Compound microbial inoculant and coating preparation and application thereof in preservation of cucumber

Abstract

The application discloses a composite microbial inoculant and a coating preparation and application thereof in cucumber preservation. The composite microbial inoculant provided by the application is composed of Lactobacillus brevis and Lactobacillus pentosus. Lactobacillus brevis The Lactobacillus brevis is Lactobacillus brevis GHY-3 with a preservation registration number of CGMCC No.31924, and the Lactobacillus pentosus is Lactobacillus pentosus GHY-5 with a preservation registration number of CGMCC No.36297. Lactobacillus pentosus The application further provides a cucumber preservation method, which comprises the following steps: spraying the coating preparation on cucumbers and then storing the cucumbers. The application is helpful for the preservation of cucumbers during storage after picking.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a compound microbial agent and a coating preparation and their application in cucumber preservation. Background Technology

[0002] With the rapid development of fresh food e-commerce, the post-harvest preservation of cucumbers, as high-value fruits and vegetables, has attracted increasing attention. Cucumbers have thin, delicate skins and a high metabolic rate, making them prone to dehydration, wilting, yellowing, rotting, and nutrient loss during post-harvest storage and transportation, severely impacting their commercial value and consumer experience. While cold chain logistics can delay fruit senescence to some extent, factors such as temperature fluctuations, humidity changes, and microbial contamination still threaten fruit quality. Therefore, developing efficient, safe, and biodegradable coating preservation technologies suitable for cold chain environments is of great significance for improving the quality of e-commerce logistics for cucumbers.

[0003] Chitosan, a natural cationic polysaccharide, possesses excellent film-forming and antibacterial properties and has been widely used in fruit and vegetable preservation. However, the antibacterial spectrum and preservation effect of chitosan coatings alone are limited. In recent years, antimicrobial peptides derived from lactic acid bacteria have become a hot topic in natural preservative research due to their advantages such as broad-spectrum antibacterial activity, safety and non-toxicity, and low likelihood of inducing drug resistance. Summary of the Invention

[0004] The purpose of this invention is to provide a compound microbial agent and a coating preparation, and their application in cucumber preservation.

[0005] The compound microbial agent provided by this invention is composed of *Lactobacillus brevis* and *Lactobacillus pentosus*; the *Lactobacillus brevis* is *Lactobacillus brevis* (…). Lactobacillus brevis GHY-3, its accession number is CGMCC No. 31924; the Lactobacillus pentosus is Lactobacillus pentosus ( Lactobacillus pentosus GHY-5, its accession number is CGMCC No.36297.

[0006] Specifically, in the compound microbial agent, the CFU ratio of Lactobacillus brevis to Lactobacillus pentosus is 1-10:1-10.

[0007] Specifically, in the compound microbial agent, the CFU ratio of Lactobacillus brevis to Lactobacillus pentosus is 1:1.

[0008] This invention also provides a method for preparing a coating formulation, comprising the following steps: mixing *Lactobacillus brevis* bacterial suspension, *Lactobacillus pentosus* bacterial suspension, and chitosan solution to obtain a coating formulation; wherein the *Lactobacillus brevis* is *Lactobacillus brevis* (…). Lactobacillus brevis GHY-3, its accession number is CGMCC No. 31924; the Lactobacillus pentosus is Lactobacillus pentosus ( Lactobacillus pentosusGHY-5, its accession number is CGMCC No.36297.

[0009] Specifically, the amount of *Lactobacillus brevis* added to every 100 mL of the coating formulation is 1 × 10⁻⁶. 9 CFU-10×10 9 The amount of CFU, specifically the *Lactobacillus pentosaccharide*, added is 1 × 10⁻⁶. 9 CFU-10×10 9 CFU. Specifically, the amount of *Lactobacillus brevis* added to each 100 mL of the coating formulation is 5 × 10⁻⁶. 9 The amount of CFU, specifically the *Lactobacillus pentosaccharide*, added was 5 × 10⁻⁶. 9 CFU.

[0010] Specifically, the amount of chitosan added in each 100 mL of the coating formulation is 0.5 g to 1.5 g. Specifically, the amount of chitosan added in each 100 mL of the coating formulation is 1.0 g.

[0011] Specifically, the amount of *Lactobacillus brevis* added to every 100 mL of the coating formulation is 5 × 10⁻⁶. 9 The amount of CFU, specifically the *Lactobacillus pentosaccharide*, added was 5 × 10⁻⁶. 9 CFU, wherein the amount of chitosan added is 1.0g.

[0012] Specifically, the *Lactobacillus brevis* bacterial suspension is a bacterial suspension obtained by resuspending *Lactobacillus brevis* in water. Specifically, the bacterial density in the *Lactobacillus brevis* bacterial suspension is 1 × 10⁻⁶. 9 cfu / mL.

