CN106070586A - A kind of complex biological antistaling agent and application thereof - Google Patents
A kind of complex biological antistaling agent and application thereof Download PDFInfo
- Publication number
- CN106070586A CN106070586A CN201610438169.4A CN201610438169A CN106070586A CN 106070586 A CN106070586 A CN 106070586A CN 201610438169 A CN201610438169 A CN 201610438169A CN 106070586 A CN106070586 A CN 106070586A
- Authority
- CN
- China
- Prior art keywords
- antistaling agent
- complex biological
- jerusalem artichoke
- lactococcus garvieae
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
- 241000194040 Lactococcus garvieae Species 0.000 claims abstract description 52
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 49
- 240000008892 Helianthus tuberosus Species 0.000 claims abstract description 39
- 235000003230 Helianthus tuberosus Nutrition 0.000 claims abstract description 39
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 29
- 239000000469 ethanolic extract Substances 0.000 claims abstract description 27
- 238000004321 preservation Methods 0.000 claims abstract description 20
- 229920001661 Chitosan Polymers 0.000 claims abstract description 17
- 235000021028 berry Nutrition 0.000 claims abstract description 15
- 239000000284 extract Substances 0.000 claims abstract description 10
- 230000001580 bacterial effect Effects 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 239000002609 medium Substances 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 22
- 238000000855 fermentation Methods 0.000 claims description 20
- 230000004151 fermentation Effects 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 8
- 239000003755 preservative agent Substances 0.000 claims description 8
- 238000000605 extraction Methods 0.000 claims description 7
- 241000194036 Lactococcus Species 0.000 claims description 6
- 229920002261 Corn starch Polymers 0.000 claims description 5
- 229920001202 Inulin Polymers 0.000 claims description 5
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 5
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 5
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims description 5
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- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 claims description 5
- 229940029339 inulin Drugs 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 claims description 5
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 claims description 5
- 230000002335 preservative effect Effects 0.000 claims description 4
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- UFLHIIWVXFIJGU-ARJAWSKDSA-N (Z)-hex-3-en-1-ol Chemical compound CC\C=C/CCO UFLHIIWVXFIJGU-ARJAWSKDSA-N 0.000 claims description 2
- 230000001476 alcoholic effect Effects 0.000 claims description 2
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- 238000002481 ethanol extraction Methods 0.000 claims description 2
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- 230000002401 inhibitory effect Effects 0.000 description 3
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- 230000035764 nutrition Effects 0.000 description 3
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- 241001478240 Coccus Species 0.000 description 2
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
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- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 108010066097 lactococcin A Proteins 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- XJAJBFWPYSQCKP-UHFFFAOYSA-N methyl 2-(6,7-dimethyl-3-oxo-4h-quinoxalin-2-yl)-4-(4-methoxy-2-nitroanilino)-3,4-dioxobutanoate Chemical compound N=1C2=CC(C)=C(C)C=C2NC(=O)C=1C(C(=O)OC)C(=O)C(=O)NC1=CC=C(OC)C=C1[N+]([O-])=O XJAJBFWPYSQCKP-UHFFFAOYSA-N 0.000 description 2
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
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- 235000002722 Dioscorea batatas Nutrition 0.000 description 1
- 235000006536 Dioscorea esculenta Nutrition 0.000 description 1
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- 101100117236 Drosophila melanogaster speck gene Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
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- 229930014669 anthocyanidin Natural products 0.000 description 1
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- 239000004310 lactic acid Substances 0.000 description 1
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229940099908 multivitamin and calcium Drugs 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/16—Coating with a protective layer; Compositions or apparatus therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicines Containing Plant Substances (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a kind of complex biological antistaling agent and application thereof.Complex biological antistaling agent is made up of Lactococcus garvieae element, jerusalem artichoke leaves ethanol extract, water-soluble chitosan.Described Lactococcus garvieae element (Lactococcus garvieae) by Lactococcus garvieae SY 1 fermented after again extract be prepared from, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, culture presevation number is CGMCC No.9485, preservation date July 28 in 2014.Described jerusalem artichoke leaves ethanol extract uses ultrasonic extraction to be prepared from.It is fresh-keeping that this antistaling agent is applied to small berries, can form one layer of safe and nontoxic, edible transparent membrane at fruit surface, fresh-keeping in conjunction with conventional freezer, can effectively keep color and luster and nutritional labeling, the various small berries shelf lifes of notable prolongation of fruit.
