CN109182212A - A kind of apple endophyte and its fresh-keeping liquid - Google Patents
A kind of apple endophyte and its fresh-keeping liquid Download PDFInfo
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- 239000007788 liquid Substances 0.000 title claims abstract description 158
- 238000000855 fermentation Methods 0.000 claims abstract description 76
- 230000004151 fermentation Effects 0.000 claims abstract description 76
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 52
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 46
- 239000011248 coating agent Substances 0.000 claims abstract description 33
- 238000000576 coating method Methods 0.000 claims abstract description 33
- 238000013329 compounding Methods 0.000 claims abstract description 31
- 229920001661 Chitosan Polymers 0.000 claims abstract description 22
- 239000000661 sodium alginate Substances 0.000 claims abstract description 22
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 22
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 21
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 21
- 244000141359 Malus pumila Species 0.000 claims description 65
- 235000011430 Malus pumila Nutrition 0.000 claims description 63
- 235000015103 Malus silvestris Nutrition 0.000 claims description 63
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000006228 supernatant Substances 0.000 claims description 26
- 239000001963 growth medium Substances 0.000 claims description 19
- 230000001580 bacterial effect Effects 0.000 claims description 15
- 230000001954 sterilising effect Effects 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 14
- 239000012153 distilled water Substances 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 13
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 8
- 238000003760 magnetic stirring Methods 0.000 claims description 8
- 239000008223 sterile water Substances 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 229920002472 Starch Polymers 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 239000008107 starch Substances 0.000 claims description 7
- 235000019698 starch Nutrition 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 241000726221 Gemma Species 0.000 claims description 5
- 244000061456 Solanum tuberosum Species 0.000 claims description 5
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 5
- 229960000583 acetic acid Drugs 0.000 claims description 5
- 230000008485 antagonism Effects 0.000 claims description 5
- 239000000470 constituent Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 239000012362 glacial acetic acid Substances 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 240000004922 Vigna radiata Species 0.000 claims description 3
- 235000010721 Vigna radiata var radiata Nutrition 0.000 claims description 3
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 claims description 3
- 230000003042 antagnostic effect Effects 0.000 claims description 3
- 241000956151 bacterium T09 Species 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims 1
- 238000002156 mixing Methods 0.000 abstract description 23
- 230000000694 effects Effects 0.000 abstract description 14
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- 235000012055 fruits and vegetables Nutrition 0.000 abstract description 9
- 238000011161 development Methods 0.000 abstract description 8
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 22
- 239000002253 acid Substances 0.000 description 13
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 11
- 229930003268 Vitamin C Natural products 0.000 description 11
- 235000019154 vitamin C Nutrition 0.000 description 11
- 239000011718 vitamin C Substances 0.000 description 11
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- 150000001875 compounds Chemical class 0.000 description 6
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- 230000029058 respiratory gaseous exchange Effects 0.000 description 6
- 238000003860 storage Methods 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 239000007888 film coating Substances 0.000 description 3
- 238000009501 film coating Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000193417 Brevibacillus laterosporus Species 0.000 description 2
- 229920002101 Chitin Polymers 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- RAHZWNYVWXNFOC-UHFFFAOYSA-N Sulphur dioxide Chemical compound O=S=O RAHZWNYVWXNFOC-UHFFFAOYSA-N 0.000 description 2
- 235000010724 Wisteria floribunda Nutrition 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 244000273928 Zingiber officinale Species 0.000 description 2
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
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- 239000003795 chemical substances by application Substances 0.000 description 2
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- 235000008397 ginger Nutrition 0.000 description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
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- 229910052760 oxygen Inorganic materials 0.000 description 2
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- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
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- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 244000241235 Citrullus lanatus Species 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
- 238000010159 Duncan test Methods 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 241000366676 Justicia pectoralis Species 0.000 description 1
- 241000190142 Neofusicoccum ribis Species 0.000 description 1
- 206010064458 Penicilliosis Diseases 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 238000002479 acid--base titration Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000031016 anaphase Effects 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 241000956155 bacterium T07 Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000004320 controlled atmosphere Methods 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 238000011981 development test Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009920 food preservation Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 230000005068 transpiration Effects 0.000 description 1
Classifications
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- 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
- 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
-
- 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
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- 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
- C12N1/02—Separating microorganisms from their culture media
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- 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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Abstract
The invention belongs to biological way of keeping fresh fields, and in particular to a kind of apple endophyte and its fresh-keeping liquid.The fresh-keeping liquid includes bacillus licheniformis T-07, the extracting solution of bacillus amyloliquefaciens T-09 fermentation liquid and compounding coating liquid.The present invention with 1%~3% chitosan, 1%~3% sodium alginate compounding prepare coating liquid, then plus different proportion endophyte fermentation liquid extracting solution, develop mixing fresh-keeping liquid.The mixing fresh-keeping liquid can not only promote the development of fruits and vegetables industry, reduce expenses for orchard worker to rear apple is adopted with good fresh-keeping effect；It can also promote the development of China's Advance in Microbial Preservation Technology at this stage simultaneously, the system research for postharvest fruit and vegetable biological control and preservation and freshness lays the foundation.
