CN109984189B - Fresh-cut fruit and vegetable fresh-keeping agent compounded with bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens - Google Patents
Fresh-cut fruit and vegetable fresh-keeping agent compounded with bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens Download PDFInfo
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- 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
-
- 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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- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
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Abstract
The invention belongs to the technical field of microorganism application, and provides a fresh-cut fruit and vegetable fresh-keeping agent compounded by bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens. Bacillus licheniformis, Bacillus atrophaeus and Bacillus amyloliquefaciens with strong antibacterial activity are selected from Shanxi mature vinegar fermented grains and preserved in China general microbiological culture Collection center with the serial numbers of CGMCC 169909, 1699 and 15732 respectively. The supernatant obtained by fermenting the strain is prepared into 1.0mg/mL composite preservative after ammonium sulfate precipitation, dialysis and freeze drying, and is used for preserving fresh cut apples and lettuce, and the result shows that after the fresh cut apples and the lettuce sprayed with the pathogenic bacteria mixed solution are treated by the composite preservative, the composite preservative has obvious bacteriostatic action compared with a control group, the colony numbers of staphylococcus aureus, escherichia coli, salmonella enteritidis, shigella flexneri and listeria monocytogenes are all obviously reduced, and the putrefaction rate of the fresh cut apples and the lettuce is reduced.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to a fresh-cut fruit and vegetable fresh-keeping agent compounded by bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens.
Background
The fresh-cut fruits and vegetables are instant fruit and vegetable products formed by selecting, cleaning, peeling, cutting, sterilizing, packaging and other production processes of fresh fruits and vegetable raw materials, and are also called micro-processing fruits and vegetables. The fresh-cut fruits and vegetables keep the original fresh state of the fruits and vegetables, and the products are clean and sanitary after processing, belong to the category of clean vegetables, are natural, nutritional, fresh and convenient, and can meet the requirements of people on pursuing natural, nutritional and fast-paced life styles and the like. The market demand is gradually increased, and the fresh-cut fruit and vegetable industry is bound to develop rapidly.
The original tissue state of fresh fruits and vegetables is destroyed by the cutting process, the physiology of the fresh fruits and vegetables is changed remarkably, the juice characteristics of the fruits and vegetables can provide propagation conditions for microorganisms, the infected microorganisms mainly comprise pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella enteritidis, listeria monocytogenes and the like, and if the fresh fruits and vegetables are infected by the pathogenic bacteria in the processes of processing, transportation and sale, the fresh fruits and vegetables can be quickly rotted, so that the nutrition and the economic value of the products are influenced, and food-borne diseases can be caused to harm the health of the public. Therefore, effective bacteriostasis measures should be implemented on fresh-cut fruit and vegetable products in the production and storage processes to ensure the product quality.
At present, the chemically synthesized bacteriostat is widely applied to fresh-keeping of fresh-cut fruits and vegetables, and although the chemically synthesized bacteriostat is low in price and rapid in sterilization, the bacteriostat has some unsafe problems of cancer induction, teratogenicity, chronic poisoning of food and the like, so that the research and development of natural and safe food sterilization and preservative are particularly important. Compared with chemically synthesized bacteriostats, natural bacteriostats have the characteristics of wide action range, no toxicity and strong bacteriostasis. Wherein, the microbial source preservative is a research hotspot of the current novel food bacteriostatic agent. The spore bacteria are bacteria capable of producing various important metabolites including high-efficiency antibacterial substances, and have the advantages of fast growth and reproduction, easy survival, strong stress resistance, simple nutritional requirement and the like, so the spore bacteria have good application prospect in the aspect of development of food fresh-keeping preservatives. The publication No. CN 106939289A discloses that the amylospore strain and the product thereof can effectively inhibit the postharvest putrefaction of various fruits, and can be used for preparing a biocontrol preservative which can inhibit the putrefaction bacteria such as penicillium, alternaria and the like and has a wider bacteriostatic spectrum. At present, most of spore bacteria are developed into a single strain, 3 strains of spore bacteria respectively having strong bacteriostatic activity on staphylococcus aureus (gram positive bacteria), escherichia coli (gram negative bacteria) and aspergillus niger (pathogenic fungi) are separated from Shanxi mature vinegar fermented grains, and bacteriostatic substances of the spore bacteria are prepared and then are compounded and applied to fresh-cut fruits and vegetables, so that the spore bacteria have wider bacteriostatic application range and better bacteriostatic effect.
