Complex microbial inoculant, preparation method thereof and application of complex microbial inoculant in fermentation of pickled Chinese cabbages

Abstract

The invention belongs to the technical field of microorganisms and food fermentation, and particularly relates to a complex microbial inoculant, a preparation method thereof and application of the complex microbial inoculant in fermentation of pickled Chinese cabbages. The complex microbial inoculant provided by the invention comprises leuconostoc citreum, lactobacillus clavuliformis Z5-1 and yarrowia lipolytica Z6-2, the preservation number of the lactobacillus clavuliformis Z5-1 is CGMCC (China General Microbiological Culture Collection Center) No.31547; the preservation number of the yarrowia lipolytica Z6-2 is CGMCC (China General Microbiological Culture Collection Center) No. 31548. According to the invention, different microorganisms are compounded, so that the leuconostoc citreum can rapidly produce acid, rapidly breed and inhibit the growth of harmful microorganisms; according to the present invention, with the Yarrowia lipolytica Z6-2, the Lactobacillus clavuliformis Z5-1 can produce the terpenoid flavor substance under the synergy of the Yarrowia lipolytica Z6-2, and the Yarrowia lipolytica Z6-2 can inhibit the fermentation peak of the Lactobacillus clavuliformis Z5-1 so as to control the pH value at about 4.5-5, such that the taste and the crispness of the pickled Chinese cabbage can be ensured;

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and food fermentation, and particularly relates to a composite bacterial agent and a preparation method thereof and application thereof in fermenting sauerkraut. Background Art

[0002] Pickled cabbage is a traditional fermented vegetable product in northern my country. It has a unique and refreshing flavor and can increase appetite. The fermented cabbage liquid is rich in lactic acid bacteria, and the aromatic substances produced during its growth and metabolism give the sauerkraut a unique flavor. The fermentation cycle of traditional naturally fermented sauerkraut is relatively long, generally 30 to 45 days, while artificial inoculation of lactic acid bacteria can quickly acidify the fermentation environment and accelerate the fermentation cycle of sauerkraut.

[0003] During the fermentation of sauerkraut, flavor substances are mainly formed through different metabolic pathways of microorganisms. When sauerkraut is naturally fermented, a wide variety of microorganisms cross-promote each other through different metabolic pathways to produce rich metabolites, so the aroma components are relatively complex. However, compared with natural fermentation, the sauerkraut prepared by the existing inoculation of lactic acid bacteria fermentation method uses conventional lactic acid bacteria as the dominant bacteria and controls the degree of fermentation, inhibits the metabolism of other microorganisms in the early stage of fermentation, and reduces the formation of metabolites. Therefore, the components of flavor substances are obviously less, and organic acids and amino acids are different in type and content, resulting in the flavor quality of sauerkraut prepared by the existing lactic acid bacteria fermentation method being significantly lower than that of naturally fermented sauerkraut, which in turn affects its consumption level and consumer acceptability. Therefore, improving the flavor quality of fermented sauerkraut has become a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0004] The purpose of the present invention is to provide a composite bacterial agent and a preparation method thereof and application in fermented sauerkraut, so as to improve the flavor quality of the fermented sauerkraut, ensure the consistency and safety of the sauerkraut, and guarantee the taste and crispness of the sauerkraut.

[0005] The present invention provides a composite bacterial agent, including Leuconostoc citrinum, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2;

[0006] The deposit number of the coryneform Lactobacillus putrefaciens Z5-1 is CGMCC No.31547;

[0007] The deposit number of the Yarrowia lipolytica Z6-2 is CGMCC No.31548.

[0008] Preferably, the viable bacterial count of the composite bacterial agent is ≥ 10 8 CFU / mL.

[0009] Preferably, the ratio of the number of live bacteria of Leuconostoc citrinum, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 in the composite bacterial agent is (1-3): (1-3): (1-2).

[0010] Preferably, the composite bacterial agent includes a degradable adsorption carrier.

[0011] The present invention also provides a method for preparing the composite bacterial agent described in the above technical solution, comprising the following steps:

[0012] Leuconostoc lemon, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 are cultured respectively to obtain Leuconostoc lemon culture solution, Lactobacillus putrefaciens Z5-1 culture solution and Yarrowia lipolytica Z6-2 culture solution;

[0013] The Leuconostoc citrinum culture solution, the Lactobacillus putrefaciens Z5-1 culture solution and the Yarrowia lipolytica Z6-2 culture solution are mixed to obtain the composite bacterial agent.

