CN108497058A - Biological fresh-keeping microbial inoculum fermented by bean curd yellow slurry and application thereof in fruit and vegetable fresh-keeping - Google Patents

Abstract

The invention discloses a biological fresh-keeping bacterial agent fermented with tofu yellow pulp water and its application in fruit and vegetable fresh-keeping. The biological fresh-keeping bacterial agent is a fermentation liquid obtained by fermenting Bacillus subtilis with tofu yellow pulp water as a fermentation raw material Directly used as a fresh-keeping bacterial agent, the concentration of bacteria in the fermentation broth reaches 1×10 ¹³ ~2.7×10 ¹³ CFU/ml, the concentration of γ-polyglutamic acid reaches 30~50g/L, and the output of antimicrobial peptides reaches 1.2~2g /l. Compared with other biological preservatives, the acquisition method of the biological antistaling bacteria agent is more cost-effective and more environmentally friendly. It can use waste resources such as tofu yellow pulp water to reduce the safety of pathogenic bacteria in yellow pulp water in soybean product processing. At the same time, the content of γ-polyglutamic acid and antibacterial substances in the fermentation product is high, so it has a good fresh-keeping effect on harvested fruits and vegetables and is non-polluting and pollution-free. It has long-term development prospects and industrial utilization value.

Description

A kind of biological fresh-keeping bacteria agent fermented with tofu yellow pulp water and its application in fruit and vegetable fresh-keeping Applications

technical field

The invention belongs to the field of food preservation, and in particular relates to the application of Bacillus subtilis in post-harvest preservation of fruits and vegetables.

Background technique

The fruit and vegetable industry has long faced the problem of spoilage and deterioration of fruits and vegetables. The spoilage of fruits and vegetables after picking is mainly caused by two factors. On the one hand, the internal physiological activities of fruits and vegetables are still continuing after picking, including respiration, oxidation, and water evaporation. Wait. These life metabolic activities of fruits and vegetables lead to the loss of fruit nutrients and the shortening of storage life to a certain extent. On the other hand, the infection of pathogenic bacteria can also accelerate the spoilage of fruits and vegetables after picking. Therefore, inhibiting the respiration of fruits and vegetables can slow down their spoilage. Post-harvest spoilage of fruits and vegetables is usually dealt with by chemical preservatives and physical methods, but these methods are costly and cannot be guaranteed to be non-toxic.

Coating fresh-keeping technology is to coat a layer of film on the surface of fruits and vegetables to provide selective gas barrier, moisture barrier, content loss resistance and harmful influence of the external environment, inhibit breathing, delay ripening and aging, and inhibit the growth of microorganisms on the surface , improve storage quality and other functions, so as to achieve the purpose of fruit and vegetable preservation.

Bacillus subtilis is a microorganism with a wide range of functions. It can secrete a large amount of γ-polyglutamic acid, produce heat-resistant and stress-resistant spores, and secrete various antibacterial substances in the early stage of spore formation. It has specific control effect on pathogenic fungi.

Using the characteristics of γ-polyglutamic acid in the fermentation liquid of Bacillus subtilis to preserve the freshness of picked fruits and vegetables by coating film can effectively avoid the pollution of chemical preservatives, inedible and other problems, and has attracted much attention in the field of fruit and vegetable preservation. "201410452988.5 A γ-polyglutamic acid-Cu(II) preservative and its application in fruit and vegetable preservation" discloses the application of γ-polyglutamic acid fermented by Bacillus licheniformis in fruit and vegetable preservation.

Patent 200910192821.9 is a biopolymer coating preservative for lychees. It is found that γ-polyglutamic acid or γ-polyglutamate compounded and then coated can slow down the loss of moisture from the lychee peel and effectively prolong the lychee preservation period. Its biological antistaling bacteria agent has defects such as long fermentation period, complicated processing technology and high operating cost. At present, there are cumbersome processing techniques and high costs in the existing fresh-keeping technologies, and it is urgent to develop new biological preservatives suitable for application.

Contents of the invention

The invention utilizes the fresh-keeping effect of γ-polyglutamic acid in the Bacillus subtilis fermentation liquid and the antibacterial substance secreted by bacteria to keep fruits and vegetables fresh, effectively prolonging the fresh-keeping time of fruits and vegetables.

