Complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention discloses a complex microbial inoculant as well as a preparation method and application thereof. When the complex microbial inoculant is used for preparing the fermented vegetables, the fermentation period can be shortened, the fragrance is increased, and the prepared fermented vegetables are stable in quality, safe and mellow in flavor. The complex microbial inoculant has a wide application prospect in preparation of fermented vegetables.

Description

Technical Field

[0001] The present invention belongs to the field of biological fermentation, and particularly relates to a composite bacterial agent, a preparation method thereof, and an application thereof. Background Art

[0002] Fermented vegetables are traditional fermented foods in China, with a long culture and history. Fermented vegetables are deeply loved by consumers due to their unique sour and fragrant taste, flavor, and nutritional value. Common categories include pickled cabbages, pickled radishes, peppers, cowpeas, etc., which are widely used in various dishes, such as pickled fish with pickled cabbage, shredded pork with pickled cabbage, and luosifen. Their unique salty and sour flavor forms the core flavor of the dishes. The production of fermented vegetables is gradually changing from small-scale workshop production to industrialized and large-scale production. With the expansion of the industrial production scale, artificial inoculation fermentation is considered to be able to shorten the fermentation cycle and stabilize the quality of fermented vegetable products.

[0003] Most existing studies have explored the effects of lactic acid bacteria inoculation fermentation on the quality of pickled cabbages, and mostly used multiple lactic acid bacteria for compound inoculation to improve the flavor of fermented vegetables. However, relevant studies on direct-inoculation lactic acid bacteria fermentation have shown that when inoculating lactic acid bacteria for fermentation, the flavor of fermented vegetables is not as mellow as that of natural fermentation. In addition, vegetables themselves contain nitrates, which will be converted into nitrites by bacteria during the pickling process. It is often difficult to obtain pickled vegetables with stable quality and control the nitrite content in pickled vegetables through natural fermentation. The selection and ratio of excellent strains can not only shorten the production cycle of pickled vegetable products, determine the flavor and quality of fermented vegetables, but also avoid or reduce the content of nitrites and biogenic amines, thus avoiding food safety problems. Therefore, there is an urgent need to obtain a composite bacterial agent that can endow fermented vegetables with excellent flavor and low nitrite content. Summary of the Invention

[0004] To solve the problems in the prior art that the quality of natural fermentation is unstable, the flavor is insufficient, and it is difficult to control the content of harmful substances such as nitrites, and the flavor of existing direct-inoculation lactic acid bacteria fermentation is not as mellow as that of natural fermentation, the present invention provides a composite bacterial agent, which can shorten the fermentation cycle and increase the fragrance when used to prepare fermented vegetables, and the prepared fermented vegetables have stable quality, are safe, and have a mellow flavor.

[0005] The present invention solves the above technical problems through the following technical solutions:

[0006] The present invention provides a composite bacterial agent, which is selected from any one of the following:

[0007] (1) The composite bacterial agent includes Lactobacillus plantarum, Leuconostoc mesenteroides, and Wickerhamomyces anomalus;

[0008] (2) The composite bacterial agent includes Lactobacillus plantarum, Leuconostoc mesenteroides, and Pichia kudriavzevii.

[0009] In the present invention, the preservation number of Lactobacillus plantarum is CCTCC M2025931; the preservation number of Leuconostoc mesenteroides is CCTCC M2025932; and / or, the preservation number of Wickerhamomyces anomalus is CCTCC M2025930.

[0010] In some embodiments of the present invention, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Wickerhamomyces anomalus in the composite bacterial agent is (80 - 120):(0.5 - 5):(5 - 20).

[0011] Preferably, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Wickerhamomyces anomalus in the composite bacterial agent is (90 - 100):(0.5 - 2):(8 - 12).

[0012] More preferably, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Wickerhamomyces anomalus in the composite bacterial agent is 100:1:10.

[0013] In some embodiments of the present invention, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Pichia kudriavzevii in the composite bacterial agent is (80 - 120):(0.5 - 5):(5 - 20).

[0014] Preferably, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Pichia kudriavzevii in the composite bacterial agent is (90 - 100):(0.5 - 2):(8 - 12).

[0015] More preferably, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Pichia kudriavzevii in the composite bacterial agent is 100:1:10.

[0016] The ratio is a quantity ratio, referring to the ratio of the viable bacteria numbers. In the present invention, this ratio can also be the cell activity ratio of the bacteria.

[0017] In the present invention, the composite bacterial agent is a liquid preparation, powder, granule, tablet, immobilized bacterial agent, or biochar bacterial agent.

[0018] The present invention also provides a method for preparing a compound bacterium agent, which includes compounding Lactiplantibacillus plantarum, Leuconostoc mesenteroides and Wickerhamomyces anomalus in a quantity ratio of (80 - 120):(0.5 - 5):(5 - 20); or,

[0019] The method includes compounding Lactiplantibacillus plantarum, Leuconostoc mesenteroides and Pichia kudriavzevii in a quantity ratio of (80 - 120):(0.5 - 5):(5 - 20);

[0020] In the present invention, the preservation number of the Lactiplantibacillus plantarum is CCTCC M2025931; the preservation number of the Leuconostoc mesenteroides is CCTCC M2025932; and / or, the preservation number of the Wickerhamomyces anomalus is CCTCC M2025930.

[0021] Preferably, the method includes compounding Lactiplantibacillus plantarum, Leuconostoc mesenteroides and Wickerhamomyces anomalus in a quantity ratio of (90 - 100):(0.5 - 2):(8 - 12); or,

[0022] The method includes compounding Lactiplantibacillus plantarum, Leuconostoc mesenteroides and Pichia kudriavzevii in a quantity ratio of (90 - 100):(0.5 - 2):(8 - 12).

[0023] More preferably, the method includes compounding Lactiplantibacillus plantarum, Leuconostoc mesenteroides and Wickerhamomyces anomalus in a quantity ratio of 100:1:10; or,

[0024] The method includes compounding Lactiplantibacillus plantarum, Leuconostoc mesenteroides and Pichia kudriavzevii in a quantity ratio of 100:1:10.

