CN109984189A - A kind of fresh-cut fruit and vegetable antistaling agent by bacillus licheniformis, atrophy bacillus and the produced bateriostatics compounding of bacillus amyloliquefaciens - Google Patents
A kind of fresh-cut fruit and vegetable antistaling agent by bacillus licheniformis, atrophy bacillus and the produced bateriostatics compounding of bacillus amyloliquefaciens Download PDFInfo
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- CN109984189A CN109984189A CN201910354887.7A CN201910354887A CN109984189A CN 109984189 A CN109984189 A CN 109984189A CN 201910354887 A CN201910354887 A CN 201910354887A CN 109984189 A CN109984189 A CN 109984189A
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- 241000366676 Justicia pectoralis Species 0.000 title claims abstract description 113
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 62
- 235000012055 fruits and vegetables Nutrition 0.000 title claims abstract description 45
- 241000193744 Bacillus amyloliquefaciens Species 0.000 title claims abstract description 26
- 241000194108 Bacillus licheniformis Species 0.000 title claims abstract description 26
- 206010003694 Atrophy Diseases 0.000 title claims abstract description 24
- 230000037444 atrophy Effects 0.000 title claims abstract description 24
- 238000013329 compounding Methods 0.000 title claims abstract description 9
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
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- 235000012333 Vitis X labruscana Nutrition 0.000 description 2
- 240000006365 Vitis vinifera Species 0.000 description 2
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- 230000004913 activation Effects 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
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- 239000008103 glucose Substances 0.000 description 2
- LHGVFZTZFXWLCP-UHFFFAOYSA-N guaiacol Chemical compound COC1=CC=CC=C1O LHGVFZTZFXWLCP-UHFFFAOYSA-N 0.000 description 2
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- 239000007788 liquid Substances 0.000 description 2
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
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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
- A23B7/14—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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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to technical field of microbe application, provide a kind of fresh-cut fruit and vegetable antistaling agent by bacillus licheniformis, atrophy bacillus and the produced bateriostatics compounding of bacillus amyloliquefaciens.Bacillus licheniformis, atrophy bacillus, bacillus amyloliquefaciens with stronger bacteriostatic activity are filtered out from Shanxi mature vinegar vinegar fermented grain, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, number is respectively CGMCC 16909,16911,15732.Strain fermentation supernatant is made to the composite preservative of 1.0mg/mL after ammonium sulfate precipitation, dialysis, freeze-drying, with on Fresh-cut Apples and romaine lettuce are fresh-keeping, after the Fresh-cut Apples of sprinkling pathogen mixed liquor and romaine lettuce are handled with composite preservative as the result is shown, compared with the control group, there is apparent bacteriostasis, staphylococcus aureus, Escherichia coli, Bacterium enteritidis, shigella flexneri, Listeria monocytogenes clump count have significant decrease, reduce the spoilage rate of Fresh-cut Apples and romaine lettuce.
Description
Technical field
The invention belongs to technical field of microbe application, and in particular to one kind is by bacillus licheniformis, atrophy bacillus
With the fresh-cut fruit and vegetable antistaling agent of the produced bateriostatics compounding of bacillus amyloliquefaciens.
Background technique
Fresh-cut fruit and vegetable be fresh fruit and vegetable raw-material by selecting, cleaning, removing the peel, cutting, disinfection, the production work such as packaging
The instant fruit and vegetable product that skill is formed, also referred to as micro Process fruits and vegetables.Fresh-cut fruit and vegetable had not only maintained the original fresh state of fruits and vegetables, but also passed through
Processing makes product sanitation and hygiene, belongs to clean vegetables scope, natural, nutrition, fresh, conveniently, can meet people pursue natural, nutrition,
The demand of allegro life style etc..The market demand is gradually increased, and fresh-cut fruit and vegetable industry will welcome flourishing at full speed
Development.
Due to the original structural state of cutting processing damage fresh fruit of vegetables, significant changes, fruits and vegetables itself occur for physiology
The juice characteristic of generation can provide Reproduction Conditions to microorganism, and infecting microorganism mainly includes pathogenic bacterium such as large intestine bar
Bacterium, staphylococcus aureus, Bacterium enteritidis, Listeria monocytogenes etc., if fresh-cut fruit and vegetable is in processing, transport, sales process
Middle to be infected by pathogenic bacteria, fresh-cut fruit and vegetable product meeting fast rotting can also cause food source to influence product nutrition and economic value
Property disease and the health for endangering the public.Therefore, effective suppression should be implemented to fresh-cut fruit and vegetable product in production and storage
Bacterium measure is to ensure product quality.