Description
Technical field
Present invention relates particularly to a kind of complex biological antistaling agent and be applied to the method that small berries is fresh-keeping, belonging to agricultural product
Storage technique field.
Background technology
Small berries refers to that fruit is less, fruit trees seeds of succulence, mainly includes blue berry, Fructus Rubi, black currant, red adds
Logical sequence, mossberry, blackberry, Fructus Myricae rubrae, Fructus Fici etc..Its fruit is vivid, sarcocarp succulence, unique flavor, and fragrance acid strong, sweet is tasty and refreshing,
Rich in trace element, organic acid, anthocyanidin, flavonoid isoreactivity materials such as multivitamin and calcium, phosphorus, zinc, have antioxidation,
The multiple medicines such as defying age, protection cardiovascular, anticancer, invigorating the spleen and regulating the stomach, the lung moistening and production of body fluid promoting, nourishing are enriched blood, facilitating digestion, enhancing immunity
Reason health-care effect, can effectively prevent the generation of multiple disease, the development such as anemia, hypertension, hyperlipidemia, cancer, heart disease.Can be wide
General being applied to the fields such as food, medicine, health product, supply falls short of demand in the international market.But owing to small berries peel is thin, it is soft to organize
Soft, be weak to accumulating, easily rot, frequently result in fruit production and marketing and seriously disconnect, a large amount of fruits are rotten in the edge of a field, stand, past
Toward causing serious economic loss to orchard worker.
Generally adopt rear Repiration, moisture transpiration effect, antibacterial mycete invasion cause small berries to rot
Main cause.Small berries preservation method has preservation by low temperature, controlled atmosphere, fresh-keeping, the coating preservation of chemical preservation etc. the most both at home and abroad.
Wherein the fresh-keeping means being combined with cryostat of chemical preservation are easy to use, strong adaptability, be widely used, but chemical preservative
Remain the often serious consequence such as environmental pollution, food safety, carcinogenic teratogenesis, and coating preservation can be many by chitosan etc.
Glucide can form the characteristic of protecting film at fruit surface, strengthens the protective effect of fruit surface, suppresses pore Repiration
Invade with moisture evaporation, suppression microorganism thus reach fresh-keeping purpose.But Edible coatings generally exists in the market
Shortcomings such as antibacterial effect is poor, tunicle is unstable, use cost is high, therefore are that exploitation is more suitable for, safe efficient, edibility is multiple
Join the main direction of studying that bio-preservative is fruit fresh-keeping field.
Summary of the invention
Goal of the invention: the present invention is directed to the deficiency that prior art exists, its object is to provide a kind of complex biological fresh-keeping
Agent, this antistaling agent can form protecting film, and safe and nontoxic, edible, fresh-keeping effect simultaneously at fruit surface after being applied to fruit
Good, can significantly extend the shelf life of fruit particularly small berries.
To achieve these goals, technical scheme is as follows:
A kind of complex biological antistaling agent, including Lactococcus garvieae element, jerusalem artichoke leaves ethanol extract and water-soluble chitosan.
The mass ratio of described Lactococcus garvieae element, jerusalem artichoke leaves ethanol extract and water-soluble chitosan is 100:2～5:45～65.
Described complex biological antistaling agent can contain or not contain solvent.
When described complex biological antistaling agent contains solvent, solvent can be water, the concentration of jerusalem artichoke leaves ethanol extract be 2～
5g/L, the concentration of water-soluble chitosan is 45～65g/L.
Described Lactococcus garvieae element extracts after being fermented by Lactococcus garvieae and prepares, and described Lactococcus garvieae is grignard breast
Coccus SY-1, this bacterial strain is obtained from the separation of Rhizoma Dioscoreae epidermis by present inventor, is preserved in Chinese microorganism strain preservation management
Committee's General Microbiological Culture preservation center, culture presevation number is CGMCC No.9485, preservation date July 28 in 2014.
Studying discovery through present inventor, Lactococcus garvieae SY-1 has stronger bacteriocinogeny activity, Jerusalem artichoke folic alcohol
Extract also has certain anti-bacterial disease, and the two is used in combination the stability that both can improve Lactococcus garvieae element with chitosan,
It is significantly expanded again the antimicrobial spectrum of three, small berries soft rot and gray mold, yeast have been respectively provided with preferable biocontrol effect, and
Can significantly inhibit weight-loss ratio and the respiratory intensity of small berries, fresh-keeping effect is the most notable.
