Preparation and application of compound fermentation inoculant and fermentation liquor

Abstract

The invention relates to a compound fermentation bacterial agent, in particular to a compound fermentation bacterial agent, which is a compound bacterial agent consisting of lactococcus lactis, trichoderma harzianum, bacillus subtilis and candida aspera, and is characterized in that the compound fermentation bacterial agent is a compound bacterial agent consisting of bacillus subtilis and candida aspera, and the compound fermentation bacterial agent is a compound bacterial agent consisting of bacillus subtilis, trichoderma harzianum, bacillus subtilis and candida aspera. According to the invention, the efficient antibacterial acidic fermentation liquor is prepared through fermentation of the microbial composite inoculant, the efficient antibacterial acidic fermentation liquor is compounded with a mixed liquor with a film-forming characteristic, when the efficient antibacterial acidic fermentation liquor is sprayed on the surface of the kiwi fruit, the efficient antibacterial acidic fermentation liquor has high adhesive force and can form a complete package on the surface of the kiwi fruit, and an antibacterial component obtained through fermentation isolates invasion of pathogenic microorganisms to the kiwi fruit; the erosion of the antibacterial components to the kiwi fruits can be prevented, and the flavor and taste of the kiwi fruits are effectively guaranteed.

Description

[0001] The present invention is a divisional application of patent 202411004705.0, with the invention name of “A microbial composite agent and its application”. Technical Field

[0002] The present invention relates to the technical field of microbial fermentation, and in particular to the preparation and application of a composite fermentation agent and fermentation liquid. Background Art

[0003] Kiwifruit gray mold is a disease caused by Botrytis cinerea that occurs in kiwifruit. The disease invades the entire growth process of kiwifruit, from the beginning of spring growth to the flowering period, young fruit period and storage period.

[0004] Soft rot, anthracnose and gray mold of kiwifruit during storage are usually caused by pathogens in the early growth stage and occur within 2 months after harvest. The pathogens have strong survival ability and long infection time. After the disease occurs, the yield of kiwifruit can be greatly reduced, causing huge economic losses to fruit farmers. During picking, transportation, storage and sale, the fruit is very easy to be bumped and squeezed, causing wounds on the peel. The disease can also invade through the wounds on the surface of the fruit, causing the fruit to rot and deteriorate, causing losses to kiwifruit producers and operators. In order to extend the storage time of kiwifruit after harvest and improve the preservation effect, preservatives are generally sprayed on the surface of kiwifruit. Preservatives such as ethylene inhibitors have little effect on the prevention and control of infectious pathogens during the storage of kiwifruit. Moreover, when most preservatives are sprayed on the surface of kiwifruit, due to the large number of fine hairs on the surface of kiwifruit, the binding force of the preservatives is poor and they are easy to fall off, resulting in unsatisfactory preservation effect. Summary of the invention

[0005] Based on the above technical problems, the present invention aims to provide a composite fermentation agent. The fermentation liquid prepared by fermentation of the composite fermentation agent can effectively prevent and control the pathogenic bacteria of gray mold.

[0006] Another object of the present invention is to apply the composite fermentation agent. Specifically, it is applied to the antibacterial effect on the surface of kiwi fruit. The object of the present invention is achieved by the following technical solution: A microbial composite fermentation agent, characterized in that: it is composed of Lactococcus lactis ( Lactococcus lactis )、Trichoderma harzianum( Trichoderma harzianum ), Bacillus subtilis ( Bacillus subtilis ) and Candida lucidii ( Candida lusitania ) composed of a composite bacterial agent.

[0007] Furthermore, in the composite fermentation bacterial agent, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida lusicola are composed according to a live bacteria mass ratio of 1-1.5:1.2-1.5:2-3:2-3.

[0008] Furthermore, the above-mentioned composite fermentation bacteria agents were all purchased from Guangdong Provincial Culture Collection Center (GDMCC), among which the preservation number of Lactococcus lactis is GDMCC1.1803, the preservation number of Trichoderma harzianum is GDMCC NO.3.442, the preservation number of Bacillus subtilis is GDMCC NO.1.131, and the preservation number of Candida portugalensis is GDMCC NO.2.145.

