A compound microbial inoculant, a preparation method thereof and application thereof in fermented pickled cabbage

Abstract

This invention belongs to the field of microbial and food fermentation technology, specifically relating to a compound microbial agent, its preparation method, and its application in fermented sauerkraut. The compound microbial agent provided by this invention includes *Leuconostoc mesenteroides*, *Lactobacillus corynebacterium* Z5-1, and *Jeromea lipolytica* Z6-2; the preservation number of *Lactobacillus corynebacterium* Z5-1 is CGMCC No. 31547; the preservation number of *Jeromea lipolytica* Z6-2 is CGMCC No. 31548. This invention combines different microorganisms. *Leuconostoc mesenteroides* can rapidly produce acid and multiply rapidly, inhibiting the growth of harmful microorganisms; *Lactobacillus corynebacterium* Z5-1 can produce terpenoid flavor substances in synergy with *Jeromea lipolytica* Z6-2, while *Jeromea lipolytica* Z6-2 inhibits the fermentation peak of *Lactobacillus corynebacterium* Z5-1, keeping the pH controlled at around 4.5-5, thus ensuring the taste and crispness of the sauerkraut.

Description

Technical Field

[0001] This invention belongs to the field of microbial and food fermentation technology, specifically relating to a compound microbial agent, its preparation method, and its application in fermented sauerkraut. Background Technology

[0002] Sauerkraut, made from Chinese cabbage, is a traditional fermented vegetable product in northern my country. It has a unique, refreshing flavor and can stimulate the appetite. The fermentation liquid contains abundant lactic acid bacteria, and the aromatic substances produced during their growth and metabolism give sauerkraut its distinctive flavor. Traditional natural fermentation of sauerkraut has a relatively long fermentation period, generally 30-45 days, while artificial inoculation with lactic acid bacteria can quickly acidify the fermentation environment and accelerate the fermentation cycle.

[0003] During the fermentation of sauerkraut, flavor compounds are mainly formed through different metabolic pathways of microorganisms. In natural fermentation, a wide variety of microorganisms interact and promote each other through different metabolic pathways, resulting in a rich array of metabolites and thus a complex aroma composition. However, compared to natural fermentation, sauerkraut prepared using existing lactic acid bacteria inoculation methods, due to the use of conventional lactic acid bacteria as the dominant bacteria and the control of the fermentation degree, inhibits the metabolism of other microorganisms in the early stages of fermentation, reducing the formation of metabolic products. Therefore, the composition of flavor compounds is significantly lower, and there are differences in the types and amounts of organic acids and amino acids. Consequently, the flavor quality of sauerkraut prepared using existing lactic acid bacteria fermentation methods is significantly lower than that of naturally fermented sauerkraut, thus affecting its consumption level and consumer acceptance. Therefore, improving the flavor quality of fermented sauerkraut has become a pressing technical problem that needs to be solved by those skilled in the art. Summary of the Invention

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

[0005] This invention provides a compound microbial agent comprising Leuconostoc mesenteroides, Lactobacillus corynebacterium Z5-1, and Jeromea lipolytica Z6-2;

[0006] The accession number of the Lactobacillus putrefactivee Z5-1 is CGMCC No. 31547;

[0007] The preservation number of the described *Yerovia lipophila* Z6-2 is CGMCC No. 31548.

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

[0009] Preferably, the ratio of viable bacteria of Leuconostoc mesenteroides, Lactobacillus putrefactivee Z5-1 and Yerovia lipolytica Z6-2 in the compound microbial agent is (1~3):(1~3):(1~2).

[0010] Preferably, the composite microbial agent includes a biodegradable adsorbent carrier.

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

[0012] Leuconostoc melanocytoxin, Lactobacillus putrefactivee Z5-1 and Jeroensis lipolytica Z6-2 were cultured separately to obtain Leuconostoc melanocytoxin culture medium, Lactobacillus putrefactivee Z5-1 culture medium and Jeroensis lipolytica Z6-2 culture medium;

[0013] The compound bacterial agent is obtained by mixing the culture medium of Leuconostoc mesenteroides, the culture medium of Lactobacillus putrefactiveus Z5-1, and the culture medium of Yersinia lipolytica Z6-2.

[0014] Preferably, after mixing the Leuconostoc mesenteroides culture medium, Lactobacillus putrefactive strain Z5-1 culture medium and Yersinia lipolyticis Z6-2 culture medium, the mixture further includes: adsorbing the mixture using a biodegradable adsorption carrier to obtain the composite microbial agent.

