阅读说明：本技术 一种利用芽孢杆菌发酵产物复配的水果保鲜剂及其应用 (Fruit preservative compounded by using bacillus fermentation product and application thereof ) 是由 郝宁 史靓 郭格格 李涛 刘兆星 欧阳平凯 于 2021-08-23 设计创作，主要内容包括：本发明公开了一种利用芽孢杆菌发酵产物复配的水果保鲜剂及其应用,所述芽孢杆菌为枯草芽孢杆菌、地衣芽孢杆菌、解淀粉芽孢杆菌、凝结芽孢杆菌、巨大芽孢杆菌、短小芽孢杆菌六种芽孢杆菌。将六种芽孢杆菌分别单独发酵,再将发酵液用硫酸铵沉淀,溶于无菌水后冷冻干燥,再按照质量比(1-5):(1-5):(1-5):(1-5):(1-5):(1-5)复配制成水果保鲜剂。其为天然益生菌微生物发酵获得,用在新鲜水果上有明显的抑菌作用,安全有效地降低了腐败速率,延长了水果的贮藏期。(The invention discloses a fruit preservative compounded by using a bacillus fermentation product and application thereof, wherein bacillus is six types of bacillus including bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus. Respectively fermenting the six bacillus, precipitating the fermentation liquor by ammonium sulfate, dissolving in sterile water, freeze-drying, and compounding according to the mass ratio of (1-5) to prepare the fruit preservative. The probiotic microorganism is obtained by fermenting natural probiotic microorganisms, has obvious bacteriostatic action when being used on fresh fruits, safely and effectively reduces the putrefaction rate, and prolongs the storage period of the fruits.)

1. A fruit preservative compounded by using bacillus fermentation products is characterized by being prepared by compounding bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus fermentation products in a mass ratio of (1-5) to (1-5) after vacuum freeze drying.

2. The fruit preservative compounded by using the Bacillus fermentation products as claimed in claim 1, wherein the Bacillus subtilis is Bacillus subtilis with the collection number of CGMCC No.13932, the Bacillus licheniformis (Bacillus licheniformis) with the collection number of CGMCC No.9688, the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) with the collection number of CGMCC No.15732, the Bacillus coagulans (Bacillus coagulans) with the collection number of CGMCC No.19487, the Bacillus megaterium (Bacillus megaterium) with the collection number of CGMCC No.1.10466 and the Bacillus pumilus (Bacillus pumilus) with the collection number of CGMCC No. 1.8167.

3. The use of the preservative of claim 1 for the preservation of fruits and vegetables.

4. The application of claim 3, wherein the preservative is prepared into a solution of 0.1-10 mg/ml by using sterile water, and the solution is sprayed or soaked on the fruits for 10-30 min.

5. The preparation method of the fruit preservative compounded by using the bacillus fermentation product as claimed in claim 1 comprises the following steps:

(1) respectively coating the frozen and preserved bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus in a fresh LB solid culture medium, and performing constant-temperature overnight activation culture at the temperature of 27-45 ℃;

(2) respectively selecting a ring from activation plates of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus, inoculating the ring into a seed liquid culture medium, and independently culturing at the temperature of 27-45 ℃ and the rpm of 200 +/-20 for 8-12 hours to obtain seed liquid;

(3) respectively inoculating the seed liquid obtained in the step (2) into a fermentation liquid culture medium according to the inoculation amount of 3-7%, independently culturing for 10h at the temperature of 27-45 ℃ and the rpm of 100 +/-20, and increasing the rotating speed to 200 +/-20 rpm to continuously culture for 10-18h to obtain a bacterial suspension;

(4) centrifuging the bacterial suspension obtained in the step (3), taking supernatant and filtering to obtain a fermentation filtrate without thalli;

(5) adjusting the filtrate obtained in the step (4) to be neutral by using HCl solution, adding solid ammonium sulfate while stirring, and standing overnight in a refrigerator at 4 ℃ when the saturation reaches 80%;

(6) centrifuging at 4000r/min at normal temperature for 20min, collecting precipitate, dissolving in sterile water, vacuum freeze-drying, and mixing according to the mass ratio of (1-5) to obtain the compound preservative.

