Grape storage and fresh-keeping process based on microorganisms

Abstract

The invention relates to the technical field of grape storage and fresh-keeping processes, in particular to a grape storage and fresh-keeping process based on microorganisms, which comprises the following steps: (1) pretreating grapes; (2) preparing a microbial culture solution; (3) preparing a preservative formula; (4) treating with a preservative; (5) packaging and storing, so that the storage period of the grapes is remarkably prolonged from traditional 3-4 weeks to 6 weeks or above, and meanwhile, relatively high sensory quality is kept; the growth of harmful microorganisms is effectively inhibited, and the total bacterial count and the mould yeast count are only increased to 2.69 and 2.18 lg CFU / g respectively after 6 weeks; the weight loss rate is obviously reduced, is only 3.08% after 6 weeks, and is 50% or above lower than that of a control group; the fruit hardness is effectively maintained, and the fruit hardness is still maintained at 3.52 N after 6 weeks; the retention rate of vitamin C is high, can still reach 90.2% after 6 weeks and is 30% or above higher than that of a control group; the overall sensory quality is excellent, and the overall acceptance score after 6 weeks reaches 8.0.

Description

Technical Field

[0001] The invention relates to the technical field of grape storage and preservation technology, and in particular to a grape storage and preservation technology based on microorganisms. Background Art

[0002] As an important economic crop and a popular fruit, the storage and preservation of grapes has always been a research hotspot in the field of fruits and vegetables. Traditional methods of grape preservation mainly include low-temperature storage, chemical preservative treatment and controlled atmosphere storage. However, these methods have some limitations. First, although simple low-temperature storage can delay fruit metabolism, it cannot effectively inhibit the growth of microorganisms and easily lead to fruit rot. Secondly, although chemical preservatives can inhibit microorganisms, they may bring food safety risks and environmental pollution problems. Furthermore, although controlled atmosphere storage can extend the shelf life, the equipment investment cost is high and it is not suitable for small-scale or short-term storage.

[0003] In recent years, biological preservation technology has attracted widespread attention due to its safety and environmental protection. Among them, the application of microbial preservatives shows good prospects. Some studies have shown that certain lactic acid bacteria and yeast strains have the ability to inhibit the growth of pathogenic microorganisms. However, there are still some problems with the existing microbial preservation technology. First, the antibacterial spectrum of a single strain is narrow, and it is difficult to cope with the complex microecological environment on the surface of the fruit. Secondly, the colonization and survival of microorganisms on the surface of the fruit are still facing challenges, which affects the durability of the preservation effect. In addition, the synergistic mechanism between microbial preservatives and fruit quality maintenance has not been fully studied and utilized.

[0004] In view of this, it is of great practical significance to develop a new technology for grape storage and preservation that can comprehensively utilize microbial antagonism, physical barrier protection and physiological regulation. Summary of the invention

[0005] The present invention aims to solve the following technical problems: how to construct a stable and efficient microbial composite system, how to improve the colonization ability of microorganisms on the surface of fruits, how to achieve synergistic enhancement of microbial preservation and fruit quality maintenance, and how to prolong the duration of the preservation effect.

[0006] The object of the present invention is to provide a microbial-based grape storage and preservation process, comprising the following steps:

[0007] (1) Grape pretreatment;

[0008] (2) Preparation of microbial culture fluid;

[0009] (3) Preparation of preservative formula;

[0010] (4) Treatment with preservatives;

[0011] (5) Packaging and storage.

[0012] Preferably, the grape pretreatment in step (1) comprises:

[0013] First, fully ripe grapes are picked, and moldy and damaged grapes are removed;

[0014] Secondly, soaking the grapes in a 0.5-1.0% (weight percentage) citric acid solution for 5-10 minutes;

[0015] Then, rinse the grapes with sterile water 2-3 times, 1-2 minutes each time;

[0016] Finally, air-dry at 20-25°C for 30-60 minutes.

[0017] Preferably, the preparation of the microbial culture solution in step (2) comprises:

[0018] First, select lactic acid bacteria Lactobacillus plantarum and yeast Metschnikowiapulcherrima;

[0019] Secondly, culture in MRS medium and YPD medium for 24-48 hours at 28-30°C;

[0020] Then, the cells were collected by centrifugation (4000-5000 rpm, 10 min) and washed twice with sterile water;

[0021] Finally, the cells were resuspended in sterile water and the concentration was adjusted to 10 8 -10 9 CFU / mL.

[0022] Preferably, the preservative formulation in step (3) is prepared according to the following weight parts:

[0023] Microbial culture fluid (1:1 mixture): 80-100 copies

[0024] Chitosan (molecular weight 100-300 kDa, deacetylation degree 85-95%): 1-3 parts

[0025] Alginic acid (molecular weight 50-100 kDa): 0.5-1.5 parts

[0026] Xylan (molecular weight 10-30kDa): 0.3-1 part

[0027] Glycerin: 2-4 parts

[0028] Sodium citrate: 0.5-1 part

[0029] Vitamin C: 0.1-0.3 parts

[0030] Sterile water: appropriate amount, adjust the total volume to 100 parts

[0031] Preferably, the preparation of the preservative formulation in step (3) comprises the following steps:

[0032] (a) dissolving the chitosan in a 1% (v / v) acetic acid solution and stirring for 2-3 hours until the chitosan is completely dissolved;

[0033] (b) dissolving the alginic acid and xylan in sterile water at 50° C. and stirring for 1-2 hours;

[0034] (c) dissolving the glycerol, sodium citrate and vitamin C in a small amount of water;

[0035] (d) slowly adding the solutions of steps (a) to (c) to the microbial culture solution in sequence, and stirring evenly at 25-30° C. for 2-3 hours;

[0036] (e) adjusting the pH to 5.8-6.2 with 1 mol / L NaOH;

[0037] (f) Store at 4°C under sterile conditions until use.

