CN113373087A

CN113373087A - Microbial inoculum for preparing complete-plant corn fine silage

Info

Publication number: CN113373087A
Application number: CN202110654063.9A
Authority: CN
Inventors: 吴哲
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2021-09-10
Anticipated expiration: 2041-06-11
Also published as: CN113373087B

Abstract

The invention provides a microbial inoculum for preparing complete fine corn silage. 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 inoculum¹⁰And (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

Microbial inoculum for preparing complete-plant corn fine silage

Technical Field

The invention belongs to the field of feeds, relates to a microbial inoculum for preparing complete-strain corn fine silage, and particularly provides lactobacillus for complete-strain corn fine silage, and more particularly provides lactobacillus buchneri which can reduce resistance genes and viruses in various silage prepared from corn.

Background

Ensiling and semi-dry ensiling are processing means for cutting green feed with water content of 45-75%, sealing and fermenting with lactobacillus as main material to realize effective preservation of nutrients, and the ensiling feed has the functions of reducing weed seed germination rate and reducing virus and can be preserved for a long time.

The silage additive is a substance which is added in the silage process and promotes lactic acid fermentation or inhibits undesirable microorganism fermentation, such as lactic acid bacteria, an enzyme preparation, a compound bacteria preparation, a chemical additive and the like. Generally, the method can play a role in reducing nutrient loss and improving fermentation speed.

Corn is the most widely used feed at present, and can be prepared into whole-plant corn silage, corn kernel silage and corn silage with different stubble heights.

Silage accounts for a large part of the composition of the coarse ration feed, and in recent years, because superbacteria have a strong resistance to antibiotics, the risk of killing can be avoided, and the propagation of the silage poses a threat to human health. Thus, there is an increasing interest in resistance genes, but there has been less research on resistance genes in silage. The bacteria in the intestinal tract of livestock can take up the resistance genes in the feed, and can become bacteria capable of resisting various medicines, namely super bacteria, after excretion. Since the breeding farm has become the largest place for producing the super bacteria except for hospitals, reducing the types and the number of the resistance genes in the feed has the possibility of reducing the number of the super bacteria in the breeding farm, and therefore, screening and applying the additive capable of reducing the resistance genes in the feed is an emerging research hotspot at present.

Disclosure of Invention

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

The Lactobacillus buchneri (Lactobacillus brevis) LB is preserved in China general microbiological culture Collection center (CGMCC for short; address: No. 3 of Xilu No.1 of Beijing Korean district, Microbiol research institute of Chinese academy of sciences; zip code: 100101) in 6.6.22.2017, with the preservation number of CGMCC No. 14269; abbreviated as lactobacillus buchneri LB.

The new application of the lactobacillus buchneri LB provided by the invention is as follows: application of lactobacillus buchneri LB in preparation of fine forage grass silage of whole corn plants.

The application specifically comprises the following steps: the application of lactobacillus buchneri LB in the reduction of resistance genes in the fine pasture silage of whole corn plants, more specifically: application of lactobacillus buchneri LB in reducing the variety of resistance genes in fine pasture silage of whole corn.

The whole corn plant comprises a whole corn plant, grains and corn harvested at different stubble heights.

The other purpose of the invention is to provide a microbial inoculum.

The microbial inoculum provided by the invention is prepared by mixing lactobacillus buchneri LB and a probiotic protective agent.

The microbial inoculum is prepared by mixing and freeze-drying 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 inoculum¹⁰CFU。

Specifically, each gram of the microbial inoculum contains 1 × 10¹²CFU Lactobacillus buchneri LB.

The probiotic protecting agent can be skim milk.

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

The solid milk powder can be high-protein defatted high-calcium milk powder, and more specifically can be high-protein defatted high-calcium milk powder purchased from inner Mongolia Yili industry group GmbH with the product number of 6907992440071.

The microbial inoculum is prepared by a method comprising the following steps: mixing Lactobacillus buchneri LB and probiotic protectant.

Specifically, the microbial inoculum is prepared by a method comprising the following steps: and mixing the Lactobacillus buchneri LB thallus with a probiotic protective agent, and freeze-drying to obtain the microbial inoculum.