6. The preparation method of the fruit preservative compounded by using the bacillus fermentation product as claimed in claim 5, wherein the seed liquid culture medium is: 2-10g/L beef extract, 5g/L sodium chloride, 5-15g/L peptone and 7.0-7.2 pH.

7. The preparation method of the fruit preservative compounded by using the bacillus fermentation product according to claim 5, wherein the fermentation liquid culture medium is: 20-40g/L of molasses, 5-20g/L of bean flour, 2-10g/L of corn steep liquor powder, 10-30g/L of yeast extract, 1-5g/L of urea, 1-10g/L of sodium chloride, 0.1-1g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate monohydrate, 1-3g/L of zinc sulfate, 0.5-1.5g/L of monopotassium phosphate and pH of 6.8-7.5.

8. The preparation method of the fruit preservative compounded by using the bacillus fermentation product according to claim 5, wherein the vacuum freeze-drying condition is that the temperature is-40 ℃, the vacuum degree is 100Pa, and the drying time is 35-40 h.

Technical Field

The invention belongs to the technical field of bacillus application, and particularly relates to a fruit preservative compounded by bacillus fermentation products and application thereof.

Background

Fruits are easy to rot and deteriorate before and after being harvested due to physiological aging, microbial invasion, mechanical damage and other reasons, and are not easy to store and transport across regions. With the improvement of living standard and the transformation of consumption concept, people have higher and higher requirements on fruit preservation, and the problems of keeping the fresh quality of fruits and prolonging the storage time of the fruits become common concerns of current consumers. According to investigation, due to the technical problem of storage and preservation, the annual loss rate of fruits in China is about 25%.

The research personnel can carry out deep research on the fresh-keeping technologies such as ice temperature fresh-keeping, modified atmosphere storage, coating fresh-keeping, ozone and carbon dioxide treatment and the like, and make certain progress. To date, effective methods for preserving fruit are generally divided into two types, physical and chemical: the chemical method generally uses a chemical preservative to perform treatments such as soaking or film coating on fruits, so that serious hidden dangers exist for human health; physical methods generally require specialized equipment, are complex to operate, are technically robust, and are costly to operate and maintain. The biological fresh-keeping substance is directly derived from the self-constituent components of organisms or metabolites thereof, has the characteristics of no smell, no toxicity, safety and the like, and can be generally biodegraded without causing secondary pollution. Therefore, research on biological preservatives is an important issue at present.

The biological antistaling agent of animal and plant sources is limited by raw materials and has higher cost, so the antistaling agent is often prepared by taking a microorganism metabolite as a raw material in the industrial development so as to achieve the effect of 'bacteria inhibition'.

The bacillus is a very representative biocontrol bacterium, has the advantages of fast growth and reproduction, easy survival, strong stress resistance, wide nutrition spectrum and the like, and mainly comprises bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus megaterium, bacillus pumilus, bacillus mycoides, bacillus coagulans and the like. They can colonize the surfaces of fruits and vegetables, compete for nutrients with pathogenic bacteria, and secrete a plurality of important metabolites, wherein polypeptide active substances with high-efficiency bacteriostatic action are not lacked. At present, the application of bacillus licheniformis, bacillus subtilis and bacillus coagulans in food preservation has been reported, but the problems of single strain, difficult long-term effective storage of prepared bacteriostatic agent and incapability of realizing breakthrough improvement of bacteriostatic efficiency exist.

According to the invention, the six bacillus fermentation products are compounded and freeze-dried to prepare the dry powder state easy to store, so that the corrosion resistance can be improved on the basis of safety and no toxicity, and the fruit preservation and fresh keeping are facilitated. In addition, the fruit preservative adopts a spraying or soaking use method, so that the mechanical damage of fragile fruits such as strawberries can be reduced, and the integrity is kept.

