Microorganism, microbial inoculant and application thereof

Abstract

This invention discloses a microorganism, a microbial agent, and their applications. The microorganism is *Zygosaccharomyces rouxii*, which was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30794. The novel *Zygosaccharomyces rouxii* strain obtained by this invention exhibits rapid growth, strong salt and acid tolerance, and a strong ability to metabolize nitrates and nitrites. Food or feed production methods developed using this novel strain and microbial agent demonstrate high salt tolerance, high fermentation efficiency, and a short production cycle, effectively reducing nitrate and nitrite content during fermentation. Fermented foods made using this novel strain are rich in yeast, low in nitrates and nitrites, and are nutritionally healthy with a unique flavor.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to microorganisms, microbial agents, and their applications. Background Technology

[0002] Traditional fermented chili peppers rely on microorganisms attached to the surface of the food raw materials for fermentation, and salt is usually added to reduce the water activity of the chili peppers, inhibit the growth of spoilage bacteria, extend the shelf life of the chili peppers, and improve the flavor. With the global advocacy for low-sodium diets, reduced-sodium fermentation is gaining increasing attention. Reduced-sodium fermented chili peppers typically contain 5-12% salt, but during the fermentation process, most yeasts still cannot avoid a decrease in viable cell count due to salt stress. Therefore, under natural fermentation conditions, microbial growth is slow, and the production cycle of fermented foods is long, making it difficult to meet the needs of large-scale production of reduced-sodium fermented foods. Therefore, screening for yeasts with good salt and acid tolerance and good fermentation performance is of great significance for the development of reduced-sodium fermented foods and their fermentation processes.

[0003] Furthermore, nitrates are a common contaminant in the food industry, especially in pickled vegetables and certain fermented foods. High concentrations of nitrates not only pose a threat to human health but can also affect the flavor and quality of food. Therefore, identifying microorganisms that can efficiently metabolize nitrates and nitrites, and developing microbial agents that can effectively metabolize nitrates and nitrites, is of great significance for improving the safety and quality of fermented products. Summary of the Invention

[0004] This invention aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, this invention provides a microorganism, *Gnaphalium rouxense* (…). Zygosaccharomyces rouxii This invention relates to microbial agents and their applications. The novel *Zygosacchariformis* strain screened in this invention exhibits rapid growth, strong salt and acid tolerance, and a high capacity for metabolizing nitrates and nitrites. Food or feed production methods developed using this novel strain and microbial agents demonstrate high salt tolerance, high fermentation efficiency, and a short production cycle, effectively reducing nitrate and nitrite content during fermentation. Fermented foods prepared using this novel strain are rich in yeast, low in nitrates and nitrites, and are nutritionally healthy with a unique flavor.

[0005] In a first aspect, the present invention provides a microorganism. According to an embodiment of the invention, the microorganism is *Zygosacchariformis* (…). Zygosaccharomyces rouxii It was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30794.

[0006] Preservation information:

[0007] Strain name: FVPHBJ24271

[0008] Category Naming: Zygosaccharomyces rouxii

[0009] Deposit date: May 27, 2024

[0010] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0011] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China

[0012] Accession number: CGMCC No. 30794

[0013] The *Rhodotorula roux* strain obtained by screening in this invention ( Zygosaccharomyces rouxii The novel strain FVPHBJ24271 exhibits rapid growth, strong salt and acid tolerance, and a high capacity for metabolizing nitrates and nitrites. Food or feed production methods developed using this novel strain and microbial agents demonstrate high salt tolerance, high fermentation efficiency, and a short production cycle, while also reducing nitrate and nitrite content during fermentation. Fermented foods prepared using this novel strain are rich in yeast, low in nitrates and nitrites, and are nutritionally healthy with a unique flavor.

[0014] In a second aspect, the present invention provides a microbial inoculant. According to embodiments of the invention, it includes the aforementioned microorganisms.

[0015] Those skilled in the art will understand that the features and advantages described above for microorganisms also apply to this microbial agent, and will not be repeated here.

[0016] In a third aspect, the present invention provides a food additive or feed additive. According to embodiments of the invention, it includes the aforementioned microorganisms. The food additive or feed additive according to embodiments of the present invention, during the preparation of food or feed, can be used to adjust the acidity of the food or feed, improve the flavor characteristics of the food, effectively reduce the nitrate and nitrite content in the product, improve product quality, adjust the yeast content in the food or feed, and improve product safety.

