Preparation method and application of sodium alginate-gellan gum based antagonistic yeast bioactive membrane

Abstract

The invention belongs to the field of biological control of postharvest diseases of fruits and vegetables, and particularly relates to a preparation method and application of a sodium alginate-gellan gum based antagonistic yeast bioactive membrane. The preservation number of the candida linangbi is CGMCC (China General Microbiological Culture Collection Center) No.33800 Firstly, a yeast suspension is prepared; then preparing a basic membrane solution from sodium alginate, gellan gum, glycerol and water, and adding the yeast suspension to obtain a bacterial-containing membrane solution; and finally, the fruits and vegetables are sequentially immersed in a mycoderm-containing solution and a calcium chloride solution, and after the fruits and vegetables are taken out and aired, a stable sodium alginate-gellan gum base antagonistic yeast bioactive membrane can be formed on the surfaces of the fruits and vegetables, so that the antibacterial and fresh-keeping purposes of the fruits and vegetables are achieved. The candida linangbi has originality, and meanwhile, the antagonistic yeast is encapsulated by adopting an edible coating film, so that the candida linangbi is safe and environment-friendly, the survival rate of the yeast in the film is high, long-acting release of bacteriostatic activity is realized, the shelf life is prolonged, the edible safety is guaranteed, and the candida linangbi has a wide market application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biological control of postharvest diseases of fruits and vegetables, and particularly relates to a preparation method and application of an alginate-gellan gum-based antagonistic yeast bioactive film. Background Art

[0002] Traditional control methods for postharvest diseases of fruits and vegetables mainly include chemical pesticides and low-temperature storage technology. As the most widely used control means at present, long-term use of chemical pesticides is likely to cause the enhancement of drug resistance of pathogenic bacteria, and there are also potential safety hazards such as excessive pesticide residues, environmental pollution and damage to ecological balance. Although low-temperature storage technology can effectively delay the senescence of fruits and vegetables, the construction and maintenance costs of cold chain facilities are high, and it is easy to cause the phenomenon of chilling injury of chilling-sensitive fruits and vegetables. Therefore, developing new preservation technologies that are safe, efficient, economical and feasible is of great significance for breaking through the existing chemical dependence and low-temperature storage bottlenecks.

[0003] In recent years, the antagonistic microorganism control technology, as a new direction of green preservation, has attracted much attention. Among them, antagonistic yeasts show significant application potential due to their unique advantages: ① low nutritional requirements and short growth cycle, suitable for industrial cultivation; ② no production of fungal toxins and allergenic spores, with excellent biological safety; ③ inhibition of the growth of pathogenic bacteria through multiple mechanisms such as nutritional competition, space occupation and induced resistance. Research has confirmed that strains such as Candida oleophila and Meyerozyma guilliermondii have antagonistic effects on postharvest pathogenic fungi such as Penicillium spp. and Botrytis cinerea.

[0004] The edible coating technology forms a selective barrier on the surface of fruits and vegetables through natural matrices such as polysaccharides and proteins, and has preservation functions such as blocking water vapor exchange and delaying respiratory metabolism. However, the antibacterial performance of a single coating material is limited, and it is difficult to effectively prevent and control the infection of pathogenic microorganisms. Therefore, compounding the coating matrix with antibacterial active ingredients has become an important technical path to improve the preservation effect.

[0005] In addition, sodium alginate (SA) is a natural polysaccharide compound extracted from kelp, fungi, and algae plants. Its basic structure is composed of 1-4-β-D-mannuronic acid and α-L-guluronic acid through α-1,4 glycosidic bonds. Sodium alginate is rich in sources, non-toxic, odorless, and low in price, and has the characteristics of degradability, safety, moisture retention, film-forming property and biocompatibility, showing good application potential in the field of fruit and vegetable preservation. However, there are few reports on the preparation of active films by combining with antagonistic yeasts.

[0006] Therefore, the development of a bioactive film combining an edible coating and antagonistic yeast has important practical application value and broad market prospects for the prevention and control of post-harvest diseases of fruits and vegetables. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the present invention aims to provide a new and effective solution to solve the above problems. A preparation method and application of a sodium alginate-gellan gum-based antagonistic yeast bioactive film are proposed. The antagonistic yeast is encapsulated in an edible coating and applied to fruit and vegetable preservation, which can not only exert the antibacterial performance of the antagonistic yeast but also the preservation performance of the edible coating; it can specifically inhibit fruit and vegetable pathogenic bacteria and significantly reduce the occurrence of post-harvest diseases; at the same time, it does not damage the quality of fruits and vegetables and has broad application prospects.