Disclosure of Invention

The invention aims to provide a fruit preservative compounded by using a bacillus fermentation product and application thereof, which can effectively inhibit the rotting phenomenon caused by mechanical damage in the transportation process and microbial propagation in the preservation process of fresh fruits so as to maintain the quality of the fruits and prolong the storage time.

The invention provides a fruit preservative which is prepared by vacuum freeze-drying and compounding fermentation products of six bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus according to the mass ratio of (1-5) to (1-5), and is applied to the preservation of fruits such as strawberries, grapes and winter jujubes.

The inventor firstly finds that the bacteriostatic and fresh-keeping effects of the combined use of fermentation products of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus on fruits are far better than that of single use.

The invention is realized by the following technical scheme:

a fruit preservative compounded by bacillus fermentation products is prepared by compounding six bacillus fermentation products of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus after vacuum freeze drying.

And after the fermentation products of the bacillus subtilis, the bacillus licheniformis, the bacillus amyloliquefaciens, the bacillus coagulans, the bacillus megaterium and the bacillus pumilus are subjected to vacuum freeze drying, the mass ratio of the fermentation products is (1-5): (1-5):(1-5):(1-5): (1-5) compounding (1-5); preferably according to the mass ratio of 2:1:2:1:1: 1.

The Bacillus subtilis is Bacillus subtilis with a collection number of CGMCC No.13932, the Bacillus licheniformis with a collection number of CGMCC No.9688, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens with a collection number of CGMCC No.15732, the Bacillus coagulans is Bacillus coagulans with a collection number of CGMCC No.19487, the Bacillus megaterium is Bacillus megaterium with a collection number of CGMCC No.1.10466, and the Bacillus pumilus is Bacillus pumilus with a collection number of CGMCC No. 1.8167.

The preservative is applied to the preservation of fruits and vegetables.

The fruit preservative compounded by using the bacillus fermentation product is obtained by the following steps:

(1) respectively coating the bacillus subtilis, the bacillus licheniformis, the bacillus amyloliquefaciens, the bacillus coagulans, the bacillus megaterium and the bacillus pumilus which are preserved at the temperature of-80 ℃ in a fresh LB solid culture medium, and performing activation culture at the constant temperature of 27-45 ℃ overnight.

(2) Respectively selecting a ring from activated plates of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus, inoculating the ring into a seed liquid culture medium, and independently culturing at the temperature of 27-45 ℃ and the rpm of 200 +/-20 for 8-12h to obtain seed liquid.

(3) Respectively inoculating the seed liquid obtained in the step (2) into a fermentation liquid culture medium according to the inoculation amount of 3-7%, independently culturing for 10h at the temperature of 27-45 ℃ and the rpm of 100 +/-20, and increasing the rotating speed to 200 +/-20 rpm to continue culturing for 10-18h to obtain a bacterial suspension.

(4) And (4) centrifuging the bacterial suspension obtained in the step (3) for 10min at the temperature of 4 ℃ and at the speed of 8000r/min, and filtering the supernatant by using a 0.22-micron filter membrane to obtain a thallus-free fermentation filtrate.

(5) Adjusting the filtrate obtained in the step (4) to be neutral by using 6mol/L HCl solution, adding solid ammonium sulfate while stirring, and standing at 4 ℃ in a refrigerator overnight when the saturation degree reaches 80%.

(6) Centrifuging at 4000r/min at normal temperature for 20min, collecting precipitate, dissolving in sterile water, vacuum freeze-drying, and mixing according to the mass ratio of (1-5) to obtain the compound preservative.

The LB solid culture medium is: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 20g/L of agar powder.

The seed liquid culture medium comprises: 2-10g/L beef extract, 5g/L sodium chloride, 5-15g/L peptone and 7.0-7.2 pH.

The culture medium of the fermentation liquid is as follows: 20-40g/L of molasses, 5-20g/L of bean flour, 2-10g/L of corn steep liquor powder, 10-30g/L of yeast extract, 1-5g/L of urea, 1-10g/L of sodium chloride, 0.1-1g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate monohydrate, 1-3g/L of zinc sulfate, 0.5-1.5g/L of monopotassium phosphate and pH of 6.8-7.5.

