阅读说明：本技术 一种利用病死猪制备的生物酵素及其制备方法 (Biological enzyme prepared from pigs died of diseases and preparation method thereof ) 是由 闫志英 蒋心茹 何光赞 吕青阳 毕列锋 许力山 张云祥 高胜 黄亮 唐颖玲 于 2021-09-29 设计创作，主要内容包括：本发明属于微生物复合菌剂领域,具体涉及一种病死猪生物酵解用酵素及制备方法。具体技术方案为：以死亡动物为发酵底物,先进行3～5天的好氧发酵,再进行3～5天的厌氧发酵。本发明提供的发酵工艺操作简单、能耗低、产物安全、无二次污染,在病死猪无害化处理的基础上实现了资源的回收利用,具有很好的推广前景。(The invention belongs to the field of microbial composite inoculants, and particularly relates to a ferment for biological glycolysis of dead pigs and a preparation method thereof. The specific technical scheme is as follows: and taking dead animals as fermentation substrates, firstly carrying out aerobic fermentation for 3-5 days, and then carrying out anaerobic fermentation for 3-5 days. The fermentation process provided by the invention is simple to operate, low in energy consumption, safe in product and free of secondary pollution, realizes the recycling of resources on the basis of harmless treatment of dead pigs, and has a good popularization prospect.)

1. A biological ferment, which is characterized in that: and taking dead animals as fermentation substrates, firstly carrying out aerobic fermentation for 3-5 days, and then carrying out anaerobic fermentation for 3-5 days.

2. The bioferment according to claim 1, wherein: and (3) adding 1-5% of sugar during anaerobic fermentation.

3. The bioferment according to claim 1, wherein: in the aerobic fermentation, the addition amount of the aerobic microbial agent is 1-5% by volume; the concentration of viable microorganism in aerobic microbial agent is 1 × 10⁸～1×10⁹CFU/g。

4. The bioferment according to claim 3, wherein: the aerobic microorganism includes: pseudomonas aeruginosa, bacillus amyloliquefaciens and bacillus subtilis.

5. The bioferment according to claim 4, wherein: according to the viable bacteria ratio, the pseudomonas aeruginosa: b, bacillus amyloliquefaciens: 1-2 parts of bacillus subtilis: 2-3: 1 to 3.

6. The bioferment according to claim 1, wherein: in the anaerobic fermentation, the addition amount of the anaerobic microbial agent is 1-5% by volume; the concentration of viable microorganism in the anaerobic microbial agent is 1 × 10⁸～1×10⁹CFU/g。

7. The bioferment according to claim 6, wherein: the anaerobic microorganism includes: lactobacillus plantarum and lactobacillus casei.

8. The bioferment according to claim 7, wherein: according to the viable bacteria ratio, the lactobacillus plantarum: lactobacillus casei ═ (3-5): (1-3).

9. The bioferment according to any one of claims 1 to 8, wherein: the dead animals are pigs dead of diseases.

10. A method for preparing the bioferment according to any one of claims 1 to 9, wherein: the method comprises the following steps:

(1) pretreatment: crushing dead animals, sterilizing the crushed dead animals at high temperature and high pressure, and after sterilization, treating the dead pigs according to the mass ratio: adding sterile water in a ratio of 1: 1;

(2) aerobic fermentation: adding aerobic microorganisms according to the volume ratio of 1-5%, and fermenting for 3-5 days;

(3) anaerobic fermentation: adding anaerobic microorganisms according to the volume ratio of 1-5%, and fermenting for 3-5 days.

Technical Field

The invention belongs to the field of microbial composite inocula, and particularly relates to a biological enzyme prepared from dead pigs and a preparation method thereof.

Background

In the live pig breeding, the death rate of the pigs is about 5 to 10 percent; in the face of such huge dead pigs, if timely, effective and scientific treatment is not carried out, epidemic diseases can be spread, and great threats are brought to public health environment and livestock and poultry safety.

