Preparation method of composite film suitable for fresh keeping of instant vegetable mixed salad

Abstract

The invention belongs to the technical field of fresh-cut vegetable preservation, and particularly relates to a preparation method of a composite film suitable for preservation of instant vegetable mixed salad. According to the invention, a nano silver particle (AgNPs)-citrus essential oil compound core layer, an epigallocatechin gallate (EGCG)-chitosan middle layer and an enzyme crosslinked protein-polysaccharide network outer layer are respectively prepared, and a supercritical CO2 emulsification technology is utilized to realize the preparation of a stable multi-layer composite microcapsule emulsion. Citrus essential oil / EGCG / nano-silver particle ternary composite nano-microcapsule powder is obtained after freeze-drying, polyvinyl alcohol (PVA) is used as a film-forming agent, the composite preservative film with antibacterial, antioxidant, uvioresistant and slow-release functions for food preservation is prepared, the composite preservative film is applied to preservation of instant vegetable mixed salad, and the shelf life of the instant vegetable mixed salad can be remarkably prolonged.

Description

Technical Field

[0001] The invention belongs to the technical field of fresh-cut vegetable preservation, and more specifically, relates to a method for preparing a composite film suitable for preserving ready-to-eat mixed vegetable salad. Background Art

[0002] Fresh-cut fruits and vegetables, such as fruit platters and vegetable salads, have become the favorites of consumers, especially those who love beauty, weight loss, and fitness, because of their fresh quality, rich taste, rich dietary fiber, and relatively low fat content. However, fresh fruits and vegetables need to go through a long process from picking, transportation, processing, storage, and consumption. Even if they are partially cleaned and sterilized during this period, they are still susceptible to infection by foodborne microorganisms, causing them to easily produce unpleasant sensory effects such as browning, odor, and softening within 2 to 3 days of storage after processing, and the quality deteriorates rapidly. Therefore, how to effectively extend the shelf life of fresh-cut fruits and vegetables has become a key issue that the industry needs to solve urgently.

[0003] In traditional preservation technology, the preservation of fresh-cut fruits and vegetables mainly relies on technologies such as modified atmosphere packaging, low-temperature refrigeration, and chemical preservatives, but these technologies are difficult to maintain the quality of fresh-cut fruits and vegetables for a long time. Patent Publication No. CN103892404A discloses a method for low-temperature preservation of fresh salads in modified atmosphere packaging, which includes placing fresh salads in a packaging container; filling a mixed gas of oxygen, carbon dioxide, and nitrogen into the packaging container, and sealing the packaging container; and storing the above packaging container in an environment of 3-5°C. The invention uses a suitable gas ratio and microporous film packaging materials to achieve an atmosphere-controlled equilibrium state that is conducive to the preservation of fruits and vegetables and maintains weak aerobic respiration. The shelf life of fresh salads can be extended to 6-8 days. However, the invention does not take into account the impact of microbial growth on the storage quality of vegetable salads, and the shelf life is limited, and long-term preservation cannot be achieved.

[0004] In order to overcome the shortcomings of traditional preservation technology, researchers have actively explored emerging preservation technologies in recent years, mainly including: preparing biological preservatives from natural antibacterial substances, which can inhibit the growth of microorganisms and reduce chemical residues; using natural antibacterial substances to prepare edible coatings to form a protective film on the surface of fresh-cut fruits and vegetables, reducing water loss and oxygen contact, and delaying browning and corruption; using non-thermal sterilization technology, such as ultraviolet rays, high-voltage pulsed electric fields, etc., to kill microorganisms without affecting quality; adding active ingredients such as antioxidants and antibacterial agents to packaging materials to further extend the shelf life of the product, or combining multiple preservation technologies to achieve the effect of extending the shelf life.

[0005] Patent Publication No. CN107259295A discloses a biophysical dual fresh-keeping and storage method for salads. Natural biological antibacterial, antiseptic, and antioxidant active substances such as chitosan, non-starch polysaccharides, tea polyphenols, and flavonoids are dissolved in water, sprayed on the inner walls of packaging bags and boxed containers, and dried by blowing air at room temperature to form a natural fresh-keeping film. The salad is placed in a boxed container. The boxed container is placed in a packaging bag, filled with a mixed gas such as nitrogen, carbon monoxide, and carbon dioxide, and then sealed. After sealing, the oxygen content in the package is reduced to less than 0.1-0.5%, and stored at low temperature, which can extend the shelf life of fruit salad, vegetable salad, cooked meat product salad, or mixed salad from 5-7 days to 8-12 days. However, since a large amount of water in the tissues of vegetable salad products will evaporate during storage, the natural fresh-keeping film attached to the inner wall of the packaging box and dried is extremely susceptible to the influence of the evaporated water and falls off from the inner wall of the packaging box, resulting in a deterioration of the fresh-keeping effect. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and provide a preparation method of nano-microcapsules.

[0007] The object of the present invention is to provide the nano-microcapsules prepared by the above preparation method.

[0008] Another object of the present invention is to provide a preparation method of a composite fresh-keeping film.

[0009] Another object of the present invention is to provide the composite fresh-keeping film prepared by the above preparation method.

[0010] Another object of the present invention is to provide the application of the composite fresh-keeping film in the fresh-keeping of ready-to-eat vegetable mixed salad.

[0011] Another object of the present invention is to provide a fresh-keeping method.

[0012] The above objects of the present invention are achieved by the following technical solutions:

[0013] The present invention provides a preparation method of nano-microcapsules, comprising the following steps:

[0014] S1. Preparation of the core layer: Mix citrus essential oil and nano-silver solution, and perform ultrasonic treatment to obtain the core layer;

[0015] S2. Preparation of the intermediate layer: Add epigallocatechin gallate to the polysaccharide solution, and adjust the pH value to 5.4-6.2 to obtain the intermediate layer;

[0016] S3. Preparation of the outer layer: Mix the protein aqueous solution and the polysaccharide solution, add transglutaminase, and adjust the pH value to 6.0-7.0. After the reaction is complete, terminate the reaction to obtain the outer layer;

[0017] S4. Preparation of nano-microcapsules: Under the condition of supercritical CO2 with a pressure of 10 - 12 MPa, the core layer prepared in S1, the intermediate layer prepared in S2, and the outer layer prepared in S3 are mixed evenly to obtain a composite nano-microcapsule emulsion, which is then freeze-dried to obtain the product;

[0018] The concentration ratio of nano-silver to citrus essential oil in the core layer is 0.03 - 0.05 mol / L : 6.4 - 12.8 g / L;

[0019] The concentration of epigallocatechin gallate in the intermediate layer is 0.5 - 1 mg / mL;

[0020] The volume ratio of the core layer : intermediate layer : outer layer is 3 : 3 : 3 - 4.

[0021] The nano-microcapsules of the present invention use active substances with antibacterial and antioxidant properties - silver nanoparticles (AgNPs), citrus essential oil, and epigallocatechin gallate (EGCG) as the main active ingredients. First, a nano-silver - citrus essential oil composite core layer is prepared, then an intermediate layer is prepared by binding epigallocatechin gallate (EGCG) with polysaccharides through hydrogen bonds, and subsequently, an outer layer of a protein - polysaccharide network is prepared using an enzyme cross-linking technique. Finally, the above system solution is emulsified by supercritical CO2 to form a stable composite nano-microcapsule emulsion, and freeze-drying is carried out to form nano-microcapsules. The present invention adopts the supercritical CO2 emulsification technique to avoid residual organic solvents, and the prepared microcapsules have a high encapsulation rate for essential oils (≥90%). At the same time, transglutaminase (TGase enzyme) is used to cross-link polysaccharides and proteins to form a covalent network structure, and the prepared microcapsules have high thermal stability (the retention rate of essential oil is ≥85% after being treated at 80°C for 1 hour).

[0022] In addition, the present invention uses the supercritical CO2 emulsification technique to form a homogeneous and stable nano-microcapsule emulsion. On the one hand, it can improve the dispersibility of the composite nano-emulsion, enabling the active ingredients with effective antibacterial and antioxidant properties to be evenly distributed. On the other hand, it lays a foundation for the subsequent application in the preparation of composite fresh-keeping films to form excellent mechanical, water-permeable, and air-permeable properties.