The vacuum freeze drying condition is that the temperature is-40 ℃, the vacuum degree is 100Pa, and the drying time is 35-40 h.

The application method of the preservative comprises the following steps: preparing the preservative into 0.1-10 mg/ml solution by using sterile water, and spraying or soaking the solution on fruits for 10-30 min; preferably, the fruit juice is prepared into 1mg/ml solution, and sprayed or soaked on the fruit for 20 min.

Before use, the preservative is prepared into 0.1-10 mg/ml solution by using sterile water, and fresh fruits are treated by spraying or soaking, so that soft and fragile fruits such as strawberries and the like cannot be damaged, and compared with the existing similar fruit preservatives, the preservative has the advantages of longer effective storage time and better preservation effect, ensures the fruit quality in the storage and long-distance transportation processes, reduces huge waste caused by spoilage, and has important significance in the field of modern agriculture.

Has the advantages that:

according to the invention, the six bacillus fermentation liquids are compounded, so that the ratio is reasonable, the use method is simple, the effect is uniform, the antibacterial effect is obviously improved compared with the antibacterial effect of single use, the preservation period of fruits at normal temperature is prolonged, and the waste caused by spoilage is reduced. The method utilizes industrial production byproducts such as molasses, bean flour and the like as fermentation carbon sources, realizes the reutilization of waste resources, reduces the environmental pollution, greatly reduces the production cost, is economical and feasible, and has wide market application value.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific process conditions and results described in the examples are merely illustrative of the invention and should not, nor should they, limit the invention as described in detail in the claims.

The strains referred to in the following examples are: bacillus subtilis with the preservation number of CGMCC No. 13932; bacillus licheniformis (Bacillus licheniformis) with preservation number of CGMCC No. 9688; bacillus amyloliquefaciens (Bacillus amyloliquefaciens) with the preservation number of CGMCC No. 15732; bacillus coagulans (Bacillus coagulons) with the preservation number of CGMCC No. 19487; bacillus megaterium (Bacillus megaterium) with the preservation number of CGMCC No. 1.10466; bacillus pumilus (Bacillus pumilus) with the preservation number of CGMCC No. 1.8167.

Example 1

In the embodiment, the freshness retaining effect of the preservatives of different strains is considered by taking the strawberries as the acting objects.

A fruit preservative compounded by using a bacillus fermentation product is obtained by the following steps:

(1) preparing a culture medium:

the LB solid medium is: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 20g/L of agar powder.

The seed liquid culture medium comprises: 3g/L of beef extract, 5g/L of sodium chloride, 10g/L of peptone and 7.0-7.2 of pH value.

The culture medium of the fermentation liquid comprises: 40g/L of molasses, 10g/L of bean flour, 4g/L of corn steep liquor powder, 20g/L of yeast extract, 2.5g/L of urea, 5g/L of sodium chloride, 0.4g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate monohydrate, 2g/L of zinc sulfate, 1g/L of monopotassium phosphate and pH 7.

The autoclaving condition is 121 deg.C for 20 min.

(2) Spreading Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus megaterium, and Bacillus pumilus stored at-80 deg.C in fresh LB solid culture medium, and performing activation culture at 37 deg.C, 35 deg.C, 27 deg.C, 40 deg.C, 37 deg.C, and 30 deg.C overnight.

(3) Respectively selecting a ring from an activation plate of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus, inoculating the ring into a seed solution culture medium, and independently culturing at 37 ℃, 35 ℃, 27 ℃, 40 ℃, 37 ℃, 30 ℃ and 200rpm for 8 hours to obtain a seed solution.

(4) And (4) respectively inoculating the seed liquid obtained in the step (3) into a fermentation liquid culture medium according to the inoculation amount of 5%, respectively and independently culturing for 10h at respective culture temperature and 100rpm, and increasing the rotation speed to 200rpm to continue culturing for 14h to obtain bacterial suspension.

(5) And (4) centrifuging the bacterial suspension obtained in the step (4) for 10min at the temperature of 4 ℃ and at the speed of 8000r/min, and filtering the supernatant by using a 0.22-micron filter membrane to obtain a thallus-free fermentation filtrate.