[0017] In a fourth aspect, the present invention provides a food or feed. According to embodiments of the present invention, the food comprises at least one of the aforementioned microorganisms, the aforementioned microbial agents, or the aforementioned food additives; the feed comprises at least one of the aforementioned microorganisms, the aforementioned microbial agents, or the aforementioned feed additives.

[0018] Those skilled in the art will understand that the features and advantages described above for microorganisms, microbial agents, food additives or feed additives also apply to this food or feed, and will not be repeated here.

[0019] In a fifth aspect, the present invention provides for the use of the aforementioned microorganisms, the aforementioned microbial agents, the aforementioned food additives or feed additives in the preparation of food or feed.

[0020] Those skilled in the art will understand that the features and advantages described above for microorganisms, microbial agents, food additives or feed additives also apply to this use, and will not be repeated here.

[0021] In a sixth aspect, the present invention provides a method for preparing food or feed. According to embodiments of the invention, the method includes: fermenting at least one of the aforementioned microorganisms, the aforementioned microbial agents, or the aforementioned food additives or feed additives with food raw materials or feed raw materials to obtain the food or feed. The preparation method according to embodiments of the present invention exhibits high salt tolerance, high fermentation efficiency, short production cycle, reduced nitrate and nitrite content during fermentation, and high application value during the fermentation process.

[0022] In a seventh aspect, the present invention provides a method for reducing nitrates and / or nitrites in a sample. According to embodiments of the invention, the method includes co-culturing the aforementioned microorganisms, the aforementioned microbial agents, or the aforementioned food additives or feed additives with a sample containing nitrates and / or nitrites. According to the method of embodiments of the invention, *Zygosaccharomyces rouxii* FVPHBJ24271 has a high capacity for metabolizing nitrates and / or nitrites and can be used to reduce nitrates and / or nitrites in a sample.

[0023] In an eighth aspect, the present invention proposes the application of the aforementioned microorganisms, the aforementioned microbial agents, or the aforementioned food additives or feed additives in the denitrification of nitrogen-containing water bodies. According to embodiments of the present invention, the nitrogen-containing water body contains nitrates and / or nitrites. The application according to embodiments of the present invention can effectively reduce the content of nitrates and / or nitrites in the water body, contributing to improved water quality and enhanced safety. Attached Figure Description

[0024] 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:

[0025] Figure 1 The morphological identification results of *Zygosaccharomyces rouxii* CGMCC No. 30794 in Example 2 of this invention;

[0026] Figure 2 The growth curve of *Zygosaccharomyces rouxii* CGMCC No. 30794 in Example 3 of this invention;

[0027] Figure 3 The absorbance (OD) values ​​of *Zygosaccharomyces rouxii* CGMCC No. 30794 before and after culturing in different pH media in Example 3 of this invention are shown. 600 ) Inspection results;

[0028] Figure 4 The results of investigating the viable cell count of *Zygosaccharomyces rouxii* CGMCC No. 30794 in culture media at different pH values ​​in Example 3 of this invention;

[0029] Figure 5 The absorbance (OD) values ​​of *Zygosaccharomyces rouxii* CGMCC No. 30794 before and after culturing in culture media with different salt concentrations in Example 3 of this invention are shown. 600 ) Inspection results;

[0030] Figure 6 The results of the study on the viable count of *Zygosaccharomyces rouxii* CGMCC No. 30794 in culture media containing different salt concentrations in Example 3 of this invention;

[0031] Figure 7 This is the viable cell count during the preparation of fermented vegetables using *Zygosacchariformis* CGMCC No. 30794 vegetable starter in Example 4 of the present invention.

[0032] Figure 8 The results of the nitrite metabolism ability study of *Zygosaccharomyces rouxii* CGMCC No. 30794 in Example 5 of this invention;

[0033] Figure 9 The results of the nitrate metabolism ability study of *Zygosacchariformis* CGMCC No. 30794 in Example 5 of this invention are shown. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Terms and Definitions

[0039] In this article, the term "YPD medium" refers to yeast culture medium containing yeast extract, peptone, and glucose. Depending on the content of the solidifying agent (usually agarose), it is divided into two types: YPD solid medium and YPD liquid medium. In this article, the terms "YPD solid medium" and "YPD agar medium" are synonymous.

