CN113373087B - Microbial inoculum for preparing complete-plant corn fine silage - Google Patents

Abstract

The invention provides a method for preparing whole-plant corn fine greenMicrobial inoculum for feed storage. The microbial inoculum is prepared by mixing lactobacillus buchneri LB and a probiotic protective agent. The content of the Lactobacillus buchneri LB is more than 1 x 10 per gram of the microbial inoculum ¹⁰ And (4) CFU. The experimental results show that: the lactobacillus buchneri LB can be used as a whole-plant corn fine silage additive, so that the lactic acid output is obviously improved, the pH value is obviously reduced, and the whole-plant corn fine silage fermentation quality is effectively improved; and can reduce the variety of resistance genes in the silage; meanwhile, the lactobacillus buchneri LB has the advantages of high fermentation efficiency, low cost and the like, and can be applied to the production of green and environment-friendly biological feed.

Description

A kind of bacterial agent for preparing fine silage of whole plant corn

technical field

The invention belongs to the field of feed, and relates to a bacterial agent for preparing fine silage of whole-plant corn, in particular, it provides a lactic acid bacterium for fine silage of whole-plant corn, more specifically, it provides a kind of Lactobacillus Brucella, which Lactobacillus can reduce resistance genes and viruses in various silages prepared from corn.

Background technique

Silage and semi-dry silage is a processing method to effectively preserve nutrients by chopping green feed with a water content of 45%-75% and sealing it through lactic acid bacteria-based fermentation. Silage has the ability to reduce weed seeds. Germination rate, reduce the effect of virus, and can be preserved for a long time.

Silage additives are substances added during the silage process to promote lactic acid fermentation or inhibit the fermentation of undesirable microorganisms, such as lactic acid bacteria, enzyme preparations, compound bacterial preparations, and chemical additives. Generally, it can reduce nutrient loss and increase the fermentation speed.

Corn is currently the most widely used feed. Corn can be prepared into whole plant corn silage, corn grain silage, and corn silage with different stubble heights.

Silage accounts for a large part of the roughage in the diet. In recent years, because super bacteria have strong resistance to antibiotics and can escape the danger of being killed, their spread has caused a threat to human health. Therefore, more attention has been paid to resistance genes, but less research has been done on resistance genes in silage. Bacteria in the intestines of livestock can ingest resistance genes in feed, and after excretion, may become bacteria that can tolerate multiple drugs, that is, superbugs. Farms have become the largest birthplace of superbugs other than hospitals, so reducing the types and quantities of resistance genes in feed may reduce the number of superbugs in farms. Therefore, screening and application of antibiotics that can reduce the resistance genes in feed Additives are currently emerging research hotspots.

Contents of the invention

One of the objects of the present invention is to provide a new application of Lactobacillus brevis (Lactobacillus brevis) LB.

The Lactobacillus brevis (Lactobacillus brevis) LB has been preserved on June 22, 2017 in the General Microbiology Center of the China Committee for the Collection of Microorganisms (CGMCC for short; address: No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Zip Code: 100101), the preservation number is CGMCC No.14269; referred to as Lactobacillus Brucella LB.

The new application of the Lactobacillus brucelli LB provided by the present invention is: the application of the Lactobacillus brucei LB in the preparation of whole-plant corn fine pasture silage.

Said application can specifically be: the application of Lactobacillus Brucella LB in reducing the resistance gene in whole-plant corn fine pasture silage, and more specifically can be: the application of Lactobacillus Brucella LB in reducing whole-plant corn fine pasture silage Application of resistance gene types.

The whole plant corn includes the whole corn plant, grains and corn harvested with different stubble heights.

The second object of the present invention is to provide a bacterial agent.

The microbial agent provided by the invention is prepared by mixing Lactobacillus Brucella LB with a probiotic protective agent.

Specifically, the microbial agent can be prepared by mixing and freeze-drying Lactobacillus Brucella LB and a probiotic protective agent.