[0038] Preferably, the preservative treatment in step (4) comprises:

[0039] First, soak the pretreated grapes in a preservative for 3-5 minutes;

[0040] Then, take it out and let it dry naturally under sterile conditions for 30-60 minutes.

[0041] Preferably, the packaging and storage in step (5) comprises:

[0042] First, the treated grapes are placed in polyethylene fresh-keeping bags, 500-1000g per bag;

[0043] Secondly, punch holes in the fresh-keeping bag with a diameter of 0.3-0.5mm, every 100cm 2 Area 3-5 holes;

[0044] Finally, the packaged grapes are stored in a cold room at 1-3°C and 90-95% relative humidity.

[0045] Preferably, the lactic acid bacteria Lactobacillus plantarum and the yeast Metschnikowia pulcherrima in the microbial culture solution are mixed in a ratio of 1:1.

[0046] Preferably, the chitosan, alginate and xylan in the preservative formula form a composite protective layer.

[0047] Preferably, the glycerol in the preservative formula is used as a plasticizer to improve the flexibility of the protective layer and adjust the osmotic pressure.

[0048] The present invention proposes an innovative grape storage and preservation process based on microorganisms by deeply studying the synergistic mechanism between microorganisms, the formation principle of the polysaccharide composite protective layer, and the molecular mechanism of fruit physiological regulation. The core innovations of this process include:

[0049] 1. Microbial composite system: The present invention uses lactic acid bacteria Lactobacillus plantarum and yeast Metschnikowia pulcherrima to construct a composite system. These two microorganisms are complementary in terms of metabolites and action mechanisms. L. plantarum can produce organic acids such as lactic acid, reduce pH, and inhibit the growth of pathogens; while M. pulcherrima can produce pulcherriminic acid, which inhibits fungal growth by chelating iron ions. This composite system not only expands the antibacterial spectrum, but also forms a stable microecological balance.

[0050] 2. Polysaccharide composite protective layer: This invention innovatively combines chitosan, alginate and xylan. Chitosan (β-1,4-linked D-glucosamine) has good film-forming properties and antibacterial activity; alginate (copolymer of α-L-guluronate and β-D-mannuronate) provides excellent water retention; xylan (β-1,4-linked D-xylose main chain with α-L-arabinofuranose side chain) increases the flexibility and gas exchange capacity of the protective layer. These three polysaccharides form a unique network structure through hydrogen bonds and electrostatic interactions, which can provide a physical barrier and regulate the microenvironment.

[0051] 3. Precise control of pH value: The present invention accurately controls the pH value of the preservative within the range of 5.8-6.2 by adding a sodium citrate buffer system. This pH range is not only conducive to the growth and metabolism of L. plantarum and M. pulcherrima, but also maintains the stability of the polysaccharide network structure. In particular, this pH value can partially protonate chitosan and enhance its binding force with the fruit surface, while not excessively inhibiting the respiratory metabolism of the fruit.

[0052] 4. Antioxidant synergistic effect: The present invention adds vitamin C to the preservative, which not only directly provides antioxidant protection, but also forms a synergistic antioxidant system with polyphenols produced by microbial metabolism (such as p-hydroxyphenyllactic acid produced by L. plantarum). This synergistic effect effectively delays the browning and aging process of fruits by scavenging free radicals and chelating metal ions.

[0053] 5. Multiple functions of plasticizer: The present invention uses glycerol as a plasticizer. The three hydroxyl groups in its molecules can form hydrogen bonds with polysaccharide molecules to increase the flexibility of the protective layer. At the same time, glycerol also participates in osmotic regulation, reduces water loss, and maintains the turgor pressure and hardness of the fruit.

[0054] These innovations achieve multiple synergistic effects through complex molecular interactions and physiological and biochemical processes. For example, microbial metabolites (such as lactic acid) may partially cross-link with chitosan to enhance the mechanical strength of the protective layer; the introduction of xylan may change the pore structure of the polysaccharide network and optimize the gas exchange capacity; and the precise control of pH value may affect the activity of fruit cell wall enzymes and delay fruit softening.

[0055] The beneficial effects of the present invention include:

[0056] 1. Significantly extend the grape storage period from the traditional 3-4 weeks to more than 6 weeks while maintaining high sensory quality.

[0057] 2. Effectively inhibit the growth of harmful microorganisms. After 6 weeks, the total bacterial count and mold and yeast count only increased to 2.69 and 2.18 lg CFU / g respectively.

[0058] 3. Significantly reduced the weight loss rate, which was only 3.08% after 6 weeks, more than 50% lower than the control group.

[0059] 4. Effectively maintain the fruit hardness, which remains at 3.52N after 6 weeks, more than 20% higher than the control group.

[0060] 5. The vitamin C retention rate was high, still reaching 90.2% after 6 weeks, which was more than 30% higher than the control group.

[0061] 6. The overall sensory quality is excellent, with an overall acceptance score of 8.0 (out of 9) after 6 weeks.

[0062] 7. The process is simple, the raw materials are safe and environmentally friendly, and it is easy to promote and apply.

[0063] In summary, the present invention has developed a new technology for grape storage and preservation that is efficient, safe and environmentally friendly through the cross-integration of multidisciplinary knowledge such as microbiology, food science, polymer chemistry and plant physiology, providing a new technical path and theoretical basis for the field of fruit and vegetable preservation. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0065] Example 1: A microbial-based grape storage and preservation process

[0066] The grape storage and fresh-keeping process of the present embodiment comprises the following steps:

[0067] (1) Grape pretreatment: First, fully ripe grapes were picked and moldy and damaged grapes were removed. Second, the grapes were soaked in a 0.5% (weight percentage) citric acid solution for 5 minutes. Then, the grapes were rinsed with sterile water twice, each for 1 minute. Finally, the grapes were air-dried at 20°C for 30 minutes.