[0040] In this article, the term "Roughlykia rouxae" refers to a yeast belonging to the phylum Ascomycota (…). Ascomycota Yeasts (Class A) Saccharomycetes ) Spore-forming yeasts ( Sporobolomycetales ) Saccharomyces family ( Sporobolomycetaceae ) genus *Lourdes* Zygosaccharomyces rouxii ), colonies are small, round, and white.

[0041] In this paper, the term "NM" refers to nitrate medium, which is used to investigate the strain's ability to remove nitrates. The term "EM" refers to enrichment medium, which provides sufficient carbon and nitrogen sources to sustain the growth of the strain.

[0042] In this article, the term "OD" is used. 600 "This is a standard method for determining microbial concentration. The number of bacteria is estimated by comparing the turbidity of the bacterial suspension with a standard curve."

[0043] In this paper, the term "denitrification" refers to the removal of nitrates (NOx) from water bodies through microbial metabolic activities. 3- ) and nitrite (NO 2- ) is converted into nitrogen (N) 2 The process of ).

[0044] This invention proposes a microorganism, a microbial agent, a food additive or feed additive, and its uses and applications, food or feed, and a method for preparing food or feed, which will be described in detail below.

[0045] strains

[0046] This invention proposes a microorganism. According to an embodiment of the invention, the microorganism is *Zygosaccharomyces rouxii* (…). Zygosaccharomyces rouxii It was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30794.

[0047] According to an embodiment of the present invention, the ITS rDNA sequencing results of the microorganism are shown in SEQ ID NO: 1.

[0048] The *Rhodotorula roux* strain obtained by screening in this invention ( Zygosaccharomyces rouxii The novel strain FVPHBJ24271 exhibits rapid growth, strong salt and acid tolerance, and a high capacity for metabolizing nitrates and nitrites. Food or feed production methods developed using this novel strain and microbial agents demonstrate high salt tolerance, high fermentation efficiency, and a short production cycle, while effectively reducing nitrate and nitrite content during fermentation. Fermented foods prepared using this novel strain are rich in yeast, low in nitrates and nitrites, and are nutritionally healthy with a unique flavor.

[0049] In this article, the term "Russian conjugation yeast" ( Zygosaccharomyces rouxii "FVPHBJ24271" and "FVPHBJ24271" and "CGMCC No.30794" are synonymous.

[0050] Microbial agents

[0051] This invention proposes a microbial inoculant. According to embodiments of the invention, it includes the aforementioned microorganisms.

[0052] Those skilled in the art will understand that the features and advantages described above for microorganisms also apply to this microbial agent, and will not be repeated here.

[0053] 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.

[0054] It should be noted that in the microbial agent of the present invention, the *Zygosacchariformis* CGMCC No. 30794 exists in the form of live cells and / or non-live cells.

[0055] In this article, "living cell" refers to a cell that has the ability to metabolize, reproduce, or replicate.

[0056] 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.

[0057] 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.

[0058] In some specific embodiments, the *Zygosacchariformis* CGMCC No. 30794 exists as live cells, dry cells, immobilized cells, or in any other form.

[0059] In some specific embodiments, the dried bacterial cells are obtained by freeze-drying the *Zygosacchariformis* CGMCC No. 30794.

[0060] In some specific embodiments, the microbial agent may also contain strains acceptable for use in food and feed.

[0061] In some specific embodiments, the microbial agent further includes food-grade excipients or carriers, or animal feed-grade excipients or carriers.

[0062] 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.

[0063] 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.

[0064] Food additives or feed additives

[0065] This invention proposes a food additive or feed additive. According to embodiments of the invention, it includes the aforementioned microorganisms. The food additive or feed additive according to embodiments of the invention can be used in the preparation of food or feed to adjust the acidity of the food or feed, improve the flavor characteristics of the food, effectively reduce the content of nitrates and nitrites in the product, improve product quality, adjust the yeast content in the food or feed, and improve product safety.

[0066] food or feed

[0067] This invention provides a food or feed. According to embodiments of the invention, the food includes at least one of the aforementioned microorganisms, the aforementioned microbial agents, or the aforementioned food additives; the feed includes at least one of the aforementioned microorganisms, the aforementioned microbial agents, or the aforementioned feed additives.

[0068] Those skilled in the art will understand that the features and advantages described above for microorganisms, microbial agents, food additives or feed additives also apply to this food or feed, and will not be repeated here.

[0069] According to embodiments of the present invention, the product further includes food- or feed-acceptable excipients or carriers. This results in food or feed products that are nutritious, healthy, and have a unique flavor.