The preparation method of described Lactococcus garvieae element includes:
(1) by after Lactococcus garvieae actication of culture, it is inoculated in culture medium and cultivates, prepare seed liquor；
(2) seed liquor is inoculated in fermentation medium carries out fermentation culture, after fermentation culture terminates, collect fermentation liquid；
(3) described fermentation liquid is centrifuged, takes supernatant, be to precipitate after 2～3 with acid regulation PH, take precipitation, freezing dry
Dry, it is described Lactococcus garvieae element.
In step (1), described culture medium is MRS fluid medium, and the time of cultivation is 24～36h, the temperature of cultivation
It it is 28～30 DEG C.
In step (2), inoculum concentration is 2～5% (v/v)；Fermentation medium is by bean cake, corn starch, inulin, Fructus Lycopersici esculenti
Juice, sodium carboxymethyl cellulose and sterilized water composition, each composition respectively is with the mass volume ratio of sterilized water: 15～20g/L,
8～12g/L, 1～2g/L, 0.1～0.5g/L and 0.5～1.2g/L；The temperature of fermentation culture is 28～30 DEG C, the time be 18～
24h。
In step (3), described in carry out precipitating temperature be 4～8 DEG C, the time is 12～18h.
Described jerusalem artichoke leaves ethanol extract is jerusalem artichoke leaves ethanol extraction.
The preparation method of described jerusalem artichoke leaves ethanol extract includes: take jerusalem artichoke leaves, is placed according to the solid-liquid ratio of 1:20～25
Carrying out supersound extraction in 40%～60% alcoholic solution, supersound extraction is centrifugal after terminating, and takes supernatant, dried described jerusalem artichoke leaves
Ethanol extract.
During supersound extraction, ultrasonic power is 350～500w, and ultrasonic time is 40～50min.
The preparation method of described complex biological antistaling agent includes: by Lactococcus garvieae element, jerusalem artichoke leaves ethanol extract and water solublity
Chitosan directly mixes.
Present invention also offers the application in fruit freshness preserving of the described complex biological antistaling agent.
Described fruit is small berries.
Described application includes: by complex biological preservative dilute with water 30～60 times, be placed on by fruit precooling compounding
Bio-preservative diluent impregnates, carries out fresh-keeping in conjunction with conventional freezer preservation method.
Concrete, described application includes: chilling room put into by fruit after gathering, and uses cold wind to be cooled to 2～5 DEG C, wind speed
10～15m/s, humidity 80～85%, the time 1～3h, place into and complex biological antistaling agent diluent impregnates 1～3min, cold wind
Dry up, be placed in freezer carry out fresh-keeping.The condition that wherein freezer is fresh-keeping selects according to the kind of fruit.
Compared with prior art, the invention have the benefit that
1) the complex biological antistaling agent that the present invention provides is a kind of brand-new complex biological antistaling agent, has efficient, wide spectrum
Antibacterial protection effect, soft rot, gray mold etc. can be carried out associating prevention and control.This complex biological antistaling agent also has user
Just, safety, edible, significantly reduce small berries weight-loss ratio and respiratory intensity, holding fruit commodity, extend the useful effect of shelf
Really.
2) present invention employs Lactococcus garvieae SY-1 and prepare Lactococcus garvieae element, this bacterium is preserved in Chinese microorganism strain
(culture presevation number is CGMCC No.9485, preservation date 2014 at preservation administration committee General Microbiological Culture preservation center
July 28), there is stronger bacteriocinogeny function, and has a broad antifungal spectrum, with jerusalem artichoke leaves extract, the collaborative use of water-soluble chitosan
After, it has had significant enhancing to the protection effect of the antistaling agent that the antibacterial protection effect of small berries is made than single component, and
The film formed is uniform, lasting, stable.
3) present invention is when preparing jerusalem artichoke leaves extract, have employed ultrasound assisted extraction technique, the extractive content prepared is high,
Biological activity is high, and antibacterial effect is obvious, and can significantly inhibit color and luster brown stain, flavor deterioration after Fruit, well maintains fruit
Real nutrition and active component, improve the commodity value of fruit.
4) the complex biological antistaling agent that prepared by the present invention is safe efficient, nontoxic, edible, will not be to environment and human body
Damage, in conjunction with conventional freezer preservation technique, have more incomparable than chemical preservative and common coating antistaling agent excellent
Point.
5) complex biological antistaling agent of the present invention be applied to small berries fresh-keeping time, can fruit show formed layer of transparent vivid
Edible film, fruit respiratory intensity can be suppressed, reduce weight-loss ratio, keep the nutrition of fruit and active component, prolongation fruit shelf
Phase, bigger economic benefit can be produced.