Currently, chemical synthesis class bacteriostatic agent in fresh-cut fruit and vegetable is fresh-keeping using more, although chemically synthesized bacteriostatic agent
Rapidly, but such bacteriostatic agent lures carcinous, teratogenesis and easily causes food slow poisoning etc. there are some for cheap, sterilization
Unsafe problems, thus, the food sterilization and preservative for researching and developing natural safety are particularly important.Relative to chemical synthesis
For bacteriostatic agent, natural bacteriostatic agent has the characteristics that wide, non-toxic, the strong biocidal property of sphere of action.Wherein, microbial source antistaling agent
It is the research hotspot of current novel foodstuff bacteriostatic agent.Bacillus is that a kind of to generate a variety of important metabolites include Efficient antibacterial
The bacterium of substance, because it has many advantages, such as that growth and breeding is fast, be easy to survive, resistance is strong, nutritional requirement is simple, in food fresh keeping
Preservative exploitation aspect has a good application prospect.Publication No. be CN106939289 A announce understand starch gemma bacterial strain and
Corruption, which generates, after its product can adopt various fruits effectively inhibits, and can be used for preparing the spoilage organisms such as a kind of inhibition mould, rod method
And the biological and ecological methods to prevent plant disease, pests, and erosion antistaling agent with wider antimicrobial spectrum.The antibacterial exploitation of Bacillus at present is mostly single bacterial strain, and the present invention is old from Shanxi
It is isolated in mature vinegar vinegar fermented grain to staphylococcus aureus (gram-positive bacteria), Escherichia coli (Gram-negative bacteria), aspergillus niger
(disease fungus) has 3 plants of Bacillus of strong bacteriostatic activity respectively, and carries out composite usage in fresh-cut fruit and vegetable after preparing its bateriostatics
In fresh-keeping, and thereby it is made to have wider antibacterial application range and better fungistatic effect.
Summary of the invention
The purpose of the present invention is to provide a kind of safe and effective fresh-cut fruit and vegetable antistaling agent and preparation method thereof and users
Method can effectively inhibit in fresh-cut fruit and vegetable preservation because the rotten and brown stain as caused by enzymatic reaction caused by microbial reproduction is existing
As extending the shelf life of product to keep the quality of fresh-cut fruit and vegetable.
The present invention provides a kind of bacillus licheniformis CGMCC 16909 being isolated from Shanxi mature vinegar fermentation process,
The Substance that atrophy bacillus CGMCC 16911, bacillus amyloliquefaciens CGMCC 15732 are generated is prepared compound
Antistaling agent, and applied Fresh-cut Apples and Fresh-cut Lettuce it is fresh-keeping in.Technical scheme is as follows:
Screening with high bacteriostatic activity Bacillus: acquiring the vinegar fermented grain sample in Shanxi mature vinegar acetic fermentation stage respectively, uses
MRS culture medium is diluted coating, finally isolates 50 plants of Bacillus, carries out having suppression to it using Oxford cup inhibition zone method
The active screening of bacterium, wherein Bacillus 297 is preferable to Gram-negative bacteria fungistatic effect, wherein to the fungistatic effect of Escherichia coli
Most significant, antibacterial circle diameter is 20.54 mm；Bacillus 1671 is preferable to gram-positive bacteria fungistatic effect, wherein to golden yellow
Staphylococcic fungistatic effect is most significant, and antibacterial circle diameter is 21.82 mm；Bacillus 2014 is to the antibacterial effect of fruits and vegetables Sapromyces
Fruit is preferable, and wherein the fungistatic effect of aspergillus niger is most significant, and antibacterial circle diameter is 23.48 mm.
The MRS culture medium the preparation method comprises the following steps: glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g,
K2HPO42g, sodium acetate 2g, Triammonium citrate 2g, Tween 80 1g, MgSO40.2g, MnSO40.05g, distilled water 1000mL.
PH to 6.2 6.6 is adjusted, 121 DEG C of sterilizing 20min are spare.
16Sr DNA sequencing, primer are as follows: upstream: 5 '-CAGATGGGAGCTT are carried out to Bacillus 297,1671,2014
GCTCCCTG-3 ', downstream: 5 '-CGACTTCACCCAATCATCTG-3 ', identified Bacillus 297 are atrophy bacillus
(Bacillus atrophaeus), Bacillus 1671 be bacillus licheniformis (Bacillus licheniformis), Bacillus
2014 for bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and it is deposited in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center is hidden, deposit number is atrophy bacillus CGMCC 16911, bacillus licheniformis
CGMCC 16909, bacillus amyloliquefaciens CGMCC 15732.