[0070] In some specific embodiments, the aforementioned microorganisms, microbial agents, or food additives are added to or inoculated into food or feed.

[0071] For example, the food includes, but is not limited to, fermented chili peppers, fermented cabbage, fermented cucumbers, and fermented radishes.

[0072] use

[0073] This invention proposes the use of the aforementioned microorganisms, the aforementioned microbial agents, and the aforementioned food additives or feed additives in the preparation of food or feed.

[0074] Those skilled in the art will understand that the features and advantages described above for microorganisms, microbial agents, food additives or feed additives also apply to this use, and will not be repeated here.

[0075] Methods for preparing food or feed

[0076] This invention proposes a method for preparing food or feed. According to embodiments of the invention, the method includes: fermenting at least one of the aforementioned microorganisms, the aforementioned microbial agents, or the aforementioned food additives or feed additives with food raw materials or feed raw materials to obtain the food or feed. The preparation method according to embodiments of the invention exhibits high salt tolerance, high fermentation efficiency, short production cycle, reduced nitrate and nitrite content during fermentation, and high application value during the fermentation process.

[0077] According to an embodiment of the present invention, before fermentation, the food raw material or feed raw material is mixed with sodium chloride, and the amount of sodium chloride added is 5 to 25 wt% based on the mass of the food raw material or feed raw material.

[0078] According to an embodiment of the present invention, the pH of the fermentation treatment is 2.5 to 6.5, preferably 3 to 6, and more preferably 4 to 6.

[0079] Further optimization of processing conditions during fermentation is needed to obtain higher quality food or feed products.

[0080] Methods to reduce nitrate and / or nitrite in samples

[0081] This invention proposes a method for reducing nitrates and / or nitrites in a sample. According to embodiments of the invention, the method includes co-culturing the aforementioned microorganisms, microbial agents, or food or feed additives with a sample containing nitrates and / or nitrites. According to the method of this invention, *Zygosaccharomyces rouxii* FVPHBJ24271 has a high capacity for metabolizing nitrates and / or nitrites and can be used to reduce nitrates and / or nitrites in a sample.

[0082] For example, the samples include, but are not limited to, soil samples, water samples, and food samples.

[0083] Application in nitrogen removal from nitrogen-containing water bodies

[0084] This invention proposes the application of the aforementioned microorganisms, microbial agents, or food or feed additives in denitrification of nitrogen-containing water bodies. According to embodiments of the invention, the nitrogen-containing water body contains nitrates and / or nitrites. The application according to embodiments of the invention can effectively reduce the content of nitrates and / or nitrites in the water body, contributing to improved water quality and enhanced safety.

[0085] For example, the nitrogen-containing water bodies include, but are not limited to, rivers, lakes, reservoirs, groundwater, industrial wastewater and agricultural wastewater, and contain high levels of nitrates and / or nitrites.

[0086] Unless otherwise specified, the YPD agar medium used in the embodiments of the present invention has the following formulation: 20g peptone, 10g yeast extract, 20g glucose, 10g agar, and 1L distilled water.

[0087] Unless otherwise specified, the YPD liquid culture medium used in the embodiments of the present invention has the following formulation: 20g peptone, 10g yeast extract, 20g glucose, and 1L distilled water.

[0088] Unless otherwise specified, the formulations of the enrichment medium (EM) and nitrate medium used in the embodiments of the present invention are shown in Table 1. After preparation, the pH of the medium was adjusted to 7.0~7.3. The formulations of the added trace elements are shown in Table 2.

[0089] Table 1. Enrichment medium (EM) and its components

[0090]

[0091] Table 2 Trace Element Composition Table

[0092]

[0093] 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.

[0094] Example 1: Obtaining *Zygosaccharomyces rouxii* CGMCC No. 30794

[0095] The *Zygosaccharomyces rouxii* CGMCC No. 30794 of this invention was isolated from naturally fermented chili sauce from Hebei Province.

[0096] Strain collection and isolation process: Naturally fermented chopped chili peppers were added to sterilized physiological saline, mixed thoroughly, diluted, and spread onto YPD solid medium. The mixture was then incubated statically at pH 6.0±0.2 and 30℃ for 24–48 h. Single colonies of varying sizes and morphologies were then picked and collected from fresh YPD solid medium. body Streak the culture medium on agar plates and incubate at 30°C for 24-48 hours. Repeat the streaking purification culture multiple times until the colony morphology in the plates is consistent. After microscopic examination and no contamination, the strain is named FVPHBJ24271 and identified.