[0014] Preferably, after mixing the culture solution of Leuconostoc citrinum, the culture solution of Lactobacillus putrefaciens Z5-1 and the culture solution of Yarrowia lipolytica Z6-2, the method further comprises: using a degradable adsorption carrier to adsorb the mixed solution to obtain the composite bacterial agent.

[0015] The present invention also provides application of the composite bacterial agent described in the above technical solution in fermenting sauerkraut.

[0016] The present invention also provides a method for preparing fermented sauerkraut, comprising the following steps: fermenting raw materials for preparing sauerkraut using the composite bacterial agent described in the above technical solution to obtain fermented sauerkraut.

[0017] Preferably, the fermentation temperature is 15-25°C and the fermentation time is 21-35 days.

[0018] Preferably, the inoculation amount of the composite bacterial agent is 5 to 8 wt.%.

[0019] Beneficial effects:

[0020] The present invention provides a composite bacterial agent, comprising Leuconostoc citrinum, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2; the preservation number of the Lactobacillus putrefaciens Z5-1 is CGMCC No. 31547; the preservation number of the Yarrowia lipolytica Z6-2 is CGMCC No. 31548. The present invention compounds different microorganisms, Leuconostoc citrinum produces acid quickly, reproduces quickly, occupies an ecological niche, plays a role in the early stage of fermentation, and inhibits the growth of harmful microorganisms, and the Lactobacillus putrefaciens Z5-1 can produce terpenoid flavor substances in cooperation with the Yarrowia lipolytica Z6-2, and the Yarrowia lipolytica Z6-2 inhibits the fermentation peak of the Lactobacillus putrefaciens Z5-1, so that the pH is controlled at about 4.5 to 5, and the taste and crispness of the sauerkraut are guaranteed.

[0021] Furthermore, the present invention utilizes a degradable adsorption carrier to adsorb Leuconostoc citrinum, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2, and can release live bacteria immediately when put into sauerkraut fermentation raw materials during fermentation.

[0022] Biological deposit information

[0023] Loigolactobacillus coryniformis was deposited on August 5, 2024 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 31547.

[0024] Yarrowia lipolytica Z6-2, biologically classified as Yarrowia lipolytica, was deposited on August 5, 2024 at the China General Microbiological Culture Collection Center, address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, with the deposit number CGMCC No.31548. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 These are the growth curves of Leuconostoc citrinum, Lactobacillus coryneformis (Lactobacillus coryneformis Z5-1) and Yarrowia lipolytica (Yarrowia lipolytica Z6-2). DETAILED DESCRIPTION

[0027] The present invention provides a composite bacterial agent, including Leuconostoc citrinum, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2;

[0028] The deposit number of the coryneform Lactobacillus putrefaciens Z5-1 is CGMCC No.31547;

[0029] The deposit number of the Yarrowia lipolytica Z6-2 is CGMCC No.31548.

[0030] As an embodiment, the viable bacterial count of the composite bacterial agent of the present invention is ≥10 8 CFU / mL; As another embodiment, the viable bacterial count of the composite bacterial agent of the present invention is 10 8 CFU / mL. As an embodiment, the ratio of the number of live bacteria of Leuconostoc citrinum, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 in the composite bacterial agent of the present invention is 2:2:1. As an embodiment, the Leuconostoc citrinum of the present invention is Leuconostoc citrinum BNCC194779. The present invention has no strict requirements on the source of the Leuconostoc citrinum, and conventional purchase is sufficient.

