Lactobacillus plantarum, lactobacillus paracasei and application of complex microbial inoculant and preparation thereof in cherry fruit preservation

Abstract

The invention provides lactobacillus plantarum, lactobacillus paracasei, a complex microbial inoculant thereof and application of the complex microbial inoculant and the complex microbial inoculant in cherry fruit preservation, and belongs to the technical field of fruit and vegetable biological preservation. The lactobacillus plantarum TZ28 and the lactobacillus paracasei TZ13, which are screened out by the invention, have broad-spectrum antibacterial effects and have safety and excellent probiotic characteristics. The whole culture solution, the bacterial suspension and the cell-free fermentation supernatant of the lactobacillus plantarum TZ28 and the lactobacillus paracasei TZ13 can inhibit the alternaria alternata separated from the rotten cherries. A bacterial suspension formed by combining lactobacillus plantarum TZ28 and lactobacillus paracasei TZ13 according to a ratio of 1: 1 is used as a biological fresh-keeping agent, so that the decay rate and weight loss rate of cherry fruits can be effectively reduced, the content and hardness of soluble solids are maintained, the aging decay of cherries in the storage period is effectively slowed down, the quality of the cherries is obviously improved, the storage period of the fruits is prolonged, and the quality of the cherries is improved. The nutritional value and the health benefits of the cherry fruits are enhanced.

Description

Technical Field

[0001] This invention belongs to the field of fruit and vegetable biological preservation technology, and in particular relates to the application of a strain of Lactobacillus plantarum, Lactobacillus paracasei and their compound bacterial agents and preparations in the preservation of cherry fruits. Background Technology

[0002] Cherries belong to the genus *Prunus* in the family Rosaceae. PrunusL. ) Subgenus Cherry ( subgen.Cerasus Deciduous fruit trees, widely distributed throughout the world, mainly including the Chinese cherry (Prunus cerasifera). P.pseudocerasus European sweet cherry (P.avium European sour cherry () P.vulgaris ), hairy cherry ( P.tomentosa With the continuous expansion of cherry cultivation area and yield in my country, post-harvest storage and transportation, as well as fruit decay during shelf life, have become bottlenecks in the development of the cherry industry. During post-harvest storage, cherries are susceptible to mechanical and physical damage, metabolic disorders, and pathogenic microorganisms, with fungal infection being the primary cause of post-harvest decay. Related studies have shown that post-harvest decay losses caused by pathogenic fungi such as *Penicillium expansum*, *Mucor*, and *Alternaria* can reach over 50%, resulting in significant economic losses for the cherry industry chain.

[0003] Alternaria ( Alternaria spp Alternariol (Alternariol) is a widely distributed fungus in nature, belonging to the subfamily Alterninae of the family Alternariaceae. It includes saprophytic and endophytic fungi and is a pathogenic factor for many grains, fruits, and vegetables. Because it can survive at low temperatures, ordinary cold chain technologies and storage methods are insufficient to control the spoilage of agricultural products caused by it. Alternariol produces various metabolites, many of which are toxic to plants and animals, including alternariol (AOH), alternariol methyl ether (AME), tenuzonicacid (TeA), and alternarimycin (…). altenuene ALT), bactericides I, II, III altertoxin I, II, III, ATX-I, ATX-II, ATXIII) and AAL toxin (AAL toxins Alternaria alternata, the main pathogen causing cherry black spot disease, primarily infects cherry fruits, forming black patches of varying sizes on the fruit surface. As the disease progresses, a dense black mold layer gradually develops on the surface of the lesions. Because the pathogen's early symptoms are not obvious and difficult to identify with the naked eye, consumers may unknowingly consume cherries contaminated with Alternaria alternata and its toxins, posing a potential health risk. Therefore, inhibiting the reproduction of Alternaria alternata and reducing toxin production is of great significance in the field of food safety.

[0004] Currently, cherry preservation primarily employs physical, chemical, and biological preservation technologies to maintain post-harvest cherry quality and extend their shelf life. Common physical preservation techniques include low-temperature storage, modified atmosphere storage, low-pressure storage, irradiation preservation, heat treatment preservation, packaging materials, ultrasonic treatment, ozone treatment, and temperature intensification treatment. However, physical preservation technologies are limited by high costs, expensive equipment, strict requirements, and difficulty in widespread adoption, contributing to the high price of fruit. Chemical preservation technologies mainly utilize coatings, fumigants, and preservatives to treat cherries through processes such as coating, fumigation, and soaking, killing or inhibiting the growth and reproduction of microorganisms to achieve preservation. In production practice, chemical fungicides are largely relied upon to prevent post-harvest diseases. While chemical preservatives are simple to use and inexpensive, long-term use can lead to antibiotic resistance in pathogens, reducing their effectiveness. Furthermore, frequent and high-concentration use of chemical preservatives increases pesticide residues on fruits and vegetables, threatening human health and posing potential safety hazards to the environment and human well-being. Biological preservation utilizes biological extracts and biotechnology to create biological preservatives to achieve preservation. Biological preservatives inhibit microbial growth, delay food spoilage, extend shelf life, and minimize the loss of nutritional value and sensory attributes. They are typically natural, low in toxicity, and easily degradable, posing less harm to humans. Therefore, developing non-toxic, harmless, residue-free, and environmentally friendly green biological preservatives is crucial for the development of the cherry industry.

[0005] In recent years, microbial antagonistic preservation, touted as a "green" method, has become a research hotspot both domestically and internationally. Microbial antagonistic preservation refers to the process by which one microorganism produces antibacterial metabolites during its life activities, inhibiting or killing another microorganism, or competing with harmful microorganisms for nutrients, thus achieving the goal of preservation. The mechanisms of microbial antagonistic preservation are diverse, including competition, parasitism, induction of host tissue resistance, and the production of volatile metabolites. Being non-toxic, harmless, and residue-free, it does not pollute the environment and has gradually become a research focus for controlling postharvest diseases of fruits and vegetables. It is also gaining attention because it holds promise as a new postharvest disease control measure, potentially replacing chemical methods.

