A fermenting *Lactobacillus mucilaginosus* strain that inhibits the growth of *Aspergillus ochraceus* and its application

Abstract

This invention discloses a fermenting *Lactobacillus mucilaginosus* strain that inhibits the growth of *Aspergillus ochraceus* and its applications. The fermenting *Lactobacillus mucilaginosus* is named *Lactobacillus mucilaginosus* (…). Limosilactobacillus leaven The strain A119 is deposited at the China Center for Type Culture Collection (CCTCC); accession number CCTCC NO: M 2025001. This strain is resistant to Aspergillus ochraceus. Aspergillus ochraceus BNCC144159, Penicillium Penicillium expansum BNCC117714 exhibits strong antibacterial effects against pathogenic microorganisms such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella, and Pseudomonas aeruginosa, and can be used for food preservation.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to a fermenting Lactobacillus that inhibits the growth of Aspergillus ochraceus and its application. Background Technology

[0002] Coffee cherries, due to their high water content, sugars, proteins, and other nutrients, are susceptible to mold infection during harvesting, processing, and storage, leading to the production of various mycotoxins. Studies have shown that most coffee samples contain multiple mycotoxins, among which ochratoxin A is the most toxic, widely distributed, and closely related to human health. Ochratoxin A OTA (Oatmea oleifera) is a structurally similar secondary metabolite produced by fungi. It primarily damages the liver and kidneys of animals, exhibiting nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, genotoxicity, and immunotoxicity, thus endangering human health. The International Agency for Research on Cancer (IARC) classifies it as a Group 2B carcinogen. Furthermore, coffee consumption is a major route of OTA intake, and because its toxicity is difficult to completely avoid during production and consumption, OTA has become a major key risk factor for health. According to data from the Codex Alimentarius Commission, the food categories posing the greatest threat to humans from OTA are: cereals > grapes and their products > coffee beans and their products > others. In the national food safety standard GB 2761-2017, "Limits of Mycotoxins in Food," the limit for OTA in roasted coffee beans is 5.0 ug / kg, and in instant coffee it is 10.0 ug / kg. Therefore, inhibiting the growth of Aspergillus ochraceus (Oatmea oleifera) in fresh coffee cherries and their products is crucial. Aspergillus ochraceus Abbreviation: A.ochraceus Antibacterial agents that inhibit the proliferation and growth of bacteria while suppressing the formation of ochratoxin have broad application prospects.

[0003] Currently, the main methods to prevent or reduce ochratoxin contamination of fresh coffee cherries are adding preservatives, improving storage equipment, optimizing the storage environment, and packaging measures. However, adding preservatives poses potential hazards, and improving storage equipment, optimizing the storage environment, and packaging measures have the drawback of significantly increasing production costs, while also damaging the integrity of the coffee cherry's outer skin and reducing its storability.

[0004] Lactic acid bacteria ( lactic acid bacteriaLactic acid bacteria (LAB) are a collective term for Gram-positive bacteria that ferment sugar compounds to produce lactic acid. Lactic acid bacteria play a crucial role in many foods, not only because they produce unique flavors, but more importantly because lactic acid bacteria and their metabolites, such as bacteriocins, organic acids, hydrogen peroxide, extracellular polysaccharides, siderophores, and biosurfactants, possess excellent antibacterial activity. Furthermore, lactic acid bacteria can produce various organic acids through sugar fermentation, mainly including lactic acid, acetic acid, and phenyllactic acid, as well as hydroxyphenyllactic acid and indole-3-lactic acid, all of which have certain antibacterial effects. They also possess natural antibacterial, preservative, and freshness-preserving effects. Nisin, a bacteriocin produced by *Streptococcus lactis*, can inhibit various Gram-positive bacteria that cause food spoilage. It is safe, highly effective, non-toxic, and has no side effects, therefore it is widely used as a food preservative in the food industry. Currently, antimicrobial substances derived from lactic acid bacteria are mainly metabolites in cell-free supernatant (CFS), which can inhibit the growth of some fungi. However, to date, no antimicrobial substances derived from lactic acid bacteria (LAB) that inhibit the growth of Aspergillus ochraceus and the production of OTA have been reported. Therefore, screening for LABs that inhibit the growth of Aspergillus ochraceus can provide a material and theoretical basis for developing green, safe, and healthy antimicrobial agents. Summary of the Invention

[0005] To address the above problems, the purpose of this invention is to provide a strain that inhibits... A.ochraceus (Ochratus) grows fermenting lactobacillus ( Limosilactobacillus fermentum A119 (abbreviation: L. fermentum A119). This invention uses a microplate method to initially screen a lactic acid bacterium with good antibacterial ability— L. fermentum A119, the lactic acid bacteria were isolated and purified from fresh milk from Jinning District, Kunming City. (Discovery) L. fermentum A119 and its CFS have good antibacterial ability, especially against... A.ochraceus It has a significant inhibitory effect and can be used for the preservation of fresh coffee cherries, effectively reducing... A.ochraceus Contamination of fresh coffee cherries.