[0015] The present invention also provides the application of the compound microbial agent described in the above technical solution in fermented sauerkraut.

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

[0017] Preferably, the fermentation temperature is 15~25℃ and the time is 21~35 days.

[0018] Preferably, the inoculation amount of the compound microbial agent is 5-8 wt.%.

[0019] Beneficial effects:

[0020] This invention provides a compound microbial agent comprising *Leuconostoc mesenteroides*, *Lactobacillus corynebacterium* Z5-1, and *Jeromea lipolytica* Z6-2; the preservation number of *Lactobacillus corynebacterium* Z5-1 is CGMCC No. 31547; and the preservation number of *Jeromea lipolytica* Z6-2 is CGMCC No. 31548. This invention combines different microorganisms. *Leuconostoc mesenteroides* rapidly produces acid, multiplies rapidly, occupies an ecological niche, and plays a role in the early stage of fermentation, inhibiting the growth of harmful microorganisms. *Lactobacillus corynebacterium* Z5-1 can produce terpenoid flavor substances in synergy with *Jeromea lipolytica* Z6-2, while *Jeromea lipolytica* Z6-2 inhibits the fermentation peak of *Lactobacillus corynebacterium* Z5-1, keeping the pH at around 4.5-5, thus ensuring the taste and crispness of the sauerkraut.

[0021] Furthermore, this invention utilizes a biodegradable adsorbent carrier that can adsorb Leuconostoc mesenteroides, Lactobacillus putrefactiveus Z5-1, and Jeromea lipolytica Z6-2. When added to the sauerkraut fermentation raw materials during fermentation, the live bacteria are released immediately.

[0022] Biological Preservation Information

[0023] Lactobacillus corynebacterium Z5-1, biologically classified as Loigolactobacillus coryniformis It 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 accession number CGMCC No. 31547.

[0024] Yersinia lipolyticis Z6-2 is biologically classified as Yersinia lipolyticis. Yarrowia lipolytica It 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 accession number CGMCC No. 31548. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0026] Figure 1 The growth curves are for Leuconostoc melanogaster, Lactobacillus corynebacterium (Lactobacillus corynebacterium putrefactivee Z5-1), and Yersinia lipolytica (Yersinia lipolytica Z6-2). Detailed Implementation

[0027] This invention provides a compound microbial agent comprising Leuconostoc mesenteroides, Lactobacillus corynebacterium Z5-1, and Jeromea lipolytica Z6-2;

[0028] The accession number of the Lactobacillus putrefactivee Z5-1 is CGMCC No. 31547;

[0029] The preservation number of the described *Yerovia lipophila* Z6-2 is CGMCC No. 31548.

[0030] As one embodiment, the viable count of the compound microbial agent of the present invention is ≥10. 8 CFU / mL; as another embodiment, the viable count of the compound bacterial agent of the present invention is 10. 8 CFU / mL. As one embodiment, the viable cell ratio of *Leuconostoc tumefaciens*, *Lactobacillus putrefactiveus* Z5-1, and *Yerovia lipolyticis* Z6-2 in the compound microbial agent of this invention is 2:2:1. As one embodiment, the *Leuconostoc tumefaciens* mentioned in this invention is *Leuconostoc tumefaciens* BNCC194779. This invention does not have strict requirements regarding the source of the *Leuconostoc tumefaciens*; it can be purchased conventionally.

[0031] In one embodiment, the composite microbial agent of the present invention includes a biodegradable adsorbent carrier. The present invention utilizes a biodegradable adsorbent carrier to adsorb *Leuconostoc tumefaciens*, *Lactobacillus corynebacterium* Z5-1, and *Yerovia lipolyticis* Z6-2, which immediately release live bacteria when added to the sauerkraut fermentation raw material during fermentation. In another embodiment, the biodegradable adsorbent carrier of the present invention includes biodegradable paper. In another embodiment, the thickness of the biodegradable paper of the present invention can be 500 μm, providing sufficient surface area and adsorption space. The present invention does not have strict requirements on the composition of the biodegradable paper, as long as it has biodegradable 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. In another embodiment, the cellulose of the present invention is derived from renewable resources such as wood, reeds, and bamboo, and has good biodegradability. In another embodiment, the starch or its derivatives of the present invention are used as additives to increase the biodegradation rate. In yet another embodiment, the natural polymer materials of the present invention not only have good biocompatibility but also improve the strength and adsorption performance of the biodegradable paper. In one embodiment, the biodegradable paper of the present invention may further include biodegradation promoters, antibacterial agents, or surfactants to improve specific properties of the paper. The present invention utilizes biodegradable paper, which is added to the sauerkraut fermentation raw materials according to the specified quantity during fermentation.