[0025] The present invention also provides a method for preparing fermented vegetables, which includes contacting the compound bacterium agent with raw materials for fermentation.

[0026] In some embodiments of the present invention, the fermentation temperature is 20 - 30 °C. For example, it is 20 - 25 °C, 21 - 25 °C, 22 - 25 °C, 23 - 25 °C, 24 - 25 °C, 25 - 30 °C, 25 - 29 °C, 25 - 28 °C, 25 - 27 °C or 25 - 26 °C.

[0027] In some specific embodiments of the present invention, the fermentation temperature is 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C or 30 °C.

[0028] In some embodiments of the present invention, the fermentation time is 2 - 15 days. For example, it can be 2 - 8 days, 2 - 6 days, 2 - 4 days, 4 - 6 days, 4 - 8 days, 6 - 8 days, 4 - 10 days, 2 - 15 days, 4 - 15 days, 2 - 10 days or 4 - 10 days.

[0029] In some specific embodiments of the present invention, the fermentation time is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 15 days.

[0030] In some embodiments of the present invention, the inoculation amount of the compound bacterium agent is: for Lactiplantibacillus plantarum, it is (0.8 - 1.2)×10 9 CFU / mL, for Leuconostoc mesenteroides, it is (0.5 - 5)×10 7 CFU / mL, and for Wickerhamomyces anomalus, it is (0.5 - 2)×10 8 CFU / mL; or,

[0031] for Lactiplantibacillus plantarum, it is (0.8 - 1.2)×10 9 CFU / mL, for Leuconostoc mesenteroides, it is (0.5 - 5)×10 7 CFU / mL, and for Pichia kudriavzevii, it is (0.5 - 2)×10 8 CFU / mL.

[0032] Preferably, the inoculation amount of the compound bacterium agent is: for Lactiplantibacillus plantarum, it is (0.9 - 1)×10 9 CFU / mL, for Leuconostoc mesenteroides, it is (0.5 - 2)×10 7 CFU / mL, and for Wickerhamomyces anomalus, it is (0.8 - 1.2)×10 8 CFU / mL; or,

[0033] for Lactiplantibacillus plantarum, it is (0.9 - 1)×10 9 CFU / mL, for Leuconostoc mesenteroides, it is (0.5 - 2)×10 7 CFU / mL, and for Pichia kudriavzevii, it is (0.8 - 1.2)×10 8 CFU / mL.

[0034] More preferably, the inoculation amount of the compound bacterium agent is: for Lactiplantibacillus plantarum, it is 1×10 9 CFU / mL, for Leuconostoc mesenteroides, it is 1×10 7 CFU / mL, and for Wickerhamomyces anomalus, it is 1×10 8 CFU / mL; or,

[0035] for Lactiplantibacillus plantarum, it is 1×10 9CFU / mL, and the Leuconostoc mesenteroides is 1×10 7 CFU / mL and the Pichia kudriavzevii is 1×10 8 CFU / mL.

[0036] In some embodiments of the present invention, the raw material is a vegetable.

[0037] Preferably, the vegetable is a radish, mustard, green vegetable or Chinese cabbage.

[0038] In some embodiments of the present invention, the method further includes the step of adding auxiliary materials, and the auxiliary materials include water and spices.

[0039] In some embodiments of the present invention, the method further includes the step of adding seasonings.

[0040] In some embodiments of the present invention, the seasonings are white granulated sugar and / or salt.

[0041] The present invention also provides the application of the compound bacterium agent in the preparation of fermented vegetables.

[0042] In the present invention, "fermented vegetables" refers to the use of beneficial microorganisms for co-fermentation, which changes the color and texture of vegetables by producing enzymes and other substances. At the same time, microorganisms participate in the metabolism of carbohydrates, amino acids, lipids and glucosinolates to form the special flavor of fermented vegetables, and the finished product flavor of the finished product can be further enhanced through various ingredients. According to the differences in raw materials and pickling time, it can be divided into "pickled vegetables", "pickled Chinese cabbages" and "preserved pickles", etc. When not specifically stated, "fermented vegetables", "pickled vegetables", "pickled Chinese cabbages" and "preserved pickles" in the present invention can be used interchangeably.

[0043] Wickerhamomyces anomalus is one of the most important ester-producing strains in Chinese liquor brewing, and Pichia kudriavzevii is a non-saccharomycete yeast, which is famous for its ability to enhance the aroma quality in fermented foods.

[0044] This study was to explore the effects of the co-fermentation of yeasts and lactic acid bacteria on the flavor and quality during the fermentation of pickled Chinese cabbages. Wickerhamomyces anomalus and Pichia kudriavzevii were used in combination with lactic acid bacteria for fermentation, and the changes in pH value, total acid, texture properties, nitrite content, total ester content, microbial colony count, amino acids, organic acids and volatile components during the fermentation of pickled Chinese cabbages were measured. And the differences in volatile flavors were further clarified by principal component analysis (PCA), providing a theoretical basis for the safety of industrial production of pickled Chinese cabbages and improving product quality.

[0045] On the basis of conforming to the common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0046] The reagents and raw materials used in the present invention are all commercially available.

[0047] The positive and progressive effects of the present invention are as follows: when the compound bacterium agent is used for preparing fermented vegetables, it can alleviate the excessive acid production of fermented vegetables, significantly reduce the peak value of nitrite, and improve the safety of fermented vegetables. In addition, it can shorten the fermentation period, increase the fragrance, and the total content of volatile substances in the prepared fermented vegetables is high, and the flavor is mellow.

[0048] Biological Material Deposit Information

[0049] The Lactobacillus plantarum of the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on April 28, 2025. The deposit address is Wuhan University, Wuhan, China, with the postcode 430072. The deposit number is CCTCC M2025931, the culture name is LP10, and the taxonomic name is Lactobacillus plantarum.

[0050] The Leuconostoc mesenteroides of the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on April 28, 2025. The deposit address is Wuhan University, Wuhan, China, with the postcode 430072. The deposit number is CCTCC M2025932, the culture name is LM427, and the taxonomic name is Leuconostoc mesenteroides.

