Fermented hot cabbage with antioxidant, sleep improvement and blood pressure health maintenance function and preparation method thereof

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to Leuconostoc mesenteroides and fermented spicy cabbage with effects of soothing nerves, helping sleep and relieving anxiety and a preparation method thereof.The Leuconostoc mesenteroides NCU018046 provided by the present application was preserved in the China General Microbiological Culture Collection Center on September 5, 2024, and the preservation number thereof is CGMCC NO.31870.The strain has strong acid and bile salt resistance, strong inhibition capacity for common foodborne pathogenic bacteria, and meets safety standards.The compound microbial agent is prepared from the strain and Lactobacillus plantarum NCU0011098.The fermented spicy cabbage prepared by fermentation of the single microbial agent of the Leuconostoc mesenteroides NCU018046 or the compound microbial agent has good flavor, low harmful substance content, and multiple biological activities such as soothing nerves, helping sleep, relieving anxiety, resisting hypertension, resisting oxidation, reducing uric acid, resisting colon cancer and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a fermented hot cabbage which is helpful for antioxidation, sleep improvement and blood pressure health level maintenance and a preparation method thereof. BACKGROUND

[0002] Fermented food is a kind of food in which sugars, proteins and other organic substances in food are converted into acids, alcohols, esters and other substances by the action of microorganisms. The fermentation process makes it have unique flavor and nutritional value, and produces active substances that are beneficial to health. Fermented foods are diverse, such as yogurt, pickles, soy sauce, fermented soybean, fermented bean curd, and hot cabbage.

[0003] Hot cabbage is a kind of characteristic fermented vegetable in Northeast China, and is one of the most famous traditional fermented vegetables in China, and is a common food on people's table. Hot cabbage is rich in dietary fiber, vitamins, and calcium, copper, phosphorus, iron and other rich nutrients. The high content of cellulose in hot cabbage can prevent constipation and other intestinal diseases. The traditional method of making hot cabbage in Northeast China is to ferment Chinese cabbage at low temperature with chili powder, apples, pears, ginger, garlic and other ingredients. Industrial production of hot cabbage usually uses natural fermentation, and the initial microorganisms are usually derived from microorganisms remaining on the vegetables. During the fermentation process, the strain is often not fixed, which not only may cause changes in the fermentation flavor of different batches of hot cabbage products, but also may cause large batch-to-batch differences in their health care functions. In addition, if the fermentation process is not properly controlled, harmful microorganisms may grow, the content of nitrite may be high, the fermentation period may be unstable, and the health care function may be poor. Therefore, it is of great significance to develop fermented probiotics with the functions of improving product flavor, reducing harmful substance content, and improving health care function. SUMMARY

[0004] In view of the above technical problems, the present application provides a Leuconostoc mesenteroides and a fermented hot cabbage which is helpful for antioxidation, sleep improvement and blood pressure health level maintenance and a preparation method thereof. The Leuconostoc mesenteroides has strong acid and bile salt tolerance, strong inhibition ability to common foodborne pathogenic bacteria, and meets safety standards, and has high application value. In addition, the Leuconostoc mesenteroides or the compound microbial agent containing the strain can be used to prepare fermented food, such as hot cabbage, so that the product has multiple biological activities such as helping to resist oxidation, helping to improve sleep, and helping to maintain blood pressure health level.

[0005] To achieve the above-mentioned application purposes, the technical scheme adopted by the present application is as follows:

[0006] The present application provides a Leuconostoc mesenteroides NCU018046 in the first aspect, and the classification name of the Leuconostoc mesenteroides is Leuconostoc mesenteroides (Cohn) Esders 1923. Leuconostoc mesenteroidesLactobacillus intestinalis NCU018046 was deposited at China General Microbiological Culture Collection Center (CGMCC) on September 5, 2024, and the deposit number is CGMCC NO. 31870. The deposit address is No. 1, Yihuangyuan, Beichenxi Road, Beijing, China, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences.

[0007] The Lactobacillus intestinalis NCU018046 strain provided by the present application is isolated and screened from pickled spicy cabbage. The strain has strong acid and bile salt tolerance, is sensitive to erythromycin, tetracycline, ampicillin, penicillin G and cephalothin, is resistant to gentamicin, streptomycin, ciprofloxacin and kanamycin, and has no hemolytic activity, which meets the basic standards generally considered safe (GRAS). In addition, the strain has strong inhibitory ability to common foodborne pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and Salmonella murium. Therefore, the strain has high application value.

[0008] In addition, through experimental research on fermented spicy cabbage, it is found that Lactobacillus intestinalis NCU018046 can reduce the content of biogenic amines, nitrite and salinity in the product, inhibit Escherichia coli and Staphylococcus aureus, increase the content of amino acids and short-chain fatty acids beneficial to intestinal regulation, and convert glutamic acid into gamma-aminobutyric acid during the fermentation production of spicy cabbage. In addition, the fermented spicy cabbage prepared by Lactobacillus intestinalis NCU018046 has the effects of helping to resist oxidation and helping to maintain a healthy level of blood pressure. Therefore, the strain has wide application prospects in the field of fermented food and can be used to produce fermented food such as spicy cabbage.

[0009] The second aspect of the present application provides the application of the above-mentioned Lactobacillus intestinalis NCU018046, including any one of the following:

[0010] Application in the production of fermented food;

[0011] Application in the preparation of single-strain agent for producing fermented food;

[0012] Application in the preparation of composite microbial agent for producing fermented food.

[0013] The skilled in the art can use any available fermentation and preparation method to obtain a single-strain fermenting agent or a composite microbial agent containing live bacteria, as long as the number of live bacteria meets the requirements. The present application provides a preparation method of a single-strain agent as an example, but it is not used to limit the preparation method of the composite microbial agent:

[0014] The Leuconostoc mesenteroides NCU018046 is inoculated into MRS liquid medium for continuous activation three times, inoculated into MRS liquid medium at a 2% v / v inoculation amount, and cultured at 37 DEG C for 24 hours. After centrifugation (4 DEG C, 6000 xg, 10 min), the bacterial cells are mixed with 10% sterile skim milk of the bacterial cell mass, pre-frozen at -80 DEG C for 2 hours, and then transferred to a vacuum freeze dryer for freezing for 24 hours to obtain the single bacterial agent of the Leuconostoc mesenteroides NCU018046. Before use, the viable cell count of the single bacterial agent can be adjusted to (1-9) x 10 10 CFU / g by using glucose dry powder.

