Microorganism, microbial agent and application thereof

Abstract

The invention discloses a microorganism, a microbial agent and application thereof. The microorganism is zygosaccharomyces rouxii, is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on May 27, 2024, and has the preservation number of CGMCC No.30794. The invention further discloses a preparation method of the saccharomyces rouxii strain. The new strain of the zygosaccharomyces rouxii obtained through screening is high in growth speed, high in salt resistance and acid resistance and high in nitrate and nitrite metabolism capacity; a food or feed production method developed by utilizing the new strain and the microbial agent is high in salt tolerance, high in fermentation efficiency and short in production period, and the content of nitrate and nitrite in the fermentation production process can be effectively reduced; fermented food prepared from the new strain is rich in yeast content, low in nitrate and nitrite content, nutritional, healthy and unique in flavor.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to microorganisms, microbial agents and applications thereof. Background Art

[0002] Traditional fermented peppers rely on microorganisms attached to the surface of food raw materials for fermentation, and salt is usually added to reduce the water activity of peppers, inhibit the growth of spoilage bacteria, extend the shelf life of peppers, and improve the flavor of peppers. With the global advocacy of a low-sodium diet, salt-reduced fermentation has received more and more attention. Reduced-salt fermented peppers usually add 5-12% salt, but during the fermentation process, most yeasts still cannot avoid the reduction in the number of viable bacteria caused by 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-salt fermented foods. Therefore, screening yeasts with good salt resistance, acid resistance, and good fermentation performance is of great significance to meet the development of reduced-salt fermented foods and their fermentation processes.

[0003] In addition, nitrates are common pollutants 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 may also affect the flavor and quality of food. Therefore, finding microorganisms that can efficiently metabolize nitrates and nitrites, and developing microbial agents that can effectively metabolize nitrates and nitrites are of great significance for improving the safety and quality of fermented products. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a microorganism Zygosaccharomyces rouxii, a microbial agent and its application. The new strain of Zygosaccharomyces rouxii screened by the present invention has a fast growth rate, strong salt and acid resistance, and a strong ability to metabolize nitrates and nitrites; the production method of food or feed developed using the new strain and microbial agent of the present invention has high salt tolerance, high fermentation efficiency, short production cycle, and can effectively reduce the content of nitrates and nitrites in the fermentation production process; the fermented food prepared using the new strain has rich yeast content, low nitrate and nitrite content, is nutritious and healthy, and has a unique flavor.

[0005] In a first aspect of the present invention, the present invention provides a microorganism. According to an embodiment of the present invention, the microorganism is Zygosaccharomyces rouxii, which was deposited in the General Microbiological Center of the China Microorganism Culture Collection Administration on May 27, 2024, with a deposit number of CGMCC No.30794.

[0006] Collection information:

[0007] Strain name: FVPHBJ24271

[0008] Taxonomic nomenclature: Zygosaccharomyces rouxii

[0009] Date of deposit: May 27, 2024

[0010] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0011] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0012] Deposit number: CGMCC No.30794

[0013] The new strain of Zygosaccharomyces rouxii FVPHBJ24271 screened by the invention has fast growth speed, strong salt and acid resistance, and strong ability to metabolize nitrate and nitrite; the production method of food or feed developed by using the new strain and microbial agent of the invention has high salt tolerance, high fermentation efficiency, short production cycle, and reduces the content of nitrate and nitrite in the fermentation production process; the fermented food prepared by using the new strain has rich yeast content, low nitrate and nitrite content, is nutritious and healthy, and has unique flavor.

[0014] In a second aspect of the present invention, the present invention provides a microbial agent. According to an embodiment of the present invention, the microorganism is included.

[0015] Those skilled in the art will appreciate that the features and advantages described above for microorganisms are also applicable to the microbial agent and will not be described in detail here.

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

[0017] In a fourth aspect of the present invention, the present invention provides a food or feed. According to an embodiment 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, and the aforementioned feed additives.

[0018] Those skilled in the art will appreciate that the features and advantages described above for microorganisms, microbial agents, food additives or feed additives are also applicable to the food or feed and will not be elaborated herein.

[0019] In the fifth aspect of the present invention, the present invention proposes 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 appreciate that the features and advantages described above for microorganisms, microbial agents, food additives or feed additives are also applicable to this use and will not be described in detail here.

