Preparation and application of a compound fermentation agent and fermentation broth

Abstract

A compound fermentation agent, which is composed of Lactococcus lactis ( Lactococcus lactis Trichoderma harzianum ( Trichoderma Harzian ), Bacillus subtilis ( Bacillus subtle ) and Candida Portugueseis ( White Portuguese The invention utilizes a compound microbial agent composed of the aforementioned microbial compound microbial agent to prepare a highly effective antibacterial acidic fermentation broth. This broth is then combined with a mixture possessing film-forming properties. When sprayed onto the surface of kiwifruit, it exhibits high adhesion, forming a complete coating on the kiwifruit surface. The antibacterial components obtained through fermentation isolate pathogenic microorganisms from invading the kiwifruit and also prevent the antibacterial components themselves from eroding the kiwifruit, effectively preserving the flavor and texture of the kiwifruit.

Description

[0001] This invention is a divisional application of patent 202411004705.0, entitled "A Microbial Compound Agent and Its Application". Technical Field

[0002] This invention relates to the field of microbial fermentation technology, specifically to a compound fermentation agent, the preparation of fermentation broth, and their applications. Background Technology

[0003] Gray mold of kiwifruit is a disease caused by Botrytis cinerea. This disease can affect kiwifruit throughout its entire growth cycle, from the beginning of spring growth through flowering, the young fruit stage, and storage.

[0004] Soft rot, anthracnose, and gray mold, common diseases affecting kiwifruit during storage, typically infect the fruit early in its growth stages and manifest within two months post-harvest. These pathogens are highly resilient and have a long infection period, leading to a significant drop in kiwifruit yield and substantial economic losses for growers. During harvesting, transportation, storage, and sales, the fruit is easily bruised and squeezed, creating wounds on the peel. These wounds can allow the disease to enter, causing the fruit to rot and spoil, resulting in losses for kiwifruit producers and sellers. To extend the post-harvest shelf life and improve preservation, preservatives are usually sprayed onto the surface. However, preservatives like ethylene inhibitors are largely ineffective against pathogens infecting kiwifruit during storage. Furthermore, most preservatives have poor adhesion to the kiwifruit surface due to its numerous fine hairs, making them prone to peeling off and resulting in unsatisfactory preservation effects. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a compound fermentation agent. The fermentation broth prepared using this compound fermentation agent can effectively control gray mold pathogens.

[0006] Another objective of this invention lies in the application of the aforementioned compound fermentation agent. Specifically, it is applied to the antibacterial action on the surface of kiwifruit. This objective is achieved through the following technical solution:

[0007] A microbial compound fermentation agent, characterized in that: it is composed of Lactococcus lactis (… Lactococcus lactis Trichoderma harzianum ( Trichoderma harzianum ), Bacillus subtilis ( Bacillus subtilis ) and Candida Portugueseis ( Candida lusitania A compound microbial agent composed of )

[0008] Furthermore, the compound fermentation agent is composed of Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida lucida in a live cell count ratio of 1~1.5:1.2~1.5:2~3:2~3.

[0009] Furthermore, all of the above-mentioned compound fermentation agents were purchased from the Guangdong Provincial Microbial Culture Collection Center (GDMCC). Among them, 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 lucida is GDMCC NO.2.145.

[0010] The application of the above-mentioned compound fermentation agent is characterized in that it is used for fermentation to prepare antibacterial agents, specifically for fermentation to prepare compound fermentation broth.

[0011] Furthermore, the fermentation process is carried out in two steps. In the first step, Lactococcus lactis and Trichoderma harzianum are used for fermentation. After the first step, Bacillus subtilis and Candida lucida are used for the second step of fermentation.

[0012] The above-mentioned compound microbial agent is used to prepare a compound fermentation broth, characterized in that: Sophora flavescens, corn flour, Phellodendron chinense, Portulaca oleracea, and sugarcane are first fermented with Lactococcus lactis (… Lactococcus lactis ) and Trichoderma harzianum ( Trichoderma Harzian The first stage of fermentation is carried out. After sterilization, the supernatant and fermentation precipitate are collected. Bacillus subtilis ( ) is added to the fermentation precipitate. Bacillus subtilis ) and Candida Portugueseis ( Candida lusitania The second fermentation step is carried out. After the fermentation is completed, the supernatant is sterilized and collected. The two supernatants are combined to obtain a compound fermentation broth.