[0013] Specifically, the *Lactobacillus pentosus* bacterial suspension is a bacterial suspension obtained by resuspending *Lactobacillus pentosus* in water. Specifically, the bacterial density in the *Lactobacillus pentosus* bacterial suspension is 1 × 10⁻⁶. 9 cfu / mL.

[0014] Specifically, the chitosan solution is obtained by dissolving chitosan in a 1% (volume percentage) aqueous acetic acid solution.

[0015] Specifically, the method is as follows: 0.5 parts by volume of Lactobacillus brevis bacterial solution, 0.5 parts by volume of Lactobacillus pentosolic bacterial solution and 9 parts by volume of chitosan solution are mixed to obtain a coating preparation.

[0016] Specifically, the mixing process involves magnetic stirring at room temperature for 20 minutes.

[0017] This invention also protects coating formulations prepared by any of the methods described above.

[0018] The present invention also provides a coating formulation comprising *Lactobacillus brevis*, *Lactobacillus pentosus*, and chitosan; wherein the *Lactobacillus brevis* is *Lactobacillus brevis* (… Lactobacillus brevis GHY-3, its accession number is CGMCC No. 31924; the Lactobacillus pentosus is Lactobacillus pentosus ( Lactobacillus pentosus GHY-5, its accession number is CGMCC No.36297.

[0019] Specifically, the amount of *Lactobacillus brevis* added to every 100 mL of the coating formulation is 1 × 10⁻⁶. 9 CFU-10×10 9 The amount of CFU, specifically the *Lactobacillus pentosaccharide*, added is 1 × 10⁻⁶. 9 CFU-10×10 9 CFU. Specifically, the amount of *Lactobacillus brevis* added to each 100 mL of the coating formulation is 5 × 10⁻⁶. 9 The amount of CFU, specifically the *Lactobacillus pentosaccharide*, added was 5 × 10⁻⁶. 9 CFU.

[0020] Specifically, the amount of chitosan added in each 100 mL of the coating formulation is 0.5 g to 1.5 g. Specifically, the amount of chitosan added in each 100 mL of the coating formulation is 1.0 g.

[0021] Specifically, the amount of *Lactobacillus brevis* added to every 100 mL of the coating formulation is 5 × 10⁻⁶. 9 The amount of CFU, specifically the *Lactobacillus pentosaccharide*, added was 5 × 10⁻⁶. 9 CFU, wherein the amount of chitosan added is 1.0g.

[0022] This invention also protects the application of any of the above-described compound microbial agents in the preparation of cucumber preservatives.

[0023] The present invention also protects the application of any of the above-described coating formulations, as follows (a) or (b): (a) Preparation of cucumber preservative; (b) As a cucumber preservative.

[0024] This invention also protects the use of any of the above-described coating formulations in cucumber preservation or cucumber cold chain transportation.

[0025] Specifically, the conditions for preserving cucumbers are: temperature 9-11℃ and relative humidity 70%-80%.

[0026] Specifically, the conditions for cold chain transportation are: temperature 9-11℃ and relative humidity 70%-80%.

[0027] Cold chain transportation can refer to cold chain transportation in e-commerce logistics.

[0028] The present invention also provides a method for preserving cucumbers, comprising the following steps: spraying cucumbers with any of the coating preparations described above, and then storing them.

[0029] Specifically, the storage conditions are: temperature 9-11℃ and relative humidity 70%-80%.

[0030] Specifically, any of the chitosans mentioned above are chitosans with a weight-average molecular weight (Mv) of 720,000-880,000.

[0031] Specifically, any of the chitosans mentioned above are chitosans with a weight-average molecular weight (Mv) of 800,000.

[0032] Specifically, any of the chitosans mentioned above are chitosans with a degree of deacetylation ≥ 85%.

[0033] Specifically, any of the chitosans mentioned above are chitosans with a degree of deacetylation of 85%-100%.

[0034] Specifically, any of the cucumbers mentioned above refers to fruit cucumbers.

[0035] Specifically, any of the cucumbers mentioned above are fruit-type hybrid cucumbers.

[0036] Specifically, any of the cucumbers mentioned above is a type of "Ikkyu-style" cucumber.

[0037] The effects of any of the cucumber preservatives mentioned above are manifested in: inhibiting weight loss and / or rotting and / or reducing firmness and / or reducing vitamin C content and / or reducing chlorophyll content during cucumber storage.

[0038] This invention combines *Lactobacillus brevis* GHY-3 and *Lactobacillus pentosus* GHY-5, and then combines them with chitosan to prepare a coating formulation. When sprayed onto cucumbers, this coating formulation can improve multiple indicators of cucumbers during storage (sensory quality, weight loss rate, rot rate, firmness, vitamin C, titratable acid, and chlorophyll content, etc.), greatly contributing to the preservation of cucumbers during the post-harvest storage process. This invention provides an efficient and green preservation solution for the cold chain e-commerce logistics of cucumbers.