[0009] The application of the above-mentioned composite fermentation bacteria agent is characterized in that it is used for fermentation to prepare antibacterial agents, specifically for fermentation to prepare composite fermentation liquid.

[0010] Furthermore, the fermentation is divided into two steps. The first step of fermentation uses Lactococcus lactis and Trichoderma harzianum. After the fermentation is completed, the second step of fermentation uses Bacillus subtilis and Candida lusicola.

[0011] The composite bacterial agent is used to prepare a composite fermentation liquid by fermentation, which is characterized in that: Sophora flavescens, corn flour, Phellodendron amurense, Purslane and sugarcane are firstly treated with Lactococcus lactis ( Lactococcus lactis ) and Trichoderma harzianum ( Trichoderma Harzian ) for the first fermentation, and after sterilization, the supernatant and fermentation precipitate were collected, and Bacillus subtilis ( Bacillus subtilis ) and Candida lucidii ( Candida lusitania ) for the second fermentation, sterilize and collect the supernatant after the fermentation, and combine the two supernatants to obtain the composite fermentation liquid.

[0012] Furthermore, in the first step of fermentation, the fermentation temperature is 30-35°C, the fermentation is for 7-9 days, and the supernatant is collected after sterilization. The second step of fermentation is performed at a temperature of 28-32°C, the fermentation is for 5-7 days, and the supernatant is collected after sterilization after the fermentation is completed, and the two supernatants are combined to obtain a fermentation liquid.

[0013] Furthermore, in parts by weight, the mixed powder comprises 10 to 15 parts of Sophora flavescens, 20 to 30 parts of corn flour, 8 to 12 parts of Phellodendron amurense, 10 to 12 parts of Portulaca oleracea, 8 to 10 parts of sugarcane, and 50 to 70 parts of purified water, and the mass ratio of the number of viable bacteria of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis, and Candida portugal is 100: 0.10 to 0.15: 0.12 to 0.15: 0.20 to 0.30: 0.20 to 0.30.

[0014] Furthermore, the mixed powder is prepared by mixing Sophora flavescens, corn flour, Phellodendron amurense, Purslane and sugarcane, placing the mixture in a universal grinder, grinding the mixture through a 16-mesh sieve, and then mixing the mixture at 20-25 rpm for 30-40 min.

[0015] More specifically, the application of the above-mentioned composite fermentation bacteria agent is to use the above-mentioned composite fermentation bacteria agent to ferment and prepare fermentation liquid, which is further used to prepare a kiwi fruit preservative.

[0016] Furthermore, the kiwi fruit preservative is prepared by sequentially adding gelatin, polyethylene glycol and polyvinyl alcohol into an ethanol solution to form a mixed solution, adding a composite fermentation liquid into the mixed solution, mixing, and filtering to obtain the biological preservative.

[0017] Furthermore, the mixed solution is prepared by heating a 70-80% ethanol solution to 45-50° C., adding gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence under stirring at 40-60 rpm, and continuing stirring for 50-60 min.

[0018] Furthermore, the mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:20-25:15-18:10-12.

[0019] Furthermore, the mass ratio of the fermentation liquid to the mixed liquid is 1:3-5.

[0020] The present invention also provides a preparation method of the biological preservative, which is characterized by: taking Sophora flavescens, corn flour, Phellodendron amurense, Purslane and sugar cane and fermenting them with Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida portugalensis, sterilizing and collecting the fermentation liquid after the fermentation, adding gelatin, polyethylene glycol and polyvinyl alcohol in turn to the ethanol solution to form a mixed liquid, adding the fermentation liquid to the mixed liquid, mixing, and filtering to obtain the biological preservative.