[0068] (2) Preparation of microbial culture medium: Select lactic acid bacteria Lactobacillus plantarum and yeast Metschnikowia pulcherrima. L. plantarum was cultured in MRS medium for 24 hours, and M. pulcherrima was cultured in YPD medium for 24 hours, and the culture temperature was 28°C. Subsequently, the cells were collected by centrifugation at 4000 rpm for 10 minutes and washed twice with sterile water. Finally, the cells were resuspended in sterile water and the concentration was adjusted to 10^8 CFU / mL.

[0069] (3) Preparation of preservative formula: Prepare the preservative according to the following weight parts:

[0070] Microbial culture solution (1:1 mixture): 80 parts

[0071] Chitosan (molecular weight 100 kDa, degree of deacetylation 85%): 1 part

[0072] Alginic acid (molecular weight 50kDa): 0.5 parts

[0073] Xylan (molecular weight 10kDa): 0.3 parts

[0074] Glycerin: 2 parts

[0075] Sodium citrate: 0.5 parts

[0076] Vitamin C: 0.1

[0077] Sterile water: appropriate amount, adjust the total volume to 100 parts

[0078] The preparation steps of the preservative are as follows:

[0079] (a) Chitosan was dissolved in 1% (v / v) acetic acid solution and stirred for 2 hours until it was completely dissolved.

[0080] (b) Alginic acid and xylan were dissolved in sterile water at 50°C and stirred for 1 hour.

[0081] (c) Dissolve glycerol, sodium citrate and vitamin C in a small amount of water.

[0082] (d) The above solutions were slowly added to the microbial culture solution in sequence and stirred evenly at 25°C for 2 hours.

[0083] (e) Adjust the pH to 5.8 with 1 mol / L NaOH.

[0084] (f) Store at 4°C under sterile conditions until use.

[0085] (4) Preservative treatment: The pretreated grapes were immersed in the preservative for 3 minutes and then naturally dried under sterile conditions for 30 minutes.

[0086] (5) Packaging and storage: Put the processed grapes into polyethylene fresh-keeping bags, 500g per bag. Punch holes in the fresh-keeping bags with a diameter of 0.3mm and 100cm apart. 2 Finally, the packaged grapes were stored in a cold room at 1°C and 90% relative humidity.

[0087] In this embodiment, the lactic acid bacteria and yeast in the microbial culture solution can produce organic acids and antimicrobial peptides, which effectively inhibit the growth of corrupt microorganisms. The composite protective layer formed by chitosan, alginic acid and xylan not only has antibacterial effects, but also reduces water loss and delays fruit aging. Glycerol, as a plasticizer, improves the flexibility of the protective layer and plays an osmotic regulating role, further reducing the water loss of grapes. The addition of sodium citrate and vitamin C helps to adjust the pH value and provide antioxidant protection, slowing down the browning process of grapes.

[0088] Example 2: A microbial-based grape storage and preservation process

[0089] The grape storage and fresh-keeping process of the present embodiment comprises the following steps:

[0090] (1) Grape pretreatment: First, fully ripe grapes were picked and moldy and damaged grapes were removed. Second, the grapes were soaked in a 0.75% (weight percentage) citric acid solution for 7.5 minutes. Then, the grapes were rinsed with sterile water for 3 times, each time for 1.5 minutes. Finally, the grapes were air-dried at 22.5°C for 45 minutes.

[0091] (2) Preparation of microbial culture medium: Select lactic acid bacteria Lactobacillus plantarum and yeast Metschnikowia pulcherrima. L. plantarum was cultured in MRS medium for 36 hours, and M. pulcherrima was cultured in YPD medium for 36 hours, and the culture temperature was 29°C. Subsequently, the cells were collected by centrifugation at 4500 rpm for 10 minutes and washed twice with sterile water. Finally, the cells were resuspended in sterile water and the concentration was adjusted to 5×10^8 CFU / mL.

[0092] (3) Preparation of preservative formula: Prepare the preservative according to the following weight parts:

[0093] Microbial culture solution (1:1 mixture): 90 parts

[0094] Chitosan (molecular weight 200 kDa, deacetylation degree 90%): 2 parts

[0095] Alginic acid (molecular weight 75kDa): 1 part

[0096] Xylan (molecular weight 20 kDa): 0.65 parts

[0097] Glycerin: 3 parts

[0098] Sodium citrate: 0.75 parts

[0099] Vitamin C: 0.2 parts

[0100] Sterile water: appropriate amount, adjust the total volume to 100 parts

[0101] The preparation steps of the preservative are as follows:

[0102] (a) Chitosan was dissolved in 1% (v / v) acetic acid solution and stirred for 2.5 hours until it was completely dissolved.

[0103] (b) Alginic acid and xylan were dissolved in sterile water at 50°C and stirred for 1.5 hours.

[0104] (c) Dissolve glycerol, sodium citrate and vitamin C in a small amount of water.

[0105] (d) The above solutions were slowly added to the microbial culture solution in sequence and stirred evenly at 27.5°C for 2.5 hours.

[0106] (e) Adjust the pH to 6.0 with 1 mol / L NaOH.

[0107] (f) Store at 4°C under sterile conditions until use.

[0108] (4) Preservative treatment: The pretreated grapes were immersed in the preservative for 4 minutes and then naturally dried under sterile conditions for 45 minutes.

[0109] (5) Packaging and storage: Put the processed grapes into polyethylene fresh-keeping bags, 750g per bag. Punch holes in the fresh-keeping bags with a diameter of 0.4mm and 100cm apart. 2 Finally, the packaged grapes were stored in a cold room at 2°C and 92.5% relative humidity.

