, and in the process group of lactic acid bacteria microbial inoculum, lactic acid bacteria can stably be survived.Table 4 is to add different microbial inoculum to rye grass
The impact of Silage Quality.Compared with matched group, commodity microbial inoculum, Lactobacillus plantarum ZRR and lactic acid bacteria microbial inoculum all can significantly improve green grass or young crops
The lactic acid content of storage feedstuff, significantly reduces the content of the organic acid such as ammoniacal nitrogen and ethanol, propanoic acid, butanoic acid, wherein Lactobacillus plantarum
The lactic acid content of ZRR and lactic acid bacteria microbial inoculum group is above commodity microbial inoculum group.The acetic acid content of lactic acid bacteria microbial inoculum group is the highest simultaneously, aobvious
Write higher than commodity microbial inoculum, Lactobacillus plantarum ZRR group.Fig. 3-1,3-2 be after different microbial inoculum ensiling rye grass aerobic exposes micro-
Biodynamic changes.After aerobic exposes, owing to lactic acid bacteria microbial inoculum group produces acetic acid and the existence of a large amount of lactic acid bacteria, can effectively suppress
Mycete, the growth of yeast, improve aerobic stability.
Table 4 adds the different microbial inoculum impact (%/dry) on rye grass Silage Quality
Note: with column data subscript difference letter representation significant difference (P < 0.05)
Therefore, shown with the result of table 4 by Fig. 2-1,2-2,2-3,2-4, Fig. 3-1,3-2, with matched group, commodity bacterium
Agent, Lactobacillus plantarum ZRR compares, and by adding lactic acid bacteria microbial inoculum of the present invention in ensiling raw material, can be effectively improved institute
Obtain the total content of lactic acid in ensilage, reduce ammoniacal nitrogen, propanoic acid, the content of butanoic acid, thus be conducive to improving ensilage
Palatability, improves nutritive value；Significantly reduce the content of ethanol, improve acetic acid content, mycete, yeast etc. in suppression ensilage
The growth of fungus, improves its aerobic stability, thus is conducive to extending the pot-life of ensilage, achieve unexpected
Technique effect.
<110>OrganizationName: Jiangsu Province Agriculture Science Institute
Application Project
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<120>Title: a kind of lactic acid bacteria microbial inoculum being applicable to grass silage and application thereof
<130> AppFileReference :
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atggtggggt aacggctcac catggcaatg atacgtagcc gacctgagag ggtaatcggc 300
cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg gaatcttcca 360
caatggacga aagtctgatg gagcaacgcc gcgtgagtga agaagggttt cggctcgtaa 420
aactctgttg ttaaagaaga acatatctga gagtaactgt tcaggtattg acggtattta 480
accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540
tgtccggatt tattgggcgt aaagcgagcg caggcggttt tttaagtctg atgtgaaagc 600
cttcggctca accgaagaag tgcatcggaa actgggaaac ttgagtgcag aagaggacag 660
tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca gtggcgaagg 720
cggctgtctg gtctgtaact gacgctgagg ctcgaaagta tgggtagcaa acaggattag 780
ataccctggt agtccatacc gtaaacgatg aatgctaagt gttggagggt ttccgccctt 840
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. the lactic acid bacteria microbial inoculum being applicable to grass silage, it is characterised in that described lactic acid bacteria microbial inoculum is by Lactobacillus plantarum
Lyophilized powder and Lactobacillus buchneri lyophilized powder composition, described Lactobacillus plantarum and Lactobacillus buchneri all separate from rye grass straw
Obtaining, Lactobacillus plantarum is homofermentative lactic bacteria, and Lactobacillus buchneri is heterofermentation lactobacillus, and two strain bacterial strains were in 2016 5
The moon is preserved in China typical culture collection center on the 25th, and the bacterial strain of described Lactobacillus plantarum is L.plantarumZRR, preservation
Number being CCTCC No:M2016281, the bacterial strain of described Lactobacillus buchneri is L.buchneriBR2, and preserving number is CCTCC No:
M2016280；
In described lactic acid bacteria microbial inoculum, the weight ratio of Lactobacillus plantarum lyophilized powder and Lactobacillus buchneri lyophilized powder is 2:1, plant breast
Viable count in bacillus lyophilized powder reaches 8 × 109Cfu/g, the viable count in Lactobacillus buchneri lyophilized powder reaches 4 × 109Cfu/g,
The viable count of lactic acid bacteria microbial inoculum reaches 1.2 × 1010cfu/g；
Described lactic acid bacteria microbial inoculum is 2.4 × 10 at the addition of grass silage feedstuff5cfu/g。
The lactic acid bacteria microbial inoculum being applicable to grass silage the most according to claim 1, it is characterised in that: described Lactobacillus plantarum
16S rDNA gene order as shown in SEQ ID No.1, the 16S rDNA gene order such as SEQ ID of described Lactobacillus buchneri
Shown in No.2.
The most as claimed in claim 1 be applicable to grass silage lactic acid bacteria microbial inoculum preparation grass silage feedstuff application.
Application the most according to claim 3, it is characterised in that described grass silage feed producing method is as follows:
1) raw material prepares: after herbage cradles, it is ensured that cleaning, nothing are gone rotten rotten, and are cut to 2-3cm；
2) bacterial strain activation: L.plantarumZRR, L.buchneriBR2 of freezen protective are inoculated in MRS liquid culture respectively
In base, cultivating 18-24h at temperature 37 DEG C, such Secondary Culture obtains described activated spawn for 2-3 time；
3) preparation of mycopowder: the strain inoculation fermentation respectively of above-mentioned activation, lyophilization, pulverizing, adjuvant are mixed and made into lyophilizing
Mycopowder, the lyophilized powder of L.plantarumZRR, L.buchneriBR2 mixes according to the part by weight of 2:1, makes lactic acid bacteria microbial inoculum freeze
The viable count of dry powder reaches 1.2 × 1010cfu/g；
4) modulation of grass silage: moisture during herbage raw material ensiling controls at 65%-70%, by the mycopowder of above-mentioned preparation
With 2.4 × 105The inoculum concentration of cfu/g is inoculated in raw material, makes ensiling herbage by fermentation.
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| CN106834167A (en) * | 2016-12-29 | 2017-06-13 | 中国农业大学 | A kind of corn straw silage composite bacteria agent |
| CN107259127A (en) * | 2017-07-12 | 2017-10-20 | 华南农业大学 | Wooden ensilage of a kind of a variety of millet and preparation method and application |
| CN107259074A (en) * | 2017-08-11 | 2017-10-20 | 广西农垦西江乳业有限公司奶牛场 | A kind of solid fungicide and its preparation and application for being used to make silage corn |
| CN107354115A (en) * | 2017-09-04 | 2017-11-17 | 本农国际有限公司 | Lactobacillus plantarum for ensilage fermentation |
| CN107603923A (en) * | 2017-11-06 | 2018-01-19 | 四川省草原科学研究院 | 3 strains of lactic acid bacteria of Alpine-arctic Pastoral ensiling |
| 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 |
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