The preparation of fresh-cut fruit and vegetable composite preservative: by the bacillus licheniformis CGMCC 16909 of activation, atrophy bacillus
CGMCC 16911, bacillus amyloliquefaciens CGMCC 15732 press 5% inoculum concentration, are inoculated in MRS fluid nutrient medium respectively,
37 DEG C, after 160rpm/min is cultivated for 24 hours respectively, 4 DEG C, 10000 r/min are centrifuged 10 min, take supernatant；With the HCl of 6mol/L
Supernatant is tuned into neutrality by solution, and solid ammonium sulfate is added while stirring, and when saturation degree reaches 80%, 4 DEG C of refrigerators are stood overnight；
4000r/min collects precipitating after being centrifuged 20 min, is dissolved with the phosphate buffer of pH=7.2, desalination of dialysing, and vacuum refrigeration is dry
After dry, compound according to mass ratio 1:1:1 to get composite preservative.
The preparation method of phosphate buffer described above are as follows: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, chlorine
Change sodium 8.0g, potassium chloride 0.2g, 1000mL, pH=7.2.
Vacuum freeze drying described above be -40 DEG C, vacuum degree be 100 Pa under the conditions of dry 40 h.
Prepare composite preservative according to above-mentioned technical proposal, applied Fresh-cut Apples it is fresh-keeping in, method particularly includes:
Apple is cut into small pieces (2cm × 2cm × 2cm) according to shape, by the apple after fresh-cut be immersed in 0.5% ascorbic acid and
It takes out and drains after 2min in 1.0% citric acid, be put into the disease for first uniformly spraying 250 μ L in sterilized petri dishes with the small watering can of 50mL
Opportunistic pathogen mixed liquor (concentration 104 Cfu/mL the composite preservative (concentration 1.0mg/mL) of 500 μ L), then is uniformly sprayed, later
Preservative film is covered after the drying of super-clean bench sterile wind, is placed in 4 DEG C of environment, to spoilage organisms golden yellow grape common during preservation
Coccus, Escherichia coli, Listeria monocytogenes, Bacterium enteritidis and shigella flexneri clump count are detected, and measure guarantor
The variation of the physical and chemical indexes such as the weight-loss ratio of Fresh-cut Apples, total phenol, vitamin C, MDA content, POD and PPO enzyme activity during hiding.
Prepare composite preservative according to above-mentioned technical proposal, applied Fresh-cut Lettuce it is fresh-keeping in, method particularly includes:
It romaine lettuce is torn into piece is cleaned up in the chlorine water of 120mg/L and drained, is put into first uniform with the small watering can of 50mL in sterilized petri dishes
Spray the pathogen mixed liquor (concentration 10 of 250 μ L4 Cfu/mL), then uniformly (concentration is the composite preservative of 500 μ L of sprinkling
1.0mg/mL), it is put into after super-clean bench air-dries and is encapsulated with preservative film.Be placed in it is fresh-keeping under 4 DEG C of environment, observe composite preservative to fresh-cut
The fresh-keeping effect of romaine lettuce, to spoilage organisms staphylococcus aureus common during preservation, Escherichia coli, Listeria monocytogenes, intestines
Scorching salmonella and shigella flexneri clump count are detected, and measure the weight-loss ratio of Fresh-cut Lettuce, total phenol, dimension during preservation
The variation of the physical and chemical indexes such as raw element C, MDA content, POD and PPO enzyme activity.
Applying the fresh-cut fruit and vegetable composite fresh-keeping agent solution for being 1.0mg/mL in the fresh-keeping middle concentration of fresh-cut fruit and vegetable is with sterile
What water was configured to.
After the Fresh-cut Apples of sprinkling pathogen mixed liquor and romaine lettuce are handled with composite preservative as the result is shown, with control group phase
Than having apparent bacteriostasis, staphylococcus aureus, Escherichia coli, Bacterium enteritidis, shigella flexneri, single increasing Lee
The clump count of this special bacterium has significant decrease, reduces the spoilage rate of Fresh-cut Apples and romaine lettuce.