In each gram of the microbial agent, the content of the Lactobacillus buchneri LB is greater than 1×10 ¹⁰ CFU.

Specifically, each gram of the bacterial agent contains 1×10 ¹² CFU of Lactobacillus Brucella LB.

The probiotic protection agent can specifically be skimmed milk.

The skimmed milk can be specifically prepared by dissolving solid milk powder in water at a mass volume ratio (w/v) of 10%.

The solid milk powder can specifically be high-protein skimmed high-calcium milk powder, more specifically high-protein skimmed high-calcium milk powder purchased from Inner Mongolia Yili Industrial Group Co., Ltd. with a product number of 6907992440071.

The bacterial agent is prepared by a method comprising the following steps: mixing Lactobacillus Brucella LB with a probiotic protective agent to obtain it.

Specifically, the bacterial preparation is prepared by a method comprising the following steps: mixing Lactobacillus Brucella LB cells with a probiotic protective agent and then freeze-drying to obtain the bacterial preparation.

The Lactobacillus brucelli LB cell is prepared by a method comprising the following steps: inoculating the Lactobacillus brucei LB into the MRS liquid medium for cultivation, centrifuging the culture system at the end of the cultivation, and collecting the precipitate, which is Lactobacillus brucei LB cells.

In the method, the OD _260nm of the culture system at the end of the culture is 4;

In the method, the initial OD _260nm of the culture system after inoculation =1.8;

In the method, the culture condition is 37°C, 250rpm shaking culture;

In each gram of the microbial agent, the content of the Lactobacillus buchneri LB is greater than 1×10 ¹⁰ CFU.

The application of Lactobacillus Brucella LB or the above bacterial agent containing Lactobacillus Brucella LB in the preparation of whole-plant corn fine silage also belongs to the protection scope of the present invention.

The present invention also protects a method (method A) for preparing silage with corn (whole plant, grain and harvested corn with different stubble heights) as raw material, which includes the following steps: applying the bacterial agent to the corn raw material, and carrying out For silage, the whole plant corn fine silage is obtained.

In the method A, the harvest period of the corn raw material can be from the milk ripening period to the wax ripening period.

In the method A, the moisture content of the corn raw material may specifically be 35%-75%.

In the method A, the corn raw material is pretreated before applying the bacterial agent;

The pretreatment method is as follows: chopping the corn stems and leaves to 2-3 cm, and breaking the corn seeds until there are no complete particles;

In the method A, before applying the bacterial agent to the corn raw material, the following steps are further included: dissolving the bacterial agent in water and activating it at room temperature for 50-70 minutes (60 minutes specifically), and the water is distilled water.

In the method A, the application amount of the bacterial agent is to inoculate 0.5-20×10 ⁵ CFU of Lactobacillus Brucella LB per gram of corn raw material; the specific application amount of the bacterial agent can be inoculated with 8×10 ⁵ per gram of corn raw material CFU of Lactobacillus Brucella LB;

In the method A, the temperature of the silage may be 20°C-40°C, specifically 20°C, 30°C or 40°C.

The time for the silage can be 30-50 days, specifically 45 days.

The invention also protects a preparation method (method B) of fine whole-plant corn silage, comprising the following steps: applying Lactobacillus Brucella LB to corn raw material for silage to obtain fine whole-plant corn silage.

In the method B, the corn raw material can be the whole plant corn, corn kernels, corn ears and part of corn stalks (including leaves) from milk ripening stage to full ripening stage.

In the method B, the moisture content of the corn raw material may specifically be 35%-75%.

In the method B, the corn raw material is pretreated before applying the bacterial agent;

The pretreatment method is as follows: chopping the corn stems and leaves to 2-3 cm, and breaking the corn seeds until there are no complete particles;

In the method B, before applying the bacterial agent to the corn raw material, the following steps are further included: dissolving the bacterial agent in water and activating it at room temperature for 50-70 minutes (60 minutes specifically), and the water is distilled water.