[0021] Furthermore, the fermentation is divided into two steps of fermentation, specifically, Sophora flavescens, corn flour, Phellodendron amurense, Purslane and sugarcane are mixed and crushed to form a mixed powder, and purified water is added and stirred evenly after sterilization. Lactococcus lactis and Trichoderma harzianum are firstly used for the first step of fermentation, the fermentation temperature is 30-35°C, the fermentation is carried out for 7-9 days, and the supernatant is collected after sterilization. Then, Bacillus subtilis and Candida portuguesa are mixed and fermented on the fermentation precipitate for the second step of fermentation, the fermentation temperature is 28-32°C, the fermentation is carried out for 5-7 days, and the supernatant is collected after sterilization after the fermentation is completed, and the two supernatants are combined to obtain the fermentation liquid.

[0022] Furthermore, in parts by weight, the mixed powder comprises 10 to 15 parts of Sophora flavescens, 20 to 30 parts of corn flour, 8 to 12 parts of Phellodendron amurense, 10 to 12 parts of Portulaca oleracea, 8 to 10 parts of sugarcane, and 50 to 70 parts of purified water, and the mass ratio of the number of viable bacteria of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis, and Candida portugal is 100: 0.10 to 0.15: 0.12 to 0.15: 0.20 to 0.30: 0.20 to 0.30.

[0023] In the present invention, Lactococcus lactis and Trichoderma harzianum are used for the first step of fermentation, and Trichoderma harzianum decomposes the lignocellulose in the Chinese medicine components to generate sugars, while Lactococcus lactis decomposes the sugars to provide small molecule nutrients for Trichoderma harzianum, forming a circular fermentation system. Through the first step of fermentation, the antibacterial components (matrine, berberine, etc.) are quickly dissolved, and the antibacterial peptides, organic acids, chitinases and β-1,3-glucanase produced by the metabolism of Lactococcus lactis and Trichoderma harzianum participate in the synergistic effect, exerting the inhibitory effect of Chinese medicine on fungi. After the second step of fermentation, the pH of the fermentation liquid is further adjusted, and the lower pH significantly improves the bactericidal ability of substances such as berberine.

[0024] Furthermore, the fermentation liquid is collected by sterilizing the first step fermentation product, centrifuging it at 8000-11000 rpm for 20-30 min, collecting the supernatant, sterilizing the second step fermentation product, centrifuging it at 8000-11000 rpm for 20-30 min, collecting the supernatant, and combining the two supernatants to obtain the fermentation liquid.

[0025] Furthermore, the mixed powder is prepared by mixing Sophora flavescens, corn flour, Phellodendron amurense, Purslane and sugarcane, placing the mixture in a universal grinder, grinding the mixture through a 16-mesh sieve, and then mixing the mixture at 20-25 rpm for 30-40 min.

[0026] Furthermore, the mixed solution is prepared by heating a 70-80% ethanol solution to 45-50° C., adding gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence under stirring at 40-60 rpm, and continuing stirring for 50-60 min.

[0027] Furthermore, the mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:20-25:15-18:10-12.

[0028] Since the surface of kiwifruit is not smooth and has a lot of hairs, the preservative cannot adhere to its surface for a long time. During transportation or collision, the preservative is easy to fall off the surface. Secondly, when the biological preservative is sprayed on the surface of post-harvest kiwifruit, a large amount of antibacterial ingredients in it will evaporate quickly, and only a small amount will remain on the surface of the kiwifruit, which cannot guarantee the continued efficacy of the drug. If the biological preservative has a strong film-forming sealing property, it will cause the antibacterial drug ingredients to penetrate into the kiwifruit, causing erosion on the kiwifruit, and affecting the flavor and taste of the kiwifruit itself.

[0029] In view of this, the present invention adopts microorganisms to ferment specific Chinese medicine raw materials to prepare a fermentation liquid with high antibacterial efficiency, and places the fermentation liquid in a mixed solution consisting of gelatin, polyethylene glycol 2000, polyvinyl alcohol and ethanol solution to form a biological preservative. When the biological preservative is sprayed on the surface of kiwi fruit, the gelatin, polyethylene glycol 2000 and polyvinyl alcohol in the preservative work synergistically to form a water-soluble soft film on the surface of kiwi fruit, and the antibacterial components in the fermentation liquid can be effectively attached to the soft film. On the one hand, the antibacterial components can achieve effective antibacterial effects, and on the other hand, the exogenous pathogenic microorganisms are isolated from attaching and growing on the surface. In addition, the soft film wraps the antibacterial components, which further inhibits the penetration of the antibacterial components into the interior of the kiwi fruit.

