CN106070586B - Compound biological preservative and application thereof - Google Patents
Compound biological preservative and application thereof Download PDFInfo
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- CN106070586B CN106070586B CN201610438169.4A CN201610438169A CN106070586B CN 106070586 B CN106070586 B CN 106070586B CN 201610438169 A CN201610438169 A CN 201610438169A CN 106070586 B CN106070586 B CN 106070586B
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- jerusalem artichoke
- preservation
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- alcohol extract
- biological preservative
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/16—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
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- 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 compound biological preservative and application thereof. The compound biological preservative consists of the grignard lactoglobulin, the jerusalem artichoke leaf alcohol extract and the water-soluble chitosan. The Lactococcus garvieae is prepared by fermenting and extracting Lactococcus garvieae SY-1, the strain is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, the preservation number is CGMCC No.9485, and the preservation date is 2014-7 and 28. The jerusalem artichoke leaf alcohol extract is prepared by an ultrasonic extraction method. The preservative is applied to the preservation of small berries, can form a layer of safe, nontoxic and edible transparent film on the surfaces of the fruits, can effectively maintain the color and the nutritional ingredients of the fruits and remarkably prolong the shelf lives of various small berries by combining the preservation of a conventional refrigerator.
Description
Technical Field
The invention particularly relates to a compound biological preservative and a method for applying the compound biological preservative to preservation of small berries, and belongs to the technical field of storage and preservation of agricultural products.
Background
Small berries generally refer to a class of fruit trees with smaller and juicy fruits, and mainly include blueberries, raspberries, blackcurrants, red currants, cranberries, blackberries, waxberries, figs and the like. The fruit is bright, the pulp is juicy, the flavor is unique, the fragrance is strong, the taste is sweet, sour and tasty, the fruit is rich in multiple vitamins, trace elements such as calcium, phosphorus and zinc, organic acid, anthocyanin, flavonoid and other active substances, the fruit wine has the pharmacological health-care effects of resisting oxidation and aging, protecting the heart and blood vessels, resisting cancer, strengthening the spleen and stomach, moistening the lung and promoting the secretion of saliva or body fluid, nourishing and enriching the blood, promoting digestion, enhancing the immunity and the like, and can effectively prevent the occurrence and development of multiple diseases such as anemia, hypertension, hyperlipidemia, cancer, heart disease and the like. Can be widely applied to the fields of food, medicine, health care products and the like, and is in short supply in the international market. However, the small berries have thin peels and soft tissues, are not resistant to storage and transportation, and are easy to rot and deteriorate, so that the fruits are usually seriously out of stock and rotten in fields and stalls, and serious economic loss is often caused to fruit growers.
Generally, postharvest respiration, water transpiration and bacterial and mold invasion are main reasons causing the small berries to rot and deteriorate. At present, the method for preserving small berries at home and abroad comprises low-temperature preservation, modified atmosphere preservation, chemical preservative preservation, coating preservation and the like. The chemical preservative and low-temperature box combined means is convenient to use, strong in adaptability and wide in application, but the residue of the chemical preservative can often cause serious consequences such as environmental pollution, food safety, carcinogenesis and teratogenesis, and the like, and the coating preservation can form a protective film on the surface of fruits by virtue of polysaccharide substances such as chitosan and the like, so that the protective effect of the fruit epidermis is enhanced, the breathing effect and water evaporation of air holes are inhibited, and the invasion of microorganisms is inhibited, thereby achieving the purpose of preservation. However, the edible film coating agents in the current market generally have the defects of poor antibacterial effect, unstable film coating, high use cost and the like, so that the development of a more suitable, safe, efficient and edible compound biological preservative is the main research direction in the field of fruit preservation.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects in the prior art, the invention aims to provide the compound biological preservative which can form a protective film on the surface of fruits after being applied to the fruits, is safe, nontoxic and edible, has good preservation effect and can obviously prolong the shelf life of the fruits, particularly small berries.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a compound biological antistaling agent comprises Grignard lactoglobulin, alcohol extract of jerusalem artichoke leaf and water-soluble chitosan.
The mass ratio of the grignard lactoglobulin to the jerusalem artichoke leaf alcohol extract to the water-soluble chitosan is 100: 2-5: 45-65.
The compound biological preservative may or may not contain a solvent.
