Microorganism, microbial agent, food, preparation method of food and method for inhibiting growth of pathogenic bacteria in vitro

Abstract

The invention discloses a microorganism, a microbial agent, food and a preparation method of the food, and relates to the field of microorganisms. The microorganism is hansenula polymorpha, and the preservation number of the microorganism is CGMCC (China General Microbiological Culture Collection Center) No.30796. The hansenula uvarum new strain obtained through screening is high in growth speed and high in acid resistance and salt resistance, especially can normally grow in a 3 mM nitrite environment, can inhibit growth of escherichia coli and staphylococcus aureus and is high in nitrate metabolism capacity; the fermented food prepared from the new strain is low in salt content, nutritional, healthy and unique in flavor; a food fermentation process developed by utilizing the new strain is high in fermentation efficiency, short in production period and high in application value.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a microorganism, a microbial agent, a food, a method for preparing food, and a method for inhibiting the growth of pathogenic bacteria in vitro. Background Technology

[0002] Salt is an essential component in the processing of fermented chili peppers. It reduces the water activity of the peppers, inhibits the growth of spoilage bacteria, extends the shelf life of the peppers, and improves their flavor. Traditional fermented chili peppers typically contain 15-25% salt, relying on microorganisms attached to the surface of the raw materials for fermentation.

[0003] With the global advocacy for low-sodium diets, reduced-sodium fermented chili peppers are gaining increasing attention. Reduced-sodium fermented chili peppers typically contain 5-12% salt; however, during fermentation, most yeasts still cannot avoid a decrease in viable bacteria due to salt stress. Therefore, under natural fermentation conditions, microbial growth is slow and the fermentation process is long, making it difficult to meet the needs of large-scale production of reduced-sodium fermented foods.

[0004] Therefore, it is still necessary to screen for yeasts with good salt and acid tolerance as well as good fermentation performance to meet the needs of developing low-salt fermented foods and their fermentation processes. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, this invention provides a *Hansenula polymorpha* strain for grape juice, FVPHBJ24146, and its applications. This invention also provides microbial agents, food or feed, food preparation methods, and methods for in vitro inhibition of pathogenic bacteria. The new *Hansenula polymorpha* strain for grape juice obtained by this invention has advantages such as rapid growth, strong acid and salt tolerance, ability to inhibit the growth of *Escherichia coli* and *Staphylococcus aureus*, and strong nitrate metabolism. Fermented foods made using this new strain have low salt content, rich yeast content, are nutritious and healthy, and have a unique flavor. The food fermentation process developed using the new strain and microbial agents of this invention has high fermentation efficiency, short production cycle, and high application value.

[0006] Therefore, in a first aspect, the present invention provides a microorganism. According to an embodiment of the present invention, the microorganism is *Hansenula polymorpha* (from grape juice). Hanseniaspora grapes It was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30796.

[0007] Preservation information: Strain name: *Hansenula polymorpha* from grape juice ( Hanseniaspora grapes )FVPHBJ24146 Classification and nomenclature: Hansenula polymorpha in grape juice Hanseniaspora grapes Deposit date: May 27, 2024 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Accession number: CGMCC No. 30796 Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China The grape juice obtained by screening in this invention contains Hansenula polymorpha ( Hanseniaspora grapes The new strain FVPHBJ24146 exhibits rapid growth, strong acid and salt tolerance, and can even grow normally in a 3 mM nitrite environment. It can also inhibit the growth of Escherichia coli and Staphylococcus aureus and has a strong ability to metabolize nitrates. Fermented foods made using this new strain have low salt content, rich yeast content, are nutritious and healthy, and have a unique flavor. The food fermentation process developed using this new strain has high fermentation efficiency, short production cycle, and high application value.

[0008] In a second aspect, the present invention provides a microbial inoculant. According to embodiments of the present invention, the microbial inoculant includes the aforementioned microorganisms.