[0030] In addition, the addition of low pH fermentation liquid to the mixed liquid adjusted the film formation rate during the film formation process, and interacted with polyethylene glycol 2000 and polyvinyl alcohol in the aqueous soft film, thereby improving the adhesion of the formed film on the surface of kiwifruit and effectively extending the action time of the preservative.

[0031] Furthermore, the mass ratio of the fermentation liquid to the mixed liquid is 1:3-5. After the fermentation liquid and the mixed liquid are mixed, they are filtered using a water system microporous filter membrane with a pore size of 0.45 microns. The filtrate is collected to obtain a biological preservative.

[0032] Most specifically, the method for preparing the above-mentioned biological preservative is characterized by comprising the following steps: Step 1: Prepare fermentation broth (1) According to weight, 10-15 parts of Sophora flavescens, 20-30 parts of corn flour, 8-12 parts of Phellodendron amurense, 10-12 parts of Portulaca oleracea, and 8-10 parts of sugar cane were mixed, and the mixture was pulverized and sieved through a 16-mesh sieve to obtain a powder. The powder was mixed and stirred at 20-25 rpm for 30-40 min to obtain a mixed powder; (2) sterilize the mixed powder at 100°C for 30 min, add 50-70 parts of purified water under a sterile environment, stir evenly, then add Lactococcus lactis and Trichoderma harzianum for the first fermentation at a temperature of 30-35°C for 7-9 days; after the fermentation, obtain the fermented product, sterilize it at 121°C for 15 min, centrifuge it at 8000-11000 rpm for 20-30 min, collect the supernatant and the centrifugal precipitate, respectively, and set aside; (3) adding Bacillus subtilis and Portuguese Candida to the centrifugal precipitate for the second fermentation, the fermentation temperature is 28-32°C, the fermentation time is 5-7 days, and the fermentation is completed to obtain a fermentation product, the fermentation product is sterilized at 121°C for 20-30 min, and the supernatant is collected, and the supernatant collected from the first fermentation and the supernatant collected from the second fermentation are combined to obtain a fermentation liquid, wherein the mass ratio of the number of live bacteria of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Portuguese Candida is 100: 0.10-0.15: 0.12-0.15: 0.20-0.30: 0.20-0.30; Step 2: Synthetic biological preservatives (1) Take an ethanol solution with a volume fraction of 70-80%, heat it to 45-50°C, add gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence while stirring at 40-60 rpm, and continue stirring for 50-60 min to obtain a mixed solution, wherein the mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:20-25:15-18:10-12; (2) Add the fermentation liquid to the mixed liquid under stirring conditions of 60-80 rpm, continue stirring for 30-40 min, and then filter with a water-based microporous filter membrane with a pore size of 0.45 μm. Collect the filtrate, which is the biological preservative. The mass ratio of the fermentation liquid to the mixed liquid is 1:3-5.

[0033] The present invention has the following technical effects: In the present invention, a highly effective antibacterial acidic fermentation liquid is prepared by combining a specific microbial composite agent with a specific fermentation step, and the acidic fermentation liquid is added to a mixed liquid with film-forming properties to form a biological preservative. When the biological preservative is sprayed on the surface of kiwi fruit, it has high adhesion and can form a complete package on the surface of kiwi fruit. The antibacterial components can isolate the invasion of pathogenic microorganisms on the kiwi fruit, and can also prevent the antibacterial components themselves from eroding the kiwi fruit, thereby effectively ensuring the flavor and taste of the kiwi fruit. DETAILED DESCRIPTION

[0034] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0035] The microorganisms used in the present invention are all commercially available and purchased from Guangdong Provincial Culture Collection Center (GDMCC). The collection number of Lactococcus lactis is GDMCC1.1803, the collection number of Trichoderma harzianum is GDMCC NO.3.442, the collection number of Bacillus subtilis is GDMCC NO.1.131, and the collection number of Candida portugalensis is GDMCC NO.2.145.

