CN108497058B - Biological fresh-keeping microbial inoculum fermented by bean curd yellow slurry and application thereof in fruit and vegetable fresh-keeping - Google Patents
Biological fresh-keeping microbial inoculum fermented by bean curd yellow slurry and application thereof in fruit and vegetable fresh-keeping Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/16—Coating with a protective layer; Compositions or apparatus therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Storage Of Fruits Or Vegetables (AREA)
Abstract
The invention discloses a biological fresh-keeping microbial inoculum fermented by yellow soybean milk and application thereof in fruit and vegetable fresh-keeping, wherein the biological fresh-keeping microbial inoculum is prepared by directly using fermentation liquor obtained by fermenting bacillus subtilis by taking yellow soybean milk as a fermentation raw material as a fresh-keeping microbial inoculum, and the concentration of thalli in the fermentation liquor reaches 1 x 1013~2.7×1013CFU/ml, the concentration of the gamma-polyglutamic acid reaches 30-50 g/L, and the yield of the antibacterial peptide reaches 1.2-2 g/L. Compared with other biological fresh-keeping agents, the method for obtaining the biological fresh-keeping agent is low in cost and more environment-friendly, waste bean curd yellow serofluid can be used, potential safety hazards such as pathogenic bacteria existing in yellow serofluid natural fermentation acid pulp in bean product processing are reduced, and meanwhile, the content of gamma-polyglutamic acid and antibacterial substances in fermentation products is high, so that the biological fresh-keeping agent has a good fresh-keeping effect on picked fruits and vegetables, is pollution-free, and has long-term development prospects and industrial utilization values without public hazards.
Description
Technical Field
The invention belongs to the field of food preservation, and particularly relates to bacillus subtilisBacillus subtilis) The application in the preservation of picked fruits and vegetables.
Background
The fruit and vegetable industry faces the problem of fruit and vegetable spoilage for a long time, the fruit and vegetable spoilage after picking is mainly caused by two factors, and on one hand, the physiological activities of the interior of the picked fruit and vegetable still include respiration, oxidation, water evaporation and the like. These vital metabolic activities of fruits and vegetables result in loss of nutritional ingredients and shortened storage life of fruits to some extent. On the other hand, the rot and deterioration of the fruits and vegetables after picking can be accelerated by the infection of pathogenic bacteria. Therefore, the fruit and vegetable can be prevented from being rotten and deteriorated by inhibiting the respiration of the fruit and vegetable. The fruit and vegetable decay problem after picking is usually treated by a chemical preservative and a physical method, but the methods have high cost and cannot ensure no toxicity.
The film coating preservation technology is to coat a film on the surface of the fruits and vegetables, so as to provide multiple functions of selectively blocking air and moisture, preventing the loss of contents, blocking the harmful influence of the external environment, inhibiting respiration, delaying after-ripening and aging, inhibiting the growth of surface microorganisms, improving the storage quality and the like, thereby achieving the purpose of preserving the fruits and vegetables.
Bacillus subtilis (A), (B) and (C)Bacillus subtilis) The bacillus subtilis is a microorganism with wide action, can secrete a large amount of gamma-polyglutamic acid, can generate spores with high heat resistance and stress resistance, and can secrete various antibacterial substances at the initial stage of spore formation. Has specific preventing and treating effect on pathogenic fungi.
The picked fruits and vegetables are coated and preserved by utilizing the characteristic that the bacillus subtilis fermentation liquor is rich in gamma-polyglutamic acid, so that the problems of pollution, inedibility and the like of a chemical preservative are effectively avoided, and the bacillus subtilis fermentation liquor is concerned in the field of fruit and vegetable preservation. 201410452988.5A Gamma-polyglutamic acid-Cu (II) antistaling agent and its application in fruit and vegetable fresh-keeping disclose the application of Gamma-polyglutamic acid fermented by Bacillus licheniformis in fruit and vegetable fresh-keeping.