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

[0033] Leuconostoc melanocytoxin, Lactobacillus putrefactivee Z5-1 and Jeroensis lipolytica Z6-2 were cultured separately to obtain Leuconostoc melanocytoxin culture medium, Lactobacillus putrefactivee Z5-1 culture medium and Jeroensis lipolytica Z6-2 culture medium;

[0034] The compound bacterial agent is obtained by mixing the culture medium of Leuconostoc mesenteroides, the culture medium of Lactobacillus putrefactiveus Z5-1, and the culture medium of Yersinia lipolytica Z6-2.

[0035] This invention involves culturing *Leuconostoc tectorum*, *Lactobacillus corynebacterium* Z5-1, and *Jeromea lipolytica* Z6-2, respectively, to obtain *Leuconostoc tectorum* culture media, *Lactobacillus corynebacterium* Z5-1 culture media, and *Jeromea lipolytica* Z6-2 culture media. As one embodiment, the *Leuconostoc tectorum*, *Lactobacillus corynebacterium* Z5-1, and *Jeromea lipolytica* Z6-2 are cultured on MRS medium at 30°C. The cultivation methods for *Leuconostoc tectorum*, *Lactobacillus corynebacterium* Z5-1, and *Jeromea lipolytica* Z6-2 are not strictly required; conventional cultivation methods in the art are sufficient.

[0036] After obtaining the Leuconostoc mesenteroides culture medium, Lactobacillus rubrum Z5-1 culture medium, and Jeromea lipolytica Z6-2 culture medium, the present invention mixes the Leuconostoc mesenteroides culture medium, Lactobacillus rubrum Z5-1 culture medium, and Jeromea lipolytica Z6-2 culture medium to obtain a mixed solution.

[0037] In one embodiment, after obtaining the mixture, the present invention uses a biodegradable adsorbent carrier to adsorb the mixture, thereby obtaining the composite microbial agent. In another embodiment, the biodegradable adsorbent carrier of the present invention includes biodegradable paper.

[0038] This invention combines different microorganisms. *Leuconostoc mesenteroides* can rapidly produce acid and undergo efficient fermentation, quickly occupying its ecological niche and playing a crucial role in the early stages of fermentation, effectively inhibiting the growth of harmful microorganisms. *Lactobacillus putrefactiveus* Z5-1, on the other hand, exhibits its unique function in the later stages of fermentation, producing abundant terpenoid flavor compounds in synergy with *Jeromes lipolyticus* Z6-2. Simultaneously, *Jeromes lipolyticus* Z6-2 forms a good symbiotic relationship with *Lactobacillus putrefactiveus* Z5-1, and its excellent lactic acid tolerance allows it to slowly produce ethanol. The produced ethanol can undergo esterification with lactic acid, not only increasing the ester-like flavor but also reducing the lactic acid content. Furthermore, the generated ethanol can be further utilized by *Lactobacillus putrefactiveus* Z5-1 to produce more flavor compounds. This process cleverly suppresses the fermentation peak of *Lactobacillus putrefactiveus* Z5-1, stabilizing the pH value between 4.5 and 5, ensuring the texture and crispness of the sauerkraut.

[0039] Given the advantages of the compound microbial agent provided by this invention, its application in fermented sauerkraut also falls within the scope of protection of this invention.

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

[0041] In one embodiment, the fermentation temperature of the present invention is 15~25℃; in another embodiment, the fermentation temperature of the present invention is 17~19℃. In one embodiment, the fermentation time of the present invention is 21~35 days; in another embodiment, the fermentation time of the present invention is 28 days. In one embodiment, the inoculum amount of the compound microbial agent of the present invention is 5~8 wt.%; in another embodiment, the inoculum amount of the compound microbial agent of the present invention is 6~7 wt.%.