[0036] Example 1 A method for preparing a biological preservative comprises the following steps: Step 1: Prepare fermentation broth (1) According to weight, 12 parts of Sophora flavescens, 25 parts of corn flour, 10 parts of Phellodendron amurense, 10 parts of Portulaca oleracea, and 8 parts of sugar cane were mixed, and the mixture was pulverized and sieved through a 16-mesh sieve to obtain a powder. The powder was mixed and stirred at 25 rpm for 30 min to obtain a mixed powder; (2) Sterilize the mixed powder at 100°C for 30 min, add 50-70 parts of purified water under a sterile environment, stir evenly, and then add Lactococcus lactis and Trichoderma harzianum for the first fermentation at a temperature of 30-35°C for 8 days; after the fermentation, obtain the fermentation product, sterilize it at 121°C for 15 min, centrifuge it at 10,000 rpm for 25 min, collect the supernatant and the centrifugal precipitate, and set aside; (3) Adding Bacillus subtilis and Portuguese Candida to the centrifugal precipitate for the second fermentation, the fermentation temperature is 28-32°C, the fermentation time is 6 days, and the fermentation is completed to obtain a fermentation product, the fermentation product is sterilized at 121°C for 25 min, the supernatant is collected, and the supernatant collected from the first fermentation step and the supernatant collected from the second fermentation step are combined to obtain a fermentation liquid, and the pH value of the fermentation liquid is 5.24; the mass ratio of the number of live bacteria of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Portuguese Candida is 100:0.12:0.14:0.25:0.25; Step 2: Synthetic biological preservatives (1) Take a 75% ethanol solution, heat it to 45°C, add gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence while stirring at 50 rpm, and continue stirring for 55 minutes to obtain a mixed solution, wherein the mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:22:16:11; (2) Add the fermentation liquid to the mixed liquid under stirring at 70 rpm, continue stirring for 35 minutes, and then filter it with a water-based microporous filter membrane with a pore size of 0.45 microns. Collect the filtrate, which is the biological preservative. The mass ratio of the fermentation liquid to the mixed liquid is 1:4.

[0037] Comparative Example 1 Compared with Example 1, when preparing the fermentation broth, Lactococcus lactis and Bacillus subtilis were first used for the first fermentation, and then Trichoderma harzianum and Candida lusicola were used for the second fermentation, and the remaining steps were the same as Example 1. The pH of the obtained fermentation broth was 6.18.

[0038] Comparative Example 2 Compared with Example 1, the difference is that the polyethylene glycol 2000 in the mixed solution is replaced with polyethylene glycol 4000, and the rest of the formula and steps are the same as Example 1.

[0039] Comparative Example 3 Compared with Example 1, the difference is that the polyethylene glycol 2000 in the mixed solution is replaced with polyethylene glycol 400, and the rest of the formula and steps are the same as Example 1.

[0040] Performance Testing: (1) Film-forming adhesion performance: Kiwifruit samples picked from the same plant with high consistency in various trait indicators were selected and divided into 4 groups. The biological preservatives prepared in Example 1, Comparative Example 1 and Comparative Example 2 were sprayed on the surfaces of kiwifruit samples in 3 groups, and the surface of the remaining group was sprayed with the mixed solution prepared in Example 1 (without fermentation liquid) as a blank control. The kiwifruit was then placed in a refrigerator for refrigeration. Every 5 days, the kiwifruit samples were taken out to observe the aqueous soft film of each preservative on the surface of the kiwifruit. The samples were then lightly rinsed with clean water to remove the fluff on the surface of the kiwifruit. The content of polyethylene glycol (400 / 2000 / 4000), polyvinyl alcohol, and antibacterial drugs on the surface of the kiwifruit were then tested. The results are shown in Table 1.