Disclosure of Invention
The invention aims to provide a safe and effective fresh-cut fruit and vegetable preservative, and a preparation method and a using method thereof, which can effectively inhibit rot caused by microorganism propagation and browning phenomenon caused by enzymatic reaction in preservation of fresh-cut fruits and vegetables so as to maintain the quality of the fresh-cut fruits and vegetables and prolong the shelf life of products.
The invention provides a composite preservative prepared by antibacterial active substances generated by bacillus licheniformis CGMCC 169909, bacillus atrophaeus CGMCC 1699 and bacillus amyloliquefaciens CGMCC 15732 in the fermentation process of Shanxi mature vinegar, and the composite preservative is applied to the preservation of fresh-cut apples and fresh-cut lettuce. The technical scheme of the invention is as follows:
screening of spore bacteria with high bacteriostatic activity: respectively collecting vinegar culture samples in the acetic acid fermentation stage of Shanxi mature vinegar, diluting and coating by adopting an MRS culture medium, finally separating 50 strains of spore bacteria, and screening the spore bacteria with bacteriostatic activity by adopting an Oxford cup bacteriostatic ring method, wherein the spore bacteria 297 has better bacteriostatic effect on gram-negative bacteria, the bacteriostatic effect on escherichia coli is most obvious, and the diameter of the bacteriostatic ring is 20.54 mm; the spore bacteria 1671 have good bacteriostatic effect on gram-positive bacteria, wherein the bacteriostatic effect on staphylococcus aureus is most obvious, and the diameter of a bacteriostatic zone is 21.82 mm; the bacillus 2014 has a good bacteriostatic effect on fruit and vegetable spoilage moulds, wherein the bacteriostatic effect of aspergillus niger is most obvious, and the diameter of a bacteriostatic zone is 23.48 mm.
The preparation method of the MRS culture medium comprises the following steps: 20g of glucose, 10g of peptone, 10g of beef extract, 5g of yeast extract and K2HPO42g, sodium acetate 2g, ammonium citrate tribasic 2g, Tween 801 g, MgSO4 0.2g，MnSO40.05g, and 1000mL of distilled water. Adjusting pH to 6.2 ‒ 6.6.6, and sterilizing at 121 deg.C for 20 min.
16Sr DNA sequencing was performed on bacillus 297, 1671, 2014 with primers: upstream: 5' -CAGATGGGAGCTT
GCTCCCTG-3', downstream: 5'-CGACTTCACCCAATCATCTG-3', Bacillus 297 is identified as Bacillus atrophaeus ((Bacillus atrophaeus))Bacillus atrophaeus) Bacillus 1671 is Bacillus licheniformis (B.licheniformis)Bacillus licheniformis) And the bacillus 2014 is bacillus amyloliquefaciens (B.amyloliquefaciens)Bacillus amyloliquefaciens) The bacillus atrophaeus strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation numbers are Bacillus atrophaeus CGMCC 1699, Bacillus licheniformis CGMCC 16909 and Bacillus amyloliquefaciens CGMCC 15732.
The preparation of the fresh-cut fruit and vegetable compound preservative: respectively inoculating activated Bacillus licheniformis CGMCC 169909, Bacillus atrophaeus CGMCC 1699 and Bacillus amyloliquefaciens CGMCC 15732 in 5% of inoculum size in MRS liquid culture medium, respectively culturing at 37 deg.C and 160rpm/min for 24h, centrifuging at 4 deg.C and 10000r/min for 10 min, and collecting supernatant; adjusting the supernatant 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 reaches 80%; and centrifuging at 4000r/min for 20min, collecting precipitate, dissolving with phosphate buffer solution with pH =7.2, dialyzing to remove salt, carrying out vacuum freeze drying, and compounding according to the mass ratio of 1:1:1 to obtain the composite preservative.
The preparation method of the phosphate buffer solution comprises the following steps: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, sodium chloride 8.0g, potassium chloride 0.2g, 1000mL, pH = 7.2.
The vacuum freeze drying is carried out for 40 h at-40 ℃ and under the vacuum degree of 100 Pa.