[0015] The single bacterial agents of other probiotics are prepared according to the method, and after the viable cell count is adjusted by using glucose dry powder, the single bacterial agents are mixed uniformly according to the proportion to obtain the compound bacterial agent.

[0016] Preferably, the fermented food is fermented spicy cabbage.

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

[0018] Preferably, the Lactobacillus plantarum is Lactobacillus plantarum (Lactobacillus plantarum) Lactiplantibacillus plantarum ) NCU0011098, which is preserved in the China General Microbiological Culture Collection Center on October 26, 2023, and the preservation number is CGMCC NO. 28774. The address of the preservation is No. 3, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology. The Lactobacillus plantarum NCU0011098 has been disclosed in the patent with the application number 202410534531.2 and the name "Lactobacillus plantarum with the effect of preventing colon cancer and its application".

[0019] The present application is found through the experimental research of fermented spicy cabbage that, compared with the fermentation using Leuconostoc mesenteroides NCU018046 alone or the fermentation using the compound bacterial agent of the strain and other Lactobacillus plantarum, the compound bacterial agent made of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 can significantly improve the effects of reducing the content of product biogenic amines, nitrite and salinity, inhibiting Escherichia coli and Staphylococcus aureus, improving the content of product amino acids and short-chain fatty acids beneficial to the regulation of intestinal tract, converting glutamic acid into gamma-aminobutyric acid, improving the antioxidant activity of the product, inhibiting xanthine oxidase, and inhibiting ACE, and can significantly increase the types of volatile aroma and the content of flavor substances, indicating that the two have synergistic effects in the above aspects.

[0020] The skilled in the art can obtain the complex microbial agent by any available fermentation and preparation method, and the number of viable bacteria is sufficient. The present application provides a preparation method as an example, but is not used to limit the preparation method of the complex microbial agent:

[0021] Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 were inoculated into MRS liquid medium for continuous activation for three times, and then inoculated into MRS liquid medium at a 2% v / v inoculation amount, and cultured at 37 ℃ for 24 h. After centrifugation (4 ℃, 6000 xg, 10 min), the bacterial bodies were collected and mixed with 10% sterile skim milk of the bacterial body weight, pre-frozen at-80 ℃ for 2 h, and then transferred to a vacuum freeze dryer for freezing for 24 h, to obtain Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 single microbial agents. The viable bacterial number of each single microbial agent was adjusted to (1~9) x 10 10 CFU / g by using glucose dry powder, and then the two single microbial starters were mixed uniformly according to the proportion to obtain the complex microbial agent.

[0022] The fourth aspect of the present application provides the use of the above-mentioned complex microbial agent in the preparation of fermented food.

[0023] Preferably, the fermented food is fermented spicy cabbage.

[0024] The fifth aspect of the present application provides a fermented spicy cabbage, which is fermented by the above-mentioned single microbial agent of Leuconostoc mesenteroides NCU018046 or the above-mentioned complex microbial agent.

[0025] The sixth aspect of the present application provides a preparation method of fermented spicy cabbage, which specifically comprises the following steps:

[0026] S1, immerse fresh cabbage in a 13%~17% salt aqueous solution, and cure at room temperature until the salt concentration is reduced to 7%~9%, and then wash and drain with clean water;

[0027] S2, evenly smear the cabbage leaves with the probiotic starter, and ferment at 4~8 ℃; the probiotic starter is the above-mentioned single microbial agent of Leuconostoc mesenteroides NCU018046 or the above-mentioned complex microbial agent.

[0028] According to the conventional operation of the spicy cabbage production process, the fresh cabbage in S1 should be free of rotten leaves and pests, and cut in half before washing with water.

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

[0030] Preferably, after curing in S1, wash and drain with clean water until the salinity of the cabbage is 1%~1.5%.

[0031] Preferably, the raw materials of the mixed material further include radish, green onion, 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 fraction of the Chinese cabbage in S2 to each raw material in the mixed material is: Chinese cabbage 80-100 parts, radish 15-20 parts, green onion 1-2 parts, garlic 1-2 parts, onion 1-2 parts, fresh ginger 0.3-0.5 parts, white pear and apple each 1-2 parts, shrimp paste 3-5 parts, fish sauce 5-7 parts, glutinous rice paste 10-20 parts, chili powder 1-2 parts, sucrose 2-4 parts, monosodium glutamate 1-3 parts, and probiotic leavening agent 0.001-0.1 parts; the probiotic leavening agent is Leuconostoc mesenteroides NCU018046 single bacterial agent or the above-mentioned compound bacterial agent.

[0033] Illustratively, the mixed material can be prepared in the following manner: radish is cut into shreds, green onion is cut into sections, garlic, onion, fresh ginger, white pear, apple, shrimp paste, fish sauce are crushed with a cell disruptor, glutinous rice powder is added to water to boil into a paste, the radish shreds, green onion sections, crushed raw materials and glutinous rice paste are mixed with chili powder, white sugar and probiotic leavening agent to obtain the mixed material.

[0034] Preferably, the viable bacterial count of the probiotic leavening agent is (1-9) × 10 10 CFU / g.

[0035] Further preferably, the probiotic leavening agent is the compound bacterial agent.

[0036] Further preferably, the viable bacterial count of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum (L. Lactiplantibacillus plantarum ) NCU0011098 in the compound bacterial agent is the same.

