CN112522349A

CN112522349A - Microbial flocculant for clarifying brewed vinegar and preparation method and application thereof

Info

Publication number: CN112522349A
Application number: CN202011518642.2A
Authority: CN
Inventors: 戴德慧; 胡伟莲
Original assignee: Zhejiang Lover Health Science and Technology Development Co Ltd
Current assignee: Zhejiang Lover Health Science and Technology Development Co Ltd; Zhejiang University of Science and Technology ZUST
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2021-03-19

Abstract

A microbial flocculant for clarifying brewed vinegar and a preparation method and application thereof belong to the technical field of microbial engineering. The method comprises the following steps: 1) inoculating Paenibacillus chestnuts to a test tube slant culture medium for activation, washing with sterile water to obtain a bacterial suspension, inoculating for seed culture, and performing microscopic examination to obtain no mixed bacteria for later use; 2) performing fermentation culture in a fermentation tank to obtain a culture solution; 3) extracting and purifying to obtain the microbial flocculant. The microbial flocculant prepared by the method can be applied to clarifying and brewing table vinegar. The microbial flocculant produced by the method has good edible safety and a wide pH application range, the extracellular polysaccharide content in the fermentation culture solution prepared by the method is more than 3.57 percent, the mucopolysaccharide content accounts for more than 90 percent, the light transmittance of the vinegar can be increased by 240.63 percent, and the microbial flocculant can be applied to clarification of brewed vinegar.

Description

Microbial flocculant for clarifying brewed vinegar and preparation method and application thereof

Technical Field

The invention belongs to the technical field of microbial engineering, and particularly relates to a microbial flocculant for clarifying brewed vinegar, and a preparation method and application thereof.

Background

The flocculant is widely applied to industrial processes such as wastewater treatment, drinking water purification, food and fermentation processing. Flocculants can be divided into inorganic flocculants such as aluminium salts (aluminium sulphate and polyaluminium chloride)) Chemical synthetic flocculants (derivatives of polyacrylamide and polyethylene) and biological flocculants. Among these flocculants, organic synthetic polymeric flocculants are widely used in practical production because of their good flocculation effect and low cost. Polyacrylamide is a common one, but has difficult degradability, and evidence shows that acrylamide monomers are strong carcinogens and have strong neurotoxicity, and the acrylamide monomers remaining in the environment are easy to cause secondary pollution. Many countries currently ban or restrict the use of such flocculants. The use of inorganic flocculant can bring a large amount of inorganic ions into treatment liquid in food and fermentation industries, and the excessive inorganic ions not only influence the flavor and taste of products, but also are not beneficial to human health, especially Al³⁺Has a direct relationship with the increasing initiation of the senile dementia at present; fe³⁺Has strong corrosivity and easy residue, and can make the water band to be treated have color and influence the water quality. Becomes a new problem for disposal, regeneration and reuse. Although natural polymeric flocculants such as chitosan, sodium alginate and chitin in the biological flocculant are nontoxic and can be safely degraded, the flocculation activity is weak, the cost is high, and the wide application of the biological flocculant is limited. The microbial flocculant is a special high-molecular polymer which is produced by microorganisms in the growth process and can cause aggregation and precipitation of solid suspended particles, thallus cells, colloid particles and the like which are not easy to degrade in a water body. The flocculant is easy to separate, high in settling efficiency, degradable, nontoxic and harmless to the environment as a degradation product, free of secondary pollution, wide in application range, good in turbidity removal and decoloration effects, and efficient and environment-friendly. Because the growth speed of the microorganism is high, industrialization is easy to realize by adopting methods such as fermentation engineering and the like. Accordingly, bioflocculants derived from microorganisms are receiving increasing attention. Microbial flocculants are expected to be applied more and more in various fields as a promising substitute of chemical flocculants.