[0023] The composite fresh-keeping film prepared by the present invention has a wide range of application scenarios. In addition to having good fresh-keeping effects under conventional light-shielding and low-temperature storage conditions, it can also delay the deterioration of fresh-cut vegetable salads under the lights of conventional supermarket display cabinets. When pure citrus essential oil is applied to fruit and vegetable preservation, its effect is often limited due to its strong hydrophobicity and volatility. In the present invention, however, EGCG gradually generates colored quinone substances during the oxidation process during storage, which can effectively block ultraviolet rays and reduce the degree of oxidation of fresh-cut vegetable tissues under photocatalysis during the storage of fresh-cut vegetable salads in display cabinets. Therefore, it can reduce the loss of nutrients such as ascorbic acid and polyphenols to a greater extent during the storage of fresh-cut vegetables. And by preparing nano-microcapsules, its hydrophilicity can be effectively improved, its volatility can be reduced, and its continuous action time can be extended, thereby achieving the effect of long-term preservation of vegetable salads.

[0024] Preferably, the particle size of silver nanoparticles in the silver nanoparticle solution is controlled within 25 - 40 nm. Preferably, the size range of the nano-microcapsules is 100 - 500 nm. The particle size of silver nanoparticles and the size of the nano-microcapsules can be tested by a nano particle size and Zeta potential analyzer.

[0025] The silver nanoparticle solution of the present invention can be prepared according to conventional methods or by purchasing commercially available silver nanoparticles and then preparing a solution with water.

[0026] Preferably, the present invention provides a preparation method of a silver nanoparticle solution for reference: Dissolve a silver salt in water, add a reducing agent, stir and react until the solution changes color, and obtain the silver nanoparticle solution after removing impurities.

[0027] Furthermore, the reducing agent includes, but is not limited to, sodium citrate, sodium borohydride, ascorbic acid, etc.

[0028] Furthermore, the concentration of the reducing agent is 0.6 - 1.0 mol / L.

[0029] Furthermore, the temperature of the stirring reaction is 20 - 25 °C.

[0030] Furthermore, the specific method for removing impurities is: centrifugation and washing.

[0031] Preferably, the citrus essential oil is one or more of bergamot essential oil, lemon essential oil, citrus essential oil, sweet orange essential oil, grapefruit essential oil.

[0032] Preferably, in step S1, the ultrasonic treatment parameters are: the power is 200 - 250 W, and the frequency is 20 - 40 KHz.

[0033] Preferably, in step S1, the ultrasonic treatment time is 15 - 25 min; the temperature is 19 - 22 °C; the working mode is a pulse working mode (working for 2 s, pausing for 1 s).

[0034] In the present invention, the solvent of the polysaccharide solution is 1% acetic acid solution. The polysaccharide is dissolved in acetic acid solution with a mass fraction of 1% to obtain the polysaccharide solution.

[0035] Preferably, in step S2, the mass fraction of the polysaccharide solution is 1% - 2%.

[0036] Preferably, the temperature during the preparation of the intermediate layer is room temperature. The room temperature in the present invention refers to 20 - 25 °C.

[0037] Preferably, in step S3, the protein is one or more of gelatin, whey protein, soy protein, zein, and wheat gluten protein.

[0038] Preferably, in step S3, the polysaccharide is one or more of chitosan, arabic gum, cellulose, corn starch, cyclodextrin, and pullulan.

[0039] Preferably, in step S3, the addition amount of transglutaminase is 0.5 - 1.5 g / L.

[0040] Preferably, in step S3, the temperature of the reaction is 37 °C - 40 °C; the reaction time is 18 - 36 h.

[0041] Preferably, in step S3, the method for terminating the reaction is: heating to 80 °C or adding ethanol to terminate the reaction.

[0042] Preferably, in step S3, the mass fraction of the protein in the protein aqueous solution is 1% - 2%; the mass fraction of the polysaccharide in the polysaccharide solution is 1% - 2%. Further preferably, the protein aqueous solution and the polysaccharide solution are mixed evenly at a volume ratio of 1:1 - 1.5; more preferably, at a volume ratio of 1:1. More specifically, the mixing operation is carried out by stirring at 55 - 65 °C for 1 - 2 h.

[0043] Preferably, in step S4, the supercritical CO2 conditions are: pressure 10 - 12 MPa, temperature 32 - 35 °C.

[0044] Preferably, in step S4, the requirements for the freeze-drying parameters are: temperature -40 ± 1 °C, pressure in the freeze-drying chamber cavity 100 ± 5 Pa, and temperature of the material heating plate 45 ± 0.5 °C.

[0045] Preferably, after freeze-drying, it can also be pulverized and sieved. Further preferably, the mesh number of the sieving is 100 - 200 meshes.

[0046] Meanwhile, the present invention also protects the nano-microcapsules prepared by the above preparation method.

[0047] Additionally, the present invention also provides a method for preparing a composite fresh-keeping film. The nano-microcapsules of the present invention are added to a polyethylene glycol solution, and after post-treatment, a composite fresh-keeping film is obtained.

[0048] Preferably, the mass fraction of the nano-microcapsules in the polyethylene glycol solution is 0.5% - 2%.

[0049] By mixing the nano-microcapsules of the present invention with a polyethylene glycol (PVA) film-forming agent, a composite fresh-keeping film based on PVA is prepared. It has excellent mechanical properties, can effectively reduce the dissolution of the packaging material caused by the volatilized water of fresh-cut vegetables during packaging and storage, and the finally formed composite fresh-keeping film has excellent water permeability and air permeability. Combined with specific modified atmosphere treatment, it can maintain the stability of the moisture and gas environment inside the package for a long time, thereby achieving the effect of medium- and long-term preservation of vegetable salad.

[0050] The composite fresh-keeping film of the present invention contains a variety of active ingredients: Nano silver particles (AgNPs) directly kill or inhibit the growth of bacteria, molds and yeasts by destroying the microbial cell membrane or inhibiting their metabolic activities, and have a continuous antibacterial effect on bacteria, molds and yeasts through contact reactions and photocatalytic reactions in food packaging; Essential oils of citrus genus contain a variety of active ingredients (such as terpenoids, phenolic compounds), which can destroy the microbial cell structure and have antioxidant effects at the same time, delaying the oxidative deterioration of fruits and vegetables; EGCG can inhibit the activity of polyphenol oxidase, reduce the occurrence of enzymatic browning, and has antioxidant and antibacterial effects at the same time.

[0051] The present invention uses AgNPs, essential oils of citrus genus and EGCG as the main active substances of the composite fresh-keeping film. The three play a synergistic role and have more excellent antibacterial and antioxidant properties. Experiments show that the combined use of nano silver ions, EGCG and essential oils of citrus genus has a synergistic antibacterial effect, and has good inhibitory or even killing effects on microorganisms such as bacteria, molds, yeasts, etc. that will cause adverse properties to the sensory properties of fresh-cut fruits and vegetables during the processing and storage of fresh-cut fruits and vegetables, such as Escherichia coli, Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa, and the effect is far better than the combination of two; In addition, the antioxidant ability of EGCG is more than 100 times that of vitamin C. In addition to directly reducing the oxidative browning of fresh-cut vegetables as a packaging material, its combined application with essential oils of citrus genus can effectively reduce the influence of external oxidation on essential oils of citrus genus during the preservation of fresh-cut vegetables, enhance its continuous action time, and the combined action between EGCG and nano silver particles can also synergistically improve the antibacterial performance of the final product, improve its stability and water solubility; Finally, the essential oils of citrus genus itself contain pleasant monoterpenoids, which can not only have antibacterial effects but also improve the flavor of vegetable salad products, bringing consumers a richer eating experience.

[0052] Preferably, the mass percentage of polyethylene glycol in the polyethylene glycol (PVA) solution is 2-4%; the solvent of the polyethylene glycol solution is water.

[0053] Further, the polyethylene glycol solution is prepared by the following method: adding polyethylene glycol into water and stirring at 60-70°C for 1-3 h to obtain the solution.

[0054] Preferably, the post-treatment is as follows: mixing and degassing, and then pouring and drying. More specifically, the mixing and degassing is specifically: stirring at 40-50°C for 0.5-1.5 h and degassing by ultrasonic wave at 400-450 W for 3-6 min. More specifically, the operation of pouring and drying is: pouring the solution after mixing and degassing into a mold and drying. The pouring amount of the solution is calculated based on the cross-sectional area of the mold at 0.4-0.6 g / cm 2 That is, 0.4-0.6 g of the solution is poured per cm 2 of the mold.