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

[0043] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a compound microbial agent, its preparation method, and its application in fermented sauerkraut, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] 1. The inventors' team isolated and screened a strain of *Lactobacillus corynebacterium* from sauerkraut liquid, named Z5-1. In MRS medium, it appears as a regularly round, grayish-white, flat, translucent bacterium with a smooth and glossy surface. The gene of strain Z5-1 was amplified and sequenced, obtaining the nucleotide sequence of its 16S rDNA gene. This sequence was then compared using the BLAST database for similarity analysis. The results showed a 99.93% similarity to the *Lactobacillus corynebacterium* fungal sequence. Therefore, strain Z5-1 was identified as *Lactobacillus corynebacterium* (*Lactobacillus corynebacterium*). Loigolactobacillus coryniformis The Lactobacillus putrefactive strain Z5-1 was deposited at the China Center for Type Culture Collection (CGMCC) on August 5, 2024, with accession number CGMCC No. 31547.

[0046] 2. The inventors' team isolated and screened a strain of *Yerovia lipolytica* from sauerkraut extract, named Z6-2. In MRS medium, it appears as a regularly round, milky-white, relatively large and thick, moist and viscous surface. The genes of strain Z6-2 were amplified and sequenced, obtaining the nucleotide sequence of its 16S rDNA gene. This sequence was then compared using the BLAST database for similarity analysis. The results showed a 99.61% similarity to the *Yerovia lipolytica* fungal sequence. Therefore, strain Z6-2 was identified as *Yerovia lipolytica*. Yarrowia lipolytica The lipolytic yeast Z6-2 was deposited at the China Center for Type Culture Collection on August 5, 2024, with accession number CGMCC No. 31548.

[0047] Example 2

[0048] Establishment of growth curves

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

[0050] according to Figure 1 It can be seen that *Lactobacillus putrefactiveus* Z5-1 (referred to as *Lactobacillus*) has a larger OD value when it reaches its stationary phase, indicating the best growth. *Leuconostoc mesenteroides* BNCC194779 (referred to as *Leuconostoc mesenteroides*) and *Yerovia lipolytica* Z6-2 (referred to as *Yerovia lipolytica*) have lower OD values ​​when they reach their stationary phase than *Lactobacillus*. *Leuconostoc mesenteroides* reaches its maximum OD value in the shortest time and has the fastest growth rate. Therefore, it can be determined that *Leuconostoc mesenteroides* has a stronger early fermentation ability and requires a shorter fermentation time than other strains, followed by *Yerovia lipolytica*. The peak growth of *Lactobacillus* occurs in the middle to late stages, at which point favorable anaerobic conditions have been established, which is conducive to the proliferation of lactic acid bacteria.

[0051] Example 3

[0052] (1) After activating Leuconostoc mesenteroides BNCC194779, Lactobacillus corynebacterium Z5-1, and Jeromea lipolytica Z6-2 for two generations in MRS liquid medium, the activated Leuconostoc mesenteroides BNCC194779, Lactobacillus corynebacterium Z5-1, and Jeromea lipolytica Z6-2 were cultured to obtain a viable count of Leuconostoc mesenteroides BNCC194779 of 10. 8 In a culture medium containing CFU / mL, the viable count of *Lactobacillus putrefactiveis* Z5-1 reached 10. 8 The viable count of CFU / mL culture medium 2 and *Yerovia lipophila* Z6-2 reached 10. 8 3. CFU / mL culture medium.

[0053] (2) Mix culture medium 1, culture medium 2 and culture medium 3 from step (1) in a mass ratio of 1:1:1 to obtain compound fermentation agent 1.

[0054] Example 4

[0055] The culture medium 1, culture medium 2 and culture medium 3 from step (1) of Example 3 were mixed in a mass ratio of 1:1:2 to obtain compound fermentation agent 2.

[0056] Example 5

[0057] The culture medium 1, culture medium 2 and culture medium 3 from step (1) of Example 3 are mixed in a mass ratio of 1:2:1 to obtain the composite fermentation agent 3.

[0058] Example 6

[0059] The culture medium 1, culture medium 2 and culture medium 3 from step (1) of Example 3 are mixed in a mass ratio of 2:1:1 to obtain the composite fermentation agent 4.

[0060] Example 7

[0061] The culture medium 1, culture medium 2 and culture medium 3 from step (1) of Example 3 are mixed in a mass ratio of 1:2:2 to obtain the composite fermentation agent 5.