[0006] Lactic acid bacteria ( Lactic acid bacteriaLactic acid bacteria (LAB) are a class of probiotics commonly found in fermented foods and the human gastrointestinal tract. During fermentation, they produce various beneficial metabolites with antibacterial, antioxidant, and gut-enhancing effects, and are widely used in the food, biological, and medical fields. As recognized as safe microorganisms, lactic acid bacteria can produce antibacterial metabolites such as organic acids, bacteriocins, peptides, and other small molecule compounds, and are considered natural preservatives that inhibit the growth of food fungi. Furthermore, the extracellular polysaccharides secreted by lactic acid bacteria can form a protective film, reducing moisture loss and maintaining nutritional components, and are now used in postharvest antibacterial preservation research on various fruits and vegetables. Martínez-Castellanos et al. studied the effects of soaking rambutan fruits in a solution of 1×10⁻⁶... 9After being soaked in a solution of Lactobacillus plantarum (CFU / mL) for 1 min and dried, the rambutan fruit was stored at 10℃ for 15 days and at 25℃ for 10 days. The results showed that the Lactobacillus plantarum treatment significantly delayed the oxidative browning of the rambutan peel, reduced the weight loss rate, maintained the firmness of the rambutan fruit well, and delayed the decrease in soluble solids and titratable acid content, demonstrating good preservation potential. (Martinez-castellanos G, Shiraik, Pelayo-zalvadar C, et al. Effect of Lactobacillus plantarum and chitosan in the reduction of browning of pericarp Rambutan (Nephelium lappaceum)[J]. Food) Microbiology, 2009, 26(4):444-449.); Shirmin et al. treated apples, grapes, and bananas with cell-free fermentation supernatant of Lactobacillus plantarum DMR14 isolated from fermented oats and found that the Lactobacillus plantarum DMR14 cell-free supernatant treatment group extended the shelf life of the fruits compared with untreated fruits (Islam S, Biswas S, Jabin T, Moniruzzaman M, Biswas J, Uddin MS, Akhtar-E-Ekram M, Elgorban AM, Ghodake G, Syed A, Saleh MA, Zaman S. Probiotic potential of Lactobacillus plantarum DMR14 for preserving and extending shelf life of fruits and fruit juice. Heliyon. 2023 Jun 19;9(6):e17382.); Li Nanyang et al. studied the use of Lactobacillus paracasei (CICC 20241) and Lactobacillus rhamnosus (CICC 20241). Lactobacillus plantarum (CICC 21805) and *Bacillus 6244* (Li N, Cheng Y, Li Z, Yue T, Yuan Y. An alginate-based edible coating containing lactic acidbacteria extends the shelf life of fresh strawberry (Fragaria × ananassa Duch.). Int J Biol Macromol.)2024 Aug;274(Pt 1):133273.). .

[0007] The Qinghai-Tibet Plateau region possesses unique geographical and climatic conditions, characterized by low temperatures, low pressure, low oxygen levels, high altitude, and strong ultraviolet radiation. This environment harbors abundant lactic acid bacteria germplasm resources. Its unique ecological environment and millennia-old traditional fermentation techniques have not only preserved a diverse range of lactic acid bacteria communities but also endowed these strains with special biological characteristics. Developing lactic acid bacteria resources from the Qinghai-Tibet Plateau region as a novel biological preservative for use in my country's massive sweet cherry production can effectively extend the shelf life of sweet cherries, reduce post-harvest losses, improve their quality and safety, and enhance their market competitiveness for higher economic benefits and regional development. Furthermore, it unlocks the potential biodiversity of the Qinghai-Tibet Plateau, promotes technological innovation, improves food safety, and contributes to sustainable agricultural development and environmental protection. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide the application of a strain of Lactobacillus plantarum, Lactobacillus paracasei, and their compound microbial agents and preparations in the preservation of cherry fruits.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a strain of Lactobacillus plantarum Lactobacillus plantarum TZ28 was deposited at the China Center for Type Culture Collection on July 22, 2024, with accession number CCTCC NO:M20241618.

[0010] This invention also provides a strain of Lactobacillus paracasei. Lacticaseibacillus paracasei TZ13 was deposited at the China Center for Type Culture Collection on July 22, 2024, with accession number CCTCC NO:M20241617.

[0011] The present invention also provides a compound microbial agent comprising the aforementioned *Lactobacillus plantarum*. Lactobacillus plantarum TZ28 and the aforementioned Lactobacillus paracasei Lacticaseibacillus paracasei TZ13.

[0012] The present invention also provides the aforementioned *Lactobacillus plantarum*. Lactobacillus plantarum TZ28, the aforementioned Lactobacillus paracasei Lacticaseibacillus paracasei Application of TZ13 or the aforementioned compound microbial agent in antibacterial activity.

[0013] Preferably, the types of bacteria inhibited include pathogenic bacteria and pathogenic fungi, wherein the pathogenic bacteria include Staphylococcus aureus, Salmonella and Escherichia coli, and the pathogenic fungi include Penicillium expansum, Botrytis cinerea, Aspergillus charcoalis and Alternaria alternata.

[0014] The present invention also provides a product containing the aforementioned *Lactobacillus plantarum*. Lactobacillus plantarum TZ28, the aforementioned Lactobacillus paracasei Lacticaseibacillus paracasei TZ13 or the aforementioned compound microbial agent formulation, wherein the formulation is in the form of a complete culture medium, a bacterial suspension, or a cell-free fermentation supernatant.

[0015] Preferably, the method for preparing the complete culture medium includes the following steps: Activated Lactobacillus plantarum Lactobacillus plantarum TZ28 or Lactobacillus paracasei Lacticaseibacillus paracasei TZ13 was inoculated into 100 mL of MRS liquid medium and cultured at 35-40℃ for 12-36 h to obtain the first generation culture medium. 2-6% of the total volume of the first generation culture medium was taken and inoculated again, and cultured under the same conditions for 12-36 h to obtain the complete culture medium. The method for preparing the bacterial suspension includes the following steps: 1) Activated Lactobacillus plantarum Lactobacillus plantarum TZ28 or Lactobacillus paracasei Lacticaseibacillus paracasei TZ13 was inoculated into 100 mL of MRS liquid medium and cultured at 35-40℃ for 12-36 h to obtain the first generation culture medium. 2-6% of the total volume of the first generation culture medium was taken and inoculated again, and cultured under the same conditions for 12-36 h to obtain the complete culture medium. 2) Centrifuge the entire culture medium to obtain bacterial cells. Wash the bacterial cells with physiological saline and resuspend them to obtain a bacterial suspension. The centrifugation speed is 6000~10000 r / min, the centrifugation time is 10~20 min, the washing number is 2~4 times, and the concentration of the bacterial suspension is 1×10⁻⁶. 8 ~1×10 10 CFU / mL; The method for preparing the cell-free fermentation supernatant includes the following steps: 1) Activated Lactobacillus plantarum Lactobacillus plantarum TZ28 or Lactobacillus paracasei Lacticaseibacillus paracasei TZ13 was inoculated into 100 mL of MRS liquid medium and cultured at 35-40℃ for 12-36 h to obtain the first generation culture medium. 2-6% of the total volume of the first generation culture medium was taken and inoculated again, and cultured under the same conditions for 12-36 h to obtain the complete culture medium. 2) Centrifuge the whole culture medium to obtain the fermentation supernatant, and filter it through a 0.22μm sterile filter membrane to obtain cell-free fermentation supernatant; the centrifugation speed is 6000~10000r / min, and the centrifugation time is 10~20min.