[0055] Meanwhile, the present invention also protects the composite fresh-keeping film prepared by the above preparation method.

[0056] Further, the present invention protects the application of the composite fresh-keeping film in the fresh-keeping of ready-to-eat vegetable mixed salad.

[0057] In addition, the present invention provides a fresh-keeping method, which includes the following steps: packaging vegetables or vegetable salad with the composite fresh-keeping film of the present invention.

[0058] The present invention uses the composite fresh-keeping film in combination with the controlled atmosphere technology to keep ready-to-eat vegetable mixed salad fresh. The whole processing and packaging process is close to the actual production process and storage process of current ready-to-eat fresh-cut vegetable salad. Due to the antibacterial and antioxidant effects of the fresh-keeping film, the growth of bacteria and other microorganisms in fresh-cut vegetables and the resulting adverse effects such as peculiar smell, browning, texture softening, and rapid reduction of active ingredients such as ascorbic acid and polyphenols are reduced.

[0059] It is found through research that the composite fresh-keeping film of the present invention is used to hold mixed fresh-cut vegetable salad, and O2, CO2, Ar, and N2 are injected in a certain proportion and heat-sealed. The shelf life reaches 14 days during storage. The weight loss rate at the end of storage (on the 14th day) is about 10%. The total colony counts of bacteria and fungi (molds, yeasts) are respectively lower than 10 6 CFU / g and 10 3 CFU / g. In addition, the nutritional quality and sensory quality of the product are well maintained. The method provided by the present invention is simple to operate, has a good fresh-keeping effect, effectively extends the storage shelf life of autumn and winter seasonal vegetable salad, and reduces the loss of product commercial and edible value.

[0060] The preservation method provided by the present invention is applicable to fresh vegetables, ready-to-eat vegetable mixed salads, etc. Preferably, the vegetables include but are not limited to lettuce, Rosa green, purple cabbage, Romaine lettuce, Rosa red, frisée, arugula, cherry radish, baby cucumber, red chicory, carrot, cherry tomato, carrot, cherry tomato, daikon radish, butterhead lettuce, Italian lettuce, cherry tomato, finger carrot.

[0061] Preferably, a mixed gas of O2, CO2, Ar, and N2 is filled during packaging. Preferably, the volume percentages of the components in the mixed gas are: O2: 3% - 7%, CO2: 3% - 7%, Ar: 5% - 10%, N2: 76% - 89%.

[0062] Preferably, the vegetables can be pre-cooled at 4°C for 10 - 14 h before packaging, then washed to remove the damaged parts caused by extrusion, and finally drained and cut into the required specifications.

[0063] Preferably, the storage condition after packaging is: stored at 4 - 10°C.

[0064] The present invention has the following beneficial effects:

[0065] (1) The nano-microcapsules prepared by the present invention adopt the supercritical CO2 emulsification technology, avoiding the residue of organic solvents, and the prepared microcapsules have a high encapsulation rate for essential oils; at the same time, the prepared microcapsules have high thermal stability.

[0066] (2) The composite fresh-keeping film prepared by the present invention is non-toxic, has high biocompatibility, is flexible, and has good film-forming properties. The composite fresh-keeping film uses polyvinyl alcohol (PVA) as the film-forming substrate and is compounded with nano-microcapsules, having the effects of long-term antibacterial, antioxidant, and anti-ultraviolet, and can be applied to the preservation of fruits and vegetables.

[0067] (3) The composite fresh-keeping film prepared by the present invention has the following characteristics: 1) Compared with the direct immersion treatment of the composite preservative, being prepared into a packaging film material can minimize the impact on the edible quality of the preservation object and ensure the color, taste, and flavor of the vegetable salad; 2) Polyvinyl alcohol (PVA) in the composite fresh-keeping film has good film-forming properties, gas barrier properties, biodegradability, and safety as a film-forming agent; the combined use of silver nanoparticles, essential oils, and EGCG has excellent antibacterial and antioxidant effects, and at the same time, these three active substances have a wide source and high food safety; proteins and polysaccharides can jointly serve as the film-forming wall material to protect the essential oils and achieve a slow-release effect, and the interaction between polyphenols (EGCG) and proteins can enhance the mechanical and barrier properties of the fresh-keeping film; 3) The composite fresh-keeping film prepared by the present invention has good air permeability, water permeability, and appropriate mechanical indexes, ensuring the feasibility of its subsequent use as a commercial food packaging film. Description of the Drawings

[0068] Figure 1 Embedding rate (a) and thermal stability (b) diagrams of citrus essential oils for different examples and comparative examples. Detailed implementation manners

[0069] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific examples, but the examples do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0070] In the examples and comparative examples of the present invention:

[0071] The preparation method of the chitosan solution is as follows: Dissolve chitosan in a 1% acetic acid solution, heat and stir to dissolve, and filter to obtain a clear chitosan solution.

[0072] In the present invention, 1% chitosan solution, 1.5% chitosan solution, 2% chitosan solution, 1% gelatin solution, etc. are all calculated by mass fraction. For example, 1% chitosan solution: that is, 1 g of chitosan is contained in every 100 g of solution; similarly, 1% gelatin solution means 1 g of gelatin is contained in 100 g of solution.

[0073] Measurement methods for relevant indicators:

[0074] 1. Weight loss rate

[0075] Obtained by comparing the proportion of the mass of the stored sample when sampled relative to the mass on the first day. The calculation formula is as follows: Weight loss rate (%) = (Initial sample mass - Sample mass at the end of storage) / Initial sample mass.

[0076] 2. pH value, conductivity, and soluble solid content

[0077] Take 15 g of the sample and grind it into a homogenate. After filtering, take the filtrate and use a pH meter to measure the pH value, use a conductivity meter to measure the conductivity, and use a handheld refractometer to measure the soluble solid content.

[0078] 3. Chlorophyll content

[0079] Homogenize 5 g of fresh cut vegetable salad in 20 mL of an ethanol aqueous solution (v:v = 80:20) using a homogenizer. The mixture is centrifuged at 11088×g, 4 °C / 20 min in a centrifuge. The supernatant is filtered through a syringe filter with a 0.45 μm pore size. Measure the absorbance at λ = 647 nm using a UV spectrophotometer. The calculation formula for chlorophyll content is as follows: Chlorophyll (mg / g) = 17.95×A647 + 7.9×A665.

[0080] 4. Ascorbic acid content

[0081] Determined by GB / T 6195—86 "2,6-Dichlorophenol Indophenol Method". Take 2.0 g of fruit and vegetable samples, add 10 mL of 20 g / L oxalic acid solution and homogenize. Centrifuge at 9000 rad / min at 4 °C for 30 min. Collect 5 mL of the supernatant and titrate with 2,6-dichlorophenol indophenol solution until it turns pink. The results are expressed as mg / 100 g.

[0082] 5. Total phenol content

[0083] Determined by colorimetry. Take 2 g of fresh-cut vegetable samples, homogenize and mix well with 10 mL of 80% ethanol solution, and centrifuge at 9000 rpm at 4 °C for 30 min. The supernatant represents the phenolic extract. Separate and filter with a polytetrafluoroethylene syringe filter (pore size 0.45 μm). Accurately measure 0.4 mL of the extract and place it in a 10 mL graduated test tube. Add 0.4 mL of 1.0 mol / L Folin-Ciocalteu reagent, then add 0.8 mL of 12% sodium carbonate solution, and add water to 8.4 mL to make up to the scale line. Heat in a water bath at 30 °C for 90 minutes, and measure the absorbance at 760 nm. All results are expressed as mg EAG / 100 g of gallic acid equivalent.

[0084] 6. Peroxidase enzyme activity

[0085] Weigh 2 g of the sample, add 7 mL of 0.05 mol / L phosphate buffer (pH 7.0), mix and grind into a homogenate in an ice bath. Centrifuge at 10000×g at 4 °C for 30 min, and collect the supernatant as the crude enzyme solution. Add 0.5 mL of the crude enzyme solution to a mixed solution of 2.2 mL of 1% guaiacol and 0.2 mL of 1.5% H2O2. The mixed solution starts to react and timing begins. Zero the absorbance value with the mixed solution without adding the enzyme solution as a reference, measure the absorbance at a wavelength of 470 nm, and read the value every 1 min. The peroxidase activity is defined as 1 enzyme activity unit for a 0.01 absorbance change per minute per gram of the sample, and the results are expressed as U / min·g.

