CN106615085B - Microbial and plant source composite fruit and vegetable fresh-keeping agent - Google Patents
Microbial and plant source composite fruit and vegetable fresh-keeping agent Download PDFInfo
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- CN106615085B CN106615085B CN201611175829.0A CN201611175829A CN106615085B CN 106615085 B CN106615085 B CN 106615085B CN 201611175829 A CN201611175829 A CN 201611175829A CN 106615085 B CN106615085 B CN 106615085B
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/16—Coating with a protective layer; Compositions or apparatus therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
-
- 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
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Microbiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to the technical field of microbial antistaling agents, and aims to develop a compound antistaling agent containing microbes and plant source active ingredients, which is used for fresh-keeping and corrosion prevention of picked fruits and vegetables and provides a microbial and plant source compound fruit and vegetable antistaling agent. The composition is prepared from a component A and a component B, wherein the component A consists of a microorganism fermentation liquor and a propolis extracting solution, and the microorganisms are bacillus amyloliquefaciens Lh-1, candida paromomea and cryptococcus laurentii; the component B consists of plant source effective component mixed liquor and film forming liquor, the plant source effective component mixed liquor is formed by mixing peony bark water extract, orange peel ethanol extract, rabdosia rubescens ethanol extract, radix zanthoxyli water extract, lycium ruthenicum ethanol extract, peony root water extract, Clinopodium chinense ethanol extract and citronella water extract, and the film forming liquor is formed by mixing konjac mannan and chitosan. Prevent the fruits and vegetables from mildewing and rotting to the maximum extent, improve the control effect of the postharvest diseases of the fruits and vegetables, reduce the water loss of the fruits and vegetables, prolong the storage time of the fruits and vegetables and create certain economic value.
Description
Technical Field
The invention belongs to the technical field of microbial preservatives, and particularly relates to a microbial and plant source composite fruit and vegetable preservative.
Background
The fresh-keeping and corrosion prevention of fruits and vegetables are an important link for storage, and how to prolong the storage time of fruits and vegetables is a major problem to be solved in storage. At present, the storage methods adopted in China are a chemical agent fresh-keeping method, a low-temperature storage method, an air-conditioning fresh-keeping method and the like, wherein the chemical agent fresh-keeping method is commonly used, but the problem of food safety is followed, so that the development of a pollution-free preservative for controlling postharvest diseases of fruits and vegetables becomes a new trend and is gradually a research hotspot.
In the storage of fruits and vegetables, diseases after picking include soft rot, gray mold, brown rot, penicillium, pear gray mold, penicillium, stalk rot, etc., so microorganisms having antagonistic action against plant pathogenic bacteria are beginning to be applied to the disease control after picking fruits and vegetables. The antagonistic microbe has high safety, can inhibit the growth of pathogenic bacteria, and does not cause damage to materials. At present, the commonly used antagonistic bacteria for inhibiting the fruit and vegetable preservation and fresh keeping comprise microzyme, bacteria and small filamentous fungi, which can be successfully propagated on the epidermis of the fruit and vegetable to form competitive inhibition with pathogenic bacteria, secrete antibacterial substances to inhibit the growth of the pathogenic bacteria, and induce a fruit and vegetable defense system to resist the invasion of the pathogenic bacteria, thereby achieving the purpose of prolonging the storage time of the fruit and vegetable.
While the microbial preservative becomes a hot point for storage research, the plant-derived preservative has become another hot point for fresh-keeping and storage of domestic fruits and vegetables by virtue of the advantages of high efficiency, naturalness, no toxicity, safety, stable performance and the like. China has a wide variety of plant resources, and at present, a plurality of experiments prove that the components in the plants have the antibacterial and fresh-keeping effects (including some spices and Chinese herbal medicines), so that the preservation time of the fruits and vegetables is effectively prolonged, and the action mechanism is as follows: the plant effective components can inhibit the activity of microorganisms attached to the surfaces of fruits and vegetables, so that the physiological activity of the fruits and vegetables is maintained at a low level, and the infection of the microorganisms to the fruits and vegetables is reduced; meanwhile, the effective components form a film on the surface of the fruits and vegetables, and the film can reduce the loss of water in the fruits and vegetables and weaken the respiratory strength of the fruits and vegetables, thereby prolonging the storage time of the fruits and vegetables. However, the microbial source preservative and the plant source preservative are used together, meanwhile, a fruit and vegetable defense system is induced to resist the invasion of pathogenic bacteria, the water loss of the fruits and the vegetables is reduced, the respiratory intensity of the fruits and the vegetables is weakened, the storage time of the fruits and the vegetables is prolonged, and few preservatives for the preservation of the fruits and the vegetables are reported at present.
Disclosure of Invention
The invention aims to develop a compound preservative containing microorganisms and plant-derived active ingredients, fully combines the advantages of the microorganisms and the plant-derived active ingredients, is used for preserving and preventing picked fruits and vegetables, prevents the fruits and vegetables from mildewing and rotting in the storage process to the maximum extent, improves the prevention and control effect of picked diseases of the fruits and vegetables, reduces the loss of water in the fruits and vegetables, prolongs the storage time of the fruits and vegetables, and creates certain economic value; provides a natural composite fruit and vegetable fresh-keeping agent which takes microorganisms and plant sources as raw materials, and also provides an application method of the composite fruit and vegetable fresh-keeping agent.