In the method B, the application amount of the bacterial agent is to inoculate 0.5-20×10 ⁵ CFU of Lactobacillus Brucella LB per gram of corn raw material; the specific application amount of the bacterial agent can be inoculated with 8×10 ⁵ per gram of corn raw material CFU of Lactobacillus Brucella LB;

In the method B, the temperature of the silage may be 20°C-40°C, specifically 20°C, 30°C or 40°C.

The time for the silage can be 30-50 days, specifically 45 days.

The whole plant corn fine silage prepared by the above method also belongs to the protection scope of the present invention.

The whole plant corn fine silage contains less types and quantities of resistance genes.

The invention provides a kind of Lactobacillus brucei LB, which can be used as an additive for fine silage of whole-plant corn, which can significantly increase the output of lactic acid, significantly reduce the pH value, effectively improve the fermentation quality of fine silage of whole-plant corn; and can reduce The types of resistance genes in silage; at the same time, Lactobacillus brauchneri LB has the advantages of high fermentation efficiency and low cost, and can be applied to the production of green and environmentally friendly biological feed.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

MRS liquid medium is made up of solute and solvent; Said solute and its concentration in MRS liquid medium are: peptone 10.0g/L, beef powder 5.0g/L, yeast powder 4.0g/L, glucose 20.0g/L , sodium acetate 5.0g/L, triammonium citrate 2.0g/L, Tween 801mL/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate 0.2g/L, manganese sulfate 0.05g/L; the solvent is water.

Whole plant corn fine fresh grass extract medium: 1kg whole plant corn fine fresh grass, chopped and added to 5L of water, 50 ° C water bath for 2 hours, to obtain the extract; first filter the extract with four layers of gauze, and then use quantitative filter paper (Hangzhou Specialty Paper Industry Co., Ltd., Nova 201) to filter and collect the filtrate; the filtrate was autoclaved at 121° C. for 15 minutes, and then dispensed into sterilized test tubes for later use.

Skimmed milk: Solid milk powder is dissolved in distilled water at a mass volume ratio (w/v) of 10%.

Solid milk powder: high-protein skimmed high-calcium milk powder, Inner Mongolia Yili Industrial Group Co., Ltd., article number: 6907992440071.

Embodiment 1, isolation, screening and identification of Lactobacillus brevis (Lactobacillus brevis) LB

1. Isolation and screening of strains

Collect the sheepskin soaking liquid in Hohhot, Inner Mongolia (the liquid after soaking raw hides in clean water during the sheepskin production process), shake it with an oscillator for 30 minutes, dilute it with distilled water 10 ^-1 -10 ^-6 gradient, and take 1mL from each dilution gradient dilution solution for inoculation Add 10-15ml of sterilized MRS solid medium to a petri dish, shake well, wait for it to cool and solidify, and let it stand for anaerobic culture for 48 hours. Pick single colonies with obvious differences in colony shape, size, color and gloss, and repeat streaking until pure colonies are obtained. Single colonies were picked for Gram staining and catalase test. All Gram-positive, catalase-negative strains were initially identified as lactic acid bacteria, and then a lactic acid bacteria strain with high growth and acid production capacity was screened out with the whole plant corn fine fresh grass extract medium, named strain LB. The growth ability and acid production ability are shown in Table 1.

Determination of strain growth ability and acid production ability: inoculate the strain to be tested into 10mL of MRS liquid medium, culture it for 2 generations, and then inoculate it into whole plant corn fine fresh herb extract for cultivation medium, 37°C, 250rpm shaking culture. The cultured samples were taken out at intervals of 12 hours for 3 times, and the last time was taken after 36 hours of cultivation. The absorbance value of all samples was measured at a wavelength of 620 nm, and the pH value of the fermentation broth was measured. Using the whole-plant corn fine extract medium without inoculation as a control, compare the maximum difference between the absorbance value of different strains in the whole-plant corn fine extract medium for 36 hours of shaking culture at a wavelength of 620nm and the control, and the fermentation The maximum difference between the pH value of the liquid and the control is used to screen out the lactic acid bacteria strain with the largest increase in absorbance value and the largest decrease in pH value.