[0021] In a sixth aspect of the present invention, the present invention proposes a method for preparing food or feed. According to an embodiment of the present invention, the method comprises: 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 an embodiment of the present invention has high salt tolerance, high fermentation efficiency, short production cycle, reduced nitrate and nitrite content during the fermentation production process, and high application value during the fermentation process.

[0022] In the seventh aspect of the present invention, the present invention proposes a method for reducing nitrate and / or nitrite in a sample. According to an embodiment of the present invention, the method comprises: co-culturing the aforementioned microorganism, the aforementioned microbial agent or the aforementioned food additive or feed additive with a sample containing nitrate and / or nitrite. According to the method of an embodiment of the present invention, Zygosaccharomyces rouxii FVPHBJ24271 has a high ability to metabolize nitrate and / or nitrite, and can be used to reduce nitrate and / or nitrite in a sample.

[0023] In an eighth aspect of the present invention, the present invention proposes the use of the aforementioned microorganisms, the aforementioned microbial agents or the aforementioned food additives or feed additives in denitrification of nitrogen-containing water bodies. According to an embodiment of the present invention, the nitrogen-containing water body contains nitrates and / or nitrites. According to the application of an embodiment of the present invention, it can effectively reduce the content of nitrates and / or nitrites in the water body, help improve the quality of the water body, and improve safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0025] Figure 1 The morphological identification result of Zygosaccharomyces rouxii CGMCC No.30794 in Example 2 of the present invention;

[0026] Figure 2 is the growth curve of Zygosaccharomyces rouxii CGMCC No.30794 in Example 3 of the present invention;

[0027] Figure 3 is the absorbance value (OD) of Zygosaccharomyces rouxii CGMCC No.30794 before and after culture in medium with different pH values ​​in Example 3 of the present invention 600 ) Investigation results;

[0028] Figure 4 The results of investigating the number of viable bacteria of Zygosaccharomyces rouxii CGMCC No.30794 in culture medium with different pH values ​​in Example 3 of the present invention are as follows;

[0029] Figure 5 The absorbance values ​​(OD) of Zygosaccharomyces rouxii CGMCC No.30794 in Example 3 of the present invention before and after cultivation in medium containing different salt concentrations 600 ) Investigation results;

[0030] Figure 6 The results of the investigation of the number of viable bacteria of Zygosaccharomyces rouxii CGMCC No.30794 in Example 3 of the present invention in culture medium containing different salt concentrations;

[0031] Figure 7 The viable bacteria counts in the process of preparing fermented vegetables using Zygosaccharomyces rouxii CGMCC No.30794 vegetable starter in Example 4 of the present invention;

[0032] Figure 8 The results of investigating the ability of Zygosaccharomyces rouxii CGMCC No.30794 to metabolize nitrite in Example 5 of the present invention are as follows;

[0033] Fig. 9 This is the result of investigating the nitrate metabolism ability of Zygosaccharomyces rouxii CGMCC No.30794 in Example 5 of the present invention. DETAILED DESCRIPTION

[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 understood as limiting the present invention.

[0035] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0036] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0037] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0038] Terms and Definitions

[0039] In this article, the term "YPD medium" is a yeast culture medium, which contains yeast extract, peptone and glucose. According to the content of coagulant (usually agarose), it is divided into YPD solid culture medium and YPD liquid culture medium. In this article, the terms "YPD solid culture medium" and "YPD agar culture medium" are synonymous.

[0040] As used herein, the term "Zygosaccharomyces rouxii" refers to a yeast belonging to the genus Zygosaccharomyces rouxii of the class Saccharomycetes of the order Sporobolomycetales and the family Sporobolomycetaceae of the phylum Ascomycota, and has small, round and white colonies.

[0041] In this article, the term "NM" refers to nitrate medium (NM), which is used to explore the nitrate removal performance of strains.

[0042] Herein, the term "EM" refers to enrichment medium (EM), which provides sufficient carbon and nitrogen sources to maintain the growth of the strain.

[0043] In this article, the term "OD 600 "It is a standard method for determining the concentration of microorganisms. The number of bacteria is estimated by comparing the turbidity of the bacterial suspension with a standard curve.