Vinegar has been widely used as an acidic condiment throughout the world for thousands of years. They are rich in nutrients and bioactive compounds, including amino acids, sugars, organic acids, polyphenols, melanin, and tetramethylpyrazine. These functional compounds not only can increase the flavor of vinegar, but also play an important role in preventing and treating human diseases, such as regulating blood lipid level, reducing weight, resisting fatigue, resisting tumor, etc. In the whole brewing process of the vinegar, because macromolecular substances such as starch, cellulose, protein, fat and the like in the raw materials are not thoroughly decomposed, the vinegar is turbid, precipitates are formed, and the quality of the vinegar is influenced, the vinegar is clarified before leaving the factory, otherwise, the components are gradually aggregated and form precipitates again in the storage process. At present, the clarification method of vinegar mainly comprises a flocculation method, a membrane filtration method and an enzyme clarification method. Among these methods, the flocculation method is widely used because of its simple process and low cost. In the clarification process of vinegar, many flocculants are reported, such as bentonite, chitosan, egg white and the like. However, the application and research of the microbial flocculant in vinegar clarification are not reported.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide a fermentation process of a bacterial strain for producing biological flocculant with high yield, and apply the fermentation process to clarification process treatment in the actual vinegar production process. The microbial flocculant is prepared by chestnut tree paenibacillus slant culture, primary seed culture, fermentation culture solution preparation, CPC complexation-alcohol precipitation extraction and purification, and is applied to vinegar clarification treatment process.

A preparation method of a microbial flocculant for clarifying brewed vinegar is characterized by comprising the following steps:

1) inoculating Paenibacillus chestnuts to a test tube slant culture medium for activation, washing with sterile water after activation to obtain bacterial suspension, inoculating to a triangular flask containing a seed culture solution for seed culture, and performing microscopic examination to obtain no mixed bacteria for later use;

2) putting the fermentation medium into a fermentation tank, carrying out autoclaving at 121 ℃ for 20 minutes, then inoculating the seeds obtained in the step 1), and carrying out fermentation culture to obtain a culture solution;

3) extracting and purifying the culture solution obtained in the step 2) to obtain the microbial flocculant.

The preparation method of the microbial flocculant for clarifying brewed vinegar is characterized in that a slant culture medium in the step 1) comprises the following components: 2.0% of soluble starch, 0.5% of peptone, 0.05% of potassium chloride, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate and 2.0% of agar, and the activation culture method comprises the following steps: adjusting pH to 7.0, autoclaving test tube with slant culture medium at 121 deg.C for 20 min, inoculating Paenibacillus chestnut, and culturing at 28 deg.C for 48 hr.

The preparation method of the microbial flocculant for clarifying brewed vinegar is characterized in that the seed culture solution in the step 1) comprises the following components: 1.0% of soluble starch, 1.5% of sucrose, 0.5% of peptone, 0.05% of potassium chloride, 0.1% of dipotassium hydrogen phosphate and 0.05% of magnesium sulfate, and the seed culture method comprises the following steps: the triangular flask containing the seed culture solution was autoclaved at 121 ℃ for 20 minutes, and then the bacterial suspension was inoculated and cultured at 28 ℃ for 30 hours.

The preparation method of the microbial flocculant for clarifying brewed vinegar is characterized in that a fermentation medium in the step 2) comprises the following components: 3.0% of glucose, 0.1% of sodium nitrate, 0.3% of peptone, 0.05% of potassium chloride, 0.1% of dipotassium hydrogen phosphate and 0.05% of magnesium sulfate.

The preparation method of the microbial flocculant for clarifying brewed vinegar is characterized in that the fermentation culture method in the step 2) is as follows: the culture temperature is 28 ℃, the ventilation rate is 1VVM, the initial pH is 7.0, the initial stirring rotation speed is 300 r/min, the later stirring rotation speed is linked with dissolved oxygen, the lowest dissolved oxygen is controlled to be 20%, the culture time is 36 hours, the OD600 is more than 2.5, 3.0% (W/V) of cane sugar in the total volume of the fermentation liquid is added, the mixture is uniformly stirred, the mixture is placed in a culture dish, the liquid depth is controlled not to exceed 2.5cm, the culture is continued for 24 hours at 26 ℃, until the culture liquid is in a jelly shape, the charging amount of the fermentation tank is 70%, and the inoculation amount is 3.0%.

The preparation method of the microbial flocculant for clarifying brewed vinegar is characterized in that the microbial flocculant mainly comprises mucopolysaccharide, and the molecular structure of the microbial flocculant contains glucosamine and uronic acid.

The microbial flocculant obtained by the preparation method.

The microbial flocculant is applied to clarifying and brewing vinegar.

The application is characterized in that the application specifically comprises the following steps: heating brewed edible vinegar to 41.5 deg.C, adding CaCl₂To a final concentration of 0.32mM, and then microbial flocculant was added to a final concentration of 3.94 mg/L.

