Fermented spicy Chinese cabbage with effects of soothing nerves, helping sleep and relieving anxiety and preparation method of fermented spicy Chinese cabbage

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to leuconostoc mesenteroides, fermented spicy cabbages with the effects of soothing the nerves, helping sleep and relieving anxiety and a preparation method of the fermented spicy cabbages. The leuconostoc mesenteroides NCU018046 provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 5, 2024, and the preservation number of the leuconostoc mesenteroides NCU018046 is CGMCC NO.31870. The strain has strong acid and cholate resistance, has strong inhibition capability on common food-borne pathogenic bacteria, and meets safety standards. The complex microbial inoculant provided by the invention is prepared from the strain and plant lactobacillus NCU0011098. The fermented spicy cabbages prepared by fermenting a single bacterial agent of leuconostoc mesenteroides NCU018046 or the complex bacterial agent are good in flavor and low in content of harmful substances, and have various biological activities of soothing the nerves, helping sleep, relieving anxiety, resisting hypertension, resisting oxidation, reducing uric acid, resisting colon cancer and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to fermented kimchi with the effects of calming nerves, promoting sleep and relieving anxiety, and a preparation method thereof. Background Art

[0002] Fermented food is a kind of food that is made by microorganisms, which convert organic substances such as sugars and proteins into substances such as acids, alcohols, and esters. The fermentation process gives it a unique flavor and nutritional value, and produces active substances that are beneficial to health. There are many kinds of fermented foods, such as yogurt, sauerkraut, soy sauce, fermented black beans, fermented bean curd, spicy cabbage, etc.

[0003] Spicy cabbage is a special fermented vegetable in Northeast China. It is one of the most famous traditional fermented vegetables in China and a common delicacy on people's tables. Spicy cabbage is rich in dietary fiber, vitamins, and rich nutrients such as calcium, copper, phosphorus, and iron. Among them, the high content of cellulose can prevent intestinal diseases such as constipation and colon cancer. The traditional method of making Northeast spicy cabbage is mainly to use Chinese cabbage as raw material, and chili powder, apples, pears, ginger, garlic and other ingredients as low-temperature fermentation. Natural fermentation is often used in industrial production of spicy cabbage, and the initial microorganisms are generally derived from microorganisms remaining on vegetables. During the fermentation process, the strains are often not fixed, which may not only cause changes in the fermentation flavor of different batches of spicy cabbage products, but also cause large batch differences in their health functions. In addition, if the fermentation process is not properly controlled, it may lead to the growth of harmful microorganisms, high nitrite content, unstable fermentation cycle, poor health functions and other problems. Therefore, it is of great significance to develop fermented probiotics that have the functions of improving product flavor, reducing the content of harmful substances, and improving health functions. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a strain of Leuconostoc mesenteroides and a fermented kimchi with the effects of calming nerves, promoting sleep and relieving anxiety, and a preparation method thereof. The Leuconostoc mesenteroides has strong acid and bile salt resistance, strong inhibitory ability against common foodborne pathogens, meets safety standards, and has high application value; and the Leuconostoc mesenteroides or a composite bacterial agent containing the strain can be used to prepare fermented foods, such as kimchi, which can give the product multiple biological activities such as calming nerves, promoting sleep, relieving anxiety, anti-hypertension, anti-oxidation, lowering uric acid, and anti-colon cancer.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a strain of Leuconostoc mesenteroides NCU018046, whose classification name is Leuconostoc mesenteroides, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 5, 2024, and its deposit number is CGMCC NO.31870; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0007] The Leuconostoc mesenteroides NCU018046 strain provided by the present invention is obtained by separation and screening from pickled spicy cabbage. The strain has strong acid and bile salt resistance, is sensitive to erythromycin, tetracycline, ampicillin, penicillin G and cephalothin, is tolerant to gentamicin, streptomycin, ciprofloxacin and kanamycin, and has no hemolytic activity, meeting the basic standard of being generally regarded as safe (GRAS). In addition, the strain has strong inhibitory ability against common foodborne pathogens such as Escherichia coli, Staphylococcus aureus and Salmonella typhimurium. Therefore, the strain has high application value.

[0008] In addition, the present invention has found through experimental research on fermented kimchi that Leuconostoc mesenteroides NCU018046 can reduce the content of biogenic amines, nitrite and salinity of the product, inhibit Escherichia coli and Staphylococcus aureus, increase the content of amino acids in the product and short-chain fatty acids that are beneficial to regulating the intestine, and convert glutamate into γ-aminobutyric acid during the fermentation process of kimchi. In addition, the fermented kimchi prepared by Leuconostoc mesenteroides NCU018046 has the effects of anti-oxidation, inhibiting xanthine oxidase, inhibiting ACE (angiotensin converting enzyme), and inhibiting cancer cell proliferation. Therefore, the strain has a wide application prospect in the field of fermented foods and can be used to produce fermented foods such as kimchi.

[0009] The second aspect of the present invention provides the use of the above-mentioned Leuconostoc mesenteroides NCU018046, including any one of the following:

[0010] Application in the production of fermented foods;

[0011] Application in the preparation of a single bacterial agent for producing fermented food;

[0012] Application in the preparation of composite bacterial agents for producing fermented foods.

[0013] Those skilled in the art can use any available fermentation and preparation method to obtain a single bacterial fermentation agent or a composite bacterial agent containing live bacteria, as long as the number of live bacteria is met. The present invention provides a preparation method of a single bacterial agent as an example, but is not intended to limit the preparation method of the composite bacterial agent:

[0014] Inoculate the enteric Leuconostoc mesenteroides NCU018046 into MRS liquid culture medium for three consecutive activations, inoculate into MRS liquid culture medium at 2% v / v inoculation amount, and culture at 37°C for 24 hours. Collect the cells by centrifugation (4°C, 6000×g, 10 min), mix evenly with sterile skim milk of 10% of the cell mass, pre-freeze at -80°C for 2 hours, and then transfer to a vacuum freeze dryer and freeze for 24 hours to obtain a single bacterial agent of enteric Leuconostoc mesenteroides NCU018046. Before use, the number of viable bacteria of the single bacterial agent can be adjusted to (1-9)×10 with glucose powder. 10 CFU / g.

[0015] After preparing single bacterial agents of other probiotics according to this method and adjusting the number of live bacteria with glucose dry powder, the composite bacterial agent can be obtained by evenly mixing the single bacterial agents in proportion.

[0016] Preferably, the fermented food is fermented kimchi.

[0017] The third aspect of the present invention provides a composite bacterial agent, comprising the above-mentioned Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum.

[0018] Preferably, the plant lactobacillus is plant lactobacillus (Lactiplantibacillus plantarum) NCU0011098, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 26, 2023, with a deposit number of CGMCC NO.28774; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Plant lactobacillus NCU0011098 has been disclosed in a patent with application number 202410534531.2 and the name "A plant lactobacillus with the effect of preventing colon cancer and its application".