200910192821.9A litchi biopolymer coating preservative is found that gamma-polyglutamic acid or gamma-polyglutamate compounded and then coated can slow down the loss of water in litchi peel and effectively prolong the litchi preservation period. The biological fresh-keeping microbial inoculum has the defects of long fermentation period, complex treatment process, high operation cost and the like. At present, the existing preservation technology has complex treatment process and high cost, and a new biological preservative suitable for application is urgently needed to be developed.
Disclosure of Invention
The invention utilizes bacillus subtilis (B.subtilis)Bacillus subtilis) The preservation effect of the gamma-polyglutamic acid in the fermentation liquor and the antibacterial substances secreted by the thalli preserve fruits and vegetables, so that the preservation time of the fruits and vegetables is effectively prolonged.
Bacillus subtilis (B) used in the inventionBacillus subtilis) The bean curd yellow serofluid is used as a nitrogen source for fermentation and growth, so that the raw material cost is effectively reduced, the waste utilization is realized, and the bean curd yellow serofluid has the functions of no toxicity and benefiting life.
The purpose of the invention is realized by the following technical scheme:
a biological fresh-keeping microbial inoculum for keeping vegetables and fruits fresh is characterized in that the biological fresh-keeping microbial inoculum is directly used as a fresh-keeping microbial inoculum by fermentation liquor obtained by fermenting bacillus subtilis in a fermentation culture medium taking yellow serofluid of bean curd as a raw material, and the concentration of thalli in the fermentation liquor reaches 1 multiplied by 1013~2.7×1013CFU/ml, the concentration of the gamma-polyglutamic acid reaches 30-50 g/L, and the yield of the antibacterial peptide reaches 1.2-2 g/L.
The fermentation medium is as follows: 10-20% (w/v) of a carbon source, 0.01-0.1% (w/v) of calcium chloride, 0.05% (w/v) of potassium dihydrogen phosphate, 0.05% (w/v) of dipotassium hydrogen phosphate, 0.01% (w/v) of magnesium sulfate, 2.5% (w/v) of sodium glutamate, 0.3% (w/v) of ammonium chloride and the balance of bean curd yellow serous water, wherein the initial pH value is 6.5-7.5.
The preparation method of the biological fresh-keeping microbial inoculum comprises the following steps: inoculating the bacillus subtilis strain into a liquid culture medium, and inoculating and shaking-bed culturing at 30 ℃ for 8-14 h to obtain a seed solution: inoculating the seed solution into a fermentation culture medium according to the volume ratio of 5-20%, performing fermentation culture at 180-220 rpm, and obtaining fermentation liquor after fermentation is finished, namely the biological fresh-keeping microbial inoculum;
the components of the liquid culture medium are as follows: 10-20% (w/v) of glucose, 10-15% (w/v) of soybean peptone,
0.01-0.1% (w/v) of calcium chloride, 0.05% (w/v) of potassium dihydrogen phosphate, and 0.05% (w/v) of dipotassium hydrogen phosphate. 0.01 percent of magnesium sulfate, (w/v) and the balance of distilled water, wherein the initial pH value is 6.5-7.5;
the fermentation medium is as follows: 10-20% (w/v) of glucose, 0.01-0.1% (w/v) of calcium chloride, 0.05% (w/v) of potassium dihydrogen phosphate, 0.05% (w/v) of dipotassium hydrogen phosphate, 0.01% (w/v) of magnesium sulfate, 2.5% (w/v) of sodium glutamate, 0.3% (w/v) of ammonium chloride and the balance of bean curd yellow serous water, wherein the initial pH value is 6.5-7.5.
The fermentation culture temperature is 10-50 ℃, and the culture time is 24-30 h. Preferably, the culture temperature is 25 ℃ or 2 ℃ and the culture time is 48 hours.
The biological fresh-keeping microbial inoculum is applied to fresh keeping of vegetables and fruits. The biological fresh-keeping agent is sprayed on the picked fruits and vegetables, and the spraying amount is that the surfaces of the fruits and vegetables are completely wet.