[0086] 7. Polyphenol oxidase enzyme activity

[0087] Weigh 2 g of the sample, add 10 mL of 0.05 mol / L phosphate buffer (pH 7.0), mix and grind into a homogenate in an ice bath. Centrifuge at 10000×g at 4 °C for 30 min, and collect the supernatant as the crude enzyme solution. Add 0.5 mL of the crude enzyme solution to a mixed solution of 3 mL of phosphate buffer and 1 mL of 0.05 mol / L catechol. The mixed solution starts to react and timing begins. Zero the absorbance value with the mixed solution without adding the enzyme solution as a reference, measure the absorbance at a wavelength of 420 nm, and read the value every 1 min. The polyphenol oxidase activity is defined as 1 enzyme activity unit for a 0.01 absorbance change per minute per gram of the sample, and the results are expressed as U / min·g.

[0088] 8. Sensory evaluation

[0089] Ten food professionals were invited to form a sensory evaluation panel to evaluate fresh-cut vegetable salads at different storage periods. The nine-point method was used to evaluate the color, texture, odor, and appearance of the vegetable salad samples (with weights of 0.3, 0.2, 0.2, and 0.3 respectively). The calculation formula for sensory scores is as follows.

[0090] Sensory score (points) = 0.3×C + 0.2×T + 0.2×F + 0.3×A

[0091] Where C, T, O, and A are the scores of each member for the color, texture, taste, and appearance of the fresh-cut vegetable salad respectively.

[0092] 9. Total number of bacteria, molds, and yeasts

[0093] Take 25 g of the sample and immerse it in 225 mL of sterile physiological saline, shake and homogenize for 2 minutes, and continuously dilute the homogenate with sterile physiological saline to different powers of 10. Take a series of dilutions (1 mL) and put them into petri dishes. Add different selective agars (about 15 mL) to the petri dishes and mix them with the diluted samples. When counting the total number of colonies (total number of bacteria), add plate count agar to the petri dish, mix it with the diluted sample, and incubate at 37 °C for 48 h. When counting molds and yeasts, add Rose Bengal Medium to the petri dish, mix it with the diluted sample, and incubate at 28 °C for 5 days. Count them separately after the incubation ends.

[0094] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0095] Example 1 Preparation of nano-silver - citrus essential oil - EGCG composite fresh-keeping bags

[0096] S1. Add sodium citrate to a 0.03 mol / L aqueous silver nitrate solution (the final concentration of sodium citrate in the aqueous silver nitrate solution is 0.6 mol / L), stir and react at room temperature until the solution turns brownish-yellow, centrifuge and wash to remove impurities to obtain a pure AgNPs solution;

[0097] S2. Mix 6.4 mg / mL citrus essential oil (add 6.4 mg of essential oil to each mL of AgNPs solution) with the AgNPs solution, and use ultrasonic treatment (power 200 W, frequency 20 KHz, treatment time 20 min, operating temperature 20 ± 0.5 °C, working mode pulse working mode (work for 2 s, pause for 1 s)) to further improve the dispersion effect and form a stable AgNPs - citrus essential oil complex;

[0098] S3. Add EGCG to a 2% chitosan solution (the concentration of EGCG in the chitosan solution is 0.5 mg / mL), adjust the pH value to 5.8 ± 0.2, stir evenly to obtain an EGCG-chitosan solution;

[0099] S4. Add a 1% chitosan solution to a 1% gelatin aqueous solution according to a volume ratio of 1:1, stir evenly, add 1.0 g / L transglutaminase (TGase), adjust the pH value to 6.0, control the temperature at 37 °C - 40 °C, react for 24 hours, and then heat to 80 °C to terminate the reaction to obtain an enzyme-crosslinked protein-polysaccharide network solution;

[0100] S5. Use the core layer (AgNPs-citrus essential oil complex) as the oil phase, the middle layer (EGCG-chitosan solution) and the outer layer (enzyme-crosslinked protein-polysaccharide network solution) as the water phase. Under the conditions of a pressure of 10 MPa and a temperature of 32 °C, first introduce supercritical CO2 as the solvent and dispersion medium into the high-pressure mixer, then mix the oil phase and the water phase (the volume ratio of the outer layer, middle layer and core layer is 3:3:4) and inject them into the high-pressure mixer, and stir at a pressure of 10 MPa for 10 - 15 minutes to form a stable composite nano-microcapsule emulsion. Release the pressure, collect the emulsion and freeze-dry it (the freeze-drying parameters require a cold trap temperature of -40 ± 1 °C, a freeze-drying chamber pressure of 100 ± 5 Pa, and a material heating plate temperature of 45 ± 0.5 °C) to obtain nano-microcapsule freeze-dried powder;

[0101] S6. Add 1.0 g of nano-microcapsule freeze-dried powder to every 100 mL of a 3% PVA solution, stir at 45 ± 0.5 °C for 1 h, and degas by ultrasonic wave at 420 W for 5 min to obtain a composite fresh-keeping film solution. Subsequently, prepare the composite fresh-keeping film by the casting and drying method. Specifically: slowly pour 160 g of the composite fresh-keeping film solution onto a 20 × 20 cm polytetrafluoroethylene plate, place it in an oven at 50 °C and dry for 8 h to prepare the composite fresh-keeping film, cut it, and prepare an 18 × 18 cm AgNPs-citrus essential oil-EGCG composite fresh-keeping bag.

[0102] Example 2 Preparation of nano-silver-citrus lemon essential oil-EGCG composite fresh-keeping bag

[0103] S1. Add sodium citrate to a 0.04 mol / L silver nitrate aqueous solution (the final concentration of sodium citrate in the silver nitrate aqueous solution is 0.8 mol / L), stir and react until the solution turns brownish-yellow; centrifuge and wash to remove impurities to obtain a pure AgNPs solution;

[0104] S2. Mix 9.6 mg / mL lemon essential oil with the AgNPs solution, and use ultrasonic treatment (power: 250 W, frequency: 30 KHz, treatment time: 20 min, operating temperature: 20 ± 0.5 °C, working mode: pulse working mode (working for 2 s and pausing for 1 s)) to further improve the dispersion effect and form a stable AgNPs-lemon essential oil complex;

[0105] S3. Dissolve 1.5% chitosan in 1% glacial acetic acid solution, heat and stir to dissolve, filter to obtain a clear chitosan solution, then add EGCG to the chitosan solution (the concentration of EGCG in the chitosan solution is 0.5 mg / mL), adjust the pH value to 5.8 ± 0.2, stir evenly to obtain the EGCG-chitosan solution;

[0106] S4. Add 1% chitosan solution to 1% gelatin solution according to a volume ratio of 1:1, stir evenly, then add 1.0 g / L transglutaminase (TGase), adjust the pH value to 6.0, control the temperature at 37 °C - 40 °C, react for 24 hours, and finally heat to 80 °C or add ethanol to terminate the reaction to obtain the enzyme-crosslinked protein-polysaccharide network solution;

[0107] S5. Use the core layer (AgNPs-lemon essential oil complex) as the oil phase, the middle layer (EGCG-chitosan solution) and the outer layer (enzyme-crosslinked protein-polysaccharide network solution) as the water phase. Under the conditions of a pressure of 12 MPa and a temperature of 35 °C, first introduce supercritical CO2 as the solvent and dispersion medium into the high-pressure mixer, then mix the oil phase and the water phase (the volume ratio of the outer layer, middle layer and core layer is 3:3:4) and inject them into the high-pressure mixer, maintain a pressure of 12 MPa and stir for 10 - 15 minutes to form a stable composite nano-microcapsule emulsion. Release the pressure, collect the emulsion and freeze-dry it (the freeze-drying parameters require a cold trap temperature of -40 ± 1 °C, a freeze-drying chamber pressure of 100 ± 5 Pa, and a material heating plate temperature of 45 ± 0.5 °C) to obtain the freeze-dried powder of nano-microcapsules;

[0108] S6. Add 1.0 g of the freeze-dried powder to every 100 mL of 3% PVA solution by mass, stir at 45 ± 0.5 °C for 1 h, and perform ultrasonic degassing at 420 W for 5 min to obtain the composite fresh-keeping film solution. Subsequently, prepare the composite fresh-keeping film by the casting and drying method, specifically: slowly pour 200 g of the composite fresh-keeping film solution onto a 20 × 20 cm polytetrafluoroethylene plate, place it in an oven at 50 °C and dry for 8 h to obtain the composite fresh-keeping film. After cutting it into a suitable shape, prepare an 18 × 18 cm AgNPs-lemon essential oil-EGCG composite fresh-keeping bag.