Accompanying drawing explanation
Fig. 1 is the antibacterial figure of Lactococcus garvieae SY-1；
Fig. 2 is Lactococcus garvieae SY-1 colonial morphology in ATB culture medium；
Fig. 3 is Lactococcus garvieae SY-1 Gram’s staining figure；
Fig. 4 is Lactococcus garvieae SY-1 16S rRNA gene order；
Fig. 5 is the different antistaling agent impacts on Fructus Fragariae Ananssae rotting rate；
Fig. 6 is the different antistaling agent impacts on Fructus Fragariae Ananssae weight-loss ratio；
Fig. 7 is the different antistaling agent impacts on Content of Soluble Solid Substance of Strawberry.
Detailed description of the invention
The following detailed description of embodiments of the invention, it should be noted that the present embodiment is narrative, it not limited
, it is impossible to limit protection scope of the present invention with this.
The isolation identification of embodiment 1 Lactococcus garvieae SY-1
Lactococcus garvieae (Lactococcus garviea) SY-1 involved in the present invention, in preservation in July 28 in 2014
In China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, (being called for short CGMCC, address Beijing is exposed to the sun
North Star West Road, district 1 institute 3, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is grignard milk-globule
Bacterium Lactococcus garviea, culture presevation number is CGMCC No.9485.Lactococcus garvieae (Lactococcus of the present invention
Garviea) SY-1 is to separate from Rhizoma Dioscoreae epidermis to obtain.
The ATB culture medium prescription of the following stated is: glucose 8g/L, yeast leaching powder 4g/L, peptone 10g/L, MgSO4·
7H2O 0.2g/L, MnSO4·4H2O 0.05g/L, Fructus Lycopersici esculenti juice 250mL/L, pH value 4.5-4.8,121 DEG C of sterilizing 20min.ATB is solid
Body culture medium: add 2% agar in ATB fluid medium.
1, separate
Take fresh Chinese yam skin (band earth) 50g, smash, mix homogeneously, take the yam skin that 10g processed and pour into containing 20
In the 250mL triangular flask of micro glass beads and 100mL sterilizing deionized water, on shaking table
150r/min, shake 1h under the conditions of 25 DEG C, after static 30min, take supernatant 1mL, with 10 times of ladders of sterilizing deionized water
Degree dilution, respectively obtains 10-1、10-2、10-3、10-4、10-5、10-6、10-77 gradients, draw 100 μ L respectively, and different gradients are dilute
Release liquid to be applied in ATB solid plate culture medium, 25-28 DEG C, static gas wave refrigerator 30-36h.Utilize inoculating loop picking list bacterium colony at ATB
Solid medium flat lining out purification 2-3 time,
Single bacterium colony of the different shape of purification is numbered and is transferred on ATB solid plate, 25-28 DEG C, static gas wave refrigerator 30-
36h, be then stored in 4 DEG C stand-by.
2, bacteriostatic test
Being cultivated in ATB fluid medium by the sublimed bacterial strain being stored on ATB solid medium, condition of culture is
25 DEG C, static gas wave refrigerator 36h.Additionally the thalline on Rhizoma Dioscoreae epidermis black speck is inoculated on ATB solid medium flat board, is uniformly coated with
Cloth, cultivates 48h at 30 DEG C.Then utilizing sterilizing card punch to punch on solid plate, aperture is 4mm, and each flat board uniformly makes a call to 6
Individual hole.Then the 40 cultured isolated and purified bacterium solution to be determined of μ L are added drop-wise in hole, at 28 DEG C of static gas wave refrigerator 36h, by pressing down
The bacterium circle assessment test strains inhibitory activity to Rhizoma Dioscoreae alternaria.Found that there are three strain bacterial strains to have bright to Rhizoma Dioscoreae alternaria
Aobvious inhibitory action, but SY-1 inhibitory activity is the strongest, and antibacterial circle diameter is 24.3mm, and result is shown in Fig. 1.Therefore below this strain bacterium is entered
Go further qualification.
3, morphologic observation
Bacterial strain SY-1 the strongest for bacteriostatic activity is inoculated in 100mL ATB fluid medium, 25 DEG C of static gas wave refrigerator 30h,
The most aseptically rule on ATB solid medium with aseptic inoculation ring, afterwards at 25 DEG C of static gas wave refrigerator 36h, observe
Colonial morphology, single bacterium colony is milky, circle, smooth surface, and edge is complete, and result is as shown in Figure 2.Picking list bacterium colony, is carried out
Gram’s staining, and examine under a microscope, this bacterium is Gram-positive coccus, and result is as shown in Figure 3.By Fig. 2 and Fig. 3
Result identifies that SY-1 is Lactococcus substantially.