When the compound biological preservative contains a solvent, the solvent can be water, the concentration of the jerusalem artichoke leaf alcohol extract is 2-5 g/L, and the concentration of the water-soluble chitosan is 45-65 g/L.
The lactococcus garvieae is prepared by fermenting lactococcus garvieae and then extracting, wherein the lactococcus garvieae is lactococcus garvieae SY-1, the lactococcus garvieae is obtained by separating the epidermis of Chinese yam by the inventor of the application and is preserved in the China general microbiological culture Collection center, the preservation number of the strain is CGMCC No.9485, and the preservation date is 2014-7 and 28.
The research of the inventor of the application shows that lactococcus garvieae SY-1 has stronger bacteriocin production activity, the ethanol extract of the jerusalem artichoke leaves also has certain bacterial disease resistance, the combined use of the lactococcus garvieae SY-1 and chitosan can improve the stability of the lactococcus garvieae and obviously expand the antibacterial spectrum of the lactococcus garvieae, the lactococcus garvieae and the chitosan, and the lactococcus garvieae extract has better biocontrol effect on soft rot, gray mold and yeast of small berries, can obviously inhibit the weight loss rate and the respiratory strength of the small berries, and has extremely obvious fresh-keeping effect.
The preparation method of the grignard lactoglobulin comprises the following steps:
(1) activating lactococcus garvieae strain, inoculating in culture medium, culturing to obtain seed liquid;
(2) inoculating the seed liquid into a fermentation culture medium for fermentation culture, and collecting fermentation liquid after the fermentation culture is finished;
(3) and centrifuging the fermentation liquor, taking the supernatant, adjusting the pH value to 2-3 with acid, then precipitating, taking the precipitate, and freeze-drying to obtain the grignard lactoglobulin.
In the step (1), the culture medium is an MRS liquid culture medium, the culture time is 24-36 h, and the culture temperature is 28-30 ℃.
In the step (2), the inoculation amount is 2-5% (v/v); the fermentation medium consists of soybean meal, corn starch, inulin, tomato juice, sodium carboxymethylcellulose and sterile water, and the mass-volume ratio of each component to the sterile water is as follows: 15-20 g/L, 8-12 g/L, 1-2 g/L, 0.1-0.5 g/L and 0.5-1.2 g/L; the temperature of the fermentation culture is 28-30 ℃, and the time is 18-24 h.
In the step (3), the temperature for precipitation is 4-8 ℃ and the time is 12-18 h.
The jerusalem artichoke leaf alcohol extract is a jerusalem artichoke leaf alcohol extract.
The preparation method of the jerusalem artichoke leaf alcohol extract comprises the following steps: putting jerusalem artichoke leaves into 40% -60% alcohol solution according to the material-liquid ratio of 1: 20-25 for ultrasonic extraction, centrifuging after the ultrasonic extraction is finished, taking supernate, and drying to obtain the jerusalem artichoke leaf alcohol extract.
And during ultrasonic extraction, the ultrasonic power is 350-500 w, and the ultrasonic time is 40-50 min.
The preparation method of the compound biological preservative comprises the following steps: directly mixing the grignard lactoglobulin, the jerusalem artichoke leaf alcohol extract and the water-soluble chitosan.
The invention also provides application of the compound biological preservative in fruit preservation.
The fruit is small berry.
The application comprises the following steps: diluting the composite biological preservative by 30-60 times with water, pre-cooling the fruits, then putting the fruits into the diluent of the composite biological preservative for dipping, and then combining with a conventional refrigeration storage preservation method for preservation.
Specifically, the application comprises the following steps: putting the harvested fruits into a cooling room, cooling to 2-5 ℃ by using cold air, cooling at the air speed of 10-15 m/s and the humidity of 80-85% for 1-3 h, soaking in a compound biological preservative diluent for 1-3 min, drying by using cold air, and putting in a cold storage for preservation. Wherein the preservation condition of the cold storage is selected according to the variety of the fruits.
Compared with the prior art, the invention has the beneficial effects that:
1) the compound biological preservative provided by the invention is a brand-new compound biological preservative, has high-efficiency broad-spectrum antibacterial and disease-preventing effects, and can be used for performing combined prevention and control on soft rot, gray mold and the like. The compound biological preservative also has the beneficial effects of convenient use, safety and edible property, remarkably reducing the weight loss rate and the breath intensity of small berries, keeping the commodity of fruits and prolonging the shelf life.