The composite preservative prepared according to the technical scheme is applied to the preservation of fresh-cut apples, and the specific method comprises the following steps: cutting fructus Mali Pumilae into small pieces (2 cm × 2cm × 2 cm), soaking fresh cut fructus Mali Pumilae in 0.5% ascorbic acid and 1.0% citric acid for 2min, draining, placing into sterile plate, spraying 250 μ L pathogenic bacteria mixed solution (10 μ L) with 50mL small spray can4 cfu/mL), then uniformly spraying 500 mu L of compound preservative (the concentration is 1.0 mg/mL), then drying by aseptic wind on an ultra-clean bench, covering a preservative film, placing in an environment at 4 ℃, detecting the colony numbers of common putrefying bacteria such as staphylococcus aureus, escherichia coli, listeria monocytogenes, salmonella enteritidis and shigella flexneri during preservation, and determining the change of physicochemical indexes such as weight loss rate, total phenol, vitamin C, MDA content, POD and PPO enzyme activity and the like of the fresh-cut apples during preservation.
The composite preservative prepared according to the technical scheme is applied to the preservation of fresh-cut lettuce, and the specific method comprises the following steps: slicing lettuce, cleaning in 120mg/L chlorine water, draining, placing into a sterile plate, spraying 250 μ L pathogenic bacteria mixed solution (with concentration of 10) with 50mL small spray can4 cfu/mL), and then homogenizedSpraying 500 mu L of compound preservative (the concentration is 1.0 mg/mL), putting the mixture into a super clean bench, air-drying the mixture, and packaging the dried mixture by using preservative films. The fresh-keeping effect of the compound preservative on the fresh-cut lettuce is observed after the fresh-keeping agent is placed in an environment with the temperature of 4 ℃, the colony numbers of staphylococcus aureus, escherichia coli, listeria monocytogenes, salmonella enteritidis and shigella flexneri, which are common putrefying bacteria, during the preservation period are detected, and the changes of the physicochemical indexes of the fresh-cut lettuce during the preservation period, such as the weight loss rate, the total phenol content, the vitamin C, MDA content, the POD enzyme activity, the PPO enzyme activity and the like, are measured.
The fresh-cut fruit and vegetable compound preservative solution with the concentration of 1.0mg/mL is prepared by sterile water when being applied to the preservation of fresh-cut fruits and vegetables.
The results show that after the fresh-cut apples and the lettuce sprayed with the pathogenic bacteria mixed solution are treated by the composite preservative, compared with a control group, the composite preservative has obvious bacteriostatic action, the colony numbers of staphylococcus aureus, escherichia coli, salmonella enteritidis, shigella flexneri and listeria monocytogenes are all obviously reduced, and the putrefaction rate of the fresh-cut apples and the lettuce is reduced.
Drawings
FIG. 1 is a colony morphology and a cell morphology diagram of Bacillus licheniformis CGMCC 169909, Bacillus atrophaeus CGMCC 1699 and Bacillus amyloliquefaciens CGMCC 15732, wherein: a is the colony morphology of Bacillus atrophaeus CGMCC 1699; b is a cell morphology diagram of Bacillus atrophaeus CGMCC 169111000 multiplied by; c is the colony form of the bacillus licheniformis CGMCC 16909; d is a cell morphology diagram of the bacillus licheniformis CGMCC 169091000 multiplied by the weight; e is the colony morphology of bacillus amyloliquefaciens CGMCC 15732; f is a cell morphology diagram of the bacillus amyloliquefaciens with the molecular weight of CGMCC 157321000 multiplied;
FIG. 2 shows a phylogenetic tree of 16S rDNA sequences of Bacillus atrophaeus CGMCC 1699, Bacillus licheniformis CGMCC 16909 and Bacillus amyloliquefaciens CGMCC 15732;
FIG. 3 is an application of the fresh-cut fruit and vegetable compound preservative in fresh-cut apple preservation, wherein: a is fresh-cut apple of 0 day of aseptic water treatment; b, fresh-cut apples on 5 th day of sterile water treatment; c, fresh-cut apples on day 8 after sterile water treatment; d, processing the fresh-cut apples on day 0 by using the fresh-cut fruit and vegetable compound preservative; e, fresh-cut apples treated by the fresh-cut fruit and vegetable compound preservative on the 5 th day are processed;
f is the fresh-cut apple processed by the fresh-cut fruit and vegetable compound preservative on the 8 th day;