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

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

[0039] (2) Leuconostoc mesenteroides NCU018046 single bacterial agent can effectively inhibit the reproduction of pathogenic bacteria during the fermentation of spicy cabbage, reduce the content of nitrite and biogenic amines, and ensure the safety of consumption; can reduce the salinity, meet the current low-salt dietary structure, avoid the health risks caused by high salt, and reduce the environmental pressure and wastewater treatment cost caused by high-salt wastewater discharge; can increase the content of organic acids and amino acids, endow spicy cabbage with sweet and sour taste and fresh and fragrant flavor, and maximize the retention of nutrients in spicy cabbage itself;

[0040] (3) The fermented cabbage fermented by the Leuconostoc mesenteroides NCU018046 single bacterial agent has a higher content of gamma-aminobutyric acid, has a health care potential to help improve sleep, has a higher content of total phenol and total flavonoids, can effectively scavenge DPPH free radicals and ABTS free radicals, has a stronger antioxidant capacity, has a better ACE inhibiting capacity, and helps maintain a healthy blood pressure level;

[0041] (4) The complex bacterial agent of the Leuconostoc mesenteroides NCU018046 and the Lactiplantibacillus plantarum NCU0011098 has a synergistic effect in the production of the fermented cabbage, can more significantly improve fermentation sourness, reduce nitrite and salinity, inhibit the reproduction of pathogenic bacteria, increase the content of organic acid, and reduce the content of biological amine during the fermentation of the fermented cabbage, so that the fermented cabbage fermented by the complex bacterial agent has a stronger fermented flavor and taste, is safer, and is more beneficial to intestinal health. At the same time, the fermented cabbage fermented by the complex bacterial agent has a higher content of gamma-aminobutyric acid, total phenol and total flavonoids, has a stronger ability to scavenge free radicals and inhibit ACE, and therefore has a more excellent health care effect in terms of antioxidant, sleep improvement and maintenance of a healthy blood pressure level.

[0042] (5) The production process of the fermented cabbage produced by the Leuconostoc mesenteroides NCU018046 single bacterial agent or the complex bacterial agent of the Leuconostoc mesenteroides NCU018046 and the Lactiplantibacillus plantarum NCU0011098 is simple, the production conditions are easy to control, the fermentation period is short, and large-scale industrial production can be easily realized. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is a bacterial body morphology chart of the Leuconostoc mesenteroides NCU018046 after gram staining in the embodiment 1 of the present application. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

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

[0046] The part of the experiment or determination method used in the present application is as follows (the rest of the methods not explicitly described are conventional determination or detection methods in the art);

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

[0048] Agilent 1260 high performance liquid chromatograph is used for detection of gamma-aminobutyric acid. Centrifuge the sample for 3 minutes at 10000 r / min, take 40 μL of supernatant, and add 120 μL of derivatization reagent and 120 μL of boric acid buffer (0.4 mol / L), then shake mix and react at room temperature for 5 min. Next, filter the mixture through a 0.22 μm filter and perform the sample loading operation. Chromatographic column: Agilent C18 column (4.6 mm x 250 mm, 5 μm); mobile phase is acetonitrile and 50 mmol / L sodium acetate; volume ratio is 35:65, isocratic elution, sample amount is 20 μL, flow rate is 0.5 mL / min, detection wavelength is 334 nm. Use gamma-aminobutyric acid standard for external standard method quantitative.

[0049] Preparation of derivatization reagent: weigh 0.1 g of o-phthaldehyde, dissolve with 1 mL of acetonitrile, then add 130 μL of β-mercaptoethanol, and dilute with water to 100 mL.

[0050] (2) The detection method of biological amine is as follows:

[0051] According to GB 5009.208-2016 "National food safety standard Determination of biological amines in food", the content of histamine, tyramine, cadaverine and putrescine in the sample is detected by high performance liquid chromatography.

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

[0053] Headspace solid phase microextraction combined with gas chromatography mass spectrometry is used to determine the flavor substance composition of the sample using Agilent triple quadrupole mass spectrometer. The specific conditions are as follows:

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

[0055] Temperature program: initial temperature 50 ℃, hold for 3 min, then increase to 100 ℃ at 2 ℃ / min and hold for 2 min, then increase to 210 ℃ at 4 ℃ / min and hold 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 spectral scan 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 amino acid analyzer is used to determine the types and contents of amino acids in the sample.

[0058] (5) The organic acid detection method is as follows:

[0059] The sample is centrifuged at 10,000 rpm for 10 min at 4 ℃, and 1 mL of supernatant is passed through a 0.22 μm water filter membrane before being determined. The Agilent 1260 high performance liquid chromatograph is used for the detection of organic acids. The chromatographic column is BIO-RAD Aminex® HPX-87H ion exchange chromatographic column C18 (5 μm, 300 mm x 7.8 mm); mobile phase: 6 mmol / L dilute sulfuric acid; detector: ultraviolet detector, wavelength: 210 nm; column temperature: 35 ℃; injection volume: 20 μL; flow rate: 0.5 mL / min; external standard method is used for quantification using organic acid standard.

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

[0061] The hot cabbage sample is homogenized and centrifuged (4 ℃, 10,000 g, 10 min), and the antibacterial activity of the hot cabbage sample on E. coli and S. aureus is determined by the punch method. 0.1 mL of various indicator bacteria suspension (10 8 CFU / mL) is evenly coated on the LB plate, and then the puncher (diameter 6 mm) is used to punch the LB plate, with three holes in each plate. 200 μL of hot cabbage sample solution is added to each hole, and incubated at 37 ℃ for 12 h. The vernier caliper is used to measure the diameter of the inhibition zone. The indicator bacteria are E. coli CMCC44350 and S. aureus CMCC26003, respectively.

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

[0063] The DPPH free radical scavenging capacity detection method is as follows:

[0064] After homogenization of the sample, 2.0 g of the sample was added to 30 mL of 80% ethanol and extracted by ultrasonication for 20 min. The supernatant was obtained by centrifugation and diluted with 80% ethanol to 50 mL. The solution was used as the sample solution (sample solution of Brassica juncea). 1 mL of the sample solution was added to 1 mL of DPPH solution (0.1 mM) and mixed well. The mixture was incubated at room temperature in the dark for 30 min. After centrifugation at 8000 g for 10 min, the absorbance of the mixture was measured at 517 nm. The scavenging ability was calculated according to the following formula:

[0065]

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

[0067] The ABTS radical scavenging ability was determined as follows:

[0068] The sample was homogenized with ultrapure water at a ratio of 1:1 and extracted by ultrasonication for 30 min. The supernatant was obtained by centrifugation and used as the sample solution. The ABTS stock solution was mixed with 2.45 mmol / L K2S2O8 and reacted at room temperature in the dark for 16 h to prepare the ABTS· ＋ solution. The ABTS· ＋ solution was prepared fresh before use. The sample solution (10 μL) or Trolox standard solution was added to 200 μL of fresh ABTS· ＋ solution and incubated at room temperature in the dark for 6 min. The absorbance of the mixture was measured. The results were expressed as μM Trolox equivalent antioxidant capacity, with Trolox as the reference standard.