The lactobacillus buchneri LB thallus is prepared by the method comprising the following steps: inoculating lactobacillus buchneri LB into MRS liquid culture medium for culture, centrifuging the culture system when the culture is finished, and collecting precipitate, namely lactobacillus buchneri LB thallus.

In the method, the OD of the culture system at the end of the culture_260nm＝4；

In the method, the initial OD of the culture system after inoculation_260nm＝1.8；

In the method, the culture condition is oscillation culture at 37 ℃ and 250 rpm;

The application of lactobacillus buchneri LB or the microbial inoculum containing lactobacillus buchneri LB in preparing the whole strain corn fine silage also belongs to the protection scope of the invention.

The invention also provides a preparation method (method A) of silage taking corns (whole plants, grains and corns harvested at different stubble heights) as raw materials, which comprises the following steps: and (3) applying the microbial inoculum to the corn raw material for ensiling to obtain the whole-plant corn fine silage.

In the method a, the harvesting period of the corn material may be from milk stage to wax stage.

In the method A, the water content of the corn raw material can be 35-75%.

In the method A, the corn raw material is pretreated before the microbial inoculum is applied;

the pretreatment method comprises the following steps: cutting corn stems and leaves into 2-3cm, and crushing corn seeds until no complete particles exist;

in the method A, before the microbial inoculum is applied to the corn raw material, the method further comprises the following steps: dissolving the microbial inoculum with water, and activating at room temperature for 50-70min (specifically 60min), wherein the water is distilled water.

In the method A, the application amount of the microbial inoculum is 0.5-20 multiplied by 10 inoculated to each gram of corn raw material⁵Lactobacillus buchneri LB of CFU; the application amount of the microbial inoculum can be specifically 8 multiplied by 10 inoculated per gram of corn raw material⁵Lactobacillus buchneri LB of CFU;

in the method A, the temperature of the ensiling can be 20-40 ℃, and specifically can be 20 ℃, 30 ℃ or 40 ℃.

The ensiling time may be 30 to 50 days, specifically 45 days.

The invention also provides a preparation method (method B) of the whole-plant corn fine silage, which comprises the following steps: and (3) applying lactobacillus buchneri LB to the corn raw material, and ensiling to obtain the whole-plant corn fine silage.

In the method B, the corn raw materials can be whole corn, corn kernels, corn ears and partial corn stalks (including leaves) from the milk stage to the complete stage.

In the method B, the water content of the corn raw material can be 35-75%.

In the method B, the corn raw material is pretreated before the microbial inoculum is applied;

in the method B, before the microbial inoculum is applied to the corn raw material, the method further comprises the following steps: dissolving the microbial inoculum with water, and activating at room temperature for 50-70min (specifically 60min), wherein the water is distilled water.

In the method B, the application amount of the microbial inoculum is 0.5-20 multiplied by 10 inoculated to each gram of corn raw material⁵Lactobacillus buchneri LB of CFU; the application amount of the microbial inoculum can be specifically 8 multiplied by 10 inoculated per gram of corn raw material⁵Lactobacillus buchneri LB of CFU;

in the method B, the temperature of the ensiling can be 20-40 ℃, and specifically can be 20 ℃, 30 ℃ or 40 ℃.

The ensiling time may be 30 to 50 days, specifically 45 days.

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

The whole-plant corn fine silage contains fewer types and quantity of resistance genes.

The lactobacillus buchneri LB can be used as a whole-plant corn fine silage additive, so that the lactic acid output is remarkably improved, the pH value is remarkably 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.

Detailed Description

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

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The MRS liquid culture medium consists of solute and solvent; the solute and the concentration thereof in the MRS liquid culture medium are as follows: 10.0g/L of peptone, 5.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 5.0g/L of sodium acetate, 2.0g/L of triammonium citrate, 801mL/L of tween, 2.0g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate and 0.05g/L of manganese sulfate; the solvent is water.