FIG. 4 is the colony number of the fresh-cut fruit and vegetable compound preservative for preserving fresh-cut apples, wherein: a is the total number of colonies of the fresh-cut apples; b is the colony number of escherichia coli of the fresh-cut apples; c is the staphylococcus aureus colony number of the fresh-cut apples; d is the number of Shigella flexneri colonies of the fresh-cut apples; e is the number of salmonella enteritidis colonies of the fresh-cut apples; f is the number of the listeria monocytogenes colonies of the fresh-cut apples;
FIG. 5 shows the physicochemical indexes of the fresh-cut fruit and vegetable compound preservative for keeping fresh of fresh-cut apples, wherein: a is the weight loss rate of the fresh-cut apples; b is the total phenol content of the fresh-cut apples; c is the vitamin C content of the fresh-cut apples; d is the MDA content of the fresh-cut apples; e is PPO enzyme activity of fresh-cut apples; f is POD enzyme activity of the fresh-cut apples;
FIG. 6 is an application of the fresh-cut fruit and vegetable compound preservative in fresh-cut lettuce preservation, wherein: a is fresh-cut lettuce of 0 day of sterile water treatment; b is fresh-cut lettuce treated by sterile water on day 5; c is fresh-cut lettuce of 7 days after sterile water treatment; d, processing fresh-cut lettuce on day 0 by the fresh-cut fruit and vegetable compound preservative; e is the fresh-cut lettuce processed by the fresh-cut fruit and vegetable compound preservative on the 5 th day;
f is the fresh-cut lettuce processed on the 7 th day by the fresh-cut fruit and vegetable compound preservative;
FIG. 7 shows the number of colonies of the fresh-cut fruit and vegetable compound antistaling agent on the fresh-cut lettuce, wherein: a is the total number of bacterial colonies of the fresh-cut lettuce; b is the number of escherichia coli colonies of the fresh-cut lettuce; c is the staphylococcus aureus colony number of the fresh-cut lettuce; d is the number of shigella flexneri colonies of the fresh-cut lettuce; e is the number of salmonella enteritidis colonies of the fresh-cut lettuce; f is the colony number of the listeria monocytogenes of the fresh-cut lettuce;
FIG. 8 is a diagram showing the physicochemical indexes of the fresh-cut fruit and vegetable compound preservative for keeping fresh of fresh-cut lettuce, wherein: a is the weight loss ratio of the fresh-cut lettuce; b is the total phenol content of the fresh-cut lettuce; c is the vitamin C content of the fresh-cut lettuce; d is the MDA content of the fresh-cut lettuce; e is PPO enzyme activity of fresh-cut lettuce; f is POD enzyme activity of fresh-cut lettuce.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
Example 1: screening, identification and preservation of spore bacteria with high bacteriostatic activity
(1) Screening of spore bacteria with high bacteriostatic activity: respectively collecting a wine mash sample in a Shanxi mature vinegar alcohol fermentation stage and a vinegar mash sample in an acetic acid fermentation stage, diluting and coating by adopting an MRS culture medium, and finally separating 50 spore bacteria. Screening the bacillus subtilis with bacteriostatic activity by adopting an oxford cup bacteriostatic circle method, wherein the bacillus 297 has a good bacteriostatic effect on gram-negative bacteria, the bacillus coli has the most remarkable bacteriostatic effect, and the diameter of a bacteriostatic circle is 20.54 mm; the spore bacteria 1671 have good bacteriostatic effect on gram-positive bacteria, wherein the bacteriostatic effect on staphylococcus aureus is most obvious, and the diameter of a bacteriostatic zone is 21.82 mm; the bacillus 2014 has a good bacteriostatic effect on fruit and vegetable spoilage moulds, wherein the bacteriostatic effect of aspergillus niger is most obvious, and the diameter of a bacteriostatic zone is 23.48 mm.
The escherichia coli, the staphylococcus aureus and the aspergillus niger are purchased to a China general microbiological culture collection center, the preservation number of the escherichia coli is CGMCC 1.1684, the preservation number of the staphylococcus aureus is CGMCC 1.184, and the preservation number of the aspergillus niger is CGMCC 3.2915.
The preparation method of the MRS solid culture medium comprises the following steps: 20g of glucose, 10g of peptone, 10g of beef extract, 5g of yeast extract and K2HPO42g, sodium acetate 2g, ammonium citrate tribasic 2g, Tween 801 g, MgSO4 0.2g，MnSO40.05g, and 1000mL of distilled water. Adjusting pH to 6.2 ‒ 6.6.6, and sterilizing at 121 deg.C for 20 min.
