Micrococcus luteus strain for inhibiting nitrite synthesis and application thereof

Abstract

The application discloses a Micrococcus luteus for inhibiting nitrite synthesis and application thereof, and belongs to the technical field of pickled vegetable food, wherein the Micrococcus luteus has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 27831; the application also discloses application of the Micrococcus luteus in inhibiting nitrite synthesis in a pickled vegetable process or shortening a pickled vegetable time. The application separates a Micrococcus luteus GXUN74703, the strain is artificially inoculated into a mustard pickling fermentation system, nitrite synthesis in the pickled vegetable process is inhibited, glucose utilization in the pickled vegetable fermentation system is accelerated, the fermentation time is shortened, and the application has a good application prospect in the pickled vegetable food.

Description

Technical Field

[0001] This invention relates to the field of vegetable pickling technology, and in particular to a strain of Micrococcus luteus that inhibits nitrite synthesis and its application. Background Technology

[0002] Pickled vegetables are low-salt, lactic acid fermented products containing high levels of probiotics, vitamins, and minerals, offering numerous benefits such as promoting weight loss, antioxidant activity, anti-cancer activity, and cholesterol reduction, making them a beneficial health food. However, the pickling process produces a certain amount of nitrites. Nitrites are permitted food additives, and their use within safe limits does not harm the human body. However, long-term consumption of foods containing nitrites can cause the acidic environment of the digestive tract to convert nitrites into nitrosamines, which have strong carcinogenic and teratogenic effects. Therefore, inhibiting nitrite synthesis during vegetable pickling and reducing the nitrite content in pickled vegetables is an urgent problem to be solved.

[0003] Currently, methods to reduce nitrite content in pickled vegetables include artificial inoculation fermentation, adding antioxidants, and seasonings. Among these, artificial inoculation fermentation is widely used in the vegetable pickling process and can effectively inhibit or reduce the synthesis or production of nitrite. Artificial inoculation fermentation controls and accelerates the reproduction of microorganisms through methods such as introducing seedlings, adding salt, seasoning, and quantitative propagation, thereby inhibiting nitrite synthesis and promoting the fermentation process. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Micrococcus luteus that inhibits nitrite synthesis and its application, in order to solve the problems existing in the prior art. The Micrococcus luteus provided by this invention not only inhibits the synthesis of nitrite during the pickling process of vegetables, but also accelerates the utilization of glucose in the vegetable pickling fermentation system and shortens the fermentation time, and has good application prospects in the field of pickled vegetable foods.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a strain of Micrococcus luteus, which was deposited on July 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27831.

[0007] The present invention also provides an application of the aforementioned Micrococcus luteus in inhibiting nitrite synthesis during vegetable pickling.

[0008] Furthermore, the *Micrococcus luteus* was inoculated into vegetables at a rate of 6% (V / V) for pickling, thereby inhibiting the synthesis of nitrites during the pickling process.

[0009] The present invention also provides an application of the aforementioned Micrococcus luteus in shortening the pickling time of vegetables.

[0010] Furthermore, the *Micrococcus luteus* is inoculated into vegetables at a rate of 6% (V / V) for pickling, thereby accelerating the utilization rate of glucose in the vegetable pickling and fermentation system and shortening the pickling time.

[0011] The present invention also provides the application of the aforementioned Micrococcus luteus in pickling vegetables or in the preparation of pickled vegetable foods.

[0012] The present invention discloses the following technical effects:

[0013] This invention isolated a strain of *Micrococcus luteus* GXUN74703. When this strain was artificially inoculated into a mustard green pickling and fermentation system, it inhibited nitrite synthesis, accelerated glucose utilization during the pickling and fermentation process, and shortened the fermentation time. The *Micrococcus luteus* strain provided by this invention not only inhibits nitrite synthesis during vegetable pickling but also accelerates glucose utilization and shortens fermentation time, showing promising application prospects in pickled vegetable products. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a colony morphology diagram of strain GXUN74703 on a culture medium.

[0016] Figure 2 Phylogenetic tree of strain GXUN74703;

[0017] Figure 3 The images show the pickling of mustard greens in Example 2, including natural fermentation (A) and artificial inoculation with GXUN74703 (B).

[0018] Figure 4 The graph shows the nitrite content detection in the mustard pickling fermentation system of natural fermentation and artificial inoculation with GXUN74703 in Example 3.

[0019] Figure 5The graph shows the glucose content detection in the mustard pickling and fermentation system of natural fermentation and artificial inoculation with GXUN74703 in Example 4. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] The culture medium formulations used in the following examples or experimental cases:

[0026] (1) Solid LB medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L, pH 7.0.