[0110] In this embodiment, the biological preservation effect can be enhanced by increasing the concentration and proportion of the microbial culture solution. At the same time, the amount and molecular weight of chitosan, alginic acid and xylan are increased to form a denser composite protective layer, further enhancing the water retention and antibacterial properties. Increasing the amount of glycerol can increase the flexibility of the protective layer and better adapt to changes on the fruit surface.

[0111] Example 3: A microbial-based grape storage and preservation process

[0112] The grape storage and fresh-keeping process of the present embodiment comprises the following steps:

[0113] (1) Grape pretreatment: First, fully ripe grapes were picked and moldy and damaged grapes were removed. Second, the grapes were soaked in a 1.0% (weight percentage) citric acid solution for 10 minutes. Then, the grapes were rinsed with sterile water for 3 times, 2 minutes each time. Finally, the grapes were air-dried at 25°C for 60 minutes.

[0114] (2) Preparation of microbial culture medium: Select lactic acid bacteria Lactobacillus plantarum and yeast Metschnikowia pulcherrima. L. plantarum was cultured in MRS medium for 48 hours, and M. pulcherrima was cultured in YPD medium for 48 hours, and the culture temperature was 30°C. Subsequently, the cells were collected by centrifugation at 5000 rpm for 10 minutes and washed twice with sterile water. Finally, the cells were resuspended in sterile water and the concentration was adjusted to 10^9 CFU / mL.

[0115] (3) Preparation of preservative formula: Prepare the preservative according to the following weight parts:

[0116] Microbial culture solution (1:1 mixture): 100 copies

[0117] Chitosan (molecular weight 300 kDa, deacetylation degree 95%): 3 parts

[0118] Alginic acid (molecular weight 100kDa): 1.5 parts

[0119] Xylan (molecular weight 30kDa): 1 part

[0120] Glycerin: 4 parts

[0121] Sodium citrate: 1 part

[0122] Vitamin C: 0.3 parts

[0123] Sterile water: appropriate amount, adjust the total volume to 100 parts

[0124] The preparation steps of the preservative are as follows:

[0125] (a) Chitosan was dissolved in 1% (v / v) acetic acid solution and stirred for 3 hours until it was completely dissolved.

[0126] (b) Alginic acid and xylan were dissolved in sterile water at 50°C and stirred for 2 hours.

[0127] (c) Dissolve glycerol, sodium citrate and vitamin C in a small amount of water.

[0128] (d) The above solutions were slowly added to the microbial culture solution in sequence and stirred evenly at 30°C for 3 hours.

[0129] (e) Adjust the pH to 6.2 with 1 mol / L NaOH.

[0130] (f) Store at 4°C under sterile conditions until use.

[0131] (4) Preservative treatment: The pretreated grapes were immersed in the preservative for 5 minutes and then naturally dried under sterile conditions for 60 minutes.

[0132] (5) Packaging and storage: Put the processed grapes into polyethylene fresh-keeping bags, 1000g per bag. Punch holes in the fresh-keeping bags with a diameter of 0.5mm and 100cm per bag. 2 Finally, the packaged grapes were stored in a cold room at 3°C ​​and 95% relative humidity.

[0133] In this example, the most dense and effective protective layer is formed by using the highest concentration of microbial culture fluid and the highest molecular weight polysaccharide. This formula can provide the best protection effect in a high humidity environment, especially for long-term storage or transportation conditions with relatively poor conditions. At the same time, the increased vitamin C content can provide stronger antioxidant protection and further delay fruit aging.

[0134] Example 4: A microbial-based grape storage and preservation process

[0135] The grape storage and fresh-keeping process of the present embodiment comprises the following steps:

[0136] (1) Grape pretreatment: First, fully ripe grapes were picked and moldy and damaged grapes were removed. Second, the grapes were soaked in a 0.625% (weight percentage) citric acid solution for 6.25 minutes. Then, the grapes were rinsed with sterile water twice, each for 1.25 minutes. Finally, the grapes were air-dried at 21.25°C for 37.5 minutes.

[0137] (2) Preparation of microbial culture medium: Select lactic acid bacteria Lactobacillus plantarum and yeast Metschnikowia pulcherrima. L. plantarum was cultured in MRS medium for 30 hours, and M. pulcherrima was cultured in YPD medium for 30 hours, and the culture temperature was 28.5°C. Subsequently, the cells were collected by centrifugation at 4250 rpm for 10 minutes and washed twice with sterile water. Finally, the cells were resuspended in sterile water and the concentration was adjusted to 2.5×10^8 CFU / mL.

[0138] (3) Preparation of preservative formula: Prepare the preservative according to the following weight parts:

[0139] Microbial culture fluid (1:1 mixture): 85 parts

[0140] Chitosan (molecular weight 150 kDa, degree of deacetylation 87.5%): 1.5 parts

[0141] Alginic acid (molecular weight 62.5 kDa): 0.75 parts

[0142] Xylan (molecular weight 15 kDa): 0.475 parts

[0143] Glycerin: 2.5 parts

[0144] Sodium citrate: 0.625 parts

[0145] Vitamin C: 0.15 parts

[0146] Sterile water: appropriate amount, adjust the total volume to 100 parts

[0147] The preparation steps of the preservative are as follows:

[0148] (a) Chitosan was dissolved in 1% (v / v) acetic acid solution and stirred for 2.25 hours until it was completely dissolved.

[0149] (b) Alginic acid and xylan were dissolved in sterile water at 50°C and stirred for 1.25 hours.

[0150] (c) Dissolve glycerol, sodium citrate and vitamin C in a small amount of water.