The invention is realized by the following technical scheme: a microbial and plant source composite fruit and vegetable fresh-keeping agent is prepared from a component A and a component B, wherein the component A is composed of microbial fermentation liquor accounting for 32% -70% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent and propolis extract accounting for 3% -8% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent, and the microorganisms are as follows: the concentration is 1X 106-1×108Bacillus at a concentration of 1X 10/mL6-1×109Mixing yeast per mL at a ratio of 1:5-1:20, wherein the Bacillus is Bacillus amyloliquefaciens Lh-1 (B)Bacillus amyloliquefaciens Lh-1) The yeast is Candida intermedia mixed at a ratio of 1:1（Candida intermedia）Cryptococcus Helioticus Hedera(Cryptococcus laurentii)(ii) a The propolis extractive solution is prepared by conventional extraction method with ethanol as extraction solvent, and has concentration of 5-50 g/L;
the component B consists of mixed liquid of plant-derived effective components accounting for 12-50% of the volume of the microorganism and plant-derived composite fruit and vegetable antistaling agent and film forming liquid accounting for 3-10% of the volume of the microorganism and plant-derived composite fruit and vegetable antistaling agent, the mixed solution of the plant source active ingredients is prepared by mixing 0.08 g/mL peony bark water extract, 0.03 g/mL citrus peel ethanol extract, 0.04g/mL rabdosia rubescens ethanol extract, 0.08 g/mL radix zanthoxyli water extract, 0.04g/mL lycium ruthenicum ethanol extract, 0.08 g/mL peony root water extract, 0.05 g/mL Clinopodium polycephalum herba Clinopodii ethanol extract and 0.1 g/mL citronella water extract, wherein the volume ratio of the extracts to the mixed solution of the plant source active ingredients is as follows: 3-30% of peony bark water extract, 1-20% of citrus peel ethanol extract, 2-18% of rabdosia rubescens ethanol extract, 1-10% of radix zanthoxyli water extract, 5-35% of lycium ruthenicum ethanol extract, 0.5-15% of peony water extract, 0.5-10% of Clinopodium chinense ethanol extract and 0.5-20% of rhizoma imperatae water extract; the film forming liquid is formed by mixing 0.5-2% of konjac glucomannan and 0.6% of chitosan according to the volume ratio of 1: 1.
The extraction method of the plant source effective components comprises the following steps: the extraction method of the tree peony bark comprises the following steps: putting 20g of tree peony bark into a 500mL round-bottom flask, adding 300mL of distilled water, soaking in a 75-85 ℃ water bath for 6h, filtering, and fixing the volume of the filtrate to 250mL to obtain 0.08 g/mL tree peony bark water extract;
the extraction method of the orange peel comprises the following steps: crushing 10g of orange peel, soaking in 300mL of 75% ethanol, and then performing microwave extraction for 7min at the microwave power of 900W and the temperature of 75 ℃ in a material-liquid ratio of 1:30 (g: mL); decolorizing the extractive solution with 10% active carbon, and filtering to obtain 0.03 g/mL ethanol extractive solution of pericarpium Citri Tangerinae;
the extraction method of rabdosia rubescens comprises the following steps: pulverizing 10g Rabdosia rubescens with a traditional Chinese medicine pulverizer, soaking in 250mL 85% ethanol for 1h, performing microwave treatment with the microwave power of 90W for 80s, transferring to a reflux device for reflux extraction for 30min, and decolorizing and filtering the extract with 10% active carbon to obtain Rabdosia rubescens ethanol extract with the concentration of 0.04 g/mL;
the extraction method of radix zanthoxyli comprises the following steps: putting 20g radix zanthoxyli into a 500mL round-bottom flask, adding 300mL of distilled water, soaking in a 75-85 ℃ water bath for 6h, filtering, and fixing the volume of the filtrate to 250mL to obtain 0.08 g/mL radix zanthoxyli water extract;
the extraction method of the lycium ruthenicum comprises the following steps: adding 2g of lycium ruthenicum mill into 30mL of 50% ethanol, leaching for 1.5h in a 50 ℃ water bath, filtering, adding 20mL of 50% ethanol into the residue, leaching for 1h, filtering, combining the filtrates, and fixing the volume to 50 mL; decolorizing the extractive solution with active carbon 10% of the extract, and filtering to obtain 0.04g/mL Lycium ruthenicum Murr ethanol extractive solution;
the extraction method of the peony comprises the following steps: putting 20g of radix paeoniae into a 500mL round-bottom flask, adding 300mL of distilled water, soaking in a water bath at 75-85 ℃ for 6h, filtering, and fixing the volume of the filtrate to 250mL to obtain a radix paeoniae aqueous extract with the concentration of 0.08 g/mL;
the extraction method of the Clinopodium chinense comprises the following steps: 10.0g of Clinopodium chinense Mill powder, adding 200mL of 80% ethanol 200mL, and ultrasonically extracting for 30min under the ultrasonic conditions: 500W, 40 Hz; filtering to obtain Clinopodium polycephalum herba Clinopodii ethanol extract with concentration of 0.05 g/mL;
the extraction method of the citronella comprises the following steps: crushing 10g of citronella by using a traditional Chinese medicine crusher, adding 100mL of distilled water, soaking for 48h at room temperature, and then filtering by using a 0.22 mu m microporous filter membrane to obtain a citronella water extract with the concentration of 0.1 g/mL.
The bacillus is cultured by adopting a conventional PDA culture medium, and the culture method comprises the following steps: A. and (3) activation: inoculating bacillus into a PDA plate culture medium, culturing at 28 ℃ for 12-16 hours, and repeatedly carrying out passage for 2 times; B. culturing: inoculating activated bacillus into a PDB liquid culture medium, culturing at 150r/min for 36h at 28 ℃; C. centrifugal separation: centrifuging Bacillus culture solution 7000r/min for 10min, discarding supernatant, washing with sterile water for 3 times, and adjusting concentration of hemocyte counting plate to 1 × 106-1×108The strain suspension per mL;
the yeast is cultured by adopting a conventional NYDA culture medium, and the specific culture method comprises the following steps: a. and (3) activation: inoculating yeast into NYDA plate culture medium, culturing at 30 deg.C for 16-20 hr, and repeatedly subculturing for 2 times; b. culturing: inoculating activated yeast into NYDB liquid culture medium, culturing at 150r/min at 30 deg.C for 22 hr; c. centrifugal separation: centrifuging yeast culture solution 7000r/min for 10min, discarding supernatant, washing with sterile water for 3 times, and adjusting concentration of hemocyte counting plate to 1 × 106-1×109And (4) preparing bacterial suspension per mL.