The harmless treatment of the pigs died of illness is a process of decomposing dead pig bodies of illness and killing viruses carried by the pig bodies by a physical, chemical or biological method. At the present stage, the common method in China comprises the following steps: 1. deep burying method. The method is most commonly used, and utilizes soil isolation to prevent virus diffusion, and utilizes natural corrosion to digest dead pig carcasses. The method can treat a large number of pigs died of illness at one time, has low cost and simple operation, but has a plurality of hidden dangers and defects, and is easy to cause secondary environmental pollution. 2. And (3) an incineration method. The method can thoroughly eliminate pathogenic bacteria, has short time consumption, but generates a large amount of unpleasant gas in the incineration process, has large energy consumption, and can not recycle pigs died of diseases. 3. The chemical preparation method is characterized in that the dead pig bodies are treated by steam pressure or dry heat pressure in a closed container, and the chemical preparation method has the advantages of short treatment time, large treatment capacity, simple treatment and the like; but the cost is high, and foul gas and sewage are easy to generate. 4. The composting method is that dead pig carcasses and auxiliary materials are mixed, aerobic microorganisms are used for fermenting and decomposing organic matters, and viruses are killed through high temperature generated by fermentation. The method has the advantages of low cost, simple operation and good sterilization effect, the compost product can be used as an organic fertilizer, but the treatment period is often as long as several months, and a large amount of malodorous gas can be generated in the treatment process.

If a new method for harmless treatment and high-efficiency utilization of dead pigs can be provided, the method has important practical significance.

Disclosure of Invention

The invention aims to provide a ferment for biological glycolysis of dead pigs and a preparation method thereof.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a biological ferment is prepared from dead animals as fermenting substrate through aerobic fermentation for 3-5 days, and anaerobic fermentation for 3-5 days.

Preferably, 1-5% of sugar is added in the anaerobic fermentation.

Preferably, in the aerobic fermentation, the addition amount of the aerobic microbial agent is 1-5% by volume; the concentration of viable microorganism in the aerobic microbial agent is 1 multiplied by 108 to 1 multiplied by 109 CFU/g.

Preferably, the aerobic microorganisms include: pseudomonas aeruginosa, bacillus amyloliquefaciens and bacillus subtilis.

Preferably, the ratio of viable bacteria of pseudomonas aeruginosa: b, bacillus amyloliquefaciens: 1-2 parts of bacillus subtilis: 2-3: 1 to 3.

Preferably, in the anaerobic fermentation, the addition amount of the anaerobic microbial agent is 1-5% by volume; the concentration of viable microorganism in the anaerobic microbial agent is 1 multiplied by 108 to 1 multiplied by 109 CFU/g.

Preferably, the anaerobic microorganism includes: lactobacillus plantarum and lactobacillus casei.

Preferably, the ratio of viable bacteria of lactobacillus plantarum: lactobacillus casei ═ (3-5): (1-3).

Preferably, the dead animal is a pig that is dead of illness.

Accordingly, a method for preparing the bioferment comprises the following steps:

(1) pretreatment: crushing dead animals, sterilizing the crushed dead animals at high temperature and high pressure, and after sterilization, treating the dead pigs according to the mass ratio: adding sterile water in a ratio of 1: 1;

(2) aerobic fermentation: adding aerobic microorganisms according to the volume ratio of 1-5%, and fermenting for 3-5 days;

(3) anaerobic fermentation: adding anaerobic microorganisms according to the volume ratio of 1-5%, and fermenting for 3-5 days.

The invention has the following beneficial effects:

the invention adopts high-protein-resistant probiotic strains, hydrolyzes pork into amino acid, soluble protein and the like through aerobic microbial fermentation, and finally obtains the animal ferment which is rich in various probiotic components such as various enzyme systems (protease, lipase and the like), polypeptide, amino acid, micromolecular acid and the like and can stimulate the growth and development of crops through anaerobic microbial fermentation. The fermentation end product has various active ingredients with rich varieties and high content, and can be stored at normal temperature for a long time without adding preservatives; can promote the growth of plants, improve the quality of crops and improve the micro-ecological environment of soil.

Specifically, the invention relates to a high-protein and high-fat resistant strain screened from various soil probiotic types. In the anaerobic fermentation, sugar is added in order to cooperate with the microorganism in the compound microbial inoculum B to play a role, so that the final product obtains more abundant micromolecular acid and longer shelf life.

The fermentation process provided by the invention is simple to operate, low in energy consumption, safe in product and free of secondary pollution, realizes the recycling of resources on the basis of harmless treatment of dead pigs, and has a good popularization prospect.