The specific method for screening the spore bacteria with the bacteriostatic activity comprises the following steps: inoculating 50 separated and purified spore bacteria to an MRS liquid culture medium, culturing at 37 ℃ at 160 r/min for 24h, centrifuging at 10000r/min for 10 min, collecting spore bacteria fermentation supernatant, and respectively adjusting to be neutral by 6.0 mol/L NaOH for later use. Escherichia coli, Staphylococcus aureus, Shigella flexneri, Listeria monocytogenes, Salmonella enteritidis, Klebsiella pneumoniae, Enterobacter hollisae, Bacillus subtilis, and Bacillus subtilis,The pseudomonas aeruginosa is respectively inoculated into BHI liquid culture medium at 37 ℃ and cultured for 12 h at 160 r/min, and then diluted 5 times (10 times) by normal saline5cfu/mL), transferring 0.1mL of diluted bacteria liquid to a BHI flat plate for coating, placing a sterilized Oxford cup on the BHI flat plate, freezing the Oxford cup in a refrigerator for 1h, taking out, transferring 200 mu L of spore bacteria fermentation supernatant to the Oxford cup, and placing the Oxford cup in a 37 ℃ incubator for culture; inoculating Aspergillus niger, Aspergillus flavus and Penicillium to a PDA culture medium plate with an Oxford cup, transferring the spore bacteria fermentation supernatant into an Oxford cup with the volume of 200 mu L, and culturing in a 30 ℃ incubator.
The Shigella flexneri, the Listeria monocytogenes, the Klebsiella pneumoniae, the Enterobacter hulsbergii and the Pseudomonas aeruginosa are purchased to the North Nara Biotechnology center, the deposit number of the Shigella flexneri is ATCC 12022, the deposit number of the Listeria monocytogenes is ATCC 19114, the deposit number of the Klebsiella pneumoniae is ATCC 10031, the deposit number of the Enterobacter hulsbergii is ATCC 700323 and the deposit number of the Pseudomonas aeruginosa is ATCC 9027; the salmonella is purchased to China medical bacteria preservation management center with the preservation number of CMCC 50041-16; the aspergillus flavus and the penicillium are purchased to China general microbiological culture collection center, the preservation number of the aspergillus flavus is CGMCC 3.11969, and the preservation number of the penicillium is CGMCC 3.15687.
(2) Identification and preservation of spore bacteria with high bacteriostatic activity
Morphological identification: after a small amount of spore bacteria 297, 1671 and 2014 are picked by an inoculating loop, streaking is carried out on an MRS plate culture medium, a single colony is separated, the colony growing on the culture medium is photographed and recorded, and then the morphological characteristics of the cell are observed through gram staining microscopic examination. The diameter of a colony formed by the spore bacteria 297 on an MRS plate culture medium is 4.42 mm, the colony is approximately circular, and the middle bulge is snowflake-shaped, milky white, opaque and large in viscosity; the spore bacteria 1671 form a colony on an MRS plate culture medium, wherein the colony diameter is 4.02 mm, the surface is rough, opaque, sticky, raised and wrinkled, the edge is irregular, and the colony is pale white; the diameter of a colony formed by the spore bacteria 2014 on the MRS plate culture medium is 4.58 mm, and the colony is circular, sticky, rough and convex in surface, regular in edge and milky whiteOpaque; the cell morphology is as follows: all gram staining is G+It is rod-shaped and has spores in the middle, as shown in FIG. 1.
16Sr DNA sequencing: extracting genomes of the spore bacteria 297, 1671 and 2014 by using the kit, and carrying out 16Sr DNA sequencing and strain identification, wherein the primers are as follows: upstream: 5'-CAGATGGGAGCTTGCTCCCTG-3' downstream: 5'-CGACTTCACCCAATCATCTG-3', homology comparison using BLAST software, shows that Bacillus 297 is Bacillus atrophaeus ((B. atrophaeus))Bacillus atrophaeus) Bacillus 1671 is Bacillus licheniformis (B.licheniformis)Bacillus licheniformis) And the bacillus 2014 is bacillus amyloliquefaciens (B.amyloliquefaciens)Bacillus amyloliquefaciens) And a phylogenetic tree is constructed, see fig. 2. The bacillus atrophaeus strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation numbers are Bacillus atrophaeus CGMCC 1699, Bacillus licheniformis CGMCC 16909 and Bacillus amyloliquefaciens CGMCC 15732.