The Bacillus subtilis used in the present invention is fermented and grown with tofu yellow pulp water as a nitrogen source, which effectively reduces the cost of raw materials and realizes waste utilization, and has the functions of non-toxicity and probiotics.

The purpose of the present invention is achieved through the following technical solutions:

A biological fresh-keeping bacterial agent for keeping vegetables and fruits fresh, characterized in that the biological fresh-keeping bacterial agent is directly used as a fresh-keeping agent by the fermentation liquid obtained by fermentation of Bacillus subtilis in a fermentation medium with tofu yellow pulp water as raw material Bacteria agent, the concentration of bacteria in the fermentation broth reaches 1×10 ¹³ ~2.7×10 ¹³ CFU/ml, the concentration of γ-polyglutamic acid reaches 30~50g/L, and the output of antimicrobial peptide reaches 1.2~2g/l.

The fermentation medium is: carbon source 10-20% (w/v), calcium chloride 0.01-0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), dipotassium hydrogen phosphate 0.05% (w/v), magnesium sulfate 0.01% (w/v), sodium glutamate 2.5% (w/v), ammonium chloride 0.3% (w/v), the balance is tofu yellow pulp water, initial pH value It is 6.5~7.5.

The preparation method of the biological fresh-keeping agent is as follows: inoculate the Bacillus subtilis strain in the liquid medium, inoculate the shaker at 30°C for 8-14 hours, and obtain the seed liquid: inoculate the seed liquid according to the volume ratio of 5%-20%. Into the fermentation medium, 180rpm/min~220rpm/min fermentation culture, after the fermentation, the fermentation liquid is obtained, which is the biological fresh-keeping agent;

The composition of the liquid medium: glucose 10-20% (w/v), soybean peptone 10-15% (w/v),

Calcium chloride 0.01~0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), dipotassium hydrogen phosphate 0.05% (w/v). Magnesium sulfate 0.01%, (w/v), the balance is distilled water, the initial pH value is 6.5~7.5;

The fermentation medium is: glucose 10-20% (w/v), calcium chloride 0.01-0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), dipotassium hydrogen phosphate 0.05% ( w/v), magnesium sulfate 0.01% (w/v), sodium glutamate 2.5% (w/v), ammonium chloride 0.3% (w/v), the balance is tofu yellow pulp water, and the initial pH value is 6.5~7.5.

The fermentation culture temperature is 10-50°C, and the culture time is 24-30h. The preferred culture temperature is 25°C±2°C, and the culture time is 48h.

The application of the biological fresh-keeping bacterium agent in the fresh-keeping of vegetables and fruits. Spray the biological antistaling agent on the picked fruits and vegetables, and the amount of spraying should be such that the surface of the fruits and vegetables is completely wet.

The specific operation steps are as follows:

Preparation of biological preservatives: Inoculate the Bacillus subtilis strain into the Bacillus subtilis liquid medium, and culture it on a shaker at 30°C for 15-18 hours to obtain the seed liquid: 5%-20% by volume Inoculate the seed solution into a large amount of fermentation medium, and cultivate at 180~220rpm/min: directly collect the fermentation solution after the fermentation is completed, and obtain the biological fresh-keeping bacteria agent.

Spray the biological fresh-keeping bacteria agent on the picked fruits and vegetables, and the amount of spraying is enough to completely wet the surface of the fruits and vegetables to achieve freshness. It is common knowledge that fruits and vegetables after picking are spoiled due to respiration and fungal viruses. The present invention utilizes the characteristics that Bacillus subtilis ( Bacillus subtilis ) can secrete γ-polyglutamic acid and antibacterial substances, and ferment the fruits and vegetables after picking. The smearing treatment overcomes the pollution and poisonous effect brought by chemical preservatives, and realizes pollution-free treatment. Moreover, the fermentation raw materials are waste resources, which are more environmentally friendly and healthy.