[0062] Example 8

[0063] The culture medium 1, culture medium 2 and culture medium 3 from step (1) of Example 3 are mixed in a mass ratio of 2:1:2 to obtain the composite fermentation agent 6.

[0064] Example 9

[0065] The culture medium 1, culture medium 2 and culture medium 3 from step (1) of Example 3 are mixed in a mass ratio of 2:2:1 to obtain the composite fermentation agent 7.

[0066] Test Example 1

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

[0068] 1. Cabbage pretreatment

[0069] Fresh yellow-hearted Chinese cabbage is sun-dried outdoors for 2-3 days (until it wilts and some moisture is removed). Rotten and withered leaves are removed, the roots are cut off and the cabbage is washed clean. The whole cabbage is then cut into quarters along its long axis.

[0070] 2. Approximately 4.5 kg of pretreated Chinese cabbage was packed into a fermentation tank, compacted, and the air was removed. About 2.9 L of water was added, along with 2.0% salt by weight. The compound fermentation agents 1-7 obtained in Examples 3-9 were inoculated at 5 wt.% of the cabbage weight, and the mixture was sealed and fermented at 17°C for 28 days. Natural fermentation without inoculation was used as a blank control: approximately 4.5 kg of pretreated Chinese cabbage was packed into a fermentation tank, compacted, and the air was removed. About 2.9 L of water was added, along with 2.0% salt by weight, and the mixture was sealed and fermented at 17°C for 28 days. Three parallel groups were set up for each treatment method. Sensory evaluation was used as an indicator, and the fermentation effect of different compound fermentation agents was evaluated according to the sauerkraut sensory scoring standard (Table 1). The results are shown in Table 2.

[0071] Table 1 Sensory Evaluation Criteria for Sauerkraut

[0072]

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

[0074]

[0075] As shown in Table 2, the total bacterial count of the compound fermentation agent reached 10. 8 The sensory effect was best when the combined ratio of Leuconostoc mesenteroides BNCC194779, Lactobacillus putrefactiveus Z5-1, and Jeromea lipolyticis Z6-2 was 2:2:1.

[0076] Test Example 2

[0077] Effects of different inoculum amounts of compound fermenting agent on sauerkraut fermentation

[0078] 1. Cabbage pretreatment

[0079] Fresh yellow-hearted Chinese cabbage is sun-dried outdoors for 2-3 days (until it wilts and some moisture is removed). Rotten and withered leaves are removed, the roots are cut off and the cabbage is washed clean. The whole cabbage is then cut into quarters along its long axis.

[0080] 2. Approximately 4.5 kg of pretreated Chinese cabbage was packed into a fermentation tank, compacted, and the air was removed. Approximately 2.9 L of water was added, along with 2.0% salt by weight. The compound fermentation agent 7 obtained in Example 9 was inoculated with different percentages of Chinese cabbage (2 wt.%, 5 wt.%, 8 wt.%, and 11 wt.%), and the mixture was sealed and fermented at 17±2℃ for 28 days. Natural fermentation without inoculation was used as a blank control: approximately 4.5 kg of pretreated Chinese cabbage was packed into a fermentation tank, compacted, and the air was removed. Approximately 2.9 L of water was added, along with 2.0% salt by weight, and the mixture was sealed and fermented at 17±2℃ for 28 days. Three parallel groups were set up for each treatment method, and the fermentation effect of different inoculation amounts was evaluated according to the method in Test Example 1. The results are shown in Table 3.

[0081] Table 3 Sensory evaluation results of different inoculum amounts of compound fermentation agent

[0082]

[0083] As shown in Table 3, the inoculum amount of the compound fermentation agent should be controlled between 5 wt.% and 8 wt.%. Although an excessively high inoculum amount can shorten the fermentation time, it may sometimes result in insufficient flavor development and a monotonous product taste. An excessively low inoculum amount may result in insufficient beneficial bacteria to dominate, allowing other bacteria in the environment to take advantage of the situation, causing poor flavor or spoilage.

[0084] Test Example 3

[0085] Experiments on different fermentation temperatures of compound fermentation agents

[0086] 1. Cabbage pretreatment

[0087] Fresh yellow-hearted Chinese cabbage is sun-dried outdoors for 2-3 days (until it wilts and some moisture is removed). Rotten and withered leaves are removed, the roots are cut off and the cabbage is washed clean. The whole cabbage is then cut into quarters along its long axis.