Example 2: preparation of fresh-cut fruit and vegetable composite antistaling agent
Respectively inoculating activated Bacillus licheniformis CGMCC 169909, Bacillus atrophaeus CGMCC 1699 and Bacillus amyloliquefaciens CGMCC 15732 in 5% of inoculum size in MRS liquid culture medium, respectively culturing at 37 deg.C and 160rpm/min for 24h, centrifuging at 4 deg.C and 10000r/min for 10 min, and collecting supernatant; adjusting the supernatant 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 reaches 80%; and centrifuging at 4000r/min for 20min, collecting precipitate, dissolving with phosphate buffer solution with pH =7.2, dialyzing to remove salt, performing vacuum freeze drying, compounding according to the weight ratio of 1:1:1, and performing vacuum packaging to obtain the composite preservative.
The preparation method of the phosphate buffer solution comprises the following steps: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, sodium chloride 8.0g, potassium chloride 0.2g, 1000mL, pH = 7.2.
The vacuum freeze drying is carried out for 40 h at-40 ℃ and under the vacuum degree of 100 Pa.
Example 3: application of composite preservative in preservation of fresh-cut apples
The composite preservative prepared according to the technical scheme is applied to the preservation of fresh-cut apples, and the specific implementation method comprises the following steps:
cutting fructus Mali Pumilae into small pieces (2 cm × 2cm × 2 cm), soaking fresh cut fructus Mali Pumilae in 0.5% ascorbic acid and 1.0% citric acid (color fixative) for 2min, draining, placing into sterile flat dish, spraying 250 μ L pathogenic bacteria mixed solution (with concentration of 10 μ L) with 50mL small spray can4 cfu/mL), then uniformly spraying 500 mu L of compound preservative (the concentration is 1.0 mg/mL), then drying by aseptic wind on an ultra-clean bench, covering a preservative film, placing in an environment at 4 ℃, detecting the colony numbers of common putrefying bacteria such as staphylococcus aureus, escherichia coli, listeria monocytogenes, salmonella enteritidis and shigella flexneri during preservation, and determining the change of physicochemical indexes such as weight loss rate, total phenol, vitamin C, MDA content, POD and PPO enzyme activity and the like of the fresh-cut apples during preservation.
As shown in fig. 3, the rate of decay of the fresh-cut apples treated with the composite antistaling agent was reduced compared to the control group; and has obvious bacteriostatic action, wherein the colony numbers of staphylococcus aureus, escherichia coli, salmonella enteritidis, shigella flexneri and listeria monocytogenes are all obviously reduced (as shown in figure 4). As can be seen from fig. 5, the weight loss rate and the MDA content increase rate of the fresh-cut apples treated by the composite preservative are slow, the vitamin C and total phenol contents decrease slowly, the peroxidase activity (POD) and the polyphenol oxidase activity (PPO) both tend to increase and decrease first, and the final enzyme activity is higher than that of the fresh-cut apples treated by the sterile water, so that the fresh-cut apples have a good fresh-keeping effect.
The specific preparation method of the pathogenic bacteria mixed liquor comprises the following steps: respectively inoculating Escherichia coli, Staphylococcus aureus, Shigella flexneri, Salmonella enteritidis and Listeria monocytogenes into BHI liquid culture medium, culturing at 37 deg.C and 160 r/min for 12 h to obtain pathogen liquid, centrifuging at 10000r/min and l 0min to discard supernatant, washing the precipitate with 0.1% sterile peptone water for 2 times, re-suspending the precipitate with 0.1% sterile peptone water, and respectively making into 104 cfu/mL of bacterial suspension, and mixing the bacterial suspension in equal volume to form a pathogenic bacteria mixed solution.
The specific method for preparing the composite preservative comprises the following steps: the compound preservative is prepared into 1.0mg/mL by sterile water.
The determination method for detecting the microorganisms comprises the following steps: weighing 10g of a sample of a fresh-cut apple under an aseptic condition, shearing the sample, adding the cut apple into 90mL of sterilized physiological saline, violently shaking, uniformly mixing, carrying out gradient dilution and coating, and then counting common putrefying bacteria of staphylococcus aureus (mannitol sodium chloride agar culture medium), escherichia coli (eosin methylene blue agar culture medium), listeria monocytogenes (PALCAM agar culture medium), salmonella enteritidis (Mackanka agar culture medium) and shigella flexneri (shigella selection culture medium).