The present invention utilizes the characteristics of γ-polyglutamic acid and antibacterial peptide produced by Bacillus subtilis to preserve the freshness of harvested fruits and vegetables, sprays the fermented liquid on the surface of harvested fruits and vegetables to form a layer of coating film, inhibits the respiration on the surface of fruits and vegetables, and keeps The humidity of fruits and vegetables is improved, and Bacillus subtilis can secrete antimicrobial substances to inhibit the growth of pathogenic bacteria, which plays a positive role in the preservation of harvested fruits and vegetables. The biological antistaling bacterial agent prepared by the present invention has a high thalline concentration and good inhibitory effect on pathogens. And the production cost is low, the present invention is fermented raw material with bean curd yellow slurry water, there are various amino acids in the tofu yellow water, organic acid comprises glutamic acid, glycine etc., and some small peptides and metal ions are beneficial to γ ~ polyglutamic acid The synthesis of acid, and because of its rich nutrition, it also has a promoting effect on the increase of the concentration of bacteria. It has low cost, simple treatment process, shorter fermentation cycle than other biological preservatives, high concentration of bacteria, and good antibacterial effect, etc. features. The spoilage of fruits and vegetables is caused by many factors, among which the four factors of physics, chemistry, enzymes and microorganisms are the main factors, among which the role of microorganisms is the most serious. The physical fresh-keeping method is costly and energy-intensive. Although the chemical fresh-keeping method can kill pathogenic bacteria and ensure antiseptic fresh-keeping effects, the abuse of chemically synthesized agents will endanger human health, and more seriously, it will cause cancer. At the same time, it will cause pesticide residues and pollute the environment. And other issues. These problems are successfully avoided by adopting the biological antistaling agent. The present invention directly produces the biological antistaling agent with the fermentation liquid, which avoids the cumbersome treatment process in other antistaling technologies, and the cost is relatively low and the application effect is good.

The use effect of the invention is remarkable, and the storage life of the picked fruits and vegetables can be increased by more than 1.8 times.

Bacillus subtilis ( Bacillus subtilis ) is a recognized non-pathogenic bacterium, approved by the Ministry of Agriculture as a probiotic that is allowed to be used in feed additives, so the preservative developed by Bacillus subtilis ( Bacillus subtilis ) has biological safety and is non-toxic , pollution-free, green and suitable for promotion.

The fermented liquid prepared by the present invention can be directly used to prepare biological fresh-keeping bacteria agent, and it is non-toxic and harmless and can be eaten directly. The discarded tofu yellow pulp water is used as the fermentation raw material, which solves the problem of difficult tofu water treatment in tofu factories, and at the same time greatly The production cost is greatly reduced, which is beneficial to the development of industrialization, and the cell concentration of the biological antistaling agent prepared by the present invention can reach 10 ¹³ , which is higher than the biological antistaling agent on the market, and has a higher inhibitory effect on harmful bacterial strains more obvious. At present, the commercially available biological fresh-keeping bacteria agents have defects such as long fermentation period, complicated treatment process, and high operating cost. Compared with other biological fresh-keeping bacteria agents in the market, the present invention has short fermentation cycle, low energy consumption, simple treatment process, and can directly prepare biological The fresh-keeping agent does not need to increase the production cost, is highly efficient and energy-saving, and has a good fresh-keeping effect.

Beneficial effect:

The biological antistaling agent of the invention is simple to prepare, and the fermentation liquid is directly used for smearing treatment, which overcomes the pollution and poisonous effect brought by the chemical antistaling agent, and realizes pollution-free treatment. Moreover, the fermentation raw materials are waste resources, which are more environmentally friendly and healthy. The use effect is remarkable, and the storage life of the picked fruits and vegetables can be increased by more than 1.8 times.

Detailed ways

The Bacillus subtilis ( Bacillus subtilis ) strain described in the following examples, No. CGMCC No. 13932, is from China General Microorganism Culture Collection and Management Center. Tofu yellow slurry water is the yellow slurry water leached from tofu production (provided by the tofu workshop in the vegetable market in Pukou District, Nanjing).

Example 1

Preparation of Biological Preservative with Bacillus subtilis No. CGMCC No.13932

Preparation of biological fresh-keeping agent: inoculate Bacillus subtilis strains into Bacillus subtilis liquid culture medium, culture on a shaking table at 30°C for 8-14 hours to obtain seed liquid: inoculate the seed liquid at a volume ratio of 10% Inoculate into a large amount of fermentation medium, cultivate at 180~220rpm/min: directly collect the fermentation liquid after the fermentation, the concentration of bacteria in the fermentation liquid reaches 10 ¹³ CFU/ml, the concentration of γ-polyglutamic acid reaches 30g/L, The antimicrobial peptide production reaches 1.2~2g/l, and the fermentation broth is directly used as a biological fresh-keeping agent.