[0051] The Wickerhamomyces anomalus of the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on April 28, 2025. The deposit address is Wuhan University, Wuhan, China, with the postcode 430072. The deposit number is CCTCC M2025930, the culture name is WA06, and the taxonomic name is Wickerhamomyces anomalus. Description of the Drawings

[0052] Figure 1 It is the change of the pH value of pickled Chinese cabbage during the fermentation process.

[0053] Figure 2 It is the change of the total acid content of pickled Chinese cabbage during the fermentation process.

[0054] Figure 3 It is the change of the hardness of pickled Chinese cabbage during the fermentation process.

[0055] Figure 4 It is the change of the chewiness of pickled Chinese cabbage during the fermentation process.

[0056] Figure 5For the change of nitrite content in pickled Chinese cabbage during the fermentation process.

[0057] Figure 6 For the change of total ester content in pickled Chinese cabbage during the fermentation process.

[0058] Figure 7 For the change of viable count of lactic acid bacteria in pickled Chinese cabbage during the fermentation process.

[0059] Figure 8 For the change of viable count of yeast in pickled Chinese cabbage during the fermentation process.

[0060] Figure 9 For the change of amino acid content in pickled Chinese cabbage during the fermentation process.

[0061] Figure 10 For the change of methionine content in pickled Chinese cabbage during the fermentation process.

[0062] Figure 11 For the change of tyrosine content in pickled Chinese cabbage during the fermentation process.

[0063] Figure 12 For the change of threonine content in pickled Chinese cabbage during the fermentation process.

[0064] Figure 13 For the change of glutamic acid content in pickled Chinese cabbage during the fermentation process.

[0065] Figure 14 For the change of volatile substance content in pickled Chinese cabbage during the fermentation process.

[0066] Figure 15 For the PCA results of volatile substances in pickled Chinese cabbage of each experimental group on the 8th day. Detailed implementation manners

[0067] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0068] Materials and methods

[0069] 1. Materials and reagents

[0070] Small-leaf mustard, pickling salt and granulated sugar were purchased from Yonghui Supermarket in Beibei District, Chongqing, China.

[0071] Lactobacillus plantarum, Leuconostoc mesenteroides and Wickerhamomyces anomalus were first isolated by the inventors and preserved in the China Center for Type Culture Collection (CCTCC). The specific information is as described in the "Biological Material Preservation Information". Pichia kudriavzevii can be selected from commercially available strains.

[0072] Sodium hydroxide, sodium nitrite, sodium tetraborate, potassium ferrocyanide, zinc acetate, p-aminobenzenesulfonic acid, naphthylethylenediamine hydrochloride, sulfuric acid, sodium acetate, potassium dihydrogen phosphate, Chengdu Kelon Chemical Reagent Factory; MRS culture medium, Bengal red agar, nutrient agar, Beijing Luqiao Technology Co., Ltd.; phenyl isothiocyanate, sodium dihydrogen phosphate, phenolphthalein, triethylamine, n-hexane, acetic acid, phosphoric acid, methyl heptanoate, methanol, acetonitrile, Chongqing Titanium New Chemical Co., Ltd.

[0073] 2. Instruments and equipment

[0074] GCMS-QP2010 gas chromatograph-mass spectrometer, Shimadzu Corporation, Japan; LC20A high performance liquid chromatograph, Shimadzu Corporation, Japan; PB-10 precision pH meter, Sartorius Company, Germany; biotek / H1MG microplate reader, BioTek Company, USA; CT325K230 physical property tester, Brookfield Company, USA; HH-4 constant temperature water bath, Changzhou Aohua Instrument Co., Ltd.; 5810 desktop high-speed centrifuge, Eppendorf Company, Germany.

[0075] 3. Experimental Methods

[0076] 3.1 Determination of pH and total acid

[0077] Take 25g of sauerkraut and mix it with water to make a homogenate, and measure the pH value with a pH meter. Add an appropriate amount of pure water, stir and heat in a boiling water bath for 30 minutes, shake 2-3 times in the middle, cool to room temperature after the end, and titrate with standard alkali solution to obtain the total acid.

[0078] 3.2 Determination of texture characteristics

[0079] A mustard sample with a root length and width of about 25 mm and a thickness of 10 mm was placed in a texture analyzer for texture measurement. Texture measurement parameters: probe TA44, test rate 1 mm / s, compression 50%, compression force 7 g.

[0080] 3.3 Determination of nitrite content

[0081] With reference to GB 5009.33-2016 “Determination of nitrite and nitrate in food”, the naphthylethylamine hydrochloride method was used for determination.

[0082] 3.4 Determination of total ester content

[0083] Take 50mL of fermentation broth and place it in a 250mL reflux flask, add phenolphthalein indicator, titrate with 0.1mol / L NaOH standard solution until it turns slightly red, add 25mL 0.1mol / L NaOH standard solution, reflux saponify in a boiling water bath for 0.5h, cool to room temperature and immediately titrate with 0.1mol / L sulfuric acid standard solution to obtain total esters.

[0084] 3.5 Determination of microbial colony count

[0085] Refer to the methods of GB 4789.35-2023 "National Food Safety Standard Food Microbiology Examination Lactic Acid Bacteria Examination" and "GB 4789.15-2016 National Food Safety Standard Food Microbiology Examination Enumeration of Molds and Yeasts" to count lactic acid bacteria and yeasts respectively.

[0086] 3.6 Determination of free amino acid content

[0087] Amino acid extraction: Take the dried and pulverized pickled Chinese cabbage, dissolve it with 0.1mol / L hydrochloric acid, ultrasonically treat it for 40min, make the volume up to 25mL with hydrochloric acid, and take the supernatant after filtration for derivatization treatment.

[0088] Derivatization: Take 200μL of the supernatant, add 100μL each of derivatizing agent A (accurately measure 250μL of phenyl isothiocyanate and make the volume up to 10mL with acetonitrile) and derivatizing agent B (accurately measure 1.4mL of triethylamine and make the volume up to 10mL with acetonitrile), incubate in the dark at room temperature for 1h, extract with 200μL of n-hexane twice, 10min each time, absorb the lower layer solution, mix it with 800μL of water, and filter through a 0.22μm filter membrane.