[0009] In a third aspect, the present invention provides a food or feed. According to embodiments of the present invention, the food or feed comprises at least one of the aforementioned microorganisms or the aforementioned microbial agents. The food of the present invention has low salt content, rich yeast content, is nutritious and healthy, and has a unique flavor; the feed of the present invention has low salt content, rich yeast content, and high nutritional value.

[0010] In a fourth aspect, the present invention proposes the use of the aforementioned microorganisms or the aforementioned microbial agents in the preparation of food or feed.

[0011] In a fifth aspect, the present invention provides a method for food preparation. According to an embodiment of the invention, the method includes: fermenting one or more of the aforementioned microorganisms and microbial agents with a food raw material to be fermented, thereby obtaining the food. The method according to the embodiment of the invention has high fermentation efficiency, short production cycle, and high application value.

[0012] In a sixth aspect, the present invention provides a method for inhibiting the growth of pathogenic bacteria in vitro. According to an embodiment of the invention, the method includes: co-culturing at least one of the aforementioned microorganisms and the aforementioned microbial agents with a sample containing pathogenic bacteria; wherein the pathogenic bacteria are selected from at least one of Staphylococcus aureus and Escherichia coli. According to the method of the embodiment of the invention, grape juice contains Hansenula polymorpha (… Hanseniaspora grapesFVPHBJ24146 has antibacterial effects and can effectively inhibit the growth of Staphylococcus aureus and Escherichia coli. It can be used to inhibit the growth of pathogens in vitro.

[0013] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0014] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The grape juice in Example 2 of this invention contains Hansenula polymorpha (Spp. spp.) Hanseniaspora grapes The morphological identification results of FVPHBJ24146 are shown in the figure. Figure 2 The grape juice in Example 3 of this invention contains Hansenula polymorpha (Spp. spp.) Hanseniaspora grapes Growth curve of FVPHBJ24146; Figure 3 The grape juice in Example 3 of this invention contains Hansenula polymorpha (Spp. spp.) Hanseniaspora grapes The absorbance (OD) values ​​of FVPHBJ24146 before and after culture in different pH media. 600 ) Observation results diagram; Figure 4 The grape juice in Example 3 of this invention contains Hansenula polymorpha (Spp. spp.) Hanseniaspora grapes Figure 1 shows the results of investigating the viable bacterial count of FVPHBJ24146 in culture media with different pH values. Figure 5 The grape juice in Example 3 of this invention contains Hansenula polymorpha (Spp. spp.) Hanseniaspora grapes The absorbance (OD) values ​​of FVPHBJ24146 before and after culture in media containing different salt concentrations were compared. 600 ) Observation results diagram; Figure 6 The grape juice in Example 3 of this invention contains Hansenula polymorpha (Spp. spp.) Hanseniaspora grapes The results of the study on the number of viable bacteria of FVPHBJ24146 in culture media with different salt concentrations are shown in the figure. Figure 7 In the fermentation process of the fermented vegetables in Example 4 of this invention, the grape juice contained Hansenula polymorpha (… Hanseniaspora grapes Figure showing the results of the viable cell count of FVPHBJ24146; Figure 8 The grape juice in Example 5 of this invention contains Hansenula polymorpha (Spp. spp.) Hanseniaspora grapes The results of the investigation on the inhibition of Escherichia coli growth in the FVPHBJ24146 experimental group and the Escherichia coli control group are shown in the figure. Figure 9The grape juice in Example 5 of this invention contains Hansenula polymorpha (Spp. spp.) Hanseniaspora grapes The results of the investigation on the inhibition of Staphylococcus aureus growth in the FVPHBJ24146 experimental group and the Staphylococcus aureus control group are shown in the figure. Figure 10 The grape juice in Example 6 of this invention contains Hansenula polymorpha (Spp. spp.) Hanseniaspora grapes Figure showing the results of the study on the ability of FVPHBJ24146 to metabolize nitrates. Detailed Implementation

[0015] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0016] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0017] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0018] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0019] In this article, the term "YPD medium" refers to yeast culture medium, which can be divided into two categories based on the content of solidifying agent (usually agarose): YPD solid medium and YPD liquid medium.