[0044] In this context, the term “denitrification” refers to the removal of nitrate (NO 3- ) and nitrite (NO 2- ) is converted into nitrogen (N 2 ) process.

[0045] The present invention provides a microorganism, a microbial agent, a food additive or a feed additive and its use and application, a food or a feed, and a method for preparing the food or the feed, which will be described in detail below.

[0046] Bacteria

[0047] The present invention proposes a microorganism. According to an embodiment of the present invention, the microorganism is Zygosaccharomyces rouxii, which was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on May 27, 2024, with a deposit number of CGMCC No.30794.

[0048] According to an embodiment of the present invention, the 16S rDNA sequencing result of the microorganism is shown as SEQ ID NO:1.

[0049] The new strain of Zygosaccharomyces rouxii FVPHBJ24271 screened by the invention has fast growth speed, strong salt and acid resistance, and strong ability to metabolize nitrate and nitrite; the production method of food or feed developed by using the new strain and microbial agent of the invention has high salt tolerance, high fermentation efficiency, short production cycle, and can effectively reduce the content of nitrate and nitrite in the fermentation production process; the fermented food prepared by using the new strain has rich yeast content, low nitrate and nitrite content, is nutritious and healthy, and has unique flavor.

[0050] As used herein, the terms "Zygosaccharomyces rouxii FVPHBJ24271" and "Zygosaccharomyces rouxii FVPHBJ24271" and "Zygosaccharomyces rouxii CGMCC No. 30794" are synonymous.

[0051] Microbial Agents

[0052] The present invention provides a microbial agent, which includes the aforementioned microorganisms according to an embodiment of the present invention.

[0053] Those skilled in the art will appreciate that the features and advantages described above for microorganisms are also applicable to the microbial agent and will not be described in detail here.

[0054] It should be noted that the microbial agent of the present invention may be a liquid microbial agent, including but not limited to fermentation products, etc.; it may also be a solid microbial agent, including but not limited to freeze-dried powder, etc.

[0055] It should be noted that, in the microbial agent of the present invention, the Zygosaccharomyces rouxii CGMCC No. 30794 exists in the form of living cells and / or non-living cells.

[0056] As used herein, "living cells" refer to cells that have the ability to metabolize, reproduce or replicate.

[0057] Exemplarily, the living cells may be immobilized cells. Herein, "immobilized cells" refer to living cells that are fixed on a carrier and can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0058] In this article, "non-living cells" refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dry bacteria. Exemplarily, the microbial agent is a freeze-dried powder.

[0059] In some specific embodiments, the Zygosaccharomyces rouxii CGMCC No. 30794 exists in the form of living cells, dried bacteria, immobilized cells or any other forms.

[0060] In some specific embodiments, the dry bacteria are obtained by freeze-drying the Zygosaccharomyces rouxii CGMCC No.30794.

[0061] In some specific embodiments, the microbial agent may also contain strains acceptable to food and feed.

[0062] In some specific embodiments, the microbial agent further includes an auxiliary material or carrier acceptable in food, or an auxiliary material or carrier acceptable in animal feed.

[0063] As used herein, "acceptable in food" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the food requirements of different countries.

[0064] In this context, "adjuvant or carrier acceptable in animal feed" refers to a substance or composition that can be consumed by animals, which may be adjusted according to the animal feed requirements of different countries.

[0065] Food additives or feed additives

[0066] The present invention provides a food additive or feed additive. According to an embodiment of the present invention, the microorganisms described above are included. The food additive or feed additive according to the embodiment of the present invention can be used to adjust the acidity of food or feed, improve the flavor characteristics of food, effectively reduce the content of nitrate and nitrite in the product, improve product quality, adjust the yeast content in food or feed, and improve product safety during the process of preparing food or feed.

[0067] Food or feed

[0068] The present invention provides a food or feed. According to an embodiment of the present 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, and the aforementioned feed additives.

[0069] Those skilled in the art will appreciate that the features and advantages described above for microorganisms, microbial agents, food additives or feed additives are also applicable to the food or feed and will not be elaborated herein.

[0070] According to an embodiment of the present invention, the food or feed may further include an auxiliary material or carrier acceptable to the food or feed, thereby further obtaining a food or feed product that is nutritious, healthy and has a unique flavor.

[0071] In some specific embodiments, the aforementioned microorganisms, the aforementioned microbial agents or the aforementioned food additives are added or inoculated into food or feed.