The specific operation steps are as follows:
preparing a biological fresh-keeping microbial agent: bacillus subtilis (A), (B) and (C)Bacillus subtilis) Inoculation of the Strain into Bacillus subtilis: (Bacillus subtilis) In a liquid culture medium, carrying out shake cultivation for 15-18 h at 30 ℃ to obtain a seed solution: inoculating the seed liquid into a large amount of fermentation culture medium according to the volume ratio of 5-20%, and culturing at 180-220 rpm/min: directly collecting fermentation liquor after fermentation to obtain the biological fresh-keeping microbial inoculum.
The biological fresh-keeping agent is sprayed on the picked fruits and vegetables, and the spraying amount is only required to be that the surfaces of the fruits and vegetables are completely wet, so that the fresh keeping is realized. The putrefaction and deterioration of picked fruits and vegetables due to respiration and fungal viruses are common knowledge, and the invention utilizes bacillus subtilis (Bacillus subtilis)Bacillus subtilis) Can secrete gamma-polyglutamic acid and antibacterial substances, and can be used for coating picked fruits and vegetables with fermentation liquor, so that the pollution and toxic effects caused by a chemical preservative are overcome, and pollution-free treatment is realized. And the fermentation raw materials are waste resources, so that the method is more environment-friendly and healthy.
The invention utilizes the characteristics of bacillus subtilis for producing gamma-polyglutamic acid and antibacterial peptide to keep fresh of the fruit and vegetable, sprays fermentation liquor on the surface of the fruit and vegetable to form a layer of coating film to inhibit the respiration action of the surface of the fruit and vegetable and keep the humidity of the fruit and vegetable, simultaneously the bacillus subtilis can secrete antibacterial substances to inhibit the growth of pathogenic bacteria and play a positive role in the fresh-keeping of the fruit and vegetable after harvesting, the biological fresh-keeping microbial inoculum prepared by the invention has high thallus concentration and good pathogenic bacteria inhibition effect and low production cost, the invention takes bean curd yellow slurry as a fermentation raw material, various amino acids exist in the bean curd yellow water, organic acids comprise glutamic acid, glycine and the like, and some small peptides and metal ions are beneficial to the synthesis of the gamma-polyglutamic acid, and the invention has the advantages of low cost and simple treatment process because the characteristics of the bacillus subtilis has rich nutrition and also has promotion effect on the increase of thallus concentration, compared with other biological fresh-keeping agents, the biological fresh-keeping agent has the characteristics of short fermentation period, high thallus concentration, good antibacterial effect and the like. Fruit and vegetable spoilage is caused by various factors, wherein 4 factors of physical, chemical, enzyme and microorganism are main factors, and the action of the microorganism is the most serious. The physical fresh-keeping method has high cost and high energy consumption, and the chemical fresh-keeping method can kill pathogenic bacteria and ensure the antiseptic and fresh-keeping effect, but the abuse of chemical synthetic agents can harm human health, can cause cancer more seriously, and can cause the problems of pesticide residue, environmental pollution and the like. The invention uses fermentation liquor to directly prepare biological fresh-keeping agent, avoids complex treatment process in other fresh-keeping technologies, and has relatively low cost and good application effect.
The invention has obvious use effect and can prolong the storage life of picked fruits and vegetables by more than 1.8 times.
Bacillus subtilis (A), (B) and (C)Bacillus subtilis) Is a recognized nonpathogenic bacterium, is approved by the agricultural department as a probiotic allowed by feed additives, and is prepared from bacillus subtilis (Bacillus subtilis)Bacillus subtilis) The developed preservative has biological safety, no toxicity, no pollution, greenness and suitability for popularization.
The fermentation liquor prepared by the invention can be directly used for preparing the biological fresh-keeping microbial inoculum, is nontoxic and harmless, can be directly eaten, takes the waste yellow soybean milk of the bean curd as the fermentation raw material, solves the problem of difficult bean curd water treatment of bean curd factories, greatly reduces the production cost, is beneficial to the industrialized development, and has the thallus concentration of 1013Compared with the biological fresh-keeping microbial inoculum on the market, the higher concentration of the thalli has more obvious effect of inhibiting harmful strains. The current commercial biological fresh-keeping microbial inoculum has the defects of long fermentation period, complex treatment process, high operation cost and the like, and compared with other biological fresh-keeping microbial inocula in the market, the invention develops the biological fresh-keeping microbial inoculumThe fermentation period is short, the energy consumption is low, the treatment process is simple, the biological preservative can be directly prepared, the production cost is not required to be increased, the efficiency and the energy conservation are high, and the preservation effect is good.