[0020] In this article, the term "Hansenula polymorpha of grape juice" refers to a yeast belonging to the phylum Ascomycota (…). Ascomycota Ascomycetes ( Saccharomycetes Hansenula order ( ) Hanseniasporales ) of the Hansenula polymorpha family ( Hanseniasporaceae ) of the genus *Hansenula* ( Hansenia sporatum ), white creamy colonies, without pseudohyphae.

[0021] This invention proposes microorganisms, microbial agents, food or feed, methods for food preparation, and methods for in vitro inhibition of pathogens, which will be described in detail below.

[0022] microorganism This invention proposes a microorganism. According to an embodiment of the invention, the microorganism is *Hansenula polymorpha* (from grape juice). Hanseniaspora grapes The specimen, named FVPHBJ24146, was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 30796.

[0023] According to an embodiment of the present invention, the 16S rDNA sequencing results of the microorganism are shown in SEQ ID NO: 1.

[0024] The grape juice obtained by screening in this invention contains Hansenula polymorpha ( Hanseniaspora grapes The new strain FVPHBJ24146 exhibits rapid growth, strong acid and salt tolerance, and can even grow normally in a 3 mM nitrite environment. It can also inhibit the growth of Escherichia coli and Staphylococcus aureus and has a strong ability to metabolize nitrates. Fermented foods made using this new strain have low salt content, rich yeast content, are nutritious and healthy, and have a unique flavor. Food fermentation processes developed using this new strain have high fermentation efficiency, short production cycles, and high application value.

[0025] In this article, the term "grape juice containing Hansenula polymorpha" is used. Hanseniaspora grapes "FVPHBJ24146" is synonymous with "Hansenula polymorpha f. sp. grape juice".

[0026] Microbial agents This invention provides a microbial inoculant. According to embodiments of the invention, the microbial inoculant includes the aforementioned microorganisms. The microbial inoculant of this invention can be used to prepare fermented foods, and upon activation, it produces acid, which can be used to adjust the acidity of the food. Furthermore, this microbial inoculant can also inhibit the growth of pathogenic bacteria such as *Escherichia coli* and *Staphylococcus aureus*.

[0027] In some specific embodiments, the microbial agent is a liquid microbial agent, and the concentration of the microorganisms is 1×10⁻⁶. 9 ~1×10 12 CFU / mL.

[0028] For example, the concentration of microorganisms in the aforementioned liquid microbial agent can be: 1×10⁻⁶ 9 CFU / mL, 5×10 9 CFU / mL, 1×10 10 CFU / mL, 5×10 10 CFU / mL, 1×10 11 CFU / mL, 5×10 11 CFU / mL, 1×10 12 CFU / mL.

[0029] It should be noted that the microbial inoculant of the present invention may be a liquid microbial inoculant, including but not limited to fermentation products; or it may be a solid microbial inoculant, including but not limited to freeze-dried powder.

[0030] It should be noted that in the microbial inoculant of the present invention, *Hansenula polysaccharide* from grape juice can exist in the form of live cells and / or non-live cells.

[0031] In this article, "living cell" refers to a cell that has the ability to metabolize, reproduce, or replicate.

[0032] For example, the living cells may be immobilized cells. In this document, "immobilized cells" refers to living cells fixed on a carrier, which can carry out life activities such as growth, development, reproduction, heredity, and metabolism within a certain spatial range.

[0033] In this document, "non-living cells" refers to cells that do not have the ability to metabolize, reproduce, and replicate, including but not limited to dried bacterial cells. For example, the microbial agent is a lyophilized powder.

[0034] In some specific embodiments, the grape juice contains Hansenula polymorpha FVPHBJ24146 as live cells, dry cells, immobilized cells, or in any other form.