[0013] Furthermore, in the first fermentation step, the fermentation temperature is 30~35℃, the fermentation time is 7~9 days, and the supernatant is collected after sterilization. In the second fermentation step, the fermentation temperature is 28~32℃, the fermentation time is 5~7 days, the supernatant is collected after sterilization after fermentation, and the two supernatants are combined to obtain the fermentation broth.

[0014] Furthermore, by weight, the mixed powder contains 10-15 parts of Sophora flavescens, 20-30 parts of corn flour, 8-12 parts of Phellodendron chinense, 10-12 parts of Portulaca oleracea, 8-10 parts of sugarcane, and 50-70 parts of purified water. The mass ratio of the live counts of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis, and Candida lucida is 100:0.10-0.15:0.12-0.15:0.20-0.30:0.20-0.30.

[0015] Furthermore, the preparation of the mixed powder involves mixing Sophora flavescens, corn flour, Phellodendron chinense, Portulaca oleracea, and sugarcane, placing them in a universal grinder, grinding them through a 16-mesh sieve, and then mixing them at 20-25 rpm for 30-40 minutes.

[0016] More specifically, the application of the above-mentioned compound fermentation agent is to use the above-mentioned compound fermentation agent to ferment and prepare fermentation broth, which is then further applied to the preparation of kiwifruit preservative.

[0017] Furthermore, the kiwi fruit preservative is prepared by sequentially adding gelatin, polyethylene glycol, and polyvinyl alcohol to an ethanol solution to form a mixture, adding a compound fermentation broth to the mixture, and then filtering to obtain the biological preservative.

[0018] Furthermore, the mixture is prepared by heating a 70-80% ethanol solution to 45-50°C, adding gelatin, polyethylene glycol 2000 and polyvinyl alcohol sequentially while stirring at 40-60 rpm, and continuing to stir for 50-60 minutes.

[0019] Furthermore, the mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000, and polyvinyl alcohol is 100:20~25:15~18:10~12.

[0020] Furthermore, the mass ratio of the fermentation broth to the mixed broth is 1:3~5.

[0021] The present invention also provides a method for preparing the above-mentioned biological preservative, characterized in that: Sophora flavescens, corn flour, Phellodendron chinense, Portulaca oleracea and sugarcane are first fermented with Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida lucida. After fermentation, the mixture is sterilized and the fermentation liquid is collected. Gelatin, polyethylene glycol and polyvinyl alcohol are added to an ethanol solution in sequence to form a mixture. The fermentation liquid is added to the mixture and mixed. The mixture is then filtered to obtain the biological preservative.

[0022] Furthermore, the fermentation is divided into two steps. Specifically, Sophora flavescens, corn flour, Phellodendron chinense, Portulaca oleracea and sugarcane are mixed and pulverized into a mixed powder. After sterilization, purified water is added and stirred evenly. The first step of fermentation is carried out using Lactococcus lactis and Trichoderma harzianum at a fermentation temperature of 30-35℃ for 7-9 days. After sterilization, the supernatant is collected. Then, the fermentation precipitate is fermented in the second step using a mixture of Bacillus subtilis and Candida lucida at a fermentation temperature of 28-32℃ for 5-7 days. After the fermentation is completed, the supernatant is collected after sterilization. The two supernatants are combined to obtain the fermentation broth.

[0023] Furthermore, by weight, the mixed powder contains 10-15 parts of Sophora flavescens, 20-30 parts of corn flour, 8-12 parts of Phellodendron chinense, 10-12 parts of Portulaca oleracea, 8-10 parts of sugarcane, and 50-70 parts of purified water. The mass ratio of the live counts of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis, and Candida lucida is 100:0.10-0.15:0.12-0.15:0.20-0.30:0.20-0.30.

[0024] In this invention, *Lactococcus lactis* and *Trichoderma harzianum* are used for the first-step fermentation. *Trichoderma harzianum* decomposes the lignocellulose in the traditional Chinese medicine components, generating sugars, while *Lactococcus lactis* decomposes these sugars, providing small-molecule nutrients to *Trichoderma harzianum*, forming a cyclical fermentation system. This first-step fermentation allows antibacterial components (such as matrine and berberine) to dissolve rapidly. Simultaneously, antimicrobial peptides, organic acids, chitinase, and β-1,3-glucanase produced by the metabolism of *Lactococcus lactis* and *Trichoderma harzianum* synergistically exert their inhibitory effect on fungi. The second-step fermentation further adjusts the pH of the fermentation broth; the lower pH significantly enhances the bactericidal ability of substances such as berberine.