Culture medium of whole-plant corn fine fresh grass extract: 1kg of whole-plant corn fine fresh grass, cutting, adding 5L of water, and carrying out water bath at 50 ℃ for 2h to obtain an extracting solution; filtering the extractive solution with four layers of gauze, then filtering with quantitative filter paper (Hangzhou special paper industry Co., Ltd., New Star type 201), and collecting filtrate; the filtrate was autoclaved at 121 ℃ for 15 minutes and then dispensed into sterilized test tubes for use.

Skim milk: the solid milk powder is dissolved in distilled water according to the mass volume ratio (w/v) of 10%.

Solid milk powder: high protein, defatted, high calcium milk powder, trade mark of the inner Mongolia Yili industry group GmbH: 6907992440071.

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

First, separating and screening bacterial strain

Collecting soaking solution of cortex Caprae Seu Ovis (obtained by soaking cortex Caprae Seu Ovis in clear water during preparation process), shaking with oscillator for 30min, and adding distilled water 10^-1-10^-6And (4) gradient dilution, namely respectively taking 1mL of each dilution gradient dilution solution, inoculating the dilution gradient dilution solution into a culture dish, adding 10-15mL of sterilized MRS solid culture medium, shaking up, cooling and solidifying the mixture, and standing for anaerobic culture for 48 hours. Single colonies with significant differences in colony morphology, size, color and gloss were picked, streaked repeatedly, and until pure colonies were obtained. Single colonies were picked for gram staining and catalase test. ValveGram-positive and catalase-negative strains are preliminarily determined as lactic acid bacteria, and then a lactic acid bacteria strain with high growth capacity and acid production capacity is screened out by using a whole-plant corn fine fresh grass extract culture medium, and is named as a strain LB, and the growth capacity and the acid production capacity of the strain are shown in Table 1.

The method for measuring the growth capacity and acid production capacity of the strain comprises the following steps: inoculating the strain to be tested into 10mL of MRS liquid culture medium, culturing for 2 generations, inoculating into a whole-plant corn fine fresh grass extract culture medium according to the inoculation amount of 3% (volume ratio), and performing shaking culture at 37 ℃ and 250 rpm. Taking out the cultured samples once every 12h, taking 3 times, taking samples after 36h of culture for the last time, measuring the absorbance values of the samples at the wavelength of 620nm by all the samples, and determining the pH value of the fermentation liquor. And (3) taking the whole-plant corn fine extract culture medium without inoculated strains as a control, comparing the maximum difference between the absorbance value of different strains in the whole-plant corn fine extract culture medium under the wavelength of 620nm after shaking culture for 36 hours and the control with the maximum difference between the pH value of the fermentation liquor and the control, and screening the lactic acid bacteria strain with the maximum absorbance value increase and the maximum pH value decrease.

TABLE 1 growth and acid productivity of Strain LB

\	Ability to grow^a	Acid production capacity^b
			Strain LB	2.44	2.61

Note: a, culturing the lactobacillus strain in a whole-plant corn fine extract culture medium for 36 hours, wherein the maximum difference value between the absorbance value at the wavelength of 620nm and a control is obtained; b, culturing the lactobacillus strain in a whole-plant corn fine extract culture medium for 36h to obtain the maximum difference value between the pH value of the fermentation liquor and the control.

II, identification of the Strain LB

Biological characteristics of strain LB: the strain LB is cultured on an MRS solid culture medium for 24 hours, the strain grows well, and milky white colonies with regular edges can be formed; the gram staining of the strain is positive, and the cell shape under a microscope is short rod-shaped and has no spore; the oxidase is negative, and the growth and acid production performance of the medium are strong in the fine extract of the whole corn.

The assimilation ability of different carbon sources of the strain is detected according to GB4789.35-2010, and the experimental results of the carbon source fermentation are shown in Table 2.

TABLE 2 carbon source fermentation test results of LB Strain

Carbon source	Strain LB
		Arabinose	+
Fructose	+
		Sucrose	+
Milk alcohol	+
		Maltose	+
Esculin	+
		Ribose	+
Cotton seed candy	+

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

And (3) amplifying and sequencing the 16S rDNA sequence of the strain LB, wherein the sequencing result is shown as a sequence 1 in a sequence table. The 16s rDNA identification showed 99% similarity of strain LB to the multiple strains of Lactobacillus buchneri in the NCBI database.