Detailed description of the invention
Fig. 1 is bacillus licheniformis CGMCC 16909, atrophy bacillus CGMCC 16911, bacillus amyloliquefaciens
The colonial morphology and cellular morphology figure of CGMCC 15732, in figure: A is the colonial morphology of atrophy bacillus CGMCC 16911；B
For atrophy bacillus CGMCC 16,911 1000 × under cellular morphology figure；C is the bacterium of bacillus licheniformis CGMCC 16909
Fall form；D be bacillus licheniformis CGMCC 16,909 1000 × under cellular morphology figure；E is bacillus amyloliquefaciens CGMCC
15732 colonial morphology；F be bacillus amyloliquefaciens CGMCC 15,732 1000 × under cellular morphology figure；
Fig. 2 is atrophy bacillus CGMCC 16911, bacillus licheniformis CGMCC 16909, bacillus amyloliquefaciens CGMCC
15732 16S rDNA sequential system develops tree；
Fig. 3 is application of the fresh-cut fruit and vegetable composite preservative in Fresh-cut Apples are fresh-keeping, in figure: A is sterile water process the 0th day fresh
Cut apple；B is the 5th day Fresh-cut Apples of sterile water process；C is the 8th day Fresh-cut Apples of sterile water process；D is fresh-cut fruit and vegetable
Composite preservative handles the 0th day Fresh-cut Apples；E is the Fresh-cut Apples that fresh-cut fruit and vegetable composite preservative handles the 5th day；
F is the Fresh-cut Apples that fresh-cut fruit and vegetable composite preservative handles the 8th day；
Fig. 4 is the fresh-cut fruit and vegetable composite preservative clump count fresh-keeping to Fresh-cut Apples, and in figure: A is the total plate count of Fresh-cut Apples；
B is the E. coli clones number of Fresh-cut Apples；C is the S. aureus colonies number of Fresh-cut Apples；D is the good fortune of Fresh-cut Apples
Family name's Shigella clump count；E is the Bacterium enteritidis clump count of Fresh-cut Apples；F is the Listeria monocytogenes bacterium of Fresh-cut Apples
Fall number；
Fig. 5 is the fresh-cut fruit and vegetable composite preservative physical and chemical index fresh-keeping to Fresh-cut Apples, and in figure: A is the weight-loss ratio of Fresh-cut Apples；
B is the total phenol content of Fresh-cut Apples；C is the Vitamin C content of Fresh-cut Apples；D is the MDA content of Fresh-cut Apples；E is fresh-cut apple
The PPO enzyme activity of fruit；F is the POD enzyme activity of Fresh-cut Apples；
Fig. 6 is application of the fresh-cut fruit and vegetable composite preservative in Fresh-cut Lettuce is fresh-keeping, in figure: A is sterile water process the 0th day fresh
Cut romaine lettuce；B is the 5th day Fresh-cut Lettuce of sterile water process；C is the 7th day Fresh-cut Lettuce of sterile water process；D is fresh-cut fruit and vegetable
Composite preservative handles the 0th day Fresh-cut Lettuce；E is the Fresh-cut Lettuce that fresh-cut fruit and vegetable composite preservative handles the 5th day；
F is the Fresh-cut Lettuce that fresh-cut fruit and vegetable composite preservative handles the 7th day；
Fig. 7 is the fresh-cut fruit and vegetable composite preservative clump count fresh-keeping to Fresh-cut Lettuce, and in figure: A is the total plate count of Fresh-cut Lettuce；
B is the E. coli clones number of Fresh-cut Lettuce；C is the S. aureus colonies number of Fresh-cut Lettuce；D is the good fortune of Fresh-cut Lettuce
Family name's Shigella clump count；E is the Bacterium enteritidis clump count of Fresh-cut Lettuce；F is the Listeria monocytogenes bacterium of Fresh-cut Lettuce
Fall number；
Fig. 8 is the fresh-cut fruit and vegetable composite preservative physical and chemical index fresh-keeping to Fresh-cut Lettuce, and in figure: A is the weight-loss ratio of Fresh-cut Lettuce；
B is the total phenol content of Fresh-cut Lettuce；C is the Vitamin C content of Fresh-cut Lettuce；D is the MDA content of Fresh-cut Lettuce；E is raw for fresh-cut
The PPO enzyme activity of dish；F is the POD enzyme activity of Fresh-cut Lettuce.
Specific embodiment
Technical solution of the present invention is described further below by specific embodiment.
Embodiment 1: screening, identification and preservation with high bacteriostatic activity Bacillus
(1) there is the screening compared with high bacteriostatic activity Bacillus: acquiring the distiller's wort sample in Shanxi mature vinegar alcoholic fermentation stage respectively
With the vinegar fermented grain sample in acetic fermentation stage, coating is diluted using MRS culture medium, finally isolates 50 plants of Bacillus.It adopts
The screening with bacteriostatic activity is carried out to it with Oxford cup inhibition zone method, wherein Bacillus 297 is to the antibacterial effect of Gram-negative bacteria
Fruit is preferable, wherein the fungistatic effect to Escherichia coli is most significant, antibacterial circle diameter is 20.54 mm；Bacillus 1671 is to gram
Positive bacteria fungistatic effect is preferable, wherein the fungistatic effect to staphylococcus aureus is most significant, antibacterial circle diameter is 21.82 mm；
Bacillus 2014 is preferable to fruits and vegetables Sapromyces fungistatic effect, and wherein the fungistatic effect of aspergillus niger is most significant, and antibacterial circle diameter is
23.48 mm。
Above-mentioned Escherichia coli, staphylococcus aureus, aspergillus niger are bought to China General Microbiological culture presevation pipe
Reason center, the deposit number of Escherichia coli is CGMCC 1.1684, the deposit number of staphylococcus aureus is CGMCC
1.184, the deposit number of aspergillus niger is CGMCC 3.2915.