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

[0070] The total flavonoid content in the sample of Lai Baicai was determined by colorimetry. The specific method is as follows: 2.0 g of the homogenized sample of Lai Baicai was added into 30 mL of 80% ethanol, and then ultrasonic extraction was performed for 20 min. The supernatant was obtained by centrifugation, and then 80% ethanol was added to make the volume 50 mL. 0.5 mL of the extract or the catechin standard solution was taken, 2.8 mL of deionized water and 0.2 mL of 5% (w / v) sodium nitrite were added, and reaction was performed for 5 min. Then, 10% (w / v) AlC13·6H2O solution 0.5 mL was added, and incubation was performed at room temperature for 6 min. Subsequently, 1 mL of 1M NaOH solution was added to the reaction system. The absorbance value relative to the blank solution was determined at 510 nm, and the total flavonoid content was calculated according to the catechin standard curve. The result was expressed as mg CE / 100 g.

[0071] The total phenol content was determined by the following method:

[0072] The total phenol content in the sample of Lai Baicai was determined by Folin phenol colorimetry. The specific method is as follows: 2.0 g of the homogenized sample of Lai Baicai was added into 30 mL of 80% ethanol, and then ultrasonic extraction was performed for 20 min. The supernatant was obtained by centrifugation, and then 80% ethanol was added to make the volume 50 mL. 0.5 mL of the sample was taken, 2.5 mL of 10% Folin phenol reagent was added, and mixing was performed. Reaction was performed for 5 min, and then 2.0 mL of 7.5% (w / v) Na2CO3 solution was added. After mixing, incubation was performed at room temperature for 1 h in the dark. The absorbance value was determined at 765 nm, and the total phenol content in the sample of Lai Baicai was calculated according to the gallic acid standard curve. The result was expressed as mg GAE / g.

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

[0074] The sample of Lai Baicai was homogenized, 2.0 g of which was added into 30 mL of 70% ethanol, and then ultrasonic extraction was performed for 20 min. The supernatant was obtained by centrifugation, filtration, rotary evaporation concentration and vacuum freeze-drying. 2 mL of PBS solution was added for reconstitution, and then the sample was used. The detection method is as follows: 10 μL of the fermented Lai Baicai sample extract and 40 μL of 5 mM HHL solution were mixed, preheated in a 37 ℃ water bath for 5 min, 30 μL of 0.02 U / mL ACE was added, and incubation was performed at 37 ℃ for 30 min, with shaking every 5 min. 150 μL of 1M HCL solution was added to terminate the reaction. The content of product HA (hippuric acid) was determined by high performance liquid chromatography. The liquid phase conditions are as follows: the sample amount is 20 μL, the chromatographic column is Kromasil 100-5-C18 (4.6×250 mm), the mobile phase is 0.05% formic acid aqueous solution: acetonitrile=80%:20%, the column oven temperature is 30 ℃, the detector is an ultraviolet detector, and the detection wavelength is 228 nm. The ACE inhibition rate is calculated according to the following formula:

[0075]

[0076] In the formula, A a : The peak area of HA in the above detection system with ACE and fermented hot cabbage extract b : The peak area of HA in the above detection system with ACE and fermented hot cabbage extract c : The peak area of HA in the above detection system with ACE and fermented hot cabbage extract

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

[0078] After homogenization of the hot cabbage sample, 2.0 g was weighed and added into 30 mL of 70% ethanol, and after ultrasonic extraction for 20 min, the supernatant was taken after centrifugation, filtered, concentrated by rotary evaporation, and vacuum freeze-dried, and 2 mL of PBS solution was used for re-dissolution and standby. The xanthine oxidase inhibition rate detection method is as follows: the reaction system is 5 mL, 0.2 mL of different fermented cowpea sample extract, 2.3 mL of PBS solution (0.2 mmol / L, pH 7.5), and 2.0 mL of xanthine solution (1.2 mmol / L) are added into the sample group, 25°C constant temperature water bath for 20 min, then 50 U / L xanthine oxidase solution is added, and the reaction is carried out for 10 min, and the absorbance value at 292 nm is measured every 1 min. In the control group, the fermented hot cabbage sample extract is replaced by PBS buffer. The xanthine oxidase inhibition rate of the sample is calculated according to the following formula, and the IC 50 value is calculated according to the inhibition rate.

[0079]

[0080] K control is the enzyme reaction rate without the addition of fermented hot cabbage sample extract (control group), and K sample is the enzyme reaction rate with the addition of hot cabbage sample extract.

[0081] The components of the culture medium used in the following examples are as follows:

[0082] MRS solid medium: proteose peptone 10.0 g, beef infusion powder 5.0 g, yeast extract powder 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 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, agar powder 15.0 g, and distilled water 1000 mL.

[0083] MRS liquid medium: proteose peptone 10.0 g, beef extract powder 5.0 g, yeast extract powder 4.0 g, glucose 20.0 g, potassium phosphate dibasic 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.

[0084] Leuconostoc mesenteroides CICC22182 used in the following examples was purchased from China General Microbiological Culture Collection Center, and Lactobacillus plantarum ATCC 8014 was a commercially available product.

[0085] Other materials, reagents, culture media and the like used in the following examples were commercially available unless otherwise specified.

[0086] The scheme of the present application is described below through specific examples.

[0087] Example 1

[0088] This example provides the screening, isolation, identification and performance investigation of Leuconostoc mesenteroides NCU018046 strain.

[0089] 1. Screening and isolation process of the strain

[0090] The commercially available fresh Chinese cabbage was washed with water, immersed in brine, and cured at room temperature for 24 h. After curing, the Chinese cabbage was washed with clean water and drained. Commercially available spicy Chinese cabbage sauce was evenly applied to the cabbage leaves, and the mixture was left to ferment at 4-8 ℃ for 30 days. During this process, samples of the pickled vegetable liquid were taken for gradient dilution (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ) and then plated on MRS solid medium with 1.5% (w / v) calcium carbonate. After 48 h of inverted culture at 37 ℃, strains with calcium solubilization rings and different colony morphologies were selected for secondary streaking. After repeated streaking for 2-3 times, the purified strains were preserved in 20% (v / v) glycerol at -80 ℃ for later use. Then, the strains were determined for their acid and bile salt tolerance, antibiotic sensitivity, hemolytic activity, and pathogenic bacteria inhibition ability, and finally a strain with high performance was obtained.