[0006] The technical solution of this invention is as follows: a fermenting *Lactobacillus mucilaginosus* strain that inhibits the growth of *Aspergillus ochraceus* (… Limosilactobacillus fermentum The fermenting *Lactobacillus* was named: *Lactobacillus fermentatus* A119. L. fermentum A119, with accession number CCTCC NO: M 2025001, accession date January 2, 2025, accession address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The present invention also provides the above. L. fermentum The cultivation method for A119 is as follows: L. fermentum A119 was inoculated in MRS medium and cultured at 37°C for 24 hours.

[0008] The present invention also provides a microbial agent containing the fermenting Lactobacillus mucilaginosus.

[0009] This invention also provides the application of the fermented *Lactobacillus mucilaginosus* or the microbial agent in the preparation of antibacterial agents, wherein the antibacterial application is for non-therapeutic purposes, and the fermented *Lactobacillus mucilaginosus* inhibits the growth of *Aspergillus ochraceus*. The metabolites in the fermentation supernatant of the fermented *Lactobacillus mucilaginosus* and the substances on the cell surface all have a significant ability to inhibit the growth of *Aspergillus ochraceus*.

[0010] Furthermore, the fermented *Lactobacillus mucinus* can also inhibit the growth of one or more of the following microorganisms: *Penicillium*, *Staphylococcus aureus*, *Listeria monocytogenes*, *Escherichia coli*, *Salmonella*, and *Pseudomonas aeruginosa*; the fermented *Lactobacillus mucinus* can be used as a natural antibacterial agent in pharmaceuticals, feed, or cosmetics.

[0011] The present invention also provides the application of the fermented Lactobacillus mucilaginosus that inhibits the growth of Aspergillus ochraceus or the microbial agent in food preservation.

[0012] Preferably, the food product is fresh coffee cherries, coffee powder, or coffee liquid. For example, adding the fermented Lactobacillus mucilaginosus to fresh coffee cherries can not only increase the shelf life of the fresh coffee cherries, but also improve the safety of the food without adding chemical antibacterial agents, shorten the production cycle, and extend the shelf life.

[0013] This invention isolates fermentable Lactobacillus myxobolus from fresh milk in Jinning District, Kunming City, Yunnan Province. Limosilactobacillus fermentum A119. The metabolites and surface substances of the fermentation supernatant of the described *Lactobacillus mucilaginosus* have a significant ability to inhibit the growth of *Aspergillus ochraceus*. Using it as a preservative or auxiliary preservative for fresh coffee cherries can delay mold growth, improve the contamination of fresh coffee cherries by *Aspergillus ochraceus*, effectively extend the storage period of fresh coffee cherries, and does not involve the potential hazards associated with chemical preservatives.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The fermented mucinous lactobacillus provided by this invention has the ability to significantly inhibit the growth of fungi (Acorus ochraceus and Penicillium), Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes), and Gram-negative bacteria (Escherichia coli, Salmonella and Pseudomonas aeruginosa).

[0016] When the fermented Lactobacillus mucilaginosus is applied to coffee preservation, the fermentation supernatant of the Lactobacillus mucilaginosus can effectively reduce the growth of Aspergillus ochraceus and destroy Aspergillus ochraceus mycelia in coffee during the storage of fresh coffee cherries, thus delaying the occurrence of mold in coffee and its products. At 28°C, the storage period of fresh coffee cherries can be extended to 5-6 days, and there are no potential hazards from chemical preservatives, which can improve the quality of fresh coffee cherries and their products. Attached Figure Description

[0017] Figure 1 The colony morphology (A), cell morphology (B), and 16S rDNA electrophoresis identification image (C) of the fermenting Lactobacillus mucinus A119 are shown.

[0018] Figure 2 The image shows the inhibitory effects of three strains of fermenting *Lactobacillus mucilaginosus* on *Ochratus ochraceus*.

[0019] Figure 3 The image shows the antibacterial spectrum of *Lactobacillus mucinus* A119 fermentation. In the image, well A contained 200 μL of MRS, well B contained 200 μL of PBS solution at pH 6.4, and wells C1, C2, and C3 each contained 200 μL of 10- PBS solution. 6 CFU / mL of pathogenic bacteria solution;

[0020] Figure 4 The growth curve and acid production curve of fermented Lactobacillus mucilaginosus A119 are shown.