The specific measuring method of the physical and chemical indexes comprises the following steps:
(1) the weight loss rate measuring method comprises the following steps: during preservation, fresh-cut apples under different treatment conditions are weighed in the same time period every day, and the weight loss rate is calculated. Weight loss rate = (weight of fruit at initial preservation-weight of fruit at final preservation)/weight of fruit at initial preservation x100%
(2) Method for determining total phenols: adopting Folin-Ciocalteau method for colorimetric determination, weighing 5g of fresh-cut apples, adding 20mL of precooled methanol solution for homogenating in ice bath, centrifuging at 4 ℃ and 12000 r/min for 20min, taking 1mL of supernatant fluid in a 25mL volumetric flask, adding 1mL of forinophenol solution, oscillating and mixing uniformly, adding 10mL of 7.5% sodium carbonate solution, and finally diluting to 25mL with deionized water. After standing at 25 ℃ for 1h, the absorbance at 750 nm was measured.
(3) The determination method of vitamin C comprises the following steps: measuring by adopting a 2, 6-dichloro indophenol titration method, weighing 5g of fresh-cut apples, adding an equivalent amount of 2% HCl solution, and smashing into homogenate. 3g of a slurried sample (made to contain 0.1-0.5mg of ascorbic acid) was weighed into a small beaker, transferred to a 100mL volumetric flask with 1% HCl solution, diluted to the mark and shaken well. The sample was filtered and the first few milliliters of filtrate discarded, then 10mL of the filtrate was quickly pipetted into a 50mL Erlenmeyer flask and titrated with calibrated 2,6 dichloroindophenol until the solution was pink in color and did not fade within 15 seconds.
(4) The method for measuring the content of Malondialdehyde (MDA) comprises the following steps: weighing 0.4g of fresh-cut apples, putting the fresh-cut apples into a mortar, adding a small amount of quartz sand and 2mL of 0.1% TCA, homogenizing the inkstone, transferring the inkstone into a test tube, washing the mortar twice by using 3mL of 0.1% TCA, combining extracting solutions, adding 5mL of 0.5% thiobarbituric acid solution into the extracting solution, shaking the mixture evenly, putting the test tube into a boiling water bath for color development reaction for 15min, immediately taking out the test tube after the time, putting the test tube into an ice bath for cooling to room temperature, after the test tube is cooled, centrifuging the test tube for 15min at 3000r/min in a 10mL centrifuge tube, taking supernatant, and measuring light absorption values at 532nm and 600 nm.
(5) Method for measuring peroxidase activity (POD): weighing 5g of fresh-cut apples, mixing with 20mL of phosphate buffer (0.2 mol/L pH 7.5) containing 2.5% of cross-linked polyvinylpyrrolidone, rapidly grinding under ice bath condition, centrifuging at 4 ℃ and 10000r/min for 15mim, and obtaining supernatant as crude enzyme. Sucking 1mL of crude enzyme solution, adding 1mL of phosphate buffer solution with pH 7.5, adding 1mL of buffer solution containing 1mmol/L of catechol, mixing, adjusting to zero with distilled water, rapidly standing at 420nm to obtain absorbance value, recording every 10s, and measuring for 2min, wherein the absorbance value change of the sample is 0.001 as 1 activity unit, and the unit is U/(g & lt, & gt min).
(6) The method for measuring polyphenol oxidase activity (PPO) comprises the following steps: weighing 5g of fresh-cut apples, mixing with 20mL of phosphate buffer (0.2 mol/L pH 7.5) containing 2.5% of cross-linked polyvinylpyrrolidone, rapidly grinding under ice bath condition, centrifuging at 4 ℃ and 10000r/m for 15mim, and obtaining supernatant as crude enzyme. Taking 2mL of 0.05% guaiacol solution (prepared by 0.2mol/L of phosphoric acid buffer solution with pH of 7.5), adding 0.5mL of crude enzyme extract, keeping the temperature in a water bath kettle at 30 ℃ for 5min, adding 1mL of 0.08% H2O2The solution was then mixed, zeroed with distilled water, rapidly placed at 470nm for absorbance value determination, recorded every 30s, and determined for 3min, with the sample absorbance change of 0.001 as 1 activity unit, in U/(g & min).
