Lysine bacillus of pepper and its application in vegetable fermentation
Patent Information
- Application Number
- CN202311468917.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-07
- Publication Date
- 2026-09-08
- Estimated Expiration
- 2043-11-07
AI Technical Summary
除此之外，传统蔬菜发酵过程复杂、难以控制，产品受外界因素影响大，质量不稳定，不同的蔬菜发酵时间不同，严重地影响了蔬菜发酵产品的品质
[0017] This invention isolated a *Bacillus capsicilysinus* strain, GXUN74704. When this strain was artificially inoculated into a mustard fermentation system, it inhibited nitrite synthesis, accelerated glucose utilization during fermentation, shortened fermentation time, and improved the quality of the fermented mustard product. The *Bacillus capsicilysinus* strain provided by this invention not only inhibits nitrite synthesis during mustard fermentation but also accelerates glucose utilization, shortens fermentation time, and improves the quality of the fermented mustard product, demonstrating promising application prospects in vegetable fermentation foods.
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Figure CN117487712B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of vegetable fermentation technology, and in particular to a strain of Bacillus capsicisae and its application in vegetable fermentation. Background Technology
[0002] Fermented vegetable foods are a type of processed vegetable products that are very popular in my country. They contain high levels of probiotics, vitamins, and minerals, and have effects such as promoting weight loss, anti-oxidation, anti-cancer activity, and lowering cholesterol, making fermented vegetable foods increasingly popular.
[0003] Traditional vegetable fermentation involves complex microbial communities, and the functions of most microorganisms are not fully understood, posing safety risks and leading to food safety issues. A certain amount of nitrite is produced during vegetable fermentation. Long-term consumption of foods containing nitrite can cause the acidic environment of the human gastrointestinal tract to convert nitrite into nitrosamines, which have strong carcinogenic and teratogenic effects. Therefore, inhibiting nitrite synthesis during vegetable fermentation and reducing the nitrite content in fermented vegetable products is an urgent problem to be solved. In addition, the traditional vegetable fermentation process is complex and difficult to control, the product is greatly affected by external factors, resulting in unstable quality. Different vegetables require different fermentation times, seriously affecting the quality of fermented vegetable products.
[0004] Currently, the main methods for reducing nitrite content and improving the quality of fermented vegetable products include artificial inoculation fermentation during the vegetable fermentation process. Artificial inoculation fermentation involves controlling and accelerating microbial reproduction through methods such as introducing new strains, adding salt, flavoring, and quantitative propagation. This inhibits nitrite synthesis, promotes the fermentation process, improves product quality, and enhances product safety. Summary of the Invention
[0005] The purpose of this invention is to provide a strain of *Bacillus capsicisae* and its application in vegetable fermentation, in order to solve the problems existing in the prior art. The *Bacillus capsicisae* provided by this invention not only inhibits the synthesis of nitrite during mustard fermentation, but also accelerates the utilization of glucose in the mustard fermentation system, shortens the fermentation time, and improves the taste and quality of fermented mustard products, and has good application prospects in vegetable fermented food.
[0006] To achieve the above objectives, the present invention provides the following solution:
[0007] This invention provides a strain of *Lysinibacillus capsici* GXUN74704, which was deposited on September 25, 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. 28530.
[0008] The present invention also provides a vegetable fermentation agent comprising the aforementioned *Bacillus capsicisae*.
[0009] The present invention also provides the application of the aforementioned Bacillus lysine-containing capsicum or the aforementioned vegetable fermentation agent in reducing the nitrite content of fermented vegetables.
[0010] Furthermore, the *Bacillus capsicilysinensis* strain or bacterial solution is inoculated into fermenting vegetables for fermentation to inhibit the amount of nitrite synthesized during the fermentation process.
[0011] The present invention also provides the application of the aforementioned Bacillus lysine capsicum or the aforementioned vegetable fermentation agent in shortening the fermentation time of pickled vegetables.
[0012] Furthermore, the *Bacillus capsicisae* strain or bacterial solution is inoculated into pickled vegetables for fermentation, thereby accelerating the utilization rate of glucose in the fermentation system and shortening the fermentation time of the pickled vegetables.