[0025] Furthermore, the fermentation broth is collected by sterilizing the first-step fermentation material, centrifuging it at 8000~11000 rpm for 20~30 min, and collecting the supernatant. The second-step fermentation material is also sterilized and centrifuged at 8000~11000 rpm for 20~30 min, and the supernatant is collected. The two supernatants are combined to obtain the fermentation broth.

[0026] Furthermore, the preparation of the mixed powder involves mixing Sophora flavescens, corn flour, Phellodendron chinense, Portulaca oleracea, and sugarcane, placing them in a universal grinder, grinding them through a 16-mesh sieve, and then mixing them at 20-25 rpm for 30-40 minutes.

[0027] Furthermore, the mixture is prepared by heating a 70-80% ethanol solution to 45-50°C, adding gelatin, polyethylene glycol 2000 and polyvinyl alcohol sequentially while stirring at 40-60 rpm, and continuing to stir for 50-60 minutes.

[0028] Furthermore, the mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000, and polyvinyl alcohol is 100:20~25:15~18:10~12.

[0029] Because kiwifruit has a rough, fuzzy surface, preservatives cannot adhere to it for long periods. During transportation or collisions, preservatives easily detach from the surface. Secondly, when biological preservatives are sprayed onto the surface of post-harvest kiwifruit, a large amount of antibacterial components evaporate quickly, leaving only a small amount on the surface, which cannot guarantee the continued effectiveness of the preservatives. If the biological preservative used has strong film-sealing properties, it can cause the antibacterial components to penetrate into the kiwifruit, causing erosion and affecting the flavor and taste of the kiwifruit itself.

[0030] In view of this, the present invention uses microorganisms to ferment specific Chinese herbal raw materials to prepare a fermentation broth with high antibacterial properties. This broth is placed in a mixture of gelatin, polyethylene glycol 2000, polyvinyl alcohol, and ethanol solution to form a biological preservative. When the biological preservative is sprayed onto the surface of kiwifruit, the gelatin, polyethylene glycol 2000, and polyvinyl alcohol in the preservative work synergistically to form a water-soluble soft film on the surface of the kiwifruit. The antibacterial components in the fermentation broth can effectively adhere to the soft film. On the one hand, the antibacterial components effectively inhibit bacteria, and on the other hand, they isolate the external pathogenic microorganisms from attaching and growing on the surface. In addition, the soft film's encapsulation of the antibacterial components further inhibits the penetration of the antibacterial components into the interior of the kiwifruit.

[0031] In addition, the addition of low-pH fermentation broth to the mixture regulates the film-forming rate during the film-forming process and generates interaction forces with polyethylene glycol 2000 and polyvinyl alcohol in the aqueous soft film, thereby improving the adhesion of the formed film to the surface of kiwifruit and effectively extending the action time of the preservative.

[0032] Furthermore, the mass ratio of the fermentation broth to the mixed liquid is 1:3~5. After the fermentation broth and the mixed liquid are mixed, they are filtered using an aqueous microporous membrane with a pore size of 0.45 micrometers. The filtrate is collected to obtain a biological preservative.

[0033] Most specifically, the preparation method of the above-mentioned biological preservative is characterized by comprising the following steps:

[0034] Step 1: Prepare fermentation broth

[0035] (1) Take 10-15 parts of Sophora flavescens, 20-30 parts of corn flour, 8-12 parts of Phellodendron chinense, 10-12 parts of Portulaca oleracea and 8-10 parts of sugarcane according to the weight, mix them, pulverize them and sieve them through a 16-mesh sieve to obtain powder, and mix and stir the powder at 20-25 rpm for 30-40 min to obtain mixed powder.

[0036] (2) Sterilize the mixed powder at 100 °C for 30 min. Under sterile conditions, add 50-70 parts of purified water, stir evenly, add Lactococcus lactis and Trichoderma harzianum for the first step of fermentation. The fermentation temperature is 30-35 °C and the fermentation time is 7-9 days. After the fermentation is completed, the fermentation product is obtained. Sterilize at 121 °C for 15 min. Centrifuge the fermentation product at 8000-11000 rpm for 20-30 min and collect the supernatant and centrifuged precipitate separately for later use.