[0109] Example 3 Preparation of AgNPs-bergamot essential oil-EGCG composite fresh-keeping bag

[0110] S1. Add an aqueous silver nitrate solution of 0.04 mol / L to sodium citrate (the final concentration of sodium citrate in the aqueous silver nitrate solution is 0.6 mol / L), stir and react until the solution turns brownish-yellow, centrifuge and wash to remove impurities to obtain a pure AgNPs solution;

[0111] S2. Mix 12.8 mg / mL bergamot essential oil with the AgNPs solution, and use ultrasonic treatment (power 200 W, frequency 30 KHz, treatment time 20 min, operating temperature 20 ± 0.5 °C, working mode is pulse working mode (working for 2 s, pausing for 1 s)) to further improve the dispersion effect and form a stable AgNPs-bergamot essential oil complex;

[0112] S3. Dissolve 1% chitosan in a 1% glacial acetic acid solution, heat and stir to dissolve, filter to obtain a clear chitosan solution, then add EGCG to the chitosan solution (the concentration of EGCG in the chitosan solution is 0.5 mg / mL), adjust the pH value to 5.8 ± 0.2, stir evenly to obtain an EGCG-chitosan solution;

[0113] S4. Add a 1% chitosan solution to a 1% gelatin aqueous solution according to a volume ratio of 1:1, stir evenly, then add 1.0 g / L transglutaminase (TGase), adjust the pH value to 6.0, control the temperature at 37 °C - 40 °C, react for 24 hours, and finally heat to 80 °C or add ethanol to terminate the reaction to obtain an enzyme-crosslinked protein-polysaccharide network solution;

[0114] S5. Use the core layer (AgNPs-bergamot essential oil complex) as the oil phase, the middle layer (EGCG-chitosan solution) and the outer layer (enzyme-crosslinked protein-polysaccharide network solution) as the water phase. Under the conditions of a pressure of 10 MPa and a temperature of 35 °C, first introduce supercritical CO2 as the solvent and dispersion medium into the high-pressure mixer, then mix the oil phase and the water phase (the volume ratio of the outer layer, middle layer and core layer is 3:3:4) and inject them into the high-pressure mixer, maintain a pressure of 10 MPa and stir for 10 - 15 minutes to form a stable composite nano-microcapsule emulsion. Release the pressure, collect the emulsion and freeze-dry (the freeze-drying parameters require a cold trap temperature of -40 ± 1 °C, a freeze-drying chamber cavity pressure of 100 ± 5 Pa, and a material heating plate temperature of 45 ± 0.5 °C) to obtain a nano-microcapsule freeze-dried powder;

[0115] S6. Add 1.0 g of freeze-dried powder to 100 mL of 3% PVA solution by mass fraction, stir at 45 ± 0.5 °C for 1 h, and perform ultrasonic degassing at 420 W for 5 min to obtain a composite fresh-keeping film solution. Subsequently, prepare the composite fresh-keeping film by the casting and drying method, specifically: slowly pour 240 g of the composite fresh-keeping film solution onto a 20×20 cm polytetrafluoroethylene plate, place it in an oven at 50 °C for 8 h to obtain the composite fresh-keeping film, cut it into a suitable shape, and then prepare an 18×18 cm AgNPs-bergamot essential oil-EGCG composite fresh-keeping bag.

[0116] Preparation of nano-silver-citrus essential oil-EGCG composite fresh-keeping bag in Comparative Example 1

[0117] The difference from Example 1 is that in step S5, the pressure in the specific parameters of the supercritical CO2 condition is different, and the rest of the operation steps and condition parameters are the same as those in Example 1.

[0118] In this comparative example, the pressure of supercritical CO2 is 8 MPa.

[0119] Preparation of nano-silver-citrus essential oil-EGCG composite fresh-keeping bag in Comparative Example 2

[0120] The difference from Example 1 is that in step S5, the pressure in the specific parameters of the supercritical CO2 condition is different, and the rest of the operation steps and condition parameters are the same as those in Example 1.

[0121] In this comparative example, the pressure of the supercritical CO2 condition is 18 MPa.

[0122] Preparation of nano-silver-citrus essential oil-EGCG composite fresh-keeping bag in Comparative Example 3

[0123] The difference from Example 1 is that in step S5, the volume ratio of the outer layer, the middle layer, and the core layer is different, and the rest of the operation steps and condition parameters are the same as those in Example 1.

[0124] In this comparative example, the volume ratio of the outer layer, the middle layer, and the core layer is 3:3:2.

[0125] Preparation of nano-silver-citrus essential oil-EGCG composite fresh-keeping bag in Comparative Example 4

[0126] The difference from Example 1 is that in step S5, the volume ratio of the outer layer, the middle layer, and the core layer is different, and the rest of the operation steps and condition parameters are the same as those in Example 1.

[0127] In this comparative example, the volume ratio of the outer layer, the middle layer, and the core layer is 3:3:5.

[0128] Preparation of nano-silver-citrus essential oil-EGCG composite fresh-keeping bag in Comparative Example 5

[0129] The difference from Example 1 lies in that: the specific preparation method of the nano-microcapsule freeze-dried powder in steps S2-S4 is different, and the remaining operation steps and condition parameters are the same as those in Example 1.

[0130] The preparation method of the nano-microcapsule freeze-dried powder in this comparative example is to replace steps S2-S5 in Example 1 with the following steps S2-S4:

[0131] S2. Mix 6.4 mg / mL citrus essential oil with the AgNPs solution, and stir magnetically at 20 ± 0.5 °C for 1 h to form a relatively stable AgNPs-citrus essential oil complex;

[0132] S3. Dissolve 2% chitosan in 1% glacial acetic acid solution, heat and stir to dissolve, filter to obtain a clear chitosan solution, and then add the chitosan solution dropwise to the mixture of the AgNPs-citrus essential oil complex prepared in S2 in a volume ratio of 1:1, and continue to stir at a constant temperature in a magnetic stirrer to obtain a chitosan suspension;

[0133] S4. Add a 1% gelatin aqueous solution with a volume ratio of 1:1 to the chitosan suspension, and then add a certain mass of EGCG powder (the concentration of EGCG in the mixed solution is 0.5 mg / mL), stir magnetically at room temperature for 2 h to obtain a relatively stable mixed emulsion and freeze-dry it (the freeze-drying parameter requirements are a cold trap temperature of -40 ± 1 °C, a freeze-drying chamber cavity pressure of 100 ± 5 Pa, and a material heating plate temperature of 45 ± 0.5 °C) to obtain the nano-microcapsule freeze-dried powder.

[0134] Preparation of the nano-silver-citrus essential oil-EGCG composite fresh-keeping bag in Comparative Example 6

[0135] The difference from Example 1 lies in that: the specific preparation method of the nano-microcapsule freeze-dried powder in steps S2-S5 is different, and the remaining operation steps and condition parameters are the same as those in Example 1.

[0136] The preparation method of the nano-microcapsule freeze-dried powder in this comparative example is to replace steps S2-S5 in Example 1 with the following steps S2-S4:

[0137] S2. Mix 6.4 mg / mL citrus essential oil with the AgNPs solution, and then add a certain mass of EGCG powder (the concentration of EGCG in the mixed solution is 0.5 mg / mL), stir magnetically at 20 ± 0.5 °C for 1 h to form a relatively stable AgNPs-citrus essential oil-EGCG complex solution;

[0138] S3. Dissolve 2% chitosan in 1% glacial acetic acid solution, heat and stir to dissolve, filter to obtain a clear chitosan solution, add a 1% gelatin aqueous solution with a volume ratio of 1:1 to the chitosan solution, and continue to stir at a constant temperature in a magnetic stirrer to obtain a chitosan-gelatin mixed solution;

[0139] S4. The chitosan-gelatin mixed solution was dropped into the mixture solution of the AgNPs-citrus essential oil-EGCG complex prepared in S2 at a volume ratio of 1:1, and magnetically stirred at room temperature for 2 h to obtain a relatively stable mixed emulsion, which was then freeze-dried (the freeze-drying parameters required were a cold trap temperature of -40 ± 1°C, a freeze-drying chamber pressure of 100 ± 5 Pa, and a material heating plate temperature of 45 ± 0.5°C) to obtain the freeze-dried powder of nano-microcapsules.

