CN114480186B

CN114480186B - Compound fermentation inoculant for fermenting mycotoxin contaminated feed and application thereof

Info

Publication number: CN114480186B
Application number: CN202210058221.9A
Authority: CN
Inventors: 朱伟; 冯政夫; 刘冬美
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2022-01-19
Filing date: 2022-01-19
Publication date: 2023-02-03
Anticipated expiration: 2042-01-19
Also published as: CN114480186A; CN116656527A

Abstract

The invention provides a compound fermentation inoculant for fermenting polluted feed and application thereof, belonging to the technical field of microorganisms. The composite fermentation microbial inoculum provided by the invention is prepared from lysine bacillus HQ08 (HQ)Bacillus lysinosus) And Sinorhizobium meliloti CM 77: (Sinorhizobium meliloti) The preservation number of the lysine bacillus HQ08 is CGMCC No.24221, and the preservation number of the sinorhizobium meliloti CM77 is CGMCC No.24222. The composite fermentation inoculant provided by the invention can effectively reduce zearalenone toxin and aflatoxin B1 in the feed at the same time, and has excellent effects.

Description

Compound fermentation inoculant for fermenting mycotoxin contaminated feed and application thereof

Technical Field

The invention belongs to the technical field of microbiology, and particularly relates to a compound fermentation inoculant for fermenting polluted feed and application thereof.

Background

Crops such as corn, wheat, barley and peanut and byproducts thereof are often polluted by mycotoxin in harvesting, airing, storing and other links. In 2015-2020, the detection rates of aflatoxin, zearalenone, vomitoxin and fumonisin agricultural and sideline products are continuously increased. From 2016, the detection rate of aflatoxin B1 in corn byproducts is increased rapidly, the corn byproducts are in a high pollution level for 3 years continuously, and the phenomenon that aflatoxin B1 and zearalenone are severely polluted by bran products in Shandong, jiangsu, yunnan, anhui and other places exists. Meanwhile, in recent years, the pollution condition of zearalenone in China is very serious, the standard exceeding rate of zearalenone is in a remarkable rising trend compared with the past year, and the detection rate of zearalenone in corn byproducts and pet food reaches 100 percent.

Therefore, the problem that the pollution of aflatoxin B1 and zearalenone in the feed is effectively reduced is a problem which needs to be solved at present. Among them, the biological detoxification method includes microbial degradation and enzymolysis, and has the characteristics of high efficiency, strong specificity, environmental friendliness and little pollution, so that the biological detoxification method becomes a hot point of research of people in recent years. However, the prior art is mostly lack of complex microbial inoculum capable of removing a plurality of toxins simultaneously aiming at single toxin microbial inoculum.

Disclosure of Invention

The invention aims to provide a compound fermentation inoculant for fermenting polluted feed and application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a compound fermentation microbial inoculum which is a microbial compound microbial inoculum consisting of lysine bacillus HQ08 and sinorhizobium meliloti CM 77;

wherein, the lysine bacillus HQ08 (HQ)Bacillus lysinosus) The culture medium is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the address is No. 3 of Xilu No.1 of Beijing, chaoyang district, the preservation number is CGMCC No.24221, and the preservation date is 2021, 12 months and 30 days; sinorhizobium meliloti CM 77: (Sinorhizobium meliloti) The culture medium is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the address is No. 3 of Xilu No.1 of Beijing, chaoyang district, the preservation number is CGMCC No.24222, and the preservation date is 2021, 12 months and 30 days.

Preferably, the weight parts of the bacillus lysimachiae HQ08 and the sinorhizobium meliloti CM77 are respectively 1-9 parts and 1-9 parts.

Preferably, the content of the lysine bacillus HQ08 is 4-6 parts by weight, and the content of the sinorhizobium meliloti CM77 is 4-6 parts by weight.

Preferably, the complex microbial inoculum also comprises 5-20 parts of trehalose, 1-10 parts of glycine, 5-25 parts of glycerol and 20-45 parts of sterile water.