Above-mentioned MRS solid medium the preparation method comprises the following steps: glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g,
K2HPO42g, sodium acetate 2g, Triammonium citrate 2g, Tween 80 1g, MgSO40.2g, MnSO40.05g, distilled water 1000mL.
PH to 6.2 6.6 is adjusted, 121 DEG C of sterilizing 20min are spare.
Above-mentioned screening has bacteriostatic activity Bacillus method particularly includes: isolate and purify 50 plants of Bacillus are connected to MRS
Fluid nutrient medium, 37 DEG C, 160 r/min cultivate 24 h, and 10000 r/min are centrifuged 10 min and collect Bacillus fermented supernatant fluid,
It is spare after being adjusted to neutrality with the NaOH of 6.0 mol/L respectively.By Escherichia coli, staphylococcus aureus, Fu Shi shiga
Bacterium, Listeria monocytogenes, Bacterium enteritidis, Friedlander's bacillus, Huo Shi enterobacteria, pseudomonas aeruginosa are respectively connected to
37 DEG C in BHI fluid nutrient medium, after 160 r/min cultivate 12 h, with 5 times (10 of normal saline dilution5Cfu/mL), pipette
The diluted bacterium solution of 0.1mL to BHI plate is coated, and after placing on BHI plate the Oxford cup to 1 h of refrigerator freezing of sterilizing
It takes out, Bacillus fermented supernatant fluid is pipetted into 200 μ L into Oxford cup, puts 37 DEG C of incubator cultures；By aspergillus niger, aspergillus flavus,
Mould point is connected to the PDA culture medium plate for being placed with Oxford cup, and Bacillus fermented supernatant fluid is pipetted 200 μ L into Oxford cup,
And put 30 DEG C of incubator cultures.
Above-mentioned shigella flexneri, Listeria monocytogenes, Friedlander's bacillus, Huo Shi enterobacteria, P. aeruginosa
Bacterium is bought to north and receives biotechnology center, and the deposit number of shigella flexneri is ATCC 12022, Listeria monocytogenes
Deposit number is ATCC 19114, the deposit number of Friedlander's bacillus is ATCC 10031, the preservation of Huo Shi enterobacteria is compiled
It number is ATCC 700323, the deposit number of pseudomonas aeruginosa is ATCC 9027；Salmonella is bought to Chinese medicine bacterium
Preservation administrative center, deposit number are CMCC 50041-16；Aspergillus flavus, mould are bought to China General Microbiological strain and are protected
Administrative center is hidden, the deposit number of aspergillus flavus is CGMCC 3.11969, the deposit number of mould is CGMCC 3.15687.
(2) there is the identification and preservation compared with high bacteriostatic activity Bacillus
Morphological Identification: in MRS plate culture after using oese picking Bacillus 297, Bacillus 1671, Bacillus 2014 a little
It crosses on base, isolates single colonie, the bacterium colony grown on culture medium is taken pictures, is recorded, then contaminated by gram
Look mirror inspection, observes its cell morphological characteristic.Bacillus 297 forms 4.42 mm of colony diameter, bacterium colony on MRS plating medium
Approximate circle, intermediate projections at flakes, milky, opaque, stickiness is larger；Bacillus 1671 is on MRS plating medium
4.02 mm of colony diameter is formed, rough surface, opaque, sticky, the more folds of protuberance, edge are irregular, bacterium colony is in shallow white；
Bacillus 2014 forms 4.58 mm of colony diameter on MRS plating medium, and bacterium colony is rounded, more sticky, rough surface is convex
It rises, is neat in edge, milky, opaque；Its cellular morphology: Gram's staining is in G+, in the shape of a rod, there are gemma, such as Fig. 1 in centre
It is shown.
16Sr DNA sequencing: extracting the genome of Bacillus 297, Bacillus 1671, Bacillus 2014 with kit, carries out
16Sr DNA sequencing and strain idenfication, primer are as follows: upstream: the downstream 5 '-CAGATGGGAGCTTGCTCCCTG-3 ': 5 '-
CGACTTCACCCAATCATCTG-3 ', using BLAST software through tetraploid rice, the results showed that Bacillus 297 is atrophy gemma
Bacillus (Bacillus atrophaeus), Bacillus 1671 be bacillus licheniformis (Bacillus licheniformis), bud
Spore bacterium 2014 be bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and systematic evolution tree is constructed, see Fig. 2.