[0031] As an embodiment, the composite bacterial agent of the present invention includes a degradable adsorption carrier. The present invention utilizes a degradable adsorption carrier to adsorb Leuconostoc citrinum, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2, and can release live bacteria immediately when placed in sauerkraut fermentation raw materials during fermentation. As an embodiment, the degradable adsorption carrier of the present invention includes degradable paper. As an embodiment, the thickness of the degradable paper of the present invention can be 500 μm, providing sufficient surface area and adsorption space. The present invention has no strict requirements on the composition of the degradable paper, and it only needs to have degradable properties, for example, it can include cellulose, starch or its derivatives, or natural polymer materials; the natural polymer materials include chitosan and / or sodium alginate. As an embodiment, the cellulose of the present invention is derived from renewable resources such as wood, reeds, bamboo, etc., and has good biodegradability. As an embodiment, the starch or its derivatives of the present invention are used as additives to increase the biodegradation rate. As an embodiment, the natural polymer material of the present invention not only has good biocompatibility, but also can improve the strength and adsorption performance of the degradable paper. As an embodiment, the degradable paper of the present invention may further include a biodegradation promoter, an antibacterial agent or a surfactant to improve the specific properties of the paper. The present invention utilizes degradable paper, and during fermentation, the amount of paper can be put into the sauerkraut fermentation raw material.

[0032] The present invention also provides a method for preparing the composite bacterial agent described in the above technical solution, comprising the following steps:

[0033] Leuconostoc lemon, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 are cultured respectively to obtain Leuconostoc lemon culture solution, Lactobacillus putrefaciens Z5-1 culture solution and Yarrowia lipolytica Z6-2 culture solution;

[0034] The Leuconostoc citrinum culture solution, the Lactobacillus putrefaciens Z5-1 culture solution and the Yarrowia lipolytica Z6-2 culture solution are mixed to obtain the composite bacterial agent.

[0035] The present invention respectively cultures Leuconostoc lemon, rod-shaped Lactobacillus putrefactive Z5-1 and Yarrowia lipolytica Z6-2 to obtain Leuconostoc lemon culture solution, rod-shaped Lactobacillus putrefactive Z5-1 culture solution and Yarrowia lipolytica Z6-2 culture solution. As an embodiment, the culture of Leuconostoc lemon, rod-shaped Lactobacillus putrefactive Z5-1 and Yarrowia lipolytica Z6-2 described in the present invention is MRS culture medium, and the culture temperature is 30°C. The present invention has no strict requirements on the culture methods of Leuconostoc lemon, rod-shaped Lactobacillus putrefactive Z5-1 and Yarrowia lipolytica Z6-2, and the culture can be carried out by conventional culture methods in the art.

[0036] After obtaining the Leuconostoc citrinum culture solution, the rod-shaped Lactobacillus putrefaciens Z5-1 culture solution and the Yarrowia lipolytica Z6-2 culture solution, the present invention mixes the Leuconostoc citrinum culture solution, the rod-shaped Lactobacillus putrefaciens Z5-1 culture solution and the Yarrowia lipolytica Z6-2 culture solution to obtain a mixed solution.

[0037] As an embodiment, after obtaining the mixed solution, the present invention uses a degradable adsorption carrier to adsorb the mixed solution to obtain the composite bacterial agent. As an embodiment, the degradable adsorption carrier of the present invention includes degradable paper.

[0038] The present invention compounds different microorganisms, and Leuconostoc citrinum can quickly produce acid and perform efficient fermentation, thereby quickly occupying an ecological niche, playing a key role in the early stage of fermentation, and effectively inhibiting the growth of harmful microorganisms. The rod-shaped Lactobacillus putrefaciens Z5-1 exhibits its unique function in the late stage of fermentation, and produces rich terpene flavor substances under the synergistic effect with Yarrowia lipolytica Z6-2. At the same time, Yarrowia lipolytica Z6-2 forms a good symbiotic relationship with the rod-shaped Lactobacillus putrefaciens Z5-1, and its excellent lactic acid resistance enables it to slowly produce ethanol. The generated ethanol can undergo an esterification reaction with lactic acid, which not only increases the flavor of the ester aroma, but also reduces the content of lactic acid. In addition, the generated ethanol can also be further utilized by the rod-shaped Lactobacillus putrefaciens Z5-1 to produce more flavor substances. This process cleverly suppresses the fermentation peak of the rod-shaped Lactobacillus putrefaciens Z5-1, stably controls the pH value between 4.5 and 5, and ensures the taste and crispness of the sauerkraut.

[0039] In view of the advantages of the composite bacterial agent provided by the present invention, the application of the composite bacterial agent in fermenting sauerkraut also falls within the protection scope of the present invention.