[0021] Figure 5 Acid tolerance curve for fermentation of Lactobacillus mucilaginosus A119;

[0022] Figure 6 Hemolytic activity test for fermenting Lactobacillus mucilaginosus A119 cells;

[0023] Figure 7 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of fermenting Lactobacillus mucilaginosus A119 against Aspergillus ochraceus were determined.

[0024] Figure 8 The effect of CFS on the surface destruction of Aspergillus ochraceus by fermenting Lactobacillus mucilaginosus A119;

[0025] Figure 9 Changes in the exocarp of coffee cherries during the preservation of fermented Lactobacillus mucilaginosus A119;

[0026] Figure 10 Changes in the pericarp of coffee cherries during the preservation of Lactobacillus fermentum A119. Detailed Implementation

[0027] This invention is based on A. ochraceusUsing Aspergillus ochraceus (BNCC144159) as an indicator bacterium, a lactic acid bacterium with strong antibacterial ability was initially screened using the microplate method. Further... P.expansum (Penicillium) BNCC117714, S.aureus (Staphylococcus aureus) ATCC25923, L. monocytogenes (Listeria monocytogenes) CMCC21633, E.coli (E. coli) CICC10389, Salmonella (Salmonella) WX29 and P.aeruginosa Using *Pseudomonas aeruginosa* ATCC27853 as an indicator bacterium, the broad-spectrum antibacterial activity of the screened LAB and its CFS was determined by double-layer plate method and well method. It was found that... L. fermentum A119 and its CFS exhibit good antibacterial activity, and were subsequently identified as *Lactobacillus fermentans* through morphological and 16S rDNA analysis. Limosilactobacillus fermentum The antibacterial properties of its fermentation supernatant, the antibacterial ability of its bacterial cells, and its preservation ability were determined.

[0028] The relevant culture medium and reagent formulations and test methods in the following examples are as follows.

[0029] 1. Test strain

[0030] Fermented Lactobacillus mucinus E036-A-02 ( L. fermentum E036-A-02) was isolated from pear trees in Dehong, Yunnan, and fermented with Lactobacillus mucilage (… L. fermentum The two strains (A138-A-10) were isolated from pickled vegetables in Fuyuan, Yunnan. They were identified as Lactobacillus fermentans by morphology and 16S rDNA analysis, respectively.

[0031] Indicator bacteria: Aspergillus ochraceus ( Aspergillus ochraceus BNCC144159, abbreviated as BNCC144159 A. ochraceus BNCC144159), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ATCC27853, abbreviated as P.aeruginosa ATCC27853) and Penicillium ( Penicillium expansum BNCC117714 Abbreviation: P.expansum BNCC117714 originates from Beina Chuanglian Biotechnology Co., Ltd.

[0032] Listeria monocytogenes ( Listeria monocytogenes CMCC21633, abbreviated as L. monocytogenes CMCC21633 originates from Guangdong Huankai Microbial Technology Co., Ltd.

[0033] Staphylococcus aureus ( Staphylococcus aureusATCC25923, or S. aureus ATCC25923 for short, was purchased from the U.S. Model Cell / Strain Collection Center and Escherichia coli (E. coli). Escherichia coli CICC10389, abbreviated as E.coli CICC10389) and Salmonella ( Salmonella WX29 was purchased from the China Industrial Microbial Culture Collection Center.

[0034] 2. Formulas for experimental culture media and reagents

[0035] MRS medium (1L): 20.0g glucose, 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 1mL Tween, 0.2g magnesium sulfate and 0.04g manganese sulfate, add distilled water to 1L, pH 7.0; add 15.0g agar for solid medium.

[0036] PDA medium (1L): 12 g potato extract powder and 20 g glucose, add distilled water to 1L for mold culture; add 20.0 g agar for solid culture.

[0037] LB medium (1L): 5.0g yeast extract, 10.0g tryptone and 10.0g sodium chloride, add distilled water to 1L, pH 7.0; add 20.0g agar to make a solid medium.

[0038] TSB medium (1L): 17.0g tryptone, 3.0g plant peptone, 5.0g sodium chloride, 2.5g dipotassium hydrogen phosphate and 2.5g glucose, pH 7.0; add 20.0g agar to make a solid medium.

[0039] Columbia blood agar medium (1L): 10 g casein trypsin digest, 5 g yeast extract, 1 g corn starch, 3 g cardiac trypsin digest, 5 g vesicular trypsin digest, 5 g NaCl, 50-70 mL rabbit blood and 13-15 g agar; add distilled water to 1L.

[0040] 3. Test Methods

[0041] (1) Determination of antibacterial ability

[0042] The optimal concentration of indicator bacteria for antibacterial experiments is determined based on the diameter of the inhibition zone and the regularity of its edges.