[0039] Preservation Instructions Strain name: Lactobacillus pentosus Latin name: Lactobacillus pentosus Classification and nomenclature: Lactobacillus pentosus ( Lactobacillus pentosus ) Strain number: GHY-5 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Abbreviation of depositary institution: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: October 22, 2025 CGMCC Registration Number: CGMCC No. 36297 Preservation Instructions Strain name: Lactobacillus brevis Latin name: Lactobacillus brevis Classification and nomenclature: Lactobacillus brevis ( Lactobacillus brevis ) Strain number: GHY-3 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Abbreviation of depositary institution: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: September 10, 2024 CGMCC Registration Number: CGMCC No. 31924 Attached Figure Description Figure 1 The sensory rating results.

[0040] Figure 2 This is the result of the weight loss rate measurement.

[0041] Figure 3 The results are from the determination of the decay rate.

[0042] Figure 4 This is the result of the hardness measurement.

[0043] Figure 5 This is the result of the vitamin C content determination.

[0044] Figure 6 This represents the results of the titratable acid content determination.

[0045] Figure 7 The results show the determination of chlorophyll content in the fruit peel. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0047] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the water used in the examples is sterile distilled water, and the aqueous solutions used in the examples are solutions prepared using sterile distilled water as a solvent. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are averaged. Room temperature means 23℃±2℃. The experimental results were processed and analyzed using Excel software, and graphs were generated using Origin 2018 software. The chitosan used in the examples is a product of Shanghai Maclean Biochemical Technology Co., Ltd., with product catalog number C660110, CAS number 9012-76-4, a weight-average molecular weight (Mv) of approximately 800,000 (720,000-880,000), and a degree of deacetylation ≥85% (85%-100%). The cucumber used in the examples is Ikkyu-Liang Gua (Ikkyu-Liang Gua is a fruit-type hybrid cucumber cultivar of the Cucurbitaceae family).

[0048] Liquid MRS medium (pH 6.2±0.2): Add 10.0g peptone, 5.0g beef extract, 4.0g yeast extract, 20.0g glucose, 1.0ml Tween-80, 2.0g dipotassium hydrogen phosphate, 5.0g sodium acetate, 2.0g triammonium citrate, 15.0g agar, 0.2g MgSO4•7H2O, 0.05g MnSO4•4H2O per liter, with the remainder being distilled water.

[0049] Example 1: Isolation, identification and preservation of Lactobacillus pentosus GHY-5 The strain GHY-5 was obtained by screening traditional Gansu slurry.

[0050] Morphological characteristics of strain GHY-5: round-ended straight bacillus, about 0.8μm×3.6μm, single; surface colony diameter about 2.2mm, raised, round, smooth, dense, and white.

[0051] Physiological and biochemical characteristics of strain GHY-5: Gram-positive, lactic acid produced.

[0052] Genomic DNA was extracted from strain GHY-5 and amplified by PCR using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The amplified products were then recovered and sequenced. Primers 27F and 1492R are universal primers targeting the coding gene of 16S rRNA. The sequencing results are shown in SEQ ID NO: 1. Homology alignment of the sequencing results was performed using NCBI.

[0053] Based on the above morphological characteristics, physiological and biochemical characteristics, and molecular identification results, strain GHY-5 belongs to Lactobacillus pentosus (… Lactobacillus pentosus It was named Lactobacillus pentosolicus GHY-5.

[0054] Lactobacillus pentosus ( Lactobacillus pentosus GHY-5 was deposited on October 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 36297.

[0055] Example 2: Obtaining, identifying and preserving Lactobacillus brevis GHY-3 The strain GHY-3 was obtained by screening traditional Gansu slurry.

[0056] Morphological characteristics of strain GHY-3: short rod-shaped, about 0.8 μm × 6.0 μm, arranged in short chains, with raised colonies on nutrient agar and a surface colony diameter of about 2 mm.

[0057] Physiological and biochemical characteristics of strain GHY-3: Gram-positive, lactic acid produced.

[0058] Molecular identification of strain GHY-3: The gene encoding 16S rRNA in the genomic DNA is shown in SEQ ID NO: 2, and homology comparison was performed in NCBI.

[0059] Based on the above morphological characteristics, physiological and biochemical characteristics and molecular identification results, strain GHY-3 belongs to Lactobacillus brevis and is named Lactobacillus brevis GHY-3.

[0060] Lactobacillus brevis ( Lactobacillus brevis GHY-3 was deposited on September 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 31924.

[0061] Example 3: Preparation of bacterial suspension 1. Inoculate the test bacteria into liquid MRS medium and incubate at 37°C and 180 rpm for 24 h with shaking.