[0091] 2. Strain identification

[0092] 2.1 Morphology and characteristics of the bacterial cells

[0093] The bacterial cell morphology of the strain after Gram staining is shown in Figure 1 .

[0094] 2.2 16S rDNA sequence determination

[0095] DNA extraction, PCR, 16S rDNA sequencing were performed on the strain on July 1, 2024, and the 16S rRNA sequence is as follows (SEQ ID NO. 1):

[0096]

[0097] The 16S rDNA sequence was analyzed by BLAST comparison in the NCBI database, and the identification result showed that the strain was Leuconostoc mesenteroides (ATCC 8293T). Leuconostoc mesenteroides )。

[0098] It was named Leuconostoc mesenteroides NCU018046 and preserved in the China General Microbiological Culture Collection Center (CGMCC, located at No. 1, Yikhinxi Lu, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences), with the preservation number CGMCC NO. 31870 and the preservation date September 5, 2024.

[0099] 3. Performance of Leuconostoc mesenteroides NCU018046

[0100] 3.1 Acid and bile salt tolerance of Leuconostoc mesenteroides NCU018046

[0101] The activated NCU018046 was collected, centrifuged (4 ℃, 8000xg, 5 min), and washed twice with sterile PBS, and then resuspended in MRS liquid medium at pH 2.5, incubated at 37 ℃ for 3 h, and the viable cell count was determined at 0, 1, 2, 3 h, with three replicates for each sample. At the same time, the acid tolerance of Leuconostoc mesenteroides CICC22182 was determined under the same conditions.

[0102] According to the above method, NCU018046 bacterial bodies were collected and washed, and then resuspended in MRS liquid medium containing 0.3% bovine bile salt at pH 8.0, incubated at 37 ℃ for 4 h, and the viable cell count of the culture was determined at 0, 2, 4 h, with three 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:

[0103]

[0104] Where X1 is the number of surviving bacteria after incubation, and X0 is the initial number of bacteria.

[0105] The results are shown in Tables 1 and 2.

[0106] Table 1 Acid tolerance of Leuconostoc mesenteroides

[0107]

[0108] Table 2 Bile salt tolerance of Leuconostoc mesenteroides

[0109]

[0110] The results show that the survival rate of Leuconostoc mesenteroides NCU018046 is still above 90% after 3 h of treatment in an environment with pH 2.5, and the survival rate is as high as above 95% after 4 h of treatment in an environment with a bile salt concentration of 0.3%. It can be seen that Leuconostoc mesenteroides NCU018046 has strong acid and bile salt resistance.

[0111] 3.2 Antibiotic sensitivity and hemolytic activity of Leuconostoc mesenteroides NCU018046

[0112] A K-B (disk diffusion method) antibiotic sensitivity test was used to evaluate the antibiotic sensitivity of Leuconostoc mesenteroides NCU018046. The overnight culture of Leuconostoc mesenteroides NCU018046 was adjusted to a cell concentration of 10 8 CFU / mL, and 100 µL was taken and spread on an MRS plate. Streptomycin (10 mg / mL), ampicillin (10 mg / mL), erythromycin (15 mg / mL), tetracycline (30 mg / mL), gentamicin (10 mg / mL), kanamycin (30 mg / mL), penicillin (10 mg / mL), cephalothin (15 mg / mL), ciprofloxacin (5 mg / mL), and amoxicillin (30 mg / mL) were placed on the MRS plate with the spread Leuconostoc mesenteroides NCU018046 fermentation broth, and the plate was incubated at 37 ℃ for 24 h. 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.

[0113] Table 3 Antibiotic sensitivity and hemolytic activity of Leuconostoc mesenteroides NCU018046

[0114]

[0115] Note: R represents resistance, S represents sensitivity, and “γ” represents no hemolysis.

[0116] As shown in Table 3, Leuconostoc mesenteroides NCU018046 is sensitive to erythromycin, tetracycline, ampicillin, penicillin G, and cephalothin, and is resistant to gentamicin, streptomycin, ciprofloxacin, and kanamycin, and has no hemolytic activity, which meets the basic standards of GRAS.

[0117] 3.3 Inhibition ability of Leuconostoc mesenteroides NCU018046 on common pathogenic bacteria

[0118] The inhibition activities of the fermentation supernatant of Leuconostoc mesenteroides NCU018046 on Escherichia coli, Salmonella typhimurium and Staphylococcus aureus were determined by the punch method and the co-culture method. Leuconostoc mesenteroides NCU018046 (and Leuconostoc mesenteroides CICC22182) was continuously subcultured twice (inoculated into fresh MRS medium at a 2% inoculation amount, and incubated at 37°C for 24 h), centrifuged (at 4°C, 7000 x g for 10 min) to obtain the supernatant, and filtered through a 0.22 µm filter membrane for use. The bacterial suspensions of the various indicator bacteria prepared in advance were inoculated into sterilized LB solid medium (10 7 -10 8 CFU / mL), shaken, and then poured into sterile semi-solid culture dishes. After the medium cooled and solidified, the puncher (diameter 6 mm) was used to punch holes on the LB plates, three holes per plate. 200 μL of the fermentation supernatant of Leuconostoc mesenteroides NCU018046 and Leuconostoc mesenteroides CICC22182 prepared above was added to each hole, and incubated at 37°C for 12-16 h. The diameters of the inhibition zones were measured using a vernier caliper. The indicator bacteria were Escherichia coli CMCC44350, Staphylococcus aureus CMCC26003, and Salmonella typhimurium ATCC13311. The inhibition ability of Leuconostoc mesenteroides CICC22182 on pathogenic bacteria was also determined by the same method.

[0119]

[0120] wherein A c is the absorbance value after 24 h of the control group; A c0 is the absorbance value at 0 h of the control group, A t is the absorbance value after 24 h of the treatment group, A t0 is the absorbance value at 0 h of the treatment group.

[0121] The results are shown in Table 4.

[0122] Table 4 Inhibition ability of lactic acid bacteria on common pathogenic bacteria

[0123]

[0124] The results of the pathogenic bacteria showed that the fermentation supernatant of Leuconostoc mesenteroides NCU018046 had strong inhibition ability on Escherichia coli, Staphylococcus aureus and Salmonella typhimurium, which are three common foodborne pathogenic bacteria.

[0125] Example 2

[0126] This example provides a Leuconostoc mesenteroides NCU018046 single bacterium agent.