[0041] Table 1:

[0042] During the testing process, it was found that after the biological preservative prepared in Example 1 was sprayed on the surface of kiwifruit, no antibacterial drugs and film-forming components were found to penetrate into the kiwifruit within 30 days. After the biological preservative was sprayed, a thin film layer could be observed on the fuzz on the surface of the kiwifruit. In the blank control group, there was no antibacterial component in the preservative film, and the adhesion ability of the film layer formed on the surface of the kiwifruit was worse than that in Example 1. The film layer on some areas of the kiwifruit surface disappeared within 10 days. In Comparative Examples 1 and 3, the antibacterial component and film-forming component had infiltration to varying degrees, and the film layer also fell off in Comparative Examples 1 and 2. Although the film-forming components in Comparative Example 1 are the same as those in Example 1, the change in the fermentation process results in a large difference in the final fermentation liquid system, and the pH of the fermentation liquid is significantly increased. Since the overall pH value of the preservative is relatively high, the film-forming rate is significantly different, resulting in changes in the performance of the film layer formed on the surface of the kiwifruit, and the adhesion performance of the film layer on the surface of the kiwifruit is reduced. Therefore, both the film layer shedding phenomenon and obvious component infiltration occur.

[0043] (2) Verification of preservation effect: Take 400 fully ripe kiwifruits from the same plant with high consistency of trait indicators (fruit hardness is 4.5±0.1kg / cm 2 ), the weight deviation does not exceed 5g, divided into four groups, each group of 100, namely the blank control group (no spraying of any preservative, normal placement), preservative comparative example 1 (spraying the preservative prepared by comparative example 1), preservative comparative example 2 (spraying the preservative prepared by comparative example 2) and the preservative group of the present invention (spraying the preservative of the present invention), and placed at a temperature of 10-20°C for storage at the same time, and all taken out for testing of water loss rate, decay rate, and antibacterial rate at 5 days, 10 days, 20 days, and 30 days respectively. After the test is completed, continue to place samples, and at 0 days, measure the weight of each fruit and record it. The results are shown in Table 2.

[0044] Table 2:

[0045] As can be seen from the table above, the blank control group did not adopt any preservation measures. On the 5th day of placement, the fruit turned black and soft, and rotted. On the 10th day, bacteria grew. On the 20th day, all kiwi samples were rotten. The kiwis prepared in Example 1 had good moisture retention after spraying with the biological preservative, and no bacteria were detected within 30 days. Only 4% of the fruits were slightly rotten, indicating that the biological preservative prepared by the present invention has excellent preservation effect, which can effectively ensure that the moisture and nutrition of kiwis are not lost, while inhibiting the attachment and erosion of pathogenic microorganisms by kiwi fruits. In Comparative Example 1, since the preparation method of the fermentation liquid is significantly different from that of the present invention, the composition of the antibacterial components in the obtained fermentation liquid and the pH are different, resulting in differences in the adhesion characteristics of the final biological preservative on the surface of the kiwi after film formation, as well as the antibacterial effect on pathogenic microorganisms. On the 20th day, obvious fruit rot and bacteria growth occurred, and the kiwi water loss was faster than that in Example 1. In Comparative Example 2, polyethylene glycol 2000 in the mixed solution was replaced with polyethylene glycol 4000 with a larger molecular weight. However, due to the special villi structure on the surface of kiwifruit, the biological preservative could not adhere to the surface of kiwifruit for a long time after forming a film. As the storage time increased, the water-based soft film fell off the surface of kiwifruit, resulting in more obvious problems such as water loss and fruit rot in the later stage. As the water-based soft film fell off, the traditional Chinese medicine components inside were lost rapidly, which also led to a serious decrease in the antibacterial effect.