Has the advantages that:
the biological fresh-keeping agent of the invention is simple to prepare, directly smeared by fermentation liquor, overcomes the pollution and toxic action brought by chemical fresh-keeping agents, and realizes pollution-free treatment. And the fermentation raw materials are waste resources, so that the method is more environment-friendly and healthy. The using effect is obvious, and the storage life of the picked fruits and vegetables can be prolonged by more than 1.8 times.
Detailed Description
Bacillus subtilis described in the following examples (B.), (B.) (B.subtilis)Bacillus subtilis) The strain has the number of CGMCC No.13932 and is from the China general microbiological culture Collection center. The bean curd yellow serofluid is the yellow serofluid drained in bean curd production (provided by bean curd workshops in Putou district of Nanjing).
Example 1
Using Bacillus subtilis (B.) (Bacillus subtilis) Biological preservative prepared by using serial number of CGMCC No.13932
Preparing a biological fresh-keeping microbial agent: bacillus subtilis (A), (B) and (C)Bacillus subtilis) Inoculation of the Strain into Bacillus subtilis: (Bacillus subtilis) In a liquid culture medium, carrying out shake cultivation for 8-14 h at 30 ℃ to obtain a seed solution: inoculating the seed solution into a large amount of fermentation culture medium according to the volume ratio of 10%, and culturing at 180-220 rpm/min: directly collecting fermentation liquor after fermentation, wherein the thallus concentration in the fermentation liquor reaches 1013CFU/ml, the concentration of the gamma-polyglutamic acid reaches 30g/L, the yield of the antibacterial peptide reaches 1.2-2 g/L, and the fermentation liquor is directly used as a biological fresh-keeping microbial inoculum.
The seed culture medium comprises the following components: glucose 10% (w/v), Soy peptone 15% (w/v), calcium chloride 0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), dipotassium hydrogen phosphate 0.05% (w/v). 0.01 percent of magnesium sulfate, (w/v) and the balance of distilled water, wherein the initial pH value is 6.5-7.5.
The components of a large amount of fermentation culture medium: glucose 10% (w/v), calcium chloride 0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), and dipotassium hydrogen phosphate 0.05% (w/v). 0.01% (w/v) of magnesium sulfate, 2.5% (w/v) of sodium glutamate, 0.3% (w/v) of ammonium chloride and the balance of bean curd yellow serofluid, wherein the initial pH value is 6.5-7.5.
Example 2
Fresh-keeping effect of biological fresh-keeping agent on picked bananas
Mature bananas which are free of mechanical damage, equal in size and free of speckles and are good in color and luster are purchased from the market. The biological preservative prepared in the embodiment 1 is fully shaken up, sprayed on the surface of fruit to be treated by a spray can, the spraying amount is suitable for completely wetting the fruit, the treated fruit is placed in a fresh-keeping bag, meanwhile, sterilized water with the same volume is sprayed as a control group, the treated group and the control group are stored under the same condition (25-30 ℃), the freshness of the fruit is regularly observed, the freshness is graded according to the following standard, and the freshness at different time points (the average value/the highest freshness grade of all the fruits) is calculated, and the results are shown in the table 1
The grading standard of banana freshness (evaluated by subjective judgment of individuals) comprises 0 grade of severe rot, 1 grade of starting rotten (yellow area of peel is more than 50 percent) and being kneaded by hand to be moved, the peel is shrunk, 2 grade of local turning yellow and softening, the hand feeling of the fruit is hard, 3 grade of local turning yellow (yellow area of peel is less than 50 percent) and uneven color, 4 grade of basically normal, slightly yellow peel (yellow area is less than 10 percent), and 5 grade of dark green, hard texture, no scab and white and clean pulp cut.