[0040] The present invention also provides a method for preparing fermented sauerkraut, comprising the following steps: fermenting raw materials for preparing sauerkraut using the composite bacterial agent described in the above technical solution to obtain fermented sauerkraut.

[0041] As an embodiment, the fermentation temperature of the present invention is 15-25°C; as another embodiment, the fermentation temperature of the present invention is 17-19°C. As an embodiment, the fermentation time of the present invention is 21-35 days; as another embodiment, the fermentation time of the present invention is 28 days. As an embodiment, the inoculation amount of the composite bacterial agent of the present invention is 5-8wt.%; as another embodiment, the inoculation amount of the composite bacterial agent of the present invention is 6-7wt.%.

[0042] As an embodiment, the raw materials for preparing sauerkraut of the present invention include Chinese cabbage, salt and water. As an embodiment, the mass volume ratio of the Chinese cabbage and water of the present invention is 4.5kg:2.9-3.0L. As an embodiment, the mass ratio of the total mass of the Chinese cabbage and water of the present invention to the salt is 2.0-3.0:100; as another embodiment, the mass ratio of the total mass of the Chinese cabbage and water of the present invention to the salt is 2.5:100.

[0043] In order to further illustrate the present invention, a composite bacterial agent provided by the present invention, a preparation method thereof and an application thereof in fermenting sauerkraut are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] 1. The inventor team isolated and screened a rod-shaped lactobacillus from sauerkraut liquid, named Z5-1, which was regular and round in MRS medium, off-white, flat, translucent, and smooth and shiny on the surface. The gene of the strain Z5-1 was amplified and sequenced to obtain the nucleotide sequence of the 16s rDNA gene of the strain Z5-1; the sequence was subjected to similarity retrieval and comparative analysis through the BLAST database, and after comparison, the sequence was obtained to have a similarity of 99.93% with the rod-shaped lactobacillus fungal sequence; therefore, the strain Z5-1 was determined to be a rod-shaped corrupt lactobacillus (Loigolactobacillus coryniformis). The rod-shaped corrupt lactobacillus Z5-1 was deposited in the China Center for Type Culture Collection on August 5, 2024, with the deposit number of CGMCCNo.31547.

[0046] 2. The inventor team isolated and screened a strain of Yarrowia lipolytica from the sauerkraut liquid, named Z6-2, which was regular and round in MRS medium, milky white, large and thick, with a moist and sticky surface. The gene of the strain Z6-2 was amplified and sequenced to obtain the nucleotide sequence of the 16s rDNA gene of the strain Z6-2; the sequence was subjected to a similarity search and comparative analysis through the BLAST database, and after comparison, the sequence was found to have a similarity of 99.61% with the Yarrowia fungal sequence; therefore, the strain Z6-2 was determined to be Yarrowia lipolytica. The Yarrowia lipolytica Z6-2 was deposited in the China Center for Type Culture Collection on August 5, 2024, with the deposit number of CGMCCNo.31548.

[0047] Example 2

[0048] Growth curve construction

[0049] Referring to the literature (Wang Yuan, Han Yanqiu, Lv Lianying, et al. Screening and process optimization of citric acid lactic acid bacteria for degrading hawthorn juice [J]. Liaoning Agricultural Science, 2023 (4): 17-24. DOI: 10.3969 / j.issn.1002-1728.2023.04.004.), the growth curves of Leuconostoc citrinum BNCC194779 (purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd.), Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 were drawn respectively. The results are as follows Figure 1 shown.

[0050] according to Figure 1 It can be seen that the rod-shaped putrefactive Lactobacillus Z5-1 (referred to as rod-shaped Lactobacillus) has a larger OD value in the stable period and the best growth condition. The OD values ​​of lemon Leuconostoc BNCC194779 (referred to as lemon Leuconostoc) and Yarrowia lipolytica Z6-2 (referred to as Yarrowia) in the stable period are lower than those of rod-shaped Lactobacillus. When Leuconostoc lemon reaches the maximum OD value, it takes the shortest time and grows the fastest. Therefore, it can be judged that the early fermentation ability of Leuconostoc lemon is stronger, and the fermentation time required is shorter than that of other strains, followed by Yarrowia. The growth peak of rod-shaped Lactobacillus occurs in the middle to late stages, when good anaerobic conditions have been formed, which is conducive to the growth of lactic acid bacteria.