Preferably, in the composite microbial agent, the viable count of lysine bacillus HQ08 is more than or equal to 0.5 x 10 ⁹ cfu/g, the viable count of the Sinorhizobium meliloti CM77 is more than or equal to 0.5 x 10 ⁹ cfu/g。

Preferably, the complex microbial inoculum is used for treating aflatoxin B1 and zearalenone polluted feed.

Secondly, the invention provides an application of a composite microbial inoculum in reducing the pollution of feed aflatoxin B1 and zearalenone, wherein the composite fermentation microbial inoculum is a microbial composite microbial inoculum consisting of lysine bacillus HQ08 and sinorhizobium meliloti CM 77;

wherein, the lysine bacillus HQ08 (HQ)Bacillus lysinosus) The culture medium is preserved in the China general microbiological culture Collection center with the address of No. 3 Xilu No.1 of Beijing, chaoyang district, the preservation number of CGMCC No.24221 and the preservation date of 2021, 12 months and 30 days; sinorhizobium meliloti CM 77: (Sinorhizobium meliloti) The culture medium is preserved in the China general microbiological culture Collection center with the address of No. 3 Xilu No.1 of Beijing, chaoyang, the preservation number of CGMCC No.24222 and the preservation date of 2021, 12 months and 30 days.

Preferably, in the composite microbial inoculum, according to the mass parts, the lysine bacillus HQ08 is 4-6 parts, and the sinorhizobium meliloti CM77 is 4-6 parts.

Preferably, the complex microbial inoculum further comprises 5-20 parts of trehalose, 1-10 parts of glycine, 5-25 parts of glycerol and 20-45 parts of sterile water.

Preferably, in the composite microbial inoculum, the viable count of lysine bacillus HQ08 is more than or equal to 0.5 x 10 ⁹ cfu/g, the viable count of the Sinorhizobium meliloti CM77 is more than or equal to 0.5 x 10 ⁹ cfu/g。

The invention has the beneficial effects that:

the composite fermentation inoculant provided by the invention can effectively reduce zearalenone toxin and aflatoxin B1 in the feed at the same time, and has excellent effects.

Detailed Description

In order to clearly explain the technical features of the present solution, the present solution is explained by the following detailed description.

Example 1

Separation and screening of lysine bacillus HQ08

(1) Preparing a screening medium 1L: 5g of ammonium sulfate, 2.5g of potassium dihydrogen sulfate, 1g of magnesium sulfate, 0.5g of disodium hydrogen phosphate and 15-20g of agar powder, adjusting the pH value to 7, sterilizing at 120 ℃ for 30min, cooling to room temperature, adding an aflatoxin B1 standard substance, and adjusting the final concentration to be 1 mu g/ml;

(2) Weighing 1g of soil, dissolving in 100ml of sterile distilled water, standing at room temperature for 5min, taking supernatant, diluting with sterile distilled water to obtain different concentration gradients, taking 1ml, coating on a screening culture medium, and culturing at 37 ℃;

(3) Selecting single colonies with different forms, performing amplification culture with LB liquid culture medium, diluting with sterilized distilled water, spreading 1ml of the diluted solution in a screening culture medium, and culturing at 37 deg.C;

(4) Selecting a single bacterial colony, purifying the bacterial strain by adopting a scribing method, and selecting bacterial colonies with consistent growth for degradation rate detection;

(5) Thus obtaining the strain with higher capability of degrading the aflatoxin B1: lysine bacillus HQ08.

Example 2

Isolation and screening of Sinorhizobium meliloti CM77 strain

(1) Primary screening:

1) Preparing an improved basic salt culture medium (1L): 0.5g of ammonium nitrate, 0.5g of potassium dihydrogen phosphate, 1.5 g of dipotassium hydrogen phosphate, 1g of sodium chloride, 0.02 g of magnesium sulfate, 1000 mL of distilled water and pH =7.4; adding 15-20g of agar, mixing and sterilizing;

2) Preparing zearalenone with the final concentration of 1 mug/mL, adding the zearalenone into a culture medium, uniformly mixing, and pouring into a flat plate;

3) Weighing soil in a natural environment, dissolving the soil in sterile distilled water or physiological saline, uniformly mixing and standing, respectively taking supernatant, semi-turbidity and turbidity, and inoculating the supernatant, semi-turbidity and turbidity into the flat plate;

4) Culturing in 37 deg.C constant temperature incubator until the colony in the plate reaches 70-80%, and selecting single colony;

5) The propagation was carried out at 37 ℃ and 170 rpm.