[0008] In order to achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0009] The present invention first provides an antagonistic yeast, which was self-screened by the inventor in 2019, isolated and purified by the inventor from the intertidal zone marine sediment of the Xisha Islands in the South China Sea, and is now preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number of CGMCC No. 33800 and the preservation date of March 12, 2025. The proposed taxonomic name is Candida pseudolambica.

[0010] Secondly, the present invention provides a preparation method for preparing a sodium alginate-gellan gum-based antagonistic yeast bioactive film based on the above antagonistic yeast, which is carried out according to the following steps:

[0011] (1) Preparation of the antagonistic yeast C. pseudolambica bacterial suspension

[0012] Take the antagonistic yeast Candida pseudolambica and inoculate it into a nutrient yeast dextrose broth (NYDB) medium for the first activation culture to obtain an activation solution; then transfer the activation solution into a new NYDB medium for the second activation culture to obtain a culture solution; then centrifuge the culture solution to obtain a cell precipitate, and then wash it several times by centrifugation with sterile water to obtain a washed yeast cell precipitate. Finally, resuspend the yeast cell precipitate with sterile water to obtain a yeast suspension for standby;

[0013] (2) Preparation of the sodium alginate-gellan gum-based basic film solution

[0014] S1. Components of the basic film solution: It consists of sodium alginate, gellan gum, glycerol and water;

[0015] S2. Weigh sodium alginate and add it to water, stir evenly to form a sodium alginate mixed solution; then place it under water bath conditions for heat treatment, and continuously stir during the heat treatment to completely dissolve sodium alginate, finally obtaining a sodium alginate solution;

[0016] S3. Weigh gellan gum and add it to water, stir evenly to form a gellan gum mixed solution; then place it under water bath conditions for heat treatment, and continuously stir during the heat treatment to completely dissolve sodium gellan gum, finally obtaining a gellan gum solution;

[0017] S4. Dissolve glycerol in water to obtain a glycerol aqueous solution; then mix the gellan gum solution with the sodium alginate solution, stir and then add the glycerol aqueous solution, stir evenly to obtain a sodium alginate - gellan gum based basic film solution;

[0018] (3) Preparation of sodium alginate - gellan gum based antagonistic yeast composite biofilm

[0019] Add the yeast suspension obtained in step (1) to the sodium alginate - gellan gum based basic film solution, stir and mix evenly to obtain a bacteria - containing film solution, which is the sodium alginate - gellan gum based antagonistic yeast bioactive film solution. It has good film - forming stability and can be dried to obtain the sodium alginate - gellan gum based antagonistic yeast bioactive film.

[0020] Preferably, the components of the NYDB medium in step (1) are, based on 1 L: 5 g of yeast extract, 10 g of glucose, 8 g of beef extract, made up to 1 L with distilled water, natural pH, sterilized at 121 °C for 20 min.

[0021] Preferably, the conditions for both the first activation culture and the second activation culture in step (1) are: temperature 28 °C, rotation speed 180 - 200 rpm, time 20 - 24 h.

[0022] Preferably, the dosage of the activated liquid transfer in step (1) is 0.01% - 0.02% of the volume of the nutritional yeast glucose broth medium.

[0023] Preferably, the conditions for centrifugation in step (1) are: 6000 rpm, 4 °C, 5 - 10 min; the number of centrifugation washes is 2 - 4 times.

[0024] Preferably, the concentration of the yeast suspension in step (1) is 1×10 8 cells / mL.

[0025] Preferably, in step (2) S1, every 1000 g of the sodium alginate - gellan gum based basic film solution contains 12.5 g of sodium alginate, 0.3 g of gellan gum, 4 g of glycerol, and the balance is water;

[0026] Preferably, the sodium alginate in step (2), S2 is added to water at a temperature of 80 - 90°C; the heating treatment is carried out under water bath conditions at a temperature of 90 - 100°C for 50 - 70 min.

[0027] Preferably, the gellan gum in step (2), S3 is added to water at a temperature of 70 - 90°C; the heating treatment is carried out under water bath conditions at a temperature of 90 - 100°C for 15 - 25 min.

[0028] Preferably, the stirring rate in step (2), S4 is 2400 rpm / min.

[0029] Preferably, the stirring rate in step (3) is 11000 rpm / min for 3 - 5 min; the final concentration of yeast in the biofilm - containing liquid is 1×10 8 cells / mL; the drying includes natural drying at room temperature or drying at 30 - 40°C.