[0051] Example 3

[0052] (1) After activating lemon BNCC194779, rod-shaped Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 in MRS liquid medium for 2 generations, the activated lemon BNCC194779, rod-shaped Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 were cultured respectively to obtain a live cell count of 10 lemon BNCC194779. 8 CFU / mL of culture medium 1, the number of viable bacteria of coryneform putrefactive Lactobacillus Z5-1 reached 10 8 The number of viable cells of culture medium 2 and Yarrowia lipolytica Z6-2 reached 10 8 CFU / mL of culture medium 3.

[0053] (2) The culture solution 1, culture solution 2 and culture solution 3 in step (1) are mixed in a mass ratio of 1:1:1 to obtain a composite starter culture 1.

[0054] Example 4

[0055] The culture solution 1, culture solution 2 and culture solution 3 in step (1) of Example 3 were mixed in a mass ratio of 1:1:2 to obtain a composite starter culture 2.

[0056] Example 5

[0057] The culture solution 1, culture solution 2 and culture solution 3 in step (1) of Example 3 were mixed in a mass ratio of 1:2:1 to obtain a composite starter culture 3.

[0058] Example 6

[0059] The culture solution 1, culture solution 2 and culture solution 3 in step (1) of Example 3 were mixed in a mass ratio of 2:1:1 to obtain a composite starter culture 4.

[0060] Example 7

[0061] The culture solution 1, culture solution 2 and culture solution 3 in step (1) of Example 3 were mixed in a mass ratio of 1:2:2 to obtain a composite starter culture 5.

[0062] Example 8

[0063] The culture solution 1, culture solution 2 and culture solution 3 in step (1) of Example 3 were mixed in a mass ratio of 2:1:2 to obtain a composite starter culture 6.

[0064] Example 9

[0065] The culture solution 1, culture solution 2 and culture solution 3 in step (1) of Example 3 were mixed in a mass ratio of 2:2:1 to obtain a composite starter culture 7.

[0066] Test Example 1

[0067] Effects of different compound fermentation agents on sauerkraut fermentation

[0068] 1. Cabbage pretreatment

[0069] Dry fresh yellow-heart Chinese cabbage outdoors for 2 to 3 days (to wilt and remove some moisture), remove rotten and dead leaves, cut off the roots and wash them clean, and cut the whole cabbage into four halves along the long axis.

[0070] 2. About 4.5 kg of pretreated Chinese cabbage was loaded into a fermentation tank and compacted and the air was exhausted, about 2.9 L of water was added, and salt was added according to 2.0% of the total weight. The composite fermentation agents 1 to 7 obtained in Examples 3 to 9 were inoculated at an inoculation amount of 5 wt.% of the cabbage mass, respectively, and sealed and fermented at 17°C for 28 days. The natural fermentation treatment without inoculation of the fermentation agent was used as a blank control, that is, about 4.5 kg of pretreated Chinese cabbage was loaded into a fermentation tank and compacted and the air was exhausted, about 2.9 L of water was added, and salt was added according to 2.0% of the total weight, and sealed and fermented at 17°C for 28 days; three parallel groups were set for each treatment method. Taking sensory evaluation as an indicator, the fermentation effect of different composite fermentation agents was evaluated according to the sauerkraut sensory scoring standard (Table 1), and the results are shown in Table 2.

[0071] Table 1 Sensory scoring standards for pickled cabbage

[0072]

[0073] Table 2 Sensory evaluation results of different strain compound ratios

[0074]

[0075]

[0076] According to Table 2, the total bacterial count of the composite fermentation agent reached 10 8 CFU / mL, and the composite ratio of Leuconostoc citrinum BNCC194779, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 was 2:2:1, the sensory effect was the best.

[0077] Test Example 2

[0078] Effects of different inoculation amounts of composite fermentation agents on sauerkraut fermentation

[0079] 1. Cabbage pretreatment

[0080] Dry fresh yellow-heart Chinese cabbage outdoors for 2 to 3 days (to wilt and remove some moisture), remove rotten and dead leaves, cut off the roots and wash them clean, and cut the whole cabbage into four halves along the long axis.