The method provided by the invention is novel and easy to operate, and the produced microbial flocculant has good edible safety and a wide pH application range, is applicable to the pH range of 2.0-9.0, and has important practical value in industrial processes such as brewing food of table vinegar and the like, drinking water purification, wastewater treatment and the like. The fermentation culture solution prepared by the method has the extracellular polysaccharide content of more than 3.57 percent and the mucopolysaccharide content of more than 90 percent, can increase the light transmittance (lambda is 900nm) of rice vinegar by more than 240.63 percent, and can be applied to clarification of brewed vinegar (pH2.9-3.5).

Drawings

FIG. 1 is a graph of the effect of microbial flocculant dosage on the rate of increase in permeability;

FIG. 2 is a graph of the effect of flocculation system temperature on the rate of increase of permeability;

FIG. 3 is a graph showing the effect of metal ions on the rate of increase in transmittance;

FIG. 4 shows Ca²⁺Influence on the rate of increase of transmittance.

Detailed Description

The present invention will be further illustrated by the following examples.

Example 1:

before inoculation of the chestnut tree bacillus (Paenibacillus castanea), activation is firstly carried out on a test tube slant, the chestnut tree bacillus (Paenibacillus castanea) is cultured for 48 hours at the temperature of 28 ℃, and a slant culture medium used for culture comprises the following components: 2.0% of soluble starch, 0.5% of peptone, 0.05% of potassium chloride, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 2.0% of agar and pH 7.0; then, the bacterial cells were washed with sterile water and inoculated into a flask (500mL) containing 100mL of a seed culture medium (1.0% soluble starch, 1.5% sucrose, 0.5% peptone, 0.05% potassium chloride, 0.1% dipotassium hydrogen phosphate, and 0.05% magnesium sulfate) to perform seed culture (30 hours at 28 ℃ C.). After the seeds are grown, microscopic examination is carried out to ensure that no mixed bacteria exist for standby.

A7L fermentation medium is prepared, and the formula of the medium comprises 3.0% of glucose, 0.1% of sodium nitrate, 0.3% of peptone, 0.05% of potassium chloride, 0.1% of dipotassium hydrogen phosphate and 0.05% of magnesium sulfate. The mixture was placed in a 10L fermenter, autoclaved at 121 ℃ for 20 minutes, and 200mL of cultured Erlenmeyer flask seeds were inoculated. The initial pH of the medium was adjusted to 7.0. Controlling culture conditions: the temperature is 28 ℃, the ventilation rate is 1VVM, the pH is controlled to be 7.0, the initial stirring speed is 300 r/min, the later stirring speed is linked with dissolved oxygen, the lowest dissolved oxygen is controlled to be 20%, the culture time is 36 hours until the OD600 is above 2.5, 3% of sucrose (W/V) of the total volume of the fermentation liquor is added and uniformly stirred, the culture is placed in a pot to a culture dish, the liquid depth is controlled not to exceed 2.5cm, and the culture is continued for 24 hours at 26 ℃ until the culture solution is in a jelly shape.

And collecting the culture solution, adding 3-fold water for dilution, filtering by using filter cloth, and removing somatic cells. Cetyl Pyridinium Chloride (CPC) was added at 2.0% (w/v) until no more insoluble CPC-bioflocculant complex formed. And adding 0.6mol/L NaCl brine into the precipitate until the precipitate is completely dissolved, adding 3 times of ethanol by volume, carrying out ethanol precipitation for 6 hours, removing supernatant, and freeze-drying the ethanol precipitate to obtain the purified microbial flocculant.

The application method of microbial flocculant in vinegar clarifying treatment process comprises heating vinegar to 41.5 deg.C, and adding CaCl with final concentration of 0.32mM₂And then adding the mixture to a microbial flocculant with a final concentration of 3.94 mg/L. Under these conditions, the light transmittance (λ 900nm) of vinegar can be increased by 240.63% or more.

Example 2: optimization of application conditions of microbial flocculant in vinegar brewing

1. Method for measuring flocculation effect of vinegar

Collecting deep liquid fermentation vinegar, and taking the transmittance as an index for measuring the flocculation effect of the purified biological flocculant in the rice vinegar. 1mL of the purified bioflocculant solution was added to 100mL of rice vinegar. Stirring for 2min, standing for 10min, and collecting supernatant for analysis. The transmittance of the supernatant was measured at a wavelength of 900 nm. The control group was prepared in the same manner except that the sample solution was used instead of distilled water.

The rate of Increase of transmittance (IRT) was calculated as follows:

a is an initial value; b: post-flocculation treatment value

2. Single-factor optimization method for flocculation conditions of vinegar

2.1 Effect of the amount of microbial flocculant added on the flocculation Effect

The effect of the microbial flocculant on the vinegar transmittance increase rate is examined within the range of 1-11 mg/L, and the result is shown in figure 1.