[0030] Finally, the present invention provides the use of sodium alginate - gellan gum - based antagonistic yeast bioactive film in the antibacterial preservation of fruits and vegetables, and the steps are as follows:

[0031] First, immerse the fruits and vegetables in the prepared sodium alginate - gellan gum - based antagonistic yeast bioactive film liquid. After soaking, take out the fruits and vegetables from the sodium alginate - gellan gum - based antagonistic yeast bioactive film liquid; then immerse the fruits and vegetables in calcium chloride solution. After soaking, take out the fruits and vegetables and let them dry naturally at room temperature, and a stable sodium alginate - gellan gum - based antagonistic yeast bioactive film will be formed on the surface of the fruits and vegetables, thereby realizing the use of antibacterial preservation of fruits and vegetables.

[0032] Preferably, the fruits and vegetables include cherry tomatoes; the soaking time in the sodium alginate - gellan gum - based antagonistic yeast bioactive film liquid is 2 - 3 min; the concentration of the calcium chloride solution is 1% - 2%, and the soaking time in the calcium chloride solution is 1 - 2 min.

[0033] Compared with the existing technology, the advantages of the present invention are as follows:

[0034] (1) By screening and preserving specific strains (CGMCC No.33800) from the marine intertidal zone environment, the present invention obtains a Candida parapsilosis that is adapted to complex environments and has strong antagonistic activity, which is original. And this strain has higher environmental tolerance (such as salt tolerance, temperature fluctuation tolerance) and broad - spectrum antibacterial activity compared with conventional strains, and can specifically inhibit pathogenic bacteria of fruits and vegetables, significantly reducing the occurrence of post - harvest diseases.

[0035] (2) The film - forming materials used in the present invention are sodium alginate, gellan gum, and glycerol, all of which are food - grade safe materials, non - toxic, harmless, and biodegradable, safe and environmentally friendly, and have no harm to human health.

[0036] (3) The formulation and process parameters of the film-forming solution are strictly optimized, the process steps are standardized, the raw materials are easily available and the cost is controllable, with the potential for large-scale production; at the same time, the preparation method is simple, the film-forming properties are stable, and it has good application prospects and high market value.

[0037] (4) The present invention uses an edible coating to encapsulate antagonistic yeast, enabling the yeast to maintain a high survival rate within the film and achieving a long-term release of antibacterial activity. The alginate-gellan gum-based antagonistic yeast bioactive film combines the fresh-keeping function of the edible coating and the antibacterial effect of the antagonistic yeast, and can effectively control the postharvest diseases of cherry tomatoes, extend the shelf life and ensure food safety. Description of the Drawings

[0038] Figure 1 Shows the effect of the film-forming solution components on the growth of antagonistic yeast.

[0039] Figure 2 Are the morphology diagrams of the base film and the film containing bacteria.

[0040] Figure 3 Are the scanning electron microscope images of the base film and the film containing bacteria; note: A is the base film, B is the film containing bacteria.

[0041] Figure 4 Shows the growth dynamics of yeast at the wound of cherry tomatoes in the yeast suspension and the film solution containing bacteria; note: Yeast is the treatment group with yeast suspension, Film+Yeast is the treatment group with film solution containing bacteria, and the yeast concentration of both is 1×10 8 cells / mL.

[0042] Figure 5 Shows the inhibitory effect of the alginate-gellan gum-based antagonistic yeast composite biofilm on the growth of Botrytis cinerea hyphae; note: Film is the treatment group with the base film without yeast, Yeast is the treatment group with yeast suspension, Film+Yeast is the treatment group with the film containing bacteria, and the yeast concentration of all is 1×10 8 cells / mL.

[0043] Figure 6 Shows the control effect of the alginate-gellan gum-based antagonistic yeast composite biofilm on gray mold of cherry tomatoes; among them, Figure A is the incidence rate; Figure B is the lesion diameter; CK is the treatment group with sterile water, Film is the treatment group with the base film solution without yeast; Yeast is the treatment group with yeast suspension, Film+Yeast is the treatment group with the film solution containing bacteria, and the yeast concentration of all is 1×10 8 cells / mL.

[0044] Figure 7Effect of alginate-gellan gum-based antagonistic yeast composite biofilm on the weight loss rate of cherry tomato fruits; Note: CK is the sterile water treatment group, Film is the basic film solution treatment group without yeast; Yeast is the yeast suspension treatment group, Film+Yeast is the film solution treatment group containing bacteria, and the concentration of yeast is 1×10 8 cells / mL. Detailed implementation manners

[0045] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0046] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0049] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0050] The antagonistic yeast used in the present invention was self-screened by the inventor in September 2019. The inventor isolated and purified it from the intertidal marine sediments of the Xisha Islands in the South China Sea. It is currently preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number of CGMCC No. 33800. The preservation date is March 12, 2025, and the proposed taxonomic name is Candida pseudolambica.