And it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is atrophy bacillus
CGMCC 16911, bacillus licheniformis CGMCC 16909, bacillus amyloliquefaciens CGMCC 15732.
Embodiment 2: the preparation of fresh-cut fruit and vegetable composite preservative
By the bacillus licheniformis CGMCC 16909 of activation, atrophy bacillus CGMCC 16911, bacillus amyloliquefaciens
CGMCC 15732 presses 5% inoculum concentration, is inoculated in MRS fluid nutrient medium respectively, 37 DEG C, 160rpm/min is cultivated for 24 hours respectively
Afterwards, 4 DEG C, 10000 r/min are centrifuged 10 min, take supernatant；Supernatant is tuned into neutrality with the HCl solution of 6mol/L, while stirring
It mixes side and solid ammonium sulfate is added, when saturation degree reaches 80%, 4 DEG C of refrigerators are stood overnight；4000r/min is collected after being centrifuged 20 min
Precipitating is dissolved with the phosphate buffer of pH=7.2, desalination of dialysing, multiple for 1:1:1 according to weight ratio after vacuum freeze drying
Match, is composite preservative after vacuum packaging.
The preparation method of phosphate buffer described above are as follows: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, chlorine
Change sodium 8.0g, potassium chloride 0.2g, 1000mL, pH=7.2.
Vacuum freeze drying described above be -40 DEG C, vacuum degree be 100 Pa under the conditions of dry 40 h.
Embodiment 3: application of the composite preservative in Fresh-cut Apples are fresh-keeping
Prepare composite preservative according to above-mentioned technical proposal, applied Fresh-cut Apples it is fresh-keeping in, specific implementation method is such as
Under:
Apple is cut into small pieces (2cm × 2cm × 2cm) according to shape, the apple after fresh-cut is immersed in 0.5% ascorbic acid
It is drained with being taken out after 2min in 1.0% citric acid (color stabilizer), is put into sterilized petri dishes and is first uniformly sprayed with the small watering can of 50mL
Pathogen mixed liquor (the concentration 10 of 250 μ L4 Cfu/mL composite preservative (the concentration 1.0mg/ of 500 μ L), then is uniformly sprayed
ML), preservative film is covered after the drying of super-clean bench sterile wind later, be placed in 4 DEG C of environment, to spoilage organisms gold common during preservation
Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Bacterium enteritidis and shigella flexneri clump count are detected,
And measure the physical and chemical indexes such as weight-loss ratio, total phenol, vitamin C, MDA content, POD and PPO enzyme activity of Fresh-cut Apples during preservation
Variation.
As shown in figure 3, compared with the control group, the spoilage rate of the Fresh-cut Apples handled with composite preservative reduces；And
There is apparent bacteriostasis, wherein staphylococcus aureus, Escherichia coli, Bacterium enteritidis, shigella flexneri, single increasing Lee
The clump count of this special bacterium is significantly reduced (such as Fig. 4).As shown in Figure 5, the Fresh-cut Apples handled with composite preservative, it is weightless
Rate, MDA content climbing speed are slower, and vitamin C and total phenol content decline slower, peroxidase activity (POD), polyphenol oxidase
(PPO) living in downward trend after first rising, last enzyme activity is higher than sterile water process, has good fresh-keeping effect.
Pathogen mixed liquor described above specific the preparation method comprises the following steps: respectively by Escherichia coli, staphylococcus aureus,
Shigella flexneri, Bacterium enteritidis and Listeria monocytogenes are inoculated in BHI fluid nutrient medium, in 37 DEG C, 160 r/min
It cultivates 12 h and obtains cause of disease bacterium solution, supernatant is abandoned in 10000 r/min, l0 min centrifugation, heavy with 0.1% sterile peptone water washing
It forms sediment 2 times, and precipitating is resuspended with 0.1% sterile peptone water, be respectively prepared 104 The bacteria suspension of cfu/mL, and it is isometric
It is mixed into pathogen mixed liquor.
What composite preservative described above was prepared method particularly includes: composite preservative is configured to 1.0mg/ with sterile water
mL。
The measuring method of microorganism detection described above are as follows: Fresh-cut Apples aseptically weigh 10g sample, after shredding
It is incorporated in 90mL sterile saline, acutely shakes, gradient dilution is coated with after mixing, then to common spoilage organisms golden yellow Portugal
Grape coccus (mannitol sodium chloride agar medium), Escherichia coli (EMB agar medium), single monocytogenes
(will congratulates selection culture for (PALCAM agar culture medium), Bacterium enteritidis (maconkey agar culture medium), shigella flexneri
Base) it is counted.