TABLE 1
The treated bananas are stored at the temperature of 25-30 ℃, the average freshness grade of the treated bananas is still close to the highest freshness grade number within 15 days, namely the freshness preservation rate within 15 days reaches over 96 percent, and the freshness preservation rate of untreated bananas is reduced to below 90 percent after 5 days under the same storage condition, which indicates that the freshness preservation time of the treated bananas is prolonged by over 3 times.
Example 3
Biological fresh-keeping agent for keeping cucumber fresh after picking
The cucumber which is pure in variety, free of mechanical damage, equal in size, free of specks and good in color and luster is purchased from the market. The biological preservative prepared in the embodiment 1 is fully shaken up, sprayed on the surface of the vegetable to be treated by a spray can, the spraying amount is suitable for completely wetting the vegetable, the treated vegetable is placed in a fresh-keeping bag, meanwhile, the sterilized water with the same volume is sprayed as a control group, the treated group and the control group are stored under the same condition (25-30 ℃), the freshness of the fruit is regularly observed, the freshness is graded according to the following standard, and the freshness at different time points (the average value of freshness/the highest freshness grade of all the vegetables) is calculated, and the results are shown in Table 2
The grading standard of the freshness of the cucumbers (by taking subjective judgment of individuals as a judgment standard) comprises 0 grade of severe rot, 1 grade of beginning to become thoroughly decomposed (lesion area is more than 50 percent) and being capable of being pinched by hands to shrink the cucumber peel, 2 grade of local lesion (lesion area of the cucumbers is less than 50 percent) and uneven color, 3 grade of basically normal color and slight color change of the cucumbers (yellow area is less than 10 percent), and 4 grade of cucumbers are uniform in color, greenish and full.
TABLE 2
The treated cucumbers are stored at 25-30 ℃, the average freshness grade of the treated cucumbers is still close to the highest freshness grade in 15 days, namely, the freshness keeping rate of the treated cucumbers reaches over 90% in 15 days, and the freshness keeping rate of the untreated cucumbers is reduced to below 90% after 5 days under the same storage condition, which indicates that the freshness keeping time of the treated bananas is prolonged by over 3 times.
Example 4
Postharvest fresh-keeping effect of biological fresh-keeping microbial inoculum on apples
Apples with pure color, no mechanical damage, equal size and no disease spots are purchased from the market. The biological preservative prepared in the embodiment 1 is fully shaken up and sprayed on the surface of an apple to be treated by a spray can, the spraying amount is suitable for completely wetting the surface of the apple, the apple is placed in a fresh-keeping bag after being treated, meanwhile, sterilized water with the same volume is sprayed as a control group, the treated group and the control group are stored under the same condition (25-30 ℃), the freshness of the fruit is regularly observed, the freshness is graded according to the following standards, and the freshness (the average value/the highest freshness grade of all the fruits) at different time points is calculated, and the results are shown in the table 3
The apple freshness grading standard (evaluated by subjective judgment of individuals) is 0 grade, serious rotting, 1 grade, starting rotting (the lesion area of the fruit peel is greater than 50 percent), the fruit peel is shrunk, 2 grade, local blackening (the black area of the fruit peel is less than 50 percent), uneven color, 3 grade: local black spots (the black area of the peel is less than 5 percent), and the 4-grade apple has good color and luster and is bright.
TABLE 3
The processed apples are stored at 25-30 ℃, the average freshness grade of the processed apples is still in the highest freshness grade within 30 days, namely, the freshness preservation rate reaches 100 within 30 days, and the freshness preservation rate of the unprocessed apples is reduced to below 80% after 20 days under the same storage condition, which indicates that the freshness preservation time of the processed apples is prolonged by more than 1.8 times.