[0081] 2. About 4.5 kg of the pretreated Chinese cabbage was loaded into a fermentation tank, compacted and air was exhausted, about 2.9 L of water was added, salt was added according to 2.0% of the total weight, the composite fermentation agent 7 obtained in Example 9 was inoculated according to different cabbage mass percentages (2wt.%, 5wt.%, 8wt.% and 11wt.%), and the mixture was sealed and fermented at 17±2°C for 28 days. The natural fermentation treatment without inoculation of the fermentation agent was used as a blank control, that is, about 4.5 kg of the pretreated Chinese cabbage was loaded into a fermentation tank, compacted and air was exhausted, about 2.9 L of water was added, salt was added according to 2.0% of the total weight, and the mixture was sealed and fermented at 17±2°C for 28 days; three parallel groups were set for each treatment method, and the fermentation effect of different inoculation amounts was evaluated in the manner of Test Example 1, and the results are shown in Table 3.

[0082] Table 3 Sensory evaluation results of different inoculation amounts of composite starter

[0083]

[0084] It can be seen from Table 3 that the inoculation amount of the composite leavening agent should be controlled at 5wt.%~8wt.%. Although a high inoculation amount can shorten the fermentation time, it may sometimes make the flavor development insufficient, resulting in a single taste of the product; a low inoculation amount may result in insufficient number of beneficial bacteria to dominate, allowing miscellaneous bacteria in the environment to take advantage of the situation and cause poor flavor or corruption problems.

[0085] Test Example 3

[0086] Experiment on different fermentation temperatures of compound starter

[0087] 1. Cabbage pretreatment

[0088] Dry fresh yellow-heart Chinese cabbage outdoors for 2 to 3 days (to wilt and remove some moisture), remove rotten and dead leaves, cut off the roots and wash them clean, and cut the whole cabbage into four halves along the long axis.

[0089] 2. About 4.5 kg of the pretreated Chinese cabbage was loaded into a fermentation tank and compacted and the air was exhausted, about 2.9 L of water was added, and salt was added according to 2.0% of the total weight, and the composite fermentation agent 7 obtained in Example 9 was inoculated at an inoculum amount of 5 wt.% of the cabbage mass, and sealed and fermented for 28 days at different temperatures (14°C, 17°C, 20°C and 23°C). The natural fermentation treatment without inoculation of the fermentation agent was used as a blank control, that is, about 4.5 kg of the pretreated Chinese cabbage was loaded into a fermentation tank and compacted and the air was exhausted, about 2.9 L of water was added, and salt was added according to 2.0% of the total weight, and sealed and fermented for 28 days at different temperatures (14°C, 17°C, 20°C and 23°C); three parallel groups were set for each treatment method, and the fermentation effect of different fermentation temperatures was evaluated in accordance with the method of step test example 1, and the results are shown in Table 4.

[0090] Table 4 Sensory evaluation results of composite starter at different fermentation temperatures

[0091]

[0092] According to Table 4, it is ideal to control the temperature between 15 and 19°C. This range is conducive to the growth of lactic acid bacteria and yeasts but not conducive to the reproduction of harmful microorganisms. Too high a temperature may speed up the fermentation process, but it may also cause contamination by bacteria or make the product too soft; too low a temperature will slow down the fermentation process and may not achieve the ideal sourness level as expected.

[0093] Test Example 4

[0094] Experiment on different fermentation time of compound leavening agent

[0095] 1. Cabbage pretreatment

[0096] Dry fresh yellow-heart Chinese cabbage outdoors for 2 to 3 days (to wilt and remove some moisture), remove rotten and dead leaves, cut off the roots and wash them clean, and cut the whole cabbage into four halves along the long axis.

[0097] 2. About 4.5 kg of the pretreated Chinese cabbage was loaded into a fermentation tank and compacted and the air was exhausted, about 2.9 L of water was added, salt was added according to 2.0% of the total weight, the composite fermentation agent 7 obtained in Example 9 was inoculated at an inoculum amount of 5 wt.% of the cabbage mass, and the mixture was sealed and fermented at 17°C. The natural fermentation treatment without inoculation of the fermentation agent was used as a blank control, that is, about 4.5 kg of the pretreated Chinese cabbage was loaded into a fermentation tank and compacted and the air was exhausted, about 2.9 L of water was added, salt was added according to 2.0% of the total weight, and the mixture was sealed and fermented at 17°C for different time periods (21d, 28d, 35d and 42d); three parallel groups were set for each treatment method, and the fermentation effect of different fermentation times was evaluated in the manner of Test Example 1, and the results are shown in Table 5.