[0013] Furthermore, the inoculum amount of the capsicum lysine spores is 6% (V / V).
[0014] The present invention also provides the application of the aforementioned Bacillus capsicum lysine or the aforementioned vegetable fermentation agent in vegetable fermentation foods or in the preparation of vegetable fermentation foods.
[0015] Furthermore, the vegetables mentioned include mustard greens.
[0016] The present invention discloses the following technical effects:
[0017] This invention isolated a *Bacillus capsicilysinus* strain, GXUN74704. When this strain was artificially inoculated into a mustard fermentation system, it inhibited nitrite synthesis, accelerated glucose utilization during fermentation, shortened fermentation time, and improved the quality of the fermented mustard product. The *Bacillus capsicilysinus* strain provided by this invention not only inhibits nitrite synthesis during mustard fermentation but also accelerates glucose utilization, shortens fermentation time, and improves the quality of the fermented mustard product, demonstrating promising application prospects in vegetable fermentation foods. Attached Figure Description
[0018] 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.
[0019] Figure 1 Gram staining image of strain GXUN74704;
[0020] Figure 2 Phylogenetic tree of strain GXUN74704;
[0021] Figure 3 The images show the fermentation of mustard greens in Example 2, including natural fermentation (A) and artificial inoculation with GXUN74704 (B).
[0022] Figure 4 This is a pH value measurement graph of the mustard fermentation system in Example 2, which involved natural fermentation and artificial inoculation with GXUN74704.
[0023] Figure 5 The graph shows the detection of lactic acid content in the mustard fermentation system of natural fermentation and artificial inoculation with GXUN74704 in Example 2;
[0024] Figure 6 The graph shows the nitrite content detection in the mustard fermentation system of natural fermentation and artificial inoculation with GXUN74704 in Example 2;
[0025] Figure 7 The graph shows the glucose content detection in the mustard fermentation system of natural fermentation and artificial inoculation with GXUN74704 in Example 2. Detailed Implementation
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] The culture medium formulations used in the following examples or experimental cases:
[0032] (1) Solid LB medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L, pH 7.0.
[0033] (2) Liquid LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0.
[0034] Example 1: Isolation and Identification of Strains
[0035] 1. Test sample
[0036] The test sample was collected from sauerkraut juice purchased from Luowen Farmers Market in Xixiangtang District, Nanning City, Guangxi Zhuang Autonomous Region.
[0037] 2. Experimental Methods
[0038] (1) Strains isolation and purification
[0039] Take 20 mL of sample and place it in a 50 mL centrifuge tube. Shake at 180 rpm for 30 min. Dilute the bacterial suspension to 10⁻⁶. -2 10 -3 and 10 -4 100 μL of each culture was spread onto solid LB medium and incubated at 30°C for 1-2 days. Single colonies were picked and streaked to purify the strain. The pure colonies were stored at 4°C or -80°C. A strain was obtained and named GXUN74704.
[0040] (2) Classification and identification
[0041] Based on the differences in the composition and structure of bacterial cell walls, bacteria are divided into two categories: Gram-positive bacteria and Gram-negative bacteria. Gram staining includes four steps: primary staining, mordanting, destaining, and counterstaining. The specific steps are as follows: fix the smear; primary stain with crystal violet for 1 min, wash with water; mordant with iodine solution for 1 min, wash with water; destain with ethanol for 30 s, wash with water; counterstain with safranin for 1 min, wash with water; allow to dry, and examine under a microscope. Under a microscope, strain GXUN74704 appears as a rod shape and stains purple with Gram stain, therefore this bacterium is a Gram-positive strain (see...). Figure 1 ).
[0042] (3) Molecular biological identification
[0043] Genomic DNA was extracted from strain GXUN74704, 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, revealing 100% homology with *Lysinibacillus capsici*. A phylogenetic tree was constructed using the Neighbor-Joint Linking (NJ) method (see [link to phylogenetic tree]). Figure 2 The results showed that GXUN74704 is most closely related to Lysinibacillus capsici, thus it can be determined that GXUN74704 belongs to the taxonomic category of Lysinibacillus capsici.