(2) Re-screening:

1) Preparing an LB liquid culture medium, adding zearalenone with the adjusted final concentration of 2 mug/mL, uniformly mixing, and performing shake culture;

2) Selecting a plate streaking method to streak the strain into a re-screening culture medium, culturing under the same culture conditions until an observable single colony grows out, selecting the single colony in an LB liquid culture medium, and carrying out amplification propagation culture at 37 ℃ and 170rpm to obtain the strain with the zearalenone degradation capability: sinorhizobium meliloti CM77 strain.

Example 3

Identification of the strains

(1) Extracting total DNA of the bacterial liquid, using the total DNA as a PCR template of the bacterial liquid, and performing PCR amplification by using primers 27F and 1492R;

(2) The PCR amplification reaction (10. Mu.l) was as follows:

mu.l cDNA, 1. Mu.l 10 XBuffer, 0.8. Mu.l 2.5 mM dNTP, 0.5. Mu.l each of 10. Mu.M forward and reverse primers, 0.05. Mu.l rTaq enzyme, 2H ₂ O 6.15μl；

(3) The sequences of the designed PCR primers are as follows

Name (R)	Sequence (5 '-3')
		27F16s	AGAGTTTGATCCTGGCTCAG
1492R16s	GGTTACCTTGTTACGACTT
		M13(-47)	CGCCAGGGTTTTCCCAGTCACGAC
M13(-48)	AGCGGATAACAATTTCACACAGGA

(4) The PCR reaction conditions are as follows:

5min at 94 ℃; 30 cycles of 94 ℃ 30s,51 ℃ 30s,72 ℃ 1min, 72 ℃ 10min;

(5) After the whole reaction is finished, detecting the reaction product by using 1.0% agarose gel electrophoresis, cutting back a target band under a gel imaging system, and recovering and purifying DNA according to the instruction of the SanPrep column type kit of Shanghai Biotechnology;

(6) Connecting the recovered product with a PMD18-T cloning vector, connecting overnight at 16 ℃ to obtain a target strain gene sequence PMD18-T recombinant vector, wherein the connection system is as follows: the recovered product was 4.5. Mu.l, PMD was 18-T0.5. Mu.l,

(7) Transferring all the ligation products into prepared Escherichia coli competence DH5 alpha, crushing ice bath for 30min, performing heat shock at 42 ℃ for 30s, and placing on ice;

(8) Adding 650 mu L of LB culture medium preheated to 37 ℃, shaking and culturing at 37 ℃ for 1h, then centrifuging at 5000 rpm for 3 min, discarding most of supernatant, blowing and beating the rest 150 mu L of supernatant and precipitate uniformly, coating on an LB plate containing 100 mg/mL ampicillin sodium (Amp), culturing at 37 ℃ for 1h, and then culturing for 12-16h in an inverted manner;

(9) Single colonies growing well on the plate were picked, cultured at 37 ℃ for 6 hours, screened for positive clones with specific primers 27F and 1492R, and the correctly ligated bacterial solution was sent to Hipposony Biometrics for sequencing. Finally, performing Blast comparison on the cDNA sequence of the target strain to determine the strain species;

(10) Wherein, lysine bacillus HQ08 (II)Bacillus lysinosus) The bacillus thuringiensis HQ08 gene is preserved in the common microorganism center of China general microbiological culture Collection center, the address is No. 3 of Xilu No.1 of Beijing Korean-Yang district, the preservation number is CGMCC No.24221, the preservation date is 2021 year, 12 months and 30 days, and the 16s DNA sequence of the bacillus lysinate HQ08 is shown as SEQ ID No. 1;

sinorhizobium meliloti CM77 (Sinorhizobium meliloti) Deposited in China general microbiological culture Collection center (CGMCC) and addressed toNo. 3 of Xilu No.1 of Beijing republic of Chaoyang, the preservation number is CGMCC No.24222, the preservation date is 12 months and 30 days in 2021, and the 16s DNA sequence of the Sinorhizobium meliloti CM77 is shown as SEQ ID NO. 2.