[0051] Example 1:

[0052] 1. Strain description:

[0053] (1) Preparation of the antagonistic yeast C. pseudolambica bacterial suspension

[0054] Take the glycerol bacterial solution of the antagonistic yeast Candida pseudolambica (C. pseudolambica) stored in a -80°C refrigerator. According to an inoculation amount of 0.01%, that is, take 5 μL and transfer it into 50 mL of NYDB medium. Incubate it on a shaker at 28°C and 180 rpm for 24 h to complete the first activation. Then take 5 μL of the bacterial solution from the activated solution of the first activation and transfer it into 50 mL of NYDB medium (inoculation amount is 0.01%). Incubate it on a shaker at 28°C and 180 rpm for 24 h to complete the second activation. Take the bacterial suspension after the second activation and centrifuge it at 6000 rpm and 4°C for 5 min. Remove the supernatant and collect the yeast precipitate. Resuspend the yeast precipitate with sterile water, centrifuge, and remove the supernatant. Repeat this step 2 times. Finally, resuspend the yeast precipitate with sterile water and adjust the concentration of the yeast with a hemocytometer to obtain a yeast suspension for standby.

[0055] (2) The pathogenic bacterium used in the present invention is B. cinerea, purchased from the China Agricultural Culture Collection Center, with the number of ACCC 36028, and glycerol preserved in a -80°C refrigerator. When in use, inoculate the glycerol-preserved B. cinerea on potato dextrose agar medium (PDA) and culture it in a constant temperature and humidity incubator at 25°C for 5 d for activation. Activate it continuously twice before use. Scrape the spores into sterile physiological saline and adjust the concentration to 1×10 5 spores / mL.

[0056] 2. Influence of the film-forming liquid components on the growth of yeast

[0057] Add 500 μL of the solution with a concentration of 1×10 6The antagonistic yeast suspension of cells / mL was inoculated into 250 mL conical flasks containing 50 mL of the following two types of solutions: (1) NYDB medium containing 0%, 0.01%, 0.03%, and 0.05% gellan gum; (2) NYDB medium containing 0%, 0.2%, 0.4%, and 0.6% glycerol. The conical flasks inoculated with the antagonistic yeast suspension were placed in a shaker at 28 °C and 180 rpm for cultivation. After 48 h of cultivation, samples were taken, and the absorbance values of the samples were measured at a wavelength of 600 nm using an ultraviolet spectrophotometer. During the measurement, a blank medium without yeast corresponding to the sample to be measured was used for zero adjustment to eliminate the background interference of the medium components and the film-forming solution components on the absorbance measurement.

[0058] Figure 1 For the effect of the film-forming solution components on the growth of antagonistic yeast, where Figure A is gellan gum and Figure B is glycerol; as Figure 1 shown, the OD values of the treatment groups with different mass concentrations of gellan gum and glycerol were significantly higher than those of the blank control group, indicating that a certain concentration of gellan gum and glycerol can promote the growth of antagonistic yeast C. pseudolambica, which is beneficial for the survival of antagonistic yeast in the biofilm and conducive to its biocontrol effect.

[0059] 3. Preparation of sodium alginate-gellan gum-based basic film solution

[0060] The formula of the basic film solution: Each 1000 g of the film solution contains 12.5 g of sodium alginate, 0.3 g of gellan gum, 4 g of glycerol, and the balance is water.

[0061] Preparation was carried out according to 1000 g of the film solution: Weigh 12.5 g of sodium alginate with a balance, dissolve it in 650 mL of hot water (80 °C), stir evenly with a magnetic stirrer for 5 min, place the beaker in a water bath at 95 °C, heat for 50 min, and continue magnetic stirring for 15 min until evenly mixed after the sodium alginate is completely dissolved (no particles, powder lumps, or agglomerates) to obtain a sodium alginate solution.

[0062] Weigh 0.3 g of gellan gum, dissolve it in a beaker containing 300 mL of hot water (80 °C) and stir evenly, place the beaker in a water bath, heat for 20 min, and quickly pour it into the dissolved sodium alginate solution after the gellan gum is completely dissolved, and stir magnetically for 5 min to mix evenly to obtain a mixed film solution.