Table 1 Growth ability and acid production ability of bacterial strain LB

\ Growth capacity ^a Acid production capacity^b strainLB 2.44 2.61

Note: a, the maximum difference between the absorbance value of lactic acid bacteria strains cultured in whole plant corn fine extract medium for 36 hours and the control at an intrinsic wavelength of 620nm; b, lactic acid bacteria strains cultured in whole plant corn fine extract medium for 36 hours The maximum difference between the pH value of the internal fermentation broth and the control.

2. Identification of strain LB

Biological characteristics of strain LB: strain LB was cultured on MRS solid medium for 24 hours, the strain grew well, and could form milky white colonies with neat edges; Gram staining of the strain was positive, and the cell morphology under the microscope was short rod-shaped, without spores ; Negative for oxidase, has strong growth and acid production performance in the whole plant corn fine extract medium.

According to GB4789.35-2010, the assimilation ability of different carbon sources of the strains was tested, and the experimental results of the fermentation of carbon sources are shown in Table 2.

Table 2 Carbon source fermentation experiment results of LB strain

carbon source strainLB Arabic candy + fructose + sucrose + Lactol + maltose + Escin + ribose + Raffinose +

Note: "+" is positive; "-" is negative

The 16S rDNA sequence of strain LB was amplified and sequenced, and the sequencing result is shown in sequence 1 of the sequence table. The 16srDNA identification results showed that the similarity between the strain LB and the multiple strains of Lactobacillus Brucella in the NCBI database was 99%.

After the above identification, it can be determined that the strain LB belongs to Lactobacillus Brucella, so it was renamed as Lactobacillus Brucella LB.

3. Preservation of Lactobacillus Brucella LB

Lactobacillus brevis (Lactobacillus brevis) LB has been preserved in the General Microbiology Center of China Committee for Microbial Culture Collection (CGMCC for short) on June 22, 2017; address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences; Zip code: 100101), the deposit number is CGMCC No.14269. Lactobacillus brevis (Lactobacillus brevis) LB is referred to as Lactobacillus brevis LB.

4. Determination of the growth of Lactobacillus Brucella LB under different pH, temperature and salt concentration environmental conditions

1. Inoculate Lactobacillus Brucella LB in MRS liquid medium with different initial pH (3, 4, 4.5, 5, 6, 7, and 8), and culture it statically at 37°C for 2 days. After the culture, mix with blank MRS Liquid media versus growth. The results are shown in Table 3. The results showed that Lactobacillus brauchneri LB can grow under the culture condition with initial pH value of 4.0-8.0.

Table 3 The growth of Lactobacillus brucelli LB in different initial pH environments

Medium initial pH strainLB 3.0 + 4.0 + 4.5 + 5.0 + 6.0 + 7.0 + 8.0 +

Note: "+" is positive; "-" is negative

2. Inoculate Lactobacillus Brucella LB into MRS liquid medium, and perform the following grouping operations:

Group A: static culture at 4°C for 14 days;

Group B: Static culture at 10°C for 2 days;

Group C: Static culture at 20°C for 2 days;

Group D: Static culture at 30°C for 2 days;

Group F: Static culture at 40°C for 2 days;

Group G: Static culture at 50°C for 7 days;

Group H: static culture at 60°C for 7 days.

After the end of the culture, the growth was compared with that of the blank MRS liquid medium. The results are shown in Table 4. The results showed that Lactobacillus Brucella LB could not grow at 4°C, 50°C and 60°C, but grew well at 10°C-40°C.