Description
Technical field
The invention belongs to biological way of keeping fresh fields, and in particular to a kind of apple endophyte and its fresh-keeping liquid.
Background technique
Apple is primary one of the industrial crops in China, and contained nutriment is abundant.It is easy in growth period green by extension
Mould (Pennicilum expansum's) the mechanicalnesses damage such as infects, and is inevitably vibrated, squeezes during transportation
Wound, to provide chance to pathogen infection, and then causes to be easy to happen the lesions such as penicilliosis in anaphase storage, influences economy
Benefit.
The physical methods such as existing deepfreeze, controlled atmosphere, heat treatment, radiation treatment in the market, sulfur dioxide, bicarbonate
The chemical methodes such as sodium processing there are also biological control, carry out fresh-keeping biological method using gene.With Modern Preservation Technology
Continuous development and people to the remaining worry of chemical preservative harmful substance, the development and utilization of safe crude antistaling agent is drawn
The most attention of people is played, especially edible freshness-keeping thin coat is applied in fresh preservation.
Chitosan (Chitosan, CTS) is that one kind for obtaining after chitin (Chitin) deacetylation is nontoxic, biological
Degradable polysaccharide has film forming and broad spectrum antibacterial, is widely used in field of food preservation.Studies have shown that concentration is
1.5%~2% chitosan can the effectively maturation of delayed fruit, aging, can be used as fruits and vegetables film forming agent for fresh preservation, but
Its anti-corrosion effect of single chitosan is limited.Sodium alginate (Sodium alginate), have excellent moisture retention, film forming,
Many advantages, such as antibiotic property, non-toxic and tasteless, biodegradable, good biocompatibility, is applied using ginger extract-sodium alginate
Film studies the fresh-keeping effect of red fuji apple, the results showed that, it is mentioned using 1% sodium alginate and 0.1g/mL ginger
Liquid coating problems are taken, can make that breathing peak value can be effectively reduced, respiratory climacteric evening is made 15d or so occur.1% sodium alginate
It is able to maintain the good quality of Fresh-cut Watermelon and sensory properties.Chitosan can form one layer of very thin film, this film in fruit surface
With preferable film forming and permeability, sodium alginate can form film and can inhibit the respiration of fruits and vegetables.So chitosan with
Sodium alginate is widely used in the postharvest handling of fruits and vegetables, has good fresh-keeping effect.
Since there are permeabilities to oxygen for edible film-coating, the oxygen concentration of apple surface can be made to maintain lower water
Flat, edible film-coating, which is not only able to inhibit brown stain also, can drop low-moisture transpiration, the respiration of cell and ethylene
It generates.Therefore the hot spot direction in major laboratory or each research institution research apple biological preservation is had become, but also rarely people will
The extracting solution and edible film-coating liquid of the fermentation liquid of apple endophyte, the supernatant of endophyte fermentation liquid or endophyte fermentation liquid
It combines, the fresh-keeping effect of research mixing fresh-keeping liquid.In consideration of it, the present invention is with 1%~3% chitosan, 1%~3% alginic acid
Sodium compounding prepare coating liquid, then plus different proportion endophyte, the fermentation liquid of endophyte or the extracting solution of endophyte fermentation liquid,
Optimal mixed proportion is selected according to that can extend the fresh keeping time for adopting rear apple and improve anti-mildew ability, develops mixing
Fresh-keeping liquid.The mixing fresh-keeping liquid can not only promote the development of fruits and vegetables industry, be to rear apple is adopted with good fresh-keeping effect
Orchard worker reduces expenses；It can also promote the development of China's Advance in Microbial Preservation Technology at this stage simultaneously, be postharvest fruit and vegetable biological control
System research with preservation and freshness lays the foundation.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of apple endophyte and its fresh-keeping liquids.
Specific technical solution are as follows: a kind of apple endophyte, the endophyte include that bacillus licheniformis T-07 reconciliation is formed sediment
Afnyloliquefaciens T-09, the endophyte has to be obtained by following step:
Step 1: Apple is cut mung bean block size with the blade of sterilizing in superclean bench gnotobasis
Pulp is placed in PDA culture medium and beef-protein medium, after cultivating 2d in 28 DEG C of incubators, after growing bacterium colony
Plate streak culture is carried out, is then isolated and purified in PDA culture medium, then saves inclined-plane, be numbered；
Step 2: apple stem portion, root 3-5 hours are impregnated with sterile water, and after impregnating 2min with 95% ethyl alcohol, sterile water
Rinse 3 times, later again use 0.1% mercury chloride surface sterilization 30s, continue use rinsed with sterile water 5 times, by last time flushing liquor
Whether be coated on plate as control thorough to detect surface sterilization.It takes the thorough apple tree of surface sterilization respectively to organize, uses scissors
Blade is cut into 0.5cm2 size, stem, root are cut into 0.25cm long, the tissue block cut is coupled with PDA culture medium
On plate, four tissue blocks of each board joint, 28 DEG C of culture 72h, after surrounding grows bacterium colony, according to colonial morphology, size, face
The different feature such as color carries out initial gross separation purifying, after obtaining purifying bacterial strain, is connected to inclined-plane and is placed in 4 DEG C of refrigerators and save backup, adopts
With tablet face-off method, after cultivating 2d in 28 DEG C of incubators, measures bacterial strain radius and calculates bacteriostasis rate, see formula (1):
By the bacteriostasis rate of comparison endophyte, colony radius < 3cm is picked out, and bacteriostasis rate is more than or equal to 58% bacterial strain,
It is bacillus licheniformis T-07, bacillus amyloliquefaciens T-09 respectively.