[0037] (3) Add Bacillus subtilis and Candida lucida to the centrifuged precipitate for the second fermentation. The fermentation temperature is 28-32℃ and the fermentation time is 5-7 days. After the fermentation is completed, the fermentation product is obtained. The fermentation product is sterilized at 121℃ for 20-30 minutes. The supernatant is collected. The supernatant collected in the first fermentation and the supernatant collected in the second fermentation are combined to obtain the fermentation broth. The mass ratio of viable cells of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida lucida is 100:0.10-0.15:0.12-0.15:0.20-0.30:0.20-0.30.

[0038] Step 2: Synthesize biological preservatives

[0039] (1) Take an ethanol solution with a volume fraction of 70-80%, heat it to 45-50℃, and add gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence while stirring at 40-60 rpm. Continue stirring for 50-60 min to obtain a mixture. The mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:20-25:15-18:10-12.

[0040] (2) Add the fermentation broth to the mixture under stirring at 60-80 rpm, continue stirring for 30-40 min, and then filter it with a water-based microporous membrane with a pore size of 0.45 micrometers. Collect the filtrate, which is the biological preservative. The mass ratio of fermentation broth to mixture is 1:3-5.

[0041] The present invention has the following technical effects:

[0042] In this invention, a highly effective antibacterial acidic fermentation broth is prepared by combining a specific microbial compound agent with a specific fermentation step. This broth is then added to a mixture with film-forming properties to form a biological preservative. When sprayed onto the surface of kiwifruit, this biological preservative has high adhesion and can form a complete coating on the kiwifruit surface. The antibacterial components isolate pathogenic microorganisms from invading the kiwifruit and also prevent the antibacterial components themselves from eroding the kiwifruit, effectively ensuring the flavor and taste of the kiwifruit. Detailed Implementation

[0043] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed 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 description.

[0044] All microorganisms used in this invention are commercially available and were purchased from the Guangdong Provincial Microbial Culture Collection Center (GDMCC). The accession number of Lactococcus lactis is GDMCC1.1803, the accession number of Trichoderma harzianum is GDMCC NO.3.442, the accession number of Bacillus subtilis is GDMCC NO.1.131, and the accession number of Candida lucida is GDMCC NO.2.145.

[0045] Example 1

[0046] A method for preparing a biological preservative includes the following steps:

[0047] Step 1: Prepare fermentation broth

[0048] (1) Take 12 parts of Sophora flavescens, 25 parts of corn flour, 10 parts of Phellodendron chinense, 10 parts of Portulaca oleracea and 8 parts of sugarcane according to the weight, mix them, pulverize them and sieve them through a 16-mesh sieve to obtain powder. Mix the powder at 25 rpm for 30 min to obtain mixed powder.

[0049] (2) Sterilize the mixed powder at 100 °C for 30 min. Under sterile conditions, add 50-70 parts of purified water, stir evenly, add Lactococcus lactis and Trichoderma harzianum for the first step of fermentation. The fermentation temperature is 30-35 °C and the fermentation time is 8 days. After the fermentation is completed, the fermentation product is obtained. Sterilize at 121 °C for 15 min. Centrifuge the fermentation product at 10000 rpm for 25 min and collect the supernatant and centrifuged precipitate separately for later use.

[0050] (3) Add Bacillus subtilis and Candida lucida to the centrifuged precipitate for the second fermentation. The fermentation temperature is 28-32℃ and the fermentation time is 6 days. After the fermentation is completed, the fermentation product is obtained. The fermentation product is sterilized at 121℃ for 25 min. The supernatant is collected. The supernatant collected in the first fermentation and the supernatant collected in the second fermentation are combined to obtain the fermentation broth. The pH of the fermentation broth is 5.24. The mass ratio of viable cells of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida lucida is 100:0.12:0.14:0.25:0.25.

[0051] Step 2: Synthesize biological preservatives

[0052] (1) Take an ethanol solution with a volume fraction of 75%, heat it to 45°C, and add gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence while stirring at 50 rpm. Continue stirring for 55 min to obtain a mixture. The mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:22:16:11.

[0053] (2) Add the fermentation broth to the mixture under stirring at 70 rpm, continue stirring for 35 min, and then filter it with a water-based microporous membrane with a pore size of 0.45 micrometers. Collect the filtrate, which is the biological preservative. The mass ratio of fermentation broth to mixture is 1:4.