[0035] In some specific embodiments, the dried mycelium is obtained by freeze-drying the grape juice Hansenula polymorpha FVPHBJ24146.

[0036] In some specific implementations, the microbial agent may also contain at least one acceptable strain of bacteria found in food or feed.

[0037] In some specific embodiments, the microbial agent further includes an excipient or carrier acceptable for use in food or animal feed.

[0038] In this article, "acceptable in food" refers to substances or compositions that are edible for human consumption, which may be adjusted according to the food requirements of different countries.

[0039] In this article, “acceptable adjuvants or carriers in animal feed” refers to substances or compositions that can be consumed by animals, and these can be adjusted according to the animal feed requirements of different countries.

[0040] It should be noted that the characteristics and advantages described above for the microorganisms also apply to this microbial agent, and will not be repeated here.

[0041] food or feed This invention provides a food or feed. According to embodiments of the invention, the food or feed includes at least one of the aforementioned microorganisms or microbial agents. The food or feed of this invention has low salt content, is rich in yeast, is nutritious and healthy, and has a unique flavor.

[0042] According to embodiments of the present invention, it further includes food or feed-acceptable excipients or carriers.

[0043] In some specific embodiments, the aforementioned microorganisms or microbial agents are added to or inoculated into food or feed. This further yields food or feed that is low in salt, rich in yeast, nutritious, and has a unique flavor.

[0044] For example, food includes, but is not limited to: probiotic tablets, fermented dairy products (such as probiotic yogurt), probiotic solid beverages, probiotic milk powder, probiotic cheese, probiotic soy products, probiotic candies, probiotic fermented vegetables, etc.

[0045] For example, food includes, but is not limited to, probiotic fermented feed.

[0046] It should be noted that the characteristics and advantages described above for microorganisms also apply to this food or feed, and will not be repeated here.

[0047] use The present invention also proposes the use of the aforementioned microorganisms or the aforementioned microbial agents in the preparation of food or feed.

[0048] It should be noted that the characteristics and advantages described above for microorganisms also apply to this application, and will not be repeated here.

[0049] method This invention proposes a method for preparing food. According to an embodiment of the invention, the method includes: fermenting one or more of the aforementioned microorganisms and the aforementioned microbial inoculant, along with the food raw material to be fermented, to obtain the food.

[0050] According to embodiments of the present invention, food ingredients include, but are not limited to: chili peppers, cabbage, cucumbers, and radishes.

[0051] The present invention also proposes a method for inhibiting the growth of pathogenic bacteria in vitro. According to an embodiment of the present invention, the method includes: co-culturing at least one of the aforementioned microorganisms and the aforementioned microbial agents with a sample containing pathogenic bacteria.

[0052] According to an embodiment of this invention, the pathogen is selected from at least one of Staphylococcus aureus and Escherichia coli.

[0053] It should be noted that the characteristics and advantages described above for microorganisms also apply to this method, and will not be repeated here.

[0054] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0055] Example 1: Obtaining Hansenula polymorpha FVPHBJ24146 from grape juice The grape juice containing Hansenula polymorpha FVPHBJ24146 of this invention was isolated from fermented chili sauce from Hebei.

[0056] Strain collection and isolation process: Naturally fermented chopped chili peppers were added to sterilized physiological saline, mixed well, and then diluted and spread on YPD medium (10.0g peptone, 100g yeast extract, 200g glucose). The medium was incubated at pH 6.0±0.2 and 30℃ for 24-48 h. Single colonies of different sizes and morphologies were picked and streaked on fresh YPD medium plates and incubated at 30℃ for 24-48 h. The streaking purification culture was repeated multiple times until the colonies in the plates had a uniform morphology. After microscopic examination to find no contaminating bacteria, the culture was identified.

[0057] Strain preservation: Mix the bacterial culture with sterile 50% glycerol in a cryovial at a 1:1 ratio and store at -80℃.