Preferably, the constituent of the PDA culture medium are as follows: potato 200g, glucose 20g, agar 20g, distilled water
1000mL, pH are natural；The composition of the beef-protein medium becomes: beef extract 3g, peptone 5g, glucose 10g, fine jade
Rouge 18g, distilled water 1 000mL, pH 7.2.
Preferably, the bacillus licheniformis T-07, bacillus amyloliquefaciens T-09 are combined, and are carried out by tablet face-off method
Antagonistic effect can be carried out compounding without antagonism between strain Bacillus licheniformis T-07 and bacillus amyloliquefaciens T-09.
A kind of fermentation liquid, the fermentation liquid are fermented by above-mentioned bacillus licheniformis T-07, bacillus amyloliquefaciens T-09
It is made, method particularly includes: picking is stored in bacillus licheniformis T-07, bacillus amyloliquefaciens T-09 on test tube, fills whole
A PDA culture medium is punched the bacteria cake for obtaining diameter as 0.6cm after covering with plate, while preparing PDA liquid medium, often
The PDB culture medium of addition 250mL, is put into 5 bacteria cakes for every bottle after sterilization and cooling, in 200r/min, 28 DEG C in the triangular flask of 500mL
Shaking table in cultivate 3d, as bacillus licheniformis T-07 fermentation liquid and bacillus amyloliquefaciens T-09 fermentation liquid.
A kind of supernatant of fermentation liquid, the supernatant of the fermentation liquid are specific the preparation method comprises the following steps: by liquid bacterial strain 3
Supernatant, the as supernatant of fermentation liquid are taken after being centrifuged 10min in 500r/min centrifuge.
A kind of extracting solution of fermentation liquid, the extracting solution the preparation method comprises the following steps: by resulting fermentation liquid anhydrous acetic acid second
Ester is extracted according to the volume ratio of 1:1, and gained upper layer is the extracting solution of fermentation liquid after standing 12h.
A kind of fresh-keeping liquid, the fresh-keeping liquid include the extracting solution of above-mentioned bacillus licheniformis T-07 fermentation liquid, solution starch
The extracting solution and compounding coating liquid of bacillus T-09 fermentation liquid.
Preferably, the extracting solution and bacillus amyloliquefaciens of the compounding coating liquid, bacillus licheniformis T-07 fermentation liquid
The ratio of the extracting solution of T-09 fermentation liquid is 2:1:1.
Preferably, it is described compounding coating liquid the preparation method comprises the following steps:
1%~3% chitosan: weighing 1g~3g chitosan, adds 100ml, 1% glacial acetic acid, is carried out using magnetic stirring apparatus
Stirring；
1%~3% sodium alginate: weighing 1g~3g sodium alginate, adds 100ml distilled water, is carried out using magnetic stirring apparatus
Stirring；
1%~3% chitosan, 1%~3% sodium alginate are mixed according to the volume ratio of 1:1 and applied to get to compounding
Film liquid.
A kind of purposes of fresh-keeping liquid, the fresh-keeping liquid are used for adopting the fresh-keeping of rear apple.
Beneficial effects of the present invention: the present invention prepares film with 1%~3% chitosan, 1%~3% sodium alginate compounding
Liquid, then plus different proportion endophyte fermentation liquid extracting solution, develop mixing fresh-keeping liquid.The mixing fresh-keeping liquid is to adopting rear apple
With good fresh-keeping effect, the development of fruits and vegetables industry can not only be promoted, reduced expenses for orchard worker；It can also promote me simultaneously
The development of state's Advance in Microbial Preservation Technology at this stage, the system research for postharvest fruit and vegetable biological control and preservation and freshness lay the foundation.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is the change curve of apple weight-loss ratio after different disposal；
Fig. 2 is the change curve of apple titratable acid content after different disposal；
Fig. 3 is the change curve of apple total sugar content after different disposal；
Fig. 4 is the change curve of apple Vitamin C content after different disposal；
Fig. 5 is the change curve of apple MDA content after different disposal.
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
1, material
(1) source of apple: purchase is selected in the Shang Jia supermarket of Yan'an Xinzhou small town and has no mechanical damage, invade without disease pest
Evil, free from extraneous odour, rich 3 red fuji apples of cigarette that nothing is rotted, maturity is almost the same, size is essentially identical.