(6) Adjusting the filtrate obtained in the step (5) to be neutral by using 6mol/L HCl solution, adding solid ammonium sulfate while stirring, and standing at 4 ℃ in a refrigerator overnight when the saturation degree reaches 80%.

(7) Centrifuging at 4000r/min at normal temperature for 20min, collecting precipitate, dissolving in sterile water, vacuum freeze-drying, and compounding according to the mass ratio of 1:1:1:1:1:1 to obtain the compound preservative.

Commercially available fresh strawberries that were disease free, wound free, uniform in maturity and size were divided into A, B, C, D, E, F, G, H eight groups: group A is treated by the compound preservative prepared by the embodiment, specifically, the compound preservative is prepared into 1mg/ml solution by using sterile water and is sprayed on fresh strawberries, and the spraying amount is one thousandth of the mass of the fresh strawberries; b group sprays bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens and bacillus coagulans fermentation liquor to be mixed according to the mass ratio of 1:1:1:1, and the treatment method and the spraying amount are consistent with those of A group; C. d, E, F, G separately spraying Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus megaterium, and Bacillus pumilus fermentation liquid, wherein the treatment method and spraying amount are consistent with those of group A; group H did not spray preservative. Each group of strawberries was divided into two parts, wherein one part of eight groups of strawberries were stored in the same environment at 4 ℃ and a relative humidity of 85% -90% for 4 days, and the results are shown in Table 1; another eight groups of strawberries were stored in the same environment at 4 ℃ and a relative humidity of 85% -90%, and the average decay time was calculated as an indication of the occurrence of white hairs, and the results are shown in Table 2.

TABLE 1 influence of antistaling agents of different bacterial components on strawberry fresh-keeping effect

TABLE 2 extension of strawberry freshness by antistaling agents with different bacterial components

	Decay time (h)	Prolonged preservation period (h)	Extension of shelf life (%)
				Group A	127.9	78.8	160.49％
Group B	92.7	43.6	88.80％
				Group C	78.2	29.1	59.27％
Group D	71.7	22.6	46.03％
				Group E	76.5	27.4	55.80％
Group F	68.3	19.2	39.10％
				Group G	64.4	15.3	31.16％
Group H	49.1	0	0.00％

As can be seen from tables 1 and 2, the shelf life of the strawberry added with the preservative is effectively prolonged compared with that of the control group without the preservative. The fresh-keeping effect of the compound fresh-keeping agent of the six bacillus fermentation liquids is superior to that of the single use of each bacillus fermentation liquid or the mixed use of a plurality of bacillus fermentation liquids.

Example 2

In the embodiment, the fresh-keeping effect of the fresh-keeping agents with different proportions is investigated.

A fruit preservative compounded by using a bacillus fermentation product is obtained by the following steps:

(1) preparing a culture medium:

the LB solid medium is: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 20g/L of agar powder.

The seed liquid culture medium comprises: 3g/L of beef extract, 5g/L of sodium chloride, 10g/L of peptone and 7.0-7.2 of pH value.

The culture medium of the fermentation liquid comprises: 40g/L of molasses, 10g/L of bean flour, 4g/L of corn steep liquor powder, 20g/L of yeast extract, 2.5g/L of urea, 5g/L of sodium chloride, 0.4g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate monohydrate, 2g/L of zinc sulfate, 1g/L of monopotassium phosphate and pH 7.

The autoclaving condition is 121 deg.C for 20 min.

(2) Spreading Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus megaterium, and Bacillus pumilus stored at-80 deg.C in fresh LB solid culture medium, and performing activation culture at 37 deg.C, 35 deg.C, 27 deg.C, 40 deg.C, 37 deg.C, and 30 deg.C overnight.

(3) Respectively selecting a ring from an activation plate of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus, inoculating the ring into a seed solution culture medium, and independently culturing at 37 ℃, 35 ℃, 27 ℃, 40 ℃, 37 ℃, 30 ℃ and 200rpm for 8 hours to obtain a seed solution.