[0016] The present invention also provides the use of the aforementioned formulation in inhibiting Alternaria.

[0017] The present invention also provides the aforementioned *Lactobacillus plantarum*. Lactobacillus plantarum TZ28, the aforementioned Lactobacillus paracasei Lacticaseibacillus paracasei TZ13, the application of the compound microbial agent or the preparation thereof in the preservation of fruits and vegetables.

[0018] Preferably, the fruit or vegetable includes cherry fruit.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 strains screened in this invention exhibit broad-spectrum antibacterial effects, capable of inhibiting a variety of common pathogenic bacteria and fungi, which is particularly important for fruit and vegetable preservation. Furthermore, the safety and excellent probiotic properties of these two strains indicate their potential value in industrial applications, especially in the development of novel biopreservatives. These characteristics make *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 a promising green and environmentally friendly post-harvest preservation solution for fruits and vegetables, helping to reduce the use of chemical preservatives while improving food quality and safety.

[0020] This invention isolated five putrefactive fungi from rotten cherries, among which *Alternaria alternata* exhibited the strongest pathogenicity. The whole culture, suspension, and cell-free fermentation supernatant of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 all inhibited *Alternaria alternata*, with the suspension of TZ28 and TZ13 in a 1:1 ratio showing the strongest inhibition rate of 74.03%.

[0021] This invention utilizes a bacterial suspension of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 in a 1:1 ratio as a biological preservative. This suspension effectively reduces the spoilage and weight loss rate of cherry fruit, maintains the content and firmness of soluble solids, effectively slows down aging and spoilage during storage, significantly improves cherry quality, and extends the fruit's shelf life. Simultaneously, treatment with the combined *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* suspension enhances the nutritional value and health benefits of cherry fruit. Using this probiotic for post-harvest treatment of cherry fruit can be an effective strategy to improve the market value of cherry fruit and enhance consumer health benefits. Attached Figure Description

[0022] Figure 1 The inhibitory effects of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 on Penicillium expansum; Figure 2 The inhibitory effects of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 on Botrytis cinerea; Figure 3 The inhibitory effects of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 on Aspergillus charcoalis; Figure 4 The hemolysis test is for Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13. Figure 5 The hydrophobicity evaluation of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13; Figure 6 This is an evaluation of the self-aggregation ability of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13; Figure 7 The evaluation of the co-aggregation ability of Lactobacillus plantarum TZ28, Lactobacillus paracasei TZ13 and pathogenic bacteria; Figure 8 The acid tolerance of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 (where a is TZ28 and b is TZ13). Figure 9 It refers to the bile salt tolerance of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 (where a is TZ28 and b is TZ13). Figure 10 The sodium chloride tolerance of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 (where a is TZ28 and b is TZ13). Figure 11 The hydrogen peroxide tolerance of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 (where a is TZ28 and b is TZ13). Figure 12 The temperature tolerance of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 (where a is TZ28 and b is TZ13). Figure 13 This is the result of strain identification of Lactobacillus plantarum TZ13; Figure 14 This is the result of strain identification of Lactobacillus paracasei TZ28; Figure 15 This refers to the colony morphology of postharvest rotten fungi in cherries (where A is strain A1-8, B is strain A6-2, C is strain A7-12, D is strain A7-14, E is strain A1-5, A1~E1 represent the front view of the fungal colony morphology growing on the culture medium, A2~E2 represent the reverse view of the fungal colony morphology growing on the culture medium, and A3~E3 represent the spore and hyphae morphology of the fungal colony under a microscope). Figure 16 This describes the pathogenicity of postharvest rot fungi in cherries; Figure 17 This is a morphological observation of A6-2, the main rot-causing fungus of cherry (the left image shows the colony morphology of A6-2, and the right image shows the microscopic morphology of A6-2). Figure 18This is a phylogenetic tree constructed based on ITS sequences of the main decay fungi of cherry; Figure 19 The inhibitory effect of complete lactic acid bacteria culture on Alternaria alterniflora (where A is the control and B is the control). Lacticaseibacillus paracasei TZ13, C is Lactobacillus plantarum TZ28, D is Lactobacillus plantarum TZ28+ Lacticaseibacillus paracasei TZ13); Figure 20 The inhibitory effect of lactic acid bacteria suspension on Alternaria alterniflora (where A is the control and B is the control). Lacticaseibacillus paracasei TZ13, C is Lactobacillus plantarum TZ28, D is Lactobacillus plantarum TZ28+ Lacticaseibacillus paracasei TZ13); Figure 21 This study investigated the inhibitory effect of cell-free fermentation supernatant (CFS) of *Alternaria alternata* on Alternaria (where AD represents the inhibitory effect of 24-hour cell-free fermentation supernatant (CFS) on *Alternaria alternata*, ad represents the inhibitory effect of 48-hour cell-free fermentation supernatant (CFS) on *Alternaria alternata*, A, a: control, B, b: control). Lacticaseibacillus paracasei TZ13, C, c: Lactobacillus plantarum TZ28, D, d: Lactobacillus plantarum TZ28+ Lacticaseibacillus paracasei TZ13); Figure 22 The effect of high-altitude lactic acid bacteria treatment on the rot rate of cherries during storage; Figure 23 The effect of high-altitude lactic acid bacteria treatment on the weight loss rate of cherries during storage; Figure 24 The effect of high-altitude lactic acid bacteria treatment on the hardness of cherries during storage; Figure 25 The effect of high-altitude lactic acid bacteria treatment on titratable acidity and soluble solids content of cherries during storage; Figure 26 The effect of high-altitude lactic acid bacteria treatment on the color of cherries during storage; Figure 27 This is a sensory evaluation of cherries during storage.