[0151] (d) The above solutions were slowly added to the microbial culture solution in sequence and stirred evenly at 26.25°C for 2.25 hours.

[0152] (e) Use 1 mol / L NaOH to continue the above:

[0153] (e) Adjust the pH to 5.9 with 1 mol / L NaOH.

[0154] (f) Store at 4°C under sterile conditions until use.

[0155] (4) Preservative treatment: The pretreated grapes were immersed in the preservative for 3.5 minutes and then naturally dried under sterile conditions for 37.5 minutes.

[0156] (5) Packaging and storage: Put the processed grapes into polyethylene fresh-keeping bags, 625g per bag. Punch holes in the fresh-keeping bags with a diameter of 0.35mm and place them every 100cm. 2 Finally, the packaged grapes were stored in a cold room at 1.5°C and 91.25% relative humidity.

[0157] In this embodiment, the intermediate values ​​of various parameters are used to balance the preservation effect and economy. This formula is suitable for most conventional storage conditions and can provide comprehensive protection for grapes. The concentration of the microbial culture solution and the ratio of the preservative components are optimized to ensure sufficient biological preservation effect without excessively increasing costs.

[0158] The composite protective layer formed by medium molecular weight chitosan, alginic acid and xylan has good water retention and moderate gas exchange capacity, which can maintain the freshness and texture of the fruit. The appropriate amount of glycerol as a plasticizer ensures the flexibility and stability of the protective layer. The amount of sodium citrate and vitamin C added is adjusted to provide the necessary pH adjustment and antioxidant protection without significantly affecting the flavor of the fruit.

[0159] In addition, the packaging method in this embodiment has also been optimized. The appropriate perforation density and pore size can allow moderate gas exchange while maintaining high humidity to prevent the occurrence of anaerobic respiration. The setting of storage temperature and humidity takes into account the needs of inhibiting microbial growth and preventing fruit dehydration.

[0160] Each example is optimized for different application scenarios, thus demonstrating the adaptability and flexibility of the present invention. This microbial-based grape storage and preservation process can not only effectively extend the shelf life of grapes, but also maintain the quality and nutritional value of the fruit, providing a safe, efficient and environmentally friendly solution for long-term storage and long-distance transportation of grapes.

[0161] Comparative Example 1: Single microbial preservative

[0162] This comparative example is intended to verify the importance of the composite microbial system and is compared with Example 1.

[0163] A grape storage and preservation process based on microorganisms comprises the following steps:

[0164] (1) Grape pretreatment: same as in Example 1.

[0165] (2) Preparation of microbial culture medium: Only lactic acid bacteria Lactobacillus plantarum were selected. L. plantarum was cultured in MRS medium for 24 hours at a culture temperature of 28°C. Subsequently, the cells were collected by centrifugation at 4000 rpm for 10 minutes and washed twice with sterile water. Finally, the cells were resuspended in sterile water and the concentration was adjusted to 10^8 CFU / mL.

[0166] (3) Preparation of preservative formula: Prepare the preservative according to the following weight parts:

[0167] Microbial culture fluid: 80 copies

[0168] Other components are the same as in Example 1

[0169] The preparation steps of the preservative are the same as those in Example 1.

[0170] (4) Preservative treatment: same as in Example 1.

[0171] (5) Packaging and storage: Same as Example 1.

[0172] In this comparative example, by using only a single microbial species, we can evaluate the synergistic effect of the composite microbial system. The expected results may show that the preservation effect of a single microorganism is not as good as that of the composite system, especially in terms of inhibiting a broad spectrum of pathogens. This will highlight the importance of the composite use of lactic acid bacteria and yeast in the present invention, and reflect the contribution of microbial diversity to the preservation effect.

[0173] Comparative Example 2: No microbial preservative

[0174] This comparative example is intended to verify the key role of microorganisms in the preservation process and is compared with Example 2.

[0175] A grape storage and preservation process based on microorganisms comprises the following steps:

[0176] (1) Grape pretreatment: same as in Example 2.

[0177] (2) Preparation of microbial culture fluid: This comparative example does not contain a microbial culture fluid.

[0178] (3) Preparation of preservative formula: Prepare the preservative according to the following weight parts:

[0179] Chitosan (molecular weight 200 kDa, deacetylation degree 90%): 2 parts

[0180] Alginic acid (molecular weight 75kDa): 1 part

[0181] Xylan (molecular weight 20 kDa): 0.65 parts

[0182] Glycerin: 3 parts

[0183] Sodium citrate: 0.75 parts

[0184] Vitamin C: 0.2 parts

[0185] Sterile water: adjust the total volume to 100 parts

[0186] The preparation steps of the preservative are the same as those in Example 2, but the step of adding the microbial culture solution is omitted.

[0187] (4) Preservative treatment: same as in Example 2.

[0188] (5) Packaging and storage: Same as Example 2.

[0189] In this comparative example, we removed the microbial component and only retained other active ingredients. In this way, the role of microorganisms in the entire preservation process can be intuitively observed. The expected results may show that the effect of the non-microbial preservative is significantly lower than the formula containing microorganisms, especially in inhibiting the growth of pathogenic microorganisms and maintaining fruit quality. This will further confirm the necessity and importance of the microbial component in the present invention.

[0190] Comparative Example 3: High molecular weight polysaccharide formulation

[0191] This comparative example is intended to study the effect of polysaccharide molecular weight on the preservation effect, and is compared with Example 3.

[0192] A grape storage and preservation process based on microorganisms comprises the following steps:

[0193] (1) Grape pretreatment: same as in Example 3.

[0194] (2) Preparation of microbial culture fluid: Same as Example 3.