[0097] Obtaining bacterial culture: Pick a single colony with an inoculation loop and place it in YPD liquid medium, then incubate at 37°C until the bacterial culture reaches OD. 600 When the concentration is 1.0~1.5, the strain is preserved.

[0098] Preservation of bacterial strains: The above OD 600 After mixing the bacterial culture (1.0~1.5) with sterile 50% glycerol in a cryovial at a 1:1 ratio, store at -80℃.

[0099] Example 2: Identification of Zygosaccharomyces rouxii CGMCC No. 30794

[0100] 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 ITS rDNA gene were performed.

[0101] 1. Morphological and physiological / biochemical identification

[0102] Refer to the "Handbook for Systematic Identification of Common Fungi" to identify the morphological characteristics and physiological and biochemical properties of the test strains.

[0103] Morphological identification results are as follows Figure 1 .

[0104] The identification results showed that the colony morphology on YPD agar medium was characterized by small, round, and white colonies. Figure 1 In YPD liquid medium, its final pH is 3.0.

[0105] 2. PCR amplification and sequencing analysis of the ITS rDNA gene

[0106] The ITS rDNA sequence of strain FVPHBJ24271 was identified. PCR amplification conditions: 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 3.

[0107] Table 3 PCR reaction system

[0108]

[0109] The strain FVPHBJ24271 was sent to Sangon Biotech (Shanghai) Co., Ltd. for ITS rDNA sequence detection. The obtained sequence was compared with GenBank, and a phylogenetic tree was constructed using MEGA11.0.13 to compare the sequence of the test strain with the sequences of other strains in the same genus.

[0110] The results showed that the isolated strain FVPHBJ24271 belonged to the same branch as other strains of *Zygosaccharomyces rouxii*. Zygosaccharomyces , Zygosaccharomyces rouxii The ITS rDNA sequence homology was 99%.

[0111] Based on the physiological and biochemical identification results, strain FVPHBJ24271 was further confirmed as *Zygosacchari rouxae*, with the sequence accession number CGMCC No. 30794.

[0112] The final identification confirmed that the isolated strain FVPHBJ24271 was a strain of Zygosacchari roux, named Zygosacchari roux FVPHBJ24271 (hereinafter referred to as "Zygosacchari roux CGMCC No. 30794"), and was deposited at the China General Microbiological Culture Collection Center on May 27, 2024, with the accession number CGMCC No. 30794.

[0113] The ITS rDNA sequencing results of *Zygosaccharomyces rouxii* CGMCC No. 30794 are shown in SEQ ID NO: 1.

[0114] (SEQ ID NO: 1)

[0115] Example 3: Investigation of the growth characteristics, acid production capacity, acid tolerance, and salt tolerance of Zygosaccharomyces rouxii CGMCC No. 30794

[0116] Examining the basic characteristics of strains, such as growth characteristics, acid production capacity, acid tolerance, and salt tolerance, helps to screen out microbial strains with specific application potential.

[0117] In this embodiment, the experimental strain was prepared as follows: Zygosacchariformis CGMCC No. 30794 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.

[0118] 1. Investigation of the growth characteristics and acid production capacity of the strain

[0119] The activated strain was inoculated into YPD liquid medium at a 1% inoculum and incubated statically at 30°C. The absorbance was measured at 600 nm 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 ).

[0120] The results showed that: (1) In the first 24 h of culture, the *Zygosacchariformis* CGMCC No. 30794 was in the growth adaptation period and its growth and metabolism were slow; after 24 h, it entered the logarithmic growth phase. (2) The pH of the bacterial culture when it reached the stationary phase was basically maintained below 4.0.

[0121] 2. Investigation of the acid resistance characteristics of the strain

[0122] The activated strain was inoculated at a rate of 1% into YPD liquid medium with different pH values ​​(pH values ​​investigated were 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0), and incubated statically at 30°C for 24 h. The absorbance (OD) of the activated strain at 600 nm was measured before and after incubation in different pH media. 600 () Figure 3 The number of viable yeast cells before and after culturing activated strains was determined using a culturable microbial assay. Figure 4 ).