[0023] Biological Preservation Instructions The Lactobacillus plantarum provided by this invention Lactobacillus plantarum TZ28 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:M20241618, deposited on July 22, 2024, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0024] The Lactobacillus paracasei provided by this inventionLacticaseibacillus paracasei TZ13 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:M20241617, deposited on July 22, 2024, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] The standard pathogenic strains of this invention are mainly used for bacterial inhibition experiments, including enteroaggregative Escherichia coli (E. coli). Escherichia coli EAEC CICC 25044), Staphylococcus aureus ( Staphylococcus aureus CICC23926 was purchased from the China Industrial Microbial Culture Collection Center and Salmonella enteritidis subsp. enteritidis (CICC23926). Salmonelia enterica BNCC 292614 was purchased from Wuhan AmyJet Technology Co., Ltd. The indicator bacteria in the mold inhibition test included: Penicillium expansum (…). Penicillium expansum ,bio-25700), Botrytis cinerea ( Botrytis cinerea ,bio-81601), Aspergillus charcoalis ( Aspergillus carbonarius (bio-52150), all purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0027] The culture media involved are as follows: Table 1. Basic MRS medium (solid medium with 15 g / L agar powder added)

[0028] Table 2 PDA solid culture medium

[0029] Example 1 1. Isolation, purification, and identification of lactic acid bacteria According to the standard GB 4789.35-2023 National Food Safety Standard, Microbiological Examination of Food, Lactic Acid Bacteria Examination, samples of yak milk and traditionally fermented yak milk products from the Qinghai-Tibet Plateau region of Gansu and Qinghai provinces were cultured using diluted MRS solid medium. The diluted samples were incubated at 37℃ for 48-72 hours. Colonies with distinct characteristics were repeatedly streaked from the plates until colonies of uniform morphology and size were isolated. The purified lactic acid bacteria were then identified as lactic acid bacteria using MALDI-TOF. A total of 1236 lactic acid bacteria strains were obtained in the experiment.

[0030] 2. Screening of antibacterial lactic acid bacteria (1) Inhibition test of common pathogenic bacteria Intestinal aggregation of Escherichia coli ( Escherichia coli EAEC CICC 25044), Staphylococcus aureus ( Staphylococcus aureus CICC 23926), Salmonella enteritis subsp. enteritidis ( Salmonelia enterica Single colonies of BNCC 292614 were inoculated into LB liquid medium and incubated at 37°C for 12-16 hours. The indicator bacteria were inoculated at 1% in LB semi-solid medium at 45-50°C. After mixing, the mixture was transferred into petri dishes. After the medium solidified, sterile Oxford cups were placed in the dishes. 200 μL of overnight culture of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 was added to each well. The dishes were incubated at 37°C for 16 hours. The size of the inhibition zone was measured using calipers.

[0031] Experimental results are shown in Table 3.

[0032] Table 3. Inhibitory effect of lactic acid bacteria on common pathogenic bacteria.

[0033] Note: The inner diameter of the Oxford cup is 6mm.

[0034] As shown in Table 3, Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 have varying degrees of inhibitory effects on Staphylococcus aureus, Salmonella enteritidis subsp. enteritidis, and Escherichia coli aggregation in the intestine, with Lactobacillus plantarum TZ28 showing the strongest inhibitory effect.

[0035] (2) Fungal inhibition test Activated Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13, after two generations, were inoculated into MRS solid medium (two parallel lines about 2 cm long and 1.5 cm apart, located in the center of the medium). The medium was incubated at 37°C for 48 hours, and then 15 mL of a mixture containing Penicillium expansum was added. Penicillium expansum Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea ), Aspergillus charcoalis ( Aspergillus carbonarius The spore suspension was poured onto MRS medium after 48 hours of incubation in PDA medium. The culture dishes containing the double-layer medium were incubated at 25°C for 5 days.

[0036] Experimental results: such as Figures 1-3 As shown, *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13, which have strong inhibitory effects on both pathogenic bacteria and fungi, were obtained.

[0037] 3. Evaluation of the safety and probiotic properties of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 (1) Safety evaluation ① Hemolytic activity test After activating *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13, a small amount of culture medium was taken with an inoculation loop and streaked in four zones on a Columbia blood agar plate. The blood agar plates were incubated at 37°C for 48 hours, and the presence or absence of a hemolytic zone around the colonies was observed. The absence of a hemolytic zone indicates that the strain possesses γ-hemolytic activity, meaning it is non-hemolytic and safe for humans. *Staphylococcus aureus* was used as a positive control strain.

[0038] Experimental results: such as Figure 4 As shown, no hemolytic ring appeared on the blood agar plates of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13, indicating that they are non-hemolytic.

[0039] ② Antibiotic susceptibility testing The paper disc diffusion method was used to determine the susceptibility of tested lactic acid bacteria to different antibiotics on MRS solid medium. 100 μL of bacterial suspension was evenly spread onto sterile MRS solid medium, and after absorption for 30 min, it was incubated at 37°C for 48 h. The size of the inhibition zone (mm) was measured after incubation, and the lactic acid bacteria were classified for antibiotic resistance according to CLSI 2021 parameters. *Lactobacillus rhamnosus* (Lactobacillus rhamnosus) was used as an example. Lacticaseibacillus rhamnosus CICC6001 was the positive control strain, purchased from the China Industrial Microbial Culture Collection Center.

[0040] Experimental results are shown in Table 4.

[0041] Table 4. Antibiotic sensitivity of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13

[0042] Note: R - drug resistance; I - moderate sensitivity; S - sensitive.

[0043] As shown in Table 4, the antibiotic susceptibility test indicates that TZ28 is resistant to ciprofloxacin only, TZ13 is resistant to trimethoprim-sulfamethoxazole and gentamicin, while CICC6001 is resistant to trimethoprim-sulfamethoxazole, gentamicin and ciprofloxacin. Neither TZ28 nor TZ13 are multidrug-resistant bacteria, and their antibiotic susceptibility is stronger than that of CICC6001.

[0044] (2) Evaluation of probiotic characteristics ① Evaluation of self-aggregation, hydrophobicity, and co-aggregation ability with pathogenic bacteria A. Hydrophobicity Evaluation After activating *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 for two generations, the culture medium was centrifuged at 10,000 r / min and 4℃ for 15 minutes to collect the bacterial cells. The bacterial suspension concentration was adjusted to 1×10⁻⁶ using sterile physiological saline. 8The absorbance at 600 nm was measured using CFU / mL and recorded as A1. Two mL of the bacterial suspension was added to an equal volume of xylene / ethyl acetate / chloroform, mixed for 5 min, and allowed to stand at room temperature for 1 h. The absorbance at 600 nm of the lower aqueous phase was then measured and recorded as A2. The positive control strain was... Lacticaseibacillus rhamnosus CICC6001, used to calculate surface hydrophobicity.

[0045] Hydrophobicity (%) = ×100% Experimental results: such as Figure 5 As shown, under the conditions of ethyl acetate and xylene, the hydrophobicity of Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 in both organic solvents was significantly higher than that of CICC6001 (P<0.05).