[0027] (2) Liquid LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0.

[0028] Example 1: Strain Isolation and Screening

[0029] 1. Test sample

[0030] The test sample was collected from sauerkraut juice purchased from Luowen Farmers Market in Xixiangtang District, Nanning City, Guangxi Zhuang Autonomous Region.

[0031] 2. Experimental Methods

[0032] 2.1 Separation and Screening

[0033] (1) Strain isolation and purification: Take 20 mL of sample and place it in a 50 mL centrifuge tube. Shake at 180 r / min for 30 min. Dilute the bacterial suspension to 10. -2 10 -3 and 10 -4 Take 100 μL of each culture and spread it on solid LB medium. Incubate at 30°C for 1-2 days. Pick a single colony and streak it to purify the strain. Store the obtained pure colonies at 4°C or -80°C.

[0034] (2) Initial screening: The isolated and purified strains were inoculated into liquid LB medium and cultured until OD500. 600 When the value is 1.0, pipette 10 μL of bacterial culture onto the functional plate and select strains that have no cellulase or amylase activity but have protease activity.

[0035] (3) Secondary screening: Select strains that showed no cellulase or amylase activity but possessed protease activity in the initial screening. Inoculate the strains into Erlenmeyer flasks containing 30 mL of liquid LB medium and incubate at 30°C and 180 r / min until OD. 600 =1.0 was used as seed culture, and 2% (v / v) of the culture was transferred to 300 mL of LB medium and cultured at 30 °C and 180 r / min. 10 μL of the bacterial culture was pipetted onto a protein functional plate, and the strain with the strongest decomposition ability was selected. Using this method, a bacterial strain was obtained and named GXUN74703.

[0036] 2.2 Identification

[0037] (1) Morphological identification

[0038] On the culture medium, strain GXUN74703 forms round, yellow colonies with a smooth, moist surface and regular edges. Figure 1 ).

[0039] (2) Physiological and biochemical identification

[0040] Physiological and biochemical identification of strain GXUN74703 was performed with reference to Bergey's Manual of Bacterial Identification, Ninth Edition and the Manual of Systematic Identification of Common Bacteria. The results are shown in Table 1.

[0041] As shown in Table 1, the results indicate that strain GXUN74703 is a Gram-positive bacterium that can produce catalase and protease, but not amylase and cellulase.

[0042] Table 1. Physiological and biochemical identification results of strain GXUN74703

[0043]

[0044] Note: "+" indicates presence, "-" indicates absence, "G" indicates presence. + "Indicates Gram-positive."

[0045] (3) Molecular biological identification

[0046] Genomic DNA was extracted from strain GXUN74703, and its 16S rDNA was amplified using PCR. Electrophoresis of the amplified PCR product revealed a distinct band at 1500 bp, consistent with the size of the 16S rDNA. This PCR product was sent to Shanghai Sangon Biotech for sequencing. The sequence obtained was compared with 16S-based IDs on the Ezbiocloud website, showing 100% homology with *Micrococcus luteus*. A phylogenetic tree was constructed using the Neighbor-Joint Linking (NJ) method (see [link to phylogenetic tree]). Figure 2 The results showed that GXUN74703 was most closely related to Micrococcus luteus, thus it can be determined that GXUN74703 belongs to the taxonomic category of Micrococcus luteus.

[0047] The strain GXUN74703 was classified as Micrococcus luteus and was deposited on July 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27831.

[0048] Example 2: Pickled Mustard Greens

[0049] Based on the cultivation conditions of Example 1, mustard greens were pickled and subjected to natural fermentation. Figure 3 (A) and artificial inoculation fermentation ( Figure 3 (B)

[0050] (1) Dry the mustard greens in the sun one day in advance, about 500g;

[0051] (2) Prepare about 2L of rice water by boiling and cooling it the night before.

[0052] (3) Boil a pot of water, add an appropriate amount of peppercorns to the water, blanch the mustard greens in the boiling water until softened, and then put them into the rice water prepared in advance.

[0053] (4) Sprinkle 3% (W / V) salt and 2% (W / V) brown sugar into the mustard greens, mix well, and carry out natural fermentation; or inoculate GXUN74703 at a 6% (V / V) inoculation rate for artificial inoculation and fermentation.

[0054] (5) The rice water should cover the mustard greens, weigh them down with a heavy object, seal the jar, and start pickling.