[0054] Comparative Example 1

[0055] Compared to Example 1, in preparing the fermentation broth, Lactococcus lactis and Bacillus subtilis were used for the first fermentation step, followed by Trichoderma harzianum and Candida lucida for the second fermentation step. The remaining steps were the same as in Example 1. The pH of the obtained fermentation broth was 6.18.

[0056] Comparative Example 2

[0057] Compared with Example 1, the difference is that polyethylene glycol 2000 in the mixture is replaced with polyethylene glycol 4000, while the rest of the formulation and steps are the same as in Example 1.

[0058] Comparative Example 3

[0059] Compared with Example 1, the difference is that polyethylene glycol 2000 in the mixture is replaced with polyethylene glycol 400, while the rest of the formulation and steps are the same as in Example 1.

[0060] Performance testing:

[0061] (1) Film adhesion properties:

[0062] Kiwi fruit samples harvested from the same plant with high consistency in various phenotypic indicators were divided into 4 groups. The biopreservatives prepared in Example 1, Comparative Example 1, and Comparative Example 2 were sprayed onto the surface of 3 of the kiwi fruit samples. The remaining 1 group was sprayed with the mixture prepared in Example 1 (without fermentation liquid) as a blank control. The kiwi fruit samples were then refrigerated. Every 5 days, the kiwi fruit samples were taken out to observe the water-based soft film formed by each preservative on the surface of the kiwi fruit. The samples were then rinsed lightly with water to remove the fuzz on the surface of the kiwi fruit. The content of polyvinyl glycol (400 / 2000 / 4000), polyvinyl alcohol, and antibacterial drugs on the surface of the kiwi fruit were then tested. The results are shown in Table 1.

[0063] Table 1:

[0064]

[0065] During the testing process, it was found that after the bio-preservative prepared in Example 1 was sprayed onto the surface of kiwifruit, no antibacterial drugs or film-forming components were observed to penetrate into the kiwifruit within 30 days. After spraying the bio-preservative, a thin film layer could be observed on the fuzzy surface of the kiwifruit. In the blank control group, there were no antibacterial components in the preservative film, and the adhesion of the film layer formed on the surface of the kiwifruit was worse than that in Example 1. The film layer disappeared in some areas of the kiwifruit surface after 10 days. In Comparative Examples 1 and 3, antibacterial components and film-forming components penetrated to varying degrees. Film layer peeling also occurred in Comparative Examples 1 and 2. Although the film-forming components in Comparative Example 1 were the same as those in Example 1, the changes in the fermentation process resulted in significant differences in the final fermentation broth system. The pH of the fermentation broth was significantly higher. Due to the overall higher pH value of the preservative, the film-forming rate was significantly different, which led to changes in the performance of the film layer formed on the kiwifruit surface. Furthermore, the adhesion performance of the film layer on the kiwifruit surface decreased, resulting in both film layer detachment and significant infiltration of components.

[0066] (2) Verification of preservation effect:

[0067] Take 400 fully ripe kiwifruit from the same plant that are harvested normally, and whose phenotypic indicators are highly consistent (fruit firmness 4.5 ± 0.1 kg / cm²). 2 The samples, with a weight deviation not exceeding 5g, were divided into four groups of 100 samples each: a blank control group (no preservative sprayed, left to stand normally), preservative comparison example 1 (sprayed with the preservative prepared in comparison example 1), preservative comparison example 2 (sprayed with the preservative prepared in comparison example 2), and the preservative group of this invention (sprayed with the preservative of this invention). All samples were stored at 10-20℃. After 5, 10, 20, and 30 days, all samples were removed for testing of water loss rate, rot rate, and antibacterial rate. After testing, the samples were placed back in the storage area. On day 0, the weight of each fruit was measured and recorded. The results are shown in Table 2.