[0089] Chromatographic conditions: C18 special analytical column for amino acids (250mm×4.6mm, 5μm), mobile phase A is 50mmol / L anhydrous sodium acetate (pH 6.5), mobile phase B is a methanol-acetonitrile mixture (methanol:acetonitrile:water = 20:60:20, V / V), flow rate is 0.8mL / min, detection wavelength is 254nm, column temperature is 35℃, injection volume is 20μL, gradient elution: 0 - 39min, 100% - 95% A, 0% - 5% B; 39 - 40min, 51% - 0% A, 49% - 100% B; 40 - 50min, 0% - 0% A, 100% - 100% B; 50 - 51min, 0% - 95% A, 100% - 5% B; 51 - 60min, 95% - 95% A, 5% - 5% B.

[0090] 3.7 Determination of organic acid content

[0091] Organic acid extraction: Weigh 5g of pickled Chinese cabbage homogenate, add 15mL of 80% ethanol-aqueous solution, water bath at 75℃ for 0.5h, make the volume up to 25mL with pure water, centrifuge at 9000r / min for 30min, accurately absorb the supernatant, and filter through a 0.22μm filter membrane for determination.

[0092] Chromatographic conditions: C18 chromatographic column (250 mm × 4.6 mm, 5 μm), ultraviolet detector, mobile phase A is 0.01 mol / L sodium dihydrogen phosphate - water (3:97, V / V) (pH 2.8), mobile phase B is methanol, wavelength is 210 nm, column temperature is 35 °C.

[0093] 3.8 Analysis of volatile components

[0094] Accurately weigh 5 g of pickled Chinese cabbage homogenate into a 15 mL headspace vial, add 1 g of NaCl and 5 μL of methyl heptanoate (17.58 g / L), mix well and seal. Equilibrate at 75 °C for 45 min, insert the extraction head into the vial for extraction for 20 min, take out the extraction head and insert it into the GC-MS injection port for desorption for 5 min.

[0095] GC conditions: Chromatographic column type: RTX-WAX chromatographic column (30 m × 0.25 mm, 0.25 μm); Temperature programming: Initial temperature is 40 °C, hold for 3 min, increase at a rate of 5 °C / min to 150 °C, hold for 3 min, then increase at a rate of 10 °C / min to 230 °C, hold for 2 min; Carrier gas is helium, flow rate is 0.8 mL / min, splitless injection.

[0096] MS conditions: Ionization mode is electron ionization, ion source temperature is 230 °C, quadrupole temperature is 150 °C, interface temperature is 250 °C, energy is 70 eV; Mass scanning range is 25 - 450 m / z.

[0097] Qualitative and quantitative analysis: The chromatograms obtained by GC-MS are compared and retrieved by computer in the standard spectral library NIST8.0. Substances with a similarity > 80 (maximum value is 100) are selected for qualitative analysis, and the volatile components of each sample are accurately identified. At the same time, methyl heptanoate (17.58 g / L) is used as the internal standard for semi-quantitative analysis to obtain the mass concentration of each component.

[0098] 4. Data processing

[0099] Use Excel software to statistically analyze and process the experimental data; Use Origin software to draw charts and perform principal component analysis mapping, and use SPSS software to analyze the significant differences between the data. P < 0.05 indicates significant differences; Each texture determination is repeated 8 times, and the rest of the experiments are repeated 3 times for each group.

[0100] Example 1 Preparation of pickled Chinese cabbage

[0101] Pickled Chinese cabbage preparation process: Fresh mustard → Wash → Weigh → Add sugar brine prepared with 4% sugar and 7% salt [m(pickled Chinese cabbage):m(sugar brine) = 1:2] → Adjust the salinity of the pickled Chinese cabbage water so that the final salinity is stable at 7% → Inoculate and ferment (inoculation amount of Lactiplantibacillus plantarum is 10 9CFU / mL, the inoculation amount of Leuconostoc mesenteroides was 10 7 CFU / mL, the inoculation amounts of Wickerhamomyces anomalus and Pichia kudriavzevii were 10 8 CFU / mL) → Ferment at a constant temperature of 25°C.

[0102] Set up 4 experimental groups:

[0103] (1) L+W: Lactiplantibacillus plantarum 10 9 CFU / mL, the inoculation amount of Leuconostoc mesenteroides was 10 7 CFU / mL, and the inoculation amount of Wickerhamomyces anomalus was 10 8 CFU / mL;

[0104] (2) L+P: Lactiplantibacillus plantarum 10 9 CFU / mL, the inoculation amount of Leuconostoc mesenteroides was 10 7 CFU / mL, and the inoculation amount of Pichia kudriavzevii was 10 8 CFU / mL;

[0105] (3) L0: Lactiplantibacillus plantarum 10 9 CFU / mL, and the inoculation amount of Leuconostoc mesenteroides was 10 7 CFU / mL;

[0106] (4) N0: Natural fermentation (without the inoculation fermentation step).

[0107] After fermentation, store in a -80°C refrigerator for subsequent index measurement. Analyze the pickled cabbages on the 0th, 2nd, 4th, 6th, and 8th days respectively.

[0108] Example 2 Effects of Co-fermentation of Different Yeasts and Lactic Acid Bacteria on the pH Value and Total Acid of Pickled Cabbage

[0109] As Figure 1 shown, during the fermentation process, the pH values of the pickled cabbages in each group significantly decreased (P < 0.05) from the 0th to the 6th day of fermentation, and then gradually leveled off. The pH value of the L0 group was lower than that of other groups throughout the process, but at the end of fermentation, the pH values of the four groups were all within the range of 3.21 - 3.32, and there was no significant difference between groups (P > 0.05).

[0110] As Figure 2As shown, the total acid content of the L+P group and the N0 group increased slowly from day 0 to day 2, and increased significantly from day 2 to day 8 (P < 0.05). The L+W group increased gently in the initial stage and increased significantly in the later stage (P < 0.05). It is worth noting that the final total acid content of the L0 group reached (5.10 ± 0.16) g / Kg, which was significantly higher than that of the other three groups (3.5 - 3.7 g / Kg) (P < 0.05). It can be seen that the interaction between yeast and lactic acid bacteria helps to alleviate the excessive acid production of lactic acid bacteria during fermentation, thus improving the product quality and extending the shelf life.