[0127] Leuconostoc mesenteroides NCU018046 was inoculated into MRS liquid medium for continuous activation three times, inoculated into MRS liquid medium at a 2% v / v inoculation amount, and cultured at 37 ℃ for 24 h. After centrifugation (4 ℃, 6000 x g, 10 min) to collect the bacterial cells, the bacterial cells were mixed with 10% sterile skim milk of the bacterial cell mass, pre-frozen at -80 ℃ for 2 h, and then transferred to a vacuum freeze dryer for freezing for 24 h to obtain the single bacterial starter of Leuconostoc mesenteroides NCU018046. The bacterial activity of the single bacterial starter was adjusted to 1 x 10 10 CFU / g by using glucose dry powder.

[0128] Example 3

[0129] The present example provides a composite microbial inoculant, and a preparation method thereof is as follows:

[0130] Leuconostoc mesenteroides NCU018046 and Lactiplantibacillus halli NCU0011098 were respectively inoculated into MRS liquid medium for continuous activation three times, inoculated into MRS liquid medium at a 2% (v / v) inoculation amount, and cultured at 37 ℃ for 24 h. After centrifugation (4 ℃, 6000 x g, 10 min) to collect the bacterial cells, the bacterial cells were respectively mixed with 10% sterile skim milk of the bacterial cell mass, pre-frozen at -80 ℃ for 2 h, and then transferred to a vacuum freeze dryer for freezing for 24 h to obtain the single bacterial starters of Leuconostoc mesenteroides and Lactiplantibacillus halli. The bacterial activity of the single bacterial starters was adjusted to (1-9) x 10 10 CFU / g by using glucose dry powder, and then the two single bacterial starters were mixed at a ratio of 1:1 of the viable bacterial number to obtain the composite microbial inoculant. The bacterial activity of the mixed composite microbial inoculant was 1 x 10 10 CFU / g.

[0131] Example 4

[0132] The present example provides a fermented spicy cabbage, and a preparation method thereof is as follows:

[0133] Select fresh cabbage without rotten leaves 90 parts, wash clean with clean water, cut in half, then immerse in brine with a concentration of 15%, cure at room temperature for 24 hours (the concentration of brine decreases to 8%), after curing, wash clean with clean water until the salinity of the cabbage is 1.2%, then drain; cut radish 18 parts into shreds, cut scallion 1.5 parts into sections, add glutinous rice powder into water to boil into paste, smash garlic 1.5 parts, onion 1.5 parts, fresh ginger 0.4 parts, white pear 1.5 parts, apple 1.5 parts, shrimp paste 4 parts, fish sauce 6 parts with a cell disruptor, mix the shredded radish, scallion sections and smashed raw materials, then add chili powder 1.5 parts, white granulated sugar 3 parts, monosodium glutamate 2 parts, glutinous rice paste 15 parts and probiotic starter 0.01 parts, mix evenly to make the seasoning, evenly spread between the leaves of the cabbage, ferment at 4-8 ℃ for 30 days, after fermentation, vacuum package the spicy cabbage and store at 4 ℃, then the finished product is obtained. The probiotic starter is the Leuconostoc mesenteroides NCUM018046 single bacterial agent (viable bacterial count 1×10 10 CFU / g) of Example 2.

[0134] Example 5

[0135] This example provides a fermented spicy cabbage, the preparation method of which is the same as that of Example 4, except that the probiotic starter is the compound bacterial agent of Example 3, and the amount used is 0.001 parts.

[0136] Example 6

[0137] This example provides a fermented spicy cabbage, the preparation method of which is:

[0138] Select fresh cabbage without rotten leaves 80 parts, wash clean with clean water, cut in half, then immerse in brine with a concentration of 15%, cure at room temperature for 24 hours (the concentration of brine decreases to 8%), after curing, wash clean with clean water until the salinity of the cabbage is 1.2%, then drain; cut radish 15 parts into shreds, cut scallion 1 part into sections, add glutinous rice powder into water to boil into paste, smash garlic 1 part, onion 1 part, fresh ginger 0.3 parts, white pear 1 part, apple 1 part, shrimp paste 3 parts, fish sauce 5 parts with a cell disruptor, mix the shredded radish, scallion sections and smashed raw materials, then add chili powder 1 part, white granulated sugar 2 parts, monosodium glutamate 1 part, glutinous rice paste 10 parts and probiotic starter 0.1 parts, mix evenly to make the seasoning, evenly spread between the leaves of the cabbage, ferment at 4-8 ℃ for 20 days, after fermentation, vacuum package the spicy cabbage and store at 4 ℃, then the finished product is obtained. The probiotic starter is the compound bacterial agent of Example 3.

[0139] Example 7

[0140] This example provides a fermented spicy cabbage, the preparation method of which is:

[0141] Select no rotten leaf fresh cabbage 100 parts, with clean water, cut in half after immersion in the concentration of 15% salt water, room temperature curing 24 h (salt concentration decreased to 8%), after curing with clean water to wash the salt of the cabbage is 1.2%, drain; radish 20 parts into silk, green onions 2 parts into section, glutinous rice powder into water and boil into paste, garlic 2 parts, onion 2 parts, fresh ginger 0.5 parts, white pear 2 parts, apple 2 parts, shrimp paste 5 parts, fish sauce 7 parts with broken wall machine broken, radish silk, green onion section, broken into mixed with chili powder 2 parts, white sugar 4 parts, monosodium glutamate 3 parts, glutinous rice paste 20 parts and probiotic starter 0.001 parts mixed evenly into the dressing, evenly spread between the leaves of cabbage, at 4 -8 ℃ fermentation 40 days, after fermentation, vacuum packaging and 4 ℃ cold storage of spicy cabbage, can obtain finished product. The probiotic starter is the composite microbial inoculum of example 3.

[0142] Comparative example 1

[0143] The present comparative example provides a fermented spicy cabbage, the preparation method of which is as follows:

[0144] The preparation method is the same as example 4, the difference is that after the dressing is evenly spread between the leaves of cabbage, it is directly vacuum packaged and stored at 4 ℃, and the finished product can be obtained.

[0145] Comparative example 2

[0146] The present comparative example provides a fermented spicy cabbage, the preparation method of which is as follows:

[0147] The preparation method is the same as example 4, the difference is that the probiotic starter is Lactobacillus plantarum NCU0011098 single microbial inoculum (preparation method is the same as example 2, viable bacterial count is 1×10 10 CFU / g).