The composition of the seed medium: glucose 10% (w/v), soybean peptone 15% (w/v), calcium chloride 0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), Dipotassium hydrogen phosphate 0.05% (w/v). Magnesium sulfate 0.01%, (w/v), the balance is distilled water, the initial pH value is 6.5~7.5.

Mass fermentation medium composition: glucose 10% (w/v), calcium chloride 0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), dipotassium hydrogen phosphate 0.05% (w/v). Magnesium sulfate 0.01% (w/v), sodium glutamate 2.5% (w/v), ammonium chloride 0.3% (w/v), the balance is tofu yellow pulp water, and the initial pH value is 6.5~7.5.

Example 2

Effects of Biological Preservatives on Preservation of Bananas after Picking

Buy bananas from the market that are ripe, have no mechanical damage, are of equal size, and have no disease spots and good color. After fully shaking the biological antistaling agent prepared in Example 1, spray it on the surface of the fruit to be treated with a watering can. Sterilized water was used as the control group, and the treatment group and the control group were stored under the same conditions (25-30°C). The freshness of the fruits was regularly observed, and the freshness was graded according to the following standards, and the freshness preservation rate at different time points was calculated (all Fruit freshness mean/highest freshness series), the results are shown in Table 1

Banana freshness grading standard (based on personal subjective judgment): Grade 0: Severely rotten; Grade 1: Beginning to rot (yellow area of the peel > 50%), can be pinched with hands, and the peel shrinks; Grade 2: Partial yellowing and softening, fruit feels hard; Grade 3: partial yellowing (yellow area of pericarp<50%), uneven color; Grade 4: basically normal, slightly yellow skin (yellow area<10%); Grade 5: fruit Green, hard texture, no disease spots, white flesh incision.

Table 1

The treated bananas were stored at 25-30°C, and the average freshness level was still close to the highest freshness level within 15 days, that is, the freshness rate reached more than 96% within 15 days, while the untreated bananas were stored under the same storage conditions. , after the 5th day, its fresh-keeping rate drops below 90%, indicating that the fresh-keeping time of the processed bananas has been extended by more than 3 times.

Example 3

Post-harvest Fresh-keeping Effect of Biological Preservatives on Cucumber

Buy cucumbers with pure varieties, no mechanical damage, equal size, no disease spots and good color from the market. After the biological antistaling agent prepared in Example 1 is fully shaken, spray it on the surface of the vegetables to be treated with a watering can. The amount of spraying is advisable to completely wet the vegetables. Sterilized water was used as the control group, and the treatment group and the control group were stored under the same conditions (25-30°C). The freshness of the fruits was regularly observed, and the freshness was graded according to the following standards, and the freshness preservation rate at different time points was calculated (all Vegetable freshness average/highest freshness series), the results are shown in Table 2

Cucumber freshness grading standard (based on personal subjective judgment): Grade 0: severe rot; Grade 1: beginning to rot (lesion area > 50%), can be pinched with hands, and the skin is shriveled; Grade 2: Local lesions (cucumber lesion area <50%), uneven color; Grade 3: basically normal, slightly discolored cucumbers (yellow area <10%); Grade 4 cucumbers are uniform in color, green and plump.

Table 2

The average grade of freshness of treated cucumbers stored at 25-30°C within 15 days was still close to the highest grade of freshness, that is, the freshness rate reached more than 90% within 15 days, while untreated cucumbers were stored under the same storage conditions , after the 5th day, its fresh-keeping rate drops below 90%, indicating that the fresh-keeping time of the processed bananas has been extended by more than 3 times.