A kind of apple endophyte, the endophyte include bacillus licheniformis T-07 and bacillus amyloliquefaciens T-09, institute
It states endophyte and has and obtained by following step:
Step 1: Apple is cut mung bean block size with the blade of sterilizing in superclean bench gnotobasis
Pulp is placed in PDA culture medium and beef-protein medium, after cultivating 2d in 28 DEG C of incubators, after growing bacterium colony
Plate streak culture is carried out, is then isolated and purified in PDA culture medium, then saves inclined-plane, be numbered；
Step 2: apple stem portion, root 3-5 hours are impregnated with sterile water, and after impregnating 2min with 95% ethyl alcohol, sterile water
Rinse 3 times, later again use 0.1% mercury chloride surface sterilization 30s, continue use rinsed with sterile water 5 times, by last time flushing liquor
Whether be coated on plate as control thorough to detect surface sterilization.It takes the thorough apple tree of surface sterilization respectively to organize, uses scissors
Blade is cut into 0.5cm2 size, stem, root are cut into 0.25cm long, the tissue block cut is coupled with PDA culture medium
On plate, four tissue blocks of each board joint, 28 DEG C of culture 72h, after surrounding grows bacterium colony, according to colonial morphology, size, face
The different feature such as color carries out initial gross separation purifying, after obtaining purifying bacterial strain, is connected to inclined-plane and is placed in 4 DEG C of refrigerators and save backup, adopts
With tablet face-off method, after cultivating 2d in 28 DEG C of incubators, measures bacterial strain radius and calculates bacteriostasis rate, see formula (1):
By the bacteriostasis rate of comparison endophyte, colony radius < 3cm is picked out, and bacteriostasis rate is more than or equal to 58% bacterial strain,
It is bacillus licheniformis T-07, bacillus amyloliquefaciens T-09 respectively.
Further, the constituent of the PDA culture medium are as follows: potato 200g, glucose 20g, agar 20g, distillation
Water 1000mL, pH are natural；The composition of the beef-protein medium becomes: beef extract 3g, peptone 5g, glucose 10g,
Agar 18g, distilled water 1 000mL, pH 7.2.
Further, the bacillus licheniformis T-07, bacillus amyloliquefaciens T-09 combine, by tablet face-off method into
Row antagonistic effect can be carried out compounding without antagonism between strain Bacillus licheniformis T-07 and bacillus amyloliquefaciens T-09.
A kind of fermentation liquid, the fermentation liquid are fermented by above-mentioned bacillus licheniformis T-07, bacillus amyloliquefaciens T-09
It is made, method particularly includes: picking is stored in bacillus licheniformis T-07, bacillus amyloliquefaciens T-09 on test tube, fills whole
A PDA culture medium is punched the bacteria cake for obtaining diameter as 0.6cm after covering with plate, while preparing PDA liquid medium, often
The PDB culture medium of addition 250mL, is put into 5 bacteria cakes for every bottle after sterilization and cooling, in 200r/min, 28 DEG C in the triangular flask of 500mL
Shaking table in cultivate 3d, as bacillus licheniformis T-07 fermentation liquid and bacillus amyloliquefaciens T-09 fermentation liquid.
The constituent of the PDA liquid medium are as follows: potato 200g, glucose 20g, distilled water 1000mL, pH are certainly
So；The constituent of PDB culture medium are as follows: potato 200g, sucrose 20g, agar 20g, distilled water 1000mL, pH are natural.
A kind of supernatant of fermentation liquid, the bacillus licheniformis T-07 fermentation liquid and bacillus amyloliquefaciens T-09 fermentation
The supernatant of liquid it is specific the preparation method comprises the following steps: liquid bacterial strain is centrifuged 10min in 3 500r/min centrifuges after take supernatant, as
The supernatant of bacillus licheniformis T-07 fermentation liquid and bacillus amyloliquefaciens T-09 fermentation liquid.
A kind of extracting solution of fermentation liquid, the extracting solution the preparation method comprises the following steps: by resulting bacillus licheniformis T-07 send out
Zymotic fluid and bacillus amyloliquefaciens T-09 fermentation liquid are extracted with anhydrous ethyl acetate according to the volume ratio of 1:1 respectively, are stood
Gained upper layer is the extracting solution of bacillus licheniformis T-07 fermentation liquid and bacillus amyloliquefaciens T-09 fermentation liquid after 12h.
A kind of fresh-keeping liquid, the fresh-keeping liquid include the extracting solution of above-mentioned bacillus licheniformis T-07 fermentation liquid, solution starch
The extracting solution and compounding coating liquid of bacillus T-09 fermentation liquid.
Further, the extracting solution reconciliation starch gemma bar of the compounding coating liquid, bacillus licheniformis T-07 fermentation liquid
The ratio of the extracting solution of bacterium T-09 fermentation liquid is 2:1:1.