The application method of the prepared microbial and plant source composite fruit and vegetable fresh-keeping agent comprises the following steps: the two-step spraying method comprises the following steps: spraying the component A, and spraying in the second step when the air drying degree of the surfaces of the fruits and the vegetables reaches 80%; the second step is that: spraying the component B, and picking and storing the fruits and vegetables after the fruits and vegetables are completely aired; wherein: the film forming thickness of the component A is 2-25 mu m, the film forming thickness of the component B is 3-25 mu m, and the total thickness is 5-50 mu m.
Among the strains, the bacillus amyloliquefaciens Lh-1 is disclosed as having the application number of 201410735508.6, the application date of 2014-12-08 and the publication (announcement) number of 104531559A, and the bacillus amyloliquefaciens Lh-1 disclosed in the invention patent of the bacillus amyloliquefaciens Lh-1 and the application thereof is separated from the test field soil of Shanxi province agricultural academy of sciences, and the preservation number of the bacillus amyloliquefaciens Lh-1 in the China general microbiological culture Collection center is CGMCC No. 8548. The address is as follows: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; the Candida syndiotactic and Cryptococcus laurentii are purchased from China general microbiological culture Collection center (CGMCC), and the specifications of the Candida syndiotactic and Cryptococcus laurentii conform to the national industrial standard.
The bacillus amyloliquefaciens Lh-1 used in the invention has good bacteriostatic action, wide bacteriostatic spectrum and stable bacteriostatic effect, and is used for animal feed additives. The candida species and cryptococcus laurentii are yeasts with bacteriostatic effects, are commonly used for postharvest control of fruits, are reported at home and abroad, can be purchased from the market, and meet the national industrial standard in specification. Propolis contains various components, and flavonoid compounds such as rutin, quercetin, galangin, caffeic acid, alpha-catechin and the like have strong antioxidation, and particularly, the flavonoid compounds in the propolis have the effects of antibiosis, antioxidation and the like. The cortex moutan extract can inhibit the growth of pathogenic bacteria of gray mold, early blight, etc.; the citrus peel extract has a strong antibacterial effect on penicillium; rabdosia rubescens has antibacterial and antioxidant effects, and can delay fruit onset and reduce morbidity; chemical components with antibacterial activity and antioxidant activity in radix Zanthoxyli extract, Lycium ruthenicum Murr extract, radix Paeoniae extract and Clinopodium polycephalum herba Clinopodii extract; the citronella extract has good antibacterial effect on rotten germs.
According to the invention, the mixture of the component A microbial fermentation liquor and the propolis extract forms a first layer of protective film to inhibit growth and propagation of microbes attached to the surfaces of fruits and vegetables in the growth process, and the mixture of the component B plant source active ingredient mixed liquor and the film forming liquor forms a layer of film on the surfaces of the fruits and vegetables at first, so that the respiratory strength of the fruits and vegetables is inhibited, the water evaporation in the fruits and vegetables is reduced, and the hardness of the fruits and vegetables is kept, thereby avoiding the fruits and vegetables from being damaged mechanically and preventing the fruits and vegetables from being infected by harmful microbes to a certain extent. Meanwhile, the plant source effective components have good antibacterial effect, a second layer of protective film is formed, growth and propagation of microorganisms attached to the surface after picking are inhibited, evaporation of water of the fruits and vegetables is inhibited under the combined action of the plant source effective components and the protective film, damage of harmful substances is reduced, growth and propagation of harmful microorganisms are inhibited, the fresh-keeping function of the fruits and vegetables is achieved, safety, high efficiency, simplicity in operation and low cost are achieved, rotting and water loss of the stored fruits and vegetables are remarkably reduced, the shelf life is prolonged, and the service life of the fruits and vegetables is prolonged.
Detailed Description
Example 1: a microbial and plant source composite fruit and vegetable fresh-keeping agent is prepared from a component A and a component B, wherein the component A is composed of a microbial fermentation liquid accounting for 62% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent and a propolis extracting solution accounting for 5% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent, and the microorganisms are as follows: the concentration is 1X 108Bacillus at a concentration of 1X 10/mL6Mixing yeast strains per mL at a ratio of 1:5, wherein the Bacillus is Bacillus amyloliquefaciens Lh-1 (Bacillus amyloliquefaciens L)Bacillus amyloliquefaciens Lh-1) The yeast is Candida intermedia mixed at a ratio of 1:1（Candida intermedia）Cryptococcus Helioticus Hedera(Cryptococcus laurentii)(ii) a The propolis extract is prepared by using ethanol as an extraction solvent and adopting a conventional extraction method, wherein the concentration of the propolis extract is 50 g/L;
the component B consists of a plant source effective component mixed solution accounting for 30% of the volume of the microorganism and plant source composite fruit and vegetable antistaling agent and a film forming solution accounting for 3% of the volume of the microorganism and plant source composite fruit and vegetable antistaling agent, wherein the plant source effective component mixed solution is formed by mixing a peony bark water extract with the concentration of 0.08 g/mL, a citrus peel ethanol extract with the concentration of 0.03 g/mL, a rabdosia rubescens ethanol extract with the concentration of 0.04g/mL, a radix zanthoxyli water extract with the concentration of 0.08 g/mL, a lycium ruthenicum ethanol extract with the concentration of 0.04g/mL, a peony water extract with the concentration of 0.08 g/mL, a Clinopodium chinense ethanol extract with the concentration of 0.05 g/mL and a citronella water extract with the concentration of 0.1 g/mL, and the volume ratios of the extracts to the plant source effective component mixed solution are as follows: 6% of peony bark water extract, 20% of citrus peel ethanol extract, 18% of rabdosia rubescens ethanol extract, 1% of radix zanthoxyli water extract, 25% of lycium ruthenicum ethanol extract, 2% of peony water extract, 10% of calamus margaritae ethanol extract and 18% of rhizoma imperatae water extract; the film forming liquid is formed by mixing 2% of konjac glucomannan and 0.6% of chitosan according to the volume ratio of 1: 1.