As shown in figure 1, the IRT of the microbial flocculant in the addition range of 1-11 mg/L exceeds 48%, and when the optimal addition of the microbial flocculant is 3.0mg/L, the maximum IRT reaches 133.62%. When the bioflocculant is insufficient, the microbial flocculant bridging phenomenon cannot be effectively formed. Conversely, too much negatively charged bioflocculant will cause repulsion of the negatively charged particles, which will also reduce the effective volume, resulting in poor stability. This is consistent with the microbial mucopolysaccharide flocculant properties.

2.2 influence of flocculation System temperature on flocculation Effect

The effect of flocculation system temperature on flocculation effect is shown in figure 2. The flocculation activity gradually increases with increasing temperature, reaches a maximum at 40 ℃ and then gradually decreases. This may be due to the high temperature changing the spatial structure of the bioflocculant, resulting in a decrease in flocculation efficiency.

2.3 Effect of Metal ion type and concentration on flocculation Effect

To achieve higher flocculation activity, metal cations are often added during flocculation to enhance bridging and neutralization flocculation of the flocculant. The effect of cationic species on flocculation is shown in FIG. 3, Ca²⁺The effect of concentration on the flocculation effect is shown in figure 4.

As shown in FIG. 3, trivalent ions (Al)³⁺And Fe³⁺) Anddivalent ion (Ca)²⁺、Mg²⁺And Fe²⁺) Monovalent ion (Na)⁺And K⁺) Is more effective. Considering high cost and other considerations (Fe)²⁺Easily oxidized and colored system, Al³⁺Easily causing alzheimer disease), Ca²⁺Is an ideal coagulant aid. As can be seen from FIG. 4, when Ca is used²⁺The flocculation efficiency is higher when the concentration is 0.3 mM.

Box-Behnken design for further optimizing vinegar flocculation conditions

These factors were further optimized using the Box-Behnken design based on a single factor experiment. The test was performed in triplicate, with the average IRT at each run as the response variable. And analyzing the data acquired by the RSM according to a quadratic polynomial equation by adopting Design-Expert 10.0 software. The test results and the analysis of variance are shown in tables 1 and 2, respectively.

TABLE 1 Box-Behnken design and measurements thereof

TABLE 2 analysis of flocculation Condition variance

The adding amount of the biological flocculant, the temperature of a flocculation system and calcium chloride are important factors influencing the flocculation effect. The Box-Behnken design optimizes these factors with the average IRT as the response variable. The actual response value obtained by the analysis of variance is compared with the predicted response value, and the result shows that the model probability P (Pmodel 0.0009) has statistical significance and is very reliable for predicting the flocculation efficiency. The absence of fitted values was not significant (p-0.1292), and the flocculation efficiency was well predicted. Determining the coefficient (R)²0.9504) represents the sufficiency of the model, accounting for 95.04% of the response variability. The optimal conditions for bioflocculants were obtained using the "numerical optimization" function in Design-Expert 10.0. When the flocculating agent is 3.94mg/L, CaCl₂Is 032mM, the maximum increase in light transmittance at 41.5 ℃ of the flocculation system is 241.04%. To verify the reproducibility of the model, all experiments were performed under optimal conditions. The results showed that the transmittance was improved by 240.63%, 242.51%, and 243.64%, respectively. The model can reasonably optimize the flocculation condition of the rice vinegar and predict the flocculation efficiency of the rice vinegar clarification.

Example 3: safety test of products prepared by the method of the invention

1. Acute oral toxicity test in mice

Uniformly dispersing the prepared sample homogenizer in distilled water of 70 ℃ to prepare the solution with the concentration of 0.1 g/ml^-1The solution was cooled to 40 ℃ for further use. Selecting clean-grade ICR mice with weight of 18-24g, 10 male and female respectively, fasting for 16h before gavage, and feeding the test object for 24h after weighing by gavage for three times, wherein the gavage volume is 40 ml/kg^-1The total dosage is 12 g/kg^-1. The food is fed 4h after the gavage, and the poisoning and death conditions are recorded after continuously observing for 14 d.