[0072] Exemplarily, the food includes but is not limited to fermented peppers, fermented cabbage, fermented cucumbers, and fermented radishes.

[0073] use

[0074] The present invention provides the use of the aforementioned microorganism, the aforementioned microbial agent, the aforementioned food additive or feed additive in the preparation of food or feed.

[0075] Those skilled in the art will appreciate that the features and advantages described above for microorganisms, microbial agents, food additives or feed additives are also applicable to this use and will not be described in detail here.

[0076] Method for preparing food or feed

[0077] The present invention provides a method for preparing food or feed. According to an embodiment of the present invention, the method comprises: 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 the embodiment of the present invention has high salt tolerance, high fermentation efficiency, short production cycle, reduced nitrate and nitrite content in the fermentation production process, and high application value in the fermentation process.

[0078] 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 5-25 wt % based on the mass of the food raw material or feed raw material.

[0079] According to an embodiment of the present invention, the pH of the fermentation treatment is 2.5-6.5, preferably 3-6, and more preferably 4-6.

[0080] Further optimize the processing conditions during the fermentation process to obtain better quality food or feed products.

[0081] Methods for reducing nitrate and / or nitrite in samples

[0082] The present invention proposes a method for reducing nitrate and / or nitrite in a sample. According to an embodiment of the present invention, the method comprises: co-culturing the aforementioned microorganism, the aforementioned microbial agent or the aforementioned food additive or feed additive with a sample containing nitrate and / or nitrite. According to the method of an embodiment of the present invention, Zygosaccharomyces rouxii FVPHBJ24271 has a high ability to metabolize nitrate and / or nitrite, and can be used to reduce nitrate and / or nitrite in a sample.

[0083] Exemplarily, the samples include but are not limited to soil samples, water source samples, and food samples.

[0084] Application in denitrification of nitrogen-containing water

[0085] The present invention proposes the use of the aforementioned microorganisms, the aforementioned microbial agents or the aforementioned food additives or feed additives in denitrification of nitrogen-containing water bodies. According to an embodiment of the present invention, the nitrogen-containing water body contains nitrate and / or nitrite. According to the application of the embodiment of the present invention, it can effectively reduce the content of nitrate and / or nitrite in the water body, help improve the quality of the water body, and improve safety.

[0086] Exemplarily, 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 nitrate and / or nitrite.

[0087] Unless otherwise specified, the formula of the YPD agar medium used in the embodiments of the present invention is: 20 g of peptone, 10 g of yeast extract, 20 g of glucose, 10 g of agar, and 1 L of distilled water.

[0088] Unless otherwise specified, the formula of the YPD liquid culture medium used in the embodiments of the present invention is: 20 g of peptone, 10 g of yeast extract, 20 g of glucose, and 1 L of distilled water.

[0089] Unless otherwise specified, the formulas of the enrichment medium (EM) and nitrate medium (NM) used in the embodiments of the present invention are shown in Table 1. After the preparation, the pH of the medium is adjusted to 7.0-7.3. The configuration of the added trace elements is shown in Table 2.

[0090] Table 1 Composition of enriched medium (EM) and nitrate medium (NM)

[0091]

[0092] Table 2 Trace element composition

[0093] Element Content (g / L) Element Content (g / L) FeCl3·6H2O 1.50 ZnSO4·7H2O 0.12 H3BO3 0.15 KI 0.18 CuSO4·5H2O 0.03 MnCl2·4H2O 0.12 Na2MoO4·2H2O 0.06 CoCl2·6H2O 0.15 Disodium EDTA 10.00 NaHCO3 3.00

[0094] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0095] Example 1: Obtaining Zygosaccharomyces rouxii CGMCC No.30794

[0096] The Zygosaccharomyces rouxii CGMCC No.30794 of the invention is separated and obtained from the naturally fermented chili sauce of Hebei Province.

[0097] The collection and isolation process of the strain: take naturally fermented chopped pepper and add it to sterilized physiological saline, mix it well and then dilute it and spread it on YPD solid culture medium, and culture it at pH 6.0±0.2 and 30°C for 24 to 48 hours. Pick out single colonies of different sizes and shapes and streak them on fresh YPD solid culture medium plates, and culture them at 30°C for 24 to 48 hours. Repeat the streak purification culture several times until the colonies in the plate are consistent in morphology. After no foreign bacteria are found under microscopic examination, the strain is named FVPHBJ24271 and identified.