[0058] Example 2: Identification of Hansenula polymorpha f. sp. FVPHBJ24146 in grape juice After further confirming that the culture isolated and purified in Example 1 was a pure culture by streak and smear microscopy, morphological and physiological biochemical identification of the bacterial strain, PCR amplification and sequencing analysis of the 16S rDNA gene were performed.

[0059] 1. Morphological and physiological / biochemical identification Referring to the "Handbook for Systematic Identification of Common Fungi", FVPHBJ24146 was identified in terms of morphological characteristics and physiological and biochemical properties.

[0060] The identification results showed that the colony morphology on YPD agar medium was creamy, with no pseudohyphae. Figure 1 The final pH in YPD liquid medium was 3.5.

[0061] 2. PCR amplification and sequencing analysis of the 16S rDNA gene The 16S rDNA sequence of *Hansenula polymorpha* FVPHBJ24146 was identified using grape juice. PCR amplification conditions were as follows: pre-denaturation at 95℃ for 10 min, followed by 30 cycles: denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 1.5 min (1 kb / min), and final extension at 72℃ for 5 min; storage at 4℃. The obtained PCR products were detected by 1% agarose gel electrophoresis. The PCR reaction system is shown in Table 1.

[0062] Table 1 PCR reaction system

[0063] The samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA sequencing. The obtained sequences were compared with those in GenBank, and a phylogenetic tree was constructed using MEGA11.0.13 to compare the sequences of the tested strain with those of other strains in the same genus.

[0064] The results showed that the isolated strain FVPHBJ24146 belonged to the same branch as other strains of *Hansenula polymorpha* found in grape juice. Hanseniaspora grapes The 16S rDNA sequence showed 99% homology. Combined with physiological and biochemical identification, FVPHBJ24146 was further confirmed to be *Hansenula polymorpha*, with the sequence accession number CGMCC No. 30796.

[0065] The final identification confirmed that the isolated strain was a strain of *Hansenula polymorpha* from grape juice, named *Hansenula polymorpha* FVPHBJ24146, which was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30796.

[0066] The 16S rDNA sequencing results of *Hansenula polymorpha* FVPHBJ24146 from grape juice are shown in SEQ ID NO: 1.

[0067] (SEQ ID NO: 1).

[0068] Example 3: Investigation of the growth characteristics, acid production capacity, acid tolerance, and salt tolerance of Hanseniaspora uvarum FVPHBJ24146 in grape juice. In this embodiment, the experimental strain was prepared as follows: Hanseniasporauvarum FVPHBJ24146 was streaked on YPD agar plates for 24–48 h, and single colonies were picked and grown in YPD liquid medium for 16–20 h to activate the strain.

[0069] 1. Growth characteristics and acid production capacity of the strain The activated strain was inoculated into YPD liquid medium at a 1% inoculum and incubated statically at 30°C. The absorbance at 600 nm was measured every 1 hour until the stationary phase. A growth curve was plotted with the incubation time on the x-axis and the corresponding absorbance value on the y-axis. Figure 2 ).

[0070] The results showed that the *Hanseniaspora uvarum* FVPHBJ24146 cultured in grape juice was in the growth adaptation phase during the first 24 hours of culture, and then entered the logarithmic growth phase. The pH of the bacterial culture at the time of reaching the stationary phase was measured and remained basically below 4.0.

[0071] 2. Acid resistance characteristics of the strain The activated yeast strain was inoculated at a rate of 1% into YPD liquid medium at different pH values ​​(pH values ​​of 3, 4, 5, and 6), and incubated statically at 30°C for 24 h. The absorbance (OD600) of the yeast was measured before and after incubation in different pH mediums. Figure 3 At the same time, the number of viable yeast cells before and after culturing was determined using a culturable bacteria assay method. Figure 4 ).