[0019] The present invention finds through experimental research on fermenting kimchi that, compared with fermentation with Leuconostoc mesenteroides NCU018046 alone or fermentation with a composite bacterial agent of the strain and other Lactobacillus plantarum, the composite bacterial agent prepared by Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 has significantly improved effects in reducing the content of biogenic amines, nitrite and salinity of the product, inhibiting Escherichia coli and Staphylococcus aureus, increasing the content of amino acids and short-chain fatty acids in the product that are beneficial to regulating the intestine, converting glutamate into gamma-aminobutyric acid, increasing the antioxidant activity of the product, inhibiting xanthine oxidase, inhibiting ACE and inhibiting the proliferation of cancer cells, and can significantly increase the types of volatile aromas and improve the content of flavor substances, indicating that the two have a synergistic effect in the above aspects.

[0020] Those skilled in the art can use any available fermentation and preparation method to obtain the composite bacterial agent, as long as the viable bacterial count is met. The present invention provides a preparation method as an example, but is not intended to limit the preparation method of the composite bacterial agent:

[0021] Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 were inoculated into MRS liquid culture medium for three consecutive activations, respectively, and inoculated into MRS liquid culture medium at a 2% v / v inoculation amount, and cultured at 37°C for 24 h. The cells were collected by centrifugation (4°C, 6000×g, 10 min), and then mixed evenly with sterile skim milk (10% of the cell mass), pre-frozen at -80°C for 2 h, and then transferred to a vacuum freeze dryer and frozen for 24 h, respectively, to obtain single bacterial agents of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098. The number of viable bacteria of each single bacterial agent was adjusted to (1-9)×10- 10 CFU / g, and then the two single bacteria fermentation agents are evenly mixed in proportion to obtain the composite bacterial agent.

[0022] A fourth aspect of the present invention provides use of the composite bacterial agent in the preparation of fermented food.

[0023] Preferably, the fermented food is fermented kimchi.

[0024] A fifth aspect of the present invention provides a fermented kimchi obtained by fermenting the above-mentioned single bacterial agent of Leuconostoc mesenteroides NCU018046 or the above-mentioned composite bacterial agent.

[0025] A sixth aspect of the present invention provides a method for preparing fermented kimchi, which specifically comprises the following steps:

[0026] S1. Wash the fresh Chinese cabbage and immerse it in a 13% to 17% salt solution, pickle it at room temperature until the salt concentration drops to 7% to 9%, then wash it with clean water and drain it;

[0027] S2. Evenly spread the mixture containing the probiotic fermentation agent between the leaves of the Chinese cabbage, and let it stand and ferment at 4-8° C.; the probiotic fermentation agent is the single bacterial agent of the above-mentioned Leuconostoc mesenteroides NCU018046 or the above-mentioned composite bacterial agent.

[0028] According to the conventional operation of the kimchi production process, the fresh Chinese cabbage in S1 should be free of rotten leaves, diseases and insect pests, and should be cut in half and washed with water before pickling.

[0029] Preferably, the salt concentration of the saline solution in S1 is 15%.

[0030] Preferably, after the pickling in S1 is completed, the cabbage is washed and drained with clean water until the salinity of the cabbage is 1% to 1.5%.

[0031] Preferably, the raw materials of the dressing also include radish, shallot, garlic, onion, fresh ginger, white pear, apple, shrimp paste, fish sauce, glutinous rice paste, chili powder, sucrose and monosodium glutamate.

[0032] Further preferably, the mass proportions of the Chinese cabbage and each raw material in the mixing material in S2 are: 80-100 parts of Chinese cabbage, 15-20 parts of radish, 1-2 parts of shallot, 1-2 parts of garlic, 1-2 parts of onion, 0.3-0.5 parts of fresh ginger, 1-2 parts of white pear and apple respectively, 3-5 parts of shrimp paste, 5-7 parts of fish sauce, 10-20 parts of glutinous rice paste, 1-2 parts of chili powder, 2-4 parts of sucrose, 1-3 parts of monosodium glutamate, and 0.001-0.1 parts of probiotic fermentation agent; the probiotic fermentation agent is a single bacterial agent of Enterococcus mesenteroides NCU018046 or the above-mentioned composite bacterial agent.

[0033] Exemplarily, the dressing can be prepared in the following manner: cut radish into shreds, cut shallot into segments, crush garlic, onion, fresh ginger, white pear, apple, shrimp paste, and fish sauce with a wall breaking machine, add glutinous rice flour into water and boil it into a paste, mix the shredded radish, shallot segments, the crushed raw materials and glutinous rice paste with chili powder, white sugar and probiotic fermentation agent to obtain the dressing.

[0034] Preferably, the number of viable bacteria in the probiotic fermentation agent is (1-9)×10 10 CFU / g.

[0035] Further preferably, the probiotic fermentation agent is the composite bacterial agent.

[0036] Further preferably, the viable counts of the Leuconostoc mesenteroides NCU018046 and the Lactobacillus plantarum NCU0011098 in the composite bacterial agent are the same.

[0037] The beneficial effects of the present invention are:

[0038] (1) The present invention provides a strain of Leuconostoc mesenteroides NCU018046 that has strong acid and bile resistance and the ability to inhibit common pathogenic bacteria and meets safety standards. The strain can be used to prepare fermented products, such as kimchi;

[0039] (2) The single bacterial agent of Leuconostoc mesenteroides NCU018046 can effectively inhibit the reproduction of pathogenic bacteria and reduce the content of nitrite and biogenic amines during the fermentation of kimchi, ensuring food safety; it can reduce salinity, which is in line with the current low-salt diet structure, avoids the health risks caused by high salt, and reduces the environmental pressure and wastewater treatment costs caused by high-salt wastewater discharge; it increases the content of organic acids and amino acids, giving the kimchi a sweet and sour taste and a fresh and fragrant flavor, while retaining the nutrients in the kimchi to the maximum extent;

[0040] (3) The fermented kimchi obtained by fermenting with the single bacterial agent of Leuconostoc mesenteroides NCU018046 has a high content of γ-aminobutyric acid, which has the health care potential of calming the nerves, promoting sleep, and relieving anxiety; the total phenol and total flavonoid content is high, which can effectively scavenge DPPH free radicals and ABTS free radicals, and has strong antioxidant capacity; it can inhibit the activity of xanthine oxidase and has good uric acid lowering potential; it has good ACE inhibition ability and has good potential antihypertensive activity; it can inhibit the proliferation of colon cancer cells and has the potential to prevent colon cancer;

[0041] (4) The composite bacterial agent provided by the present invention, which is composed of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098, has a synergistic effect in the production of fermented kimchi. In the process of fermenting kimchi, the fermented sour taste can be more significantly improved, nitrite and salinity can be reduced, the reproduction of pathogenic bacteria can be inhibited, the organic acid content can be increased, and the biogenic amine content can be reduced. Therefore, the kimchi obtained by fermenting with the composite bacterial agent has a stronger fermentation flavor and taste, higher safety, and is more conducive to intestinal health. At the same time, the fermented kimchi obtained by fermenting with the composite bacterial agent has higher gamma-aminobutyric acid, total phenol, and total flavonoid content, and has a stronger ability to scavenge free radicals, inhibit xanthine oxidase, and inhibit ACE, and the ability to inhibit colon cancer cells is also more prominent, so it has a more excellent health care effect in calming the nerves and helping sleep, relieving anxiety, anti-oxidation, lowering uric acid, anti-hypertension, and anti-colon cancer.