[0062] 2. Inoculate the bacterial culture obtained in step 1 into liquid MRS medium at an inoculum of 2% and culture at 37°C and 180 rpm for 48 hours with shaking.

[0063] 3. After completing step 2, centrifuge at 4℃ and 8000rpm for 15min and collect the bacterial precipitate.

[0064] 4. Take the bacterial precipitate obtained in step 3, wash it with sterile physiological saline, and then resuspend it in water to obtain a bacterial density of 1×10⁻⁶. 9 A bacterial suspension of cfu / mL. Store the bacterial suspension at 4°C and use within 4 hours.

[0065] When the test bacterium is Lactobacillus brevis GHY-3, the bacterial suspension obtained by performing the above steps is named GHY-3 bacterial suspension.

[0066] When the test bacterium is Lactobacillus pentosus GHY-5, the bacterial suspension obtained by the above steps is named GHY-5 bacterial suspension.

[0067] Example 4: Optimization of the composition of the coating formulation To determine the optimal amounts of chitosan and bacterial cells in the coating formulation, a single-factor experiment was conducted.

[0068] Chitosan was dissolved in a 1% (volume percentage) aqueous acetic acid solution to obtain a chitosan solution. Equal volumes of the GHY-3 bacterial suspension prepared in Example 3 and the GHY-5 bacterial suspension prepared in Example 3 were mixed to obtain a compound bacterial solution. The compound bacterial solution and chitosan solution were mixed and magnetically stirred at room temperature for 20 minutes to obtain a coating formulation. Different amounts of chitosan were added, resulting in 0.50 g, 0.75 g, 1.00 g, 1.25 g, or 1.50 g of chitosan per 100 mL of the coating formulation. Different amounts of the compound bacterial solution were also added, resulting in 5.0 mL, 7.5 mL, 10.0 mL, 12.5 mL, or 15.0 mL of the compound bacterial solution per 100 mL of the coating formulation.

[0069] The cucumbers were stored for 18 days under simulated cold chain storage conditions (10℃±1℃, relative humidity 70%-80%). The effects of different chitosan and compound bacterial solutions added on the sensory quality of cucumbers (sensory evaluation method is described in step 3.1 of Example 5) and on weight loss and decay rate (weight loss determination method is described in step 3.2 of Example 5, and decay rate determination method is described in step 3.3 of Example 5). The results are shown in Table 1. The results indicate that the coating preparation exhibits the best overall preservation effect when the chitosan content is 1.00 g and the compound bacterial solution content is 10 mL per 100 mL.

[0070] Table 1

[0071] Example 5: Preparation and efficacy verification of the coating formulation I. Preparation of Coating Formulations Chitosan is dissolved in a 1% (volume percentage) aqueous solution of acetic acid to obtain a chitosan solution.

[0072] One volume of the GHY-3 bacterial suspension prepared in Example 3 was mixed with nine volumes of chitosan solution, and the mixture was magnetically stirred at room temperature for 20 min to obtain the GHY-3 coating formulation. In 100 mL of the GHY-3 coating formulation, the amount of chitosan added was 1.00 g, and the amount of *Lactobacillus brevis* GHY-3 added was 10 × 10⁻⁶ g. 9 CFU.

[0073] One volume of the GHY-5 bacterial suspension prepared in Example 3 was mixed with nine volumes of chitosan solution, and the mixture was magnetically stirred at room temperature for 20 min to obtain the GHY-5 coating formulation. In 100 mL of the GHY-5 coating formulation, the amount of chitosan added was 1.00 g, and the amount of *Lactobacillus pentosus* GHY-5 added was 10 × 10⁻⁶ g. 9 CFU.

[0074] 0.5 volume parts of the GHY-3 bacterial suspension prepared in Example 3, 0.5 volume parts of the GHY-5 bacterial suspension prepared in Example 3, and 9 volume parts of chitosan solution were mixed and magnetically stirred at room temperature for 20 min to obtain a compound coating formulation (also known as the GHY-3+5 coating formulation). In 100 mL of the GHY-3+5 coating formulation, the amount of chitosan added was 1.00 g, and the amount of *Lactobacillus brevis* GHY-3 added was 5 × 10⁻⁶ g. 9 The dosage of CFU and Lactobacillus pentosaccharide GHY-5 was 5 × 10⁻⁶. 9 CFU.

[0075] Mix 1 volume of water with 9 volumes of chitosan solution and stir magnetically for 20 minutes at room temperature to obtain the control coating formulation (also known as the CH coating formulation). In 100 mL of the CH coating formulation, the amount of chitosan added is 1.00 g.

[0076] All coating formulations are prepared and used immediately.

[0077] II. Grouping Select freshly picked cucumbers that are uniform in size, free from mechanical damage, pests and diseases, and have a consistent color, and randomly divide them into 5 groups of 45 cucumbers each.