Drawings

FIG. 1 is a schematic diagram showing the change of soluble protein after pork is fermented by different aerobic microorganisms;

FIG. 2 is a schematic diagram of the solid content of pork fermented by different aerobic microorganisms;

FIG. 3 is a schematic diagram of the acid-producing capacity of different anaerobic microorganisms;

FIG. 4 is a graph showing the pH change in fermentations of groups 4, 21, 23, 24 and the blank control of Table 5.

Detailed Description

The invention provides a microbial compound microbial inoculum for glycolysis of pigs died of diseases. The microbial compound bacteria agent consists of a compound bacteria agent A and a compound bacteria agent B.

The compound microbial inoculum A is a microorganism used for aerobic fermentation, and comprises the following components: pseudomonas aeruginosa, bacillus amyloliquefaciens and bacillus subtilis. The preferable scheme is as follows: according to the viable bacteria ratio, the pseudomonas aeruginosa: b, bacillus amyloliquefaciens: 1-2 parts of bacillus subtilis: 2-3: 1-3; the viable bacteria of pseudomonas aeruginosa, bacillus amyloliquefaciens and bacillus are all 1 multiplied by 10⁸～1×10⁹CFU/g。

The more preferable scheme is as follows: the Pseudomonas aeruginosa (Pseudomonas aeruginosa) is Pseudomonas aeruginosa X7 and is preserved in China general microbiological culture Collection center (CGMCC); the address of the depository: the preservation number of the Xilu No.1 Hospital No. 3 of Beijing Chaoyang district is: CGMCC No. 8983. The culture temperature of the pseudomonas aeruginosa is 25-42 ℃, and the optimal growth temperature is 25-30 ℃.

The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is Bacillus amyloliquefaciens JX-6 and is preserved in China general microbiological culture Collection center (CGMCC); the address of the depository: the preservation number of the Xilu No.1 Hospital No. 3 of Beijing Chaoyang district is: CGMCC No. 13715. Vegetative cells of the strain in an LB culture medium are rod-shaped, gram-positive, 2-3 mu m long and 0.7-0.9 mu m wide; the bacterial colony is cultured on a beef extract peptone agar culture medium in a flat plate mode, the surface of the bacterial colony is convex, smooth and wrinkle-free, the bacterial colony is transparent, the edge of the bacterial colony is regular and circular, the viscosity of the bacterial colony is large, and the bacterial colony is in a filamentous shape when being picked up. The culture temperature of the bacillus amyloliquefaciens is 30-37 ℃.

The Bacillus subtilis (Bacillus subtilis) is Bacillus subtilis 8-2, and the preservation number is as follows: CGMCC No. 17215. The strain can grow in a large amount in 1d after being cultured at 25-35 ℃ on a beef extract peptone agar medium plate and an E-type fermentation medium agar plate, the surface of a bacterial colony is convex, the surface of the bacterial colony is not smooth and has wrinkles, the bacterial colony is white and translucent, the edge of the bacterial colony is irregular and circular, the surface of the bacterial colony has transparent liquid drops, the viscosity of the bacterial colony is high, and the bacterial colony is in a filamentous shape after being picked up. The culture temperature of the bacillus subtilis is 28-35 ℃.

The compound microbial inoculum B is a microorganism used for anaerobic fermentationAn organism, comprising: lactobacillus plantarum and lactobacillus casei; the microorganisms used are facultative anaerobes. The preferable scheme is as follows: the lactobacillus plantarum consists of lactobacillus plantarum and lactobacillus casei. The preferable scheme is as follows: according to the viable bacteria ratio, the lactobacillus plantarum: lactobacillus casei ═ (3-5): (1-3); the viable bacteria of Lactobacillus plantarum and Lactobacillus casei are both 1 × 10⁸～1×10⁹CFU/g。

The lactobacillus casei can be obtained by commercial or self-screening. The Lactobacillus plantarum is Lactobacillus (preferably a microorganism preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation date of 2012, 9 and 3 days and the preservation number of CGMCC No. 6495. When the strain is observed under a microscope, the strain cells are short rod-shaped, the surfaces of bacterial colonies are raised, the bacterial colonies are milky white, the surfaces are rough, and the edges are irregular. Colonies turned yellow, gram positive with increasing incubation time. The optimal growth pH is 6.5-7, and the growth cannot be carried out in 6.5% NaCl. The strain does not produce indole, does not hydrolyze starch, does not utilize citrate, does not reduce nitrate, and does not produce H₂S, VP test is negative. Glucose, fructose, sucrose, xylose, arabinose, mannitol, lactose, maltose are used. The culture temperature of the lactobacillus plantarum is 30-35 ℃.