[0044] The strain GXUN74704 was classified as Lysinibacillus capsici and was deposited on September 25, 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. 28530.
[0045] Example 2: Pickled Mustard Greens
[0046] I. Preparation of Fermentation Inoculum:
[0047] (1) The *Bacillus capsicilysinensis* 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;
[0048] (2) Transfer the bacterial culture to 120 mL of culture medium at a rate of 2% (V / V), and incubate 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.
[0049] 2. Pickling mustard greens and allowing them to ferment naturally (see...) Figure 3 (A) and artificial inoculation fermentation (see 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 peppercorns to the water, blanch the mustard greens in the boiling water until soft, and put them into the rice water that has been prepared in advance.
[0053] (4) Sprinkle 3% (W / V) salt and 2% (W / V) brown sugar into the mustard greens, mix well, and let them ferment naturally as a control.
[0054] Another batch of mustard greens was treated as described above and then inoculated with the above-mentioned GXUN74704 fermentation agent at an inoculation rate of 6% (V / V) for artificial inoculation and fermentation, which was the experimental group;
[0055] (5) The rice water should cover the mustard greens, weigh them down with a heavy object, seal the jar, and start pickling.
[0056] III. Detection of indicators during the fermentation process
[0057] 1. Determination of pH in the mustard fermentation system
[0058] 10 mL of fermentation broth samples from the control and experimental groups were collected on days 1, 3, 5, 7, 11, 15, 19, and 23 after inoculation. The pH was measured and recorded using a portable pH meter.
[0059] The results show (see) Figure 4 The pH of the mustard fermentation system artificially inoculated with strain GXUN74704 was lower than that of the natural fermentation system in the early stage of fermentation. In the later stage, the pH of the natural fermentation system was slightly lower than that of the mustard fermentation system artificially inoculated with strain GXUN74704, but there was no significant difference.
[0060] 2. Determination of lactic acid content in mustard fermentation system
[0061] 20 μL of mustard fermentation broth samples were collected from the control group and the experimental group on days 1, 3, 5, 7, 11, 15, 19 and 23 after inoculation.
[0062] With NAD + As a hydrogen acceptor, LDH catalyzes the dehydrogenation of lactate to produce pyruvate, thus enabling NAD+ to be released. + It is converted to NADH, in which PMS transfers hydrogen to reduce NBT to a purple complex. The absorbance of the chromogenic compound is linearly related to the lactic acid content at 530 nm.
[0063] The results show (see) Figure 5 There was no significant difference in lactic acid content between the naturally fermented and artificially inoculated mustard fermentation systems using strain GXUN74704. This indicates that artificial inoculation with strain GXUN74704 does not affect the content of beneficial components in the mustard fermentation system.
[0064] 3. Determination of nitrite content in mustard fermentation system
[0065] 0.5 mL of mustard fermentation broth samples were collected from the control group and the experimental group on days 1, 3, 5, 7, 11, 15, 19 and 23 after inoculation.
[0066] The nitrite content is determined by colorimetry based on the principle that nitrite can react with a colorimetric reagent to form a pale red azo compound.
[0067] The results show (see) Figure 6 When strain GXUN74704 was inoculated into fresh pickled mustard greens at an inoculation rate of 6% (V / V), the nitrite content in the fermentation system was extremely low at 1, 3, 5, 7, 11, 15, 19, and 23 days after inoculation, and was basically undetectable after 5 days. In contrast, in the natural fermentation (control group), nitrite was synthesized at different levels at 1, 3, 5, 7, 11, 15, and 19 days of fermentation. At 3 days of fermentation, the nitrite content in the fermentation system reached approximately 308.6 μmol / L, and was not detectable until 23 days later.
[0068] 4. Determination of glucose content in mustard fermentation system
[0069] 10 μL of mustard fermentation broth samples were collected from the control group and the experimental group on days 1, 3, 5, 7, 11, 15, 19 and 23 after inoculation.