4, molecular biology identification
Utilize the sterilizing toothpick mono-bacterium colony of picking SY-1 in the 250mL triangular flask containing 50mL ATB fluid medium,
25 DEG C of static gas wave refrigerator 24h, then utilize bacterial genomes DNA extraction kit to extract the genomic DNA of this bacterium.Utilize 16S
RRNA universal primer fd2 (5 '-AGAGTTTGATCATGGCTCAG-3 ') and rpl (5 '-ACGGTTACCTTGTTACGACTT-3 ')
Pcr amplified DNA fragment, send gene sequencing company to check order by PCR primer.This bacterium 16S rRNA gene sequencing result such as Fig. 4
Shown in.According to the 16S rRNA gene order obtained, utilize genebank data base to compare in ncbi database, draw
It is up to 99.5% with Lactococcus garvieae gene similarity, therefore identifies that this bacterium is Lactococcus garvieae (Lactococcus
And to name this bacterium be Lactococcus garvieae SY-1 garviea),.
Embodiment 2
A kind of complex biological antistaling agent and the method for strawberry preservation thereof, step is:
(1) preparation of jerusalem artichoke leaves ethanol extract: take dry jerusalem artichoke leaves (Jiangsu Bi Qingyuan marine organisms Science and Technology Ltd.),
Join according to the solid-liquid ratio (g/L) of certain 1:20 in the ethanol water of 50%, put into ultrasonic washing instrument, carry out ultrasonic
Extract, supersound extraction 40min under the conditions of ultrasonic power 350w, be then placed in centrifuge, centrifugal under 3000rpm/min
20min, supernatant Rotary Evaporators concentrates 15 times, then is dried to constant weight with freeze dryer.
(2) spawn culture: take Lactococcus garvieae SY-1 and (be preserved in China Committee for Culture Collection of Microorganisms the most micro-
Biological inoculum preservation center, culture presevation number is CGMCC No.9485, preservation date July 28 in 2014) glycerol preserves strain,
On MRS (lactic acid bacteria culture medium) solid plate that streak inoculation is the freshest, activation culture 24h under the conditions of 30 DEG C, then exist
During on solid plate, picking one single colony inoculation to contains the 250mL conical flask of 50mL MRS fluid medium, 30 DEG C
Lower cultivation 24h, is seed liquor；
(3) prepared by fermentation medium: by bean cake, corn starch, inulin (the Jiangsu Bi Qingyuan marine organisms limited public affairs of science and technology
Department), Tomato juice, sodium carboxymethyl cellulose (being commercially available) and sterilized water form, each composition and the mass volume ratio of sterilized water
It is followed successively by: 15g/L, 8g/L, 2g/L, 0.2g/L, 0.5g/L.
(4) preparation of Lactococcus garvieae cellulose solution: by step 2) seed liquor prepared is inoculated in the ratio of 2% (v/v)
In fermentation medium, at 30 DEG C, cultivate 20h.Cultivation terminates rear 4000r/min and is centrifuged 20min, takes supernatant, (commercially available with citric acid
Food stage) adjust pH value to 3,4 DEG C of overnight precipitation, the next day in 9000r/min, 4 DEG C of centrifugal 10min, the pellet frozen extracted do
With the deionized water dissolving of 10 times of quality after dry, it is Lactococcus garvieae cellulose solution.
(5) compounding: by jerusalem artichoke leaves ethanol extract and water-soluble chitosan (the Jinan limited public affairs of Hai get Bei marine biotechnology
Department, goods number is syx001) join in Lactococcus garvieae cellulose solution, stirring, to being completely dissolved, is complex biological fresh-keeping
Agent.Wherein jerusalem artichoke leaves ethanol extract and water-soluble chitosan shared mass volume ratio in Lactococcus garvieae cellulose solution is respectively 5g/L
And 60g/L.
(6) the complex biological antistaling agent extension rate after compounding is 3 times, standby；
(7) after Fructus Fragariae Ananssae (academy of agricultural sciences of Jiangsu Province Lishui base of plant) is gathered, it is immediately placed in chilling room, uses air-cooler to force
It is cooled to 2 DEG C, wind speed 10m/s, humidity 80%, time 1h, place in the complex biological antistaling agent diluent diluting 3 times and soak
Stain 1min, pulls out, dries up with cold wind, enters according still further to conventional freezer preservation method (temperature of ice house 0～4 DEG C, humidity 90～95%)
Row is fresh-keeping.