The growth situation of table 4 Lactobacillus brucelli LB in different initial pH environments

Note: "+" is positive; "-" is negative

3. Inoculate Lactobacillus Brucella LB into the MRS liquid medium containing 3% (mass percentage) sodium chloride and the MRS liquid medium containing 6.5% (mass percentage) sodium chloride respectively, and culture statically at 37°C for 4 Day, observe the growth status of lactic acid bacteria. The results are shown in Table 5. The results showed that Lactobacillus brauchneri LB could grow under the conditions of 3% (mass percentage) and 6.5% (mass percentage) sodium chloride.

The growth situation of table 5 Lactobacillus brucelli LB in salt environment

Note: "+" is positive; "-" is negative

Embodiment 2, Lactobacillus brevis (Lactobacillus brevis) LB prepare whole plant corn fine silage

1. Bacteria powder preparation

Lactobacillus Brucella LB is inoculated in 10ml MRS liquid medium, 37 DEG C, 250rpm shaking culture, after cultivating 2 generations, inoculate in 60ml MRS liquid medium by the inoculum size of 3% (volume ratio) (bacterial liquid initial OD _260nm =1.8), shake culture at 37°C and 250rpm for 18-24h (the final OD _260nm of the bacterial solution =4), centrifuge the culture system, collect the bacterial precipitate, mix the bacterial precipitate with 10ml sterilized skim milk, and freeze-dry , to obtain a solid microbial agent, the concentration of Lactobacillus Brucella in the solid microbial agent is 1×10 ¹² CFU g ^-1 (need to be greater than 1×10 ¹⁰ CFU g ^-1 ).

Second, the whole plant corn fine silage preparation

Whole-plant corn fine: Whole-plant corn includes whole-plant corn, corn kernels, corn ears, corn ears and part of the stalks (including leaves). Chopped to 2-3cm respectively, the grains are crushed, and the water content is 35%-75%.

1. Take the solid bacterial agent prepared in step 1, dissolve it in 5ml of distilled water, and activate it at room temperature for 25 minutes to obtain a liquid bacterial agent (the content of Lactobacillus Brucella LB in 5ml of liquid bacterial agent is 2×10 ⁹ CFU).

2. Spray all the 5ml liquid bacterial agent obtained in step 1 on 2500g corn raw material (water content is 35%-75%), and inoculate 8×10 ⁵ CFU of Lactobacillus Brucella LB per gram of corn raw material. The control group (CK) was sprayed with 5 ml sterile water. Adopt polyethylene bag (180 * 260mm) to vacuumize and seal to make silage, about 250g of every bag, then store respectively in the incubator at 20, 30 and 40 ℃, silage 45 days.

3. Detection of fine silage effect of whole plant corn

1. Open each bag of silage samples respectively, weigh 20 g of fully mixed silage, add 180 mL of distilled water, grind for 1 min with a mixer, filter with 4 layers of gauze and qualitative filter paper (Hangzhou Special Paper Co., Ltd., Xinxing 102) successively, Collect the filtrate.

The filtrate was tested for the following indicators: pH value, lactic acid (LA), acetic acid (AA), propionic acid (PA), butyrate (BA), ammonia nitrogen (ammonia nitrogen) , AN) and free amino acid nitrogen (AA-N).

The pH value was measured with a Leici PHS-3C pH meter.

Lactic acid, acetic acid, propionic acid and butyric acid were analyzed by high performance liquid chromatography. Instrument: Shimadzu 20A high performance liquid chromatography; detector: Shimadzu SPD-M20A detector; chromatographic column: ShodexRSpak KC-811 chromatographic column (8mm × 300mm); mobile phase: 3mmol L ^-1 perchloric acid (excellent grade purity); detection wavelength: 210nm; flow rate: 1mL·min ^-1 ; injection volume: 5μL; column temperature: 50°C. The filtrate to be tested was filtered with a 0.45 μm water filter membrane and tested on the machine.

Lactic acid, acetic acid, propionic acid and butyric acid standard (Geel, Belgium) were purchased from Beijing Bailingwei Chemical Technology Co., Ltd.

The peak time of lactic acid standard is 8.1min; under the same conditions, the peak position is within ±0.2min, which can be identified as the same substance.