[0046] B. Evaluation of self-cohesion After activating *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 for two generations, the culture medium was centrifuged at 10,000 r / min and 4℃ for 15 minutes to collect the bacterial cells. The bacterial suspension concentration was adjusted to 1×10⁻⁶ using sterile physiological saline. 8 The CFU / mL concentration was measured, and its absorbance at 600 nm was recorded as A0. 5 mL of the resuspended bacterial culture was taken and incubated at 37°C for 2 hours, 4 hours, 6 hours, and 24 hours. 200 μL of the culture was then added to each well of a 96-well plate, and the OD600 nm value at different time points was measured using a microplate reader and recorded as At. The self-aggregation was then calculated using the formula.

[0047] Self-aggregation (%) = ×100% Experimental results: such as Figure 6 As shown, the self-aggregation rates of TZ28 and TZ13 were significantly higher than those of CICC6001 at all detection times (P<0.05).

[0048] C. Evaluation of Coagulation Ability After activating *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 for two generations, the culture medium was centrifuged at 10,000 r / min and 4℃ for 15 minutes to collect the bacterial cells. The bacterial suspension concentration was adjusted to 1×10⁻⁶ using sterile physiological saline. 8 The absorbance at 600 nm was measured using CFU / mL and denoted as A1. After activation and two generations of single colonies of *E. coli* and *Staphylococcus aureus*, the culture medium was centrifuged at 10000 r / min at 4℃ for 15 minutes, and the bacterial cells were collected. The bacterial suspension concentration was adjusted to 1×10⁻⁶ using sterile physiological saline. 8The absorbance at 600 nm was measured using CFU / mL and denoted as A2. 2 mL of bacterial suspension and 2 mL of *Escherichia coli* / *Staphylococcus aureus* bacterial suspension were mixed for 5 min and incubated at 37℃ for 5 h. The supernatant was then measured and its absorbance at 600 nm was denoted as A2. mix Substitute the values ​​into the formula to calculate the copolymerization of the lactic acid bacteria strains.

[0049] Copolymerization (%) = ×100%.

[0050] Experimental results: such as Figure 7 As shown, *Lactobacillus paracasei* TZ13 showed the strongest co-aggregation rate against intestinal aggregated *Escherichia coli*, followed by *Lactobacillus plantarum* TZ28, with CICC6001 showing the weakest. TZ28 and TZ13 showed significantly higher co-aggregation rates against *Staphylococcus aureus* than CICC6001.

[0051] It is evident that Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 exhibit superior performance in terms of self-aggregation, co-aggregation, and hydrophobicity, and both possess strong adhesion and colonization potential.

[0052] ②Tolerance evaluation A. Acid tolerance assessment The pH of the MRS liquid medium was adjusted to 2.5, 3.5, 4.5, and 9.0 using HCl (1 mol / L) and NaOH (1 mol / L). Activated *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 were inoculated at a 4% inoculum in the media at different pH values, with unadjusted MRS liquid medium serving as a control. After incubation at 37°C for 0, 6, 12, 18, and 24 hours, the optical density of the bacterial suspension was measured at 600 nm.

[0053] Experimental results: such as Figure 8 As shown, *Lactobacillus plantarum* TZ28 exhibited the strongest growth ability at a culture medium pH of 9.0; *Lactobacillus casei* TZ13 exhibited the strongest growth ability at a culture medium pH of 5.7, i.e., the original pH of the culture medium. *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 could also grow under acidic conditions.

[0054] B. Evaluation of bile salt tolerance Activated *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 were inoculated at 4% in MRS liquid medium containing 0.3% and 0.5% ox bile salts, respectively, with MRS liquid medium without added bile salts serving as a control. After incubation at 37°C for 0, 6, 12, 18, and 24 hours, the optical density of the bacterial suspension at a wavelength of 600 nm was measured.

[0055] Experimental results: such as Figure 9As shown, both strains exhibited poor tolerance to bile salts.

[0056] C. Sodium chloride tolerance evaluation Activated *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 were inoculated at 4% in MRS liquid medium containing 3%, 5%, 8%, and 10% NaCl, respectively. MRS liquid medium without added sodium chloride served as a control. After incubation at 37°C for 0, 6, 12, 18, and 24 hours, the optical density of the bacterial suspensions was measured at 600 nm.

[0057] Experimental results: such as Figure 10 As shown, when 3% NaCl was added, Lactobacillus plantarum TZ28 showed the strongest tolerance, and the concentration was not much different from the cell concentration in the control medium. When an additional 5% NaCl was added, the growth of Lactobacillus paracasei TZ13 was significantly inhibited, but TZ28 still showed the strongest tolerance.

[0058] D. Hydrogen peroxide tolerance assessment A 30% hydrogen peroxide solution was filtered through a membrane for sterilization and then added to MRS liquid culture medium to achieve final hydrogen peroxide concentrations of 1.0 mmol / L, 2.0 mmol / L, and 3.0 mmol / L. Activated *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 were inoculated at a 4% inoculum into the media with different hydrogen peroxide concentrations, with MRS liquid culture medium without added hydrogen peroxide serving as a control. After incubation at 37°C for 0, 6, 12, 18, and 24 hours, the optical density of the bacterial suspension was measured at 600 nm.

[0059] Experimental results: such as Figure 11 As shown, *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 exhibited good tolerance to hydrogen peroxide, showing good growth at various hydrogen peroxide concentrations after 24 hours and being able to tolerate 3.0 mmol / L of hydrogen peroxide, demonstrating good antioxidant capacity.

[0060] E. Temperature tolerance evaluation Activated Lactobacillus plantarum TZ28 and Lactobacillus paracasei TZ13 were inoculated into MRS liquid medium at an inoculation rate of 4% and cultured in incubators at 4℃, 25℃, 37℃ and 40℃, respectively. The optical density of the bacterial solution at a wavelength of 600 nm was measured at 0, 6, 12, 18 and 24 h after inoculation.

[0061] Experimental results: such as Figure 12 As shown, the optimal growth temperature for both *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 is 37℃, and neither is tolerant to low temperatures. However, TZ28 has a wider temperature tolerance range.

[0062] 4. Identification of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 The method was based on GB / T 33682-2017, "General Rules for Identification of Microorganisms by Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry," with slight modifications. LAB colonies were isolated using the four-zone streak method. A single colony was picked up with a 1 μL inoculation loop and spread evenly in the well of the sample target plate. After air-drying at room temperature, 1 μL of 50% formic acid solution and 1 μL of matrix solution were mixed with the sample, and the mixture was allowed to air-dry to form crystals. The sample target plate was then placed in a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF MS) for identification of the LAB strains. The obtained microbial fingerprint spectral data were compared with a database, and the identification results were displayed after comparison with the fingerprint spectral data.