Further, it is described compounding coating liquid the preparation method comprises the following steps:
1% chitosan: weighing 1g chitosan, adds 100ml, 1% glacial acetic acid, is stirred using magnetic stirring apparatus；
1% sodium alginate: weighing 1g sodium alginate, adds 100ml distilled water, is stirred using magnetic stirring apparatus；
Chitosan, sodium alginate are mixed according to the volume ratio of 1:1 to get compounding coating liquid is arrived.
Embodiment 2
It is described compounding coating liquid the preparation method comprises the following steps: 3% chitosan: weigh 3g chitosan, add 100ml, 1% glacial acetic acid,
It is stirred using magnetic stirring apparatus；
3% sodium alginate: weighing 3g sodium alginate, adds 100ml distilled water, is stirred using magnetic stirring apparatus；
3% chitosan, 3% sodium alginate are mixed according to the volume ratio of 1:1 to get compounding coating liquid is arrived.
A kind of purposes of fresh-keeping liquid, the fresh-keeping liquid are used for adopting the fresh-keeping of rear apple.
Embodiment 3
1, experimental design
22 processing are arranged in this test altogether, and each processing is mixed according to the volume ratio in the following table 1, and A processing is blank
Control；Bacillus licheniformis T-07 group (A+B+C+D) is in proportion to addition distilled water and lichens gemma bar in compounding coating liquid
Bacterium T-07 fermentation liquid, the supernatant of fermentation liquid and the extracting solution of fermentation liquid；Bacillus amyloliquefaciens T-09 group (A+E+F+G) be by
Ratio is to the supernatant and fermentation liquid that distilled water and bacillus amyloliquefaciens T-09 fermentation liquid, fermentation liquid are added in compounding coating liquid
Extracting solution；Mixed fermentation liquid group (A+B+E+H+I+J+K+L) be will compound coating liquid, bacillus licheniformis T-07 fermentation liquid and
Bacillus amyloliquefaciens T-09 fermentation liquid is mixed according to special ratios；Mixing supernatant group (A+C+F+M+N+O+P+Q) is
Compounding coating liquid, bacillus licheniformis T-07 supernatant and bacillus amyloliquefaciens T-09 supernatant are carried out according to special ratios
Mixing；Mixed extract group (A+D+G+R+S+T+U+V) is will to compound coating liquid, bacillus licheniformis T-07 extracting solution reconciliation shallow lake
Afnyloliquefaciens T-09 extracting solution is mixed according to special ratios.Each processing shares 6 apples, and does 3 repetitions.
Table 1 respectively handles contained additive and ratio
In the table 1 of the present embodiment, 22 English alphabets indicate 22 processing ,+indicate to contain this content in this processing,
Ratio is volume ratio.
Embodiment 4
1, apple is handled
Chosen in supermarket have no mechanical damage, without disease pest infringement, free from extraneous odour, without rotten, maturity is almost the same, size is basic
Identical apple, washes dirt with tap water after buying back, with originally after the hypochlorite disinfectant 1.5min for being 0.1% with concentration
Water rinses out remaining sodium hypochlorite, then with time naturally dry of distilled water flushing, and random grouping is tested, and every group in correspondence
Compounding fresh-keeping liquid in impregnate 5min, naturally dry puts into hermetic bag in numerical order.In 25 DEG C, the ring that water content is 65%
It is stored in border, surveys an index every 5d.
2, measurement index
(1) weight-loss ratio:
Weight-loss ratio (%)=[(fruit quality when fruit quality-survey before storing)/fruit quality before storing] × 100
(2) titratable acid content: acid-base titration is used
(3) total sugar content: anthrone colorimetry is used
(4) Vitamin C content: spectrocolorimetry is used
(5) mda content: thiobarbituricacidα- method is used
Embodiment 5
1, result and analysis
(1) the selection result of endophyte
20 plants of endophytes are obtained by tablet face-off method, respectively to the inhibitory effect of canker of apple fruit (bacteriostasis rate) such as table 2
It is shown.
The bacteriostasis rate of 2 apple endophyte of table
Note: numerical value indicates that average bacteriostasis rate ± standard deviation, different letters represent different significant differences in table
(Duncan-test,P<0.05)。
According to the size of colony radius and bacteriostasis rate in table 2, colony radius < 3cm is picked out, and bacteriostasis rate is 58% or more
Bacterial strain.Satisfactory only four plants of bacterial strains, the i.e. bacteriostasis rate of bacillus licheniformis T-07 are 61.6%, solve starch gemma bar
The bacteriostasis rate equal 58.7% of bacterium T-09, bacillus laterosporus T-13, bacillus laterosporus T-15.
In this four plants of bacterial strains, combination of two, by tablet face-off method, only one group of discovery without obvious isolation strip, i.e., without
Antagonism is T-07 and T-09.Since two plants of test strains do not have antagonism, therefore can be compounded.