The extraction method of the plant source effective components comprises the following steps: the extraction method of the tree peony bark comprises the following steps: putting 20g of tree peony bark into a 500mL round-bottom flask, adding 300mL of distilled water, soaking in a 75-85 ℃ water bath for 6h, filtering, and fixing the volume of the filtrate to 250mL to obtain 0.08 g/mL tree peony bark water extract;
the extraction method of the orange peel comprises the following steps: crushing 10g of orange peel, soaking in 300mL of 75% ethanol, and then performing microwave extraction for 7min at the microwave power of 900W and the temperature of 75 ℃ in a material-liquid ratio of 1:30 (g: mL); decolorizing the extractive solution with 10% active carbon, and filtering to obtain 0.03 g/mL ethanol extractive solution of pericarpium Citri Tangerinae;
the extraction method of rabdosia rubescens comprises the following steps: pulverizing 10g Rabdosia rubescens with a traditional Chinese medicine pulverizer, soaking in 250mL 85% ethanol for 1h, performing microwave treatment with the microwave power of 90W for 80s, transferring to a reflux device for reflux extraction for 30min, and decolorizing and filtering the extract with 10% active carbon to obtain Rabdosia rubescens ethanol extract with the concentration of 0.04 g/mL;
the extraction method of radix zanthoxyli comprises the following steps: putting 20g radix zanthoxyli into a 500mL round-bottom flask, adding 300mL of distilled water, soaking in a 75-85 ℃ water bath for 6h, filtering, and fixing the volume of the filtrate to 250mL to obtain 0.08 g/mL radix zanthoxyli water extract;
the extraction method of the lycium ruthenicum comprises the following steps: adding 2g of lycium ruthenicum mill into 30mL of 50% ethanol, leaching for 1.5h in a 50 ℃ water bath, filtering, adding 20mL of 50% ethanol into the residue, leaching for 1h, filtering, combining the filtrates, and fixing the volume to 50 mL; decolorizing the extractive solution with active carbon 10% of the extract, and filtering to obtain 0.04g/mL Lycium ruthenicum Murr ethanol extractive solution;
the extraction method of the peony comprises the following steps: putting 20g of radix paeoniae into a 500mL round-bottom flask, adding 300mL of distilled water, soaking in a water bath at 75-85 ℃ for 6h, filtering, and fixing the volume of the filtrate to 250mL to obtain a radix paeoniae aqueous extract with the concentration of 0.08 g/mL;
the extraction method of the Clinopodium chinense comprises the following steps: 10.0g of Clinopodium chinense Mill powder, adding 200mL of 80% ethanol 200mL, and ultrasonically extracting for 30min under the ultrasonic conditions: 500W, 40 Hz; filtering to obtain Clinopodium polycephalum herba Clinopodii ethanol extract with concentration of 0.05 g/mL;
the extraction method of the citronella comprises the following steps: crushing 10g of citronella by using a traditional Chinese medicine crusher, adding 100mL of distilled water, soaking for 48h at room temperature, and then filtering by using a 0.22 mu m microporous filter membrane to obtain a citronella water extract with the concentration of 0.1 g/mL.
Among the strains, the bacillus amyloliquefaciens Lh-1 is the Bacillus amyloliquefaciens Lh-1 with the application number of 201410735508.6, the application date of 2014-12-08 and the publication (announcement) number of 104531559A, and the bacillus amyloliquefaciens Lh-1 is separated from the test field soil of Shanxi province agricultural academy of sciences, and the preservation number of the bacillus amyloliquefaciens Lh-1 in the China general microbiological culture center of the culture Collection Committee is CGMCC No. 8548. The address is as follows: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; the Candida syndiotactic and Cryptococcus laurentii are purchased from China general microbiological culture Collection center (CGMCC), and the specifications of the Candida syndiotactic and Cryptococcus laurentii conform to the national industrial standard.
Wherein: the bacillus amyloliquefaciens Lh-1 is preserved at 4 ℃ by adopting a conventional PDA slant culture medium which comprises the following components: peeled potato 200g, sucrose 20g, agar 20g, water 1000mL, natural pH. The culture method comprises the following steps: and (3) activation: inoculating Bacillus amyloliquefaciens Lh-1 into PDA plate culture medium, culturing at 28 deg.C for 12-16 hr, and repeating for passage 2 times. Culturing: the PDB liquid culture medium (200 g of peeled potato, 20g of cane sugar, 1000mL of water and natural pH) is inoculated with activated bacillus amyloliquefaciens Lh-1 by an inoculating loop and cultured for 36h at the temperature of 28 ℃ at 150 r/min. Centrifugal separation: centrifuging culture solution 7000r/min of Bacillus amyloliquefaciens Lh-1 for 10min, discarding supernatant, washing with sterile water for 3 times, and adjusting to concentration of 1 × 10 with blood counting plate8Per mL in bacterial suspension.