TABLE 3 record of weight gain in acutely toxic mice

Note: significant difference from the control group (P <0.05) and significant difference from the control group (P <0.01)

The appearance hair color of the mice is bright after administration, and the phenomena of excitation, uneasiness, drunkenness, jumping, hair erection, lacrimation, salivation, convex eyes and writhing reaction do not occur; after administration, no obvious abnormality occurs in food intake, water intake, defecation and the like. No animals died during the observation period and the weight gain was recorded in table 3. After 14 days of administration, mice are killed by dislocation of cervical vertebrae, and pathological examination is carried out on important visceral organs such as heart, liver, spleen, lung, kidney, uterus, testis, stomach, intestine and the like, and no obvious pathological changes such as hemorrhage, congestion, edema, exudation, ulcer, perforation, no effusion and the like are found in the thoracic cavity, abdominal cavity and pericardium. The results show that the maximum oral tolerance of the mucopolysaccharide powder male and female amphoterics is more than 12 g/kg^-1. According to the acute toxicity classification, the sample belongs to the non-toxic grade.

2. Mouse bone marrow cell chromosome aberration test

50 healthy ICR mice of 25-30g were selected and randomly divided into 5 groups of 10 mice each with half of males and females. The prepared samples were divided into 1.0, 2.0, 4.0 g.kg^-1The positive control group of three dose groups was treated with Cyclophosphamide (CP)40 mg/kg^-1And preparing the mixture to the required concentration by using distilled water. The solvent control group is administered with distilled water for 3 times at an interval of 24 hr, the material is taken 24 hr after the last administration, and the dose is 4 mg/kg 2 hr before killing the animal^-1Colchicine is injected into the abdominal cavity of the body weight. Mice are dislocated to sacrifice the cervical vertebra, femurs are taken, bone marrow is washed into a 10ml centrifuge tube, and after repeated washing for a plurality of times, 1.0ml cell suspension is prepared by uniformly mixing the materials by a suction tube. After staining, metaphase chromosome analysis was performed with an oil lens, and 100 metaphase cells were analyzed per animal.

TABLE 4 mouse bone marrow cell chromosomal aberration test results

As can be seen from Table 4, the samples of each dose group have no significant difference compared with the negative control group, have larger difference with the positive control group (CP group), and the chromosome aberration rate of each test result has no dose response relationship. The sample is shown to have no effect of causing chromosome aberration of mouse bone marrow cells.

3. Mouse teratospermia test

Male cleaning grade ICR mice 25, 28-35g weight, randomly divided into 5 groups of 5 mice each. The dosage is 1.0, 2.0, 4.0 g/kg^-1Three groups were separately provided with a negative control group and a CP positive control group (50 mg. kg)^-1) The test substance was administered by gavage for 5 days continuously until 35d after the test substance was administered for the first time, the mice were sacrificed by cervical dislocation, and the epididymis on both sides was taken out and placed in a dish containing 2ml of physiological saline. Cutting the epididymis longitudinally with an ophthalmic scissors for 1-2 times, standing for 3-5min, and gently shaking. Filtering with synthetic fiber blood bag, sucking filtrate and smearing. Air drying, fixing with methanol for more than 5min, drying, dyeing with 1.5% eosin for 1 hr, rinsing with water, and drying. Each group counted 5 animals and 1000 animals eachThe incidence of aberrant spermatozoa (in percent) was calculated for each structurally intact sperm.

TABLE 5 results of teratospermia test

As can be seen from Table 5, the sample dose groups showed no significant difference from the negative control group, the difference from the positive control group (CP group) was large, and the chromosome aberration rate of each test result showed no dose-response relationship. The sample has no aberration on mouse sperm under the experimental condition.

4.30d feeding test

Clean grade ICR mice 80, weight 18-24g, randomly divided into 4 groups of 20 each, each male and female. The experimental components are 1.0, 2.0 and 4.0 g/kg^-1Three dose groups of equal height, medium and low dose. And (5) setting one control group, and performing intragastric administration on the same dose of distilled water every day. The general state, signs, food intake, water intake and fecal status of the mice were observed daily during the experiment. The body weight was measured every 7 days, and the dose was adjusted according to the change in body weight and continuously observed for 30 days. After administration period of 30 days, after fasting and water deprivation for 12h, taking blood from eyeball, and examining hematology index and blood biochemistry index. Immediately after blood collection, organs such as the liver, kidney, spleen, stomach, testis or ovary of the animal are dissected and weighed, the organ weight coefficient is calculated according to the body weight, and the morphological change is observed by naked eyes.

4.1 general toxic reaction

Mice of the high, medium and low dose administration groups and the control group all move normally within a test period of 30d, have lively behaviors, bright hair color, normal eating and drinking, and do not change abnormally in urination or urination, and die.