[0068] Table 2:

[0069]

[0070] As shown in the table above, the blank control group, without any preservation measures, showed signs of rotting, including blackening and softening of the fruit, on the 5th day. Bacterial growth appeared on the 10th day, and by the 20th day, all kiwifruit samples had rotted. In contrast, the kiwifruit sprayed with the bio-preservative prepared in Example 1 maintained good moisture retention, with no bacterial growth detected within 30 days, and only 4% of the fruit showing slight rot. This indicates that the bio-preservative prepared in this invention has excellent preservation effects, effectively ensuring that the kiwifruit retains its moisture and nutrients while inhibiting the adhesion and erosion of pathogenic microorganisms. In Comparative Example 1, the fermentation broth preparation method is significantly different from that in this invention, resulting in different compositions of antibacterial components and different pH levels. This leads to differences in the adhesion characteristics of the bio-preservative film on the kiwifruit surface and its antibacterial effect against pathogenic microorganisms. Significant fruit rot and bacterial growth appeared on the 20th day, and the kiwifruit lost moisture faster than in Example 1. In Comparative Example 2, the polyethylene glycol 2000 in the mixture was replaced with polyethylene glycol 4000, which has a larger molecular weight. However, due to the special velvety structure of the kiwi fruit surface, the biopreservative could not adhere to the kiwi fruit surface for a long time after forming a film. As the storage time increased, the water-based soft film fell off the kiwi fruit surface, resulting in more obvious problems such as water loss and fruit rot. With the fall off of the water-based soft film, the rapid loss of the internal Chinese herbal components also led to a serious decrease in the antibacterial effect.

[0071] Example 2

[0072] A method for preparing a biological preservative includes the following steps:

[0073] Step 1: Prepare fermentation broth

[0074] (1) Take 10 parts of Sophora flavescens, 20 parts of corn flour, 8 parts of Phellodendron chinense, 10 parts of Portulaca oleracea and 10 parts of sugarcane according to the weight, mix them, pulverize them and sieve them through a 16-mesh sieve to obtain powder. Mix the powder at 20 rpm for 40 min to obtain mixed powder.

[0075] (2) Sterilize the mixed powder at 100 °C for 30 min. Under sterile conditions, add 60 parts of purified water, stir evenly, add Lactococcus lactis and Trichoderma harzianum for the first step of fermentation. The fermentation temperature is 30~35 °C and the fermentation time is 7 days. After the fermentation is completed, the fermentation product is obtained. Sterilize at 121 °C for 15 min. Centrifuge the fermentation product at 8000 rpm for 30 min and collect the supernatant and centrifuged precipitate separately for later use.

[0076] (3) Add Bacillus subtilis and Candida lucida to the centrifuged precipitate for the second fermentation. The fermentation temperature is 28~32℃ and the fermentation time is 7 days. After the fermentation is completed, the fermentation product is obtained. The fermentation product is sterilized at 121℃ for 30 min. The supernatant is collected. The supernatant collected in the first fermentation and the supernatant collected in the second fermentation are combined to obtain the fermentation broth. The pH of the obtained fermentation broth is 5.16. The mass ratio of viable cells of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida lucida is 100:0.10:0.15:0.20:0.30.

[0077] Step 2: Synthesize biological preservatives

[0078] (1) Take an ethanol solution with a volume fraction of 70%, heat it to 50°C, and add gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence while stirring at 40 rpm. Continue stirring for 50 min to obtain a mixture. The mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:20:15:10.

[0079] (2) Add the fermentation broth to the mixture under stirring at 60 rpm, continue stirring for 30-40 min, and then filter it with a water-based microporous membrane with a pore size of 0.45 micrometers. Collect the filtrate, which is the biological preservative. The mass ratio of fermentation broth to mixture is 1:3.

[0080] The biological preservative prepared in this embodiment is sprayed onto the surface of kiwifruit, forming a stable, attached water-based soft film that effectively inhibits the growth of pathogenic microorganisms on the kiwifruit surface and reduces its moisture loss. After 30 days, the water loss rate is less than 1.5%, thus extending the shelf life of the kiwifruit.

[0081] Example 3

[0082] A method for preparing a biological preservative includes the following steps:

[0083] Step 1: Prepare fermentation broth

[0084] (1) Take 15 parts of Sophora flavescens, 30 parts of corn flour, 12 parts of Phellodendron chinense, 12 parts of Portulaca oleracea and 9 parts of sugarcane according to the weight, mix them, pulverize them and sieve them through a 16-mesh sieve to obtain powder. Mix the powder at 25 rpm for 30 min to obtain mixed powder.

[0085] (2) Sterilize the mixed powder at 100 °C for 30 min. Under sterile conditions, add 70 parts of purified water, stir evenly, add Lactococcus lactis and Trichoderma harzianum for the first step of fermentation. The fermentation temperature is 30~35 °C and the fermentation time is 9 days. After the fermentation is completed, the fermentation product is obtained. Sterilize at 121 °C for 15 min. Centrifuge the fermentation product at 11000 rpm for 20 min and collect the supernatant and centrifuged precipitate separately for later use.