[0042] (5) The production process of kimchi produced by using a single bacterial agent of Enterobacterium mesenteroides NCU018046 or a composite bacterial agent of Enterobacterium mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 is simple, the production conditions are easy to control, the fermentation cycle is short, and it is easy to realize large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a bacterial morphology diagram of Leuconostoc mesenteroides NCU018046 after Gram staining in Example 1 of the present invention. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0045] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0046] Some of the experimental or determination methods used in the present invention are as follows (the remaining methods not explicitly described are conventional determination or detection methods in the art);

[0047] (1) The detection method of γ-aminobutyric acid is as follows:

[0048] Agilent 1260 high performance liquid chromatograph was used for the detection of γ-aminobutyric acid. The sample was centrifuged for 3 minutes at a speed of 10000r / min, 40μL of the supernatant was taken, and 120μL of the derivatization reagent and 120μL of boric acid buffer (0.4mol / L) were added, then shaken and mixed and reacted at room temperature in the dark for 5min. Next, the mixture was filtered through a 0.22μm filter membrane and loaded. Chromatographic column: Agilent C18 column (4.6mm×250mm, 5μm); the mobile phase was acetonitrile and 50mmol / L sodium acetate; the volume ratio was 35:65, isocratic elution, the injection volume was 20μL, the flow rate was 0.5mL / min, and the detection wavelength was 334nm. γ-aminobutyric acid standards were used for external standard method quantification.

[0049] Preparation of derivatization reagent: Weigh 0.1 g of o-phthalaldehyde, dissolve it in 1 mL of acetonitrile, then add 130 μL of β-mercaptoethanol, and make up to 100 mL with water.

[0050] (2) The detection method of biogenic amines is as follows:

[0051] According to the liquid chromatography method in GB 5009.208-2016 “National Food Safety Standard Determination of Biogenic Amines in Foods”, high performance liquid chromatography was used to detect the content of histamine, tyramine, cadaverine and putrescine in the samples.

[0052] (3) The detection method of volatile flavor substances is as follows:

[0053] The flavor composition of the samples was determined by headspace solid phase microextraction combined with gas chromatography-mass spectrometry using an Agilent triple quadrupole gas chromatography-mass spectrometer tandem time-of-flight mass spectrometer. The specific conditions were:

[0054] Chromatographic column: HP-5MS quartz capillary column (30m×0.25mm, 0.25μm).

[0055] Temperature program: initial temperature 50℃, maintained for 3 min, then increased to 100℃ at 2℃ / min and maintained for 2 min, then increased to 210℃ at 4℃ / min and maintained for 5 min; carrier gas flow rate 1.0 mL / min, pressure 7.6522 psi; split ratio 1:50; EI ionization source, ionization energy 70 eV, temperature 200℃; injection port temperature 250℃; transfer line 230℃; mass spectrometry scanning range m / z 35-450, 2 scans / s.

[0056] (4) The free amino acid detection method is as follows:

[0057] According to GB 5009.124-2016 "Determination of Amino Acids in Foods", the types and contents of amino acids in samples were determined using an amino acid analyzer.

[0058] (5) The method for detecting organic acids is as follows:

[0059] The sample was centrifuged at 10000 rpm for 10 min at 4°C, and 1 mL of the supernatant was filtered through a 0.22 μm water filter for determination. An Agilent 1260 high performance liquid chromatograph was used for the detection of organic acids. The chromatographic column was BIO-RAD HPX-87H ion exchange chromatography column C18 (5μm, 300mm×7.8mm); mobile phase: 6mmol / L dilute sulfuric acid; detector: UV detector, wavelength: 210nm; column temperature: 35℃; injection volume: 20μL; flow rate: 0.5mL / min; organic acid standards were used for external standard quantification.

[0060] (6) The antibacterial activity detection method is as follows:

[0061] The kimchi samples were homogenized and centrifuged (4°C, 10000 g, 10 min), and the antibacterial activity of the kimchi samples against Escherichia coli and Staphylococcus aureus was determined by the punch method. 0.1 mL of each indicator bacterial suspension (10 8 CFU / mL) was evenly spread on the LB plate, and then a hole puncher (6 mm in diameter) was used to punch three holes on each plate. 200 μL of spicy cabbage sample solution was added to each well, incubated at 37°C for 12 hours, and the diameter of the inhibition zone was measured using a vernier caliper. The indicator bacteria were Escherichia coli CMCC44350 and Staphylococcus aureus CMCC26003.

[0062] (7) The antioxidant capacity detection method is as follows:

[0063] The DPPH free radical scavenging ability test method is as follows:

[0064] Take 2.0g of the kimchi sample after homogenization and add 30mL of 80% ethanol. After ultrasonic extraction for 20min, centrifuge and take the supernatant and make it up to 50mL with 80% ethanol for standby use (kimchi sample solution). 1mL of kimchi sample is added to 1mL of ethanol DPPH free radical solution (0.1mM), incubate in dark environment at room temperature for 30min after mixing. After 8000g centrifugation for 10min, use the mixed solution of 1mL 80% ethanol and 1mL DPPH solution as control, and the mixed solution of 1mL ethanol and 1mL kimchi sample solution as blank, and measure the absorbance of the resulting solution at 517nm. The scavenging ability is expressed by the following formula:

[0065]

[0066] Among them A s , A b and A c are the absorbance of sample, blank and control solution at 517 nm, respectively.

[0067] The ABTS free radical scavenging ability test method is as follows:

[0068] The kimchi sample was mixed with ultrapure water in a ratio of 1:1 and homogenized. After ultrasonic extraction for 30 min, the supernatant was centrifuged and used for later use. The ABTS stock solution was mixed with 2.45 mmol / L K2S2O8 and reacted in the dark at room temperature for 16 h to obtain ABTS· + The resulting solution was then diluted with PBS and the absorbance at 734 nm was adjusted to 0.70 ± 0.01. + The solution is prepared and used immediately. Add the extracted sample (10 μL) or Trolox standard solution to 200 μL of fresh ABTS. + The solution was incubated in the dark at room temperature for 6 min and the absorbance of the mixture was measured. Trolox was used as a reference standard and the results were expressed as μM Trolox equivalent antioxidant capacity.