The Candida syndiotactic and Cryptococcus laurentii are purchased from China general microbiological culture Collection center (CGMCC), and are preserved at 4 ℃ by adopting a conventional NYDA slant culture medium, wherein the NYDA culture medium is as follows: 0.8% of beef extract, 0.5% of yeast extract, 1% of glucose and 2% of agar. The culture method comprises the following steps: and (3) activation: inoculating Candida syndiotactic and Cryptococcus laurentii to NYDA plate culture medium, culturing at 30 deg.C for 16-20 hr, and repeating passage for 2 times. Culturing: NYDB liquid culture medium (glucose 20g dissolved in 100mL distilled water, yeast extract 5g and beef extract 8g dissolved in 900mL distilled water, two bottles of solution sterilized by high pressure steam at 121 deg.C for 20min, when using, the two bottles of solution are mixed uniformly), inoculating activated Candida syndiotactic and Cryptococcus laurentii with inoculating loop respectively in NYDB liquid culture medium, culturing at 150r/min and 30 deg.C for 22 hr. Centrifugal separation: centrifuging culture solution of Candida syndiotactic and Cryptococcus laurentii at 7000r/min for 10min, discarding supernatant, washing with sterile water for 3 times, and adjusting concentration to 1 × 10 with blood counting plate6one/mL yeast suspension.
The application method of the prepared microbial and plant source composite fruit and vegetable fresh-keeping agent comprises the following steps: the two-step spraying method comprises the following steps: spraying the component A, and spraying in the second step when the air drying degree of the surfaces of the fruits and the vegetables reaches 80%; the second step is that: and spraying the component B, and picking and storing the fruits and vegetables after the fruits and vegetables are completely aired.
Strawberry plants with consistent growth and similar maturity are selected for experiment, and a control group and three treatment groups are set. The control group is that the strawberry fruits are directly picked and boxed after being ripe, and are refrigerated at low temperature for 10 days; treatment group 1: spraying the component A when 75% of strawberry fruits are ripe, wherein the film forming thickness of the component A is 2 mu m, picking and boxing the strawberry fruits when the surfaces of the strawberries are naturally air-dried, and refrigerating the strawberry fruits at a low temperature for 10 days; treatment group 2: spraying the component B when 75% of strawberry fruits are ripe, wherein the film forming thickness of the component B is 3 mu m, picking and boxing the strawberry fruits when the surfaces of the strawberries are naturally air-dried, and refrigerating the strawberry fruits at a low temperature for 10 days; treatment group 3: spraying the component A when the strawberry fruits are 75% ripe, wherein the film forming thickness of the component A is 2 mu m, spraying the component B when the surfaces of the strawberries are naturally air-dried, the film forming thickness of the component B is 3 mu m, the total thickness is 5 mu m, picking and boxing are carried out after the strawberry fruits are air-dried again, and the strawberry fruits are refrigerated at a low temperature for 10 d. The results are shown in Table 1. The result shows that the component A and the component B are used together, the good fruit rate reaches 94.7 percent after the low-temperature refrigeration is carried out for 10 days, the fresh-keeping effect of the strawberries is obviously improved, the weight loss rate is only 2.0 percent, and the water loss rate of the strawberries is obviously reduced.
TABLE 1
| Good fruit rate | Weight loss ratio | |
| Control group | 80.3% | 7.4% |
| Treatment group 1 (A) | 90.6% | 2.5% |
| Treatment group 2 (B) | 91.0% | 2.6% |
| Treatment group 3 (A + B) | 94.7% | 2.0% |
Example 2: a microbial and plant source composite fruit and vegetable fresh-keeping agent is prepared from a component A and a component B, wherein the component A is prepared by taking the microbial and plant source composite fruit and vegetable fresh-keeping agentMicroorganism fermentation liquor accounting for 32% of the volume of the fruit and vegetable composite fruit and vegetable preservative and propolis extract accounting for 8% of the volume of the fruit and vegetable composite preservative, wherein the microorganisms are as follows: the concentration is 1X 107Bacillus at a concentration of 1X 10/mL7Mixing yeast strains per mL at a ratio of 1:10, wherein the Bacillus is Bacillus amyloliquefaciens Lh-1 (Bacillus amyloliquefaciens L)Bacillus amyloliquefaciens Lh-1) The yeast is Candida intermedia mixed at a ratio of 1:1（Candida intermedia）Cryptococcus Helioticus Hedera(Cryptococcus laurentii)(ii) a The propolis extract is prepared by using ethanol as an extraction solvent by a conventional extraction method, and the concentration of the propolis extract is 40 g/L;
the component B consists of a plant source effective component mixed solution accounting for 50% of the volume of the microorganism and plant source composite fruit and vegetable antistaling agent and a film forming solution accounting for 10% of the volume of the microorganism and plant source composite fruit and vegetable antistaling agent, wherein the plant source effective component mixed solution is formed by mixing a peony bark water extract with the concentration of 0.08 g/mL, a citrus peel ethanol extract with the concentration of 0.03 g/mL, a rabdosia rubescens ethanol extract with the concentration of 0.04g/mL, a radix zanthoxyli water extract with the concentration of 0.08 g/mL, a lycium ruthenicum ethanol extract with the concentration of 0.04g/mL, a peony water extract with the concentration of 0.08 g/mL, a Clinopodium chinense ethanol extract with the concentration of 0.05 g/mL and a citronella water extract with the concentration of 0.1 g/mL, and the volume ratios of the extracts to the plant source effective component mixed solution are as follows: 3% of peony bark water extract, 18% of citrus peel ethanol extract, 16% of rabdosia rubescens ethanol extract, 7% of radix zanthoxyli water extract, 35% of lycium ruthenicum ethanol extract, 0.5% of peony water extract, 0.5% of calamus ethanol extract and 20% of rhizoma imperatae water extract; the film forming liquid is formed by mixing 1.8 percent of konjac glucomannan and 0.6 percent of chitosan according to the mass percent ratio of 1: 1.
The method for extracting the plant-derived active ingredients is the same as that described in example 1. The method of culturing the microorganism was the same as described in example 1.