[0086] (3) Add Bacillus subtilis and Candida lucida to the centrifuged precipitate for the second fermentation. The fermentation temperature is 28~32℃ and the fermentation time is 5 days. After the fermentation is completed, the fermentation product is obtained. The fermentation product is sterilized at 121℃ for 20 min. The supernatant is collected. The supernatant collected in the first fermentation and the supernatant collected in the second fermentation are combined to obtain the fermentation broth. The pH of the obtained fermentation broth is 5.09. The mass ratio of viable cells of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida lucida is 100:0.15:0.12:0.30:0.20.

[0087] Step 2: Synthesize biological preservatives

[0088] (1) Take an ethanol solution with a volume fraction of 80%, heat it to 45°C, and add gelatin, polyethylene glycol 2000 and polyvinyl alcohol in sequence while stirring at 60 rpm. Continue stirring for 60 min to obtain a mixture. The mass ratio of the ethanol solution, gelatin, polyethylene glycol 2000 and polyvinyl alcohol is 100:25:18:12.

[0089] (2) Add the fermentation broth to the mixture under stirring at 80 rpm, continue stirring for 30 min, and then filter it with a water-based microporous membrane with a pore size of 0.45 micrometers. Collect the filtrate, which is the biological preservative. The mass ratio of fermentation broth to mixture is 1:5.

[0090] The biological preservative prepared in this embodiment is sprayed onto the surface of kiwifruit, forming a stable, attached water-based soft film that effectively inhibits the growth of pathogenic microorganisms on the kiwifruit surface and reduces its moisture loss. After 30 days, the water loss rate is less than 1.5%, thus extending the shelf life of the kiwifruit.

Claims

1. Use of a fermentation broth in the preparation of a kiwifruit bio-preservation agent, characterized in that: is a mixed powder of Sophora flavescens, corn flour, Phellodendron amurense, Portulaca oleracea and Saccharum officinarum, which is subjected to a first fermentation with Lactococcus lactis ( Lactococcus lactis ) and Trichoderma harzianum ( Trichoderma harzianum ), and then subjected to a second fermentation with Bacillus subtilis ( Bacillus subtilis ) and Candida utilis ( Candida lusitaniae ) after sterilization and collection of the supernatant and the fermentation precipitate, and then subjected to sterilization and collection of the supernatant after the fermentation is completed, and then subjected to a combination of the supernatants to obtain a compound fermentation liquor, and then subjected to a combination of the compound fermentation liquor with a mixed liquor obtained by sequentially adding gelatin, polyethylene glycol and polyvinyl alcohol into an ethanol solution, and then subjected to filtration to obtain the biological preservative, and the preservation number of the Lactococcus lactis is GDMCC1.1803, the preservation number of the Trichoderma harzianum is GDMCC NO.3.442, the preservation number of the Bacillus subtilis is GDMCC NO.1.131, and the preservation number of the Candida utilis is GDMCC NO.2.

145.

2. Use of a fermentation broth according to claim 1, characterized in that: The first step fermentation is carried out at a temperature of 30-35℃ for 7-9 days, and the supernatant is collected after sterilization; the second step fermentation is carried out at a temperature of 28-32℃ for 5-7 days, and the supernatant is collected after sterilization; and the two supernatants are combined to obtain the fermentation liquor.

3. Use of a fermentation broth according to claim 1 or 2, characterized in that: The mixed powder comprises, by weight, 10-15 parts of Sophora flavescens, 20-30 parts of corn flour, 8-12 parts of Phellodendron, 10-12 parts of Portulaca oleracea, 8-10 parts of sugarcane, and 50-70 parts of purified water; and the mass ratio of the viable bacteria of the mixed powder, Lactococcus lactis, Trichoderma harzianum, Bacillus subtilis and Candida lusitaniae is 100:0.10-0.15:0.12-0.15:0.20-0.30:0.20-0.

30.

4. Use according to claim 3, wherein: The mixed solution is prepared by heating an ethanol solution with a volume fraction of 70-80% to 45-50℃, and then adding gelatin, polyethylene glycol 2000 and polyvinyl alcohol under stirring at 40-60 rpm, and continuing to stir for 50-60 min.

5. The use according to claim 4, wherein: 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 liquor and the mixed solution is 1:3-5.

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