2) The invention adopts the lactococcus garvieae SY-1 to prepare the lactococcus garvieae, the bacteria are preserved in the China general microbiological culture Collection center (the culture collection number is CGMCC No.9485, the preservation date is 7/28/2014), the bacteria have stronger bacteriocin production function and wide antibacterial spectrum, after the bacteria are used with the jerusalem artichoke leaf extract and the water-soluble chitosan, the antibacterial and disease-preventing effect of the bacteria on small berries is obviously enhanced compared with the disease-preventing effect of the preservative prepared from single component, and the formed film is uniform, durable and stable.
3) When the jerusalem artichoke leaf extract is prepared, an ultrasonic extraction technology is adopted, the prepared extract has high content, high biological activity and obvious antibacterial effect, can obviously inhibit the browning of the picked fruit and the deterioration of the flavor, well keeps the nutrition and active ingredients of the fruit and improves the commodity value of the fruit.
4) The compound biological preservative prepared by the invention is safe, efficient and nontoxic, can be eaten, does not cause harm to the environment and human bodies, and has the advantages that the compound biological preservative is incomparable with chemical preservatives and common coating preservatives by combining the conventional refrigeration storage preservation technology.
5) When the compound biological preservative is applied to the preservation of small berries, a layer of transparent and bright edible film is formed on the surface of fruits, so that the respiration intensity of the fruits can be inhibited, the weight loss rate is reduced, the nutrition and active ingredients of the fruits are maintained, the shelf life of the fruits is prolonged, and great economic benefit can be generated.
Drawings
FIG. 1 is a diagram of the SY-1 bacteriostasis of lactococcus garvieae;
FIG. 2 shows the colony morphology of lactococcus garvieae SY-1 on ATB medium;
FIG. 3 is a gram stain of lactococcus garvieae SY-1;
FIG. 4 shows the gene sequence of the SY-116S rRNA of lactococcus garvieae;
FIG. 5 is a graph showing the effect of different preservatives on the decay rate of strawberries;
FIG. 6 shows the effect of different preservatives on the strawberry weight loss rate;
FIG. 7 is a graph of the effect of different preservatives on soluble solids content of strawberries.
Detailed Description
The following detailed description is of embodiments of the present invention, and the embodiments are illustrative, not restrictive, and should not be construed as limiting the scope of the present invention.
Example 1 isolation and identification of lactococcus garvieae SY-1
The Lactococcus garviea SY-1 is preserved in China general microbiological culture Collection center (CGMCC for short, No. 3 of the institute of microbiology, China academy of sciences, Microbiol research institute of China) in 7.28.2014, is named as Lactococcus garviea by classification, and has the strain preservation number of CGMCC No. 9485. The lactococcus garviea SY-1 is separated from the epidermis of Chinese yam.
The ATB culture medium formula comprises the following components: 8g/L glucose, 4g/L yeast extract powder, 10g/L peptone and MgSO4·7H2O 0.2g/L，MnSO4·4H2O0.05 g/L, tomato juice 250mL/L, pH 4.5-4.8, sterilizing at 121 deg.C for 20 min. ATB solid medium: 2% agar was added to the ATB liquid medium.
1. Separation of
Collecting fresh cortex Dioscoreae 50g, crushing, mixing, pouring 10g processed cortex Dioscoreae into 250mL triangular flask containing 20 fine glass beads and 100mL sterilized deionized water, and placing on shaking table
Shaking at 25 deg.C for 1h at 150r/min, standing for 30min, collecting supernatant 1mL, and gradient diluting with sterilized deionized water 10 times to obtain 10-1、10-2、10-3、10-4、10-5、10-6、10-7Sucking 100 μ L of 7 gradients, respectively, spreading different gradient dilutions on ATB solid plate culture medium, and standing at 25-28 deg.C for 30-36 h. Selecting single colony by inoculating loop, streaking and purifying on ATB solid culture medium plate for 2-3 times,
transferring the purified single colony with different forms to ATB solid plate, static culturing at 25-28 deg.c for 30-36 hr, and storing at 4 deg.c for further use.