The invention also provides a preparation process of the microbial compound inoculant, which comprises the following steps:

1. culturing each microorganism in the composite microbial inoculum A to 1 multiplied by 10⁸～1×10⁹CFU/g, and uniformly mixing according to the proportion;

2. culturing each microorganism in the compound bacterial agent B to 1 × 10⁸～1×10⁹CFU/g, and mixing in proportion.

The invention also provides an application method of the microbial compound inoculant in the glycolysis of pigs died of diseases, which comprises the following steps:

1. pretreatment: mechanically crushing the dead pigs to 1-2 cm; and then sterilizing the dead pigs after crushing treatment at high temperature and high pressure, wherein the sterilization conditions are preferably as follows: sterilizing at 134 deg.c and 0.1-0.2 MPa for 90 min. After the sterilization is finished, pigs died of diseases according to the mass ratio: sterile water 1:1 sterile water was added.

2. Aerobic fermentation: adding the compound microbial inoculum A according to 1-5% (V/V), fermenting for 3-5 days, preferably fermenting until the solid content is reduced to below 30%.

3. Anaerobic fermentation: adding the compound microbial inoculum B according to 1-5% (V/V), fermenting for 3-5 days, preferably fermenting until the pH value is reduced to 5.0 or below.

The preferable scheme is as follows: in the anaerobic fermentation, 1 to 5 percent of sugar is added according to the mass ratio; glucose is preferably added.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The examples relate to the media and reagents as follows:

(1) beef extract peptone medium: 2g of yeast powder, 5g of beef extract, 10g of peptone and 5g of sodium chloride, wherein the pH value is 7.0, water is supplemented to 1000mL, and the sterilization conditions are as follows: sterilizing at 121 deg.C for 20 min.

(2) The MRS culture medium has water as solvent, the following solutes and concentrations: 5g of yeast powder, 10g of peptone, 8g of beef extract, 20g of glucose, 5g of sodium acetate, 2g of diammonium citrate, 801 mL of Tween and K₂HPO₄ 2g、MgSO₄·7H₂O 0.2g、CaCO₃ 16g、MnSO₄·H₂0.05g of O, 6.3-6.8 of pH, supplementing water to 1000mL, and sterilizing under the following conditions: sterilizing at 121 deg.C for 20 min.

The first embodiment is as follows: screening of microorganisms in complex microbial inoculum

1. And (4) selecting microorganisms in the compound microbial inoculum A.

(1) Screening for the ability to degrade proteins. Setting 8 treatment groups, wherein each group of lean pork of pigs died of illness is crushed to 50 meshes by a meat grinder according to the mass ratio of pork to water of 1:1, adding sterilized water, and sterilizing the crushed materials at 133 ℃ for 90min by using high temperature and high pressure. Centrifuging at 10000 r 4 deg.C, removing upper fat solid, making the rest into 200g system,sterilizing at 121 deg.C for 20min to obtain pork culture medium. Respectively adding bacillus amyloliquefaciens (bacterium 1 in figure 1), bacillus thuringiensis (bacterium 2 in figure 1), paenibacillus polymyxa (bacterium 3 in figure 1), bacillus subtilis (bacterium 4 in figure 1), pseudomonas aeruginosa (bacterium 5 in figure 1), saccharomyces cerevisiae (bacterium 6 in figure 1), candida utilis (bacterium 7 in figure 1), lactic acid bacteria (bacterium 8 in figure 1) 8 microorganisms (wherein the pseudomonas aeruginosa is CGMCC NO.8983, the bacillus amyloliquefaciens is CGMCC NO.13715 and the bacillus subtilis is CGMCC NO.17215) into each group of pork culture medium; all microorganisms are added in the form of microorganism culture solution, and the viable bacteria concentration in the culture solution is 3 × 10⁸CFU/mL, the volume of broth addition was 5% of the total fermentation substrate volume. Simultaneously setting a blank group (adding the same amount of sterile water) for aerobic fermentation. After 3 days of aerobic fermentation, soluble protein in each group of pork material was measured. Soluble protein assay methods: using BCA protein concentration determination kit; increase and decrease (soluble protein content in pork after 3 days of fermentation-soluble protein content in pork before fermentation)/soluble protein content in pork before fermentation. The increase and decrease of soluble protein in each group of materials are shown in FIG. 1. The results show that: bacteria 1 (bacillus amyloliquefaciens), bacteria 4 (bacillus subtilis), bacteria 5 (pseudomonas aeruginosa) and bacteria 8 (lactic acid bacteria) can obviously increase the soluble protein of the material.