The peak eluting time of the acetic acid standard is 9.6 minutes; the peak eluting position is within ±0.2 min under the same conditions, which can be identified as the same substance.

The peak time of propionic acid standard is 11.2min; under the same conditions, the peak position is within ±0.2min, which can be identified as the same substance.

The peak time of butyric acid standard is 13.8min; under the same conditions, the peak position is within ±0.2min, which can be identified as the same substance.

AN was determined by the phenol-hypochlorous acid colorimetric method.

AA-N was determined by the ninhydrin copper colorimetric method.

DNA samples extracted from silage were transported at low temperature (below 0°C) to a gene sequencing company. The company will conduct sample testing on the samples received.

Qualified DNA samples are tested for library construction and library detection. The qualified library will be sequenced using Illumina PE150, and the off-machine data (Raw Data) obtained by sequencing will be used for later information analysis.

Qualified DNA samples were randomly broken into fragments with a length of about 350 bp using a Covaris ultrasonic breaker, and the entire library was prepared through steps such as end repair, A-tailing, sequencing adapters, purification, and PCR amplification.

After the library construction is completed, first use Qubit2.0 for preliminary quantification, dilute the library to 2ng/ul, and then use Agilent 2100 to detect the insert size of the library. After the insert size meets the expectation, use the Q-PCR method to check the effective concentration of the library. Accurate quantification (library effective concentration > 3nM) to ensure library quality.

After the library inspection is qualified, different libraries are pooled according to the effective concentration and the target off-machine data volume, and then Illumina PE150 sequencing is performed.

Data quality control: There will be a certain proportion of low-quality data in the raw data (Raw Data) obtained by sequencing. In order to ensure the accuracy and reliability of subsequent information analysis results, the quality control and host filtering of the raw data must first be performed to obtain valid data (Clean Data );

Metagenome assembly: Starting from the Clean Data of each sample after quality control, Metagenome assembly is performed, and the unused reads of each sample are put together for mixed assembly, in order to discover the information of low-abundance species in the sample; gene prediction: from Starting from the scaftigs after single sample and mixed assembly, MetaGeneMark is used for gene prediction, and the genes generated by each sample and mixed assembly prediction are put together to remove redundancy and construct a gene catalog. Starting from the gene catalog, the Clean Data of each sample is synthesized , the abundance information of the gene catalog in each sample can be obtained; resistance gene annotation: using the gene catalog and the antibiotic resistance gene database CARD (TheComprehensive Antibiotic Resistance Database) to annotate, the abundance distribution of resistance genes and these resistance genes can be obtained. Species affiliation of sex genes and resistance mechanisms.

The results are shown in Table 6.

Table 6 Effects of Lactobacillus brauchneri LB on the fermentation quality of whole-plant maize fine silage

additive silage raw material The number of types of resistance genes pH CK whole plant corn 138 3.99 CK corn kernels 135 4.01 CK ears of corn 124 4.08 CK Ears of corn and part of the stalk (including leaves) 117 3.94 LB whole plant corn 105 3.91 LB corn kernels 116 4.11 LB ears of corn 93 4.05 LB Ears of corn and part of the stalk (including leaves) 87 3.87 LP whole plant corn 119 3.91 LP corn kernels 122 4.03 LP ears of corn 108 4.05 LP Ears of corn and part of the stalk (including leaves) 104 3.88

LP is Lactobacillus plantarum (LP, Lactobacillus plantarum, see The effects of stage of maturity and lactic acid bacteria inoculants on the ensiling characteristics, aerobic stability and in vitro digestibility of whole-crop oat silages, Tingting Jia, Bing Wang, Zhu Yu, Zhe Wu), the preparation method and addition amount are the same as that of Lactobacillus brevis (Lactobacillus brevis) LB.

In conclusion, Lactobacillus brazineri LB can be used as an additive for whole-plant corn fine silage, which can significantly reduce the pH value and effectively improve the fermentation quality of whole-plant corn fine silage; reduce the types of resistance genes in whole-plant corn fine silage; Lactobacillus LB has the advantages of low cost and can be applied in the production of green and environment-friendly biological feed.