[0072] The results showed that *Hansenula polymorpha* FVPHBJ24146 in grape juice exhibited high tolerance to acid. Growth was optimal when the pH of the YPD liquid medium was between 4 and 6. Figure 3 , Figure 4 Even when the pH value drops to 3.0, it maintains a high survival rate, with the viable bacterial count remaining at 10^6. 7 CFU / g or higher ( Figure 4 ).

[0073] 3. Salt tolerance characteristics of strains The activated yeast strain was inoculated at a rate of 1% into YPD liquid medium containing different NaCl concentrations (0%, 4%, 8%, 10%, 15%, 20%) and incubated at 3°C ​​for 24 h. The absorbance (OD600) of the yeast was measured before and after incubation in media with different salt concentrations. Figure 5 At the same time, the number of viable yeast cells before and after culturing was determined using a culturable bacteria assay method. Figure 6 ).

[0074] The results showed that *Hansenula polymorpha* FVPHBJ24146 in grape juice exhibited high tolerance to NaCl. The strain could still grow in liquid medium with a salt concentration of 15%; and in liquid medium with a salt concentration of 20%, the viable cell count remained above 10 CFU / g. Figure 6 ).

[0075] Example 4: Grape juice containing Hansenula polymorpha ( Hanseniaspora grapes Application of FVPHBJ24146 In this embodiment, grape juice containing Hansenula polymorpha ( Hanseniaspora grapes FVPHBJ24146 produced fermented vegetables. The specific method is as follows: 1. Grape juice contains Hansenula polymorpha (Saccharomyces cerevisiae). Hanseniaspora grapes Preparation of FVPHBJ24146 fermentation antagonist Grape juice contains Hansenula polymorpha ( Hanseniaspora grapes FVPHBJ24146 was streaked on YPD agar plates for 24–48 h. Single colonies were picked and grown in YPD liquid medium for 16–20 h for activation. The activated *Hansenula polymorpha* FVPHBJ24146 was then inoculated into YPD liquid medium at a 1% inoculum and cultured for 16–20 h. The culture was centrifuged, washed with sterile distilled water until no culture medium residue remained, and resuspended in sterile distilled water at a cell density of 1 × 10⁻⁶. 10 ~1×10 11 CFU / mL.

[0076] 2. Preparation of Fermented Vegetables Fermented vegetables are prepared according to the following steps: 50.0 kg of fresh vegetables are selected, washed, and drained. After crushing or not crushing, 4.5-7.5 kg of salt is added and stirred evenly. The amount of salt added accounts for 8%-12% of the total vegetable weight. Grape juice Hansenula polysaccharide FVPHBJ24146 fermentation agent is inoculated at 1% of the total vegetable weight. The mixture is sealed and fermented at room temperature for 30 days to obtain fermented vegetables made using grape juice Hansenula polysaccharide FVPHBJ24146 as a fermentation agent.

[0077] The number of viable yeast cells was examined, and the results showed that the number of viable yeast cells remained at 10 throughout the entire fermentation process. 5 CFU / mL or higher ( Figure 7 ).

[0078] For example, vegetables suitable for fermentation using grape juice containing Hansenula polysaccharide FVPHBJ24146 include, but are not limited to: chili peppers, cabbage, cucumbers, and radishes.

[0079] Example 5: Grape juice containing Hansenula polymorpha ( Hanseniaspora grapes Antibacterial properties of FVPHBJ24146 1. Inhibits the growth of Escherichia coli Add 200 μL of distilled water and 10 μL of fermentation broth supernatant to wells A1-F1 of a 96-well plate and mix well. Add 100 μL of LB medium to each of the remaining wells. Aspirate 100 μL of the mixture from wells A1-F1 and add it to wells A2-F2, then mix well. Repeat this process for wells A10-F10. Take the cultured *E. coli* and prepare a bacterial suspension with a concentration equivalent to 0.5 McFarland standard using sterile water. Dilute this suspension 1:1000 with LB medium and add 100 μL of the suspension to wells 1-10 and well 12 of each row, mixing well well. Incubate at 37°C for 16-20 h. Measure the OD value every h until the bacterial density stabilizes and measurements are stopped.