After 8 days of aerobic fermentation, the solid content in the materials of the bacterium 1 group, the bacterium 4 group, the bacterium 5 group and the bacterium 8 group which increase the soluble protein of the material is determined. The solid content determination method comprises the following steps: after fermentation of each group, the bottles are disassembled, centrifugation is carried out at 10000 r and 4 ℃, solid and liquid are separated, and the solid content is equal to the weight of the solid/the total weight. The results are shown in FIG. 2. The results show that: the lowest solid content is the bacterium 4 group, and is reduced to 29.43 percent; the strain 1 group is reduced to 32.70%, and the strain 5 group is reduced to 35.38%. Shows that the bacteria 1 (bacillus amyloliquefaciens), the bacteria 4 (bacillus subtilis) and the bacteria 5 (pseudomonas aeruginosa) can grow and propagate on a high-protein pork culture medium, and the hydrolysis effect is better than that of other microorganisms. In addition, the strain 7 (Candida utilis) can synthesize protein, and after a certain period of fermentation, the strain 7 synthesizes free amino acids in the culture medium into protein solids, so that the solid content tends to increase.

After 8 days of aerobic fermentation, the change of the amino acid content in the supernatant of the pork culture medium was measured, and the results are shown in Table 1. The data in Table 1 are in mg/mL.

TABLE 1 comparison table of amino acid content change after fermentation of pork culture medium by each microorganism

Name (R)	Blank space	Bacterium 1	Bacterium 4	Bacterium 5	Bacterium 7	Bacterium 8
							Cysteic acid (Cya)	0.015	0.024	0.024	0.038	0.021	0.018
L-aspartic acid (Asp)	0.038	0.217	0.197	0.176	0.002	0.022
							L-threonine (Thr)	0.037	0.020	0.270	0	0.001	0.051
L-serine (Ser)	0.036	0.074	0.116	0.383	0	0.057
							L-glutamic acid (Glu)	0.054	1.189	1.210	0.914	0	0.056
Glycine (Gly)	0.077	0.200	0.427	0.266	0	0.028
							L-alanine (Ala)	0.169	1.168	1.059	0.517	0.010	0.058
L-cystine ((Cys)2)	0.056	0.206	0.267	0.215	0.076	0.058
							L-valine (Val)	0.046	1.882	1.066	0.565	0	0.093
L-methionine (Met)	0.015	1.523	1.052	0.432	0.007	0.058
							L-isoleucine (Ile)	0.015	1.956	0.996	0.449	0.001	0.080
L-leucine (Leu)	0.031	3.215	1.990	0.91	0.003	0.128
							L-tyrosine (Tyr)	0.009	1.112	1.066	0.505	0.001	0.045
L-phenylalanine (Phe)	0.018	2.102	1.366	0.541	0.003	0.083
							L-histidine (His)	0.188	1.137	0.854	0.325	0.135	0.228
L-lysine (Lys)	0.034	0.186	2.303	0.912	0.010	0.085
							L-arginine (Arg)	0	0.149	0.151	0	0.003	0.021
L-proline (Pro)	0.081	0.790	0.364	0.244	0.054	0.060
							Total amount (mg/mL)	0.919	17.15	14.778	7.392	0.461	1.229

The results show that: the total content of amino acids in the culture medium is the highest after the fermentation of the bacteria 1 and 4. Meanwhile, by combining the increase condition of the soluble protein after fermentation and the result of the solid content rate, the bacteria 1 and the bacteria 4 can hydrolyze the sterilized pork solid into the soluble protein and the amino acid.

(2) Screening high grease resistance. The pork culture medium is added with pig large intestines with different mass ratios (10%, 20% and 100%) to prepare culture media with different grease contents. The 8 types of microorganisms in step (1) were streaked on each of the above-mentioned media, and the strains that grew normally were marked as "√" and those that did not grow normally were marked as "×", and the results are shown in Table 2.