[0111] Example 3 Effects of Co-fermentation of Different Yeasts and Lactic Acid Bacteria on the Texture Characteristics of Pickled Chinese Cabbage

[0112] Hardness and chewiness can reflect the changes in the texture characteristics of pickled Chinese cabbage. As Figure 3 shown, except for the L0 group, the other three groups showed a significant decrease throughout the fermentation process (P < 0.05). At 0 - 6 days of fermentation, the L0 group showed the lowest hardness during fermentation, indicating that the inoculation of lactic acid bacteria can accelerate the reduction of the hardness of pickled Chinese cabbage. Protopectin is hydrolyzed into soluble pectic acid under the action of pectinase and acid, dissolving the middle lamella of the cell wall and decreasing the adhesion between cells, resulting in a decrease in hardness. At the end of fermentation, the hardness of the four groups ranged from 497 to 678 g, among which the hardness of the L0 group increased to the highest while the L+P group was the lowest. The hardness of the N0 group was relatively high at the initial stage of fermentation and decreased slowly. The hardness of the L+W group and the L+P group was between that of the N0 group and the L0 group, indicating that yeast has a certain promoting effect on the softening of pickled Chinese cabbage but is weaker than lactic acid bacteria, which provides some ideas for the control of texture softening in the later stage of pickled Chinese cabbage fermentation. From Figure 4 it can be seen that the chewiness of all treatment groups showed a downward trend. The L+W group had the fastest rate of decrease at the initial stage of fermentation, reaching the lowest value (1.40 ± 0.29) mJ on the 8th day of fermentation. The L0 group had the slowest rate of decrease at the initial stage of fermentation and finally maintained the highest chewiness (2.80 ± 0.21 mJ). The chewiness of the L+P group and the N0 group was between that of the L+W group and the L0 group.

[0113] Example 4 Effects of Co-fermentation of Different Yeasts and Lactic Acid Bacteria on the Nitrite Content and Total Ester Content of Pickled Chinese Cabbage

[0114] 4.1 Effects of Co-fermentation of Different Yeasts and Lactic Acid Bacteria on the Nitrite Content of Pickled Chinese Cabbage

[0115] Nitrite is an important indicator for evaluating the edible safety of pickled Chinese cabbage. From Figure 5It can be seen that in the initial stage of fermentation, the nitrite content in the L+P group and the L+W group increased significantly (P<0.05), and nitrite peaks appeared on the 2nd day of fermentation (2.87 mg / Kg and 1.37 mg / Kg) respectively, while the change in the N0 group was gentle. In the middle stage of fermentation, a nitrite peak (3.82 mg / Kg) appeared in the L0 group on the 4th day of fermentation, and the nitrite content in the N0 group increased significantly (P<0.05). In the later stage of fermentation, the nitrite content in the L+P group and the L0 group decreased significantly (P<0.05). The N0 group reached the peak value (3.55 mg / Kg) on the 6th day of fermentation and then decreased slightly, while the change in the L+W group remained stable all the time. The nitrite reductase produced by lactic acid bacteria during fermentation can degrade nitrite into NH4 + or N2, effectively reducing nitrite. Compared with the L0 group and the N0 group, the nitrite peak values in the L+P group and the L+W group were relatively low, indicating that the mixed inoculation of lactic acid bacteria and yeast can effectively reduce the generation of nitrite. In addition, it was also observed that the nitrite peak value in the L0 group was the highest, because the N0 group, as an uninoculated control group, had a relatively lagging natural fermentation rate, and nitrite might continue to accumulate in its subsequent fermentation stage. The nitrite content in all experimental groups during the fermentation process was lower than the national standard limit of 20 mg / kg.

[0116] 4.2 Effects of Co-fermentation of Different Yeasts and Lactic Acid Bacteria on the Total Ester Content of Pickled Cabbage

[0117] The total ester content is a direct measure of the total ester production of aroma-producing yeasts and a key indicator of the flavor of pickled cabbage. As Figure 6 shown, in the initial stage of fermentation, the total ester content in all experimental groups decreased significantly (P<0.05), which means the rapid generation of volatile ester substances. The total ester content in the L+P group and the L+W group was always lower than that in the L0 group, indicating that the co-fermentation of yeast and lactic acid bacteria can promote the enrichment of flavor compounds. The total ester content in the L0 group changed smoothly in the middle stage of fermentation and decreased significantly in the final stage (P<0.05), while the other three groups showed a phenomenon of total ester accumulation in the middle and later stages of fermentation. The total ester content in the N0 group showed fluctuating changes during the fermentation process, which was attributed to the complexity and variability of the microbial community composition in the natural fermentation system.

[0118] Example 5 Effects of Co-fermentation of Different Yeasts and Lactic Acid Bacteria on the Microbial Colony Counts of Pickled Cabbage

[0119] As Figure 7As shown in the figure, the number of lactic acid bacteria colonies in all experimental groups increased significantly during the initial stage of fermentation (P < 0.05), and the number of lactic acid bacteria colonies in the N0 group increased the fastest. During the natural fermentation process, complex metabolic networks and interactions may have formed among other microorganisms, promoting the growth of lactic acid bacteria and maintaining a relatively high level. In the later stage of fermentation, the number of lactic acid bacteria colonies in the N0 group was higher than that in the L+W group, L+P group, and L0 group. During the fermentation of sauerkraut, lactic acid accumulates continuously, and at this time, the growth of lactic acid bacteria itself is inhibited, resulting in a slow growth rate or even inhibited growth of lactic acid bacteria in the later stage of fermentation. In addition, yeasts inhibit the growth of lactic acid bacteria by competing for carbon sources and metabolizing ethanol to affect the cell structure products of lactic acid bacteria. As Figure 8 shown in the figure, the number of yeast colonies in the L0 group and N0 group showed an overall upward trend, while the L+W group and L+P group showed an overall downward trend. In the later stage of fermentation, the number of yeast colonies in the L0 group and N0 group was higher than that in the L+W group and L+P group. The same phenomenon also occurred among lactic acid bacteria, indicating that the presence of yeasts can promote the growth of lactic acid bacteria. This is because there is a metabolic complementary mechanism between lactic acid bacteria and yeasts. The metabolic substances produced by yeasts can induce the synthesis of aminopeptidase in lactic acid bacteria, increasing the amino acid content, and lactic acid bacteria use it to promote their own growth. In addition, the pyranomannose produced by yeast autolysis can absorb medium-chain fatty acids, relieve their toxic effects on lactic acid bacteria, and may also increase the activities of α-glucosidase, β-glucosidase, N-acetyl-β-glucosidase dehydratase, and peptidase in lactic acid bacteria, providing a stable carbon source to promote the growth of lactic acid bacteria. Lactic acid bacteria can metabolize lactic acid to form lactate to provide a carbon source for yeasts and can also decompose lactose, and the resulting glucose can also be utilized by yeasts. The levels of lactic acid bacteria and yeast colonies in the L+W group and L+P group were significantly lower than those in the N0 group in the later stage of fermentation. This is because the direct inoculation of starter cultures makes the differences in the quality of the two microbial communities obvious, and the competition between lactic acid bacteria and yeasts is more direct and prominent.