2, the supernatant and extracting solution of fermentation liquid, fermentation liquid
It can be obtained after shaking table culture: the fermentation liquid of the fermentation liquid of bacillus licheniformis T-07, bacillus amyloliquefaciens T-09
It can be obtained after centrifugation: the supernatant of bacillus licheniformis T-07 fermentation liquid, bacillus amyloliquefaciens T-09 fermentation liquid
Supernatant
It can be obtained after extraction: the extracting solution of bacillus licheniformis T-07 fermentation liquid, bacillus amyloliquefaciens T-09 fermentation liquid
Extracting solution
3, coating liquid is compounded
1%~3% chitosan, 1%~3% sodium alginate are mixed according to the volume ratio of 1:1, compounding can be obtained
Coating liquid.
4, the result of measurement index
(1) variation of apple weight-loss ratio
This is tested each processing group and all there is different degrees of influence to apple weight-loss ratio, and generally mixed extract group is weightless
The rate of change of rate is lower than blank control lower than mixed fermentation liquid group lower than mixing supernatant group, and bacillus licheniformis T-07 group is lost
The rate of change of rate and the almost equal below blank control of bacillus amyloliquefaciens T-09 group again.In Fig. 1, chooses every group and lose
The minimum processing of rate again is analyzed, and is being stored between 5~10d, the variation of the weight-loss ratio of each processing group slowly, from storage the
10d starts, and weight-loss ratio obviously rises, when storing 30d, T processing (compounding coating liquid+bacillus licheniformis T-07 extracting solution+
Bacillus amyloliquefaciens T-09 extracting solution according to 2:1:1 volume ratio mix) weight-loss ratio be only 7.4%, compare blank control group
Reduce 38.8%.Show that the endophyte extracting solution being added can effectively keep the moisture in apple, and coating liquid, lichens will be compounded
Bacillus T-07 extracting solution is prepared fresh-keeping after mixing with bacillus amyloliquefaciens T-09 extracting solution according to the volume ratio of 2:1:1
Liquid fresh-keeping effect is best.
(2) variation of apple titratable acid content
In this test, all there is influence to the variation of titratable acid content in apple in each processing, and generally mixing is extracted
The wear rate of liquid group titratable acid content is lower than blank control, lichens bud lower than mixed fermentation liquid group lower than mixing supernatant group
The almost equal below blank pair of the wear rate and bacillus amyloliquefaciens T-09 group of spore bacillus T-07 group titratable acid content
According to.It in Fig. 2, chooses every highest processing of group's titratable acid content and is analyzed, storing between 5~10d, blank control group
Existing significant change, each test group difference is unobvious, and since storing 10d, titratable acid content is decreased obviously, and is being stored
When 30d, T processing (compounding coating liquid+bacillus licheniformis T-07 extracting solution+bacillus amyloliquefaciens T-09 extracting solution according to
The volume ratio of 2:1:1 mixes) titratable acid content be 0.251%, be higher by 33.5% than blank control group.Show to be added interior
Raw bacterium extracting solution can effectively inhibit the respiration of apple, reduce the conversion of titratable acid, and will compound coating liquid, lichens bud
The fresh-keeping liquid that spore bacillus T-07 extracting solution is prepared after mixing with bacillus amyloliquefaciens T-09 extracting solution according to the volume ratio of 2:1:1
Fresh-keeping effect is best.
(3) variation of apple total sugar content
In this test, all there is influence, generally mixed extract group to the variation of total sugar content in apple in each processing
The wear rate of total sugar content is lower than blank control, bacillus licheniformis T- lower than mixed fermentation liquid group lower than mixing supernatant group
The almost equal below blank control of the wear rate and bacillus amyloliquefaciens T-09 group of 07 group of total sugar content.In Fig. 3, choose
Every highest processing of group's total sugar content is analyzed, and is being stored between 5~10d, and blank control group has significant change, each to try
It is unobvious to test group difference, since storing 10d, each processing group total sugar content is decreased obviously, when storing 30d, T processing
(coating liquid+bacillus licheniformis T-07 extracting solution+bacillus amyloliquefaciens T-09 extracting solution is compounded according to the volume ratio of 2:1:1
Mixing) total sugar content be 17.3%, be higher by 37.3% than blank control group.Show that the endophyte extracting solution being added can reduce
The wear rate of total reducing sugar in apple slows down the consumption of nutriment, and will compound coating liquid, bacillus licheniformis T-07 extracting solution
The fresh-keeping liquid fresh-keeping effect prepared after mixing with bacillus amyloliquefaciens T-09 extracting solution according to the volume ratio of 2:1:1 is best.
(4) variation of apple Vitamin C content
In this test, all there is influence, generally mixed extract to the variation of Vitamin C content in apple in each processing
The wear rate of group Vitamin C content is lower than blank control, lichens gemma lower than mixed fermentation liquid group lower than mixing supernatant group
The almost equal below blank control of the wear rate and bacillus amyloliquefaciens T-09 group of bacillus T-07 group Vitamin C content.