[0043] Table 1. Determination of indicator bacteria concentration

[0044]

[0045] When the dilution factor is 100, 180 μL of indicator bacteria is spread on the plate. After diffusion culture, the thickness and size of the inhibition zone are moderate, and the number of indicator bacteria spread on the plate is approximately 10. 6 CFU / mL. Therefore, the indicator bacterium for the antibacterial experiment in this invention (Asteris ochraceus) A. ochraceus BNCC144159, Penicillium P.expansum BNCC117714, Staphylococcus aureus S.aureus ATCC25923, Listeria monocytogenes L. monocytogenes CMCC21633, Escherichia coli E.coli CICC10389, Salmonella Salmonella WX29 and Pseudomonas aeruginosa P.aeruginosa The concentration of ATCC27853 was fixed at 10. 6 CFU / mL.

[0046] Microplate method was used: 100 μL of sterile lactic acid bacteria fermentation broth and 100 μL of 10% sodium hydroxide solution were added to each well of a 96-well plate. 6 CFU / mL A. ochraceus BNCC144159 bacterial suspension was used as an indicator bacterium. After incubation at 37℃ for 12 hours, its absorbance was measured at 600 nm using a microplate reader. The antibacterial activity of lactic acid bacteria was assessed based on its effect against the indicator bacterium. A. ochraceus The antibacterial rate of BNCC144159 is expressed.

[0047]

[0048] When determining the antibacterial properties of bacteria and CFS in this invention, the following were selected: A. ochraceus BNCC144159 P.expansum BNCC117714 S.aureus ATCC25923 L. monocytogenes CMCC21633 E.coli CICC10389 Salmonella WX29 and P.aeruginosa The most typical pathogenic bacteria of ATCC27853 are indicator bacteria. After activation, the indicator bacteria were inoculated at a 1% inoculum into PDA liquid medium, LB liquid medium, and TSB medium, and cultured in a shaker at 28℃ and 37℃ for 72 h and 18 h, respectively. The cultured indicator bacterial solutions were serially diluted as stock solutions, and viable cell counts were performed. Antimicrobial inhibition experiments were conducted using the double-layer agar method and the perforation method.

[0049] Double-layer plate method: Pour 28 mL of MRS solid culture medium (approximately 70°C) evenly into a plate as the lower layer. After complete solidification, draw two horizontal lines (3.5 cm long and 1.5 cm wide) in the center of the petri dish using an inoculation needle dipped in lactic acid bacteria solution. Incubate upside down in a 37°C incubator for 24 hours. Add a 10% concentration of... 6 CFU pathogens ( A. ochraceus BNCC144159 P.expansum 25 mL of PDA semi-solid medium (BNCC117714, etc.) spores were evenly spread on top of MRS solid medium as the upper layer. After the PDA semi-solid medium had completely solidified, the culture was incubated at 28°C for 72 hours. Three replicates were performed for each group, and the consistency of the inhibition zone between the parallel controls was observed. The growth of indicator bacteria around the lactic acid bacteria was observed regularly, and the size of the inhibition zone was recorded and photographed on the culture dishes where inhibition zones appeared.

[0050] Perforation method: Pour 30 mL of PDA solid culture medium into each plate and blow it with sterile air in a sterile operating table for 1 hour. Quickly and evenly spread 180 μL of indicator bacteria on the plate. Use a sterile perforator to press out a 9 mm diameter hole in the plate. Remove the plate with tweezers, add 200 μL of the test sample solution, and incubate in a 37℃ incubator for 24 hours. After incubation, observe and measure the antibacterial ability. The strength of the antibacterial ability of the test sample solution is judged according to the size of the inhibition zone.

[0051] (2) Initial screening of lactic acid bacteria with antibacterial ability

[0052] Multiple strains of lactic acid bacteria preserved in the laboratory were inoculated into MRS liquid medium at a 2% inoculum and cultured at 37°C for 24 hours, activating for three generations. The culture was then centrifuged at 8000 rpm for 15 minutes at 4°C, filtered through a 0.22 μm microporous aqueous filter membrane, and the supernatant was collected for later use. A. ochraceus BNCC144159 was used as an indicator bacterium, and its antibacterial ability was detected using the microplate method.

[0053] (3) Classification and identification of lactic acid bacteria with antibacterial ability

[0054] ① Identification of strain morphology

[0055] The isolated strains were observed for colony morphology, and the bacterial cell morphology was observed under a microscope after Gram staining.

[0056] ② 16S rDNA identification

[0057] PCR amplification of the 16S rDNA gene was performed using genomic DNA as a template. Primers for PCR amplification were 27F (SEQ ID NO. 15'-AGTTTGATCMTGGCTCAG-3') and 1492R (SEQ ID NO. 2 5'-GGTTACCTTGTTACGACTT-3'). PCR reaction conditions included initial denaturation at 98℃ for 2 min, followed by 35 amplification cycles: denaturation at 98℃ for 10 s, annealing at 56℃ for 10 s, and extension at 72℃ for 10 s. The final extension was performed at 72℃ for 5 min. The amplified PCR products were subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) at 300V for 12 min. The sequenced products were then compared with nucleic acid sequence databases using BLAST.