Example 4

Postharvest preservation effect of biological preservatives on apples

Buy apples with pure color, no mechanical damage, equal size and no lesions from the market. After the biological antistaling agent prepared in Example 1 is fully shaken, spray it on the surface of the apples to be treated with a watering can. The amount of spraying is preferably to completely wet the surface of the apples. The sterilized water was used as the control group, and the treatment group and the control group were stored under the same conditions (25-30°C), the freshness of the fruit was observed regularly, and the freshness was graded according to the following standards, and the freshness preservation rate at different time points was calculated ( The average freshness/highest freshness series of all fruits), the results are shown in Table 3

Grading standards for apple freshness (based on personal subjective judgment): Grade 0: severe rot; Grade 1: beginning to rot (the lesion area of the peel > 50%), and the peel shrinks; Grade 2: partial blackening (the black area of the peel <50%), the color is uneven; Grade 3: There are black spots locally (the black area of the peel is <5%), and the color of Grade 4 apples is intact and bright.

table 3

The treated apples were stored at 25-30°C, and the average freshness level was still at the highest freshness level within 30 days, that is, the freshness rate reached 100 within 30 days, while the untreated apples were stored under the same storage conditions, the 20th day. Two days later, its preservation rate dropped below 80%, indicating that the preservation time of the processed apples was extended by more than 1.8 times.

Implementation Example 5

Post-harvest Fresh-keeping Effect of Biological Preservatives on Lettuce

Buy lettuce from the market that is neat, with fresh and tender leaves, no disease spots, no pests, no dry leaves, and no rotten lettuce. After the biological antistaling agent prepared in Example 1 is fully shaken, spray it on the surface of the lettuce to be treated with a watering can. The amount of spraying is preferably to completely wet the surface of the lettuce. The sterilized water was used as the control group, and the treatment group and the control group were stored under the same conditions (25-30°C), the freshness of the fruit was observed regularly, and the freshness was graded according to the following standards, and the freshness preservation rate at different time points was calculated ( The average freshness of all vegetables/the highest freshness series), the results are shown in Table 4

Lettuce freshness grading standard (based on personal subjective judgment): Grade 0: severe rot; Grade 1: (lesion area of lettuce >50%); Grade 2: local turning black (lesion area of lettuce <50%), color Uniform; grade 3: partial yellowing, dehydration (lettuce lesion area <5%), grade 4 lettuce leaves are fresh and tender, no disease spots, no dry leaves, no rotten leaves.

Table 4

The average grade of freshness of treated lettuce stored at 25-30°C was close to the highest grade of freshness within 8 days, that is, the freshness rate reached more than 90% within 8 days, while the untreated lettuce under the same storage conditions, After the 4th day, its fresh-keeping rate dropped to 80%, indicating that the fresh-keeping time of the treated lettuce was extended by more than 2 times.

Implementation Example 6

Post-harvest Fresh-keeping Effect of Biological Preservatives on Bitter Gourd

Buy fresh from the market, without mechanical damage, without the balsam pear of scab, after the biological antistaling agent prepared by implementation example 1 is fully shaken, spray on the balsam pear surface to be treated with a watering can, and the amount of spraying is advisable with fully wetting the balsam pear surface, Put it in a fresh fresh-keeping bag after treatment, and spray the same volume of sterilized water as the control group at the same time. The treatment group and the control group are stored under the same conditions (25~30°C), and the freshness of bitter melon is regularly observed. According to the following standards Carry out freshness grading to it, calculate the freshness preservation rate of different time points (the freshness average value/highest freshness series of all vegetables), the result is shown in Table 5

Grading standards for the freshness of bitter gourd (based on personal subjective judgment): Grade 0: severe rot; Grade 1: beginning to rot (lesion area > 50%), which can be pinched with hands, and the melon skin shrinks; Grade 2: Partial lesions (bitter melon lesion area<50%), uneven color; grade 3: basically normal, slightly discolored bitter gourd (yellow area<10%); grade 4 bitter gourd with uniform color, green and plump.

table 5

The treated bitter gourd was stored at 25-30°C, and its average freshness grade was close to the highest freshness grade within 8 days, that is, the freshness rate reached more than 90% within 8 days, while the untreated lettuce was stored under the same storage conditions. After the 4th day, its fresh-keeping rate dropped to 50%, indicating that the fresh-keeping time of the treated bitter gourd was extended by more than 2 times.