Through the identification, the strain LB belongs to the lactobacillus buchneri, so the strain LB is named again.

Preservation of Lactobacillus buchneri LB

Lactobacillus buchneri (Lactobacillus brevis) LB, which has been deposited in China general microbiological culture Collection center (CGMCC for short; address: Xilu No.1, Ministry of microbiology, China institute of microbiology, Beijing, and Inward, North Chen, No.1, and Zip code: 100101) in 6.7.22.2017, with the deposition number of CGMCC No. 14269. Lactobacillus buchneri (Lactobacillus brevis) LB is abbreviated as Lactobacillus buchneri LB.

Fourth, growth determination of Lactobacillus buchneri LB under different pH, temperature and salt concentration environmental conditions

1. Lactobacillus buchneri LB was inoculated into MRS liquid medium at different initial pH (3, 4, 4.5, 5, 6, 7 and 8), cultured statically at 37 ℃ for 2 days, and after the culture was finished, the growth was compared with that of the blank MRS liquid medium. The results are shown in Table 3. The results show that Lactobacillus buchneri LB was able to grow under culture conditions starting at a pH value of 4.0-8.0.

TABLE 3 growth of Lactobacillus buchneri LB in different initial pH environments

Initial pH of the Medium	Strain LB
		3.0	+
4.0	+
		4.5	+
5.0	+
		6.0	+
7.0	+
		8.0	+

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

2. Lactobacillus buchneri LB was inoculated in MRS liquid medium and grouped as follows:

group A: standing and culturing at 4 deg.C for 14 days;

group B: standing and culturing at 10 deg.C for 2 days;

group C: standing and culturing at 20 deg.C for 2 days;

group D: standing and culturing for 2 days at 30 ℃;

group F: standing and culturing at 40 deg.C for 2 days;

group G: standing and culturing at 50 deg.C for 7 days;

group H: the cells were cultured at 60 ℃ for 7 days.

And comparing the growth condition with a blank MRS liquid culture medium after the culture is finished. The results are shown in Table 4. The results show that the Lactobacillus buchneri LB can not grow at the temperature of 4 ℃, 50 ℃ and 60 ℃, and the growth condition is good at the temperature of 10-40 ℃.

TABLE 4 growth of Lactobacillus buchneri LB in different initial pH environments

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

3. Lactobacillus buchneri LB was inoculated into MRS liquid medium containing 3% (mass%) sodium chloride and MRS liquid medium containing 6.5% (mass%) sodium chloride, respectively, and left to stand at 37 ℃ for 4 days to observe the growth of lactic acid bacteria. The results are shown in Table 5. The results showed that Lactobacillus buchneri LB was able to grow with 3% (mass%) and 6.5% (mass%) sodium chloride.

TABLE 5 growth of Lactobacillus buchneri LB in saline

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

Example 2 preparation of Whole corn Fine silage from Lactobacillus buchneri (Lactobacillus brevis) LB

Firstly, preparation of fungus powder

Inoculating Lactobacillus buchneri LB into 10ml MRS liquid culture medium, culturing at 37 deg.C and 250rpm under shaking, and culturing 2Inoculating to 60ml MRS liquid culture medium (initial OD of bacteria liquid) with 3% (volume ratio)_260nm1.8), culturing at 37 deg.C and 250rpm for 18-24h (final OD of bacterial liquid)_260nm4), centrifuging the culture system, collecting the thallus precipitate, mixing the thallus precipitate with 10ml of sterilized skimmed milk, and freeze-drying to obtain a solid microbial inoculum with the concentration of lactobacillus buchneri in the solid microbial inoculum of 1 × 10¹²CFU g^-1(desirably greater than 1X 10)¹⁰CFU g^-1)。

Second, fine silage preparation of whole corn

Refining the whole corn: the whole corn includes whole corn, corn kernel, corn ear and partial stem (including leaves). Respectively cutting into 2-3cm, and crushing the seeds to obtain seeds with water content of 35-75%.