[0063] Weigh 4 g of glycerol, dissolve the glycerol in the remaining water to obtain an aqueous glycerol solution; then add it to the mixed film solution and stir until evenly mixed under the condition of 2400 rpm / min to obtain a sodium alginate-gellan gum-based basic film solution.

[0064] 4. Preparation of sodium alginate-gellan gum-based antagonistic yeast composite film

[0065] The yeast suspension was added to the sodium alginate-gellan gum-based membrane solution prepared in step 3 above, so that the final concentration of yeast in the membrane solution was 1×10 8 cells / mL, and then stirred with a high-speed homogenizer at 11000rpm / min for 5min to make it fully homogenized to obtain a biofilm liquid, which is the sodium alginate-gellan gum-based antagonistic yeast bioactive membrane liquid.

[0066] 5. Morphology of base membrane and biofilm

[0067] Weigh 20g of the sodium alginate-gellan gum-based basic membrane liquid and the bacterial membrane liquid into a culture dish, place in a 37°C oven and dry for 28h to obtain a yeast-free basic membrane and a yeast-containing bacterial membrane; separate the film from the culture dish with tweezers, observe its morphology, and observe its microscopic morphological characteristics with a scanning electron microscope.

[0068] like Figure 2 As shown in the figure, both the base membrane and the bacterial membrane are colorless and transparent, with a smooth surface, uniform texture, no granularity, and a certain degree of flexibility. The morphological structure of the membrane was analyzed by scanning electron microscopy. Figure 3 As shown in Figure A, the surface of the base film is smooth and continuous, indicating that sodium alginate and gellan gum are well combined, and no microbial characteristic morphology is observed on the surface of the matrix; Figure 3 As shown in Figure B, in the bacterial film, the antagonistic yeast can be clearly seen to be evenly dispersed in the membrane matrix.

[0069] 6. Growth dynamics of yeast in composite biofilm on cherry tomato wounds

[0070] A 3×3 mm wound was pierced at the equator of the cherry tomato fruit, and 10 μL (1×10 8 cells / mL) antagonistic yeast suspension (denoted as Yeast) and 10 μL (1×10 8 cells / mL) containing yeast biofilm solution (referred as Film+Yeast). After drying, it was stored in a constant temperature and humidity incubator at 20°C and a relative humidity of 85-90%. The number of yeast in the wounds of cherry tomato fruits was measured at 0, 12, 24, 48, 72, and 96 hours.

[0071] The specific operation was as follows: 3 cherry tomatoes were selected from each treatment group, and the pulp of 10×10 mm in size was cut from the wound, and ground in a mortar containing 10 mL of sterile water. The ground liquid was diluted 10 times, and 100 μL of the diluted liquid was transferred to the NYDA plate and evenly spread. The plate was incubated at 28°C for 48 hours, and the yeast was counted on the plate. The results were expressed as log 10 CFU / wound. Each treatment was replicated 3 times, and each replicate had 3 fruits.

[0072] As Figure 4 shown, the antagonistic yeast suspension treatment group and the yeast-containing film solution were respectively inoculated on the wounds of cherry tomato fruits. The results showed that the proliferation trends of yeast in the two treatment groups at the fruit wounds were highly consistent, both showing a trend of first increasing and then decreasing. This indicates that the preparation of the biological composite coating did not significantly affect the colonization and proliferation ability of the antagonistic yeast at the wounds of cherry tomato fruits.

[0073] 7. Inhibitory effect of the composite bioactive film on the growth of B. cinerea

[0074] Test method for the in vitro inhibitory effect of the composite bioactive film on the growth of B. cinerea: Use a punch to punch a 6.5-mm-diameter bacterial cake from the edge of the B. cinerea culture medium that has been cultured for 5 days and place it in the center of a newly prepared PDA medium. Then, place the film containing bacteria and the yeast-free basic film obtained in step 5 on the surface of the PDA medium containing the B. cinerea bacterial cake, which is the same size as the surface area of the medium. Then, seal the plate with two layers of sealing film and place the plate in an incubator at 25 °C for 4 days. The treatment of coating 100 μL of yeast suspension (1×10 8 cells / mL) and then inoculating the B. cinerea bacterial cake was used as the positive control. The treatment without adding the film and yeast was used as the blank control. Measure the colony diameter of B. cinerea every day and calculate the mycelial growth inhibition rate. Each treatment had 6 replicates.