When determining fresh keeping time, arrange comparison (processing without antistaling agent), Lactococcus garvieae element (100g/L) processes, jerusalem artichoke leaves
Ethanol extract (5g/L) process, lactococcin (100g/L) and jerusalem artichoke leaves ethanol extract (5g/L) Combined Treatment and Lactococcus garvieae
Element (100g/L) adds jerusalem artichoke leaves ethanol extract (5g/L) and adds 5 kinds of fresh-keeping modes of chitosan (60g/L) Combined Treatment, during process, respectively protects
Fresh dilution agent 3 times use, the rotting rate of Fructus Fragariae Ananssae, weight-loss ratio, soluble solid content in the different fresh keeping time of detection, by Fig. 1,
Fig. 2, Fig. 3 are visible, and matched group rotting rate when fresh-keeping 3 days reaches 9.2%, and when fresh-keeping 5 days, rotting rate may be up to 24%, by
This can determine that the longest fresh keeping time of matched group Fructus Fragariae Ananssae is 3～4 days, may not exceed absolutely 5 days.In same fresh keeping time, each process group
Fresh-keeping effect be followed successively by Lactococcus garvieae element and add jerusalem artichoke leaves ethanol extract and add chitosan Combined Treatment group ＞ lactococcin and Jerusalem artichoke
Alcohol extracts from the leaves Combined Treatment group ＞ Lactococcus garvieae element process group ＞ jerusalem artichoke leaves ethanol extract process group.The Fructus Fragariae Ananssae that triple combination processes
Its rotting rate and weight-loss ratio are significantly lower than other process groups, and soluble solid content is then apparently higher than other process groups.Show
Three has the effect of Synergistic to the holding of Diseases of Strawberry and strawberry nutrition composition.
Utilize this complex biological antistaling agent keeping strawberry fresh, Fructus Fragariae Ananssae can be made (not use compounding raw in fresh-keeping 7～10 days in freezer
The Fructus Fragariae Ananssae of thing antistaling agent freshness date in 0～Cool Room 4 DEG C is 3～4 days).
Embodiment 3
A kind of complex biological antistaling agent and be applied to the method that Vaccinium ashei is fresh-keeping, step is:
(1) preparation of jerusalem artichoke leaves ethanol extract: take dry jerusalem artichoke leaves (Jiangsu Bi Qingyuan marine organisms Science and Technology Ltd.),
Join according to the solid-liquid ratio (g/L) of certain 1:25 in the ethanol water of 50%, put into ultrasonic washing instrument, carry out ultrasonic
Extract, supersound extraction 45min under the conditions of ultrasonic power 400w, be then placed in centrifuge, centrifugal under 3000rpm/min
15min, supernatant Rotary Evaporators concentrates 15 times, then is dried to constant weight with freeze dryer.
(2) spawn culture: take Lactococcus garvieae SY-1 and (be preserved in China Committee for Culture Collection of Microorganisms the most micro-
Biological inoculum preservation center, culture presevation number is CGMCC No.9485, preservation date July 28 in 2014) glycerol preserves strain,
On the MRS solid plate that streak inoculation is the freshest, activation culture 40h under the conditions of 28 DEG C, then picking one on solid plate
Individual single colony inoculation to one, containing in the 250mL conical flask of 50mL MRS fluid medium, is cultivated 30h, is kind at 30 DEG C
Sub-liquid；
(3) prepared by fermentation medium: (be city by bean cake, corn starch, inulin, Tomato juice, sodium carboxymethyl cellulose
Sell) and sterilized water composition, each composition is followed successively by with the mass volume ratio of sterilized water: 15g/L, 10g/L, 1g/L, 0.5g/L, 1g/
L。
(4) preparation of Lactococcus garvieae cellulose solution: by step 2) seed liquor prepared is inoculated in the ratio of 4% (v/v)
In fermentation medium, at 30 DEG C, cultivate 20h.Cultivation terminates rear 4000r/min and is centrifuged 20min, takes supernatant, (commercially available with citric acid
Food stage) adjust pH value to 2,4 DEG C of overnight precipitation, the next day in 9000r/min, 4 DEG C of centrifugal 15min, the pellet frozen extracted do
With the deionized water dissolving of 10 times of quality after dry, it is Lactococcus garvieae cellulose solution.