[0078] Blank group (CK): No processing is performed; simply place it in the tray.

[0079] Chitosan group (CH): Spray CH coating preparation evenly (spraying amount: 0.1 mL per gram of fresh cucumber weight), let it air dry naturally, and then place it on a tray.

[0080] GHY-3 treatment group (GHY-3): The GHY-3 coating preparation was evenly sprayed (the spraying amount was 0.1 mL per gram of fresh cucumber weight), and then placed on a tray after air drying.

[0081] GHY-5 treatment group (GHY-5): GHY-5 coating preparation was evenly sprayed (spraying amount: 0.1 mL per gram of fresh cucumber weight), and then placed on a tray after air drying.

[0082] GHY-3+5 treatment group (GHY-3+5): The GHY-3+5 coating preparation was evenly sprayed (the spraying amount was 0.1 mL per gram of fresh cucumber weight), and then placed on a tray after air drying.

[0083] III. Indicator Testing After completing the grouping process in step two, all cucumbers were stored at 10℃±1℃ and 70%-80% relative humidity for 18 days. The day the grouping process was completed was recorded as storage day 0. Sensory evaluation was conducted every 3 days, evaluating all remaining cucumbers in each group. Twenty whole cucumbers were reserved from each group for dynamic monitoring of weight loss and decay rates, measured every 3 days. Testing for firmness, vitamin C content, titratable acid content, and chlorophyll content was destructive; samples were discarded after testing, and three cucumbers were sampled every 3 days for testing.

[0084] 1. Sensory evaluation Five trained sensory evaluators evaluated the cucumbers in each treatment group based on sensory scoring criteria (see Table 2), assessing aspects such as color, aroma, texture, and shape. The highest and lowest scores were discarded, and the average score was used as the sensory score for that experimental group. The cucumbers were randomly numbered before scoring, and each evaluator scored independently. Higher scores indicated better sensory quality.

[0085] Table 2

[0086] Sensory rating results are shown below Figure 1As storage time increased, the sensory quality of cucumbers in all groups showed a downward trend. During storage, the control group experienced the largest decrease in sensory score, dropping to 6.8 points on day 3, with most cucumbers in this group exhibiting shriveling and discoloration. The chitosan group, GHY-3 treatment group, GHY-5 treatment group, and GHY-3+5 treatment group, due to the chitosan coating, helped maintain the sensory characteristics of the cucumbers, resulting in a smaller decline in sensory quality compared to the control group. On day 3, their scores were concentrated between 8.20 and 9.00 points, with little difference between them. Subsequently, the cucumbers in the control group showed increasingly severe water loss and shriveling, and exhibited mold and decay. The sensory scores of the other four groups, from highest to lowest, were: GHY-3+5 treatment group > GHY-3 treatment group > GHY-5 treatment group > chitosan group. On day 18, the GHY-3+5 treatment group scored the highest at 3.83 points, followed by the GHY-3 and GHY-5 treatment groups at 2.51 and 2.33 points respectively. The chitosan group scored 2.02 points, while the blank group scored the lowest (only 0.33 points). The results indicate that the combined treatment of GHY-3 and GHY-5 with chitosan coating provides better preservation, exhibiting a synergistic effect. This combination effectively inhibits the growth and reproduction of spoilage microorganisms, reduces transpiration and respiration, thereby decreasing cucumber water loss and rot rates, delaying cucumber senescence, and maintaining good sensory quality.

[0087] 2. Determination of weight loss rate Although cucumbers are non-climacteric fruits, their physiological metabolic activities remain vigorous during post-harvest storage. Respiration and transpiration continue, leading to the continuous consumption of organic nutrients and gradual loss of water, resulting in a continuous decrease in fruit weight.

[0088] Weight loss rate (%) = (m - m1) / m × 100%; m: Mass of the cucumber at time 0, in grams; m1: The mass of cucumber after storage, in grams.

[0089] The results of the weight loss rate determination are shown in Figure 2 As storage time increased, the weight loss rate of each group gradually increased. After 18 days of storage, the weight loss rate of the control group increased by 41.27%, the chitosan group by 10.61%, and the GHY-3, GHY-5, and GHY-3+5 treatment groups by 8.14%, 8.22%, and 6.34%, respectively. The weight loss rate of each treatment group was significantly lower than that of the control group, with the GHY-3+5 treatment group exhibiting the lowest weight loss rate. This indicates that the composite coating treatment can more effectively reduce the moisture loss of cucumbers, resulting in a superior preservation effect.

[0090] 3. Determination of decay rate The main causes of cucumber rot are the combined effects of its own metabolic disorders and the invasion of external microorganisms.

[0091] The decay rate (%) = ∑(decay level × number of items at that level) / total number of items × highest level × 100%.