[0069] The total flavonoids content was determined as follows:

[0070] The total flavonoid content in the kimchi sample was determined by colorimetry. The specific method is as follows: After homogenizing the kimchi sample, take 2.0g and add 30mL of 80% ethanol. After ultrasonic extraction for 20min, centrifuge to take the supernatant and dilute it to 50mL with 80% ethanol for later use. Take 0.5mL of the extract or catechin standard solution, add 2.8mL of deionized water and 0.2mL of 5% (w / v) sodium nitrite, react for 5min, add 0.5mL of 10% (w / v) AlC13·6H2O solution and incubate at room temperature for 6min, then add 1mL of 1M NaOH solution to the reaction system. Determine the absorbance relative to the blank solution at 510nm, calculate the total flavonoid content according to the catechin standard curve, and the result is mg CE / 100g.

[0071] The total phenol content was determined as follows:

[0072] The total phenol content in the kimchi sample was determined by the Folin phenol colorimetric method. The specific method is as follows: After homogenizing the kimchi sample, take 2.0g and add 30mL of 80% ethanol. After ultrasonic extraction for 20min, centrifuge to take the supernatant and dilute to 50mL with 80% ethanol for later use. Take 0.5mL of the sample and add 2.5mL of 10% Folin phenol reagent, mix and react for 5min, then add 2.0mL of 7.5% (w / v) Na2CO3 solution, mix and incubate at room temperature in the dark for 1h, measure the absorbance at 765nm, calculate the total phenol content of the kimchi sample according to the gallic acid standard curve, and the result is expressed as mg GAE / g.

[0073] (8) The method for detecting angiotensin converting enzyme (ACE) inhibition ability is as follows:

[0074] After the kimchi sample is homogenized, 2.0 g is weighed and added to 30 mL of 70% ethanol. After ultrasonic extraction for 20 minutes, the supernatant is centrifuged, filtered, concentrated by rotary evaporation, and then vacuum-freezed and reconstituted with 2 mL of PBS solution for use. Detection method: Take 10 μL of the fermented kimchi sample extract and 40 μL of 5 mM HHL solution, mix them, preheat them in a 37°C water bath for 5 minutes, add 30 μL of 0.02 U / mL ACE, incubate at 37°C for 30 minutes, and shake once every 5 minutes; add 150 μL of 1M HCL solution to terminate the reaction. The content of the product HA (hippuric acid) is detected by high performance liquid chromatography. Liquid phase conditions: injection volume is 20 μL, chromatographic column Kromasil 100-5-C18 (4.6×250 mm), mobile phase is 0.05% formic acid aqueous solution: acetonitrile = 80%: 20%, column oven temperature is 30°C, detector is UV detector, detection wavelength is 228 nm. The ACE inhibition rate was calculated as follows:

[0075]

[0076] In the formula, Aa : The peak area of ​​HA in the presence of both ACE and fermented kimchi extract in the above detection system, A b : The peak area of ​​HA in the absence of ACE fermented kimchi extract in the above detection system, A c : The peak area of ​​HA when neither ACE nor fermented kimchi extract is present in the above detection system.

[0077] (9) The xanthine oxidase inhibition rate detection method is as follows:

[0078] After homogenizing the kimchi sample, weigh 2.0g and add 30mL of 70% ethanol. After ultrasonic extraction for 20min, centrifuge to obtain the supernatant, filter, concentrate by rotary evaporation, and then freeze-dry in vacuum. Dissolve in 2mL PBS solution and set aside. The xanthine oxidase inhibition rate detection method is as follows: the reaction system is 5mL, and different fermented cowpea sample extracts 0.2mL, 2.3mL PBS solution (0.2mmol / L, pH 7.5), and 2.0mL xanthine solution (1.2mmol / L) are added to the sample group respectively. After 20min of constant temperature water bath at 25℃, 50U / L xanthine oxidase solution is added, and the reaction is carried out for 10min. The absorbance at 292nm is measured every 1min. In the control group, PBS buffer is used instead of the fermented kimchi sample extract. The xanthine oxidase inhibition rate of the sample is calculated according to the following formula, and the IC is calculated based on the inhibition rate. 50 value.

[0079]

[0080] Among them, K control is the enzyme reaction rate when no fermented kimchi sample extract was added (control group), K sample is the enzyme reaction rate when the kimchi sample extract is added.

[0081] (10) IC of HT-29 colon cancer cells 50 The detection method is as follows:

[0082] Weigh 5.0 g of fermented kimchi juice after homogenization, add 80% ethanol, extract by ultrasonic for 30 minutes, centrifuge to obtain the supernatant, and concentrate by rotary evaporation for later use. Redissolve with RPMI complete medium and prepare different concentrations of test solutions. Use CCK-8 method to detect the inhibition rate of the sample on the proliferation of colon cancer cells, and calculate the IC value of fermented kimchi on HT-29 according to the inhibition rate.

[0083] CCK-8 method: Prepare a cell suspension of a certain concentration (1-5.0×10 5Cells / mL), add 96-well culture plates, 100 μL per well, and add 10% of the test solution at different concentrations after culturing for 24 hours; set 6 parallel wells in each group, and continue to culture for 24 hours in a cell culture incubator at 37°C and 5% CO2, then put 10 μL of CCK-8 solution in each well, continue to incubate for about 1 hour, use a multifunctional enzyme label analyzer to detect the absorbance (OD value) at 450nm, and calculate the inhibition rate of cell proliferation. The wells without the test solution were used as the control group (A c ), and the wells without cell suspension were used as blank control group (A b ).

[0084]

[0085] Among them A c is the absorbance of the control group, A t is the absorbance value of different treatment groups after 24 hours, A b is the absorbance value of the blank group.

[0086] The components of the culture medium used in the following examples are as follows:

[0087] MRS solid culture medium: peptone 10.0 g, beef extract powder 5.0 g, yeast extract powder 4.0 g, glucose 20.0 g, potassium hydrogen phosphate heptahydrate 2.0 g, sodium acetate trihydrate 5.0 g, ammonium citrate 2.0 g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate tetrahydrate 0.05 g, Tween 80 1.0 mL, agar powder 15.0 g, distilled water 1000 mL.

[0088] MRS liquid culture medium: peptone 10.0 g, beef extract powder 5.0 g, yeast extract powder 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate heptahydrate 2.0 g, sodium acetate trihydrate 5.0 g, triammonium citrate 2.0 g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate tetrahydrate 0.05 g, Tween 80 1.0 mL, distilled water 1000 mL.

[0089] The Leuconostoc mesenteroides CICC22182 used in the following examples was purchased from the China Industrial Microbiological Culture Collection Center, and Lactobacillus plantarum ATCC 8014 was a commercially available product.

[0090] Unless otherwise specified, other materials, reagents, culture media, etc. used in the following examples can be obtained from commercial sources.

[0091] The scheme of the present invention is described below through specific embodiments.

[0092] Example 1

[0093] This example provides the screening, isolation, identification and performance evaluation of Leuconostoc mesenteroides NCU018046 strain.