[0070] The glucose oxidase method involves the conversion of glucose in the fermentation broth into gluconic acid and hydrogen peroxide by glucose oxidase. The latter, under the action of peroxidase, couples reduced 4-aminoantipyrine with phenol to form a quinone compound that can be measured by a spectrophotometer, thereby detecting the glucose content.
[0071] The results show (see) Figure 7When strain GXUN74704 was inoculated into fresh pickled mustard greens at a 6% (v / v) inoculation rate, the glucose in the fermentation system was almost completely consumed after 5 days of inoculation; while in the natural fermentation (control group), the glucose in the fermentation system was not almost completely consumed until 7 days of fermentation. Therefore, it can be inferred that artificial inoculation with strain GXUN74704 accelerated glucose utilization and shortened the fermentation time during mustard green fermentation.
[0072] Example 3: Determination of the taste and quality of fermented mustard green products
[0073] The *Bacillus capsicisae* was inoculated into mustard greens at a rate of 6% (V / V) for fermentation. After 20 days of fermentation, 10 trained experts evaluated the fermented mustard greens products of naturally fermented and artificially inoculated strain GXUN74704 in a standard indoor environment (free from interference, noise, visual stimulation, and other environmental factors). This evaluation primarily assesses the color, odor, firmness, crispness, juiciness, sourness, astringency, and overall quality of fermented mustard greens. The evaluation criteria are as follows: Color: 10 points for a bluish-green or yellowish-green color, 0 points for brown; Odor: 10 points for an unpleasant odor, 0 points for a pronounced sourness or a fresh aroma; Firmness: 10 points for being easily crushed by hand, 0 points for being too soft; Crispness: 10 points for a crisp sound when chewed, 0 points for being too soft; Juiciness: 10 points for juice flowing out when chewed, 0 points for being very dry; Sourness: 10 points for a pronounced sourness, 0 points for no sourness; Astringency: 10 points for a pronounced astringency, 0 points for no astringency; Overall Quality: 10 points for being very satisfied with the overall quality of the fermented mustard greens, 0 points for being dissatisfied.
[0074] The results (see Table 1) showed that when strain GXUN74704 was inoculated into fresh pickled mustard greens at an inoculation rate of 6% (V / V), after 20 days of fermentation, an evaluation by 10 trained experts found that the overall quality evaluation of the fermented mustard greens inoculated with strain GXUN74704 was 7.5 points, while the control group scored 5 points. The product's taste and quality were superior to that of traditional naturally fermented mustard greens.
[0075] Table 1 Evaluation of the taste and quality of fermented mustard greens
[0076]
[0077] 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 strain of *Lysinibacillus capsici* GXUN74704, characterized in that, It was deposited on September 25, 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. 28530.
2. A vegetable fermentation agent, characterized in that, It contains the capsicum lysine-containing Bacillus as described in claim 1.
3. The application of the *Bacillus capsicum* as described in claim 1 or the vegetable fermentation agent as described in claim 2 in reducing the nitrite content of fermented vegetables.
4. The application according to claim 3, characterized in that, The *Bacillus capsicisae* strain or bacterial solution is inoculated into fermented vegetables for fermentation to inhibit the amount of nitrite synthesized during the fermentation process.
5. The application of the *Bacillus capsicum* as described in claim 1 or the vegetable fermentation agent as described in claim 2 in shortening the fermentation time of pickled vegetables.
6. The application according to claim 5, characterized in that, The *Bacillus capsicisae* strain or bacterial solution is inoculated into pickled vegetables for fermentation, thereby accelerating the utilization rate of glucose in the fermentation system and shortening the fermentation time of the pickled vegetables.
7. The application according to claim 6, characterized in that, The inoculation amount of the *Bacillus capsicisae* was 6% (V / V).
8. The application of the *Bacillus capsicum* as described in claim 1 or the vegetable fermentation agent as described in claim 2 in the fermentation of vegetable foods or the preparation of fermented vegetable foods.
9. The application according to claim 8, characterized in that, The vegetables mentioned include mustard greens.