(5) compounding: by Jerusalem artichoke ethanol extract and water-soluble chitosan (Jinan Haidebei Marine Organism Engineering Co., Ltd.)
Joining in Lactococcus garvieae cellulose solution, stirring, to being completely dissolved, is complex biological antistaling agent.Wherein jerusalem artichoke leaves ethanol extract and
Water-soluble chitosan shared mass volume ratio in Lactococcus garvieae cellulose solution is respectively 2g/L and 45g/L.
(6) the complex biological antistaling agent extension rate after compounding is 6 times, standby；
(7), after Vaccinium ashei (Nanjing Bai Long organic agriculture Science and Technology Development Co., Ltd.) is gathered, it is immediately placed in chilling room, adopts
With air-cooler forced cooling to 4 DEG C, wind speed 10m/s, humidity 75%, time 3h, place into that to dilute the complex biological of 6 times fresh-keeping
Dilution agent liquid impregnates 1min, pulls out, dry up with cold wind, according still further to conventional freezer method for preserving (temperature of ice house 0～5 DEG C, humidity
85～90%) carry out fresh-keeping.
Utilize this complex biological antistaling agent fresh blueberry, blue berry can be made (not use compounding in fresh-keeping 50～60 days in freezer
The blue berry of bio-preservative freshness date in 0～5 DEG C of freezer is 30～35 days).
Embodiment 4
A kind of complex biological antistaling agent and be applied to the method that Fructus Fici is fresh-keeping, step is:
(1) preparation of jerusalem artichoke leaves ethanol extract: take dry jerusalem artichoke leaves (Jiangsu Bi Qingyuan marine organisms Science and Technology Ltd.),
Join according to the solid-liquid ratio (g/L) of certain 1:20 in the ethanol water of 50%, put into ultrasonic washing instrument, carry out ultrasonic
Extract, supersound extraction 50min under the conditions of ultrasonic power 500w, be then placed in centrifuge, centrifugal under 3000rpm/min
20min, supernatant Rotary Evaporators concentrates 15 times, then is dried to constant weight with freeze dryer.
(2) spawn culture: take Lactococcus garvieae SY-1 and (be preserved in China Committee for Culture Collection of Microorganisms the most micro-
Biological inoculum preservation center, culture presevation number is CGMCC No.9485, preservation date July 28 in 2014) glycerol preserves strain,
On the MRS solid plate that streak inoculation is the freshest, activation culture 30h under the conditions of 30 DEG C, then picking one on solid plate
Individual single colony inoculation to one, containing in the 250mL conical flask of 50mL MRS fluid medium, is cultivated 24h, is kind at 30 DEG C
Sub-liquid；
(3) prepared by fermentation medium: (be city by bean cake, corn starch, inulin, Tomato juice, sodium carboxymethyl cellulose
Sell) and sterilized water composition, each composition is followed successively by with the mass volume ratio of sterilized water: 20g/L, 12g/L, 2g/L, 0.4g/L,
0.8g/L。
(4) preparation of Lactococcus garvieae cellulose solution: by step 2) seed liquor prepared is inoculated in the ratio of 5% (v/v)
In fermentation medium, at 30 DEG C, cultivate 24h.Cultivation terminates rear 4000r/min and is centrifuged 15min, takes supernatant, (commercially available with citric acid
Food stage) adjust pH value to 2.5,4 DEG C of overnight precipitation, the next day in 9000r/min, 4 DEG C of centrifugal 15min, the pellet frozen extracted
Dried with the deionized water dissolving of 10 times of quality, it is Lactococcus garvieae cellulose solution.
(5) compounding: by Jerusalem artichoke ethanol extract and water-soluble chitosan (Jinan Haidebei Marine Organism Engineering Co., Ltd.)
Joining in Lactococcus garvieae cellulose solution, stirring, to being completely dissolved, is complex biological antistaling agent.Wherein jerusalem artichoke leaves ethanol extract and
Water-soluble chitosan shared mass volume ratio in Lactococcus garvieae cellulose solution is respectively 5g/L and 55g/L.
(6) the complex biological antistaling agent extension rate after compounding is 4 times, standby；
(7), after Fructus Fici (Western Hills National modern agricultural demonstration zone, Suzhou Co., Ltd) is gathered, it is immediately placed in cold
But between, use air-cooler forced cooling to 4 DEG C, wind speed 10m/s, humidity 75%, time 40min, place into and dilute 4 times answer
Join in bio-preservative diluent dipping 1.5h, pull out, dry up with cold wind, according still further to conventional freezer method for preserving (temperature of ice house-
2～4 DEG C, humidity 85～90%) carry out fresh-keeping.