EXAMPLE 5
Postharvest fresh-keeping effect of biological fresh-keeping microbial inoculum on lettuce
The lettuce which has regular bodies, fresh and tender leaf quality, no scab, no insect pest, no dry leaf and no rot is purchased from the market. The biological preservative prepared in the embodiment 1 is fully shaken up, sprayed on the surface of the lettuce to be treated by a spray can, the spraying amount is suitable for completely wetting the surface of the lettuce, the lettuce is placed in a fresh-keeping bag after being treated, meanwhile, sterilized water with the same volume is sprayed as a control group, the treated group and the control group are stored under the same condition (25-30 ℃), the freshness of fruits is regularly observed, the freshness is graded according to the following standards, and the freshness at different time points (the average value of freshness/the highest freshness grade of all vegetables) is calculated, and the results are shown in Table 4
The grading standard of freshness of lettuce (by taking subjective judgment of individuals as the judgment standard) comprises 0 grade of serious rotting, 1 grade of (lettuce lesion area >50%), 2 grade of partial blackening (lettuce lesion area <50%), uneven color, 3 grade of: local yellowing, dehydration (the lesion area of lettuce is less than 5%), fresh and tender leaf quality of 4-grade lettuce, no disease spot, no dry leaf and no rotten leaf.
TABLE 4
The processed lettuce is stored at 25-30 ℃, the average freshness grade of the processed lettuce is close to the highest freshness grade in 8 days, namely the freshness preservation rate in 8 days reaches over 90%, and the freshness preservation rate of the unprocessed lettuce is reduced to 80% after 4 days under the same storage condition, which indicates that the freshness preservation time of the processed lettuce is prolonged by over 2 times.
EXAMPLE 6
Post-harvest fresh-keeping effect of biological fresh-keeping microbial inoculum on bitter gourds
The fresh bitter gourds without mechanical damage and spots are purchased from the market, the biological preservative prepared in the embodiment 1 is fully shaken up and then sprayed on the surface of the bitter gourds to be treated by a spray can, the spraying amount is suitable for completely wetting the surface of the bitter gourds, the bitter gourds are placed in a fresh-keeping bag after being treated, meanwhile, the sterilized water with the same volume is sprayed as a control group, the treated group and the control group are stored under the same condition (25-30 ℃), the freshness of the bitter gourds is regularly observed, the freshness is graded according to the following standard, the freshness at different time points (the average freshness/the highest freshness grade of all vegetables) is calculated, and the results are shown in table 5
The grading standard of the freshness of the balsam pears (subjective judgment of individuals is taken as a judgment standard) comprises 0 grade of severe rotting, 1 grade of rotting (lesion area is more than 50 percent) and shrinking of the melon peel by pinching with hands, 2 grade of local lesion (lesion area of the balsam pears is less than 50 percent) and uneven color, 3 grade of normal color and slight color change of the balsam pears (yellow area is less than 10 percent), and 4 grade of balsam pears are uniform in color, greenish and full.
TABLE 5
The processed bitter gourds are stored at 25-30 ℃, the average freshness grade of the processed bitter gourds is close to the highest freshness grade in 8 days, namely, the freshness preservation rate in 8 days reaches over 90%, and the freshness preservation rate of the unprocessed lettuce is reduced to 50% after 4 days under the same storage condition, which indicates that the freshness preservation time of the processed bitter gourds is prolonged by over 2 times.
EXAMPLES example 7
Research on preservation effect of bacillus subtilis fermentation liquor under different fermentation methods
The strain used in the invention is subjected to comparative fermentation under different fermentation methods, and the process of the fermentation method 1 is as follows: inoculating a bacillus subtilis strain into a liquid seed culture medium, wherein the seed culture medium comprises the following components: 10-20% (w/v) of glucose, 10-15% (w/v) of soybean peptone, 0.01-0.1% (w/v) of calcium chloride, 0.05% (w/v) of potassium dihydrogen phosphate and 0.05% (w/v) of dipotassium hydrogen phosphate. 0.01 percent of magnesium sulfate, (w/v) and the balance of distilled water, wherein the initial pH value is 6.5-7.5. Carrying out shake cultivation at 30 ℃ for 8-14 h at a rotating speed of 220r/min to obtain a seed solution, inoculating the seed solution into a large amount of fermentation medium according to a volume ratio of 5-20%, wherein the large amount of fermentation medium comprises the following components: glucose 10% (w/v), Soy peptone 15% (w/v), calcium chloride 0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), dipotassium hydrogen phosphate 0.05% (w/v). 0.01% (w/v) of magnesium sulfate, 2.5% (w/v) of sodium glutamate, 0.3% (w/v) of ammonium chloride and the balance of pure water, wherein the initial pH value is 6.5-7.5. Shaking-culturing at 30 deg.C for 28h at a rotation speed of 220r/min, and keeping fruit and vegetable fresh.