[0058] (4) Antibacterial spectrum analysis of lactic acid bacteria with antibacterial ability

[0059] Double-layer plate method for determination L. fermentum Antibacterial activity of A119: Three strains of *Lactobacillus fermentatus* (A119, E036-A-02, and A138-A-10) were inoculated into MRS liquid medium at a 2% inoculum and cultured at 37℃ for 24 h, activating for three generations. The antibacterial activity of the three *Lactobacillus fermentatus* strains was then determined using the double-layer plate method. A. ochraceus The inhibitory effect of BNCC144159 was studied, and the size of the inhibition zone was recorded and photographed.

[0060] Drilling method for determination L. fermentum A119 broad-spectrum antibacterial activity: L. fermentum A119 was inoculated at a 2% inoculum into MRS liquid medium and cultured at 37°C for 24 hours, activating for three generations. After centrifugation at 8000 rpm for 15 minutes at 4°C, the mixture was filtered through a 0.22 μL microporous aqueous filter membrane. The supernatant was collected, and its effectiveness against environmental factors was measured using both the double-layer plate method and the perforation method. P.expansum BNCC117714 S.aureus ATCC25923 L. monocytogenes CMCC21633 E.coli CICC10389 Salmonella WX29 and P.aeruginosa The inhibitory effect of ATCC27853 was studied, and the area of ​​the inhibition zone was recorded and photographed.

[0061] (5) L. fermentum Characterization of strain A119

[0062] ① L. fermentum Determination of A119 growth curve and acid production curve

[0063] Activated L. fermentumStrain A119 was inoculated into MRS liquid fermentation medium at a 2% inoculum and cultured statically at 37°C for 24 hours. Samples were taken every 2 hours to measure the changes in pH and OD600nm during the fermentation process and plotted curves.

[0064] ② L. fermentum A119 Acid Resistance Determination

[0065] The activated A119 strain was inoculated into MRS liquid fermentation medium at a 2% inoculum and cultured statically at 37°C for 24 h. MRS broths with pH values ​​of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 were prepared by resuspending 2% (v / v) bacterial weight in 1 mol / L HCl solution. These broths were then incubated statically at 37°C for 20 h, and their OD values ​​were measured at 600 nm.

[0066] ③ The Kirby–Bauer test (KB test) was used for diffusion of agar plates.

[0067] Detection L. fermentum Drug sensitivity of A119. The concentration of the activated L. fermentum A119 suspension was adjusted to 1×10⁻⁶. 6 CFU / mL, 200 μL of bacterial suspension was spread onto MRS solid medium and incubated at sterile room temperature for 10-20 min. Using sterile forceps, one drug sensitivity test strip was evenly placed on the surface of each plate, in triplicate. The plates were then anaerobically incubated at 37℃ for 24 h. The inhibition zone of the drug sensitivity test strip was measured with calipers. The drug sensitivity results were determined according to the standards of the Clinical and Laboratory Standards Institute (CLSI) as: sensitive (S), moderately sensitive (I), and resistant (R).

[0068] Table 2 Antibiotic susceptibility criteria

[0069]

[0070] (6) L. fermentum A119 hemolytic test

[0071] After three generations of activation L. fermentum A119 and Staphylococcus aureus ATCC25923 were streaked onto Columbia blood agar medium and incubated at 37°C for 48 h. The hemolytic zones around the colonies were observed and recorded.

[0072] (7) L. fermentum Determination of the antibacterial properties of A119 supernatant

[0073] ① L. fermentumDetermination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of A119 supernatant.

[0074] Will A. ochraceus Inoculate at 1% (v / v) in PDB liquid medium and incubate at 28°C and 120 rpm for 72 h. After incubation, A. ochraceus Centrifuge at 8000 rpm, 4°C for 10 min, then wash with PBS buffer, repeating 3 times. Adjust the A. ochraceus concentration to 1×10⁻⁶. 6 CFU / mL. L. fermentum A119 CFS was diluted twofold with PDB liquid medium. 100 μL of the stock solution or dilution was added to a sterile 96-well plate, and 100 μL of bacterial culture was inoculated to achieve final CFS ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32. L. fermentum CFS (Chemical Freeze-Off) of A119 was used as a positive control, and PDA medium without CFS was used as a negative control. The 96-well plates were incubated at 28°C for 48 hours. The lowest concentration at which no bacterial growth occurred was defined as the minimum inhibitory concentration (MIC). 100 μL of sample from each clear well was spread onto PDA medium and incubated at 28°C for 72 hours. The lowest concentration at which no colony growth occurred was defined as the minimum bactericidal concentration (MBC).