[0120] Example 6 Effects of Co-fermentation of Different Yeasts and Lactic Acid Bacteria on Free Amino Acids in Sauerkraut

[0121] Free amino acids are important taste substances in food, and they are classified into sweet, umami, bitter, and tasteless amino acids. A total of 3 sweet amino acids (Ala, Ser, and Thr), 2 umami amino acids (Glu and Asp), 7 bitter amino acids (Leu, Ile, Met, Lys, Tyr, Phe, and Val), and 2 tasteless amino acids (Cys and Pro) were detected in all samples. As Figure 9As shown, the total amino acid content in the L+W group, L+P group, and L0 group first increased and then decreased, with the L+W group having the highest peak total amino acid content. The total amino acid content in the N0 group generally showed an upward trend, but the peak total amino acid content was the smallest. However, at the end of fermentation, the total amino acid content in the L+W group, L+P group, and L0 group was lower than that in the N0 group. Amino acids are important nutrients for the growth and metabolism of lactic acid bacteria. During the inoculated fermentation process, lactic acid bacteria consume more amino acids in the substrate, and the introduction of yeast can promote protein degradation by secreting protease, further accumulating free amino acids. During the pickled cabbage fermentation process, bitter amino acids accounted for the highest proportion, among which tyrosine and methionine were the main bitter amino acids. As Figure 10 and Figure 11 shown, methionine in the L+W group and tyrosine in the L+P group increased sharply and then were consumed in large amounts during the fermentation process, while the methionine content in the N0 group continued to accumulate. Tyrosine and methionine are important nutrient sources for bacterial growth and reproduction. Tyrosine is catalytically deaminated to form p-hydroxyphenylpyruvic acid, which is further converted into α-keto acid, and then oxidized and decarboxylated to form aldehydes and carboxylic acids. Methionine, as a precursor of sulfur-containing compounds, can endow pickled cabbage with a unique aroma. Sweet amino acids are mainly threonine. As Figure 12 shown, the threonine content in the L+W group reached a maximum of 39.46 mg / Kg, and the content in the N0 group generally showed an upward trend. Threonine can be converted into isoleucine through an enzymatic reaction, and then converted into the corresponding α-keto acid under the catalysis of BCAT, and finally converted into volatile alcohols. It was thus found that the addition of yeast can increase the levels of bitter and sweet amino acids during the pickled cabbage fermentation process, improving the flavor of pickled cabbage. As Figure 13 shown, the glutamic acid content in all treatment groups showed a downward trend, with the L+W group having the lowest level of decrease, and the glutamic acid content in the N0 group showed fluctuating changes. During the fermentation process, the proportion of tasteless amino acids was small and the changes were not significant.

[0122] Example 7 Effects of Co-Fermentation of Different Yeasts and Lactic Acid Bacteria on Organic Acids in Pickled Cabbage

[0123] The composition and content of organic acids are important factors contributing to the sour taste of pickled Chinese cabbage. There are a total of seven organic acids, including oxalic acid, tartaric acid, lactic acid, acetic acid, succinic acid, citric acid, and malic acid. As shown in Table 1, the main organic acids in pickled Chinese cabbage are tartaric acid (1.13 mg / mL), lactic acid (0.59 mg / mL), and acetic acid (0.42 mg / mL). As fermentation progresses, the content of tartaric acid in the L+W group, L+P group, and L0 group shows a trend of first decreasing and then increasing, and there is no significant difference from the initial content at the end of fermentation (P>0.05). The content of tartaric acid in the N0 group significantly decreases to (0.69±0.04) mg / mL (P<0.05). Tartaric acid has a mellow taste, and the increase in the content of tartaric acid in the inoculated group promotes the improvement of the flavor of pickled Chinese cabbage. Lactic acid imparts a mild sour taste, and acetic acid imparts a pungent sour taste, both of which promote the formation of the flavor of pickled Chinese cabbage. The lactic acid content in the L+P group and N0 group decreases and then increases, and significantly decreases in the later stage of fermentation (P<0.05). In the initial stage of fermentation, the contents of acetic acid and citric acid in pickled Chinese cabbage are lower than the detection limit. As fermentation progresses, the contents of acetic acid and citric acid in all experimental groups significantly increase to 0.41 - 0.43 mg / mL and 0.17 - 0.22 mg / mL respectively (P<0.05), and then remain stable. Lactic acid and acetic acid are usually produced by most lactic acid bacteria, while the accumulation of citric acid may be related to some fungal communities. The succinic acid content in all experimental groups shows a trend of first increasing and then decreasing, and the L0 group is much higher than the other experimental groups at the end of fermentation. Malic acid was only detected in the middle stage of fermentation in the L+P group. Malic acid is an intermediate in the tricarboxylic acid cycle of organism metabolism. As fermentation progresses, malic acid is decomposed into lactic acid and other substances. This indicates that the antagonistic effect between lactic acid bacteria and yeast in the L+P group is relatively strong, the metabolic activity of lactic acid bacteria is weak, and the decomposition of malic acid is slow.