[0055] Example 3: Determination of nitrite content in mustard pickling and fermentation system

[0056] (1) The Micrococcus luteus was inoculated into an Erlenmeyer flask containing 30 mL of liquid LB medium and cultured at 30 °C and 180 r / min until OD. 600 =1.0 as seed solution;

[0057] (2) Transfer the bacterial culture to 120 mL of medium at a rate of 2% (V / V), and culture at 30℃ and 180 r / min for 24 h. Centrifuge the bacterial culture at 8000 rpm for 10 min, remove the supernatant, wash the bacterial cells three times with physiological saline, and then mix the bacterial cells with 50 mL of physiological saline.

[0058] (3) Inoculate the fresh pickled mustard greens from Example 2 at a rate of 6% (V / V) and mix well;

[0059] (4) Take 0.5 mL of mustard fermentation liquid samples on days 1, 3, 5, 7, 9, 11, 13, 15 and 17 after inoculation;

[0060] (5) The nitrite content is determined by colorimetry based on the principle that nitrite can react with the colorimetric reagent to form a light red azo compound.

[0061] The results show (see) Figure 4 Strain GXUN74703 was inoculated into fresh pickled mustard greens (named Q-129) at an inoculation rate of 6% (V / V). The nitrite content in the fermentation system was extremely low, almost undetectable, at 1, 3, 5, 7, 9, 11, 13, 15, and 17 days after inoculation. In contrast, the natural fermentation (control group) showed varying levels of nitrite synthesis in the fermentation system at 1, 3, 5, 7, 9, 11, 13, 15, and 17 days of fermentation. At 3 days of fermentation, the nitrite content in the fermentation system reached approximately 308.6 μmol / L.

[0062] Example 4: Determination of glucose content in mustard green pickling and fermentation system

[0063] (1) The Micrococcus luteus was inoculated into an Erlenmeyer flask containing 30 mL of liquid LB medium and cultured at 30 °C and 180 r / min until OD. 600 =1.0 as seed solution;

[0064] (2) Transfer the bacterial culture to 120 mL of medium at a rate of 2% (V / V), and culture at 30℃ and 180 r / min for 24 h. Centrifuge the bacterial culture at 8000 rpm for 10 min, remove the supernatant, wash the bacterial cells three times with physiological saline, and then mix the bacterial cells with 50 mL of physiological saline.

[0065] (3) Inoculate the fresh pickled mustard greens from Example 2 at a rate of 6% (V / V) and mix well;

[0066] (4) Take 10 μL of mustard fermentation broth samples on days 1, 3, 5, 7, 9, 11, 13, 15 and 17 after inoculation;

[0067] (5) By using glucose oxidase, glucose in the fermentation broth is converted into gluconic acid and hydrogen peroxide by glucose oxidase. The latter, under the action of peroxidase, couples and condenses reduced 4-aminoantipyrine with phenol to form quinone compounds that can be measured by spectrophotometer, thereby detecting the glucose content.

[0068] The results show (see) Figure 5 In a study, strain GXUN74703 was inoculated into fresh pickled mustard greens (named Q-129) at a 6% (v / v) inoculation rate. Five days after inoculation, the glucose in the fermentation system was completely consumed and virtually undetectable; in contrast, in the natural fermentation (control group), glucose was not completely consumed until 13 days of fermentation. Therefore, it can be inferred that artificial inoculation with strain GXUN74703 during mustard green pickling accelerates glucose utilization and shortens the fermentation time.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Micrococcus luteus ( Micrococcus luteus The application of GXUN74703 in inhibiting nitrite synthesis during vegetable pickling is characterized by, The *Micrococcus luteus* was inoculated into vegetables at a rate of 6% (V / V) for pickling, thereby inhibiting the synthesis of nitrites during the pickling process. The *Micrococcus luteus* strain was deposited on July 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27831.

2. A type of Micrococcus luteus ( Micrococcus luteus The application of GXUN74703 in accelerating the utilization rate of glucose in vegetable pickling and fermentation systems is characterized by, The *Micrococcus luteus* strain was deposited on July 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27831.

3. The application according to claim 2, characterized in that, The *Micrococcus luteus* was inoculated into the vegetables at a rate of 6% (V / V) for pickling, thereby accelerating the utilization rate of glucose in the vegetable pickling and fermentation system.

4. A type of Micrococcus luteus ( Micrococcus luteus The application of GXUN74703 in pickled vegetable foods or the preparation of pickled vegetable foods is characterized by, The *Micrococcus luteus* strain was deposited on July 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27831.

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