TABLE 2 illustrative table of results of grease resistance of each microorganism

As a result, only bacteria 2 and 5 can grow normally in all media, and thus bacteria 2 and 5 have high fat tolerance.

And (3) combining the results of the steps (1) and (2), and adopting the bacteria 1, the bacteria 4 and the bacteria 5 to form a composite microbial inoculum A.

3. And (4) selecting microorganisms in the compound microbial inoculum B. In the agricultural animal ferment system, lactic acid bacteria generate organic acids such as lactic acid, acetic acid, amino acid and the like through self metabolism, and the acidic substances have chelation, acid dissolution and bacteriostasis functions, can activate mineral nutrients in soil, can obviously reduce the pH value and Eh (oxidation-reduction potential) value of the environment, and has better inhibition effect on some putrefying bacteria and low-temperature bacteria, thereby prolonging the shelf life of the agricultural animal ferment. Therefore, the strength of lactic acid and acetic acid production capacity is used as an index for screening microorganisms in the compound microbial inoculum B.

Seven kinds of lactic acid bacteria (commercially available lactobacillus sporogenes, lactobacillus plantarum 1: CGMCC No.6495, commercially available lactobacillus plantarum 2, commercially available lactobacillus casei, commercially available lactobacillus plantarum 3, commercially available lactobacillus paracasei and commercially available lactobacillus plantarum 4) in a laboratory are activated by an MRS culture medium, then respectively inoculated into a new MRS culture medium (liquid), subjected to shake cultivation at 35 ℃ and 150rpm for 24 hours, and the content of lactic acid and acetic acid in each group of culture medium is measured by a liquid chromatograph. The results are shown in FIG. 3. Selecting bacteria C (CGMCC No.6495) and D (Lactobacillus casei) with higher lactic acid and total acid content to form a composite microbial inoculum B.

Example two: preparation of microbial composite inoculant and effect display

1. Preparing the complex microbial inoculum A. The microorganisms in the compound microbial inoculum A are activated and cultured by using a beef extract peptone culture medium until the viable bacteria concentration is 3 multiplied by 10⁸CFU/mL. Complex microbial inoculum A was prepared in the manner shown in Table 3. Each numerical value in table 2 is a volume ratio (i.e., viable bacteria ratio) of each microbial liquid; for example, group 4 refers to pseudomonas aeruginosa: b, bacillus amyloliquefaciens: bacillus subtilis 1:2: 2. Wherein, the commercially available pseudomonas aeruginosa is from shanghai auspicious biotechnology limited (ATCC 15442); bacillus amyloliquefaciens purchased in market is from Weifang Rui Bio-technology Co., Ltd; commercially available Bacillus subtilis is available from Shanghai Fuxiang Biotech, Inc. (ACCC 10627).

TABLE 3 composition comparison table of complex bacteria A of each group

And (3) fermenting the lean meat of the pigs died of the disease by using each group of the compound microbial inoculum A. The method comprises the following specific steps: crushing lean meat of pigs died of illness to 50 meshes by a meat grinder, and sterilizing the crushed material at 133 ℃ for 90min by high temperature and high pressure. According to the mass ratio of materials to water of 1:1, adding sterilized water, performing aerobic fermentation process (500 mL conical bottle is used for containing a sample, aeration is performed through a vent plug, the sample is cultured at 35 ℃ and 150rpm of a shaking table, the subsequent aerobic fermentation process is the same as the above process), adding the composite microbial inoculum A accounting for 5 percent of the total volume of the fermentation materials, and fermenting for 3 days at 35 ℃ and 150 rpm. Meanwhile, equal amount of sterile clear water is added into the materials to replace each group of the compound bacteria agent A, and the mixture is used as a blank control group. After 3 days of fermentation, the change in soluble protein in each group was measured and the results are shown in Table 4.

Table 4 table for showing fermentation conditions of each group of complex microbial agents a

Group of	Soluble protein changes
		Group 1	+25％
Group 2	+23％
		Group 3	+23％
Group 4	+26％
		Group 5	+25％
Group 6	+20％
		Group 7	+28％
Group 8	+25％
		Group 9	+29％
Group 10	+23％
		Group 11	+28％
Group 12	+30％
		Group 13	+28％
Group 14	+22％
		Group 15	+20％
Blank control group	\

2. And (4) preparing a compound microbial inoculum B. The microorganisms in the compound bacterial agent B are cultured by using MRS culture medium until the concentration of viable bacteria is 3 multiplied by 10⁸CFU/mL. Complex inoculant B was prepared as in table 5. Each numerical value in table 2 is a volume ratio (i.e., viable bacteria ratio) of each microorganism. In Table 5, Lactobacillus plantarum 1 is CGMCC No. 6495; lactobacillus plantarum 2, 3, 4 were all commercially available. The numerical values in Table 5 represent the volume ratios (i.e., viable cell ratios) of the microbial liquids.