[0094] 1. Screening and separation process of strains

[0095] The commercially available fresh Chinese cabbage was washed with water, immersed in salt water, and pickled at room temperature for 24 hours. After the pickling, it was washed with clean water and drained. The commercially available kimchi sauce was evenly applied on the cabbage leaves and fermented at 4-8°C for 30 days. During this process, the kimchi liquid samples were taken out for gradient dilution (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ) and then coated on MRS solid medium supplemented with 1.5% (w / v) calcium carbonate. After inverted culture at 37°C for 48 hours, strains with melted calcium circles and different colony morphology and size were selected for secondary streaking. After repeated streaking 2-3 times, the purified strains were stored at -80°C with 20% (v / v) glycerol for later use. After that, the acid and bile resistance, antibiotic sensitivity, hemolysis and pathogenic bacteria inhibition were measured, and finally a strain with higher performance was obtained.

[0096] 2. Strain identification

[0097] 2.1 Bacterial morphology

[0098] The bacterial morphology of the strain after Gram staining is as follows Figure 1 shown.

[0099] 2.2 16S rDNA sequence determination

[0100] On July 1, 2024, DNA extraction, PCR, and 16S rDNA sequencing were performed on the strain, and the 16S rRNA sequence is as follows (SEQ ID NO.1):

[0101]

[0102] The 16S rDNA sequence was compared with the NCBI database by BLAST analysis, and the identification results showed that the strain was Leuconostoc mesenteroides.

[0103] It was named as Leuconostoc mesenteroides NCU018046 and deposited in the General Microbiology Center of China Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with the deposit number CGMCC NO.31870 and the deposit date September 5, 2024.

[0104] 3. Performance evaluation of Leuconostoc mesenteroides NCU018046

[0105] 3.1 Acid and bile resistance of Leuconostoc mesenteroides NCU018046

[0106] The activated NCU018046 was collected, centrifuged (4°C, 8000×g, 5 min), washed twice with sterile PBS, and then resuspended in MRS liquid medium with pH 2.5, incubated at 37°C for 3 h, and the number of viable bacteria was determined at 0, 1, 2, and 3 h, with three replicates for each sample. At the same time, the acid resistance of Leuconostoc mesenteroides CICC22182 was determined under the same conditions.

[0107] The NCU018046 cells were collected and washed according to the above method, and then resuspended in MRS liquid medium with pH 8.0 and 0.3% ox bile salt, incubated at 37°C for 4 hours, and the number of viable bacteria in the culture was determined at 0, 2, and 4 hours, with 3 replicates for each sample. At the same time, the bile salt tolerance of Leuconostoc mesenteroides CICC22182 was determined under the same conditions. The survival rate of lactic acid bacteria was calculated as follows:

[0108]

[0109] Where X1 is the number of surviving bacteria after incubation, and X0 is the initial number of bacteria.

[0110] The results are shown in Tables 1 and 2.

[0111] Table 1 Acid resistance of Leuconostoc mesenteroides

[0112]

[0113] Table 2 Bile salt tolerance of Leuconostoc mesenteroides

[0114]

[0115] The results showed that the survival rate of Leuconostoc mesenteroides NCU018046 was still above 90% after being treated in a pH 2.5 environment for 3 hours, and the survival rate was as high as above 95% after being treated in an environment with a bile salt concentration of 0.3% for 4 hours. It can be seen that Leuconostoc mesenteroides NCU018046 has strong acid and bile salt resistance.

[0116] 3.2 Antibiotic sensitivity and hemolytic activity of Leuconostoc mesenteroides NCU018046

[0117] The antibiotic sensitivity of Leuconostoc mesenteroides NCU018046 was evaluated by KB (susceptibility paper agar diffusion method). The overnight culture of Leuconostoc mesenteroides NCU018046 fermentation broth was adjusted to a bacterial concentration of 10 8 CFU / mL, 100μL was drawn and coated on the MRS plate. The drug-sensitive paper of streptomycin (10mg / mL), ampicillin (10mg / mL), erythromycin (15mg / mL), tetracycline (30mg / mL), gentamicin (10mg / mL), kanamycin (30mg / mL), penicillin (10mg / mL), cephalothin (15mg / mL), ciprofloxacin (5mg / mL) and amoxicillin (30mg / mL) were gently placed on the MRS plate coated with the fermentation liquid of Leuconostoc mesenteroides NCU018046, cultured at 37℃ for 24h, and the diameter of the inhibition zone was measured with a vernier caliper to determine the sensitivity of Leuconostoc mesenteroides NCU018046 to the above antibiotics. The results are shown in Table 3.

[0118] Table 3 Sensitivity and hemolytic activity of Leuconostoc mesenteroides NCU018046 to common antibiotics

[0119]

[0120] Note: R means resistant, S means sensitive. "γ" means non-hemolytic.

[0121] As shown in Table 3, Leuconostoc mesenteroides NCU018046 was sensitive to erythromycin, tetracycline, ampicillin, penicillin G and ceftriaxone, and was resistant to gentamicin, streptomycin, ciprofloxacin and kanamycin, and had no hemolytic activity, thus meeting the basic GRAS standard.

[0122] 3.3 Inhibitory effect of Leuconostoc mesenteroides NCU018046 on common pathogens

[0123] The antibacterial activity of the fermentation supernatant of Leuconostoc mesenteroides NCU018046 against Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus was determined by the punching method and co-culture method. Leuconostoc mesenteroides NCU018046 (and Leuconostoc mesenteroides CICC22182) were subcultured twice (inoculated into fresh MRS medium at a 2% inoculum and cultured at 37°C for 24 hours), centrifuged (4°C, 7000×g, 10min), and the supernatant was taken and filtered through a 0.22μm filter membrane for later use. The various indicator bacterial suspensions prepared in advance were inoculated into the sterilized LB solid medium (10 7 -10 8 CFU / mL), shake well and pour into a semi-solid sterile culture dish. After the culture medium is cooled and solidified, use a hole puncher (6mm in diameter) to punch holes on the LB plate, with three holes on each plate. Add 200μL of the fermentation supernatant of the prepared Enterobacter mesenteroides NCU018046 and Enterobacter mesenteroides CICC22182 to each well, incubate at 37°C for 12-16h, and use a vernier caliper to measure the diameter of the inhibition zone. The indicator bacteria are Escherichia coli CMCC44350, Staphylococcus aureus CMCC26003, and Salmonella typhimurium ATCC13311. At the same time, the same method was used to determine the inhibitory ability of Enterobacter mesenteroides CICC22182 against pathogenic bacteria.

[0124]

[0125] Among them A c A is the absorbance value of the control group after 24 hours; c0 is the absorbance value of the control group at 0 h, A t is the absorbance value of the treatment group after 24 hours, A t0 It is the absorbance value of the treatment group at 0 h.

[0126] The results are shown in Table 4.

[0127] Table 4 Inhibitory ability of lactic acid bacteria against common pathogenic bacteria

[0128]

[0129] The results of pathogenic bacteria showed that the fermentation supernatant of Enterococcus mesenteroides NCU018046 had a strong inhibitory ability against three common foodborne pathogens: Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium.

[0130] Example 2

[0131] This embodiment provides a single bacterial agent of Leuconostoc mesenteroides NCU018046.