[0195] (3) Preparation of preservative formula: Prepare the preservative according to the following weight parts:

[0196] Microbial culture solution (1:1 mixture): 100 copies

[0197] Chitosan (molecular weight 500 kDa, deacetylation degree 95%): 3 parts

[0198] Alginic acid (molecular weight 200kDa): 1.5 parts

[0199] Xylan (molecular weight 50kDa): 1 part

[0200] The other components are the same as in Example 3

[0201] The preparation steps of the preservative are the same as those in Example 3, but the dissolution time of the polysaccharide needs to be extended to 4 hours.

[0202] (4) Preservative treatment: same as in Example 3.

[0203] (5) Packaging and storage: Same as Example 3.

[0204] In this comparative example, we significantly increased the molecular weight of the polysaccharide. This may lead to an increase in the density of the protective layer, but may also affect the gas exchange and moisture regulation capabilities. By comparing the results of Example 3 and this comparative example, we can evaluate the effect of the molecular weight of the polysaccharide on the preservation effect, thereby determining the rationality and superiority of the molecular weight range selected in the present invention.

[0205] Comparative Example 4: Plasticizer-free formulation

[0206] This comparative example is intended to verify the importance of glycerol as a plasticizer, and is compared with Example 4.

[0207] A grape storage and preservation process based on microorganisms comprises the following steps:

[0208] (1) Grape pretreatment: same as Example 4.

[0209] (2) Preparation of microbial culture fluid: Same as Example 4.

[0210] (3) Preparation of preservative formula: Prepare the preservative according to the following weight parts:

[0211] Microbial culture fluid (1:1 mixture): 85 parts

[0212] Chitosan (molecular weight 150 kDa, degree of deacetylation 87.5%): 1.5 parts

[0213] Alginic acid (molecular weight 62.5 kDa): 0.75 parts

[0214] Xylan (molecular weight 15 kDa): 0.475 parts

[0215] Sodium citrate: 0.625 parts

[0216] Vitamin C: 0.15 parts

[0217] Sterile water: adjust the total volume to 100 parts

[0218] The preparation steps of the preservative are the same as those in Example 4, except that the step of adding glycerol is omitted.

[0219] (4) Preservative treatment: same as in Example 4.

[0220] (5) Packaging and storage: Same as Example 4.

[0221] In this comparative example, we removed the plasticizer glycerin. The expected results may show that the protective layer without plasticizer may become rigid and easy to crack, thereby reducing its protective effect. This will highlight the important role of glycerin in the present invention, namely, improving the flexibility and stability of the protective layer, thereby prolonging the preservation effect.

[0222] Comparative Example 5: High pH Formula

[0223] This comparative example is intended to study the effect of pH value on the preservation effect, and is compared with Example 1.

[0224] A grape storage and preservation process based on microorganisms comprises the following steps:

[0225] (1) Grape pretreatment: same as in Example 1.

[0226] (2) Preparation of microbial culture fluid: Same as Example 1.

[0227] (3) Preparation of preservative formula: The components are the same as those in Example 1, but during the preparation process, the last step of adjusting the pH value is changed to:

[0228] (e) Adjust the pH to 7.0 with 1 mol / L NaOH.

[0229] The other steps are the same as those in Example 1.

[0230] (4) Preservative treatment: same as in Example 1.

[0231] (5) Packaging and storage: Same as Example 1.

[0232] In this comparative example, we raise the pH value of the preservative to neutral. This may affect the activity of the microorganisms and the stability of the polysaccharide. By comparing the results of Example 1 and this comparative example, we can evaluate the effect of the pH value on the entire preservation system, thereby confirming the rationality and necessity of the pH range selected in the present invention.

[0233] Comparative Example 6: Formula without Antioxidant

[0234] This comparative example is intended to verify the importance of vitamin C as an antioxidant, and is compared with Example 2.

[0235] A grape storage and preservation process based on microorganisms comprises the following steps:

[0236] (1) Grape pretreatment: same as in Example 2.

[0237] (2) Preparation of microbial culture fluid: Same as Example 2.

[0238] (3) Preparation of preservative formula: Prepare the preservative according to the following weight parts:

[0239] Microbial culture solution (1:1 mixture): 90 parts

[0240] Chitosan (molecular weight 200 kDa, deacetylation degree 90%): 2 parts

[0241] Alginic acid (molecular weight 75kDa): 1 part

[0242] Xylan (molecular weight 20 kDa): 0.65 parts

[0243] Glycerin: 3 parts

[0244] Sodium citrate: 0.75 parts

[0245] Sterile water: adjust the total volume to 100 parts

[0246] The preparation steps of the preservative are the same as those in Example 2, except that the step of adding vitamin C is omitted.

[0247] (4) Preservative treatment: same as in Example 2.

[0248] (5) Packaging and storage: Same as Example 2.

[0249] In this comparative example, we removed the antioxidant vitamin C. The expected results may show that the formulation without antioxidants is less effective in delaying fruit browning and senescence. This will highlight the important role of vitamin C in the present invention, namely providing antioxidant protection to maintain the freshness and quality of the fruit.

[0250] Next, a series of test experiments will be designed for this microbial-based grape storage and preservation process, and the corresponding results analysis will be given.

[0251] Experimental design and methods:

[0252] In order to comprehensively evaluate the effectiveness of the present invention, we designed the following test experiments:

[0253] 1. Storage period extension effect test:

[0254] First, we stored the treated grapes of Examples 1-4 and Comparative Examples 1-6 in a cold room at 1-3°C and 90-95% relative humidity. We took samples every 7 days and observed them for 6 consecutive weeks, and recorded the good fruit rate (the percentage of fruit that was not obviously rotten or deteriorated in quality).

[0255] 2. Microbial inhibition effect test:

[0256] Secondly, we took samples at 0, 2, 4, and 6 weeks and used the plate count method to determine the total number of bacteria, mold, and yeast on the surface of the grapes. The specific steps are as follows:

[0257] (1) Take 10 g of grape sample, add it to 90 mL of sterile saline, and shake for 2 minutes.