[0098] Table 5 Sensory evaluation results of composite leavening agents at different fermentation times

[0099]

[0100] According to Table 5, after 3 to 5 weeks of fermentation (21d to 35d), lactic acid bacteria fully act on vegetables, causing them to produce enough lactic acid to lower the pH value and develop a unique flavor. The actual time may be adjusted according to environmental conditions and personal preferences. It is recommended to check the state of the sauerkraut regularly until a satisfactory taste is achieved. After the fermentation time exceeds 5 weeks, the sauerkraut tastes too sour, the texture becomes soft, the color becomes dark, and the fermentation is over-produced. After the introduction of yeast to participate in the fermentation process, due to the production of ethanol, it undergoes an esterification reaction with lactic acid to produce aromatic substances, while making the pH value of the final product slightly higher, at around 4.0 to 4.6.

[0101] Comparative Example 1

[0102] The culture solution 1 and the culture solution 2 in step (1) of Example 3 were mixed in a mass ratio of 2:2 to obtain a composite starter culture 8.

[0103] Comparative Example 2

[0104] The culture solution 1 and the culture solution 3 in step (1) of Example 3 were mixed in a mass ratio of 2:1 to obtain a composite starter culture 9.

[0105] Comparative Example 3

[0106] The culture solution 2 and the culture solution 3 in step (1) of Example 3 were mixed in a mass ratio of 2:1 to obtain a composite starter culture 10.

[0107] Test Example 5

[0108] Fermentation test with different starter cultures

[0109] 1. Cabbage pretreatment

[0110] Dry fresh yellow-heart Chinese cabbage outdoors for 2 to 3 days (to wilt and remove some moisture), remove rotten and dead leaves, cut off the roots and wash them clean, and cut the whole cabbage into four halves along the long axis.

[0111] 2. About 4.5 kg of the pretreated Chinese cabbage was loaded into a fermentation tank, compacted and the air was exhausted, about 2.9 L of water was added, salt was added according to 2.0% of the total weight, and the composite fermentation agents 1 to 7 obtained in Examples 3 to 9, the culture solutions 1 to 3 obtained in Example 3, and the composite fermentation agents 8 to 10 obtained in Comparative Examples 1 to 3 were inoculated at an inoculation amount of 5 wt.% of the mass of the cabbage, respectively, and sealed and fermented at 17° C. for 28 days. Three parallel groups were set for each treatment method.

[0112] 3. Referring to the literature (Tian Jiaxue. Preparation of Northeastern sauerkraut composite fermentation agent and its influence on sauerkraut quality [D]. Jilin University. 2024.), the quantity of main flavor components of the fermented sauerkraut prepared above was detected, and the results are shown in Table 6.

[0113] Table 6 Quantities of main flavor components of sauerkraut fermented by different methods

[0114]

[0115] According to Table 6, it can be seen that composite starter 7 performs well in the quantity of major flavor components such as alcohols, esters, ketones, sulfur-containing compounds, nitrogen-containing compounds and amino acids, especially in esters, ketones, sulfur-containing compounds, nitrogen-containing compounds and amino acids. This shows that composite starter 7 can effectively promote the production of multiple flavor components, thereby improving the overall flavor and quality of fermented sauerkraut.

[0116] 4. Referring to the literature (Tian Jiaxue. Preparation of Northeastern sauerkraut composite fermentation agent and its influence on sauerkraut quality [D]. Jilin University. 2024.; Zhao Yujuan, Liu Caizi, Gao Yansong, et al. Screening and evaluation of lactic acid bacteria for fermentation of Northeastern sauerkraut [J]. Modern Food Science and Technology, 2024, 40(3): 121-130.), the crispness (d), nitrite (mg / Kg), sulfur dioxide residue (g / Kg), Escherichia coli content and pH of the fermented sauerkraut prepared by culture medium 1 and composite fermentation agent 7 were detected, and the natural fermentation treatment without inoculation of fermentation agent was used as the control. The results are shown in Table 7.