[0123] The results showed that *Zygosaccharomyces rouxii* CGMCC No. 30794 exhibited high acid tolerance. Growth was good at YPD liquid medium with a pH of 4–5; even when the pH dropped to 3.0, it maintained a high survival rate, with viable cell counts remaining at 102. 6 CFU / g or higher.

[0124] 3. Investigation of the salt tolerance characteristics of the strain

[0125] The activated strain was inoculated at a rate of 1% into YPD liquid medium containing different NaCl concentrations (0%, 4%, 8%, 10%, 15%, 20%, 25%) and incubated at 3℃ for 24 h. The absorbance (OD600) of the activated strain before and after incubation was measured in mediums with different salt concentrations. Figure 5Meanwhile, the number of viable yeast cells before and after culturing the activated strain was determined using a culturable bacteria assay method. Figure 6 ).

[0126] The results showed that *Zygosaccharomyces rouxii* CGMCC No. 30794 exhibited high tolerance to NaCl. In liquid medium with a salt concentration of 10%, the viable cell count remained at 10⁻⁶. 7 The strain can still grow in liquid culture medium with a salt concentration of 15% and above CFU / g.

[0127] Example 4: Application of Zygosaccharomyces rouxii CGMCC No. 30794 in the preparation of fermented vegetable foods

[0128] The metabolic activities of certain microorganisms can increase the nutritional value and flavor of fermented foods and extend their shelf life.

[0129] In this embodiment, fermented vegetables were prepared using *Zygosaccharomyces rouxii* CGMCC No. 30794, and the specific method is as follows:

[0130] 1. Preparation of *Zygosaccharomyces rouxii* CGMCC No. 30794 starter culture

[0131] Zygosacchariformis CGMCC No. 30794 was streaked on YPD agar plates for 24–48 h. Single colonies were picked and grown in YPD liquid medium for 16–20 h to activate the cells. After activation, Zygosacchariformis CGMCC No. 30794 was inoculated into YPD liquid medium at a 1% inoculum and cultured for 16–20 h. The culture was then centrifuged, washed with sterile distilled water until no culture medium residue remained, and resuspended in sterile distilled water to achieve a cell density of 1 × 10⁻⁶ cells / mL. 10 ~1×10 11 CFU / mL refers to the fermentation agent of *Zygosacchariformis* CGMCC No. 30794.

[0132] 2. Preparation of Fermented Vegetables

[0133] Take 50.0 kg of fresh vegetables, select, wash, and drain them. After crushing or leaving them whole, add 4.5–7.5 kg of salt and mix well. The salt content should be 8%–12% of the total fresh vegetable weight. Inoculate with 1% of the total vegetable weight of *Zygosaccharomyces rouxii* CGMCC No. 30794 starter culture. Ferment at room temperature in a sealed container for 30 days to obtain fermented vegetables made using *Zygosaccharomyces rouxii* CGMCC No. 30794 as the starter culture. The viable cell count during the entire fermentation process is examined, and the results are shown in […]. Figure 7 .

[0134] The results showed that the viable count of *Zygosaccharomyces rouxii* CGMCC No. 30794 remained at 10 throughout the entire vegetable fermentation process. 5 CFU / mL or higher.

[0135] For example, vegetables suitable for fermentation using *Zygosaccharomyces rouxii* CGMCC No. 30794 include, but are not limited to, chili peppers, cabbage, cucumbers, radishes, etc.

[0136] Example 5: Investigation of the ability of Zygosaccharomyces rouxii CGMCC No. 30794 to metabolize nitrite and nitrate.

[0137] In the food industry, controlling nitrate and nitrite levels is crucial for food safety. Zygosacchariformis CGMCC No. 30794, a yeast strain of the present invention, is commonly used in the preparation of fermented foods. Therefore, this embodiment investigates the ability of Zygosacchariformis CGMCC No. 30794 to metabolize nitrite and nitrate. The specific method is as follows:

[0138] 1. Preparation of bacterial strains

[0139] The *Zygosaccharomyces rouxii* CGMCC No. 30794 strain, stored at -80℃, was inoculated into an Erlenmeyer flask containing 100 mL of enrichment medium (EM). Activation culture was performed by shaking in a constant temperature shaker at 25℃ and 160 rpm for 24 h. After activation, the cell density was adjusted to approximately 2.0 (OD) using sterile water. 600 =2.0), obtain OD 600 =2.0 bacterial suspension.