[0098] Obtaining bacterial solution: Use an inoculation loop to pick up a single colony and place it in YPD liquid medium, and culture at 37°C until the bacterial solution OD 600 =1.0~1.5, the strain is preserved.

[0099] Preservation of strains: 600 =1.0~1.5 bacterial solution and sterile 50% glycerol were mixed in a freezing tube at a ratio of 1:1 and stored at -80℃.

[0100] Example 2: Identification of Zygosaccharomyces rouxii CGMCC No.30794

[0101] The culture separated and purified in Example 1 was further confirmed to be a pure culture by streaking and smear microscopy, and then the strain morphology and physiological and biochemical identification, 16S rDNA gene PCR amplification and sequencing analysis were performed.

[0102] 1. Morphological and physiological and biochemical identification

[0103] Refer to the Manual of Identification of Common Fungal Systems to identify the strains to be tested by morphological characteristics, physiological and biochemical properties, etc.

[0104] The morphological identification results are shown in Figure 1 .

[0105] The identification results showed that the colony morphology on YPD agar medium was small, round, and white ( Figure 1 ), with a final pH of 3.0 in YPD liquid medium.

[0106] 2. PCR amplification and sequencing analysis of 16S rDNA gene

[0107] The strain FVPHBJ24271 was identified by 16S rDNA sequence. PCR amplification conditions: 95℃ pre-denaturation for 10min, followed by 30 cycles: 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 1.5min (1kb / min), 72℃ post-extension for 5min; stored at 4℃. The obtained PCR products were detected by 1% agarose gel electrophoresis. The PCR reaction system is shown in Table 3.

[0108] Table 3 PCR reaction system

[0109]

[0110]

[0111] The strain FVPHBJ24271 was sent to Shanghai Sangon Biotech Co., Ltd. for 16S rDNA sequence detection. The measured sequence was compared in GenBank, and the phylogenetic tree was constructed using MEGA11.0.13 for the strain to be tested and the sequences of other strains in the genus.

[0112] The results showed that the isolated strain FVPHBJ24271 was in the same branch as other strains of Zygosaccharomyces rouxii, and had a 99% homology with the 16S rDNA sequence of strains Zygosaccharomyces and Zygosaccharomyces rouxii.

[0113] Combined with the results of physiological and biochemical identification, the strain FVPHBJ24271 was further confirmed to be Zygosaccharomyces rouxii, with the sequence accession number being CGMCC No.30794.

[0114] The final identification confirmed that the isolated strain FVPHBJ24271 was a strain of Zygosaccharomyces rouxii, named Zygosaccharomyces rouxii FVPHBJ24271 (hereinafter referred to as "Zygosaccharomyces rouxii CGMCC No.30794"), which was deposited in the "General Microbiology Center of China National Culture Collection Administration" on May 27, 2024, with the deposit number CGMCC No.30794.

[0115] The 16S rDNA sequencing result of Zygosaccharomyces rouxii CGMCC No.30794 is shown in SEQ ID NO:1.

[0116] TCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCC(SEQ ID NO:1)

[0117] Example 3: Investigation of growth characteristics, acid production ability, acid resistance and salt resistance of Zygosaccharomyces rouxii CGMCC No.30794

[0118] Examination of the basic characteristics of the strains, such as growth characteristics, acid production capacity, acid resistance, and salt resistance, can help to screen out microbial strains with potential for specific applications.

[0119] In this example, the experimental strains were prepared as follows: Zygosaccharomyces rouxii CGMCC No. 30794 was streaked on a YPD agar plate for 24 to 48 hours, and a single colony was picked and grown in a YPD liquid medium for 16 to 20 hours for activation to obtain an activated strain.

[0120] 1. Investigation of strain growth characteristics and acid production capacity

[0121] The activated strain was inoculated into YPD liquid medium at 1% inoculation rate, and cultured at 30°C. The absorbance value was measured at 600nm every 1h until the stable period. The growth curve was drawn with the culture time as the horizontal axis and the corresponding absorbance value as the vertical axis ( Figure 2 ).