Tomato plants with consistent growth and similar maturity are selected for experiment, and a control group and three treatment groups are set. The control group is obtained by directly picking the ripe tomato fruits and storing the tomato fruits at normal temperature for 15 days; treatment group 1: spraying the component A when 80% of the surfaces of the tomatoes turn red, wherein the film forming thickness of the component A is 15 mu m, picking the tomatoes when the surfaces of the tomatoes are naturally air-dried, and storing the tomatoes at normal temperature for 15 d; treatment group 2: spraying the component B when 80% of the surfaces of the tomatoes turn red, wherein the film forming thickness of the component B is 15 mu m, picking the tomatoes when the surfaces of the tomatoes are naturally air-dried, and storing the tomatoes at normal temperature for 15 d; treatment group 3: spraying the component A when the surface of the tomato turns red by 80%, wherein the film forming thickness of the component A is 15 mu m, spraying the component B after the surface of the tomato is naturally air-dried, the film forming thickness of the component B is 15 mu m, the total thickness is 30 mu m, picking after the tomato is air-dried again, and storing for 15d at normal temperature. The results are shown in table 2 and show that: the component A and the component B are used together, the rotting rate is reduced to 20.6 percent after the tomato is stored for 15 days at normal temperature, the fresh-keeping effect of the tomato is obviously improved, the weight loss rate is only 2.0 percent, and the water loss rate of the tomato is obviously reduced.
TABLE 2
| Rate of decay | Weight loss ratio | |
| Control group | 95.1% | 10.9% |
| Treatment group 1 (A) | 53.9% | 3.3% |
| Treatment group 2 (B) | 56.4% | 3.6% |
| Treatment ofGroup 3 (A + B) | 20.6% | 2.0% |
Example 3: a microbial and plant source composite fruit and vegetable fresh-keeping agent is prepared from a component A and a component B, wherein the component A is composed of microbial fermentation liquor accounting for 70% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent and propolis extract accounting for 3% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent, and the microorganisms are as follows: the concentration is 1X 106Bacillus at a concentration of 1X 10/mL9Mixing yeast strains per mL at a ratio of 1:20, wherein the Bacillus is Bacillus amyloliquefaciens Lh-1 (Bacillus amyloliquefaciens L: (L))Bacillus amyloliquefaciens Lh-1) The yeast is Candida intermedia mixed at a ratio of 1:1（Candida intermedia）Cryptococcus Helioticus Hedera(Cryptococcus laurentii)(ii) a The propolis extract is prepared by using ethanol as an extraction solvent and adopting a conventional extraction method, wherein the concentration of the propolis extract is 5 g/L;
the component B consists of a plant source effective component mixed solution accounting for 21% of the volume of the microorganism and plant source composite fruit and vegetable antistaling agent and a film forming solution accounting for 6% of the volume of the microorganism and plant source composite fruit and vegetable antistaling agent, wherein the plant source effective component mixed solution is prepared by mixing a peony bark water extract with the concentration of 0.08 g/mL, a citrus peel ethanol extract with the concentration of 0.03 g/mL, a rabdosia rubescens ethanol extract with the concentration of 0.04g/mL, a radix zanthoxyli water extract with the concentration of 0.08 g/mL, a lycium ruthenicum ethanol extract with the concentration of 0.04g/mL, a peony water extract with the concentration of 0.08 g/mL, a Clinopodium chinense ethanol extract with the concentration of 0.05 g/mL and a citronella water extract with the concentration of 0.1 g/mL, and the volume ratios of the extracts to the plant source effective component mixed solution are as follows: 30% of peony bark water extract, 15% of citrus peel ethanol extract, 2% of rabdosia rubescens ethanol extract, 10% of radix zanthoxyli water extract, 5% of lycium ruthenicum ethanol extract, 15% of peony water extract, 8% of caladium chinense ethanol extract and 15% of rhizoma imperatae water extract; the film forming liquid is formed by mixing 1.8 percent of konjac glucomannan and 0.6 percent of chitosan according to the mass percent ratio of 1: 1.
The method for extracting the plant-derived active ingredients is the same as that described in example 1. The method of culturing the microorganism was the same as described in example 1.
Cucumber plants with consistent growth and similar maturity are selected for experiment, and a control group and three treatment groups are set. The control group is obtained by directly picking cucumber fruits after they are ripe, and storing at normal temperature for 8 days; treatment group 1: spraying the component A when the cucumber fruits are 78% ripe, wherein the film forming thickness of the component A is 25 mu m, picking the cucumber fruits when the surfaces of the cucumber fruits are naturally air-dried, and storing the cucumber fruits for 8d at normal temperature; treatment group 2: spraying the component B when the cucumber fruits are 78% ripe, wherein the film forming thickness of the component B is 25 mu m, picking the cucumber fruits when the surfaces of the cucumber fruits are naturally air-dried, and storing the cucumber fruits for 8d at normal temperature; treatment group 3: spraying the component A when the cucumber fruits are 78% ripe, wherein the film forming thickness of the component A is 25 mu m, and then spraying the component B after the surfaces of the cucumbers are naturally air-dried, wherein the film forming thickness of the component B is 25 mu m, and the total thickness is 50 mu m. Picking after air drying again, and storing at normal temperature for 8 d. The results are shown in Table 3 and show that: the component A and the component B are used together, the rotting rate of the cucumber is reduced to 0.5 percent after the cucumber is refrigerated at low temperature for 8 days, the preservation effect of the cucumber is obviously improved, the weight loss rate is only 1.0 percent, and the water loss rate of the cucumber is obviously reduced.