Utilize this fresh-keeping Fructus Fici of complex biological antistaling agent, Fructus Fici can be made (not use for fresh-keeping 15～20 days in freezer
The Fructus Fici of complex biological antistaling agent freshness date in-2～Cool Room 4 DEG C is 5～7 days).
Claims (10)
1. a complex biological antistaling agent, it is characterised in that include that Lactococcus garvieae element, jerusalem artichoke leaves ethanol extract and water soluble shells are gathered
Sugar.
Complex biological antistaling agent the most according to claim 1, it is characterised in that described Lactococcus garvieae element, Jerusalem artichoke folic alcohol
The mass ratio of extract and water-soluble chitosan is 100:2～5:40～65.
Complex biological antistaling agent the most according to claim 1, it is characterised in that described Lactococcus garvieae element is derived from grignard
Lactococcus SY-1, the preserving number of this bacterial strain is CGMCC No.9485.
Complex biological antistaling agent the most according to claim 1, it is characterised in that the preparation method of described Lactococcus garvieae element
Including:
(1) by after Lactococcus garvieae actication of culture, it is inoculated in culture medium and cultivates, prepare seed liquor；
(2) seed liquor is inoculated in fermentation medium carries out fermentation culture, after fermentation culture terminates, collect fermentation liquid；
(3) described fermentation liquid is centrifuged, takes supernatant, be to precipitate after 2～3 with acid regulation PH, take precipitation, lyophilization,
It is described Lactococcus garvieae element.
Complex biological antistaling agent the most according to claim 4, it is characterised in that in step (2), fermentation medium is by bean
The dregs of rice, corn starch, inulin, Tomato juice, sodium carboxymethyl cellulose and sterilized water composition, each composition and the quality volume of sterilized water
Ratio is followed successively by: 15～20g/L, 8～12g/L, 1～2g/L, 0.1～0.5g/L and 0.5～1.2g/L；The temperature of fermentation culture is
28～30 DEG C, the time is 18～24h.
Complex biological antistaling agent the most according to claim 1, it is characterised in that described jerusalem artichoke leaves ethanol extract is jerusalem artichoke leaves
Ethanol extraction.
Complex biological antistaling agent the most according to claim 1, it is characterised in that the preparation side of described jerusalem artichoke leaves ethanol extract
Method includes: take jerusalem artichoke leaves, is placed in 40%～60% alcoholic solution according to the solid-liquid ratio of 1:20～25 and carries out supersound extraction, ultrasonic carries
Take after end centrifugal, take supernatant, dried described jerusalem artichoke leaves ethanol extract.
8. according to the application in fruit freshness preserving of the complex biological antistaling agent described in any one of claim 1～7.
Application the most according to claim 8, it is characterised in that described fruit is small berries.
Application the most according to claim 8, it is characterised in that described complex biological preservative needs with water dilute in use
Release 30～60 times；After described fruit impregnates in diluent, carry out fresh-keeping in conjunction with conventional freezer preservation method.
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| CN108029751A (en) * | 2017-12-08 | 2018-05-15 | 鲁东大学 | A kind of cherry coating antistaling agent and preparation method thereof and application method |
| CN108713585A (en) * | 2017-11-21 | 2018-10-30 | 南京农业大学 | A kind of fresh-water fishes composite preservative and the preparation method and application thereof |
| CN112136881A (en) * | 2020-09-22 | 2020-12-29 | 山东安谱检测科技有限公司 | Fruit preservative containing jerusalem artichoke extract and preparation method thereof |
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| 谌馥佳: "菊芋叶片化学成分分析及抑菌活性成分研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
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| CN107019034A (en) * | 2017-04-12 | 2017-08-08 | 浙江大学 | A kind of Herba polygoni hydropiperis extract antistaling agent and the application in red bayberry quality is improved |
| CN108713585A (en) * | 2017-11-21 | 2018-10-30 | 南京农业大学 | A kind of fresh-water fishes composite preservative and the preparation method and application thereof |
| CN108029751A (en) * | 2017-12-08 | 2018-05-15 | 鲁东大学 | A kind of cherry coating antistaling agent and preparation method thereof and application method |
| CN112136881A (en) * | 2020-09-22 | 2020-12-29 | 山东安谱检测科技有限公司 | Fruit preservative containing jerusalem artichoke extract and preparation method thereof |
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| CN106070586B (en) | 2020-02-14 |
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