Example 4

Activation of lysine bacillus strain HQ08

(1) Preparing an LB culture medium, wherein the formula of the LB culture medium is as follows: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl;

(2) Inoculating lysine bacillus strain HQ08 into LB culture medium, and culturing at 25-30 deg.C for 8-10 hr to obtain activated lysine bacillus HQ08.

Activation of Sinorhizobium meliloti strain CM77

(1) An LB culture medium is prepared, and the formula of the LB culture medium is as follows: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl;

(2) Inoculating Sinorhizobium meliloti strain CM77 into LB culture medium, and shake-culturing at 30-37 deg.C and 170rpm for 8-10 hr to obtain activated Sinorhizobium meliloti strain CM 77.

Example 5

Preparation of Complex fermentation inoculum 1

(1) Mixing 50g of lysine bacillus HQ08, 50g of alfalfa Sinorhizobium CM77, 200g of trehalose, 100g of glycine, 150g of glycerol and 450g of water to obtain a composite fermentation microbial inoculum 1;

(2) The number of viable bacteria of the lysine bacillus HQ08 in the composite microbial inoculum is 0.5 x 10 ⁹ cfu/g; the viable count of Sinorhizobium meliloti CM77 is 0.5 x 10 ⁹ cfu/g。

Example 6

Preparation of Complex fermentation inoculum 2

(1) Mixing 90g of lysine bacillus HQ08, 10g of sinorhizobium meliloti CM77, 200g of trehalose, 100g of glycine, 150g of glycerol and 450g of water to obtain a composite fermentation inoculant 1;

(2) The number of viable bacteria of the lysine bacillus HQ08 in the composite microbial inoculum is 0.9 x 10 ⁹ cfu/g; the viable count of Sinorhizobium meliloti CM77 is 0.1*10 ⁹ cfu/g。

Example 7

Preparation of Complex fermentation inoculum 3

(1) Mixing 10g of lysine bacillus HQ08, 90g of alfalfa Sinorhizobium CM77, 200g of trehalose, 100g of glycine, 150g of glycerol and 450g of water to obtain a compound fermentation microbial inoculum 3;

(2) The number of viable bacteria of the lysine bacillus HQ08 in the composite microbial inoculum is 0.1 x 10 ⁹ cfu/g; the viable count of Sinorhizobium meliloti CM77 is 0.9 × 10 ⁹ cfu/g。

Example 8

Detecting the effect of the composite fermentation inoculant 1 in reducing the pollution of the aflatoxin B1 and the zearalenone of the feed

(1) Adding aflatoxin B1 and zearalenone into peanut meal, wherein the content of aflatoxin B1 is 300 mug/kg, and the content of zearalenone is 1500 mug/kg;

(2) In the experimental group: mixing 50g of the composite fermentation inoculant 1 with 950g of peanut meal, and then adding 500g of sterilized water; in the control group: mixing 50g of the sterilized composite fermentation inoculant with 950g of peanut meal, and then adding 500g of sterilized water;

(2) After fermenting for 3 days at 30 ℃, detecting the contents of aflatoxin B1 and zearalenone, and detecting the change condition of the water-soluble polysaccharide.

Table 1 degradation effect of composite microbial inoculum on AFB1 and ZEN in peanut meal

Table 2 degrading effect of fermentation of composite microbial inoculum on cellulose in peanut meal

The detection result shows that the content of the aflatoxin B1 is 26.1 mu g/kg and is reduced by 90.8 percent, and the content of the zearalenone toxin is 48.7 mu g/kg and is reduced by 96.6 percent.