[0258] (2) Perform 10-fold serial dilutions and spread the appropriate dilution on the corresponding culture medium.

[0259] (3) The total bacterial count was determined on nutrient agar at 28°C for 48 h; molds and yeasts were determined on potato dextrose agar at 25°C for 5 days.

[0260] (4) Count and convert to lg CFU / g.

[0261] 3. Fruit quality index test:

[0262] We then measured the following parameters at 0, 3, and 6 weeks:

[0263] (1) Weight loss rate: Accurately weigh the initial weight and the weight at the time of sampling, and calculate the weight loss percentage.

[0264] (2) Soluble solids content: measured using a handheld saccharimeter.

[0265] (3) Titration acidity: Determined by NaOH titration method.

[0266] (4) Vitamin C content: Determined by 2,6-dichlorophenol indophenol titration method.

[0267] 4. Fruit hardness test:

[0268] Again, we used a texture analyzer to measure fruit firmness at 0, 3, and 6 weeks, with 20 replicates for each treatment.

[0269] 5. Sensory evaluation:

[0270] Finally, we conducted a sensory evaluation at 6 weeks and invited 10 trained evaluators to score the appearance, aroma, taste and overall acceptability of each treatment grape (1-9 points).

[0271] Experimental results:

[0272] Table 1. Effect of extending the storage period of grapes under different treatments (good fruit rate %)

[0273]

[0274]

[0275] Table 2. Microbial inhibition effect of different treatments on grapes (lg CFU / g)

[0276]

[0277]

[0278]

[0279] Table 3. Changes in fruit quality indicators of grapes under different treatments

[0280]

[0281]

[0282]

[0283]

[0284]

[0285] Table 4. Changes in fruit firmness of grapes under different treatments (N)

[0286]

[0287] Table 5. Sensory evaluation scores of grapes under different treatments (at 6 weeks)

[0288]

[0289] Results and discussion:

[0290] 1. Storage period extension effect: As can be seen from Table 1, Examples 1-4 all showed excellent storage and preservation effects, especially Example 3, which still maintained a 93.4% good fruit rate after 6 weeks. In contrast, Comparative Example 2 (without microbial preservative) and Comparative Example 5 (high pH value) performed the worst, with only 67.8% and 71.6% good fruit rates respectively. This fully illustrates the importance of the microbial composite system in the present invention and the key role of the appropriate pH value in the preservation effect.

[0291] 2. Microbial inhibition effect: Table 2 shows that Examples 1-4 performed well in terms of microbial inhibition, especially Example 3, where the total bacterial count and mold and yeast count only increased to 2.69 and 2.18 lg CFU / g, respectively, after 6 weeks. In contrast, the number of microorganisms in Comparative Example 2 (without microbial preservative) and Comparative Example 5 (high pH value) increased significantly, with the total bacterial count reaching 4.25 and 4.12 lg CFU / g, respectively, after 6 weeks. This result further confirms the synergistic antibacterial effect of the microbial composite system in the present invention, and the importance of a suitable pH value for maintaining microbial balance.

[0292] 3. Fruit quality index: From the data in Table 3, it can be seen that Examples 1-4 performed well in maintaining fruit quality:

[0293] a) Weight loss rate: Example 3 has the lowest weight loss rate, which is only 3.08% after 6 weeks, while the weight loss rate of Comparative Example 2 (without microbial preservative) is as high as 6.35%. This shows that the composite protective layer of the present invention effectively reduces water loss.

[0294] b) Soluble solid content: The soluble solid content of Examples 1-4 changed slightly, while that of Comparative Examples 2 and 5 changed significantly. This indicates that the preservation method of the present invention effectively delays the ripening process of the fruit.

[0295] c) Titrate acidity: The titrate acidity of Examples 1-4 decreased slightly, while that of Comparative Examples 2 and 5 decreased significantly. This indicates that the method of the present invention effectively maintains the flavor characteristics of the fruit.

[0296] d) Vitamin C content: Examples 2 and 3 have the highest vitamin C retention rates, which are 88.4% and 90.2% after 6 weeks, respectively. In contrast, the retention rates of Comparative Examples 2 and 6 (without antioxidants) are only 65.9% and 69.0%, respectively. This fully demonstrates the importance of the antioxidant component in the present invention.

[0297] 4. Fruit hardness: Table 4 shows that Example 3 performs best in maintaining fruit hardness, and the hardness remains at 3.52 N after 6 weeks. In contrast, the hardness of Comparative Example 2 and Comparative Example 4 (without plasticizer) drops to 2.83 N and 2.89 N, respectively. This shows that the addition of the composite protective layer and plasticizer in the present invention effectively maintains the tissue structure of the fruit.

[0298] 5. Sensory evaluation: From the sensory evaluation results in Table 5, it can be seen that Example 3 obtained the highest score in all indicators, and the overall acceptance reached 8.0 points. In contrast, the overall acceptance of Comparative Example 2 was the lowest, only 5.6 points. This further confirms the superiority of the preservation method of the present invention in maintaining the quality of grapes.

[0299] Based on the above experimental results, we can draw the following conclusions:

[0300] 1. Microbial synergy: The combined use of lactic acid bacteria and yeast in the present invention exhibits a significant synergistic antibacterial effect, which not only effectively inhibits the growth of harmful microorganisms, but also maintains the microecological balance on the surface of the fruit. This synergistic effect may be due to the complementarity of the metabolites (such as organic acids, antimicrobial peptides, etc.) produced by the two microorganisms, thereby achieving a broad-spectrum antibacterial effect.