It in Fig. 4, chooses every highest processing of group's Vitamin C content and is analyzed, storing between 5~10d, blank control group is existing
Variation, each test group difference is unobvious, and since storing 10d, each processing group Vitamin C content is decreased obviously, and is being stored
When 30d, T processing (compounding coating liquid+bacillus licheniformis T-07 extracting solution+bacillus amyloliquefaciens T-09 extracting solution according to
The volume ratio of 2:1:1 mixes) Vitamin C content be 7.32mg/100g, be higher by 7% than blank control group.Show to be added interior
Raw bacterium extracting solution can reduce ascorbic oxidation in apple, slow down the decline of vitamin C mass fraction, and will compound film
Liquid, bacillus licheniformis T-07 extracting solution are matched after mixing with bacillus amyloliquefaciens T-09 extracting solution according to the volume ratio of 2:1:1
The fresh-keeping liquid fresh-keeping effect of system is best.
(5) variation of apple malonaldehyde (MDA) content
In this test, all there is influence, generally mixed extract group to the variation of apple MDA content in each processing group
MDA content is advanced the speed lower than mixing supernatant group lower than mixed fermentation liquid group lower than blank control, bacillus licheniformis T-
07 group of MDA content advance the speed and the almost equal below blank control of bacillus amyloliquefaciens T-09 group.In Fig. 5, choose
The minimum processing of every group MDA content is analyzed, and is being stored between 5~10d, blank control group has changed, each test group
Between difference it is unobvious, since storing 10d, MDA content obviously rises, when storing 30d, T processing (compounding coating liquid+
Bacillus licheniformis T-07 extracting solution+bacillus amyloliquefaciens T-09 extracting solution according to 2:1:1 volume ratio mix) MDA contain
Amount is only 0.557mmol/g, reduces 13.4% than blank control group.Show that the endophyte extracting solution being added can effectively delay
The rising of MDA content in apple, and will compounding coating liquid, bacillus licheniformis T-07 extracting solution and bacillus amyloliquefaciens T-09
Extracting solution is best according to the fresh-keeping liquid fresh-keeping effect prepared after the volume ratio mixing of 2:1:1.
Each processing group of the application will compound coating liquid, bacillus licheniformis T-07 extracting solution compared with blank control group
The mixing fresh-keeping liquid mixed with bacillus amyloliquefaciens T-09 extracting solution according to the volume ratio of 2:1:1 can be effectively reduced mistake
The increase of rate, MDA again slows down titratable acid, total reducing sugar, ascorbic reduction.Adopt rear apple will do it breathing in storage
Effect, moisture can make apple cells that normal physiological be maintained to be metabolized and guarantee preferable quality, due to thin in storage
Born of the same parents carry out respiration, lead to moisture loss, so as to cause quality mitigation.The content of titratable acid in apple is to determine apple
One key factor of flavor.The variation of total reducing sugar can intuitively reflect the consumption degree of nutriment in apple, and then reflect
The freshness of apple out.The product of Cell membrane lipids peroxidating is malonaldehyde, so in storage, MDA in apple
Changes of contents is able to reflect out the degree of damaged membrane.Since weight-loss ratio, titratable acid content, total sugar content, vitamin C contain
Amount determines that the freshness of apple, MDA content determine the rotten level of apple.So can be seen that by data analysis
The fresh-keeping liquid that this development test goes out extends the fresh keeping time of apple, anti-mildew ability is improved, so the mixing fresh-keeping liquid makes
Good fresh-keeping effect can be had by adopting rear apple,
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the scope of the present invention.It is all
Any modification, equivalent replacement, improvement and so within the spirit and principles in the present invention, are all contained in protection scope of the present invention
It is interior.
Claims (10)
1. a kind of apple endophyte, which is characterized in that the endophyte includes bacillus licheniformis T-07 reconciliation starch gemma bar
Bacterium T-09, the endophyte has to be obtained by following step:
Step 1: Apple is cut the pulp of mung bean block size with the blade of sterilizing in superclean bench gnotobasis
It is placed in PDA culture medium and beef-protein medium, after cultivating 2d in 28 DEG C of incubators, is carried out after growing bacterium colony
Plate streak culture, is then isolated and purified in PDA culture medium, and inclined-plane is then saved, and is numbered；
Step 2: apple stem portion, root 3-5 hours are impregnated with sterile water, and after impregnating 2min with 95% ethyl alcohol, aseptic water washing
3 times, later again with 0.1% mercury chloride surface sterilization 30s, continue with rinsed with sterile water 5 times, using last time flushing liquor as
Whether control is coated on plate thorough to detect surface sterilization.The thorough apple tree of surface sterilization is taken respectively to organize, with scissors by leaf
Piece is cut into 0.5cm2Stem, root are cut into 0.25cm long, the tissue block cut are coupled with PDA culture medium plate by size
On, four tissue blocks of each board joint, 28 DEG C of culture 72h, after surrounding grows bacterium colony, according to colonial morphology, size, color etc.
Different feature carries out initial gross separation purifying, after obtaining purifying bacterial strain, is connected to inclined-plane and is placed in 4 DEG C of refrigerators and save backup, using flat
Plate face-off method after cultivating 2d in 28 DEG C of incubators, measures bacterial strain radius and simultaneously calculates bacteriostasis rate, see formula (1):
By comparing the bacteriostasis rate of endophyte, colony radius < 3cm is picked out, and bacteriostasis rate is more than or equal to 58% bacterial strain, respectively
It is bacillus licheniformis T-07, bacillus amyloliquefaciens T-09.