[0117] Table 7 Crispness and safety evaluation results of sauerkraut fermented in different ways

[0118] project Natural fermentation Culture medium 1 Compound fermentation agent 7 Brittleness(N) 17 19 21 Nitrite(mg / Kg) 3.57 0.34 0.12 Sulfur dioxide residue (g / Kg) Not detected Not detected Not detected Escherichia coli (CFU / g) 3.16 Not detected Not detected pH 3.5 3.3 4.3

[0119] According to Table 7, compared with natural fermentation and fermentation using only Leuconostoc citrinum, the sauerkraut obtained by using composite fermentation agent 7 has the best crispness, reaching 21N, indicating that the sauerkraut is crisp and tender, and the residual nitrite, sulfur dioxide and Escherichia coli all meet or exceed the food safety standards, indicating that the fermented sauerkraut has excellent safety performance. Composite fermentation agent 7 promotes the esterification reaction of lactic acid and ethanol, adjusts the acidity of sauerkraut, and improves the taste. The obtained sauerkraut has moderate acidity and is more acceptable to people. Single Leuconostoc citrinum makes sauerkraut too sour, and natural fermentation acid production is difficult to control. Overall, the sauerkraut fermented with composite fermentation agent 7 not only has good crispness, but also meets high standards in terms of safety. It is a high-quality fermented sauerkraut product.

[0120] It can be seen from the above content that the composite bacterial agent provided by the present invention can improve the flavor quality of fermented sauerkraut, ensure the consistency and safety of the sauerkraut, and guarantee the taste and crispness of the sauerkraut.

[0121] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composite bacterial agent, characterized in that: including Leuconostoc citreum, Loigolactobacillus coryniformis Z5-1, and Yarrowialipolytica Z6-2; The deposit number of the coryneform Lactobacillus putrefaciens Z5-1 is CGMCC No.31547; The deposit number of the Yarrowia lipolytica Z6-2 is CGMCC No.31548.

2. The composite bacterial agent according to claim 1, characterized in that: The viable bacterial count of the composite bacterial agent is ≥10 8 CFU / mL.

3. The composite bacterial agent according to claim 2, characterized in that: The ratio of the number of live bacteria of Leuconostoc citrinum, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 in the composite bacterial agent is (1-3): (1-3): (1-2).

4. The composite bacterial agent according to any one of claims 1 to 3, characterized in that: The composite bacterial agent comprises a degradable adsorption carrier.

5. The method for preparing the composite bacterial agent according to any one of claims 1 to 4, characterized in that: The steps include: Leuconostoc lemon, Lactobacillus putrefaciens Z5-1 and Yarrowia lipolytica Z6-2 are cultured respectively to obtain Leuconostoc lemon culture solution, Lactobacillus putrefaciens Z5-1 culture solution and Yarrowia lipolytica Z6-2 culture solution; The Leuconostoc citrinum culture solution, the Lactobacillus putrefaciens Z5-1 culture solution and the Yarrowia lipolytica Z6-2 culture solution are mixed to obtain the composite bacterial agent.

6. The preparation method according to claim 5, characterized in that: After the Leuconostoc citrinum culture solution, the Lactobacillus putrefaciens Z5-1 culture solution and the Yarrowia lipolytica Z6-2 culture solution are mixed, the method further comprises: using a degradable adsorption carrier to adsorb the mixed solution to obtain the composite bacterial agent.

7. Use of the composite bacterial agent according to any one of claims 1 to 4 or the composite bacterial agent obtained by the preparation method according to claim 5 or 6 in fermenting sauerkraut.

8. A method for preparing fermented sauerkraut, characterized in that: The method comprises the following steps: fermenting raw materials for preparing sauerkraut using the composite bacterial agent described in any one of claims 1 to 4 or the composite bacterial agent obtained by the preparation method described in claim 5 or 6 to obtain fermented sauerkraut.

9. The preparation method according to claim 8, characterized in that: The fermentation temperature is 15-25°C and the fermentation time is 21-35 days.

10. The preparation method according to claim 8, characterized in that: The inoculation amount of the composite bacterial agent is 5-8wt.%.

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