The specific measuring method of physical and chemical index described above are as follows:
(1) measuring method of weight-loss ratio: during preservation, the daily same period takes the Fresh-cut Apples under the conditions of different disposal to claim
Weight, and calculate weight-loss ratio.Weight-loss ratio=(preservation initial stage fruit weight-preservation latter stage fruit weight)/preservation initial stage fruit weight
x100%
(2) measuring method of total phenol: using the colorimetric estimation of Folin-Ciocalteau method, weighs 5 g Fresh-cut Apples, is added 20
It is homogenized in the methanol solution ice bath of mL pre-cooling, 4 DEG C, 12000 r/min are centrifuged 20min, take 1mL supernatant in 25mL volumetric flask
In, mixing is vibrated after 1mL forint phenol solution is added, 7.5% sodium carbonate liquor of 10mL is added, is finally arrived with deionized water constant volume
25mL.25 DEG C of placement 1h, measure its light absorption value at 750 nm.
(3) ascorbic measuring method: using 2,6 dichloroindophenol titration measurings, weighs 5 g of Fresh-cut Apples, be added etc.
The 2%HCl solution of amount, is pounded homogenate.3g slurried sample (it is made to contain ascorbic acid 0.1-0.5mg) is weighed, small beaker is placed in
In, sample is moved into 100mL volumetric flask with 1% HCl solution, and be diluted to scale, is shaken up.Sample liquid is filtered, is discarded initially
Then several milliliters of filtrates are drawn 10mL filtrate with pipette rapidly, are placed in 50mL conical flask, with calibrated 2,6 dichloros
Indophenols titration, until solution pinkiness is until colour-fast in 15 seconds.
(4) measuring method of malonaldehyde (MDA) content: weighing 0.4g Fresh-cut Apples and be put into mortar, and a little quartz is added
The TCA of sand and 2mL 0.1%, inkstone are transferred in test tube at homogenate, then rinse mortar in two times with the TCA of 3mL 0.1%, are merged
The thiobarbituricacidα- solution of 5mL 0.5% is added in extracting solution in extracting solution, shakes up for test tube to be put into boiling water bath and develop the color instead
Answer 15min, to the time after be removed and placed in ice bath be cooled to room temperature immediately, after test tube is cooling, be transferred in 10mL centrifuge tube
3000r/min is centrifuged 15min, takes supernatant, surveys the light absorption value at 532nm and 600nm.
(5) measuring method of peroxidase activity (POD): the poly- second of crosslinking that 5g Fresh-cut Apples contain 2.5% with 20mL is weighed
The phosphate buffer (0.2mol/L pH 7.5) of alkene pyrrolidone mixes, and grinds rapidly under condition of ice bath, at 4 DEG C, 10000r/
Min is centrifuged 15mim, and supernatant is thick enzyme.1mL crude enzyme liquid is drawn, the phosphate buffer of 1mL pH 7.5, then plus 1mL is added
The buffer of catechol containing 1mmol/L after mixing, is returned to zero with distilled water, is immediately placed in 420nm and is gone out to measure absorbance value,
It is primary every 10s record, 2min is measured, 0.001 as 1 active unit is changed using sample absorbance value, unit is U/ (g ﹒
min)。
(6) measuring method of polyphenol oxidase enzyme activity (PPO): the poly- second of crosslinking that 5g Fresh-cut Apples contain 2.5% with 20mL is weighed
The phosphate buffer (0.2mol/L pH 7.5) of alkene pyrrolidone mixes, and grinds rapidly under condition of ice bath, at 4 DEG C, 10000r/m
It is centrifuged 15mim, supernatant is thick enzyme.Take the phosphoric acid buffer of 0.05% guaiacol solution 2mL(0.2mol/L pH 7.5
Solution is formulated), the thick zyme extract of 0.5mL is added, 5min is kept the temperature in 30 DEG C of water-baths, adds 1mL 0.08%
H2O2It mixes after solution, is returned to zero with distilled water, be immediately placed in measurement absorbance value at 470nm, measurement primary every 30s record
3min changes 0.001 as 1 active unit using sample absorbance value, and unit is U/ (g ﹒ min).