[0124] On the 8th day of fermentation, the contents of tartaric acid and lactic acid in the pickled Chinese cabbage of the L+W group, L+P group, and L0 group are higher than those in the N0 group.

[0125] Table 1 Changes in organic acids in pickled Chinese cabbage during fermentation

[0126]

[0127]

[0128] Note: (L+W) - Fermentation with mixed lactic acid bacteria and Wickerhamomyces anomalus; (L+P) - Fermentation with mixed lactic acid bacteria and Pichia kudriavzevii; L0 - Inoculated fermentation with Lactiplantibacillus plantarum and Leuconostoc mesenteroides; N0 - Natural fermentation. "ND" indicates not detected. Letters such as "a", "b", "c", and "d" represent significant differences in data, and there is no significant difference between the same data marked with the same letter.

[0129] Example 8 Effects of Co-fermentation of Different Yeasts and Lactic Acid Bacteria on Volatile Substances of Pickled Chinese Cabbage

[0130] Volatile substances are an important source of the flavor of pickled Chinese cabbages. A total of 101 volatile substances were detected in this study, including 30 esters, 5 acids, 20 alcohols, 20 aldehydes, 10 ketones, 5 hydrocarbons, 5 phenols, 1 ether, and 5 others.

[0131] Table 2 Volatile Substances

[0132]

[0133]

[0134]

[0135]

[0136] Note: "ND" means not detected. Letters such as "a", "b", "c", and "d" represent significant differences in data. There is no significant difference between the same kind of data marked with the same letter.

[0137] As shown in Table 2 and Figure 14As shown, the total amount of volatile substances in all treatment groups showed an overall upward trend. On the 8th day of fermentation, the content of volatile substances in the N0 group was the highest, followed by the L+W group, and the content of volatile substances in the L+P group was the lowest. Esters are important flavor components in pickled Chinese cabbage. With the progress of fermentation, the ester content in most groups showed an overall significant upward trend (P<0.05). Esters showed the highest relative content (32.75%-60.33%) during the pickled Chinese cabbage fermentation process. Among them, isothiocyanates accounted for a relatively large proportion of esters, mainly including allyl isothiocyanate, butyl isothiocyanate, and phenethyl isothiocyanate. Isothiocyanates are unique flavor substances of cruciferous plants and their fermented products, with a low threshold and a pungent and fragrant smell. The remaining esters (such as ethyl palmitate, polyunsaturated fatty acid methyl esters, etc.) contribute fruity and floral scents. The comparison between inoculated groups showed that on the 8th day of fermentation, the ester content in the L+W group was significantly higher than that in the L0 group (p<0.05), and there was no significant difference from the N0 group (p>0.05), indicating that the synergistic effect of Wickerhamomyces anomalus and lactic acid bacteria can enhance the synthesis of volatile esters, being closer to traditional natural fermentation. Alcohol substances continued to accumulate through heterofermentation of lactic acid bacteria and yeast metabolism, and the content increased significantly (P<0.05). Alcohols mainly included various alcohols such as phenethyl alcohol, n-pentanol, 1-nonanol, and 2-decyn-1-ol. The total content ranking was L+W>L0>N0>L+P, indicating that Wickerhamomyces anomalus promoted the synthesis of volatile alcohols. Acids were mainly acetic acid and octanoic acid, which had the effects of reducing the pH value of pickled Chinese cabbage and increasing its sour taste. Aldehyde substances ((Z)-2-heptenal, benzaldehyde, etc.) were produced by lipid oxidation of pickled Chinese cabbage during fermentation, contributing various scents such as fruity and floral scents, bitter almond smell, and vegetable freshness. Ketone substances were mainly dihydro-β-ionone and beta-ionone, with stable properties, giving pickled Chinese cabbage fruity and floral scents and woody scents. The aldehyde and ketone content in the L+W group was higher than that in other groups, and the ketone content in the L+P group was higher than that in the L0 group and the N0 group. Phenolic substances (such as 4-ethylphenol) had a low threshold and often produced pungent odors, and had antioxidant and antifungal effects, while the thresholds of hydrocarbons, ethers, etc. were generally high, and the total amount was small, contributing little to the flavor of pickled Chinese cabbage. PCA analysis further illustrated the flavor differences of pickled Chinese cabbage in different experimental groups, such as Figure 15As shown, a total of 2 principal components were obtained, and the cumulative variance contribution rate reached 55.1%. Among them, the typical volatile substances with a greater contribution to the PC1 principal component were 2-borneol, geraniol, 2-decyn-1-ol, 2-ethyl-1-decanol, β-cyclocitral, coconut aldehyde, etc., and the typical volatile substances with a greater contribution to the PC2 principal component were ethyl pentadecanoate, methyl 7,10,13-hexadecatrienoate, ethyl laurate, (Z)-2-heptenal, phenylacetaldehyde, n-octanal, etc., and the comprehensive scores were all above ±0.16. The differences in pickled cabbages among different experimental groups were obvious, indicating that there were obvious differences in the volatile substance flavors of pickled cabbages fermented with different inoculated strains. The L+W group was below the X-axis, the L+P group, the L0 group and the N0 group were above the X-axis, and the L+W group was the farthest from the three groups, indicating that its flavor difference was the largest. The L+P group and the L0 group were both above the X-axis and to the left of the Y-axis, and the distance was relatively close, meaning that the volatile flavors of the two groups were similar. The N0 group was relatively far from the other three groups, indicating that there were significant differences in flavor between inoculated fermentation and natural fermentation.

[0138] On the 8th day of fermentation, the total acid was lower than that of the control group and the pH was higher than that of the control group (as Figure 1 ) indicating that it could alleviate the excessive acid production in the later stage of pickled vegetable fermentation. During the fermentation process, the peak value of nitrite in the L+W group decreased significantly (as Figure 3 ), improving the safety of pickled vegetables; on the 8th day of fermentation, the total content of volatile substances in the L+W group was higher than that of the lactic acid bacteria group, and the contents of esters, alcohols (the two volatile substances with the greatest impact on the flavor of pickled vegetables), aldehydes and ketones were higher than those of the other three groups except the natural fermentation group, indicating that the L+W group could effectively improve the shortcoming of insufficient flavor in lactic acid bacteria fermentation.