[0148] Comparative example 3

[0149] The present comparative example provides a fermented spicy cabbage, the preparation method of which is as follows:

[0150] The preparation method is the same as example 4, the difference is that the probiotic starter is a composite microbial inoculum made of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum ATCC 8014 (preparation method is the same as example 3, the ratio of viable bacterial count of Leuconostoc mesenteroides to Lactobacillus plantarum ATCC 8014 is 1:1, and the viable bacterial count of the composite microbial inoculum is 1×10 10 CFU / g).

[0151] Example 8

[0152] The fermented white cabbage of Examples 4-7, Comparative Example 2 and 3 and the unfermented white cabbage of Comparative Example 1 were determined for various indexes. The specific results are as follows:

[0153] (1) γ-aminobutyric acid content

[0154] Table 5 γ-aminobutyric acid content

[0155]

[0156] Note: "-" indicates that the sample was not detected.

[0157] As shown in Table 5, after fermentation, the γ-aminobutyric acid content of the white cabbage was increased. Compared with Examples 4 and Comparative Examples 2 and 3, the fermented white cabbage prepared by Examples 5-7 had a higher γ-aminobutyric acid content, indicating that Leuconostoc mesenteroides NCU018046 had the ability to convert glutamic acid to γ-aminobutyric acid. Compared with single strain fermentation of Leuconostoc mesenteroides NCU018046 or mixed strain fermentation of Leuconostoc mesenteroides NCU018046 and other Lactobacillus plantarum, mixed strain fermentation of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 had a stronger ability to convert glutamic acid to γ-aminobutyric acid. The unfermented Comparative Example 1 did not detect γ-aminobutyric acid, indicating that the conversion of glutamic acid to γ-aminobutyric acid required fermentation by lactic acid bacteria.

[0158] (2) Main physicochemical indexes and antibacterial activity

[0159] Table 6 Main physicochemical indexes and antibacterial activity

[0160]

[0161] As shown in Table 6, the pH of the composite probiotic fermented spicy pickled cabbage prepared in Examples 4-7 is lower, and the total acid is higher, which can bring rich fermented sour taste to the product. The nitrite content of Examples 4-7 and Comparative Examples 1-3 is lower than 1 mg / kg, which is far lower than the national standard GB2762 for nitrite content of pickled vegetables (20 mg / kg). In addition, the salinity of Examples 4-7 and Comparative Examples 1-3 is lower than 4%, which is consistent with the current low-salt dietary structure, and reduces the environmental pressure caused by high-salt wastewater discharge. Compared with Comparative Example 1, Examples 4-7 and Comparative Examples 2 and 3 have inhibitory effect on common foodborne pathogenic bacteria Escherichia coli and Staphylococcus aureus, and the inhibitory ability of Examples 5-7 on Escherichia coli and Staphylococcus aureus is stronger than that of Examples 4 and Comparative Examples 1-3. The above results show that Leuconostoc mesenteroides NCU018046 can produce fermented sour taste, reduce nitrite and salinity, and inhibit the reproduction of pathogenic bacteria in fermented spicy pickled cabbage. Compared with single fermentation of Leuconostoc mesenteroides NCU018046 or mixed fermentation of Leuconostoc mesenteroides NCU018046 and other Lactobacillus plantarum, mixed fermentation of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 can achieve better results and has better food safety.

[0162] (3) Organic acid content

[0163] Table 7 Organic acid content

[0164]

[0165] Note: "-" indicates that the sample is not detected.

[0166] As shown in Table 7, compared with Comparative Example 1, after fermentation, the content of citric acid, malic acid, lactic acid, acetic acid and butyric acid in spicy pickled cabbage is increased, except for oxalic acid. Compared with Comparative Examples 1-3, the content of various organic acids in fermented spicy pickled cabbage prepared in Examples 4-7 is significantly increased, and the content of organic acids in Examples 5-7 is significantly higher than that in Example 4. Among them, lactic acid, malic acid and citric acid can provide soft and pleasant sour taste for spicy pickled cabbage products, and acetic acid and butyric acid belong to short-chain fatty acids, which can effectively regulate intestinal health. The above results show that the fermented spicy pickled cabbage prepared by Leuconostoc mesenteroides NCU018046 has good taste and can regulate intestinal health. Compared with single fermentation of Leuconostoc mesenteroides NCU018046 or mixed fermentation of Leuconostoc mesenteroides NCU018046 and other Lactobacillus plantarum, mixed fermentation of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 can achieve better taste and intestinal health regulation effect.

[0167] (4) Biogenic amine

[0168] Table 8 Biogenic amine content

[0169]

[0170] As shown in Table 8, the fermented hot cabbage prepared by Examples 4-7 has lower content of main biogenic amines (except putrescine in Example 4) than Comparative Examples 2 and 3, and the fermented hot cabbage prepared by Examples 5-7 has lower content of biogenic amines and lower content of histamine and tyramine than the European Union standard (content of histamine is lower than 100 mg / kg and content of tyramine is lower than 100-800 mg / kg). The results show that the fermented hot cabbage prepared by Leuconostoc mesenteroides NCU018046 is a green and safe fermented food, and the fermented hot cabbage prepared by the complex microbial agent of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 is safer than the fermented hot cabbage prepared by Leuconostoc mesenteroides NCU018046 or the mixed fermentation of Leuconostoc mesenteroides NCU018046 and other Lactobacillus plantarum.

[0171] (5) Antioxidant activity

[0172] Table 9 Antioxidant capacity

[0173]

[0174] As shown in Table 9, the DPPH free radical scavenging capacity of the fermented hot cabbage prepared by Examples 4-7 is obviously higher than Comparative Examples 1-3, and the ABTS free radical scavenging capacity, total phenol content and total flavonoid content are higher than Comparative Example 1. The antioxidant indexes of Examples 5-7 are obviously higher than Example 4 and Comparative Examples 1-3, which shows that the fermented hot cabbage prepared by Leuconostoc mesenteroides NCU018046 has excellent antioxidant activity, and the fermented hot cabbage prepared by the complex microbial agent of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 has better antioxidant activity than the fermented hot cabbage prepared by Leuconostoc mesenteroides NCU018046 or the mixed fermentation of Leuconostoc mesenteroides NCU018046 and other Lactobacillus plantarum.