[0075] ② Scanning electron microscopy observation

[0076] Cultured on PDA for 72 hours A. ochraceus After washing, adjust the bacterial concentration to 1×10⁻⁶ using PDA liquid culture medium. 6 CFU. Take 2 mL of the prepared indicator bacterial suspension and add 5 mL of... L. fermentum In the CFS of A119, indicator bacteria treated with PBS served as a blank control and were incubated on a shaker at 28°C for 5 days. The bacterial cells were fixed with 2.5% glutaraldehyde at 49°C for 2-4 hours, washed with PBS and ultrapure water, and then dehydrated with different concentrations of ethanol and tert-butanol to prepare a tert-butanol bacterial suspension. After pre-cooling at -80°C, the suspension was freeze-dried. After gold sputtering, the surface structural changes of *A. ochraceus* were observed using a scanning electron microscope.

[0077] (8) L. fermentum Application of A119 in the preservation of fresh coffee cherries

[0078] Fresh coffee cherries were collected from Ziyuan Coffee Factory in Pu'er City, Yunnan Province. Coffee cherries of uniform size, shape, and without damage or infection were selected based on their uniformity. The cherries, with stems attached, were cut with scissors and 5mm in diameter. The surface of the cherries was disinfected with 75% alcohol for 3 minutes, followed by rinsing three times with sterile water. The washed cherries were then air-dried in a biosafety cabinet.

[0079] The fresh coffee cherries were divided into two groups:

[0080] Group 1 (CL) simulates the storage of intact, undamaged fresh coffee cherries. The fresh coffee cherries are soaked in 100mL of... L. fermentum A119 (10) 8 Incubate at CFS (CFU / mL) for 3 min, then incubate for 2 h in a biosafety cabinet. Afterwards, inoculate with 20 μL of [unclear - likely a specific solution]. A. ochre Spore suspension (concentration of 10) 8 (CFU / mL). Fresh coffee cherries were placed in sterile containers and stored in a 28°C humidity-controlled incubator. Each treatment was performed in triplicate.

[0081] The second group (CL) simulated the storage of fresh coffee cherries with damaged skin. A 2mm hole was made in the coffee cherries using a sterile inoculation needle, and then the cherries were soaked in 100mL of [unclear - possibly a specific solution]. L. fermentum Incubate A119 in CFS for 3 min, then incubate in a biosafety cabinet for 2 h. Inoculate 20 μL of the solution into coffee-ground wells. A. ochraceus Spore suspension (concentration of 10) 8 The processed coffee was placed in a sterile container, which was then stored in a 28°C constant temperature and humidity incubator.

[0082] No vaccination L. fermentum A119, only inoculated with the same volume A. ochraceus Fresh coffee cherries in spore suspension served as the control group (Con).

[0083] Example 1: Screening of lactic acid bacteria strains with antibacterial activity

[0084] by A. ochraceus Using BNCC144159 as an indicator bacterium, 21 lactic acid bacteria strains were initially screened (Table 3). The results showed that 3 lactic acid bacteria strains exhibited inhibitory activity greater than 50% against the indicator bacterium. Among them... L. fermentum A119 against the target strain A. ochre BNCC144159 showed the most significant antibacterial effect, at 61.05%.

[0085] Table 3 shows the inhibitory effect. A. ochraceus Initial screening results of BNCC144159 probiotic strain

[0086]

[0087] Example 2: Strain Identification

[0088] (1) Morphological identification of strains

[0089] The colony morphology and Gram staining of the strain are shown in the figures below. Figure 1 A and Figure 1 B. In MRS agar medium L. fermentum A119 grew well on MRS solid medium plates. For example... Figure 1 As shown in Figure A, the strain has a circular (1-2 μm in diameter) convex shape, is milky white in color, and has a viscous texture, exhibiting typical growth characteristics of lactic acid bacteria; all strains turn purple after Gram staining, indicating that the strain is a Gram-positive bacterium. Figure 1 B).

[0090] Further 16S rDNA sequencing was performed on the strain, and extracting... L. fermentum Total DNA from A119 was amplified using 16S rDNA-specific primers and the strain's genomic DNA as a template, yielding a PCR fragment of approximately 1500 bp. The amplified fragment was then subjected to 2% agarose gel electrophoresis. Figure 1 As shown in C. The PCR products were sent to Megvii Biotechnology Co., Ltd. for sequencing, and the sequencing results were entered into the online comparison at www.NCBI.nlm.nih.gov. L. fermentum The 16S rDNA sequence of A119 (as shown in SEQ ID NO.3) and Limosilactobacillus fermentum The homology with (Lactobacillus fermentatus) was 99.859%, and the identification... L. fermentum A119 belongs to the Lactobacillus fermentans family and is named Lactobacillus fermentans A119. Limosilactobacillus fermentum A119.