[0092] Table 3

[0093] The results of the decay rate determination are shown in [the original text]. Figure 3 As storage time increased, the spoilage rate of each group gradually increased. From day 6 to 18 of storage, the difference in spoilage rate between the control group and the other four groups became increasingly significant (p<0.05), with all treatment groups showing lower spoilage rates than the control group. After 18 days of storage, the spoilage rate of the control group reached 88.41%, the chitosan group was 20.49%, the GHY-3 and GHY-5 treatment groups were 15.53% and 16.47% respectively, and the GHY-3+5 treatment group had the lowest spoilage rate at only 8.52%. The results indicate that the combination of GHY-3 and GHY-5 with a chitosan coating has the most significant antibacterial effect and the best preservation effect.

[0094] 4. Hardness Measurement During postharvest fruit storage, cell wall degrading enzymes mediate the decomposition of the primary cell wall and middle lamella structure, promoting the conversion of protopectin into water-soluble pectin, reducing intercellular adhesion, and ultimately leading to tissue softening. Therefore, cell wall enzyme activity plays a decisive role in maintaining fruit firmness.

[0095] Take three parts from each cucumber: the head, middle, and tail (each 1.0 cm wide). Remove a 0.2 cm thick layer of peel and measure and record the firmness of the flesh using a fruit firmness tester.

[0096] The results of the hardness test are shown in [the table]. Figure 4 As storage time increased, the firmness of cucumbers in all groups generally decreased. After 18 days of storage, the rate of firmness reduction in each group, from highest to lowest, was as follows: control group (89.47%) > chitosan group (51.58%) > GHY-5 treatment group (48.42%) > GHY-3 treatment group (45.26%) > GHY-3+5 treatment group (33.68%). Among them, the control group showed the fastest decrease in firmness, while the GHY-3+5 combined treatment group showed the smallest decrease in firmness, indicating that the compound treatment produced a synergistic effect, which could more effectively inhibit the activity of cell wall degrading enzymes, delay protopectin decomposition, maintain cucumber firmness, and achieve the best preservation effect.

[0097] 5. Determination of Vitamin C Content Vitamin C is an important nutritional component of cucumbers, and its content is one of the key indicators for evaluating the storage quality of the fruit. Because vitamin C has strong reducing properties, it is easily oxidized and degraded during storage due to factors such as light and oxygen, leading to a continuous decrease in its content.

[0098] Weigh 40.00g of cucumber pulp and place it in a grinder. Add 40.00g of 1% (w / w) oxalic acid aqueous solution to make a homogenate. Accurately weigh 20.00g of the homogenate into a beaker. Wash the beaker several times with 1% oxalic acid aqueous solution and transfer the entire amount to a 100mL volumetric flask. Dilute to the mark with 1% oxalic acid aqueous solution and shake thoroughly. Filter using rapid qualitative filter paper. Discard the first 5-10mL of filtrate (initial filtrate) and collect the remaining filtrate (subsequent filtrate).

[0099] Pipette 10.00 mL of the filtrate into an Erlenmeyer flask and titrate with a standardized sodium 2,6-dichlorophenolindophenol standard solution (sodium 2,6-dichlorophenolindophenol standard solution: solvent is 2 g / L sodium bicarbonate aqueous solution, freshly standardized and used immediately). Titrate until the solution turns a stable, slightly pink color that does not fade within 15 seconds; this is the titration endpoint. Record the volume consumed. Use 10.00 mL of 1% oxalic acid aqueous solution as a blank control instead of the filtrate and titrate using the same method. Perform three parallel determinations for each sample and take the average value.

[0100] X = (V - V0) × T × A / m × 100; X: Vitamin C content in the homogenate sample, in mg / 100g; V: The volume of 2,6-dichlorophenolindophenol solution consumed in the titration of the sample, in mL; V0: The volume of 2,6-dichlorophenolindophenol solution consumed in the blank control titration, in mL; T: Titration of 2,6-dichlorophenolindophenol solution, i.e., the number of milligrams of vitamin C equivalent to one milliliter of this solution, in mg / mL (T=0.082mg / mL). A: Dilution factor (A=10); m: Mass of homogenate sample, in g (m=20.00g).

[0101] See the results for vitamin C content. Figure 5The vitamin C content of cucumbers in all groups showed an overall trend of first increasing and then decreasing with prolonged storage time, reaching its peak on day 3 and then decreasing at different rates. After 18 days of storage: the vitamin C content in the control group decreased from a peak of 16.5 mg / 100g to 11.3 mg / 100g, a decrease of 31.52%; the chitosan group decreased from a peak of 17.2 mg / 100g to 11.8 mg / 100g, a decrease of 31.40%, similar to the control group, indicating that the chitosan coating alone has a limited effect on inhibiting the oxidative degradation of vitamin C. The GHY-3 and GHY-5 treatment groups showed decreases of 31.69% and 32.60%, respectively, while the GHY-3+5 treatment group showed a decrease of only 22.63%, with the highest vitamin C retention among all groups. The results showed that the combined chitosan coating of GHY-3 and GHY-5 could form a better modified atmosphere microenvironment, effectively inhibit the oxidative degradation of vitamin C, delay the loss of fruit nutrition and quality, and achieve the best preservation effect.