[0063] Experimental results: such as Figure 13 and Figure 14 As shown.

[0064] 5. Isolation, purification, and identification of postharvest-causing fungi in cherries (1) Isolation and purification of postharvest saprophytic fungi of cherry Weigh 10g of rotten cherry sample with typical black spot symptoms, place it in 90mL of sterile diluent (distilled water, physiological saline, or phosphate buffer), and shake in a shaker for 1 hour to prepare a concentration of 10. -1 The bacterial suspension, at a concentration of g / mL, was serially diluted 10-fold to a concentration of 10 g / mL. -2 g / mL, 10 -3 g / mL, 10 -4 Different concentrations of bacterial suspensions (g / mL, etc.) were prepared. Using the dilution-spreading method, 100 μL of each concentration of bacterial suspension was evenly spread onto potato dextrose agar (PDA) medium and incubated at 25°C for 3–5 days. Fungi with completely different colony morphology were selected and transferred to new PDA medium using the three-spot method. This process was repeated 2–3 times until a single colony appeared on the medium. The purified single colony was then transferred to PDA slant medium and incubated at 25°C for 5–7 days, and stored at 4°C for later use.

[0065] Experimental results: such as Figure 15 As shown, five putrefactive fungi were isolated from rotten cherry samples exhibiting typical black spot symptoms.

[0066] (2) Cultivation of postharvest saprophytic fungi in cherries and preparation of spore suspension The isolated and purified cherry-spoiling fungus was inoculated onto PDA plates and incubated at 25°C for 7 days. The spores were then scraped off the plates with sterile water, filtered through a cell filter, and a fungal spore suspension was obtained. The spores were counted using a hemocytometer, and the concentration of the spore suspension was adjusted to 1×10⁻⁶. 5 One spore / mL for use.

[0067] (3) Pathogenicity test Select cherry fruits of similar size, maturity, and free from pests, diseases, and mechanical damage. Wash them three times with sterile water, disinfect them with 1% sodium hypochlorite solution for 2 minutes, and then air-dry them on a sterile workbench. Next, use a sterile inoculation needle to make holes at the equatorial region of the cherry fruit. Before making the holes, disinfect the surface of the holes with 75% alcohol. After air-drying, inoculate the cherry fruit with a 10 μL suspension of cherry-causing fungal spores (1×10⁻⁶). 5 Inoculate the treated and control groups (number of lesions / mL) into the perforated areas of the cherries, using sterile water as a control. Place both groups in a preservation box and incubate at 25℃. Observe the disease development after 5-7 days and calculate the pathogenicity of the cherry-causing fungus. Measure the diameter of lesions using the cross-sectional method to evaluate the pathogenicity of the cherry-causing fungus. Grading criteria: Grade 0 indicates no lesions on the fruit; the lesion diameters (mm) for grades 1, 3, 5, 7, and 9 are (0,3), [3,6), [6,9), [9,12), and [12,∞), respectively. Calculate the disease index based on the disease level and classify the pathogenicity level according to the disease index. Strong pathogenicity: Disease index ≥ 60.0; Moderate pathogenicity: 30.0 ≤ Disease index < 60.0; Weak pathogenicity: Disease index < 30.0.

[0068] Fungal pathogenicity rate = (number of fruits showing pathogenic symptoms / total number of inoculated fruits) × 100%.

[0069] Disease Index (DI) = ∑(si×ni) / 9N×100 (DI = Disease Index, si = Disease Level, ni = Number of Disease Levels, N = Total Number of Surveys).

[0070] Experimental results: as shown in Table 5 and Figure 16 As shown.

[0071] Table 5. Pathogenicity determination of postharvest rot fungi in cherries.

[0072] A6-2 had a pathogenicity rate of 100% and a disease index significantly higher than the other four pathogenic fungi, exhibiting the strongest pathogenicity. Figure 16 This shows the pathogenicity of postharvest rot fungi in cherries.

[0073] (4) Morphological observation of saprophytic fungi The purified pathogen was inoculated onto PDA plates and cultured for 7 days in a constant temperature incubator with a relative humidity of 85-90% and a temperature of 25℃. The growth status and color of the colonies were observed, and the morphological characteristics of the conidia were observed using a fully automated upright fluorescence microscope.

[0074] Experimental results: such as Figure 17 As shown.

[0075] (5) Molecular biological identification of saprophytic fungi After pathogen DNA extraction, ITS and B-tubulin primers were designed, followed by PCR amplification. The amplified products were then separated by electrophoresis on a 1% agarose gel. The amplified fragments were sequenced by a testing company, and the sequencing results were analyzed using BLAST in the GenBank database. A phylogenetic tree was constructed using neighbor-joining with appropriate sequences selected using MEGA 711.0 software. Combining molecular biological and morphological identification results, the species of the main decay-causing fungus in cherry was finally determined.

[0076] Experimental results: such as Figure 18 As shown.

[0077] Based on morphological observation and molecular biological identification, it was determined that the main decay fungus of cherry, A6-2, is Alternaria.

[0078] 6. Inhibitory effects of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 on *Alternaria alterniflora*. (1) Activation of lactic acid bacteria and preparation of various components Activation of lactic acid bacteria: Take the frozen plateau lactic acid bacteria strain, inoculate the lactic acid bacteria into MRS solid medium at an inoculation rate of 4%, and incubate at 37℃ for 48h.

[0079] Preparation of complete lactic acid bacteria culture: The activated lactic acid bacteria were inoculated into 100 mL of MRS liquid medium and cultured at 37℃ for 24 h to obtain the first generation lactic acid bacteria culture. 4% of the total volume of this culture was taken and inoculated again, and cultured under the same conditions for 24 h to obtain the complete lactic acid bacteria culture.

[0080] Preparation of lactic acid bacteria suspension: Centrifuge the whole culture medium (8000 r / min, 15 min) to collect the bacterial cells. Wash the bacterial cells three times with physiological saline and resuspend them to obtain the lactic acid bacteria suspension. Adjust the concentration of the bacterial suspension to 10. 9 CFU / mL.

[0081] Preparation of cell-free fermentation supernatant of lactic acid bacteria: The whole culture medium was centrifuged (8000 r / min, 15 min) to obtain the fermentation supernatant, which was then filtered through a 0.22 μm sterile filter membrane to obtain cell-free fermentation supernatant of lactic acid bacteria.