[0140] Preparation of the citrus essential oil-EGCG composite fresh-keeping bag in Comparative Example 7

[0141] The difference from Example 1 was that step S1 was missing, and in step S2, the citrus essential oil with a concentration of 6.4 mg / mL was directly treated under the ultrasonic conditions of Example 1, and the remaining operation steps and condition parameters were the same as those in Example 1.

[0142] Preparation of the AgNPs-EGCG composite fresh-keeping bag in Comparative Example 8

[0143] The difference from Example 1 was that in step S2, no citrus essential oil was added, and the AgNPs solution was directly treated under the ultrasonic conditions of Example 1, and the remaining operation steps and condition parameters were the same as those in Example 1.

[0144] Preparation of the AgNPs-citrus essential oil composite fresh-keeping bag in Comparative Example 9

[0145] The difference from Example 1 was that in step S3, no EGCG was added, and the remaining operation steps and condition parameters were the same as those in Example 1.

[0146] Test Example 4 Comparison of the encapsulation rate and thermal stability of different composite nano-microcapsules

[0147] Experimental materials: The freeze-dried powder of nano-microcapsules prepared in Examples 1-3 and Comparative Examples 1-9.

[0148] Experimental method:

[0149] 1. Encapsulation rate: The encapsulation rate of the composite nano essential oil microcapsules was measured using a UV-visible spectrophotometer at a measurement wavelength of 264 nm. Using anhydrous ethanol as a reference sample, an anhydrous ethanol solution of citrus essential oil with a concentration of 0.25 - 2.00 μL / mL was prepared to obtain the standard curve equation Y = 3.685X + 0.0532 (R 2= 0.9968). Take the freeze-dried nano-microcapsules (2.0 g) and 95% anhydrous ethanol solution (20 mL) and put them into a centrifuge tube to grind until completely broken. After ultrasonicating the mixture for 10 min, centrifuge it at 12,000 r / min for 10 min at 4 °C. The supernatant is the sample extraction liquid. Absorb a certain amount of the sample extraction liquid and dilute it to an appropriate concentration, and measure the absorbance of the sample at 264 nm with a UV-visible spectrophotometer. Calculate the content of citrus essential oil in the freeze-dried nano-microcapsules through the standard curve and calculate the encapsulation rate.

[0150] E = (c × N × V1 × M) / (m × m1) × 100%,

[0151] In the formula: E is the encapsulation rate, %; c is the mass fraction of the measured citrus essential oil, mg / mL; N is the dilution factor of the sample extraction liquid volume during measurement; V1 is the volume of the sample extraction liquid, mL; M is the total mass of the microcapsules, g; m is the mass of the microcapsules taken, g; m1 is the added mass of citrus essential oil in the microcapsules, g.

[0152] 2. Thermal stability of nano-microcapsules: Place the nano-microcapsule sample in an environment at 80 °C for 1 hour. After treatment, release the essential oil in the same method as in the above determination of the encapsulation rate, and determine the content of the essential oil by UV-visible spectrophotometry or gas chromatography, and finally judge the strength of the thermal stability of the nano-microcapsules by calculating the essential oil retention rate.

[0153]

[0154] Experimental results: Figure 1 Figure (a) in shows the results of the encapsulation rate of citrus essential oil by the composite nano-microcapsules in different examples and comparative examples. It can be seen from the figure that the encapsulation rate of the essential oil in the composite nano-microcapsules prepared in Examples 1-3 of the present invention is high. In Comparative Examples 1-4, the supercritical pressure and the ratios of the outer layer, middle layer and core layer in the preparation process were changed, and the encapsulation rate of the essential oil in the prepared composite nano-microcapsules decreased significantly compared with the examples. In Comparative Examples 5 and 6, the preparation method of the microcapsules was changed, and the encapsulation rate of the prepared microcapsules for citrus essential oil decreased sharply. Comparative Examples 7 and 9 lacked silver nanoparticles and EGCG respectively, and their encapsulation rates of citrus essential oil in the microcapsules had no obvious influence. Therefore, from the data of the examples and comparative examples of the present invention, it can be seen that according to the preparation method described in the present invention, under appropriate supercritical carbon dioxide parameter conditions and specific ratios of the outer layer, middle layer and core layer of the microcapsules, the microcapsules with a three-layer structure prepared have a better encapsulation rate for the essential oil.

[0155] Figure 1Figure (b) shows the essential oil retention rate of the composite nano-microcapsules in different embodiments and comparative examples after being treated at 80°C for 1 hour. The higher the retention rate, the stronger the thermal stability of the microcapsules. It can be seen from the figure that the retention rate of the composite nano-microcapsules prepared in the three embodiments after being treated at 80°C for 1 hour is significantly higher than that of other comparative examples, all exceeding 85%. This result shows that the thermal stability of the composite nano-microcapsules prepared by the present invention is significantly higher than that of the composite nano-microcapsules prepared by other operations other than the present invention under the same formula.

[0156] Application Example 1

[0157] The composite fresh-keeping bag and the controlled atmosphere treatment were used to achieve the medium- and long-term preservation of a fresh-cut autumn and winter seasonal vegetable salad (three kinds of vegetable raw materials). The specific implementation steps are as follows:

[0158] (1) Purchase lettuce, rosa greens, and purple cabbage of similar maturity, uniform size, uniform color, and no mechanical damage. Transport them to the laboratory in a 4°C refrigerator within 2 hours of purchase and pre-cool for about 12 hours before use.

[0159] (2) After precooling, the three vegetables were washed with deionized water, and the leaves damaged by squeezing were manually removed, drained and prepared for cutting;

[0160] (3) Cut the three autumn and winter seasonal vegetables into slices according to the processing specifications of head lettuce (6×6cm to 2×2cm slices), rosa greens (6×6cm to 2×2cm slices), and purple cabbage (2.5mm thick shreds);

[0161] (4) according to the recipe composition that each serving of vegetable salad contains 50 g of romaine lettuce slices, 50 g of rossa green slices, and 50 g of purple cabbage shreds, the corresponding mass of raw materials are filled into a composite fresh-keeping bag;

[0162] (5) The gas components were injected into the composite fresh-keeping bag containing the vegetable salad in the proportion of O2: 3%, CO2: 7%, Ar: 10%, and N2: 80%, and then heat-sealed; when the mixed gas was injected, the parameters were set as the exhaust time of 5.0 s, the filling time of 10.0 s, and the heat-sealing time of 4.0±0.5 s.

[0163] (6) After processing, all samples were stored in a dark place at 4±0.5℃ for 2 days, and then placed in a special refrigerated display cabinet at 10±1℃ under normal fluorescent light conditions for further storage.

[0164] In step (5), the composite fresh-keeping bag is: the AgNPs-citrus essential oil-EGCG composite fresh-keeping bag prepared in Example 1, the composite fresh-keeping bag prepared in Comparative Examples 1 to 9, or a commercially available PE+PET packaging bag. All group samples (vegetable salad) were tested for relevant indicators on the 0th day and the 14th day. The results are shown in Tables 1 and 2:

[0165] Comparison of indicators of vegetable salad stored in different composite fresh-keeping bags on the 0th day

[0166]

[0167]

[0168] Comparison of indicators of vegetable salad stored in different composite fresh-keeping bags on the 14th day

[0169]

[0170]

[0171] The experimental results are shown in Table 1 - Table 2. After 14 days of storage with the composite fresh-keeping bag of Example 1, the weight loss rate is only 9.04%, which is 10.9% lower than that of the commercially available product, 2.14% - 2.17% lower than that of Comparative Example 1 and Comparative Example 2, 3.49% - 3.51% lower than that of Comparative Example 3 and Comparative Example 4, 4.05% lower than that of Comparative Example 5, and 5.21% - 5.24% lower than that of Comparative Example 6 - 9. It can be seen that the composite fresh-keeping film of the present invention can significantly reduce the weight loss rate of fresh-cut autumn and winter seasonal vegetable salad during storage, and has a significant effect compared with the vegetable salad preserved by other fresh-keeping film methods with the same formula but different processes or the same process but different formulas of the present invention.