4.2 results of body weight measurement

As can be seen from Table 6, the weight gain of the high, medium and low dose groups was normal, and no significant difference was observed between the results of the groups compared with the control group (P > 0.05).

Table 6 mouse body weight measurements

4.3 measurement results of organ coefficients of mouse

As can be seen from table 7, no significant difference in organ coefficients was observed between the dose groups and the control group after 30d administration ((P > 0.05).

TABLE 7 organ coefficient measurement results

4.4 blood general index measurement results

As can be seen from tables 8, 9 and 10, the mouse white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HB), Hematocrit (HCT), mean hematocrit (MCV), Mean Platelet Volume (MPV), mean hemoglobin content (MCH), neutrophil percentage (NE), lymphocyte percentage (LY), eosinophil percentage (EO), monocyte percentage (MO), Basophil (BA), mean hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelet count (PLT), and the like in each dose group were not significantly different from those in the control group (P > 0.05).

TABLE 8 blood routine measurement results

TABLE 9 blood routine test results

TABLE 10 blood routine test results

4.5 blood Biochemical index measurement results

As can be seen from tables 11 and 12, the blood biochemical indexes such as mouse Triglyceride (TG), total Cholesterol (CHO), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), Creatinine (CRE), urea nitrogen (BUN), Glucose (GLU), Total Protein (TP), Albumin (ALB) and the like of each dose group have no significance compared with the control group (P is more than 0.05).

TABLE 11 Biochemical assay results of blood

TABLE 12 Biochemical assay results of blood

4.630 d histopathological examination of mice fed

The sizes, shapes, colors, textures and the like of organs such as heart, liver, kidney, spleen, stomach, testis, ovary and the like of each group are observed comprehensively and carefully without abnormity. All major organs have no obvious degeneration and necrosis and normal luster.

Claims

1. A preparation method of a microbial flocculant for clarifying brewed vinegar is characterized by comprising the following steps:

2. The method of claim 1, wherein the slant culture medium in step 1) comprises: 2.0% of soluble starch, 0.5% of peptone, 0.05% of potassium chloride, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate and 2.0% of agar, and the activation culture method comprises the following steps: adjusting pH to 7.0, autoclaving test tube with slant culture medium at 121 deg.C for 20 min, inoculating Paenibacillus chestnut, and culturing at 28 deg.C for 48 hr.

3. The method according to claim 1, wherein the seed culture solution in step 1) is composed of: 1.0% of soluble starch, 1.5% of sucrose, 0.5% of peptone, 0.05% of potassium chloride, 0.1% of dipotassium hydrogen phosphate and 0.05% of magnesium sulfate, and the seed culture method comprises the following steps: the triangular flask containing the seed culture solution was autoclaved at 121 ℃ for 20 minutes, and then the bacterial suspension was inoculated and cultured at 28 ℃ for 30 hours.

4. The method according to claim 1, wherein the fermentation medium used in the step 2) comprises: 3.0% of glucose, 0.1% of sodium nitrate, 0.3% of peptone, 0.05% of potassium chloride, 0.1% of dipotassium hydrogen phosphate and 0.05% of magnesium sulfate.

5. The method according to claim 1, wherein the fermentation culture method in step 2) comprises: the culture temperature is 28 ℃, the ventilation rate is 1VVM, the initial pH is 7.0, the initial stirring rotation speed is 300 r/min, the later stirring rotation speed is linked with dissolved oxygen, the lowest dissolved oxygen is controlled to be 20%, the culture time is 36 hours, the OD600 is more than 2.5, 3.0% (W/V) of cane sugar in the total volume of the fermentation liquid is added, the mixture is uniformly stirred, the mixture is placed in a culture dish, the liquid depth is controlled not to exceed 2.5cm, the culture is continued for 24 hours at 26 ℃, until the culture liquid is in a jelly shape, the charging amount of the fermentation tank is 70%, and the inoculation amount is 3.0%.

6. The method according to claim 1, wherein the microbial flocculant comprises mucopolysaccharides as a main component, and glucosamine and uronic acid are contained in the molecular structure.

7. A microbial flocculant obtained by the production process according to any one of claims 1 to 6.

8. Use of a microbial flocculant according to claim 7 for clarifying brewed vinegar.

9. The use according to claim 8, characterized in that the use is in particular: heating brewed edible vinegar to 41.5 deg.C, adding CaCl₂To a final concentration of 0.32mM, and then microbial flocculant was added to a final concentration of 3.94 mg/L.