[0102] 6. Determination of titratable acid content Titratable acid content is an important indicator for measuring the flavor quality of cucumbers, directly affecting the fruit's taste and commercial value. The titratable acid content of cucumbers is one of the indicators for evaluating their flavor.

[0103] Weigh 40.00g of fresh cucumber pulp and place it in a grinder. Add 40.00g of carbon dioxide-free distilled water and quickly blend into a homogenate. Accurately weigh 20.00g of the homogenate sample into a beaker. Transfer the sample to a 100mL volumetric flask using carbon dioxide-free distilled water, and continue adding carbon dioxide-free distilled water to the mark. Shake well. Filter using rapid qualitative filter paper. Discard the first 5-10mL of filtrate (initial filtrate) and collect the remaining filtrate (subsequent filtrate). The first 5-10mL of filtrate flowing out during the initial filtration is the initial filtrate and should be discarded. The subsequent stable, clear filtrate is the subsequent filtrate and should be collected for later use.

[0104] Pipette 20.00 mL of the filtrate into an Erlenmeyer flask, add 2-3 drops of 1% phenolphthalein indicator, and titrate with a standardized sodium hydroxide standard aqueous solution until the solution turns a faint pink color that does not fade within 30 seconds; this is the endpoint. Record the volume consumed. Use 20.00 mL of carbon dioxide-free distilled water as a blank control, titrating in the same manner. Perform three parallel determinations for each sample and take the average value.

[0105] Y = (V - V0) × c × K × B / m × 100; Y: Organic acid content in homogenized sample (calculated as citric acid), in mg / 100g; V: The volume of standard sodium hydroxide solution consumed in the titration of the sample, in mL; V0: The volume of standard sodium hydroxide solution consumed in the titration of the blank control, in mL; c: Concentration of standard sodium hydroxide solution, in mol / L (c=0.01mol / L). K: 0.067, unit is g / mol (citric acid conversion factor); B: The ratio of the volume of filtrate taken during titration to the volume of the final volume (B=0.2). m: Mass of homogenate sample, in g (m=20.00g).

[0106] The results of titratable acid content are shown in Figure 6 During the early storage period (0-6 days), the titratable acid content of each group generally showed a decreasing trend. The GHY-3 treatment group, GHY-5 treatment group, and GHY-3+5 treatment group decreased to 0.18%, 0.19%, and 0.18%, respectively, all lower than the chitosan group (0.20%) and the control group (0.22%), indicating that treatment can reduce the loss of titratable acid in cucumbers. During the later storage period (6-18 days), the fruit gradually aged, and metabolic abnormalities intensified, and the titratable acid content of each group showed an increasing trend. Among them, the control group showed the largest increase, reaching 40.91%; the chitosan group increased by 15.00%; the GHY-3 and GHY-5 treatment groups increased by 16.67% and 10.53%, respectively; and the GHY-3+5 treatment group increased by 16.67%. Throughout the storage process, the titratable acid content of the control group fluctuated the most and showed a continuous upward trend, indicating that it had partially lost its edible value. Compared with other treatment groups, the GHY-3+5 treatment group showed a larger decrease in titratable acid content in the early stage of storage and a smaller increase in the later stage. On day 18, the titratable acid content was 0.21%, which was 8.70% lower than that of the chitosan group (0.23%). This indicates that the combination of GHY-3 and GHY-5 with chitosan coating can effectively slow down the loss of titratable acid in cucumbers, maintain the flavor and quality of the fruit, and has the best preservation effect.

[0107] 7. Determination of chlorophyll content in fruit peel The color of the cucumber peel is a direct indicator of its sensory quality and maturity. Chlorophyll, as the key pigment determining the green color of the peel, directly affects the cucumber's appearance and marketability. As cucumbers transition from ripening to senescence after harvest, the rate of chlorophyll degradation accelerates, causing the peel to gradually fade and turn yellow, thus reducing its commercial value.

[0108] Weigh 0.10 g of cucumber peel (0.2 cm thick) and place it in a test tube. Add 7 mL of dimethyl sulfoxide (DMSO), seal the tube, and incubate in a 60°C water bath for 1 hour in the dark. Remove the test tube and cool to room temperature in the dark. Transfer the liquid phase to a 10 mL stoppered graduated test tube. Wash the residue 2-3 times with a small amount of DMSO solution, transferring the washings to the same test tube. Finally, bring the volume to 10 mL with DMSO and mix well. This is the sample solution. Use DMSO as a blank control for the sample solution. Measure the absorbance of the sample solution (or blank control) at wavelengths of 665 nm and 649 nm using a UV spectrophotometer. Perform three parallel measurements for each sample and take the average value.