1. Dissolving the solid microbial inoculum prepared in the step one in 5ml of distilled water, and activating at room temperature for 25min to obtain liquid microbial inoculum (the content of Lactobacillus buchneri LB in 5ml of liquid microbial inoculum is 2 multiplied by 10)⁹CFU)。

2. 5ml of the liquid microbial inoculum obtained in the step 1 is completely sprayed on 2500g of corn raw materials (the water content is 35-75 percent), and 8 multiplied by 10 is inoculated on each gram of corn raw materials⁵CFU lactobacillus buchneri LB. Control group (CK) was sprayed with 5ml sterile water. The ensilage was prepared by vacuum-sealing polyethylene bags (180X 260mm) at about 250 g/bag and stored in thermostats at 20, 30 and 40 ℃ for 45 days.

Three, detection of fine silage effect of whole corn

1. Opening each bag of silage sample, weighing 20g of silage, adding 180mL of distilled water, mashing for 1min with a blender, filtering with 4 layers of gauze and qualitative filter paper (Hangzhou special paper industry Co., Ltd., Xinxing 102), and collecting filtrate.

And (3) detecting the following indexes of the filtrate: pH, Lactic Acid (LA), Acetic Acid (AA), Propionic Acid (PA), Butyric Acid (BA), Ammonia Nitrogen (AN), and free amino acid nitrogen (AA-N).

The pH value is measured by a thunder magnetic PHS-3C type pH meter.

Lactic acid, acetic acid, propionic acid and butyric acid were analyzed by high performance liquid chromatography. The instrument comprises the following steps: shimadzu 20A high performance liquid chromatograph; a detector: shimadzu SPD-M20A detector; a chromatographic column: shodex RSpak KC-811 column (8 mm. times.300 mm); mobile phase: 3 mmol. L^-1Perchloric acid (guaranteed purity); detection wavelength: 210 nm; flow rate: 1 mL. min^-1(ii) a Sample introduction amount: 5 mu L of the solution; the column temperature was 50 ℃. Filtering the filtrate to be detected by a 0.45 mu m water system filter membrane and detecting on a machine.

Lactic acid, acetic acid, propionic acid and butyric acid standards (gell, Belgium) were purchased from beijing bailing wafer chemical technology ltd.

The peak time of the lactic acid standard product is 8.1 min; under the same conditions, the peak positions are within +/-0.2 min, and the same substances can be identified.

The peak time of the acetic acid standard product is 9.6 min; under the same conditions, the peak positions are within +/-0.2 min, and the same substances can be identified.

The peak time of the propionic acid standard substance is 11.2 min; under the same conditions, the peak positions are within +/-0.2 min, and the same substances can be identified.

The peak time of the butyric acid standard substance is 13.8 min; under the same conditions, the peak positions are within +/-0.2 min, and the same substances can be identified.

AN was determined by phenol-hypochlorous acid colorimetry.

AA-N is measured by ninhydrin colorimetry.

DNA samples extracted from silage were transported at low temperature (below 0 ℃) to gene sequencing companies. The company will perform a sample test on the received sample.

Detecting qualified DNA samples, constructing libraries and detecting the libraries, wherein the detected qualified libraries are sequenced by Illumina PE150, and off-line Data (Raw Data) obtained by sequencing is used for later information analysis.

And randomly breaking a fragment with the length of about 350bp of a detected qualified DNA sample by using a Covaris ultrasonic disruptor, and completing the preparation of the whole library by the steps of end repair, A tail addition, sequencing joint addition, purification, PCR amplification and the like.

After the library is constructed, firstly using Qubit2.0 to carry out preliminary quantification, diluting the library to 2ng/ul, then using Agilent 2100 to detect the insert size of the library, and after the insert size meets the expectation, using a Q-PCR method to accurately quantify the effective concentration of the library (the effective concentration of the library is more than 3nM) so as to ensure the quality of the library.

And after the library is qualified, carrying out Illumina PE150 sequencing on different libraries according to the effective concentration and the requirement of the target offline data volume.