[0082] (2) Inhibitory effect of complete lactic acid bacteria culture on Alternaria alterniflora The double-layer agar plate method was used. The screened and identified lactic acid bacteria strains were streaked onto MRS agar plates with two parallel stripes and incubated at 37°C for 2 days. Then, semi-solid PDA medium containing fungal spore suspension was poured onto the plates where lactic acid bacteria were growing and incubated at 25°C for another 5 days to observe the antibacterial effect.

[0083] Experimental results: such as Figure 19 As shown, both whole cultures of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 inhibited the growth of *Alternaria alternata*.

[0084] (3) Inhibitory effect of lactic acid bacteria suspension on Alternaria alterniflora Using the solid dilution method, after the PDA medium cooled to approximately 55°C, the lactic acid bacteria suspension (final concentration 1×10⁻⁶) was diluted. 9 The experimental group consisted of CFU / mL, and the control group consisted of physiological saline. The ratio of CFU / mL to physiological saline was 1:9 (V:V) (1×10⁻⁶ CFU / mL lactic acid bacteria suspension). 10 (CFU / mL): PDA medium is 1:9) Mix the lactic acid bacteria suspension with PDA medium evenly, pour about 20 mL of the mixture into each petri dish, and after the medium cools and solidifies, inoculate 1 μL of 1×10⁻⁶ CFU / mL solution in the center of the medium. 5 Alternaria spore suspension (spores / mL). Incubated at 25°C for 7 days, colony diameter was measured using the cross-hatching method, and the colony growth inhibition rate was calculated using the following formula.

[0085] Colony growth inhibition rate = (Coronary diameter of control group - Colony diameter of experimental group) / Colony diameter of control group × 100% (4) Inhibitory effect of cell-free fermentation supernatant of lactic acid bacteria on Alternaria alterniflora An inhibitory test was conducted on the cell-free fermentation supernatant of lactic acid bacteria against postharvest putrefactive fungi of cherry, using cell-free fermentation supernatant of lactic acid bacteria as the experimental group and sterile MRS broth as the control. A solid dilution method was used. After the PDA medium cooled to approximately 55°C, the cell-free fermentation supernatant of lactic acid bacteria (as the experimental group) was mixed with the medium at a ratio of 1:9 (V:V) (cell-free fermentation supernatant = 1, PDA medium = 9), with approximately 20 mL of the mixture added to each petri dish. After the medium cooled and solidified, 1 μL of Alternaria alternata spore suspension was inoculated in the center of the medium. The mixture was incubated upright at 25°C for 7 days. The colony diameter was measured using the cross-cross method, and the colony growth inhibition rate was calculated using the formula.

[0086] Colony growth inhibition rate = (Coronary diameter of control group - Colony diameter of experimental group) / Colony diameter of control group × 100% Experimental results are shown in Table 6.

[0087] Table 6. Inhibitory effect of high-altitude lactic acid bacteria on Alternaria alterniflora.

[0088] Note: Different lowercase superscript letters in the same column indicate statistically significant differences between different groups (P < 0.05). Table 6 shows that bacterial suspensions of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13, as well as 24-hour and 48-hour cell-free fermentation supernatants (CFS), also inhibited the growth of *Alternaria alternata*. The combined use of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 showed significantly better antibacterial effects than single strains. The bacterial suspension of TZ28 and TZ13 at a 1:1 ratio exhibited the strongest antibacterial effect, significantly inhibiting the growth of *Alternaria alternata*. For specific antibacterial details, see [Table 6]. Figure 20 , 21 .

[0089] 7. Application of a 1:1 bacterial suspension of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 in cherry preservation. (1) Sample processing Select healthy cherries with no surface damage and normal color. Wash them three times with sterile water, disinfect them with 1% sodium hypochlorite solution for 2 minutes, and then air dry them on a sterile workbench. Treat the cherries at room temperature as follows: ① CK group: No treatment; ② NaCl group: Soak in physiological saline for 2 minutes; ③ LAB group: Soak in saline solution for 2 minutes. Lactobacillus plantarum TZ28 and Lacticaseibacillus paracasei The TZ13 strain was soaked in a 1:1 bacterial suspension for 2 min. After treatment, the samples were stored at room temperature, and their physicochemical properties were measured at 0, 2, 4, 6, and 8 days.

[0090] (2) Determination of physicochemical properties of cherries ①Rotten rate: Rotten rate (%) = Number of rotten cherries / Total number of cherries × 100 ②Weight loss rate: The weight of the cherry samples before storage (M0) and the weight during storage (M) were measured using an analytical balance. t The measurement interval was 2 days.

[0091] Weight loss rate (%) = (M0 - M) t ) / M0×100% In the formula, M0 is the initial weight M of the cherry. t Let t be the weight of cherries td (t=2, 4, 6, 8). ③ Hardness determination: Take cherries and use a GY-1 fruit hardness tester (8mm probe) to measure the hardness of the flesh at three equidistant locations. Each measurement is repeated ten times, and the results are expressed in Pascals (Pa).

[0092] ④ Color Change: The color of the sweet cherries was measured using a colorimeter on days 0, 2, 4, 6, and 8 of storage, including L. (Lightness), a (Redness-Greenness) and b The (Yellowness-Blueness) value is calculated using the formula to determine the total color difference (ΔE). Before use, the colorimeter should be calibrated on a white board and a black board.

[0093] ΔE=

[0094] In the formula, L0, a0, and b0 are the color values ​​for the 0th d, and L... t a t b t The color value corresponds to the storage time point.

[0095] ⑤ Determination of titratable acid and total soluble solids content Grind 15g of cherries into a homogenate, collect it in a 50mL centrifuge tube, add 45mL of distilled water, and centrifuge at 5000 r / min for 10 min at 4℃ using a low-temperature refrigerated centrifuge. Take the supernatant and determine the soluble solids and titratable acid content. The specific method is as follows: TA: Take 10 mL of supernatant, add 100 μL of 1% phenolphthalein, and titrate the filtrate with 0.1 mol / L NaOH standard solution, using phenolphthalein as an indicator, until the solution turns slightly pink and does not fade within 30 s. Record the volume of NaOH consumed and determine the pH of the mixed solution using a pH meter to determine the titration endpoint. The titration endpoint is defined as a pH between 8.1 and 8.2. Each treatment includes 3 replicates. Calculate TA using the formula.

[0096] TA(%)=V×c×(V1-V0)×f / (V s (×m)×100 In the formula, V is the total volume of the sample extract (mL). s The volume of filtrate used for titration (mL), c is the concentration of NaOH (mol / L), V1 is the volume of sodium hydroxide solution consumed in titrating the filtrate (mL), V0 is the volume of sodium hydroxide solution consumed in titrating distilled water (mL), m is the sample mass (g), and f is the conversion coefficient (g / mmol) = 0.067.