[0075] Figure 5 shows the inhibitory effect of the alginate-gellan gum-based antagonistic yeast composite biofilm on the growth of Botrytis cinerea mycelium; where Film is the yeast-free basic film treatment group; Yeast is the yeast suspension treatment group, and Film+Yeast is the film-containing bacteria treatment group. The yeast concentration in the yeast-containing groups was 1×10 8 cells / mL.

[0076] As Figure 5 shown, during the incubation period, the inhibition rate of the film-containing bacteria treatment group on the growth of B. cinerea mycelium reached over 89.0%, and the antibacterial effect was significantly higher than that of the simple film treatment group. The experimental results indicate that the antagonistic yeast encapsulated in the active film can still exert a good antibacterial effect.

[0077] 8. Control effect of the composite bioactive film on gray mold of cherry tomatoes

[0078] After disinfecting cherry tomato fruits with 75% alcohol, use a sterile punch to make a 3*3-mm wound at the equatorial part of the fruit and air-dry it at room temperature; transfer 10 μL of Botrytis cinerea spore suspension (10 5(spores / mL), and dried at room temperature. Then, the cherry tomato fruits were treated in the following four ways. (1) Control group: Soaked in sterile water for 2 min, labeled as CK; (2) Yeast treatment group: Soaked in the yeast suspension (concentration of 1×10 8 cells / mL) for 2 min, labeled as Yeast; (3) Film treatment group: Soaked in the sodium alginate-gellan gum-based basic film solution for 2 min, and then soaked in 2% calcium chloride solution for 2 min, labeled as Film; (4) Film + Yeast treatment group: Soaked in the sodium alginate-gellan gum-based antagonistic yeast bioactive film solution containing 1×10 8 cells / mL yeast for 2 min, and then soaked in 2% calcium chloride solution for 2 min, labeled as Film+Yeast. After the fruit treatment, it was dried at room temperature, and then the fruit was placed in a breathable fresh-keeping box at 20 °C, 85-90%, and stored for 5 d. From the 2nd to 5th d, the incidence rate and lesion diameter were observed every day. There were 10 fruits in each treatment, and each treatment was repeated 3 times.

[0079] As Figure 6 can be seen, during the storage period, the incidence rate (Figure A) and lesion diameter (Figure B) of the Film + Yeast treatment group were significantly lower than those of other treatment groups. The incidence rates of this treatment group on the 2nd d and 5th d were reduced by 73.3% and 27.6% respectively compared with the control group; the lesion diameters were reduced by 90.4% and 40.5% respectively compared with the control group. From Figure 6 it can be seen that the single film treatment had no control effect on the gray mold of cherry tomatoes; although the yeast treatment showed a certain control effect, the effect was limited; the Film + Yeast treatment group showed the best biocontrol effect and achieved significant results.

[0080] 9. Effects of Sodium Alginate-Gellan Gum-Based Antagonistic Yeast Composite Biofilm on the Quality of Cherry Tomato Fruits

[0081] Fresh cherry tomato fruits were soaked in (1) Control group: Soaked in sterile water for 2 min, labeled as CK; (2) Yeast treatment group: Soaked in the yeast suspension (concentration of 1×10 8 cells / mL) for 2 min, labeled as Yeast; (3) Film treatment group: Soaked in the sodium alginate-gellan gum-based basic film solution for 2 min, and then soaked in 2% calcium chloride solution for 2 min, labeled as Film; (4) Film + Yeast treatment group: Soaked in the sodium alginate-gellan gum-based antagonistic yeast bioactive film solution containing 1×10 8The cells / mL yeast in the sodium alginate-gellan gum-based antagonistic yeast bioactive film solution for 2 min, and then immersed in 2% calcium chloride solution for 2 min, labeled as Film+Yeast. After air-drying naturally at room temperature, the fruits were placed in a breathable fresh-keeping box at 20 °C, 85-90%, and stored for 12 d. The weight loss rate, hardness, total soluble solids (TSS), titratable acidity (TA), and color difference index of the fruits were measured. There were 10 fruits in each treatment, and the experiment was repeated 3 times.

[0082] The specific measurement methods are as follows:

[0083] Weight loss rate: Randomly select 10 cherry tomatoes, weigh and record the original weight, and then weigh again every 3 d. To avoid interference of the film weight on the experimental results, the outer film was torn off before weighing.

[0084] The calculation formula for the weight loss rate is as follows:

[0085] Weight loss rate = (initial weight - sampled weight) / initial weight × 100%

[0086] Hardness: Gently remove the epidermis of the cherry tomato with a surgical blade, and then measure the hardness of the equatorial part of the fruit with a hardness tester, with the unit of N.