[0122] The results showed that: (1) In the first 24 hours of culture, Zygosaccharomyces rouxii CGMCC No.30794 was in the growth adaptation period, with slow growth and metabolism; after 24 hours, it entered the logarithmic growth period. (2) The pH of the bacterial solution was basically maintained below 4.0 when it reached the stable period.

[0123] 2. Investigation of acid resistance of strains

[0124] The activated strain was inoculated into YPD liquid medium with different pH values ​​(pH values ​​were 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0) at a 1% inoculum, and cultured at 30°C for 24 h. The absorbance values ​​(OD) at 600 nm of the activated strain before and after culture in the medium with different pH values ​​were measured. 600 )( Figure 3 The number of live yeast cells before and after the activated strain culture was determined using the culturable bacteria determination method ( Figure 4 ).

[0125] The results showed that Zygosaccharomyces rouxii CGMCC No.30794 had a high tolerance to acid. When the pH value of the YPD liquid medium was 4-5, it grew well; when the pH value dropped to 3.0, it still maintained a high survival rate, and the number of viable bacteria was maintained at 10 6 CFU / g and above.

[0126] 3. Investigation of salt tolerance of strains

[0127] The activated strains were inoculated into YPD liquid medium containing different NaCl concentrations (0%, 4%, 8%, 10%, 15%, 20%, 25%) at an inoculum volume of 1%, and cultured at 3°C ​​for 24 h. The absorbance values ​​(OD600) of the activated strains before and after culture in the medium with different salt concentrations were measured respectively ( Figure 5 ), and the culturable bacteria determination method was used to determine the number of live yeast cells before and after the activated strain was cultured ( Figure 6 ).

[0128] The results showed that Zygosaccharomyces rouxii CGMCC No.30794 had a high tolerance to NaCl. In a liquid culture medium with a salt concentration of 10%, the number of viable bacteria was maintained at 10 7 CFU / g or above; the strain can still grow in liquid culture medium with a salt concentration of 15%.

[0129] Example 4: Application of Zygosaccharomyces rouxii CGMCC No.30794 in the preparation of fermented vegetable food

[0130] The metabolic activities of specific microorganisms can increase the nutritional value and flavor of fermented foods and extend their shelf life.

[0131] In this embodiment, fermented vegetables were prepared using Zygosaccharomyces rouxii CGMCC No.30794, and the specific method is as follows:

[0132] 1. Preparation of Zygosaccharomyces rouxii CGMCC No.30794 starter

[0133] Zygosaccharomyces rouxii CGMCC No.30794 was streaked on a YPD agar medium plate for 24 to 48 hours, and a single colony was picked and grown in a YPD liquid medium for 16 to 20 hours for activation. The activated Zygosaccharomyces rouxii CGMCC No.30794 was inoculated into a YPD liquid medium at a 1% inoculum amount for expansion culture for 16 to 20 hours, and the bacterial solution was centrifuged, washed with sterile distilled water until no culture medium remained, and resuspended in sterile distilled water to a bacterial density of 1×10 10 ~1×10 11 CFU / mL, that is, Zygosaccharomyces rouxii CGMCC No.30794 starter culture.

[0134] 2. Preparation of fermented vegetables

[0135] Take 50.0kg of fresh vegetables, select, wash, drain, and after crushing or not, add 4.5-7.5kg of salt and stir evenly, the amount of salt added accounts for 8%-12% of the total weight of fresh vegetables, inoculate 1% of the total weight of vegetables with Zygosaccharomyces rouxii CGMCC No.30794 fermentation agent, and ferment at room temperature for 30 days in a sealed container to obtain fermented vegetables made with Zygosaccharomyces rouxii CGMCC No.30794 as a fermentation agent. The number of viable bacteria in the whole fermentation process is investigated, and the results are shown in Figure 7 .

[0136] The results showed that during the whole vegetable fermentation process, the viable count of Zygosaccharomyces rouxii CGMCC No.30794 remained at 10 5 CFU / mL and above.

[0137] For example, vegetables suitable for making fermented vegetables using Zygosaccharomyces rouxii CGMCC No. 30794 include, but are not limited to, peppers, cabbages, cucumbers, radishes, and the like.