[0172] pH value, conductivity and soluble solids are mainly related to cell damage and tissue content exudation during the storage of fresh-cut vegetable salad. Generally, the longer the storage time, the more significant the pH change, the higher the conductivity value and the more the soluble solid content decreases. From the results, compared with the commercially available sample and the comparative example samples, on the 14th day, the pH value of the commercially available and comparative example samples drops below 4.62, the conductivity values all exceed 258 μs / cm, and the soluble solid content is all lower than 3.05, while the pH value of Example 1 still remains at 4.67, the conductivity value is 251 μs / cm, and the soluble solid content remains at 3.09. It can be seen that the composite fresh-keeping film of the present invention can avoid excessive exudation of cell contents by reducing the damage of vegetable cell tissues, ultimately affecting the product sensory. From Example 1 and Comparative Examples 1 - 4, it can be seen that when the supercritical CO2 process parameters are too high or too low and the proportion range of the volume ratios of the outer layer, middle layer and core layer is inappropriate, the pH value, conductivity and soluble solids of the prepared fresh-keeping film are significantly deteriorated. From Example 1 and Comparative Examples 5 - 6, it can be seen that when the preparation process is changed and it is not prepared in the form of the outer layer, middle layer and core layer of the present invention, the pH value, conductivity and soluble solids of the finally prepared fresh-keeping film are significantly deteriorated. From Example 1 and Comparative Examples 7 - 9, it can be seen that the combination of citrus essential oil, EGCG and AgNPs has a significant effect on the improvement of the effect.

[0173] In terms of nutritional components, the effects of different treatments on the contents of chlorophyll, ascorbic acid and total phenols in fresh-cut vegetables were mainly investigated. The results showed that during storage, Example 1 had higher contents of chlorophyll, ascorbic acid and total phenols compared with commercially available products and other comparative examples. This indicates that the composite fresh-keeping film of the present invention can reduce the loss of nutritional components in fresh-cut vegetable salads during storage. From Example 1 and Comparative Examples 1-4, it can be seen that when the supercritical CO2 process parameters are too high or too low and the proportion range of the volume ratios of the outer layer, the middle layer and the core layer is inappropriate, the contents of chlorophyll, ascorbic acid and total phenols in the prepared fresh-keeping film are significantly deteriorated. From Example 1 and Comparative Examples 5-6, it can be seen that when the preparation process is changed and it is not prepared in the form of the outer layer, the middle layer and the core layer in the present invention, the contents of chlorophyll, ascorbic acid and total phenols in the finally prepared fresh-keeping film are significantly deteriorated. From Example 1 and Comparative Examples 7-9, it can be seen that the combination of citrus essential oil, EGCG and AgNPs has a significant effect on the improvement of the effect.

[0174] In terms of microbial indicators, during storage, the bacteria, molds and yeasts in Example 1 decreased significantly compared with commercially available products and other comparative examples. Thus, it can be seen that the composite fresh-keeping film of the present invention can effectively delay the proliferation of microorganisms such as bacteria and fungi in raw materials, always maintain the microbial proliferation rate at a low level, thereby reducing the damage of microorganisms to the tissue structure of fresh-cut vegetable salads, and further maintaining the loss of moisture, nutritional components and the exudation of contents during their storage. From Example 1 and Comparative Examples 1-4, it can be seen that when the supercritical CO2 process parameters are too high or too low and the proportion range of the volume ratios of the outer layer, the middle layer and the core layer is inappropriate, the bacteria, molds and yeasts in the prepared fresh-keeping film are significantly deteriorated. Although the amount of the core layer in Comparative Example 4 was slightly increased compared with Example 1, the effect was also significantly deteriorated. The inventor speculated that it might be that the different volume ratios of the outer layer, the middle layer and the core layer damaged the multi-layer microcapsule structure, resulting in an imbalance in the release kinetics and rapid release in the short term, thus failing to play a long-term antibacterial role; in addition, high-concentration silver nanoparticles might aggregate, resulting in a decrease in the specific surface area and ultimately affecting the antibacterial effect.

[0175] From Example 1 and Comparative Examples 5-6, it can be seen that when the preparation process is changed and it is not prepared in the form of the outer layer, the middle layer and the core layer in the present invention, the bacteria, molds and yeasts in the finally prepared fresh-keeping film increase significantly. From Example 1 and Comparative Examples 7-9, it can be seen that the combination of citrus essential oil, EGCG and AgNPs has a significant effect on the improvement of the effect.

[0176] The main substrates of peroxidase are composed of phenols, which, together with polyphenol oxidase, are responsible for the development of enzymatic browning in fruits and vegetables. During the storage of fruits and vegetables, the destruction of cell structure often leads to the collapse of the enzyme-substrate system that could not be directly contacted originally, thus increasing the contact between related enzymes and substrates, resulting in an increase in enzyme activity and accelerating the aging and deterioration of fresh-cut vegetable cells. Judging from the results, on the 14th day, the peroxidase activity in Example 1 was maintained at 54 U / min·g, while that in the commercial example reached 71 U / min·g, and that in other comparative examples also exceeded 58 U / min·g; the results of polyphenol oxidase were similar to those of peroxidase, indicating that the composite fresh-keeping film of the present invention can reduce the browning and oxidation of fruits and vegetables.

[0177] In terms of the comprehensive sensory score, as the storage time increased, the sensory score of the commercial product decreased sharply, while the sensory scores of the examples and different comparative examples decreased more slowly. By the 14th day of storage, the score of the commercial example was lower than 6.2, and those of the comparative examples were also lower than 7.3, while the sensory score of Example 1 still remained at a good level (>7.5). It can be seen that the fresh-keeping film prepared by the present invention has a better overall fresh-keeping effect on autumn and winter vegetable salads than the fresh-keeping films prepared by commercial samples and comparative examples.

[0178] Application Example 2

[0179] The composite fresh-keeping film cooperates with the controlled atmosphere treatment to achieve medium and long-term fresh-keeping of a fresh-cut autumn and winter seasonal vegetable salad (5 kinds of vegetable raw materials). The specific implementation steps are as follows:

[0180] (1) Purchase romaine lettuce, Rosa green, purple cabbage, romaine lettuce and Rosa red with similar maturity, uniform size, uniform color and no mechanical damage, and transport them to a 4°C refrigerator in the laboratory for pre-cooling for about 12 h within 2 h after purchase for standby;

[0181] (2) After pre-cooling, wash these five kinds of vegetables with deionized water, manually remove the squeezed and damaged leaves, drain and prepare for cutting;

[0182] (3) Cut these five kinds of autumn and winter seasonal vegetables according to the processing specifications of romaine lettuce (sliced into pieces with sizes ranging from 6×6 cm to 2×2 cm), Rosa green (sliced into pieces with sizes ranging from 6×6 cm to 2×2 cm), purple cabbage (shredded with a thickness of 2.5 mm), romaine lettuce (sliced into pieces with sizes ranging from 6×6 cm to 2×2 cm), and Rosa red (cut into sections with a length of 2.5 cm);

[0183] (4) According to the formula composition of each portion of vegetable salad containing 30 g of romaine lettuce slices, 30 g of Rosa green slices, 30 g of purple cabbage shreds, 30 g of romaine lettuce slices and 30 g of Rosa red slices, load the corresponding quality raw materials into an 18×18 cm composite fresh-keeping bag;

[0184] (5) Flush the gas components into the composite fresh-keeping bag containing vegetable salad according to the ratio of O2: 7%, CO2: 5%, Ar: 7%, and N2: 81% and heat-seal it;

[0185] (6) After the treatment, all samples are stored in the dark at 4 ± 0.5 °C for 2 days, and then stored in a dedicated refrigerated display cabinet at 10 ± 1 °C under normal fluorescent lamp conditions.

[0186] The composite fresh-keeping bag is: the lemon essential oil-EGCG composite fresh-keeping bag prepared in Example 2 or a commercially available PE+PET packaging bag. The relevant indicators of all groups of samples (vegetable salad) are measured on the 0th day and the 14th day, and the results are shown in Table 3:

[0187] Table 3 Comparison of key node indicators of storing vegetable salad with different composite fresh-keeping bags

[0188]

[0189]

[0190] As shown in Table 3 of the experimental results, after 14 days of storage with the composite fresh-keeping bag of Example 2, the weight loss rate is only 9.02%, which is 10.85% lower than that of the commercially available product. It can be seen that the composite fresh-keeping film of the present invention can significantly reduce the weight loss rate of fresh-cut autumn and winter seasonal vegetable salad during storage.