2. Bacteriostasis test
The purified strains stored on ATB solid medium were cultured in ATB liquid medium under 25 ℃ for 36 h. In addition, the thallus on the black spot of the yam epidermis is inoculated on an ATB solid culture medium plate, evenly coated and cultured for 48h at the temperature of 30 ℃. Then, a solid plate was perforated with a sterilization perforator at a hole diameter of 4mm, and 6 holes were uniformly perforated per plate. Then, 40 mu L of the cultured separated and purified bacterial liquid to be determined is dripped into the hole, static culture is carried out for 36h at the temperature of 28 ℃, and the inhibition activity of the strain to be determined on the yam alternaria alternata is evaluated through an inhibition zone. The results show that three strains have obvious inhibition effect on the yam alternaria alternata, but the SY-1 has the strongest inhibition activity, the diameter of the inhibition zone is 24.3mm, and the results are shown in figure 1. This strain was then subjected to further characterization.
3. Morphological observation
Inoculating the strain SY-1 with the strongest bacteriostatic activity into 100mL ATB liquid culture medium, performing static culture at 25 ℃ for 30h, then scribing on the ATB solid culture medium by using a sterile inoculating loop under the aseptic condition, performing static culture at 25 ℃ for 36h, observing the colony morphology, wherein a single colony is milky and round, has a smooth surface and complete edges, and the result is shown in figure 2. A single colony was picked, gram stained and observed under a microscope, and the bacterium was a gram-positive coccus, and the results are shown in FIG. 3. SY-1 is basically identified as lactococcus lactis from the results of FIGS. 2 and 3.
4. Molecular biological identification
Single colonies of SY-1 are picked up by a sterilized toothpick in a 250mL triangular flask containing 50mL ATB liquid medium, are statically cultured for 24h at 25 ℃, and then the genome DNA of the bacterium is extracted by a bacterial genome DNA extraction kit. The 16S rRNA universal primer fd2 (5'-AGAGTTTGATCATGGCTCAG-3') and rpl (5'-ACGGTTACCTTGTTACGACTT-3') were used to PCR amplify the DNA fragment, and the PCR product was sent to the gene sequencing company for sequencing. The results of gene sequencing of 16S rRNA of this strain are shown in FIG. 4. According to the obtained 16S rRNA gene sequence, the similarity with the lactococcus garviea gene can reach up to 99.5 percent by comparison in an NCBI database by using a genebank database, so that the bacterium is identified as lactococcus garviea (lactococcus garviea), and is named as lactococcus garviea SY-1.
Example 2
A compound biological antistaling agent and a method for strawberry fresh-keeping, which comprises the following steps:
(1) preparing the jerusalem artichoke leaf alcohol extract: adding dried Jerusalem artichoke leaf (Jiangsu Biqingyuan ocean biology science and technology Co., Ltd.) into 50% ethanol water solution according to a certain material-liquid ratio (g/L) of 1:20, placing into an ultrasonic cleaning instrument, performing ultrasonic extraction for 40min under the condition of ultrasonic power of 350w, then placing into a centrifuge, centrifuging for 20min at 3000rpm/min, concentrating the supernatant by 15 times by using a rotary evaporator, and drying to constant weight by using a freeze dryer.
(2) And (3) strain culture: taking a lactococcus garvieae SY-1 (preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.9485 and the preservation date of 2014, 7 months and 28) glycerol to preserve strains, streaking and inoculating the strains on a fresh MRS (lactic acid bacteria culture medium) solid plate, carrying out activated culture for 24 hours at the temperature of 30 ℃, then selecting a single colony on the solid plate and inoculating the single colony into a 250mL conical flask containing 50mL of MRS liquid culture medium, and carrying out culture for 24 hours at the temperature of 30 ℃ to obtain a seed solution;
(3) preparing a fermentation medium: the tomato sauce consists of soybean meal, corn starch, inulin (Jiangsu Biqingyuan ocean Biotechnology Co., Ltd.), tomato juice, sodium carboxymethylcellulose (all sold in the market) and sterile water, wherein the mass-volume ratio of each component to the sterile water is as follows in sequence: 15g/L, 8g/L, 2g/L, 0.2g/L, 0.5 g/L.