The process of the fermentation method 2 is basically the same as that of the fermentation method 1, except that 10-15% (w/v) of tryptone is used as the nitrogen source in the shake flask mass fermentation medium.
Fermentation method 3 is essentially the same as fermentation method 1, except that 0.1% (w/v) glutamic acid was added to the bulk fermentation medium in the shake flask.
The process of the fermentation method 4 is basically the same as that of the fermentation method 1, except that 10-15% (w/v) of yeast powder is used as the nitrogen source in the shake flask mass fermentation medium.
The processes of the fermentation methods 5, 6 and 7 are basically the same as the fermentation method 1, except that 0.05%, 0.08% and 0.1% (w/v) tween-80 are added into a large amount of shake flask fermentation medium.
The process of fermentation method 8 is substantially the same as fermentation method 1, except that no soy peptone is added to the shake flask bulk fermentation medium, and the balance is 50% soybean curd yellow serofluid.
The process of fermentation method 9 is substantially the same as fermentation method 1, except that no soybean peptone is added to a large amount of shake flask fermentation medium, and the balance is pure soybean curd yellow serofluid.
The fermentation liquor prepared by the fermentation methods 1, 2, 3, 4, 5, 6, 7, 8 and 9 is prepared into a biological fresh-keeping microbial inoculum according to the method in the embodiment 1, and the picked fruits and vegetables are kept fresh according to the processes in the embodiments 2, 3, 4, 5 and 6. As a result, as shown in Table 6, the fermentation method 9 exhibited the best fresh-keeping effect.
Grading standard, taking personal sense as evaluation standard: grading is performed according to the difference of excellent, good and medium.
TABLE 6
Example 8
Bacteriostasis research of bacillus subtilis fermentation liquor prepared by taking bean curd yellow serofluid as raw material on different bacteria
Preparation of a crude extract of fermentation liquor:
bacillus subtilis (A), (B) and (C)Bacillus subtilis) Inoculation of the Strain into Bacillus subtilis: (Bacillus subtilis) In a liquid culture medium, carrying out shake cultivation for 8-14 h at 30 ℃ to obtain a seed solution: inoculating the seed solution into a large amount of fermentation culture medium according to the volume ratio of 10%, and culturing at 180-220 rpm/min: directly collecting fermentation liquor after fermentation, wherein the thallus concentration in the fermentation liquor reaches 1013CFU/ml, the concentration of gamma-polyglutamic acid reaches 30g/L, and the yield of the antibacterial peptide reaches 1.2-2 g/L. . Centrifuging the fermentation liquid at 4 deg.C and 10000 rpm/min/min for 10 min, and collecting supernatant; adjusting the pH of the supernatant to be 2 with 4 mol/L HCl, standing at 4 ℃ for 10-12 h, centrifuging at 4 ℃ and 10000 rpm/min/min for 10 min, collecting the precipitate, adding 5mL of methanol solution into the precipitate, and stirring for 20minStanding at 4 deg.C for 10 hr, and centrifuging at 4 deg.C at 10000 rpm/min/min for 10 min to obtain methanol extract. Vacuum freeze drying to obtain antibacterial substance, and storing at 20 deg.C.
The seed culture medium comprises the following components: glucose 10% (w/v), Soy peptone 15% (w/v), calcium chloride 0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), dipotassium hydrogen phosphate 0.05% (w/v). 0.01 percent of magnesium sulfate, (w/v) and the balance of distilled water, wherein the initial pH value is 6.5-7.5.
The components of a large amount of fermentation culture medium: glucose 10% (w/v), calcium chloride 0.1% (w/v), potassium dihydrogen phosphate 0.05% (w/v), and dipotassium hydrogen phosphate 0.05% (w/v). 0.01% (w/v) of magnesium sulfate, 2.5% (w/v) of sodium glutamate, 0.3% (w/v) of ammonium chloride and the balance of bean curd yellow serofluid, wherein the initial pH value is 6.5-7.5.