[0175] (6) Xanthine oxidase inhibitory capacity

[0176] Table 10 Xanthine oxidase inhibitory capacity

[0177]

[0178] As shown in Table 10, the xanthine oxidase inhibiting IC50values of the fermented kimchi prepared in Examples 4 to 7 and Comparative Examples 2 and 3 were lower than that of Comparative Example 1, and those of Examples 5 to 7 were much lower than those of Example 4 and Comparative Examples 1 to 3, which indicates that the fermented kimchi prepared using Leuconostoc mesenteroides NCU018046 has the ability to inhibit xanthine oxidase, and the fermented kimchi prepared using the complex microbial agent of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 has a stronger ability to inhibit xanthine oxidase than the fermented kimchi prepared using Leuconostoc mesenteroides NCU018046 alone or the fermented kimchi prepared using Leuconostoc mesenteroides NCU018046 and another Lactobacillus plantarum.

[0179] (7) Blood pressure-lowering ability

[0180] Table 11 ACE inhibiting ability

[0181]

[0182] As shown in Table 11, the ACE inhibiting IC50values of Examples 4 to 7 were lower than those of Comparative Examples 1 and 2, and those of Examples 5 to 7 were much lower than those of Example 4 and Comparative Examples 1 to 3, which indicates that the fermented kimchi prepared using Leuconostoc mesenteroides NCU018046 has the potential to help maintain a healthy level of blood pressure, and the fermented kimchi prepared using the complex microbial agent of Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum NCU0011098 has a more excellent potential to help maintain a healthy level of blood pressure than the fermented kimchi prepared using Leuconostoc mesenteroides NCU018046 alone or the fermented kimchi prepared using Leuconostoc mesenteroides NCU018046 and another Lactobacillus plantarum.

[0183] Example 9

[0184] In this example, the contents of volatile flavor substances and amino acids of the fermented kimchi of Examples 4 and 5 and Comparative Examples 2 and 3 and the unfermented kimchi of Comparative Example 1 were measured. The specific results are as follows.

[0185] (1) Effect on the content of volatile flavor substances

[0186] Table 12 Contents of main volatile flavor substances

[0187]

[0188] Note: "-" indicates that the sample was not detected.

[0189] As shown in Table 12, compared with Example 4 and Comparative Example 2 in which single strain fermentation was carried out using Leuconostoc mesenteroides NCU018046 and Bifidobacterium lactis NCU0011098 respectively, after mixed strain fermentation of Leuconostoc mesenteroides NCU018046 and Bifidobacterium lactis NCU0011098 in Example 5, the relative content of sulfides and ester compounds increased, and the types of volatile aroma were more abundant, such as new aroma substances of heptanal, diallyl disulfide, cyclooctanol, etc. Sulfides are characteristic flavor substances of spicy cabbage, which may be derived from chili powder, garlic, etc. in the ingredients, giving spicy cabbage a pungent smell of sulfur, onion and garlic, etc.

[0190] As shown in Table 12, compared with Comparative Example 3 in which mixed strain fermentation was carried out using Leuconostoc mesenteroides NCU018046 and Bifidobacterium lactis ATCC 8014, after mixed strain fermentation of Leuconostoc mesenteroides NCU018046 and Bifidobacterium lactis NCU0011098 in Example 5, the relative content of esters, aldehydes and sulfides in the product increased significantly, such as β-cyclocitral, ethyl hexanoate, ethyl acetate, diallyl disulfide and other compounds, indicating that mixed strain fermentation using Leuconostoc mesenteroides NCU018046 and Bifidobacterium lactis NCU0011098 provided by the application 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 more sweet and sweet aroma; esters are volatile substances with fruit and sweet flavors, which can bring more fruity aroma to the product.

[0191] In summary, the fermented spicy cabbage prepared by using the complex microbial agent of Leuconostoc mesenteroides NCU018046 and Bifidobacterium lactis ATCC 8014 in Example 5 has more abundant and pleasant aroma components, which is more helpful to improve the acceptance of consumers.

[0192] (2) Effect on the content of taste amino acids

[0193] Table 13: Content of main taste amino acids

[0194]

[0195] Note: "-" indicates that the sample was not detected.

[0196] As shown in Table 13, the contents of total amino acids, umami amino acids and sweet amino acids in the fermented spicy white cabbage of Examples 4 and 5 and Comparative Examples 2 and 3 are obviously higher than that of Comparative Example 1, the content of bitter amino acids is lower than that of Comparative Example 1, and compared with Examples 4 and 5 and Comparative Examples 1-3, the fermented spicy white cabbage of Example 5 has higher contents of total amino acids, umami amino acids and sweet amino acids and lower content of bitter amino acids. It can be seen that Leuconostoc mesenteroides NC U 018046 single inoculant and the complex inoculant of Leuconostoc mesenteroides NC U 018046 and Lactobacillus plantarum NC U 0011098 can impart more umami and sweet taste to spicy white cabbage and weaken the generation of bitter taste.

[0197] The above description is merely preferred embodiments of the present application, but not to limit the present application. Any modification, equivalent replacement or improvement made in the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Leuconostoc mesenteroides NCU018046, characterized in that: Its taxonomic name is Leuconostoc mesenteroides ( Leuconostoc mesenteroides ), deposited in the General Microbiology Center of China Culture Collection of Microorganisms on September 5, 2024, with the deposit number CGMCC NO. 31870; the deposit address is No. 3, Yard 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.

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

4. A composite bacterial agent, characterized in that: Including the Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) NCU0011098, the plant lactobacillus NCU0011098 was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on October 26, 2023, and its deposit number is CGMCC NO. 28774; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

5. Use of the composite bacterial agent according to claim 4 in the preparation of fermented foods.

6. The use according to claim 5, characterized in that The fermented food is fermented kimchi.

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

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

9. The preparation method according to claim 8, characterized in that The salt concentration of the saline solution in S1 is 15%; and / or After the pickling in S1 is completed, the Chinese cabbage is washed with clean water and drained until the salinity is 1% to 1.5%; and / or 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; and / or The number of viable bacteria in the probiotic fermentation agent is (1-9)×10 10 CFU / g; and / or The probiotic fermentation agent is the composite bacterial agent; and / or The number of live bacteria of the Leuconostoc mesenteroides NCU018046 and Lactobacillus plantarum in the composite bacterial agent is the same.

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