2. a kind of apple endophyte according to claim 1, which is characterized in that the constituent of the PDA culture medium are as follows:
Potato 200g, glucose 20g, agar 20g, distilled water 1000mL, pH are natural；The composition of the beef-protein medium
Become: beef extract 3g, peptone 5g, glucose 10g, agar 18g, distilled water 1 000mL, pH 7.2.
3. a kind of apple endophyte according to claim 1, which is characterized in that described bacillus licheniformis T-07, Xie Dian
Afnyloliquefaciens T-09 combination carries out antagonistic effect, strain Bacillus licheniformis T-07 and solution starch bud by tablet face-off method
Without antagonism between spore bacillus T-09, compounding can be carried out.
4. a kind of fermentation liquid, which is characterized in that the fermentation liquid is by bacillus licheniformis T-07, Xie Dian described in claim 1
Afnyloliquefaciens T-09 fermentation is made, method particularly includes: picking is stored in bacillus licheniformis T-07, solution starch bud on test tube
Spore bacillus T-09, fills entire PDA culture medium, is punched to obtain the bacteria cake that diameter is 0.6cm after covering with plate, be prepared simultaneously
PDA liquid medium, the PDB culture medium of the interior addition 250mL of the triangular flask of every 500mL, is put into 5 bacterium for every bottle after sterilization and cooling
Cake cultivates 3d, as bacillus licheniformis T-07 fermentation liquid and bacillus amyloliquefaciens T- in 200r/min, 28 DEG C of shaking table
09 fermentation liquid.
5. based on a kind of supernatant of fermentation liquid as claimed in claim 4, which is characterized in that the supernatant of the fermentation liquid is specific
The preparation method comprises the following steps: taking supernatant, the as supernatant of fermentation liquid after liquid bacterial strain is centrifuged 10min in 3 500r/min centrifuges
Liquid.
6. based on a kind of extracting solution of fermentation liquid as claimed in claim 4, which is characterized in that the preparation method of the extracting solution
Are as follows: resulting fermentation liquid is extracted with anhydrous ethyl acetate according to the volume ratio of 1:1, gained upper layer is after standing 12h
The extracting solution of fermentation liquid.
7. a kind of fresh-keeping liquid, which is characterized in that the fresh-keeping liquid includes bacillus licheniformis T-07 fermentation as claimed in claim 6
The extracting solution of liquid, the extracting solution of bacillus amyloliquefaciens T-09 fermentation liquid and compounding coating liquid.
8. a kind of fresh-keeping liquid according to claim 7, which is characterized in that the compounding coating liquid, bacillus licheniformis T-
The ratio of the extracting solution of the extracting solution and bacillus amyloliquefaciens T-09 fermentation liquid of 07 fermentation liquid is 2:1:1.
9. a kind of fresh-keeping liquid according to claim 7, which is characterized in that it is described compounding coating liquid the preparation method comprises the following steps:
1%~3% chitosan: weighing 1g~3g chitosan, adds 100ml, 1% glacial acetic acid, is stirred using magnetic stirring apparatus；
1%~3% sodium alginate: weighing 1g~3g sodium alginate, adds 100ml distilled water, is stirred using magnetic stirring apparatus；
1%~3% chitosan, 1%~3% sodium alginate are mixed according to the volume ratio of 1:1 to get compounding film is arrived
Liquid.
10. the purposes based on a kind of any fresh-keeping liquid of claim 7-9, which is characterized in that the fresh-keeping liquid for pair
Adopt the fresh-keeping of rear apple.
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| CN109161479A (en) * | 2018-08-15 | 2019-01-08 | 延安大学 | A kind of drug and preparation method thereof for preventing and treating apple tree canker |
| CN110050832A (en) * | 2019-05-08 | 2019-07-26 | 中国热带农业科学院南亚热带作物研究所 | The preparation method and gained fermentation liquid and its application method of a kind of fruit fermentation liquid |
| CN110771680A (en) * | 2019-11-15 | 2020-02-11 | 中国农业科学院果树研究所 | Method for maintaining storage quality and prolonging shelf life of apples |
| CN111990458A (en) * | 2020-08-19 | 2020-11-27 | 阿克苏优能农业科技股份有限公司 | Green antibacterial crisp-keeping type fresh-keeping packaging method for sugar-cored Fuji apples |
| CN115005270A (en) * | 2022-05-20 | 2022-09-06 | 珠海真绿色技术有限公司 | Biological fruit preservative with physiological preservation and disease prevention and control functions and preparation method thereof |
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| CN110771680B (en) * | 2019-11-15 | 2024-02-06 | 中国农业科学院果树研究所 | Method for maintaining storage quality and prolonging shelf life of apples |
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| CN115005270A (en) * | 2022-05-20 | 2022-09-06 | 珠海真绿色技术有限公司 | Biological fruit preservative with physiological preservation and disease prevention and control functions and preparation method thereof |
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