[0132] The inoculation of Leuconostoc mesenteroides NCU018046 into MRS liquid medium was activated three times continuously, and then inoculated into MRS liquid medium at a 2% v / v inoculation amount and cultured at 37°C for 24 hours. The cells were collected by centrifugation (4°C, 6000×g, 10 min) and mixed evenly with sterile skim milk of 10% of the cell mass, pre-frozen at -80°C for 2 hours, and then transferred to a vacuum freeze dryer and frozen for 24 hours to obtain a single bacterial starter of Leuconostoc mesenteroides NCU018046. The bacterial activity of the single bacterial starter was adjusted to 1×10 10 CFU / g.

[0133] Example 3

[0134] This embodiment provides a composite bacterial agent, and the preparation method thereof is as follows:

[0135] Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 were inoculated into MRS liquid culture medium for three consecutive activations, respectively, and inoculated into MRS liquid culture medium at a 2% (v / v) inoculation amount, and cultured at 37°C for 24 hours. The cells were collected by centrifugation (4°C, 6000×g, 10 min), and then mixed evenly with sterile skim milk (10% of the cell mass), pre-frozen at -80°C for 2 hours, and then transferred to a vacuum freeze dryer and frozen for 24 hours to obtain single-bacterial starter cultures of Leuconostoc mesenteroides and Lactobacillus plantarum. The bacterial activity of the single-bacterial starter culture was adjusted to (1-9)×10 10 CFU / g, and then the two single-bacteria fermentation agents were mixed evenly in a 1:1 ratio to obtain a composite bacterial agent. The active bacterial count of the mixed composite bacterial agent was 1×10 10 CFU / g.

[0136] Example 4

[0137] This embodiment provides a fermented spicy cabbage, and the preparation method thereof is as follows:

[0138] 90 parts of fresh Chinese cabbage without rotten leaves are selected, washed with clean water, cut in half and immersed in 15% salt water, pickled at room temperature for 24 hours (the concentration of the salt water is reduced to 8%), washed with clean water after pickling until the salinity of the Chinese cabbage is 1.2%, and drained; 18 parts of radish are cut into shreds, 1.5 parts of shallot are cut into sections, glutinous rice flour is added into water and boiled into a paste, 1.5 parts of garlic, 1.5 parts of onion, 0.4 part of fresh ginger, 1.5 parts of white pear, 1.5 parts of apple, 4 parts of shrimp paste and 6 parts of fish sauce are crushed with a wall breaking machine, shredded radish, shallot sections and crushed raw materials are mixed, and then 1.5 parts of chili powder, 3 parts of white sugar, 2 parts of monosodium glutamate, 15 parts of glutinous rice paste and 0.01 parts of probiotic fermentation agent are added and mixed evenly to make a dressing, which is evenly spread between leaves of the Chinese cabbage and is placed on a plate for 4 hours. -8 ° C for 30 days. After the fermentation is completed, the kimchi is vacuum packed and stored in a refrigerator at 4 ° C to obtain the finished product. The probiotic fermentation agent is the single bacterial agent of Leuconostoc mesenteroides NCU018046 (live bacteria count 1×10 10 CFU / g).

[0139] Example 5

[0140] This embodiment provides a fermented kimchi, and its preparation method is the same as that of Example 4, except that the probiotic fermentation agent is the composite bacterial agent of Example 3, and the dosage is 0.001 parts.

[0141] Example 6

[0142] This embodiment provides a fermented spicy cabbage, and the preparation method thereof is as follows:

[0143] 80 parts of fresh Chinese cabbage without rotten leaves are selected, washed with clean water, cut in half, immersed in salt water with a concentration of 15%, pickled at room temperature for 24 hours (the concentration of the salt water is reduced to 8%), washed with clean water until the salinity of the Chinese cabbage is 1.2%, and drained; 15 parts of radish are cut into shreds, 1 part of shallot is cut into sections, glutinous rice flour is added into water and boiled into a paste, 1 part of garlic, 1 part of onion, 0.3 part of fresh ginger, 1 part of white pear, 1 part of apple, 3 parts of shrimp paste and 5 parts of fish sauce are crushed with a wall breaking machine, shredded radish, shallot sections and crushed raw materials are mixed, and then 1 part of chili powder, 2 parts of white sugar, 1 part of monosodium glutamate, 10 parts of glutinous rice paste and 0.1 part of probiotic fermentation agent are added and mixed evenly to make a mixing material, which is evenly spread between leaves of the Chinese cabbage and fermented at 4-8 DEG C for 20 days. After the fermentation is completed, the spicy cabbage is vacuum-packed and refrigerated at 4 DEG C to obtain a finished product. The probiotic fermentation agent is the composite bacterial agent of Example 3.

[0144] Example 7

[0145] This embodiment provides a fermented spicy cabbage, and the preparation method thereof is as follows:

[0146] 100 parts of fresh Chinese cabbage without rotten leaves are selected, washed with clean water, cut in half and immersed in salt water with a concentration of 15%, pickled at room temperature for 24 hours (the concentration of the salt water is reduced to 8%), washed with clean water until the salinity of the Chinese cabbage is 1.2% after pickling, and drained; 20 parts of radish are cut into shreds, 2 parts of shallot are cut into sections, glutinous rice flour is added into water and boiled into a paste, 2 parts of garlic, 2 parts of onion, 0.5 parts of fresh ginger, 2 parts of white pear, 2 parts of apple, 5 parts of shrimp paste and 7 parts of fish sauce are crushed with a wall breaking machine, shredded radish, shallot sections and crushed raw materials are mixed, and then 2 parts of chili powder, 4 parts of white sugar, 3 parts of monosodium glutamate, 20 parts of glutinous rice paste and 0.001 parts of probiotic fermentation agent are added and mixed evenly to make a mixing material, which is evenly spread between leaves of the Chinese cabbage, fermented at 4-8 DEG C for 40 days, and after the fermentation is completed, the spicy cabbage is vacuum-packed and refrigerated at 4 DEG C to obtain a finished product. The probiotic fermentation agent is the composite bacterial agent of Example 3.

[0147] Comparative Example 1

[0148] This comparative example provides a fermented spicy cabbage, and the preparation method thereof is as follows:

[0149] The preparation method is the same as that of Example 4, except that the mixture is evenly spread between the leaves of the Chinese cabbage and then directly vacuum-packed and refrigerated and stored at 4° C. to obtain the finished product.

[0150] Comparative Example 2

[0151] This comparative example provides a fermented spicy cabbage, and the preparation method thereof is as follows:

[0152] The preparation method is the same as that in Example 4, except that the probiotic fermentation agent is a single bacterial agent of Lactobacillus plantarum NCU0011098 (the preparation method is the same as that in Example 2, and the number of viable bacteria is 1×10 10 CFU / g).