Meanwhile, the invention discovers that part of cellulose in the fermented peanut meal is decomposed into water-soluble polysaccharide, wherein the water-soluble polysaccharide content of a control group is 30.5%, while the water-soluble polysaccharide content of an experimental group is 35.2%, so that the nutrient value of the peanut meal is improved by 15.4%.

Example 9

Influence of aflatoxin B1 and zearalenone polluted feed fermented by composite fermentation inoculant 1 on growth of penaeus vannamei

(1) Experimental groups: after the peanut meal with the contents of aflatoxin B1 and zearalenone of 1500 mu g/kg and 7500 mu g/kg respectively is subjected to fermentation treatment by a composite microbial inoculum, the contents of aflatoxin B1 and zearalenone are respectively reduced to 142.5 mu g/kg and 283.5 mu g/kg (the degradation rates are respectively 90.5 percent and 96.3 percent). Then the white fish meal, the shrimp shell meal, the bean pulp, the high gluten flour, the fish oil, the additive and the like which are sieved by a sieve with 80 meshes are mixed according to the proportion in the table 3 to be made into balls. After mixing, the contents of aflatoxin B1 and zearalenone in the feed are respectively 28.5 mu g/kg and 56.7 mu g/kg;

TABLE 3 prawn fodder formulation

Composition (I)	Dosage per kg
		White fish meal	28
Shrimp shell powder	4
		Bean pulp	10
Peanut meal	20
		High gluten flour	34
Fish oil	1
		Additive agent	3
Is totaled	100

(2) Making into strip (diameter 1 mm) with F-26 type twin-screw extruder, oven drying at 55-60 deg.C, cutting into 2-5mm long feed particles, sealing, and drying for storage;

(3) Control group 1: aflatoxin B1 and zearalenone are not added into the peanut meal, and the peanut meal is not fermented by a complex microbial inoculum; control group 2: after aflatoxin B1 and zearalenone are added into the peanut meal, the peanut meal is not fermented by a complex microbial inoculum, and other steps are the same as those of fermentation treatment;

(4) Selecting the penaeus vannamei boone with the average weight of about 5.0g, feeding for 60 days, recording the weight of the penaeus vannamei boone after the feeding is finished, and calculating the weight gain rate of the penaeus vannamei boone, wherein the calculation formula is as follows:

weight gain ratio (WG,%) = (Wt-W0)/W0 × 100.

Wherein: wt: terminal body weight (g), W0: initial body weight (g).

(5) The results of the experiment are shown in table 4:

TABLE 4 weight gain of Penaeus vannamei after feeding different feeds

The experimental results show that the weight gain rate of the control group 2 is 210.7 percent and is reduced by 20.4 percent compared with 264.7 percent of the control group 1, while the weight gain rate of the prawns in the experimental group is 269.3 percent, and has no obvious difference compared with the control group, which indicates that after the feed is fermented by using the compound microbial inoculum, the feed for the prawns polluted by the aflatoxin B1 and the zearalenone can be recovered to a normal level, and the growth of the penaeus vannamei is not negatively influenced.

Example 10

Influence of aflatoxin B1 and zearalenone polluted feed fermented by composite fermentation inoculant 1 on growth of stichopus japonicus

(1) Experimental groups: after the peanut meal with the contents of aflatoxin B1 and zearalenone of 3000 mug/kg and 15000 mug/kg respectively is fermented by a compound microbial inoculum, the contents of aflatoxin B1 and zearalenone are respectively reduced to 295.8 mug/kg and 625.3 mug/kg (the degradation rates are 90.2 percent and 95.9 percent respectively), and the peanut meal is mixed with gulfweed, scallop rim powder, additives and the like which pass through an 80-mesh sieve according to a proportion to prepare clusters; after mixing, the contents of aflatoxin B1 and zearalenone in the feed are 29.6 mug/kg and 62.5 mug/kg respectively.