[0091] (2) L. fermentum Observation of A119's antibacterial spectrum

[0092] The inhibition zone method is a way to measure the bactericidal effect, mainly used to determine the antibacterial activity, and is a qualitative or semi-quantitative method. After subculturing, the proliferation and acid-producing abilities of the strains that passed the initial screening were further improved. Therefore, the inhibitory effects of three strains of *Lactobacillus fermentata* on *antimicrobial* activity were again determined using the double-layer plate method. A. ochraceus The antibacterial effect of BNCC144159.

[0093] The results are as follows Figure 2 As shown in Table 4, the three strains of fermenting *Lactobacillus myxobolus* have the effect on... A. ochraceus The inhibitory effects of BNCC144159 showed varying degrees of difference, among which, L. fermentum The inhibition zone area of ​​A119 reached 49.05±3.89mm. 2 To achieve the best results, L. fermentum A119> L. fermentum E036-A-02 > L. fermentum A138-A-10, the results show L. fermentum A119's A. ochraceus BNCC144159 showed the best inhibitory effect.

[0094] Table 4. Effects of three strains of fermenting *Lactobacillus mucilage* on... A. ochraceus Inhibitory effect of BNCC144159

[0095]

[0096] Simultaneous application of the inhibition zone method to determine L. fermentum A119 bacterial cells and its CFS against pathogenic strains ( P.expansum BNCC117714 S.aureus ATCC25923 E.coli CICC10389 Salmonella WX29 P.aeruginosa ATCC27853 L. monocytogenes The antibacterial ability of CMCC21633.

[0097] The results are as follows Figure 3 As shown in Table 5, L. fermentum A119 has good antifungal effects against molds and various Gram-positive and Gram-negative bacteria.

[0098] Table 5 L. fermentum A119's CFS antibacterial effect

[0099]

[0100] (3) L. fermentum A119 Features and Safety

[0101] ① L. fermentum A119 growth curve and acid production curve

[0102] Good growth and reproduction capacity is the basis for lactic acid bacteria to exert their probiotic properties. L. fermentum The growth curve of A119 is shown in Figure 4. From 0-4 h, the strain grows slowly, representing the lag phase. From 4-20 h, it is in the logarithmic growth phase, during which the strain grows rapidly. From 20-24 h... L. fermentum A119 has entered a stable growth period.

[0103] The pH change of lactic acid bacteria culture broth is closely related to the growth of the bacterial strains. During growth, lactic acid bacteria can produce organic acids such as lactic acid and acetic acid, which causes the pH of the fermentation broth to decrease. The pH change curve of the fermentation broth is shown in the figure below. Figure 4 As shown, the pH decreased rapidly from 0 to 18 h as the number of bacteria increased; after 18 h, the proliferation rate of the strain was significantly inhibited, the rate of pH decrease slowed down, and the final pH was 3.78.

[0104] ② L. fermentum A119 Acid Resistance Determination

[0105] The ability of a strain to grow within a specific pH range is an important physiological characteristic of lactic acid bacteria. This invention investigates... L. fermentum A119's acid resistance. For example... Figure 5 As shown, L. fermentum A119 grows weakly in strongly acidic regions (pH 1-3) but grows well in weakly acidic regions (pH 3-5). During the preservation of coffee, the initial pH is around 6, which can decrease to 4.5 over time. Therefore, L. fermentum A119 exhibits the potential to adapt to environmental conditions during the preservation of fresh coffee cherries, and it can be considered as a biocontrol bacterium to inhibit OTA contamination in fresh coffee cherries and their products.

[0106] ③ L. fermentum A119 antibiotic sensitivity

[0107] Multiple studies have shown that differences in antibiotic resistance may exist among lactic acid bacteria. Therefore, for L. fermentum Resistance to A119 was assessed. Table 6 shows... L. fermentum The results showed the susceptibility of A119 to 18 commonly used antibiotics. L. fermentum A119 exhibits some degree of resistance to kanamycin, streptomycin, gentamicin, doxycycline, and vancomycin. This observed resistance may be due to the limited permeability of the lactic acid bacteria cell membrane to aminoglycosides, tetracyclines, and glycopeptides.

[0108] Table 6 L. fermentum A119 antibiotic sensitivity

[0109]

[0110] Note: "S": Sensitive; "I": Moderately sensitive; "R": Resistant; "--" indicates no zone of inhibition.