[0153] Comparative Example 3

[0154] This comparative example provides a fermented spicy cabbage, and the preparation method thereof is as follows:

[0155] The preparation method is the same as that in Example 4, except that the probiotic fermentation agent is a composite bacterial agent made of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum ATCC 8014 (the preparation method is the same as that in Example 3, the ratio of the number of live bacteria of Leuconostoc mesenteroides to that of Lactobacillus plantarum ATCC 8014 is 1:1, and the number of live bacteria of the composite bacterial agent is 1×10 10 CFU / g).

[0156] Example 8

[0157] This example measured various indicators of the fermented kimchi of Examples 4 to 7, Comparative Examples 2 and 3, and the unfermented kimchi of Comparative Example 1. The specific results are as follows:

[0158] (1) GABA content

[0159] Table 5 γ-aminobutyric acid content

[0160]

[0161] Note: “-” means it was not detected in the sample.

[0162] As shown in Table 5, after fermentation, the γ-aminobutyric acid content in the spicy cabbage is increased. Compared with Example 4 and Comparative Examples 2 and 3, the γ-aminobutyric acid content of the fermented spicy cabbage prepared in Examples 5 to 7 is higher, indicating that the fermented spicy cabbage prepared by Leuconostoc mesenteroides NCU018046 has the ability to convert glutamate into γ-aminobutyric acid, and compared with the single bacterial fermentation of Leuconostoc mesenteroides NCU018046 or its mixed bacterial fermentation with other Lactobacillus plantarum, the mixed bacterial fermentation of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 has a stronger ability to convert glutamate into γ-aminobutyric acid. The fact that γ-aminobutyric acid was not detected in the unfermented Comparative Example 1 indicates that the conversion of glutamate to γ-aminobutyric acid needs to be achieved by fermentation of lactic acid bacteria.

[0163] (2) Main physical and chemical indicators and antibacterial activity

[0164] Table 6 Main physical and chemical indicators and antibacterial activity

[0165]

[0166]

[0167] As shown in Table 6, the pH of the composite probiotic fermented spicy cabbage prepared in Examples 4 to 7 is low, the total acid is high, and the product can be given a strong fermented sour taste. The nitrite content of Examples 4 to 7 and Comparative Examples 1 to 3 is lower than 1 mg / kg, which is far lower than the nitrite limit standard (20 mg / kg) for pickled vegetables in the national standard GB2762. In addition, the salinity of Examples 4 to 7 and Comparative Examples 1 to 3 is lower than 4%, which is in line with the current low-salinity diet structure and reduces the environmental pressure caused by the discharge of high-salt wastewater. Compared with Comparative Example 1, Examples 4 to 7 and Comparative Examples 2 and 3 all have an inhibitory effect on common foodborne pathogens Escherichia coli and Staphylococcus aureus, and compared with Example 4 and Comparative Examples 1 to 3, Examples 5 to 7 have a stronger inhibitory ability against Escherichia coli and Staphylococcus aureus. The above results indicate that Leuconostoc mesenteroides NCU018046 can enhance the fermentation sourness, reduce nitrite and salinity, and inhibit the reproduction of pathogenic bacteria in fermented kimchi. Moreover, compared with the single-bacterial fermentation of Leuconostoc mesenteroides NCU018046 or the mixed fermentation with other Lactobacillus plantarum, the mixed fermentation of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 can achieve better results and has better food safety.

[0168] (3) Organic acid content

[0169] Table 7 Organic acid content

[0170]

[0171] Note: “-” means it was not detected in the sample.

[0172] As shown in Table 7, compared with Comparative Example 1, after fermentation, in addition to oxalic acid, the contents of citric acid, malic acid, lactic acid, acetic acid and butyric acid in the kimchi were all increased. Compared with Comparative Examples 1 to 3, the contents of various organic acids in the fermented kimchi prepared in Examples 4 to 7 were significantly increased, and the organic acid content in Examples 5 to 7 was significantly higher than that in Example 4. Among them, lactic acid, malic acid and citric acid can provide a soft and pleasant sour taste for the kimchi product, while acetic acid and butyric acid belong to short-chain fatty acids, which can effectively regulate intestinal health. The above results show that the fermented kimchi prepared with Leuconostoc mesenteroides NCU018046 has a better taste and can regulate intestinal health, and compared with the fermentation of Leuconostoc mesenteroides NCU018046 alone or its mixed fermentation with other plant lactobacillus, the fermented kimchi obtained by mixed fermentation of Leuconostoc mesenteroides NCU018046 and plant lactobacillus NCU0011098 has a better taste and the effect of regulating intestinal health.

[0173] (4) Biogenic amines

[0174] Table 8 Biogenic amine content

[0175]

[0176] As shown in Table 8, compared with Comparative Examples 2 and 3, the fermented kimchi prepared in Examples 4 to 7 had lower contents of main biogenic amines (except putrescine in Example 4), among which the contents of biogenic amines in Examples 5 to 7 were even lower, and the contents of histamine and tyramine were lower than the EU standard (histamine content was lower than 100 mg / kg, and tyramine content was lower than 100-800 mg / kg). The results showed that the fermented kimchi prepared by using Leuconostoc mesenteroides NCU018046 was a green and safe fermented food, and the fermented kimchi prepared by using the composite bacterial agent of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 was safer than the fermentation of Leuconostoc mesenteroides NCU018046 alone or mixed fermentation with other Lactobacillus plantarum.

[0177] (5) Antioxidant activity

[0178] Table 9 Antioxidant capacity

[0179]

[0180]

[0181] As shown in Table 9, the DPPH radical scavenging ability of the fermented kimchi prepared in Examples 4 to 7 is significantly higher than that in Comparative Examples 1 to 3, and the ABTS radical scavenging ability, total phenol content and total flavonoid content are all higher than those in Comparative Example 1, wherein the various antioxidant indexes of Examples 5 to 7 are significantly higher than those of Examples 4 and Comparative Examples 1 to 3, indicating that the fermented kimchi prepared using Leuconostoc mesenteroides NCU018046 has excellent antioxidant activity, and compared with the single bacterial fermentation of Leuconostoc mesenteroides NCU018046 or its mixed bacterial fermentation with other Lactobacillus plantarum, the fermented kimchi prepared using the composite bacterial agent of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 has better antioxidant activity.

[0182] (6) Xanthine oxidase inhibition ability

[0183] Table 10 Xanthine oxidase inhibition ability

[0184]

[0185] As shown in Table 10, the xanthine oxidase inhibition IC50 values ​​of the fermented kimchi prepared by Examples 4 to 7 and Comparative Examples 2 and 3 are all lower than that of Comparative Example 1, and Examples 5 to 7 are much lower than that of Example 4 and Comparative Examples 1 to 3, which indicates that the fermented kimchi prepared by using Enterobacterium mesenteroides NCU018046 has the ability to inhibit xanthine oxidase, and compared with the single-bacterium fermentation of Enterobacterium mesenteroides NCU018046 or its mixed fermentation with other Lactobacillus plantarum, the fermented kimchi prepared by using the composite bacterial agent of Enterobacterium mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 has a stronger ability to inhibit xanthine oxidase and has a stronger potential for lowering uric acid.