[0191] The results of pH value, conductivity and soluble solids show that, compared with the commercially available product, on the 14th day, the pH value of the commercially available product drops below 4.255, the conductivity value exceeds 260 μs / cm, and the soluble solids content is lower than 2.5. On the contrary, the pH value of Example 2 still remains above 4.6, the conductivity value is below 250 μs / cm, and the soluble solids content remains above 2.8. It can be seen that the composite fresh-keeping film of the present invention can avoid excessive exudation of cell contents by reducing the damage of vegetable cell tissues, ultimately affecting the product sensory.

[0192] The results of chlorophyll, ascorbic acid and total phenol content show that during storage, Example 2 has higher chlorophyll, ascorbic acid and total phenol content compared with the commercially available product. This indicates that the composite fresh-keeping film of the present invention can reduce the loss of nutrients in fresh-cut vegetable salad during storage.

[0193] In terms of microbial indicators, during storage, the bacteria, molds and yeasts of Example 2 decreased significantly compared with the commercially available product. It can be seen that the composite fresh-keeping film of the present invention can effectively delay the proliferation of microorganisms such as bacteria and fungi in the raw materials, always maintain the microbial proliferation rate at a low level, thereby reducing the damage of microorganisms to the tissue structure of fresh-cut vegetable salad, and further maintaining the loss of moisture, nutrients and the exudation of contents during its storage.

[0194] From the results of peroxidase and polyphenol oxidase activity, when stored for 14 days, the peroxidase activity in Example 2 was always maintained below 65 U / min·g, while the commercially available product reached 70 U / min·g on the 14th day; the polyphenol oxidase result was similar to the trend of peroxidase, indicating that the composite preservative film of the present invention can reduce browning and oxidation of fruits and vegetables.

[0195] In terms of comprehensive sensory scores, as the storage time increases, the sensory scores of the commercial products drop sharply, while the sensory scores of Example 2 are still maintained at a good score.

[0196] Application Example 3

[0197] Nanosilver-bergamot essential oil-EGCG composite cling film synergistic atmosphere treatment achieves medium- and long-term preservation of a fresh-cut autumn and winter seasonal vegetable salad (7 kinds of vegetable raw materials). The specific implementation steps are as follows:

[0198] (1) Purchase lettuce, rosa green, purple cabbage, romaine lettuce, rosa red, fruit cucumbers and cherry tomatoes of similar maturity, uniform size, uniform color and no mechanical damage, and transport them to the laboratory in a 4℃ refrigerator within 2 hours of purchase for precooling for about 12 hours.

[0199] (2) After precooling, the five vegetables were washed with deionized water, and the leaves and bad fruits that were damaged by squeezing were manually removed, and then drained and prepared for cutting;

[0200] (3) Cut the seven kinds of autumn and winter seasonal vegetables according to the processing specifications of head lettuce (6×6cm~2×2cm different slices), rosa green (6×6cm~2×2cm different slices), purple cabbage (2.5mm thick shreds), romaine lettuce (6×6cm~2×2cm different slices), rosa red (2.5cm long segments), fruit cucumber (2.5mm thick slices), and cherry tomatoes (whole);

[0201] (4) According to the recipe composition of each serving of vegetable salad containing 30 g of romaine lettuce slices, 25 g of rosa green slices, 20 g of purple cabbage shreds, 25 g of romaine lettuce slices, 20 g of rosa red slices, 20 g of fruit cucumber slices, and 20 g of cherry tomatoes, the corresponding mass of raw materials were filled into a 18×18 cm composite fresh-keeping bag;

[0202] (5) injecting gas components into a packaging bag containing vegetable salad in a ratio of O2: 7%, CO2: 5%, Ar: 7%, and N2: 81% and heat-sealing the bag;

[0203] (6) After processing, all samples were stored in a dark place at 4±0.5℃ for 2 days, and then placed in a special refrigerated display cabinet at 10±1℃ under normal fluorescent light conditions for further storage.

[0204] The composite fresh-keeping bag is: the lemon essential oil-EGCG composite fresh-keeping bag prepared in Example 2 or the commercially available PE+PET packaging bag. Relevant indicators of all groups of samples (vegetable salad) were measured on the 0th day and the 14th day, and the results are shown in Table 5:

[0205] Table 4 Comparison of key node indicators of different composite fresh-keeping bags for storing vegetable salad

[0206]

[0207]

[0208] As can be seen from Table 4, compared with the commercially available PE+PET packaging bag, the composite fresh-keeping bag in Example 3 can significantly reduce the weight loss rate of vegetables and the exudation of cell contents during storage. At the same time, for nutrients such as chlorophyll, ascorbic acid, and total phenols, the packaging in Example 3 can effectively alleviate the sharp decline of nutrients during storage, reduce the growth of microorganisms and the browning of fruits and vegetables during storage, and can also maintain a better sensory score.

[0209] In summary, the composite fresh-keeping film of the present invention mainly works through: 1) The nano-silver, citrus essential oil, and EGCG active substances in the composite fresh-keeping film maintain a continuous inhibitory effect on microorganisms such as bacteria and fungi throughout the storage process of fresh-cut vegetable salad, achieving long-term control of the microbial population; 2) The combined antioxidant effect of nano-silver, essential oil, and EGCG in the composite fresh-keeping film packaging affects the enzyme activity on the surface of the cell membrane of fresh-cut fruits and vegetables. Combined with the control of microorganisms, the relative stability of the cell structure during the storage process of fresh-cut vegetable salad is achieved, thereby reducing the weight loss rate and the exudation of cell contents; 3) The composite fresh-keeping film cooperates with the modified atmosphere packaging to maintain the stability of the gas environment inside the package throughout the storage process of fresh-cut vegetable salad, reducing the accelerated rate of deterioration of the sensory quality caused by the respiration and oxidation of the cells themselves, and ultimately achieving the maintenance of the overall sensory quality of fresh-cut vegetable salad.

[0210] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing nano microcapsules, characterized in that, It includes the following steps: S1. Preparation of the core layer: Mix citrus essential oil and nano-silver solution, and perform ultrasonic treatment to obtain the core layer. S2. Preparation of the intermediate layer: Add epigallocatechin gallate to the polysaccharide solution, and adjust the pH value to 5.4 - 6.2 to obtain the intermediate layer. S3. Preparation of the outer layer: Mix the protein aqueous solution and the polysaccharide solution, add transglutaminase, and adjust the pH value to 6.0 - 7.

0. After the reaction is complete, terminate the reaction to obtain the outer layer. S4. Preparation of the nano-microcapsules: Under the condition of supercritical CO2 with a pressure of 10 - 12 MPa, mix the core layer prepared in S1, the intermediate layer prepared in S2, and the outer layer prepared in S3 to obtain a composite nano-microcapsule emulsion, and then perform freeze-drying to obtain the nano-microcapsules. In the core layer, the concentration ratio of nano-silver to citrus essential oil is 0.03 - 0.05 mol / L: 6.4 - 12.8 g / L. In the intermediate layer, the concentration of epigallocatechin gallate is 0.5 - 1 mg / mL. The volume ratio of the core layer: intermediate layer: outer layer is 3:3:3 - 4.

2. The preparation method according to claim 1, characterized in that, The preparation method of the nano-silver solution includes the following steps: Dissolve a silver salt in water, add a reducing agent, stir and react until the solution changes color, and remove impurities to obtain the nano-silver solution.

3. The preparation method according to claim 1, wherein The citrus essential oil is one or more of bergamot essential oil, lemon essential oil, citrus essential oil, sweet orange essential oil, and grapefruit essential oil.

4. The preparation method according to claim 1, characterized in that, In step S3, the protein is one or more of gelatin, whey protein, soy protein, zein, and wheat gluten protein.

5. According to the preparation method described in claim 1, characterized in that, In steps S2 and S3, the polysaccharide is one or more of chitosan, arabic gum, cellulose, corn starch, cyclodextrin, and pullulan polysaccharide.

6. The nano-microcapsules prepared by the preparation method according to any one of claims 1 - 5.

7. A preparation method of a composite fresh-keeping film, characterized in that, Add the nano-microcapsules according to claim 6 to a polyethylene glycol solution, and perform post-treatment to obtain a composite fresh-keeping film.

8. The composite fresh-keeping film prepared by the preparation method according to claim 7.

9. The application of the composite fresh-keeping film according to claim 8 in the fresh-keeping of ready-to-eat vegetable mixed salad.

10. A method for preservation, characterized in that, It includes the following steps: Package the vegetables with the composite fresh-keeping film according to claim 8.

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