(4) Preparation of a solution of lactococcus garvieae: inoculating the seed liquid prepared in the step 2) into a fermentation culture medium at a ratio of 2% (v/v), and culturing at 30 ℃ for 20 h. Centrifuging at 4000r/min for 20min after the culture is finished, taking supernatant, adjusting the pH value to 3 by using citric acid (commercially available food grade), precipitating overnight at 4 ℃, centrifuging at 9000r/min every other day for 10min at 4 ℃, freeze-drying extracted precipitate, and dissolving by using deionized water with the mass of 10 times of that of the precipitate to obtain the solution of the lactococcus garvieae.
(5) Compounding: adding the jerusalem artichoke leaf alcohol extract and water-soluble chitosan (the product number is syx001 of Jinan Haidebei ocean biological engineering Co., Ltd.) into the solution of the gelfoam lactoglobulin, and stirring until the mixture is completely dissolved to obtain the compound biological preservative. Wherein the mass volume ratio of the jerusalem artichoke leaf alcohol extract to the water-soluble chitosan in the solution of the grignard lactoglobulin is 5g/L and 60g/L respectively.
(6) Diluting the compounded biological fresh-keeping agent by 3 times for later use;
(7) the method comprises the steps of harvesting strawberries (Lishui plant base in farm courtyard of Jiangsu province), immediately putting the strawberries into a cooling room, forcibly cooling the strawberries to 2 ℃ by using an air cooler, carrying out air speed of 10m/s and humidity of 80% for 1h, then putting the strawberries into a compound biological preservative diluent diluted by 3 times for soaking for 1min, fishing out the strawberries, drying the strawberries by using cold air, and then preserving the strawberries according to a conventional cold storage preservation method (the temperature of the cold.
When the preservation time is determined, 5 preservation modes of contrast (no preservative treatment), treatment of the garlicin (100g/L), treatment of the jerusalem artichoke leaf alcohol extract (5g/L), combined treatment of the lactein (100g/L) and the jerusalem artichoke leaf alcohol extract (5g/L) and combined treatment of the garlicin (100g/L), the jerusalem artichoke leaf alcohol extract (5g/L) and the chitosan (60g/L) are set, when the treatment is carried out, each preservative is diluted by 3 times, the decay rate, the weight loss rate and the soluble solid content of the strawberries in different preservation time are detected, as can be seen from the graph 1, the graph 2 and the graph 3, the decay rate of the control group reaches 9.2 percent when the control group is preserved for 3 days, the decay rate can reach 24 percent when the control group is preserved for 5 days, therefore, the longest fresh-keeping time of the strawberries in the control group can be determined to be 3-4 days, and can never exceed 5 days. The preservation effect of each treatment group is that the combined treatment group of the garlicin and the jerusalem artichoke leaf alcohol extract and the chitosan is more than the combined treatment group of the lactucin and the jerusalem artichoke leaf alcohol extract more than the combined treatment group of the garlicin and the jerusalem artichoke leaf alcohol extract in turn within the same preservation time. The rotting rate and the weight loss rate of the strawberries treated by the three components are obviously lower than those of other treatment groups, and the content of soluble solids is obviously higher than those of other treatment groups. The three components have synergistic effect on strawberry diseases and strawberry nutrient component maintenance.
The compound biological preservative is used for preserving the strawberries, so that the strawberries can be preserved in a refrigeration house for 7-10 days (the preservation period of the strawberries without the compound biological preservative in the refrigeration house at 0-4 ℃ is 3-4 days).
Example 3
A compound biological preservative and a method for applying the same to preservation of rabbit-eye blueberries comprise the following steps:
(1) preparing the jerusalem artichoke leaf alcohol extract: adding dried Jerusalem artichoke leaf (Jiangsu Biqingyuan ocean biology science and technology Co., Ltd.) into 50% ethanol water solution according to a certain material-liquid ratio (g/L) of 1:25, placing into an ultrasonic cleaning instrument, performing ultrasonic extraction for 45min under the condition of ultrasonic power of 400w, then placing into a centrifuge, centrifuging for 15min at 3000rpm/min, concentrating the supernatant by 15 times by using a rotary evaporator, and drying by using a freeze dryer to constant weight.