TABLE 7 bacteriostatic effect of Bacillus subtilis after fermentation with bean curd yellow water
| Pathogenic bacteria | Antibacterial circle (mm) |
| Escherichia coli | 45.3±0.4 |
| Yeast | 39.8±0.3 |
| Staphylococcus aureus | 44.2±0.2 |
| Acetobacter | 46.8±0.5 |
And (3) determining the antibacterial effect: preparing 5.0 mug/ml of separated and purified antibacterial peptide by using sterile water, and performing biological activity determination by using a fungus plate confrontation experiment (the experiment process is as follows: a pathogenic fungus block is placed in the center of a 90 mm culture dish containing 15ml of PDA culture medium, holes are drilled at a position with a distance of 25 mm, a sample to be detected is injected into the holes, the sample is cultured for 3 days at 28 ℃, and the antibacterial efficiency is observed) and a bacteria inhibition zone experiment (the experiment process is as follows: preparing a bacillus subtilis aqueous solution containing 120 bacteria per ml, adding 1 ml of the bacillus subtilis sterile aqueous solution into 15ml of LB culture medium, drilling holes on the culture medium after the culture medium is solidified, injecting the sample to be detected into the holes, culturing for 8 hours at 37 ℃, and observing the size of the inhibition zone).
Claims (3)
1. A biological fresh-keeping microbial inoculum prepared by fermenting soybean curd yellow slurry is characterized in that fermentation liquor obtained by fermenting raw materials of bacillus subtilis and soybean curd yellow slurry is directly used as the biological fresh-keeping microbial inoculum, and the concentration of thalli in the fermentation liquor reaches 1 x 1013~2.7×1013CFU/mL, the concentration of the gamma-polyglutamic acid reaches 30-50 g/L, and the yield of the antibacterial peptide reaches 1.2-2 g/L;
inoculating the bacillus subtilis strain into a liquid culture medium, and inoculating and shaking-bed culturing at 30 ℃ for 8-14 h to obtain a seed solution: inoculating the seed liquid into a fermentation culture medium according to the volume ratio of 5-20%, and carrying out fermentation culture at 180-220 rpm, wherein the culture temperature is 10-50 ℃, and the culture time is 24-30 h; the fermentation liquor obtained after the fermentation is finished is the biological fresh-keeping microbial inoculum;
the components of the liquid culture medium are calculated by w/v: 10-20% of glucose, 10-15% of soybean peptone,
0.01-0.1% of calcium chloride, 0.05% of potassium dihydrogen phosphate, 0.05% of dipotassium hydrogen phosphate, 0.01% of magnesium sulfate and the balance of distilled water, wherein the initial pH value is 6.5-7.5;
the fermentation medium is measured by w/v: 10-20% of glucose, 0.01-0.1% of calcium chloride, 0.05% of potassium dihydrogen phosphate, 0.05% of dipotassium hydrogen phosphate, 0.01% of magnesium sulfate, 2.5% of sodium glutamate, 0.3% of ammonium chloride and the balance of bean curd yellow serofluid, wherein the initial pH value is 6.5-7.5.
2. The use of the biological fresh-keeping microbial inoculum prepared by fermenting yellow soybean milk of bean curd in the fresh keeping of vegetables and fruits as claimed in claim 1.
3. The application of claim 2, wherein the biological preservative is sprayed on the picked fruits and vegetables in an amount that the surfaces of the fruits and vegetables are completely wet.
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| CN104651274A (en) * | 2015-01-29 | 2015-05-27 | 湖南农业大学 | Method for performing high-density culture of bacillus |
| CN104878060A (en) * | 2015-06-25 | 2015-09-02 | 湖南农业大学 | Bacillus subtilis culture medium for producing anti-microbial peptide and application thereof |
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| CN104651274A (en) * | 2015-01-29 | 2015-05-27 | 湖南农业大学 | Method for performing high-density culture of bacillus |
| CN104878060A (en) * | 2015-06-25 | 2015-09-02 | 湖南农业大学 | Bacillus subtilis culture medium for producing anti-microbial peptide and application thereof |
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