Implementation Example 7

Study on the preservation effect of Bacillus subtilis fermentation broth under different fermentation methods

The bacterial strains used in the present invention are compared and fermented under different fermentation methods. The process of fermentation method 1 is as follows: Bacillus subtilis bacterial strains are inoculated in liquid seed culture medium, and the described seed culture medium composition: glucose 10 ~ 20% (w/ v), soybean peptone 10~15% (w/v), calcium chloride 0.01~0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), dipotassium hydrogen phosphate 0.05% (w/v) ). Magnesium sulfate 0.01%, (w/v), the balance is distilled water, the initial pH value is 6.5~7.5. Cultivate on a shaker at 30°C for 8~14h, and the rotation speed is 220r/min to obtain seed liquid, according to the volume of 5%~20% Than inoculate the seed solution into a large amount of fermentation medium, the composition of a large amount of fermentation medium: glucose 10% (w/v), soybean peptone 15% (w/v), calcium chloride 0.1% (w/v), diphosphate Potassium hydrogen 0.05% (w/v), dipotassium hydrogen phosphate 0.05% (w/v). Magnesium sulfate 0.01% (w/v), sodium glutamate 2.5% (w/v), ammonium chloride 0.3% (w/v), the balance is pure water, and the initial pH value is 6.5~7.5. The rotational speed was 220r/min, and the shaker was cultivated at 30°C for 28h, and fresh-keeping treatment was performed on the picked vegetables.

The process of fermentation method 2 is basically the same as that of fermentation method 1, except that the nitrogen source in the large-scale fermentation medium of the shake flask is replaced by tryptone 10-15% (w/v).

The process of fermentation method 3 was basically the same as that of fermentation method 1, except that 0.1% (w/v) glutamic acid was added to the large-scale fermentation medium of shake flasks.

The process of fermentation method 4 is basically the same as that of fermentation method 1, except that the nitrogen source in the large-scale fermentation medium in shake flasks is replaced by 10-15% (w/v) of yeast powder.

The processes of fermentation methods 5, 6, and 7 were basically the same as those of fermentation method 1, except that 0.05%, 0.08%, and 0.1% (w/v) Tween~80 was added to the large-scale fermentation medium in shake flasks.

The process of fermentation method 8 is basically the same as that of fermentation method 1, except that soybean peptone is not added to the large-scale fermentation medium of the shake flask, and the balance is 50% tofu yellow pulp water.

The process of fermentation method 9 is basically the same as that of fermentation method 1, except that soybean peptone is not added to the large-scale fermentation medium of shake flasks, and the balance is pure tofu yellow pulp water.

The fermented liquid prepared by fermentation method 1, 2, 3, 4, 5, 6, 7, 8, 9 is made into biological fresh-keeping bacteria agent according to the method in embodiment example 1, according to embodiment 2, 3, 4, 5, 6 The process in the process is to preserve the freshness of harvested fruits and vegetables. The results are shown in Table 6, and the fresh-keeping effect of fermentation method 9 is the best.

Grading standards, based on personal senses: graded according to excellent > good > medium > poor.

Table 6

Example 8

Bacteriostasis of Bacillus subtilis fermented liquid prepared from tofu yellow pulp water on different bacteria

Preparation of crude extract of fermentation broth:

Inoculate the Bacillus subtilis strain into the Bacillus subtilis liquid medium, and culture it on a shaker at 30°C for 8-14 hours to obtain the seed liquid: inoculate the seed liquid into a large amount of fermentation medium at a volume ratio of 10% Medium, 180~220rpm/min culture: collect the fermentation broth directly after the fermentation, the bacterial cell concentration in the fermentation broth reaches 10 ¹³ CFU/ml, the concentration of γ~polyglutamic acid reaches 30g/L, and the output of antimicrobial peptide reaches 1.2~ 2g/l. . Centrifuge the fermentation broth at 4°C and 10000 rpm/min/min for 10 min, collect the supernatant; Centrifuge at 10,000 rpm/min/min for 10 min, collect the precipitate, add 5 mL of methanol solution to the precipitate, stir for 20 min, let stand at 4°C for 10 h, centrifuge at 10,000 rpm/min/min for 10 min at 4°C, and obtain methanol extract thing. After vacuum freeze-drying, antibacterial substances were obtained and stored at 20°C.

The composition of the seed medium: glucose 10% (w/v), soybean peptone 15% (w/v), calcium chloride 0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), Dipotassium hydrogen phosphate 0.05% (w/v). Magnesium sulfate 0.01%, (w/v), the balance is distilled water, the initial pH value is 6.5~7.5.