(2) And (3) strain culture: taking a glycerol preserved strain of lactococcus garvieae SY-1 (preserved in China general microbiological culture Collection center of culture Collection center with the strain preservation number of CGMCC No.9485 and the preservation date of 2014, 7 and 28), streaking and inoculating the glycerol preserved strain onto a fresh MRS solid plate, carrying out activated culture for 40h at the temperature of 28 ℃, then selecting a single colony on the solid plate and inoculating the single colony into a 250mL conical flask containing 50mL of MRS liquid culture medium, and carrying out culture for 30h at the temperature of 30 ℃ to obtain a seed liquid;
(3) preparing a fermentation medium: the corn starch-based food additive consists of soybean meal, corn starch, inulin, tomato juice, sodium carboxymethylcellulose (all sold in the market) and sterile water, wherein the mass-volume ratio of each component to the sterile water is as follows in sequence: 15g/L, 10g/L, 1g/L, 0.5g/L, 1 g/L.
(4) Preparation of a solution of lactococcus garvieae: inoculating the seed liquid prepared in the step 2) into a fermentation medium at a ratio of 4% (v/v), and culturing at 30 ℃ for 20 h. Centrifuging at 4000r/min for 20min after the culture is finished, taking supernatant, adjusting the pH value to 2 with citric acid (commercially available food grade), precipitating overnight at 4 ℃, centrifuging at 9000r/min every other day for 15min at 4 ℃, freeze-drying the extracted precipitate, and dissolving with 10 times of deionized water to obtain the solution of the lactoglobulin.
(5) Compounding: adding Jerusalem artichoke alcohol extract and water-soluble chitosan (Jerusalem artichoke ocean biological engineering Co., Ltd.) into the Grignard lactoglobulin solution, and stirring until the mixture is completely dissolved to obtain the compound biological fresh-keeping agent. Wherein the mass volume ratio of the jerusalem artichoke leaf alcohol extract to the water-soluble chitosan in the solution of the grignard lactoglobulin is 2g/L and 45g/L respectively.
(6) Diluting the compounded biological fresh-keeping agent by 6 times for later use;
(7) after the rabbit-eye blueberries (Nanjing Bailong organic agricultural science and technology development Co., Ltd.) are harvested, the rabbit-eye blueberries are immediately placed into a cooling room, are forcibly cooled to 4 ℃ by an air cooler, the air speed is 10m/s, the humidity is 75%, the time is 3 hours, the rabbit-eye blueberries are then placed into a compound biological preservative diluent diluted by 6 times for soaking for 1min, the rabbit-eye blueberries are fished out and dried by cold air, and the blueberry preservation method is carried out according to the conventional refrigeration storage preservation method (the refrigeration storage temperature is 0-5 ℃, and the humidity is.
The blueberry preserved by the compound biological preservative can be preserved in a refrigerator for 50-60 days (the preservation period of the blueberry without the compound biological preservative in the refrigerator at 0-5 ℃ is 30-35 days).
Example 4
A compound biological antistaling agent and a method for applying the compound biological antistaling agent to fresh keeping of figs comprise the following steps:
(1) preparing the jerusalem artichoke leaf alcohol extract: adding dried Jerusalem artichoke leaf (Jiangsu Biqingyuan ocean biology science and technology Co., Ltd.) into 50% ethanol water solution according to a certain material-liquid ratio (g/L) of 1:20, placing into an ultrasonic cleaning instrument, performing ultrasonic extraction for 50min under the condition of 500w of ultrasonic power, then placing into a centrifuge, centrifuging for 20min at 3000rpm/min, concentrating the supernatant by 15 times by using a rotary evaporator, and drying to constant weight by using a freeze dryer.
(2) And (3) strain culture: taking a glycerol preserved strain of lactococcus garvieae SY-1 (preserved in China general microbiological culture Collection center of culture Collection center with the strain preservation number of CGMCC No.9485 and the preservation date of 2014, 7 and 28), streaking and inoculating the glycerol preserved strain onto a fresh MRS solid plate, performing activated culture for 30h at the temperature of 30 ℃, then selecting a single colony on the solid plate and inoculating the single colony into a 250mL conical flask containing 50mL of MRS liquid culture medium, and culturing for 24h at the temperature of 30 ℃ to obtain a seed solution;
(3) preparing a fermentation medium: the corn starch-based food additive consists of soybean meal, corn starch, inulin, tomato juice, sodium carboxymethylcellulose (all sold in the market) and sterile water, wherein the mass-volume ratio of each component to the sterile water is as follows in sequence: 20g/L, 12g/L, 2g/L, 0.4g/L and 0.8 g/L.