[0140] 2. Investigation of the strain's ability to metabolize nitrite

[0141] Take 1 mL OD 600 Add the bacterial suspension (2.0 g / L) to a new 99 mL Erlenmeyer flask containing EM, without adding OD. 600 The bacterial suspension with a concentration of 2.0 μM was used as a blank control for EM. The suspension was shaken well, and the OD value was measured at 600 nm every 1 hour until the cell density stabilized and measurements were stopped. 5 mL of OD... 600 A bacterial suspension with a concentration of 2.0 μL was added to a new 95 mL Erlenmeyer flask, and the change in nitrite content was analyzed to determine the ability of *Zygosacchariformis* CGMCC No. 30794 strain to metabolize nitrite. All experiments included a blank control and three parallel groups.

[0142] The results of the study on the ability of *Zygosaccharomyces rouxii* CGMCC No. 30794 to metabolize nitrite are shown in the table below. Figure 8 .

[0143] The results showed that within a certain period of time, *Zygosaccharomyces rouxii* CGMCC No. 30794 continuously metabolized NaNO2, and after 24 hours, its NaNO2 metabolism amount was above 15 mg / L. Figure 8 ).

[0144] 3. Investigation of the strain's ability to metabolize nitrates

[0145] Take 1 mL OD 600 Add the bacterial suspension (2.0 g / L) to a new 99 mL Erlenmeyer flask containing EM, without adding OD. 600 The bacterial suspension with a concentration of 2.0 μM was used as a blank control for EM. The suspension was shaken well, and the OD value was measured at 600 nm every 1 hour until the cell density stabilized and measurements were stopped. 5 mL of OD... 600 A bacterial suspension with a concentration of 2.0 μL was added to a new 95 mL Erlenmeyer flask, and the change in nitrate content was analyzed to determine the ability of *Zygosacchariformis* CGMCC No. 30794 strain to metabolize nitrates. All experiments included a blank control and three parallel groups.

[0146] The results of the study on the nitrate metabolism capacity of *Zygosaccharomyces rouxii* CGMCC No. 30794 are shown below. Figure 9 .

[0147] The results showed that within a certain period of time, *Zygosaccharomyces rouxii* CGMCC No. 30794 continuously metabolized NaNO3. After 24 hours, the amount of NaNO3 metabolized by *Zygosaccharomyces rouxii* CGMCC No. 30794 was above 65 mg / L. Figure 9 ).

[0148] The above results indicate that *Zygosaccharomyces rouxii* CGMCC No. 30794 has a strong ability to metabolize both nitrite and nitrate.

[0149] 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 *Zygosaccharomyces rouxii* ( Zygosaccharomyces rouxii FVPHBJ24271 was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30794.

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

3. A food additive or feed additive, characterized in that, Includes the microorganisms described in claim 1.

4. A food or feed, characterized in that, The food includes at least one of the microorganisms of claim 1, the microbial agent of claim 2, or the food additive of claim 3; the feed includes at least one of the microorganisms of claim 1, the microbial agent of claim 2, or the feed additive of claim 3.

5. The food or feed according to claim 4, characterized in that, This further includes food or feed-acceptable excipients or carriers.

6. The use of the microorganisms described in claim 1, the microbial agents described in claim 2, and the food additives or feed additives described in claim 3 in the preparation of food or feed.

7. A method for preparing food or feed, characterized in that, include: The food or feed is obtained by fermenting at least one of the microorganisms of claim 1, the microbial agent of claim 2, or the food additive or feed additive of claim 3 with food raw materials or feed raw materials.

8. The method according to claim 7, characterized in that, Before fermentation, the food or feed ingredients are mixed with sodium chloride, wherein the amount of sodium chloride added is 5 to 25 wt% based on the mass of the food or feed ingredients.

9. The method according to claim 8, characterized in that, The pH of the fermentation treatment is 2.5 to 6.

5.

10. The method according to claim 8, characterized in that, The pH of the fermentation treatment is 3 to 6.

11. The method according to claim 8, characterized in that, The pH of the fermentation treatment is 4 to 6.

12. A method for reducing nitrate and / or nitrite in a sample, characterized in that, include: The microorganisms of claim 1, the microbial agents of claim 2, or the food additives or feed additives of claim 3 are co-cultured with samples containing nitrates and / or nitrites.

13. The application of the microorganism of claim 1, the microbial agent of claim 2, or the food additive or feed additive of claim 3 in nitrogen-containing water denitrification, characterized in that, The nitrogen-containing water contains nitrates and / or nitrites.

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