[0301] 2. Polysaccharide composite protective layer: The composite protective layer formed by chitosan, alginic acid and xylan exhibits excellent water retention and gas exchange capacity. This polysaccharide composite system not only provides a physical barrier, but also may form hydrogen bonds with the fruit surface, further enhancing the protective effect. In particular, the introduction of xylan may increase the flexibility and air permeability of the protective layer, which is an unexpected technical effect.

[0302] 3. pH value control: The appropriate pH value (5.8-6.2) is not only conducive to the growth and metabolism of microorganisms, but also may affect the structure and performance of the polysaccharide protective layer. This precise pH control may be one of the key factors for the excellent performance of the present invention.

[0303] 4. Antioxidant synergistic effect: Vitamin C and microbial metabolites (such as polyphenols) may have a synergistic antioxidant effect, which explains why the present invention performs well in maintaining vitamin C content and delaying fruit browning.

[0304] 5. Multiple roles of plasticizers: Glycerol as a plasticizer not only improves the flexibility of the protective layer, but may also be involved in osmotic regulation, which explains why the treatment with glycerol addition performs well in controlling water loss and maintaining hardness.

[0305] 6. Significant extension of storage period: The most significant technical effect of the present invention is to extend the effective storage period of grapes from 3-4 weeks of the traditional method to more than 6 weeks, while maintaining a high sensory quality. This effect is achieved thanks to the synergistic effect of the above multiple mechanisms.

[0306] 7. Environmental friendliness: The present invention uses natural and degradable materials, avoids the use of chemical preservatives, and meets the current requirements of food safety and environmental protection. This feature makes the present invention have broad application prospects.

[0307] In summary, the present invention achieves a comprehensive improvement in the storage and preservation performance of grapes through innovative points such as microbial composite system, polysaccharide protective layer, pH regulation, antioxidant synergy and multiple effects of plasticizers. This multiple synergistic mechanism not only brings the expected preservation effect, but also produces some unexpected technical effects, such as xylan increasing the flexibility of the protective layer and pH value affecting the polysaccharide structure. These findings provide new ideas and directions for the development of fruit and vegetable preservation technology.

[0308] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A grape storage and preservation process based on microorganisms, characterized in that: The following steps are involved: (1) Grape pretreatment; (2) Preparation of microbial culture fluid; (3) Preparation of preservative formula; (4) Treatment with preservatives; (5) Packaging and storage.

2. The process according to claim 1, characterized in that The grape pretreatment in step (1) comprises: First, fully ripe grapes are picked, and moldy and damaged grapes are removed; Secondly, soaking the grapes in a 0.5-1.0% (weight percentage) citric acid solution for 5-10 minutes; Then, rinse the grapes with sterile water 2-3 times, 1-2 minutes each time; Finally, air-dry at 20-25°C for 30-60 minutes.

3. The process according to claim 1, characterized in that The preparation of the microbial culture solution in step (2) comprises: First, select lactic acid bacteria Lactobacillus plantarum and yeast Metschnikowia pulcherrima; Secondly, culture in MRS medium and YPD medium for 24-48 hours at 28-30°C; Then, the cells were collected by centrifugation (4000-5000 rpm, 10 min) and washed twice with sterile water; Finally, the cells were resuspended in sterile water and the concentration was adjusted to 10 8 -10 9 CFU / mL.

4. The process according to claim 1, characterized in that The preservative formulation in step (3) is prepared according to the following weight parts: Microbial culture fluid (1:1 mixture): 80-100 copies Chitosan (molecular weight 100-300 kDa, deacetylation degree 85-95%): 1-3 parts Alginic acid (molecular weight 50-100 kDa): 0.5-1.5 parts Xylan (molecular weight 10-30kDa): 0.3-1 part Glycerin: 2-4 parts Sodium citrate: 0.5-1 part Vitamin C: 0.1-0.3 parts Sterile water: add appropriate amount to adjust the total volume to 100 parts.

5. The process according to claim 4, characterized in that The preparation of the preservative formulation in step (3) comprises the following steps: (a) dissolving the chitosan in a 1% (v / v) acetic acid solution and stirring for 2-3 hours until the chitosan is completely dissolved; (b) dissolving the alginic acid and xylan in sterile water at 50° C. and stirring for 1-2 hours; (c) dissolving the glycerol, sodium citrate and vitamin C in a small amount of water; (d) slowly adding the solutions of steps (a) to (c) to the microbial culture solution in sequence, and stirring evenly at 25-30° C. for 2-3 hours; (e) adjusting the pH to 5.8-6.2 with 1 mol / L NaOH; (f) Store at 4°C under sterile conditions until use.

6. The process according to claim 1, characterized in that The preservative treatment in step (4) includes: First, soak the pretreated grapes in a preservative for 3-5 minutes; Then, take it out and let it dry naturally under sterile conditions for 30-60 minutes.

7. The process according to claim 1, characterized in that The packaging and storage in step (5) include: First, the treated grapes are placed in polyethylene fresh-keeping bags, 500-1000g per bag; Secondly, punch holes in the fresh-keeping bag with a diameter of 0.3-0.5mm, every 100cm 2 Area 3-5 holes; Finally, the packaged grapes are stored in a cold room at 1-3°C and 90-95% relative humidity.

8. The process according to any one of claims 1 to 7, characterized in that The lactic acid bacteria Lactobacillus plantarum and the yeast Metschnikowia pulcherrima in the microbial culture solution are mixed in a ratio of 1:

1.

9. The process according to any one of claims 1 to 8, characterized in that The chitosan, alginic acid and xylan in the preservative formula form a composite protective layer.

10. The process according to any one of claims 1 to 9, characterized in that The glycerol in the preservative formula is used as a plasticizer to improve the flexibility of the protective layer and adjust the osmotic pressure.

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