TABLE 5 Stichopus japonicus feed formula

Composition (I)	Dosage per kg
		Sargassum powder	65
Scallop skirt powder	22
		Peanut meal	10
Additive agent	3
		Total up to	100

(2) Making into strip (diameter 1 mm) with F-26 type twin screw extruder, oven drying at 55-60 deg.C, pulverizing, sieving to obtain 100 mesh feed granule, sealing, drying and storing;

(4) Selecting stichopus japonicus with initial weight of about 8g, feeding for 60 days, recording the quantity and weight of the stichopus japonicus after the feeding is finished, and calculating the weight gain rate of the stichopus japonicus, wherein the calculation formula is as follows:

weight gain ratio (WG,%) = (Wt-W0)/W0 × 100.

Wherein: wt: terminal body weight (g), W0: initial body weight (g).

(5) The results of the experiment are shown in table 6:

TABLE 6 weight gain of Stichopus japonicus after feeding different feeds

The experimental results show that the weight gain rate of the control group 2 is 92.5%, which is reduced by 36.1% compared with the control group 1, while the weight gain rate of the experimental group is 157.9%, which is not obviously different from the control group 1, even slightly higher than the control group 1, which indicates that the feed raw materials polluted by mycotoxins aflatoxin B1 and zearalenone are fermented by using the complex microbial inoculum, and then the feed raw materials can be recovered to the normal level, so that the stichopus japonicus can normally grow and develop.

The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.

Sequence listing

<110> Qingdao agricultural university

<120> composite fermentation inoculant for fermenting polluted feed and application thereof

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<212> DNA

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cttgctgcct gcaggtcgac gattggttac cttgttacga cttcacccca gtcatgaatc 60

acaaagtggt aagcgccctc ccgaaggtta agctacctac ttcttttgca acccactccc 120

atggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc atgctgatcc 180

gcgattacta gcgattccgg cttcatgtag gcgagttgca gcctacaatc cgaactgaga 240

acgactttat cggattagct ccctctcgcg agttggcaac cgtttgtatc gtccattgta 300

gcacgtgtgt agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc 360

cggtttgtca ccggcagtca ccttagagtg cccaactaaa tgatggcaac taagatcaag 420

ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg 480

caccacctgt caccgttgcc cccgaagggg aaactatatc tctacagtgg tcaacgggat 540

gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct ccaccgcttg 600

tgcgggcccc cgtcaattcc tttgagtttc agtcttgcga ccgtactccc caggcggagt 660

gcttaatgcg ttagctgcag cactaagggg cggaaacccc ctaacactta gcactcatcg 720

tttacggcgt ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgcgcctca 780

gcgtcagtta cagaccagaa agtcgccttc gccactggtg ttcctccaaa tctctacgca 840

tttcaccgct acacttggaa ttccactttc ctcttctgca ctcaagtccc ccagtttcca 900

atgaccctcc acggttgagc cgtgggcttt cacatcagac ttaaaggacc gcctgcgcgc 960

gctttacgcc caataattcc ggacaacgct tgccacctac gtattaccgc ggctgctggc 1020

acgtagttag ccgtggcttt ctaataaggt accgtcaagg tacagccagt tactactgta 1080

cttgttcttc ccttacaaca gagttttacg atccgaaaac cttcttcact cacgcggcgt 1140

tgctccatca ggctttcgcc cattgtggaa gattccctac tgctgcctcc cgtaggagtc 1200

tgggccgtgt ctcagtccca gtgtggccga tcaccctctc aggtcggcta cgcatcgtcg 1260

ccttggtgag ccgttacctc accaactagc taatgcgccg cgggcccatc ctatagcgac 1320

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gccgctaacg tcaaaggagc aagctccttt tctgttcgct cgacttgcat gtattaggca 1500

cgccgccagc gttcgtcctg agccaggatc aaactctaat ctctagagga tccccgggta 1560

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tggccgcgtg cttacctgca gtcgaacgat gatgcccagc ttgctgggcg gattagtggc 60