TABLE 3
| Rate of decay | Weight loss ratio | |
| Control group | 30.2% | 3.9% |
| Treatment group 1 (A) | 3.2% | 2.3% |
| Treatment group 2 (B) | 2.9% | 2.6% |
| Treatment group 3 (A + B) | 0.5% | 1.0% |
Example 4: a microbial and plant source composite fruit and vegetable fresh-keeping agent is prepared from a component A and a component B, wherein the component A is composed of microbial fermentation liquor accounting for 70% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent and propolis extract accounting for 8% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent, and the microorganisms are as follows: the concentration is 1X 106Bacillus at a concentration of 1X 10/mL8Mixing yeast strains per mL at a ratio of 1:15, wherein the Bacillus is Bacillus amyloliquefaciens Lh-1 (Bacillus amyloliquefaciens L: (L))Bacillus amyloliquefaciens Lh-1) The yeast is Candida intermedia mixed at a ratio of 1:1（Candida intermedia）Cryptococcus Helioticus Hedera(Cryptococcus laurentii)(ii) a The propolis extract is prepared by using ethanol as an extraction solvent and adopting a conventional extraction method, wherein the concentration of the propolis extract is 25 g/L;
the component B consists of a plant source effective component mixed solution accounting for 12% of the volume of the microorganism and plant source composite fruit and vegetable antistaling agent and a film forming solution accounting for 10% of the volume of the microorganism and plant source composite fruit and vegetable antistaling agent, wherein the plant source effective component mixed solution is formed by mixing a peony bark water extract with the concentration of 0.08 g/mL, a citrus peel ethanol extract with the concentration of 0.03 g/mL, a rabdosia rubescens ethanol extract with the concentration of 0.04g/mL, a radix zanthoxyli water extract with the concentration of 0.08 g/mL, a lycium ruthenicum ethanol extract with the concentration of 0.04g/mL, a peony water extract with the concentration of 0.08 g/mL, a Clinopodium chinense ethanol extract with the concentration of 0.05 g/mL and a citronella water extract with the concentration of 0.1 g/mL, and the volume ratios of the extracts to the plant source effective component mixed solution are as follows: 30% of peony bark water extract, 1% of citrus peel ethanol extract, 18% of rabdosia rubescens ethanol extract, 10% of radix zanthoxyli water extract, 20% of lycium ruthenicum ethanol extract, 15% of peony water extract, 5.5% of calamus chinensis ethanol extract and 0.5% of rhizoma imperatae water extract; the film forming liquid is formed by mixing 0.5 percent of konjac glucomannan and 0.6 percent of chitosan according to the mass percent ratio of 1: 1.
The method for extracting the plant-derived active ingredients is the same as that described in example 1. The method of culturing the microorganism was the same as described in example 1.
Apple plants with consistent growth and similar maturity are selected for experiment, and a control group and three treatment groups are set. The control group is obtained by directly picking apples after the apples are ripe, and storing the apples at normal temperature for 30 d; treatment group 1: spraying the component A when 76% of apple fruits are ripe, wherein the film forming thickness of the component A is 8 mu m, picking the apples when the surfaces of the apples are naturally air-dried, and storing the apples for 30d at normal temperature; treatment group 2: spraying the component B when 76% of the apple fruits are ripe, wherein the film forming thickness of the component B is 8 mu m, picking the apple fruits when the surfaces of the apples are naturally air-dried, and storing the apple fruits for 30d at normal temperature; treatment group 3: spraying the component A when 76% of apple fruits are ripe, wherein the film forming thickness of the component A is 8 mu m, spraying the component B after the surfaces of the apples are naturally air-dried, the film forming thickness of the component B is 8 mu m, the total thickness is 16 mu m, picking is carried out after the apples are air-dried again, and the apples are stored at normal temperature for 30 d. The results are shown in Table 4 and show that: the component A and the component B are used together, the rotting rate of the apples is reduced to 2.5 percent after the apples are stored for 30 days at normal temperature, the fresh-keeping effect of the apples is obviously improved, the weight loss rate is only 0.8 percent, and the water loss rate of the apples is obviously reduced.
TABLE 4
| Rate of decay | Weight loss ratio | |
| Control group | 40.8% | 10.8% |
| Treatment group 1 (A) | 10.7% | 5.7% |
| Treatment group 2 (B) | 9.8% | 4.9% |
| Treatment group 3 (A + B) | 2.5% | 0.8% |
Claims (3)
1. A microbial and plant source composite fruit and vegetable fresh-keeping agent is characterized in that: the microbial and plant source composite fruit and vegetable fresh-keeping agent is prepared from a component A and a component B, wherein the component A is composed of microbial fermentation liquor accounting for 32-70% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent and propolis extract accounting for 3-8% of the volume of the microbial and plant source composite fruit and vegetable fresh-keeping agent, and the microorganisms are as follows: the concentration is 1X 106-1×108Bacillus at a concentration of 1X 10/mL6-1×109Mixing yeast strains in a volume ratio of 1:5-1:20, wherein the bacillus is bacillus amyloliquefaciens Lh-1 (A)Bacillus amyloliquefaciens Lh-1) The yeast is Candida intermedia mixed in a volume ratio of 1:1（Candida intermedia）Cryptococcus Helioticus Hedera(Cryptococcus laurentii)(ii) a The propolis extractive solution is prepared by extracting with ethanol by conventional methodThe prepared concentration is 5-50 g/L;
the component B is formed by mixing plant source active ingredient mixed liquor accounting for 12-50% of the volume of the microorganism and plant source composite fruit and vegetable fresh-keeping agent with film forming liquid accounting for 3-10% of the volume of the microorganism and plant source composite fruit and vegetable fresh-keeping agent, the mixed solution of the plant source active ingredients is prepared by mixing 0.08 g/mL peony bark water extract, 0.03 g/mL citrus peel ethanol extract, 0.04g/mL rabdosia rubescens ethanol extract, 0.08 g/mL radix zanthoxyli water extract, 0.04g/mL lycium ruthenicum ethanol extract, 0.08 g/mL peony root water extract, 0.05 g/mL Clinopodium polycephalum herba Clinopodii ethanol extract and 0.1 g/mL citronella water extract, wherein the volume ratio of the extracts to the mixed solution of the plant source active ingredients is as follows: 3-30% of peony bark water extract, 1-20% of citrus peel ethanol extract, 2-18% of rabdosia rubescens ethanol extract, 1-10% of radix zanthoxyli water extract, 5-35% of lycium ruthenicum ethanol extract, 0.5-15% of peony water extract, 0.5-10% of Clinopodium chinense ethanol extract and 0.5-20% of rhizoma imperatae water extract; the film forming liquid is formed by mixing 0.5 to 2 mass percent of konjac glucomannan and 0.6 mass percent of chitosan according to the volume ratio of 1: 1;
the application method of the prepared microbial and plant source composite fruit and vegetable fresh-keeping agent comprises the following steps: the two-step spraying method comprises the following steps: firstly, spraying the component A, and carrying out second spraying when the air drying degree of the surfaces of the fruits and the vegetables reaches 75-80%; the second step is that: spraying the component B, and picking and storing the fruits and vegetables after the fruits and vegetables are completely aired; wherein: the film forming thickness of the component A is 2-25 mu m, the film forming thickness of the component B is 3-25 mu m, and the total thickness is 5-50 mu m.