[0087] Total soluble solids content: Take an appropriate amount of fruit samples and grind them into a homogeneous slurry under low-temperature conditions. After centrifugation at 4 °C and 8000 rpm for 5 min, the supernatant was aspirated. Use a digital refractometer to measure the total soluble solids content of cherry tomatoes.

[0088] Titratable acidity content: Weigh 10.0 g of the well-mixed cherry tomato samples and grind them into a homogeneous slurry in a mortar. Transfer the slurry to a 250 mL volumetric flask with distilled water and make up to the mark. Place the volumetric flask in a 75 °C water bath and heat for 30 min, shaking several times during the period. Aspirate 20.0 mL of the cooled sample solution and transfer it into a conical flask. Titrate with 0.05 mol / L NaOH standard solution until the pH reaches 8.1. Record the volume (V1) of the consumed NaOH standard titration solution. At the same time, perform the above operations, using carbon dioxide-free water instead of the sample solution for the blank test, and record the volume (V2) of the consumed NaOH standard titration solution.

[0089] Titratable acidity content (%) = (c(V1 - V2) × K × F) / M × 100

[0090] where c is the concentration of the sodium hydroxide standard titration solution (mol / L); V1 is the volume of the NaOH standard solution consumed during the titration of the test solution (mL); V2 is the volume of the NaOH standard solution consumed during the blank test (mL); F is the dilution factor of the test solution; M is the weight of the tomato sample (g); K is the conversion coefficient of the acid. The main acid in cherry tomatoes is citric acid, and K is 0.064.

[0091] Color difference: Measure the colors of two opposite sides at the equatorial part of cherry tomato fruits using a color difference meter, and record L*, a*, and b*.

[0092] Test results:

[0093] As Figure 7 shown, the weight loss rates of the film treatment group and the film + yeast treatment group are relatively low, indicating that the sodium alginate coating treatment can, to a certain extent, isolate the external environment, effectively reduce water transpiration, and thus reduce the weight loss of postharvest fruits. Among them, the weight loss rates of the film + yeast treatment group at 3, 6, 9, and 12 days of storage were reduced by 61.5%, 55.8%, 39.1%, and 17.3% respectively compared with the control group. The above results show that the film + yeast treatment group can effectively reduce the weight loss of cherry tomato fruits.

[0094] As shown in Table 1, the hardness of the film + yeast treatment group is significantly higher than that of the control group, indicating that the film + yeast treatment group can effectively maintain the fruit hardness, and there are no significant differences in other quality indicators including soluble solids content, titratable acid, and color difference values L*, a*, and b* among the treatment groups.

[0095] Table 1 Effects of coating treatments on the postharvest quality of cherry tomato fruits

[0096]

[0097] The above experimental results show that the sodium alginate-based - Candida parapsilosis complex coating treatment of the present invention can effectively inhibit the growth of B. cinerea hyphae, reduce the incidence and lesion diameter of postharvest gray mold of cherry tomato fruits, significantly reduce the weight loss rate of fruits, and maintain the fruit hardness. The sodium alginate - gellan gum-based antagonistic yeast bioactive film solution of the present invention will form an active film on the surface of fruits and vegetables, and has a better effect on the prevention and control of fruit and vegetable diseases than single film treatment and antagonistic yeast treatment. Therefore, the bioactive film of the present invention can be used for antibacterial, anti-corrosion and fresh-keeping of fruits and vegetables, and will not affect the quality of fruits and vegetables, achieving unexpected substantial effects.

[0098] Note: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims

1. An antagonistic yeast, characterized in that, The antagonistic yeast is Candida pseudolambica, deposit number: CGMCC No. 33800.