Example 4: application of composite preservative in preservation of fresh-cut lettuce
The composite preservative prepared according to the technical scheme is applied to the preservation of fresh-cut lettuce, and the specific implementation method comprises the following steps:
slicing lettuce, and washing in 120mg/L chlorine water (color fixative)Draining, placing into a sterile plate, spraying 250 μ L of pathogenic bacteria mixed solution (with concentration of 10) with a small 50mL spray can4 cfu/mL), then uniformly spraying 500 mu L of the compound preservative (the concentration is 1.0 mg/mL), putting the mixture into a super clean bench, air-drying the mixture, and packaging the dried mixture by using a preservative film. The fresh-keeping effect of the compound preservative on the fresh-cut lettuce is observed after the fresh-keeping agent is placed in an environment with the temperature of 4 ℃, the colony numbers of staphylococcus aureus, escherichia coli, listeria monocytogenes, salmonella enteritidis and shigella flexneri, which are common putrefying bacteria, during the preservation period are detected, and the changes of the physicochemical indexes of the fresh-cut lettuce during the preservation period, such as the weight loss rate, the total phenol content, the vitamin C, MDA content, the POD enzyme activity, the PPO enzyme activity and the like, are measured.
As shown in fig. 6, the rate of spoilage was reduced in the fresh-cut lettuce treated with the composite antistaling agent compared to the control group; and has obvious bacteriostatic action, wherein the colony numbers of staphylococcus aureus, escherichia coli, salmonella enteritidis, shigella flexneri and listeria monocytogenes are all obviously reduced (as shown in figure 7). As can be seen from FIG. 8, the weight loss rate of the fresh-cut lettuce treated by the composite preservative is relatively slow at the beginning, and then relatively fast, the contents of the vitamin C, MDA and the total phenol are relatively slow to decrease, the peroxidase activity (POD) and the polyphenol oxidase activity (PPO) are both in the trend of increasing firstly and then decreasing, and the final enzyme activity is higher than that of the fresh-cut lettuce treated by sterile water, so that the fresh-keeping effect is good.
Claims (4)
1. A fresh-cut fruit and vegetable fresh-keeping agent compounded with bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens is characterized in that: the bacillus licheniformis, the atrophic bacillus and the amylolytic bacillus are obtained by separating and screening the Shanxi mature vinegar culture, the preservation number of the bacillus licheniformis is CGMCC 169909, the preservation number of the atrophic bacillus is CGMCC 16915, the preservation number of the amylolytic bacillus is CGMCC 15732, and the preservation unit is as follows: china general microbiological culture Collection center (CGMCC), Address: west road No. 1 hospital No. 3, north jing, chaoyang district, preservation date: 12 and 10 days in 2018, 5 and 7 days in 2018;
the preparation method of the fresh-cut fruit and vegetable compound preservative comprises the following steps: respectively inoculating activated Bacillus licheniformis CGMCC 169909, Bacillus atrophaeus CGMCC 1699 and Bacillus amyloliquefaciens CGMCC 15732 into MRS liquid culture medium according to the inoculation amount of 3%, respectively culturing at 37 deg.C and 160rpm/min for 24h, centrifuging at 4 deg.C and 10000r/min for 10 min, and collecting supernatant; adjusting the supernatant 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 reaches 80%; and centrifuging at 4000r/min for 20min, collecting precipitate, dissolving with phosphate buffer solution with pH =7.2, dialyzing to remove salt, carrying out vacuum freeze drying, and compounding according to the mass ratio of 1:1:1 to obtain the composite preservative.
2. The fresh-cut fruit and vegetable fresh-keeping agent compounded by bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens according to claim 1, which is characterized in that: the preparation method of the phosphate buffer solution comprises the following steps: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, sodium chloride 8.0g, potassium chloride 0.2g, 1000mL, pH = 7.2.
3. The fresh-cut fruit and vegetable fresh-keeping agent compounded by bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens according to claim 1, which is characterized in that: the vacuum freeze drying is drying for 40 h at-40 ℃ and the vacuum degree of 100 Pa.
4. The application of the fresh-cut fruit and vegetable fresh-keeping agent compounded by the bacteriostatic substances produced by the bacillus licheniformis, the bacillus atrophaeus and the bacillus amyloliquefaciens in the fresh-cut fruits and vegetables in the claim 1 is characterized in that the fresh-cut fruit and vegetable fresh-keeping agent compounded solution with the concentration of 1.0mg/mL is prepared by using sterile water.
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