TABLE 5 composition comparison table of each group of composite bacterial agent B

3. Crushing lean meat of pigs died of illness to 50 meshes by a meat grinder, and sterilizing the crushed material at 133 ℃ for 90min by high temperature and high pressure. According to the mass ratio of materials to water of 1:1, adding sterilized water, performing aerobic fermentation process, adding the compound microbial inoculum A, and performing aerobic fermentation at 35 ℃ and 150 rpm. After the aerobic fermentation is finished, adding the compound microbial inoculum B, and carrying out anaerobic fermentation at 35 ℃. Or simultaneously adding the composite microbial inoculum A, B, performing aerobic fermentation at 35 ℃ and 150rpm, and performing anaerobic fermentation at 35 ℃. The complex microbial inoculum A used in the step is group 4 in the step 1.

The anaerobic fermentation specifically comprises the following steps: the operation is carried out on an ultraclean workbench, the air-permeable plug is replaced by a rubber plug (air-impermeable), and the connection part of the bottle opening and the plug is sealed by a sealing film (oxygen removal operation is not required to be carried out separately because the microorganism in the added composite microbial inoculum B is facultative anaerobe). Then placing the treated conical flask into an incubator at 35 ℃ for anaerobic fermentation, and shaking up once at a fixed point every day until the fermentation is finished.

In addition, the problem group of the inventor finds in experiments that: the pH value of the product obtained by fermenting pork in two steps approaches to neutral and is difficult to store for a long time. If sugar is added during fermentation, the generation of organic acid can be promoted, and the pH value of the final product is reduced, so that the shelf life of the product is prolonged.

Specifically, the addition of the complex microbial agents A, B and sugars in each group is shown in table 6. In table 6, each numerical value represents a volume ratio to a fermentation substrate.

TABLE 6 comparison table of complex microbial inoculum for fermentation of each group

4. The fermentation effect of each group is shown in table 7. The change in pH during fermentation for representative groups 4, 21, 22, 23, 24 of table 6 is plotted in fig. 4. Note that the maximum freshness keeping time shown in table 7 is 6 months because the inventors have verified that the freshness keeping time is only 6 months at present, and the non-freshness keeping time is only 6 months. In fact, the materials corresponding to the group with the preservation time of more than 6 months are still in a state of uniform properties and no peculiar smell when the group is 6 months, and the state difference from the state at the end of fermentation is not large. In addition, the inventors have found that although the addition of sugar can lower the pH, if sugar is added in the aerobic fermentation stage, or sugar is added to the fermentation substrate first and then the subsequent operation is performed, the microorganism tends to ferment sugar first instead of the fermentation substrate such as a dead pig, and the final pH decrease degree, soluble protein change and other results are not as good as the "addition of sugar in the anaerobic fermentation stage" treatment method.

TABLE 7 comparison table of fermentation effect of each group

The amount of each free amino acid in group 4 of Table 6 (total free amino acid content of 1.02mg/mL in the original pork culture medium, which had been ground and sterilized before any microorganism had not been inoculated) was determined to be the best results, as shown in Table 8.

TABLE 8 comparison of the free amino acid content in the material at the end of fermentation in treatment group 4

Serial number	Amino acids	Content (mg/mL)
			1	Cya	0.059
2	Asp	0.747
			3	Thr	1.450
4	Ser	0.227
			5	Glu	3.539
6	Gly	1.050
			7	Ala	2.723
8	(Cys)2	0.418
			9	Val	2.459
10	Ile	3.638
			11	Leu	5.720
12	Tyr	1.101
			13	Phe	2.944
14	His	1.912
			15	Lys	3.121
16	NH4	0.989
			17	Arg	2.334
18	Pro	0.358
			19	Met	1.952
Total up to	\	36.74

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes, modifications, alterations, and substitutions which may be made by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.