[0046] Example 2 A method for preparing a biological preservative comprises the following steps: Step 1: Prepare fermentation broth (1) According to weight, 10 parts of Sophora flavescens, 20 parts of corn flour, 8 parts of Phellodendron amurense, 10 parts of Portulaca oleracea, and 10 parts of sugar cane were mixed, and the mixture was pulverized and sieved through a 16-mesh sieve to obtain a powder. The powder was mixed and stirred at 20 rpm for 40 min to obtain a mixed powder; (2) The mixed powder was sterilized at 100°C for 30 min, 60 parts of purified water were added under a sterile environment, and after being stirred evenly, Lactococcus lactis and Trichoderma harzianum were added for the first fermentation at a temperature of 30-35°C for 7 days. After the fermentation, the fermented product was sterilized at 121°C for 15 min, and the fermented product was centrifuged at 8000 rpm for 30 min, and the supernatant and the centrifugal precipitate were collected and set aside. (3) adding Bacillus subtilis and Portuguese Candida to the centrifugal precipitate for the second fermentation, the fermentation temperature is 28-32°C, the fermentation time is 7 days, and the fermentation is completed to obtain a fermentation product, the fermentation product is sterilized at 121°C for 30 min, the supernatant is collected, and the supernatant collected from the first fermentation step and the supernatant collected from the second fermentation step are combined to obtain a fermentation broth, the pH of the obtained fermentation broth is 5.16, and the mass ratio of the number of live bacteria of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Portuguese Candida is 100: 0.10: 0.15: 0.20: 0.30; Step 2: Synthetic biological preservatives (1) Take a 70% ethanol solution, heat it to 50°C, add gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence while stirring at 40 rpm, and continue stirring for 50 min to obtain a mixed solution, wherein the mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:20:15:10; (2) Add the fermentation liquid to the mixed liquid under stirring at 60 rpm, continue stirring for 30 to 40 minutes, and then filter it with a water-based microporous filter membrane with a pore size of 0.45 microns. Collect the filtrate, which is the biological preservative. The mass ratio of the fermentation liquid to the mixed liquid is 1:3.

[0047] The biological preservative prepared in this embodiment is sprayed on the surface of kiwi fruit to form a stably attached aqueous soft film on the surface of kiwi fruit, effectively inhibiting the growth of pathogenic microorganisms on the surface of kiwi fruit and reducing its moisture loss. In 30 days, the water loss rate is within 1.5%, thereby extending the shelf life of kiwi fruit.

[0048] Example 3 A method for preparing a biological preservative comprises the following steps: Step 1: Prepare fermentation broth (1) According to weight, 15 parts of Sophora flavescens, 30 parts of corn flour, 12 parts of Phellodendron amurense, 12 parts of Portulaca oleracea, and 9 parts of sugar cane were mixed, and the mixture was pulverized and sieved through a 16-mesh sieve to obtain a powder. The powder was mixed and stirred at 25 rpm for 30 minutes to obtain a mixed powder; (2) The mixed powder was sterilized at 100°C for 30 min, 70 parts of purified water were added under a sterile environment, and after being stirred evenly, Lactococcus lactis and Trichoderma harzianum were added for the first fermentation at a temperature of 30-35°C for 9 days. After the fermentation, the fermented product was sterilized at 121°C for 15 min, and the fermented product was centrifuged at 11000 rpm for 20 min, and the supernatant and the centrifugal precipitate were collected and set aside. (3) adding Bacillus subtilis and Portuguese Candida to the centrifugal precipitate for the second fermentation, the fermentation temperature is 28-32°C, the fermentation time is 5 days, and the fermentation is completed to obtain a fermentation product, the fermentation product is sterilized at 121°C for 20 min, the supernatant is collected, and the supernatant collected from the first fermentation step and the supernatant collected from the second fermentation step are combined to obtain a fermentation liquid, the pH of the obtained fermentation liquid is 5.09, and the mass ratio of the number of live bacteria of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Portuguese Candida is 100:0.15:0.12:0.30:0.20; Step 2: Synthetic biological preservatives (1) Take an ethanol solution with a volume fraction of 80%, heat it to 45°C, add gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence while stirring at 60 rpm, and continue stirring for 60 minutes to obtain a mixed solution, wherein the mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:25:18:12; (2) Add the fermentation liquid to the mixed liquid under stirring at 80 rpm, continue stirring for 30 minutes, and then filter it with a water-based microporous filter membrane with a pore size of 0.45 microns. Collect the filtrate, which is the biological preservative. The mass ratio of the fermentation liquid to the mixed liquid is 1:5.