(4) And (4) respectively inoculating the seed liquid obtained in the step (3) into a fermentation liquid culture medium according to the inoculation amount of 5%, respectively and independently culturing for 10h at respective culture temperature and 100rpm, and increasing the rotation speed to 200rpm to continue culturing for 14h to obtain bacterial suspension.

(5) And (4) centrifuging the bacterial suspension obtained in the step (4) for 10min at the temperature of 4 ℃ and at the speed of 8000r/min, and filtering the supernatant by using a 0.22-micron filter membrane to obtain a thallus-free fermentation filtrate.

(6) Adjusting the filtrate obtained in the step (5) to be neutral by using 6mol/L HCl solution, adding solid ammonium sulfate while stirring, and standing at 4 ℃ in a refrigerator overnight when the saturation degree reaches 80%.

(7) Centrifuging at 4000r/min at normal temperature for 20min, collecting the precipitate, dissolving in sterile water, and carrying out vacuum freeze drying, and then compounding according to the mass ratio of 1:1:1:1, 2:1:1:1:1, 1:1:2:1:1, 1:1:1:1:2, 2:1:2:1:1:1 to obtain 8 groups of composite preservatives.

Placing 9 sterile oxford cups with the diameter of 7mm in an 18cm sterile flat plate, adding 80 mu L of rotten strawberry juice into 60mL LB solid culture medium precooled to 45 ℃, uniformly mixing, pouring the mixture into the flat plate, cooling, taking out the oxford cups, and marking holes 1-9. 100 mu L of sterile preservative compounded according to the mass ratio of 1:1:1:1:1:1, 2:1:1:1:1, 1:2:1:1:1, 1:1:2:1:1, 1:1:1:2:1:1, 1:1:1:1:2 and 2:1:2:1:1, and 100 mu L of sterile water is added into No.9 hole respectively. After diffusion at 4 ℃ for 1h, incubation at 37 ℃ for 24h, the zone diameter was measured with a vernier caliper, the results are given in Table 3.

TABLE 3 antistaling agent with different proportions

The fermentation liquid freeze-dried powder compounding ratio of six bacteria of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus optimized in the table 3 is 2:1:2:1:1: 1.

Example 3

In the embodiment, grapes are taken as the action objects to examine the preservation effect of the different culture medium components on the preservative.

A fruit preservative compounded by using a bacillus fermentation product is obtained by the following steps:

(1) preparing a culture medium:

the LB solid medium is: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 20g/L of agar powder.

The seed liquid culture medium comprises: 3g/L of beef extract, 5g/L of sodium chloride, 10g/L of peptone and 7.0 of pH.

The culture medium of the fermentation liquid comprises: 40g/L of molasses, 10g/L of bean flour, 4g/L of corn steep liquor powder, 20g/L of yeast extract, 2.5g/L of urea, 5g/L of sodium chloride, 0.4g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate monohydrate, 2g/L of zinc sulfate, 1g/L of monopotassium phosphate and pH 7.

The culture medium of the control group fermentation liquid is as follows: 40g/L of molasses, 20g/L of yeast extract, 2.5g/L of urea and 5g/L of sodium chloride.

The autoclaving condition is 121 deg.C for 20 min.

(2) Spreading Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus megaterium, and Bacillus pumilus stored at-80 deg.C in fresh LB solid culture medium, and performing activation culture at 37 deg.C, 35 deg.C, 27 deg.C, 40 deg.C, 37 deg.C, and 30 deg.C overnight.

(3) Respectively selecting a ring from an activation plate of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus, inoculating the activation plate into a seed solution culture medium, independently culturing for 8 hours at 37 ℃, 35 ℃, 27 ℃, 40 ℃, 37 ℃, 30 ℃ and 200rpm to obtain seed solutions, and equally dividing the seed solutions of the six types of bacillus into 1 group and 2 groups.