(4) Preparation of a solution of lactococcus garvieae: inoculating the seed liquid prepared in the step 2) into a fermentation medium at a ratio of 5% (v/v), and culturing at 30 ℃ for 24 h. Centrifuging at 4000r/min for 15min after the culture is finished, taking supernatant, adjusting the pH value to 2.5 by using citric acid (commercially available food grade), precipitating overnight at 4 ℃, centrifuging at 9000r/min every other day for 15min at 4 ℃, freeze-drying the extracted precipitate, and dissolving by using deionized water with the mass of 10 times of that of the precipitate to obtain the solution of the lactococcus garvieae.
(5) Compounding: adding Jerusalem artichoke alcohol extract and water-soluble chitosan (Jerusalem artichoke ocean biological engineering Co., Ltd.) into the Grignard lactoglobulin solution, and stirring until the mixture is completely dissolved to obtain the compound biological fresh-keeping agent. Wherein the mass volume ratio of the jerusalem artichoke leaf alcohol extract to the water-soluble chitosan in the solution of the grignard lactoglobulin is 5g/L and 55g/L respectively.
(6) Diluting the compounded biological preservative by 4 times for later use;
(7) after the figs (the Limited liability company of the national modern agriculture demonstration park of the western Suzhou mountain) are harvested, the figs are immediately placed into a cooling room, an air cooler is adopted to forcibly cool the figs to 4 ℃, the air speed is 10m/s, the humidity is 75%, the time is 40min, the figs are then placed into a compound biological preservative diluent diluted by 4 times to be soaked for 1.5h, the figs are fished out, the figs are dried by cold air, and the fresh keeping is carried out according to the conventional cold storage preservation method (the temperature of a cold storage is-2-4 ℃, and the humidity is 85-90%).
The compound biological preservative can be used for preserving the figs for 15 to 20 days in a refrigeration house (the preservation period of the figs which do not use the compound biological preservative in the refrigeration house at-2 to 4 ℃ is 5 to 7 days)
Claims (7)
1. A compound biological antistaling agent is characterized by comprising the following components of grignard lactoglobulin, alcohol extract of jerusalem artichoke leaves and water-soluble chitosan; the said Grignard lactococcus lactis originates from the lactococcus garvieae SY-1, the preservation number of the strain is CGMCC No. 9485;
the mass ratio of the grignard lactoglobulin to the jerusalem artichoke leaf alcohol extract to the water-soluble chitosan is 100: 2-5: 40-65;
the preparation method of the grignard lactoglobulin comprises the following steps:
(1) activating lactococcus garvieae strain, inoculating in culture medium, culturing to obtain seed liquid;
(2) inoculating the seed liquid into a fermentation culture medium for fermentation culture, and collecting fermentation liquid after the fermentation culture is finished;
(3) and centrifuging the fermentation liquor, taking the supernatant, adjusting the pH value to 2-3 with acid, then precipitating, taking the precipitate, and freeze-drying to obtain the grignard lactoglobulin.
2. The compound biological preservative according to claim 1, wherein in the step (2), the fermentation medium consists of soybean meal, corn starch, inulin, tomato juice, sodium carboxymethylcellulose and sterile water, and the mass-volume ratio of each component to the sterile water is as follows in sequence: 15-20 g/L, 8-12 g/L, 1-2 g/L, 0.1-0.5 g/L and 0.5-1.2 g/L; the temperature of the fermentation culture is 28-30 ℃, and the time is 18-24 h.
3. The compound biological preservative according to claim 1, wherein the jerusalem artichoke leaf alcohol extract is a jerusalem artichoke leaf alcohol extract.
4. The compound biological preservative according to claim 1, wherein the preparation method of the jerusalem artichoke leaf alcohol extract comprises the following steps: putting jerusalem artichoke leaves into 40-60% alcohol solution according to the material-liquid ratio of 1: 20-25 for ultrasonic extraction, centrifuging after the ultrasonic extraction is finished, taking supernate, and drying to obtain the jerusalem artichoke leaf alcohol extract.
5. The application of the compound biological preservative according to any one of claims 1-4 in fruit preservation.
6. The use according to claim 5, wherein the fruit is a small berry.
7. The application of claim 5, wherein the compound biological preservative needs to be diluted by 30-60 times with water when in use; and (3) soaking the fruits in the diluent, and then combining a conventional refrigeration storage preservation method for preservation.
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| 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 |
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