[0049] The biological preservative prepared in this embodiment is sprayed on the surface of kiwi fruit to form a stably attached aqueous soft film on the surface of kiwi fruit, effectively inhibiting the growth of pathogenic microorganisms on the surface of kiwi fruit and reducing its moisture loss. In 30 days, the water loss rate is within 1.5%, thereby extending the shelf life of kiwi fruit.

Claims

1. A composite fermentation agent, characterized in that: It is produced by Lactococcus lactis ( Lactococcus lactis )、Trichoderma harzianum( Trichoderma harzianum ), Bacillus subtilis ( Bacillus subtilis ) and Candida lucidii ( Candida lusitaniae ) composed of a composite bacterial agent.

2. The composite fermentation agent according to claim 1, characterized in that: In the composite fermentation bacterial agent, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida lusutica are composed according to a live bacteria mass ratio of 1-1.5:1.2-1.5:2-3:2-3.

3. A method for preparing a composite fermentation liquid based on the composite fermentation agent according to claim 1, characterized in that: Sophora flavescens, corn flour, Phellodendron amurense, Purslane and sugar cane were firstly treated with Lactococcus lactis ( Lactococcus lactis ) and Trichoderma harzianum ( Trichoderma harzianum ) for the first step of fermentation, and after sterilization, the supernatant and fermentation precipitate were collected, and Bacillus subtilis ( Bacillus subtilis ) and Candida lucidii ( Candida lusitaniae ) for the second fermentation, sterilize and collect the supernatant after the fermentation, and combine the two supernatants to obtain the composite fermentation liquid.

4. The method for preparing the composite fermentation broth according to claim 1, characterized in that: The first step of fermentation is carried out at a temperature of 30-35° C. for 7-9 days, and the supernatant is collected after sterilization. The second step of fermentation is carried out at a temperature of 28-32° C. for 5-7 days, and the supernatant is collected after sterilization after the fermentation is completed, and the two supernatants are combined to obtain a fermentation liquid.

5. The method for preparing the composite fermentation broth according to claim 3 or 4, characterized in that: In terms of weight parts, the mixed powder comprises 10-15 parts of Sophora flavescens, 20-30 parts of corn flour, 8-12 parts of Phellodendron amurense, 10-12 parts of Portulaca oleracea, 8-10 parts of sugarcane and 50-70 parts of purified water, and the mass ratio of the live bacteria count of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida portugal is 100: 0.10-0.15: 0.12-0.15: 0.20-0.30: 0.20-0.

30.

6. The method for preparing the composite fermentation broth according to claim 5, characterized in that: The deposit number of the Lactococcus lactis is GDMCC1.1803, the deposit number of the Trichoderma harzianum is GDMCC NO.3.442, the deposit number of the Bacillus subtilis is GDMCC NO.1.131, and the deposit number of the Candida lucidum is GDMCC NO.2.

145.

7. The composite fermentation liquid prepared by the method as claimed in claim 6 is used to ferment and prepare a kiwi fruit biological preservative.

8. The use according to claim 7, characterized in that: Gelatin, polyethylene glycol and polyvinyl alcohol are sequentially added to an ethanol solution to form a mixed solution, composite fermentation liquid is added to the mixed solution, and the mixture is filtered to obtain a biological preservative.

9. The use according to claim 8, characterized in that: The mixed solution is prepared by heating an ethanol solution with a volume fraction of 70-80% to 45-50° C., adding gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence under stirring at 40-60 rpm, and continuing to stir for 50-60 minutes.

10. The use according to claim 8 or 9, characterized in that: The mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:20-25:15-18:10-12, and the mass ratio of the fermentation liquid to the mixed liquid is 1:3-5.

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