[0080] The results showed that *Hansenula polymorpha* FVPHBJ24146 in grape juice could inhibit the growth of *Escherichia coli*. Within a certain time period, the concentration of *E. coli* in the experimental group was significantly lower than that in the control group. Figure 8 ).

[0081] 2. Inhibits the growth of Staphylococcus aureus Add 200 μL of distilled water and 10 μL of fermentation broth supernatant to wells A1-F1 of a 96-well plate and mix well. Add 100 μL of LB medium to each of the remaining wells. Aspirate 100 μL of the mixture from wells A1-F1 and add it to wells A2-F2, then mix well. Repeat this process for wells A10-F10. Take the cultured Staphylococcus aureus and prepare a bacterial suspension with a concentration equivalent to 0.5 McFarland standard using sterile water. Dilute this suspension 1:1000 with LB medium and add 100 μL of the suspension to wells 1-10 and well 12 of each row, mixing well well. Incubate at 37°C for 16-20 h. Measure the OD value every h until the bacterial density stabilizes and measurements are stopped.

[0082] The results showed that *Hansenula polymorpha* FVPHBJ24146 in grape juice could inhibit the growth of *Staphylococcus aureus*. Within a certain time period, the concentration of *Staphylococcus aureus* in the experimental group was significantly lower than that in the control group. Figure 9 ).

[0083] Example 6: Investigation of the nitrate metabolism capacity of Hanseniaspora uvarum FVPHBJ24146 in grape juice In this embodiment, the experimental strain was prepared as follows: the bacteria stored at -80 ℃ were inoculated into an Erlenmeyer flask containing 100 mL EM, and activated by shaking in a constant temperature shaker at 25 ℃ and 160 rpm for 24 h. After the culture was completed, the bacterial density was adjusted to about 2.0 (i.e., OD600=2.0) with sterile water.

[0084] Add 1 mL of bacterial suspension with OD600 = 2.0 to a new 99 mL Erlenmeyer flask containing EM (extra EM solution). Use an EM without added bacterial solution as a blank control. Shake well and measure the OD value at 600 nm. Measure the OD value every 1 hour until the bacterial density stabilizes and measurements are stopped. Add 5 mL of bacterial suspension with OD600 = 2.0 to a new 95 mL Erlenmeyer flask containing NM (Natural Microorganisms) and analyze the change in nitrate content to determine the strain's ability to metabolize nitrate. All experiments included a blank control and three replicates.

[0085] The results showed that *Hansenula polymorpha* FVPHBJ24146 had a strong ability to metabolize nitrates in grape juice. After 24 hours, the amount of NaNO3 metabolized reached over 30 mg / L. Figure 10 ).

[0086] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0087] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A microorganism, characterized in that, The microorganism is *Hansenula polymorpha* from grape juice. Hanseniaspora uvarum It was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30796.

2. A microbial inoculant, characterized in that, Includes the microorganisms described in claim 1.

3. A food or feed, characterized in that, It includes at least one of the microorganisms described in claim 1 or the microbial agents described in claim 2.

4. The use of the microorganism of claim 1 or the microbial agent of claim 2 in the preparation of food or feed.

5. A method for preparing a food product, characterized in that, include: The food is obtained by fermenting one or more of the microorganisms according to claim 1 and the microbial agents according to claim 2 with the food raw materials to be fermented.

6. The method according to claim 5, characterized in that, The food ingredients include, but are not limited to: chili peppers, cabbage, cucumbers, and radishes.

7. A method for inhibiting the growth of pathogenic bacteria in vitro, characterized in that, include: At least one of the microorganisms of claim 1 and the microbial agents of claim 2 is co-cultured with a sample containing pathogens; The pathogen is selected from at least one of Staphylococcus aureus and Escherichia coli.

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