[0109] Chlorophyll a content Chla=(12.19A) 665 -3.45A 649 )×V / (1000×W); Chlorophyll b content Chlb=(21.99A) 649 -5.32A 665 )×V / (1000×W); Total chlorophyll content = chlorophyll a content + chlorophyll b content.

[0110] A 665 A 649 : These represent the absorbance at the corresponding wavelengths; V: Sample solution volume, in mL (V=10mL). W: Fresh weight of the sample, in g (W=0.10g).

[0111] The results of the chlorophyll content in the fruit peel are shown below. Figure 7Throughout the storage period, the chlorophyll content of the fruit peel in all groups showed a continuous downward trend. The chlorophyll content of the control group was consistently lower than that of the treatment groups, while the content of the GHY-3+5 treatment group was the highest at all time points. From day 0 to 3 of storage, the chlorophyll content of each group decreased relatively little, with the GHY-3+5 treatment group maintaining its initial level, indicating that this treatment effectively delayed chlorophyll degradation in the early stages of storage. From day 6 to 18 of storage, the fruit entered the senescence stage, and the chloroplast structure gradually disintegrated. The chlorophyll content of each group showed a significant downward trend, and the rate of decline at this stage directly reflected the quality of the preservation effect. On day 18: the chlorophyll content in the peel of the GHY-3+5 treatment group was 0.72 mg / g, a decrease of only 34.55%; the GHY-3 and GHY-5 treatment groups were 0.60 mg / g and 0.56 mg / g, respectively, with decreases of 45.45% and 49.09%; the chitosan group had a content of 0.52 mg / g, a decrease of 52.73%; and the control group had a content of only 0.15 mg / g, a decrease as high as 86.36%. The results indicate that the combined GHY-3 and GHY-5 chitosan coating can effectively inhibit chlorophyll degradation, delay the yellowing and fading of the peel, better maintain the appearance quality of cucumbers, and achieve the best preservation effect.

[0112] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A compound microbial agent, composed of *Lactobacillus brevis* and *Lactobacillus pentosus*; wherein the *Lactobacillus brevis* is *Lactobacillus brevis* (… Lactobacillus brevis GHY-3, its accession number is CGMCC No. 31924; the Lactobacillus pentosus is Lactobacillus pentosus ( Lactobacillus pentosus GHY-5, its accession number is CGMCC No.36297.

2. The compound microbial agent as described in claim 1, characterized in that: In the compound microbial agent, the CFU ratio of Lactobacillus brevis to Lactobacillus pentosus is 1-10:1-10.

3. A method for preparing a coating formulation, comprising the following steps: mixing *Lactobacillus brevis* bacterial suspension, *Lactobacillus pentosus* bacterial suspension, and chitosan solution to obtain a coating formulation; wherein the *Lactobacillus brevis* is *Lactobacillus brevis* (… Lactobacillus brevis GHY-3, its accession number is CGMCC No. 31924; the Lactobacillus pentosus is Lactobacillus pentosus ( Lactobacillus pentosus GHY-5, its accession number is CGMCC No.36297.

4. The method as described in claim 3, characterized in that: The amount of *Lactobacillus brevis* added in every 100 mL of the coating formulation is 1 × 10⁻⁶. 9 CFU-10×10 9 The amount of CFU, specifically the *Lactobacillus pentosaccharide*, added is 1 × 10⁻⁶. 9 CFU-10×10 9 CFU, wherein the amount of chitosan added is 0.5g-1.5g.

5. The coating formulation prepared by the method of claim 3 or 4.

6. A film-forming formulation comprising *Lactobacillus brevis*, *Lactobacillus pentosus*, and chitosan; wherein the *Lactobacillus brevis* is *Lactobacillus brevis* (… Lactobacillus brevis GHY-3, its accession number is CGMCC No. 31924; the Lactobacillus pentosus is Lactobacillus pentosus ( Lactobacillus pentosus GHY-5, its accession number is CGMCC No.36297.

7. The application of the compound microbial agent according to claim 1 or 2 in the preparation of cucumber preservative.

8. The application of the coating formulation according to claim 5 or 6 is as follows (a) or (b): (a) Preparation of cucumber preservative; (b) As a cucumber preservative.

9. The application of the coating formulation according to claim 5 or 6 in cucumber preservation or cold chain transportation.

10. A method for preserving cucumbers, comprising the following steps: spraying cucumbers with the coating preparation described in claim 5 or 6, and then storing them.

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