SEQUENCE LISTING

<110> China Agricultural University

<120> A fungal agent for preparing whole-plant corn fine silage

<130> GNCAQ211742

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<170> PatentIn version 3.5

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<211> 1513

<212>DNA

<213>Lactobacillus brevis

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cgatgatacg tagccgacct gagagggtaa tcggccacat tgggactgag acacggccca 360

aactcctacg ggaggcagca gtagggaatc ttccacaatg gacgaaagtc tgatggagca 420

atgccgcgtg agtgaagaag ggtttcggct cgtaaaactc tgttgttaaa gaagaacacc 480

tttgagagta actgttcaag ggttgacggt atttaaccag aaagccacgg ctaactacgt 540

gccagcagcc gcggtaatac gtaggtggcn agcgttgtcc ggatttattg ggcgtaaagc 600

gagcgcaggc ggttttttaa gtctgatgtg aaagccttcg gcttaaccgg agaagtgcat 660

cggaaactgg gagacttgag tgcagaagag gacagtggaa ctccatgtgt agcngtggaa 720

tgcgtagata tatggaagaa caccagtggc gaaggcggct gtctagtctg taactgacgc 780

tgaggctcna aagcatgggt agcgaacagg attagatacc ctggtagtcc atgccgtaaa 840

cgatgagtgc taagtgatgg agggtttccg cccttcagtg ctgcagctaa cgcattaagc 900

actccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggccngca 960

caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac 1020

atcttctgcc aatcttagag ataagacgtt cccttcgggg acagaatgac aggtggtgca 1080

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caatggacgg tacaacgagt cgcgaagtcg tgaggctaag ctaatctctt aaagccgttc 1320

tcagttcgga ttgtaggctg caactcgcct acatgaagtt ggaatcgcta gtaatcgcgg 1380

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gagtttgtaa cacccaaagc cggtgagata accttcggga gtcagccgtc taaggtggga 1500

cagatgatta ggg 1513

Claims (1)

1. A preparation method of silage taking corns as a raw material comprises the following steps: applying the microbial inoculum to the corn raw material for ensiling to obtain the whole corn fine silage, or applying lactobacillus buchneri LB to the corn raw material for ensiling to obtain the whole corn fine silage;

the microbial inoculum is prepared by mixing lactobacillus buchneri LB and a probiotic protective agent;

in each gram of the microbial inoculum, the content of the Lactobacillus buchneri LB is more than 1 x 10 ¹⁰ CFU; the probiotic protective agent is skimmed milk; the skimmed milk is prepared by dissolving solid milk powder in water according to the mass volume ratio of 10%;

the corn raw material is the whole corn from the milk stage to the complete stage;

the Lactobacillus buchneri (Lactobacillus brevis) LB has been preserved in the China general microbiological culture Collection center (CGMCC) in 2017, 8 and 7 months; address: beijing, haoyang district, xilu No.1, ministry of microbiology, china academy of sciences, 3; and E, postcode: 100101, the preservation number is CGMCC No.14269;

the water content of the corn raw material is 35-75%;

the corn raw material is pretreated before a microbial inoculum or lactobacillus buchneri LB is applied;

the pretreatment method comprises the following steps: cutting corn stems and leaves into 2-3cm, and crushing corn seeds into complete particles;

before the microbial inoculum or the lactobacillus buchneri LB is applied to the corn raw material, the method further comprises the following steps: dissolving the microbial inoculum or Lactobacillus buchneri LB with water, and activating at room temperature for 50-70 min;

the application amount of the microbial inoculum or the Lactobacillus buchneri LB is 0.5-20 multiplied by 10 inoculated to each gram of corn raw material ⁵ Lactobacillus buchneri LB of CFU;

the temperature of the ensiling is 20-40 ℃;

the ensiling time is 30-50 days.