2. A preparation method of an alginate-gellan gum-based antagonistic yeast bioactive film, characterized in that, It is carried out according to the following steps: (1) Preparation of yeast suspension Take the antagonistic yeast Candida pseudolambica described in claim 1 and inoculate it into a nutrient yeast glucose broth medium for the first activation culture to obtain an activation solution; then transfer the activation solution into a new nutrient yeast glucose broth medium for the second activation culture to obtain a culture solution; then centrifuge the culture solution to obtain a cell precipitate, and then centrifuge and wash it with sterile water several times to obtain a washed yeast cell precipitate. Finally, resuspend the yeast cell precipitate with sterile water to obtain a yeast suspension for standby; (2) Preparation of sodium alginate-gellan gum-based basic membrane solution S1. Components of the sodium alginate-gellan gum-based basic membrane solution: It consists of sodium alginate, gellan gum, glycerol and water; S2. Weigh sodium alginate and add it to water, stir evenly to form a sodium alginate mixture; then place it under water bath conditions for heat treatment, and continuously stir during the heat treatment to completely dissolve sodium alginate, and finally obtain a sodium alginate solution; S3. Weigh gellan gum and add it to water, stir evenly to form a gellan gum mixture; then place it under water bath conditions for heat treatment, and continuously stir during the heat treatment to completely dissolve sodium gellan gum, and finally obtain a gellan gum solution; S4. Dissolve glycerol in water to obtain a glycerol aqueous solution; then mix the gellan gum solution with the sodium alginate solution, stir and then add the glycerol aqueous solution, and stir evenly to obtain a sodium alginate-gellan gum-based basic membrane solution; (3) Add the yeast suspension obtained in step (1) to the sodium alginate-gellan gum-based basic membrane solution, stir and mix evenly to obtain a bacteria-containing membrane solution, which is the sodium alginate-gellan gum-based antagonistic yeast bioactive membrane solution. It has good film-forming stability and can be dried to obtain the sodium alginate-gellan gum-based antagonistic yeast bioactive membrane.

3. The preparation method of an alginate-gellan gum-based antagonistic yeast bioactive film according to claim 2, characterized in that, The components of the nutrient yeast glucose broth medium described in step (1), based on 1 L, are: yeast extract 5 g, glucose 10 g, beef extract 8 g, made up to 1 L with distilled water, natural pH, sterilized at 121 °C for 20 min.

4. The preparation method of an alginate-gellan gum-based antagonistic yeast bioactive film according to claim 2, characterized in that, The conditions for the first activation culture and the second activation culture described in step (1) are both: temperature 28 °C, rotation speed 180 - 200 rpm, time 20 - 24 h; the dosage of the transferred activation solution is 0.01% - 0.02% of the volume of the nutrient yeast glucose broth medium; the conditions for centrifugation are: 6000 rpm, 4 °C, 5 - 10 min; the number of centrifugation washes is 2 - 4 times.

5. The preparation method of an alginate-gellan gum-based antagonistic yeast bioactive film according to claim 2, characterized in that, The concentration of the yeast suspension described in step (1) is 1×10 8 cells / mL.

6. The preparation method of an alginate-gellan gum-based antagonistic yeast bioactive film according to claim 2, characterized in that, In S1 of step (2), in every 1000 g of the sodium alginate-gellan gum-based basic film solution, there are 12.5 g of sodium alginate, 0.3 g of gellan gum, 4 g of glycerol, and the balance is water; in step S2, the sodium alginate is added to water, and the temperature of the water is 80 - 90 °C; the temperature for heat treatment under water bath conditions is 90 - 100 °C, and the treatment time is 50 - 70 min; in step S3, the gellan gum is added to water, and the temperature of the water is 70 - 90 °C; the temperature for heat treatment under water bath conditions is 90 - 100 °C, and the treatment time is 15 - 25 min; in step S4, the stirring rate is 2400 rpm / min.

7. The preparation method of an alginate-gellan gum-based antagonistic yeast bioactive film according to claim 2, wherein In step (3), the stirring rate is 11000 rpm / min, and the time is 3 - 5 min.

8. The preparation method of an alginate-gellan gum-based antagonistic yeast bioactive film according to claim 2, characterized in that, The final concentration of yeast in the bacterial film-containing solution described in step (3) is 1×10 8 cells / mL; the drying includes natural drying at room temperature or drying at 30-40°C.

9. Use of alginate-gellan gum-based antagonistic yeast bioactive film for antibacterial fresh-keeping of fruits and vegetables, characterized in that, The steps are as follows: First, immerse the fruits and vegetables in the sodium alginate-gellan gum-based antagonistic yeast bioactive film solution prepared in claim 2. After immersion, take out the fruits and vegetables from the sodium alginate-gellan gum-based antagonistic yeast bioactive film solution; then immerse the fruits and vegetables in a calcium chloride solution. After immersion, take out the fruits and vegetables and let them air-dry naturally at room temperature, and a stable sodium alginate-gellan gum-based antagonistic yeast bioactive film will be formed on the surface of the fruits and vegetables, thereby realizing the use of antibacterial and fresh-keeping of fruits and vegetables.

10. The use according to claim 9, characterized in that, The fruits and vegetables include cherry tomatoes; the immersion time in the sodium alginate-gellan gum-based antagonistic yeast bioactive film solution is 2 - 3 min; the concentration of the calcium chloride solution is 1% - 2%, and the immersion time in the calcium chloride solution is 1 - 2 min.

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