[0139] In the present invention, the physicochemical properties and volatile flavor substances of pickled cabbages fermented by combining Wickerhamomyces anomalus and Pichia kudriavzevii with lactic acid bacteria respectively were analyzed. The results showed that L+W and L+P effectively alleviated the excessive acid production of lactic acid bacteria, inhibited the accumulation of nitrite and reduced the peak value through a metabolic complementary mechanism. The metabolic activities of Wickerhamomyces anomalus promoted the formation of esters (such as allyl isothiocyanate, ethyl palmitate), alcohols (such as phenethyl alcohol, n-pentanol) and aldehydes and ketones (such as dihydro-β-ionone, 4-ethylphenol) in volatile substances. In addition, the yeast enhanced the contents of bitter (tyrosine, methionine) and sweet (threonine) amino acids through protease activity, further strengthening the taste level of pickled cabbages. In addition, on the 8th day of fermentation, the total acid of the L+W group was lower than that of the control group and the pH was higher than that of the control group (as Figure 1 ) indicating that it could alleviate the excessive acid production in the later stage of pickled vegetable fermentation. During the fermentation process, the peak value of nitrite in the L+W group decreased significantly (as Figure 3) This improves the safety of pickled vegetables. On the 8th day of fermentation, the total content of volatile substances in the L+W group was higher than that in the lactic acid bacteria group. The contents of esters, alcohols (the two volatile substances that have the greatest impact on the flavor of pickled vegetables), and aldehydes and ketones were higher than those in the other three groups except the natural fermentation group. Its flavor was significantly different from that of the other three groups, indicating that the L+W group could effectively improve the shortcoming of insufficient flavor in lactic acid bacteria fermentation. The addition of the L+P group generally reduced the content of volatile flavor substances in pickled vegetables, and its flavor was more similar to that of L0 fermentation. In summary, Wickerhamomyces anomalus can be used as a preferred strain for developing pickled vegetables with low nitrite and high ester fragrance.

Claims

1. A composite microbial agent, characterized in that, The composite bacterial agent is selected from any one of the following: (1) The composite bacterial agent comprises Lactobacillus plantarum, Leuconostoc mesenteroides, and Wickerhamomyces anomalus; (2) The composite bacterial agent comprises Lactobacillus plantarum, Leuconostoc mesenteroides, and Pichia kudriavzevii.

2. The composite microbial agent according to claim 1, wherein The preservation number of the Lactobacillus plantarum is CCTCC M2025931; the preservation number of the Leuconostoc mesenteroides is CCTCC M2025932; and / or, the preservation number of the Wickerhamomyces anomalus is CCTCC M2025930.

3. The composite bacterial agent according to claim 1 or 2, characterized in that The ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Wickerhamomyces anomalus in the composite bacterial agent is (80 - 120):(0.5 - 5):(5 - 20); or, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Pichia kudriavzevii in the composite bacterial agent is (80 - 120):(0.5 - 5):(5 - 20); Preferably, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Wickerhamomyces anomalus in the composite bacterial agent is (90 - 100):(0.5 - 2):(8 - 12); or, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides, and Pichia kudriavzevii in the composite bacterial agent is (90 - 100):(0.5 - 2):(8 - 12); The ratio is a quantitative ratio.

4. The composite bacterial agent according to any one of claims 1 to 3, characterized in that, The composite bacterial agent is a liquid preparation, powder, granule, tablet, immobilized bacterial agent, or biochar bacterial agent.

5. A method for preparing a compound microbial agent, characterized in that, The method includes compounding Lactobacillus plantarum, Leuconostoc mesenteroides, and Wickerhamomyces anomalus in a quantitative ratio of (80 - 120):(0.5 - 5):(5 - 20); or, The method includes compounding Lactobacillus plantarum, Leuconostoc mesenteroides, and Pichia kudriavzevii in a quantitative ratio of (80 - 120):(0.5 - 5):(5 - 20); The preservation number of the Lactobacillus plantarum is CCTCC M2025931; the preservation number of the Leuconostoc mesenteroides is CCTCC M2025932; and / or, the preservation number of the Wickerhamomyces anomalus is CCTCC M2025930.

6. A method for preparing fermented vegetables, characterized in that, The method includes contacting the composite bacterial agent as described in any one of claims 1 - 4 with raw materials for fermentation.

7. The method according to claim 6, wherein The temperature of the fermentation is 20 - 30 °C, and / or, the time of the fermentation is 2 - 15 days.

8. The method according to claim 6 or 7, characterized in that The inoculation amount of the compound bacterial agent is as follows: the inoculation amount of Lactiplantibacillus plantarum is (0.8 - 1.2)×10 9 CFU / mL, the inoculation amount of Leuconostoc mesenteroides is (0.5 - 5)×10 7 CFU / mL, and the inoculation amount of Wickerhamomyces anomalus is (0.5 - 2)×10 8 CFU / mL; or, The Lactiplantibacillus plantarum is (0.8 - 1.2) × 10 9 CFU / mL, the Leuconostoc mesenteroides is (0.5 - 5) × 10 7 CFU / mL, and the Pichia kudriavzevii is (0.5 - 2) × 10 8 CFU / mL; Preferably, the Lactiplantibacillus plantarum is (0.9 - 1)×10 9 CFU / mL, the Leuconostoc mesenteroides is (0.5 - 2)×10 7 CFU / mL, and the Wickerhamomyces anomalus is (0.8 - 1.2)×10 8 CFU / mL; or, The Lactiplantibacillus plantarum is (0.9 - 1)×10 9 CFU / mL, the Leuconostoc mesenteroides is (0.5 - 2)×10 7 CFU / mL, and the Pichia kudriavzevii is (0.8 - 1.2)×10 8 CFU / mL.

9. The method according to any one of claims 6-8, characterized in that, The raw material is vegetables, such as radish, mustard, green vegetables, or Chinese cabbage; The method further includes the step of adding auxiliary materials, and the auxiliary materials include water and spices; and / or, The method further includes the step of adding seasonings.

10. Use of the compound bacterial agent according to any one of claims 1 to 4 in the preparation of fermented vegetables.

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