[0138] Example 5: Investigation of the ability of Zygosaccharomyces rouxii CGMCC No.30794 to metabolize nitrite and nitrate

[0139] In the food industry, controlling the levels of nitrate and nitrite is crucial for food safety. The Zygosaccharomyces rouxii CGMCC No. 30794 of the present invention is often used in the preparation of fermented foods. Therefore, this example investigates the ability of Zygosaccharomyces rouxii CGMCC No. 30794 to metabolize nitrite and nitrate. The specific method is as follows:

[0140] 1. Strain Preparation

[0141] The strain of Zygosaccharomyces rouxii CGMCC No.30794 stored at -80°C was inoculated into a conical flask containing 100 mL of enrichment medium (EM) and shaken in a constant temperature shaker at 25°C and 160 rpm for 24 hours for activation culture. After the activation culture was completed, the cell density was adjusted to about 2.0 (i.e., OD 600 =2.0), and obtain OD 600 =2.0 bacterial suspension.

[0142] 2. Investigation of the ability of strains to metabolize nitrite

[0143] Pipette 1mL OD 600 = 2.0 bacterial suspension was added to a new 99 mL EM conical flask without adding OD 600 =2.0 bacterial suspension as blank control, shake well, measure OD value at 600nm every 1h until the bacterial density is basically stable and stop measuring. 600 = 2.0 was added into a new 95 mL nitrate medium (NM) conical flask, and the changes in nitrite content were analyzed to determine the ability of Zygosaccharomyces rouxii CGMCC No. 30794 to metabolize nitrite. The above experiments all contained blank controls and three parallel groups.

[0144] The results of the investigation on the ability of Zygosaccharomyces rouxii CGMCC No.30794 to metabolize nitrite are shown in Figure 8 .

[0145] 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 was above 15 mg / L ( Figure 8 ).

[0146] 3. Investigation of the ability of strains to metabolize nitrate

[0147] Pipette 1mL OD 600 = 2.0 bacterial suspension was added to a new 99 mL EM conical flask without adding OD 600=2.0 bacterial suspension as blank control, shake well, measure OD value at 600nm every 1h until the bacterial density is basically stable and stop measuring. 600 = 2.0 bacterial suspension was added into a new 95mL NM conical flask, and the change of nitrate content was analyzed to determine the ability of Zygosaccharomyces rouxii CGMCC No.30794 strain to metabolize nitrate. The above experiments all contained blank controls and three parallel groups.

[0148] The results of the investigation on the ability of Zygosaccharomyces rouxii CGMCC No.30794 to metabolize nitrate are shown in Fig. 9 .

[0149] The results showed that within a certain period of time, Zygosaccharomyces rouxii CGMCC No.30794 continuously metabolized NaNO3. After 24 hours, the NaNO3 metabolism of Zygosaccharomyces rouxii CGMCC No.30794 was maintained above 65 mg / L ( Fig. 9 ).

[0150] The above results show that Zygosaccharomyces rouxii CGMCC No.30794 has strong ability to metabolize nitrite and nitrate.

[0151] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A microorganism, characterized in that The microorganism is Zygosaccharomyces rouxii, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 27, 2024, with the deposit number CGMCC No.30794.

2. A microbial agent, characterized in that: The microorganism according to claim 1.

3. A food additive or feed additive, characterized in that: The microorganism according to claim 1.

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

5. The food or feed according to claim 4, characterized in that It further includes auxiliary materials or carriers acceptable to food or feed.

6. Use of the microorganism according to claim 1, the microbial agent according to claim 2, or the food additive or feed additive according to 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 microorganism according to claim 1, the microbial agent according to claim 2, or the food additive or feed additive according to claim 3 with a food raw material or a feed raw material.

8. The method according to claim 7, characterized in that 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; Optionally, the pH of the fermentation treatment is 2.5-6.5, preferably 3-6, more preferably 4-6.

9. A method for reducing nitrate and / or nitrite in a sample, characterized in that: include: The microorganism according to claim 1, the microbial agent according to claim 2, or the food additive or feed additive according to claim 3 is co-cultured with a sample containing nitrate and / or nitrite.

10. Use of the microorganism according to claim 1, the microbial agent according to claim 2, or the food additive or feed additive according to claim 3 in denitrification of nitrogen-containing water, characterized in that: The nitrogen-containing water body contains nitrate and / or nitrite.

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