(4) And (4) respectively inoculating the 1 and 2 groups of seed solutions obtained in the step (3) into a fermentation liquor culture medium and a control group fermentation liquor culture medium according to the inoculation amount of 5%, respectively and independently culturing for 10h at respective culture temperature and 100rpm, and then increasing the rotation speed to 200rpm to continue culturing for 14h to obtain 1 and 2 groups of bacterial suspensions.

(5) And (4) centrifuging the two groups of bacterial suspensions obtained in the step (4) for 10min at 4 ℃ and 8000r/min respectively, and filtering supernate with a 0.22-micron filter membrane to obtain 1 and 2 groups of fermentation filtrates without thalli.

(6) Adjusting the two groups of filter liquor obtained in the step (5) to be neutral by using 6mol/L HCl solution, adding solid ammonium sulfate while stirring, and standing overnight in a refrigerator at 4 ℃ when the saturation degree reaches 80%.

(7) Centrifuging at 4000r/min for 20min at normal temperature, collecting precipitate, dissolving in sterile water to obtain 1 and 2 groups of liquid sterile antistaling agents, vacuum freeze drying, and mixing at mass ratio of 2:1:2:1:1:1 to obtain 1 and 2 groups of composite antistaling agents.

Placing three 7mm sterile Oxford cups in a sterile plate, adding 20 μ L rotten green extract liquid into 15mL LB solid culture medium precooled to 45 ℃, uniformly mixing, pouring the mixture into the plate, cooling, taking out the Oxford cups, and marking holes No.1, No. 2 and No. 3. 100 mu L of No.1 liquid sterile preservative is added into the No.1 hole, 100 mu L of No. 2 liquid sterile preservative is added into the No. 2 hole, and 100 mu L of sterile water is added into the No. 3 hole. After diffusion at 4 ℃ for 1h, incubation at 37 ℃ for 24h, the zone diameter was measured with a vernier caliper, the results are given in Table 4.

TABLE 4 antistaling agent and bacteriostatic effect of different culture medium components

	Diameter (mm)	Diameter growth Rate (%)
			No.1 hole	27.3	77.27％
No. 2 hole	21.8	41.56％
			No. 3 hole	15.4	0

As can be seen from Table 4, the equivalent amount of the preservative was added to the wells 1 and 2, but the growth rate of the zone diameter of the No.1 well was nearly one time higher than that of the No. 2 well. Therefore, the fermentation medium obviously promotes the bacillus to produce the bacteriostatic substance.

Commercially available fresh green grape that is disease free, wound free, uniform in maturity and size was divided into A, B, C three groups: treating group A with No.1 antistaling agent, specifically preparing antistaling agent with sterile water into 1mg/ml solution, soaking whole string of fresh grape in the solution for 20min, and taking out; group B is treated by No. 2 preservative, and the implementation method is consistent with that of group A; group C was not soaked in the preservative. Three groups of grapes were stored in the same environment at 15 ℃ and 85% -90% relative humidity for 10 days, and the results are shown in Table 5.

TABLE 5 Effect of antistaling agents with different Medium Components on the fresh-keeping Effect of grapes

Example 4

In the embodiment, the preservation effect of the freeze-drying preservative is considered by taking winter jujubes as an action object.

A fruit preservative compounded by using a bacillus fermentation product is obtained by the following steps:

(1) preparing a culture medium:

the LB solid medium is: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 20g/L of agar powder.

The seed liquid culture medium comprises: 3g/L of beef extract, 5g/L of sodium chloride, 10g/L of peptone and 7.0 of pH.

The culture medium of the fermentation liquid comprises: 40g/L of molasses, 10g/L of bean flour, 4g/L of corn steep liquor powder, 20g/L of yeast extract, 2.5g/L of urea, 5g/L of sodium chloride, 0.4g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate monohydrate, 2g/L of zinc sulfate, 1g/L of monopotassium phosphate and pH 7.

The autoclaving condition is 121 deg.C for 20 min.

(2) Spreading Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus megaterium, and Bacillus pumilus stored at-80 deg.C in fresh LB solid culture medium, and performing activation culture at 37 deg.C, 35 deg.C, 27 deg.C, 40 deg.C, 37 deg.C, and 30 deg.C overnight.