And (3) data quality control: the original Data (Raw Data) obtained by sequencing has a certain proportion of low-quality Data, and in order to ensure the accuracy and reliability of the subsequent information analysis result, the original Data is firstly subjected to quality control and host filtration to obtain effective Data (clear Data);

metagenome assembly: starting from Clean Data after quality control of each sample, carrying out Metagenome assembly, putting unused reads of each sample together for mixed assembly, and discovering low-abundance species information in the samples; and (3) gene prediction: starting from single samples and mixed assembled scaftigs, adopting MetaGeneMark to carry out gene prediction, putting each sample and genes generated by mixed assembly prediction together, carrying out redundancy removal, constructing gene category, starting from the gene category, and synthesizing Clean Data of each sample to obtain abundance information of the gene category in each sample; resistance gene annotation: by using gene catalog and Antibiotic Resistance gene database CARD (the Comprehensive Antibiotic Resistance database), the abundance distribution of the Resistance genes and the species attribution and Resistance mechanism of the Resistance genes can be obtained.

The results are shown in Table 6.

TABLE 6 influence of Lactobacillus buchneri LB on the Fine silage fermentation quality of whole corn plants

LP is Lactobacillus plantarum (LP, Lactobacillus plantarum, see The effects of stage of formation and lactic acid bacteria induction on The engineering, aerobic stability and in vitro differentiation of hold-crop sites, tilling Jia, Bing Wang, Zhu Yu, Zhu Wu), with The same preparation and addition as Lactobacillus buchneri LB.

In conclusion, the lactobacillus buchneri LB can be used as an additive for fine silage of whole corn plants, so that the pH value is remarkably reduced, and the fermentation quality of the fine silage of the whole corn plants is effectively improved; reducing the types of resistance genes in the fine silage of the whole corn plant; meanwhile, the lactobacillus buchneri LB has the advantages of low cost and the like, and can be applied to the production of green and environment-friendly biological feed.

SEQUENCE LISTING

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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

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tcagttcgga ttgtaggctg caactcgcct acatgaagtt ggaatcgcta gtaatcgcgg 1380

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Claims

1. Application of Lactobacillus buchneri (Lactobacillus brevis) LB in preparation of whole-strain corn fine forage grass silage, wherein the Lactobacillus buchneri (Lactobacillus brevis) LB is preserved in the China general microbiological culture Collection center (CGMCC for short; address: No. 3 of West Lu 1 of Beijing City morning area, Navigna district, institute of microbiology, China academy of sciences; postal code: 100101) in 8-7-2017, and the preservation number is CGMCC No. 14269.

2. Use according to claim 1, characterized in that: the application is as follows: the application of lactobacillus buchneri LB in the reduction of resistance genes in fine whole-strain corn forage silage specifically comprises the following steps: application of lactobacillus buchneri LB in reducing the variety of resistance genes in fine pasture silage of whole corn.

3. A microbial preparation is prepared by mixing Lactobacillus buchneri LB and probiotic protectant.

4. The microbial inoculum of claim 3, wherein: the content of the Lactobacillus buchneri LB is more than 1 x 10 per gram of the microbial inoculum¹⁰A CFU; the probiotic protective agent is skimmed milk; the skimmed milk is prepared by dissolving solid milk powder in water at a mass volume ratio of 10%.

5. A method for producing the microbial agent of claim 4, comprising the steps of: mixing Lactobacillus buchneri LB thallus with a probiotic protective agent, and freeze-drying to obtain the microbial inoculum;

6. The method of claim 5, wherein: in the method, the culture is completedOD of culture System_260nm＝4；

7. Use of lactobacillus buchneri LB or a bacterial agent comprising lactobacillus buchneri LB as claimed in claim 3 or 4 for the preparation of fine silage for whole corn.

8. A preparation method of silage taking corns as a raw material comprises the following steps: and 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.

9. The method of claim 8, wherein: the corn raw materials are full corn, corn grains, corn ears and partial corn stalks from the milk stage to the complete stage;

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;

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 material⁵Lactobacillus buchneri LB of CFU;

the temperature of the ensiling is 20-40 ℃;

the ensiling time is 30-50 days.

10. A whole corn fine silage produced by the method of claim 8 or 9, said whole corn fine silage containing fewer species of resistance genes.