Embodiment 4: application of the composite preservative in Fresh-cut Lettuce is fresh-keeping
Prepare composite preservative according to above-mentioned technical proposal, applied Fresh-cut Lettuce it is fresh-keeping in, specific implementation method is such as
Under:
It romaine lettuce is torn into piece is cleaned up in the chlorine water (color stabilizer) of 120mg/L and drained, be put into sterilized petri dishes and first use 50mL
Small watering can uniformly spray the pathogen mixed liquor (concentration 10 of 250 μ L4 Cfu/mL the compound guarantor of 500 μ L), then is uniformly sprayed
It fresh dose (concentration 1.0mg/mL), is put into after super-clean bench air-dries and is encapsulated with preservative film.Be placed in it is fresh-keeping under 4 DEG C of environment, observe it is compound
Antistaling agent is to the fresh-keeping effect of Fresh-cut Lettuce, to spoilage organisms staphylococcus aureus common during preservation, Escherichia coli, Dan Zeng
Listeria, Bacterium enteritidis and shigella flexneri clump count are detected, and measure the mistake of Fresh-cut Lettuce during preservation
The variation of the physical and chemical indexes such as rate, total phenol, vitamin C, MDA content, POD and PPO enzyme activity again.
As shown in fig. 6, compared with the control group, the spoilage rate of the Fresh-cut Lettuce handled with composite preservative reduces；And
There is apparent bacteriostasis, wherein staphylococcus aureus, Escherichia coli, Bacterium enteritidis, shigella flexneri, single increasing Lee
The clump count of this special bacterium is significantly reduced (such as Fig. 7).As shown in Figure 8, the Fresh-cut Lettuce handled with composite preservative, it is weightless
It is slower that rate has just begun to ramp up rate, then very fast, and vitamin C, MDA and total phenol content decline slower, peroxidase activity
(POD), in downward trend after first rising, last enzyme activity is higher than sterile water process, has polyphenol oxidase enzyme activity (PPO)
Good fresh-keeping effect.
Claims (5)
1. a kind of fresh-cut fruit and vegetable by bacillus licheniformis, atrophy bacillus and the produced bateriostatics compounding of bacillus amyloliquefaciens
Antistaling agent, it is characterised in that: the bacillus licheniformis, atrophy bacillus, bacillus amyloliquefaciens are from Shanxi mature vinegar vinegar
It separated in unstrained spirits, screen gained, the bacillus licheniformis deposit number is CGMCC 16909, atrophy bacillus deposit number
For CGMCC 16911, bacillus amyloliquefaciens deposit number is CGMCC 15732, depositary institution: Chinese microorganism strain preservation
Administration committee's common micro-organisms center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date:
On December 10th, 2018, on May 7th, 2018.
2. one kind according to claim 1 is produced by bacillus licheniformis, atrophy bacillus and bacillus amyloliquefaciens
Bateriostatics compounding fresh-cut fruit and vegetable antistaling agent, it is characterised in that: the fresh-cut fruit and vegetable composite preservative the preparation method comprises the following steps: will live
Bacillus licheniformis CGMCC 16909, the atrophy bacillus CGMCC 16911, bacillus amyloliquefaciens CGMCC 15732 of change
It by 3% inoculum concentration, is inoculated in MRS fluid nutrient medium respectively, 37 DEG C, after 160rpm/min is cultivated for 24 hours respectively, 4 DEG C, 10000
R/min is centrifuged 10 min, takes supernatant；Supernatant is tuned into neutrality with the HCl solution of 6mol/L, solid sulfur is added while stirring
Sour ammonium, when saturation degree reaches 80%, 4 DEG C of refrigerators are stood overnight；4000r/min collects precipitating after being centrifuged 20 min, with pH=7.2
Phosphate buffer dissolves, desalination of dialysing, and after vacuum freeze drying, compounds according to mass ratio 1:1:1 to get composite preservative.
3. one kind according to claim 2 is produced by bacillus licheniformis, atrophy bacillus and bacillus amyloliquefaciens
The fresh-cut fruit and vegetable antistaling agent of bateriostatics compounding, it is characterised in that: the preparation method of the phosphate buffer are as follows: biphosphate
Potassium 0.24g, disodium hydrogen phosphate 1.42g, sodium chloride 8.0g, potassium chloride 0.2g, 1000mL, pH=7.2.
4. one kind according to claim 2 is produced by bacillus licheniformis, atrophy bacillus and bacillus amyloliquefaciens
The fresh-cut fruit and vegetable antistaling agent of bateriostatics compounding, it is characterised in that: the vacuum freeze drying is in -40 DEG C, vacuum degree 100
Dry 40 h under the conditions of Pa.
5. it is a kind of it is described in claim 1 produced by bacillus licheniformis, atrophy bacillus and bacillus amyloliquefaciens it is antibacterial
Application of the fresh-cut fruit and vegetable antistaling agent of object compounding in fresh-cut fruit and vegetable, the concentration are 1.0mg/mL fresh-cut fruit and vegetable composite fresh-keeping
Agent solution is formulated with sterile water.
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