2. The microbial and plant source composite fruit and vegetable fresh-keeping agent as claimed in claim 1, which is characterized in that: the extraction method of the plant source effective components comprises the following steps: the extraction method of the tree peony bark comprises the following steps: putting 20g of tree peony bark into a 500mL round-bottom flask, adding 300mL of distilled water, soaking in a 75-85 ℃ water bath for 6h, filtering, and fixing the volume of the filtrate to 250mL to obtain 0.08 g/mL tree peony bark water extract;
the extraction method of the orange peel comprises the following steps: crushing 10g of orange peel, soaking in 300mL of 75% ethanol, and then performing microwave extraction for 7min at the microwave power of 900W and the temperature of 75 ℃ in a material-liquid ratio of 1:30 (g: mL); decolorizing the extractive solution with 10% active carbon, and filtering to obtain 0.03 g/mL ethanol extractive solution of pericarpium Citri Tangerinae;
the extraction method of rabdosia rubescens comprises the following steps: pulverizing 10g Rabdosia rubescens with a traditional Chinese medicine pulverizer, soaking in 250mL 85% ethanol for 1h, performing microwave treatment with the microwave power of 90W for 80s, transferring to a reflux device for reflux extraction for 30min, and decolorizing and filtering the extract with 10% active carbon to obtain Rabdosia rubescens ethanol extract with the concentration of 0.04 g/mL;
the extraction method of radix zanthoxyli comprises the following steps: putting 20g radix zanthoxyli into a 500mL round-bottom flask, adding 300mL of distilled water, soaking in a 75-85 ℃ water bath for 6h, filtering, and fixing the volume of the filtrate to 250mL to obtain 0.08 g/mL radix zanthoxyli water extract;
the extraction method of the lycium ruthenicum comprises the following steps: adding 2g of lycium ruthenicum mill into 30mL of 50% ethanol, leaching for 1.5h in a 50 ℃ water bath, filtering, adding 20mL of 50% ethanol into the residue, leaching for 1h, filtering, combining the filtrates, and fixing the volume to 50 mL; decolorizing the extractive solution with active carbon 10% of the extract, and filtering to obtain 0.04g/mL Lycium ruthenicum Murr ethanol extractive solution;
the extraction method of the peony comprises the following steps: putting 20g of radix paeoniae into a 500mL round-bottom flask, adding 300mL of distilled water, soaking in a water bath at 75-85 ℃ for 6h, filtering, and fixing the volume of the filtrate to 250mL to obtain a radix paeoniae aqueous extract with the concentration of 0.08 g/mL;
the extraction method of the Clinopodium chinense comprises the following steps: 10.0g of Clinopodium chinense Mill powder, adding 200mL of 80% ethanol 200mL, and ultrasonically extracting for 30min under the ultrasonic conditions: 500W, 40 Hz; filtering to obtain Clinopodium polycephalum herba Clinopodii ethanol extract with concentration of 0.05 g/mL;
the extraction method of the citronella comprises the following steps: crushing 10g of citronella by using a traditional Chinese medicine crusher, adding 100mL of distilled water, soaking for 48h at room temperature, and then filtering by using a 0.22 mu m microporous filter membrane to obtain a citronella water extract with the concentration of 0.1 g/mL.
3. The microbial and plant source composite fruit and vegetable fresh-keeping agent as claimed in claim 1, which is characterized in that: the bacillus is cultured by adopting a conventional PDA culture mediumThe method comprises the following steps: A. and (3) activation: inoculating bacillus into a PDA plate culture medium, culturing at 28 ℃ for 12-16 hours, and repeatedly carrying out passage for 2 times; B. culturing: inoculating activated bacillus into a PDB liquid culture medium, culturing at 150r/min for 36h at 28 ℃; C. centrifugal separation: centrifuging Bacillus culture solution 7000r/min for 10min, discarding supernatant, washing with sterile water for 3 times, and adjusting concentration of hemocyte counting plate to 1 × 106-1×108The strain suspension per mL;
the yeast is cultured by adopting a conventional NYDA culture medium, and the specific culture method comprises the following steps: a. and (3) activation: inoculating yeast into NYDA plate culture medium, culturing at 30 deg.C for 16-20 hr, and repeatedly subculturing for 2 times; b. culturing: inoculating activated yeast into NYDB liquid culture medium, culturing at 150r/min at 30 deg.C for 22 hr; c. centrifugal separation: centrifuging yeast culture solution 7000r/min for 10min, discarding supernatant, washing with sterile water for 3 times, and adjusting concentration of hemocyte counting plate to 1 × 106-1×109And (4) preparing bacterial suspension per mL.
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