Mass fermentation medium composition: glucose 10% (w/v), calcium chloride 0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), dipotassium hydrogen phosphate 0.05% (w/v). Magnesium sulfate 0.01% (w/v), sodium glutamate 2.5% (w/v), ammonium chloride 0.3% (w/v), the balance is tofu yellow pulp water, and the initial pH value is 6.5~7.5.

Table 7 Antibacterial effect of Bacillus subtilis fermented with tofu yellow water

pathogenic bacteria Inhibition zone (mm) Escherichia coli 45.3±0.4 Yeast 39.8±0.3 Staphylococcus aureus 44.2±0.2 Acetobacter 46.8±0.5

Determination of antibacterial effect: the isolated and purified antimicrobial peptides were prepared to 5.0 μg/ml with sterile water, and the fungal plate confrontation experiment was used (the experimental process was as follows: the pathogenic fungal block was placed in the center of a 90 mm petri dish containing 15ml of PDA medium , punch a hole at a distance of 25 mm, inject the sample to be tested into the hole, incubate at 28°C for 3 days, and observe the antibacterial efficiency) and the bacterial inhibition zone experiment (the experimental process is as follows: prepare an aqueous solution of Bacillus subtilis, containing 120 bacteria per milliliter First, add 1ml of sterile aqueous solution of Bacillus subtilis to 15ml of LB medium, wait for the medium to solidify and make a hole on it, inject the sample to be tested into the hole, incubate at 37°C for 8 hours, and observe the size of the inhibition zone) to measure the biological activity .

Claims (6)

1. a kind of bio-preserving fungus agent to be fermented using bean curd yellow pulp water, which is characterized in that the bio-preserving fungus agent is by withered grass Bacillus is being directly used as preserving fungus agent using bean curd yellow pulp water as the zymotic fluid that fermentation raw material ferments, in the zymotic fluid Cell concentration reaches 1 × 10¹³~2.7×10¹³CFU/ml, gamma-polyglutamic acid concentration reach 30 ~ 50g/L, and yield of antibacterial peptides reaches 1.2~2g/l。

2. a kind of bio-preserving fungus agent to be fermented using bean curd yellow pulp water according to claim 1, which is characterized in that described Fermentation medium is：Glucose 10 ~ 20%（w/v）, calcium chloride 0.01 ~ 0.1%（w/v）, potassium dihydrogen phosphate 0.05%（w/v）, phosphoric acid Hydrogen dipotassium 0.05%（w/v）, magnesium sulfate 0.01%（w/v）, sodium glutamate 2.5%（w/v）, ammonium chloride 0.3%（w/v）Surplus is bean curd Yellow serofluid, initial pH value are 6.5 ~ 7.5.

3. a kind of bio-preserving fungus agent to be fermented using bean curd yellow pulp water according to claim 1, which is characterized in that described The preparation method of bio-preserving fungus agent is：Bacillus subtilis strain is inoculated in fluid nutrient medium, 30 DEG C of inoculation shaking table trainings 8 ~ 14h is supported, seed liquor is obtained：Seed liquor is inoculated into fermentation medium according to 5% ~ 20% volume ratio, 180 ~ 220rpm/ Min/min fermented and cultureds obtain zymotic fluid, as bio-preserving fungus agent after fermentation；

The Liquid Culture based component：Glucose 10 ~ 20%（w/v）, soy peptone 10 ~ 15%（w/v）, calcium chloride 0.01 ~ 0.1%（w/v）, potassium dihydrogen phosphate 0.05%（w/v）, dipotassium hydrogen phosphate 0.05%（w/v）, magnesium sulfate 0.01%,（w/v）, surplus is Distilled water, initial pH value are 6.5 ~ 7.5.

4. a kind of bio-preserving fungus agent to be fermented using bean curd yellow pulp water according to claim 3, which is characterized in that fermentation Cultivation temperature is 10 ~ 50 DEG C, and incubation time is 24 ~ 30h.

5. application of the bio-preserving fungus agent described in claim 1 in vegetable melon and fruit is fresh-keeping.

6. application according to claim 5, which is characterized in that bio-preserving fungus agent is sprayed onto on the fruits and vegetables after picking, The amount of spraying is moistened completely with fruit and vegetable surfaces.