[0097] TSS: The TSS content was measured using a handheld refractometer, and the unit is expressed as °Birx. Calibrate the refractometer according to the instruction manual before use.

[0098] (3) Sensory evaluation of cherries A sensory evaluation panel of 10 untrained experts (students and staff from the School of Public Health, Lanzhou University) aged 20 to 40 was used to evaluate the quality parameters of each group of cherries. Each sample was scored on color, aroma, texture, juiciness, overall perceived quality, and purchase appeal. Color scores ranged from 0 (dull, lackluster) to 10 (bright, glossy); aroma from 0 (no aroma, unpleasant) to 10 (strong, pronounced fruity aroma); juiciness from 0 (insufficient juice) to 10 (abundant juice); texture from 0 (soft) to 10 (firm); overall visual quality from 0 (not fresh) to 10 (extremely fresh); and purchase appeal from 0 (no purchase appeal at all) to 10 (extremely strong purchase appeal). An acceptance threshold of 6.0 was set; any score below 6.0 was considered past the end of shelf life. The final result was the average score of all members.

[0099] Experimental results: The cherry decay rate and weight loss rate after treatment with a bacterial suspension of *Lactobacillus plantarum* TZ28 and *Lactobacillus paracasei* TZ13 at a 1:1 ratio were significantly lower than those of the NaCl group and the control group. P <0.05) Figure 22 , 23 This indicates that treatment with a combined high-altitude lactic acid bacteria suspension significantly reduced the spoilage and weight loss rates of cherries during storage, helping to delay the spoilage process and reduce post-harvest spoilage losses. Treatment with the combined high-altitude lactic acid bacteria suspension also significantly improved cherry firmness at the end of the storage period while maintaining a low level of total soluble solids content. Figure 24 , 25 This helps maintain fruit texture, effectively slows down aging and spoilage of cherries during storage, significantly improves cherry quality, delays ripening, and thus effectively extends shelf life. The color difference of cherries treated with a suspension of high-altitude lactic acid bacteria was improved compared to the CK group and the NaCl group. Figure 26 As storage time increased, the sensory scores of cherries in all three treatment groups gradually decreased. However, the group treated with high-altitude lactic acid bacteria suspension had the highest sensory evaluation score. Cherries treated with high-altitude lactic acid bacteria maintained better appearance, color, texture, and aroma, making them more appealing to consumers. Figure 27 ).

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus plantarum Lactobacillus plantarum TZ28, characterized in that, It was deposited at the China Center for Type Culture Collection on July 22, 2024, with accession number CCTCC NO:M20241618.

2. A strain of Lactobacillus paracasei Lacticaseibacillus paracasei TZ13 is characterized by, It was deposited at the China Center for Type Culture Collection on July 22, 2024, with accession number CCTCC NO:M20241617.

3. A compound microbial agent, characterized in that, Including the Lactobacillus plantarum as described in claim 1 Lactobacillus plantarum TZ28 and the Lactobacillus paracasei as described in claim 2 Lacticaseibacillus paracasei TZ13.

4. The *Lactobacillus plantarum* as described in claim 1 Lactobacillus plantarum TZ28, Lactobacillus paracasei as described in claim 2 Lacticaseibacillus paracasei Application of the compound microbial agent as described in TZ13 or claim 3 in antibacterial activity.

5. The application according to claim 4, characterized in that, The types of bacteria to be inhibited include pathogenic bacteria and pathogenic fungi. The pathogenic bacteria include Staphylococcus aureus, Salmonella and Escherichia coli, and the pathogenic fungi include Penicillium expansum, Botrytis cinerea, Aspergillus charcoalis and Alternaria alternata.

6. A product containing the *Lactobacillus plantarum* as described in claim 1 Lactobacillus plantarum TZ28, Lactobacillus paracasei as described in claim 2 Lacticaseibacillus paracasei The formulation of TZ13 or the compound microbial agent according to claim 3 is characterized in that, The formulations include whole culture media, bacterial suspensions, and cell-free fermentation supernatants.

7. The formulation according to claim 6, characterized in that, The method for preparing the complete culture medium includes the following steps: Activated Lactobacillus plantarum Lactobacillus plantarum TZ28 or Lactobacillus paracasei Lacticaseibacillus paracasei TZ13 was inoculated into 100 mL of MRS liquid medium and cultured at 35-40℃ for 12-36 h to obtain the first generation culture medium. 2-6% of the total volume of the first generation culture medium was taken and inoculated again, and cultured under the same conditions for 12-36 h to obtain the complete culture medium. The method for preparing the bacterial suspension includes the following steps: 1) Activated Lactobacillus plantarum Lactobacillus plantarum TZ28 or Lactobacillus paracasei Lacticaseibacillus paracasei TZ13 was inoculated into 100 mL of MRS liquid medium and cultured at 35-40℃ for 12-36 h to obtain the first generation culture medium. 2-6% of the total volume of the first generation culture medium was taken and inoculated again, and cultured under the same conditions for 12-36 h to obtain the complete culture medium. 2) Centrifuge the entire culture medium to obtain bacterial cells. Wash the bacterial cells with physiological saline and resuspend them to obtain a bacterial suspension. The centrifugation speed is 6000~10000 r / min, the centrifugation time is 10~20 min, the washing is performed 2~4 times, and the concentration of the bacterial suspension is 1×10⁻⁶. 8 ~1×10 10 CFU / mL; The method for preparing the cell-free fermentation supernatant includes the following steps: 1) Activated Lactobacillus plantarum Lactobacillus plantarum TZ28 or Lactobacillus paracasei Lacticaseibacillus paracasei TZ13 was inoculated into 100 mL of MRS liquid medium and cultured at 35-40℃ for 12-36 h to obtain the first generation culture medium. 2-6% of the total volume of the first generation culture medium was taken and inoculated again, and cultured under the same conditions for 12-36 h to obtain the complete culture medium. 2) Centrifuge the whole culture medium to obtain the fermentation supernatant, and filter it through a 0.22μm sterile filter membrane to obtain cell-free fermentation supernatant; the centrifugation speed is 6000~10000r / min, and the centrifugation time is 10~20min.

8. The use of the formulation according to claims 6-7 in inhibiting Alternaria.

9. The *Lactobacillus plantarum* according to claim 1 Lactobacillus plantarum TZ28, Lactobacillus paracasei as described in claim 2 Lacticaseibacillus paracasei The application of the compound microbial agent according to claim 3 or the preparation according to any one of claims 6 to 7 in the preservation of fruits and vegetables.

10. The application according to claim 9, characterized in that, The fruits and vegetables mentioned include cherries.

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