[0186] (7) Ability to lower blood pressure

[0187] Table 11 ACE inhibition ability

[0188]

[0189] As shown in Table 11, the ACE inhibition IC50 values ​​of Examples 4 to 7 are lower than those of Comparative Examples 1 and 2, and the ACE inhibition IC50 values ​​of Examples 5 to 7 are much lower than those of Example 4 and Comparative Examples 1 to 3, indicating that the fermented kimchi prepared using Enterobacterium mesenteroides NCU018046 has the potential to lower blood pressure, and compared with the single-bacterium fermentation of Enterobacterium mesenteroides NCU018046 or the mixed fermentation with other Lactobacillus plantarum, the fermented kimchi prepared using the composite bacterial agent of Enterobacterium mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 has a better potential to lower blood pressure.

[0190] (8) IC of HT-29 colon cancer cells 50

[0191] Table 12 Antitumor effect

[0192]

[0193] As shown in Table 12, compared with Comparative Example 1, Examples 4 to 7 have lower IC 50 The IC values ​​of the fermented kimchi prepared in Examples 5 to 7 for colon cancer cells HT-29 were 0.177 and 0.227, respectively. 50The lower the value, the more effectively it can inhibit the proliferation of cancer cells, indicating that the fermented kimchi prepared by using Enterobacterium NCU018046 has good anti-colon cancer activity, and compared with the single bacterial fermentation of Enterobacterium NCU018046 or its mixed fermentation with other Lactobacillus plantarum, the fermented kimchi prepared by using the composite bacterial agent of Enterobacterium NCU018046 and Lactobacillus plantarum NCU0011098 has better anti-colon cancer activity.

[0194] Example 9

[0195] In this example, the volatile flavor substances and amino acid contents of the fermented kimchi of Examples 4 and 5, Comparative Examples 2 and 3, and the unfermented kimchi of Comparative Example 1 were measured. The specific results are as follows:

[0196] (1) Effect on the content of volatile flavor substances

[0197] Table 13 Content of main volatile flavor substances

[0198]

[0199] Note: “-” means it was not detected in the sample.

[0200] As shown in Table 13, compared with Example 4 and Comparative Example 2 in which single bacteria fermentation was performed using Leuconostoc mesenteroides NCU018046 and Bacillus lactis NCU0011098, respectively, after mixed bacteria fermentation of Leuconostoc mesenteroides NCU018046 and Bacillus lactis NCU0011098 in Example 5, the relative content of sulfide and ester compounds increased, and the types of volatile aromas were more abundant, such as new aroma substances such as heptanal, diallyl disulfide, and cyclooctanol. Sulfide is a characteristic flavor substance of kimchi, which may come from chili powder, garlic, etc. in the ingredients, giving kimchi sulfur, onion and garlic flavors and other pungent smells.

[0201] As shown in Table 13, compared with Comparative Example 3 in which mixed fermentation was performed with Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum ATCC 8014, after mixed fermentation of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 in Example 5, the relative contents of esters, aldehydes, and sulfides in the product were significantly increased, such as compounds such as β-cyclocitral, ethyl hexanoate, ethyl acetate, and diallyl disulfide, indicating that mixed fermentation of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 provided by the present invention can significantly improve the production of key volatile flavor substances. Aldehydes are volatile substances with rose and honey flavors, which can give spicy cabbage a richer sweet smell; esters are volatile substances with fruity and sweet tastes, which can bring richer fruit aroma to the product.

[0202] In summary, the fermented kimchi prepared by the composite bacterial agent of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum ATCC 8014 in Example 5 has richer and more pleasant aroma components, which is more helpful to improve consumer acceptance.

[0203] (2) Effect on the content of flavor amino acids

[0204] Table 14 Content of main flavor amino acids

[0205]

[0206]

[0207] Note: “-” means it was not detected in the sample.

[0208] As shown in Table 14, the total amino acid, umami amino acid and sweet amino acid contents in the fermented kimchi of Examples 4, 5 and Comparative Examples 2 and 3 are significantly higher than those in Comparative Example 1, and the bitter amino acid content is lower than that in Comparative Example 1. Moreover, compared with Example 4 and Comparative Examples 1 to 3, the total amino acid, umami amino acid and sweet amino acid contents of the fermented kimchi of Example 5 are higher, while the bitter amino acid content is lower. It can be seen that the single bacterial agent of Leuconostoc mesenteroides NCU018046 and the composite bacterial agent of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 can give kimchi more umami and sweetness, and reduce the production of bitterness.

[0209] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Leuconostoc mesenteroides NCU018046, characterized in that: Its classification name is Leuconostoc mesenteroides. It was deposited in the General Microbiology Center of China Culture Collection on September 5, 2024, and its deposit number is CGMCC NO.31870. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. The use of Leuconostoc mesenteroides NCU018046 according to claim 1, characterized in that: Includes any of the following: Application in the production of fermented foods; Application in the preparation of a single bacterial agent for producing fermented food; Application in the preparation of composite bacterial agents for producing fermented foods.

3. The use according to claim 2, characterized in that: The fermented food is fermented kimchi.

4. A composite bacterial agent, characterized in that: It comprises the Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum described in claim 1.

5. The composite bacterial agent according to claim 4, characterized in that: The plant lactobacillus is Lactiplantibacillus plantarum NCU0011098, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on October 26, 2023, and its deposit number is CGMCCNO.28774; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

6. Use of the composite bacterial agent according to claim 4 or 5 in the preparation of fermented food.

7. The use according to claim 6, characterized in that: The fermented food is fermented kimchi.

8. A fermented kimchi, characterized in that: Obtained by fermentation of the single bacterial agent of Leuconostoc mesenteroides NCU018046 described in claim 1 or the composite bacterial agent described in claim 4 or 5.

9. A method for preparing fermented kimchi, characterized in that: The specific steps include: S1. Wash the fresh Chinese cabbage and immerse it in a 13% to 17% salt solution, pickle it at room temperature until the salt concentration drops to 7% to 9%, then wash it with clean water and drain it; S2. Evenly spread the mixture containing the probiotic fermentation agent between the leaves of Chinese cabbage, and let it ferment at 4-8°C; the probiotic fermentation agent is the single bacterial agent of Leuconostoc mesenteroides NCU018046 described in claim 1 or the composite bacterial agent described in claim 4 or 5.

10. The preparation method according to claim 9, characterized in that: The salt concentration of the saline solution in S1 is 15%; and / or After the pickling in S1 is completed, the cabbage is washed with clean water and drained until the salinity is 1% to 1.5%; and / or The raw materials of the mixing material also include radish, shallot, garlic, onion, fresh ginger, white pear, apple, shrimp paste, fish sauce, glutinous rice paste, chili powder, sucrose and monosodium glutamate; and / or The number of live bacteria in the probiotic fermentation agent is (1 to 9)×10 10 CFU / g; and / or The probiotic fermentation agent is the composite bacterial agent; and / or The live counts of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum in the composite bacterial agent are the same.

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