[0111] ④ L. fermentum A119 hemolytic test

[0112] The safety of lactic acid bacteria strains used in coffee and coffee products must be considered, and non-hemolytic activity is one of the main safety standards for probiotics. Using Staphylococcus aureus ATCC25923 as a positive control, [the following is a separate, unrelated sentence:] ... L. fermentum A119 was subjected to a hemolysis test ( Figure 6 The results showed that the positive control *Staphylococcus aureus* ATCC25923 exhibited β-hemolytic activity, a hallmark of highly pathogenic bacteria, while... L. fermentumA119 exhibits gamma-hemolysis (i.e., no hemolytic activity), confirming that it does not produce hemolytic toxins and is suitable for food fermentation or probiotic products.

[0113] Example 3 L. fermentum Antibacterial properties of A119 supernatant

[0114] ① Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of L. fermentum A119 supernatant

[0115] by L. fermentum CFS of A119 was used as a positive control, and was assessed by measuring MIC and MBC. L. fermentum A119's CFS A. ochraceus The antibacterial ability of BNCC144159 was observed. Results were as follows... Figure 7 As shown, L. fermentum A119's CFS A. ochraceus The MIC of BNCC144159 is 1:8 (bacterial solution dilution ratio), and the MBC is 1:2 (bacterial solution dilution ratio).

[0116] ② L. fermentum Scanning electron microscopy observation of A119 CFS on A. ochraceus hyphae

[0117] Analysis using scanning electron microscopy L. fermentum A119's CFS pair A. ochraceus The influence of the micromorphology of BNCC144159 hyphae is shown in the results. Figure 8 As shown. The control group (Con group) showed that, A. ochraceus The hyphae of BNCC144159 were normal in morphology, with a uniform and dense structure, neatly arranged hyphae, and smooth cell walls. However, the hyphae of A. ochraceus BNCC144159 in the experimental group (CL group) showed significant changes, with the hyphae becoming shrunken, curled, folded, and sunken, losing their linearity.

[0118] Example 4 L. fermentum Application of A119 in the preservation of fresh coffee cherries

[0119] from Figure 9 It can be concluded that in an environment of 28℃, A.chraceus BNCC144159 began to grow mycelium on the surface of fresh coffee cherries in the control group (Con) on day 3, showing signs of mold growth. By day 7, all three coffee cherry samples were completely moldy, reaching a 100% mold rate. L. fermentum After A119 treatment, samples in the treatment group (CL) developed mold on day 6, and by day 9, all three samples were fully moldy on the surface, indicating that... L. fermentum A119's CFS pair A.chraceus It has a significant inhibitory effect, and can extend the storage period of whole, undamaged fresh coffee cherries to 5 days.

[0120] from Figure 10 It can be concluded that in an environment of 28℃, A.chraceus BNCC144159 showed Aspergillus ochraceus hyphae at the damaged areas of fresh coffee cherries in the control group (Con) starting on day 3. This experimental result is consistent with... Figure 9 The results from Group Con were consistent; by day 6, *Aspergillus ochraceus* hyphae had begun to invade the interior of the coffee fruit, and by day 9, the surface of all three fresh coffee fruit samples was completely moldy, with a mold rate of 100%. However, in the fresh coffee fruit samples treated with *L. fermentum* A119, *Aspergillus ochraceus* hyphae only appeared on the surface of the pericarp on day 7, and the hyphae only invaded the interior of the coffee pericarp on day 9, indicating that... L. fermentum A119's CFS pair A.chraceus BNCC144159 still exhibits significant inhibitory effects. L. fermentum A119's CFS can extend the storage period of fresh coffee cherries with damaged pericarps by 6 days.

[0121] The above results indicate that L. fermentum The CFS of A119 at 28°C was measured in fresh coffee cherries. A.chraceus BNCC144159 has a significant inhibitory effect, which can extend the storage period of fresh coffee cherries by about 5-6 days.

[0122] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention extends to all other methods and applications having the same function.

Claims

1. A fermenting Lactobacillus strain that inhibits the growth of Aspergillus ochraceus ( Limosilactobacillus fermentum ), characterized in that, The fermenting *Lactobacillus mucinus* was named *Lactobacillus mucinus* A119, with the accession number CCTCC NO: M2025001.

2. A microbial inoculant, characterized in that, The microbial agent contains the fermenting Lactobacillus mucinus as described in claim 1.

3. The use of the fermented *Lactobacillus mucinus* of claim 1 or the microbial agent of claim 2 in the preparation of antibacterial agents against *Aspergillus ochraceus*, *Penicillium expansum*, *Staphylococcus aureus*, *Listeria monocytogenes*, *Escherichia coli*, *Salmonella*, or *Pseudomonas aeruginosa*.

4. The application of the fermented Lactobacillus mucinus according to claim 1 or the microbial agent according to claim 2 in food preservation.

5. The application according to claim 4, characterized in that, The food product is fresh coffee fruit, coffee powder, or coffee liquid.

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