gaacgggtga gtaacacgtg agtaacctgc ccttgacttc gggataactc cgggaaaccg 120

gggctaatac cggatatgag ccgtcctcgc atgggggtgg ttggaaagtt tttcggtcag 180

ggatgggctc gcggcctatc agcttgttgg tggggtgatg gcctaccaag gcgacgacgg 240

gtagccggcc tgagagggcg accggccaca ctgggactga gacacggccc agactcctac 300

gggaggcagc agtggggaat attgcacaat gggcgcaagc ctgatgcagc gacgccgcgt 360

gagggatgaa ggccttcggg ttgtaaacct ctttcagcag ggaagaagcg caagtgacgg 420

tacctgcaga agaagcgccg gctaactacg tgccagcagc cgcggtaata cgtagggcgc 480

aagcgttgtc cggaattatt gggcgtaaag agctcgtagg cggtttgtcg cgtctggtgt 540

gaaaactcga ggctcaacct cgagcttgca tcgggtacgg gcagactaga gtgcggtagg 600

ggagactgga attcctggtg tagcggtgga atgcgcagat atcaggagga acaccgatgg 660

cgaaggcagg tctctgggcc gcaactgacg ctgaggagcg aaagcatggg gagcgaacag 720

gattagatac cctggtagtc catgccgtaa acgttgggca ctaggtgtgg ggctcattcc 780

acgagttccg tgccgcagca aacgcattaa gtgccccgcc tggggagtac ggccgcaagg 840

ctaaaactca aaggaattga cgggggcccg cacaagcggc ggagcatgcg gattaattcg 900

atgcaacgcg aagaacctta ccaaggcttg acatgcacgg gaagccacca gagatggtgg 960

tctctttgga cactcgtgca caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 1020

ttgggttaag tcccgcaacg agcgcaaccc tcgtcccatg ttgccagcgg gttatgccgg 1080

ggactcatgg gagactgccg gggtcaactc ggaggaaggt ggggatgacg tcaaatcatc 1140

atgcccctta tgtcttgggc ttcacgcatg ctacaatggc cggtacaaag ggctgcgata 1200

ccgtaaggtg gagcgaatcc caaaaagccg gtctcagttc ggattggggt ctgcaactcg 1260

accccatgaa gtcggagtcg ctagtaatcg cagatcagca acgctgcggt gaatacgttc 1320

ccgggccttg tacacaccgc ccgtcaagtc acgaaagtcg gtaacacccg aagcccatgg 1380

cccaaccgtt cgcgggggga gtgtcgaagg tcaccggcca 1420

Claims

1. The composite fermentation inoculant is characterized by being a microbial composite inoculant consisting of lysine bacillus HQ08 and Sinorhizobium meliloti CM 77;

the lysine bacillus HQ08 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.24221, the preservation date is 2021 year, 12 months and 30 days, and the 16s DNA sequence of the lysine bacillus HQ08 is shown in SEQ ID NO. 1;

the sinorhizobium meliloti CM77 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.24222 and the preservation date of 2021, 12 months and 30 days, and the 16s DNA sequence of the sinorhizobium meliloti CM77 is shown in SEQ ID No. 2.

2. The composite fermentation inoculant according to claim 1, wherein the lysine bacillus HQ08 is 1-9 parts by weight, and the sinorhizobium meliloti CM77 is 1-9 parts by weight.

3. The composite fermentation inoculant according to any one of claims 2, wherein the lysine bacillus HQ08 is 4-6 parts by weight, and the sinorhizobium meliloti CM77 is 4-6 parts by weight.

4. The complex fermentation inoculant according to any one of claims 1 to 3, wherein the complex inoculant further comprises 5-20 parts of trehalose, 1-10 parts of glycine, 5-25 parts of glycerol and 20-45 parts of sterile water.

5. The composite fermentation bacterial agent of claim 4, wherein the viable count of lysine bacillus HQ08 is not less than 0.5 x 10 ⁹ cfu/g, the viable count of the Sinorhizobium meliloti CM77 is more than or equal to 0.5 x 10 ⁹ cfu/g。