(3) Respectively selecting a ring from an activation plate of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus coagulans, bacillus megaterium and bacillus pumilus, inoculating the ring into a seed solution culture medium, and independently culturing at 37 ℃, 35 ℃, 27 ℃, 40 ℃, 37 ℃, 30 ℃ and 200rpm for 8 hours to obtain a seed solution.

(4) And (4) respectively inoculating the seed liquid obtained in the step (3) into a fermentation liquid culture medium according to the inoculation amount of 5%, respectively and independently culturing for 10h at respective culture temperature and 100rpm, and increasing the rotation speed to 200rpm to continue culturing for 14h to obtain bacterial suspension.

(5) And (4) centrifuging the bacterial suspension obtained in the step (4) for 10min at the temperature of 4 ℃ and at the speed of 8000r/min, and filtering the supernatant by using a 0.22-micron filter membrane to obtain a thallus-free fermentation filtrate.

(6) Adjusting the filtrate obtained in the step (5) to be neutral by using 6mol/L HCl solution, adding solid ammonium sulfate while stirring, and standing at 4 ℃ in a refrigerator overnight when the saturation degree reaches 80%.

(7) Centrifuging at 4000r/min for 20min at normal temperature, collecting precipitate, and dissolving in sterile water to obtain liquid compound antistaling agent. After the liquid compound preservative is subjected to vacuum freeze drying, the liquid compound preservative is compounded according to the mass ratio of 2:1:2:1:1:1 to obtain the freeze-dried powder compound preservative. The two composite preservatives are stored for 0 day, 5 days, 10 days, 15 days, 20 days, 25 days and 30 days at room temperature of 15 ℃ for later use.

Placing three 7mm sterile Oxford cups in a sterile plate, adding 20 μ L rotten Ziziphus jujube juice into 15mL LB solid culture medium precooled to 45 deg.C, mixing, pouring out the plate, cooling, taking out the Oxford cups, and marking holes No.1, No. 2 and No. 3. Preparing the freeze-dried powder sterile preservative into a solution of 1mg/ml by using sterile water, and adding 100 mu L of the solution into the No.1 hole; adding 100 mu L of liquid sterile preservative into the No. 2 hole; well 3 was filled with 100. mu.L of sterile water. After diffusion at 4 ℃ for 1h, incubation at 37 ℃ for 24h, the zone diameter was measured with a vernier caliper, the results are given in Table 6.

TABLE 6 composite antistaling agent with different forms

Dividing the fresh winter jujubes which are sold in the market and have no diseases, wounds, maturity and uniform size into A, B, C three groups: treating group A with lyophilized powder composite antistaling agent, specifically preparing 1mg/ml solution of composite antistaling agent stored at 15 deg.C at room temperature for 15 days with sterile water, and spraying onto fresh winter jujube with spraying amount of one thousandth of winter jujube quality; spraying liquid compound antistaling agent of group B for 15 days at room temperature of 15 deg.C, the spraying amount is consistent with that of group A; group C did not spray preservative. The three groups of winter jujubes were preserved in the same environment at 20 ℃ and 85% -90% of relative humidity for 10 days, and the results are shown in Table 7.

TABLE 7 influence of composite antistaling agent with different forms on winter jujube fresh-keeping effect

As can be seen from tables 6 and 7, the form of freeze-dried powder prepared from the fresh preservative does not affect the preservation effect, and compared with the liquid state, the freeze-dried powder can obviously prolong the effective period of the preservative: the preservation effect of the liquid preservative gradually tends to zero in a 30-day bacteriostatic test, and the bacteriostatic effect of the freeze-dried powder preservative is hardly weakened. Therefore, the freeze-dried powder prepared is more beneficial to maintaining the bacteriostatic effect of the preservative when the preservative is stored at normal temperature.

The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and implementations of the embodiments of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the principles of the embodiments of the present invention, and for those skilled in the art, the details and implementations of the embodiments and implementations may be changed, and in summary, the content of the present description should not be understood as a limitation to the present invention.