[0088] 2. Approximately 4.5 kg of pretreated Chinese cabbage was packed into a fermentation tank, compacted, and the air was removed. Approximately 2.9 L of water was added, along with 2.0% salt by weight. The compound fermentation agent 7 obtained in Example 9 was inoculated at an inoculation rate of 5 wt.% of the cabbage weight. The mixture was then sealed and fermented for 28 days at different temperatures (14℃, 17℃, 20℃, and 23℃). Natural fermentation without inoculation was used as a blank control. Specifically, approximately 4.5 kg of pretreated Chinese cabbage was packed into a fermentation tank, compacted, and the air was removed. Approximately 2.9 L of water was added, along with 2.0% salt by weight. The mixture was then sealed and fermented for 28 days at different temperatures (14℃, 17℃, 20℃, and 23℃). Three parallel groups were set up for each treatment method. The fermentation effect at different temperatures was evaluated according to the procedure in Example 1. The results are shown in Table 4.

[0089] Table 4 Sensory evaluation results of compound fermentation agent at different fermentation temperatures

[0090]

[0091] As shown in Table 4, a temperature range of 15-19℃ is ideal, as this range promotes the growth of lactic acid bacteria and yeast while inhibiting the reproduction of harmful microorganisms. Excessively high temperatures may accelerate fermentation but could also lead to contamination by other microorganisms or make the product too soft and mushy; conversely, excessively low temperatures will slow down fermentation and may prevent the desired acidity level from being achieved.

[0092] Test Example 4

[0093] Experiments on different fermentation times of compound fermentation agents

[0094] 1. Cabbage pretreatment

[0095] Fresh yellow-hearted Chinese cabbage is sun-dried outdoors for 2-3 days (until it wilts and some moisture is removed). Rotten and withered leaves are removed, the roots are cut off and the cabbage is washed clean. The whole cabbage is then cut into quarters along its long axis.

[0096] 2. Approximately 4.5 kg of pretreated Chinese cabbage was packed into a fermentation tank, compacted, and the air was removed. Approximately 2.9 L of water was added, along with 2.0% salt by weight. The compound fermentation agent 7 obtained in Example 9 was inoculated at an inoculation rate of 5 wt.% of the Chinese cabbage weight. The mixture was then sealed and fermented at 17°C. Natural fermentation without inoculation was used as a blank control. Specifically, approximately 4.5 kg of pretreated Chinese cabbage was packed into a fermentation tank, compacted, and the air was removed. Approximately 2.9 L of water was added, along with 2.0% salt by weight. The mixture was then sealed and fermented at 17°C for different times (21 days, 28 days, 35 days, and 42 days). Three parallel groups were set up for each treatment method. The fermentation effect of different fermentation times was evaluated according to the method in Test Example 1. The results are shown in Table 5.

[0097] Table 5 Sensory evaluation results of compound fermentation agents at different fermentation times

[0098]

[0099] As shown in Table 5, fermentation lasts 3-5 weeks (21-35 days), during which lactic acid bacteria fully act on the vegetables, producing sufficient lactic acid to lower the pH value and develop their unique flavor. The actual fermentation time may vary depending on environmental conditions and personal preference. It is recommended to check the sauerkraut regularly until a satisfactory taste is achieved. If fermentation exceeds 5 weeks, the sauerkraut will become overly sour, soft, and dark in color, indicating over-fermentation. Introducing yeast into the fermentation process produces ethanol, which reacts with lactic acid to form flavor compounds, resulting in a slightly higher pH value in the final product, around 4.0-4.6.

[0100] Comparative Example 1

[0101] The culture medium 1 and culture medium 2 from step (1) of Example 3 were mixed at a mass ratio of 2:2 to obtain the composite fermentation agent 8.

[0102] Comparative Example 2

[0103] The culture medium 1 and culture medium 3 from step (1) of Example 3 were mixed at a mass ratio of 2:1 to obtain the composite fermentation agent 9.

[0104] Comparative Example 3

[0105] The culture medium 2 and culture medium 3 from step (1) of Example 3 were mixed at a mass ratio of 2:1 to obtain the composite fermentation agent 10.

[0106] Test Example 5

[0107] Fermentation experiments with different starter cultures

[0108] 1. Cabbage pretreatment

[0109] Fresh yellow-hearted Chinese cabbage is sun-dried outdoors for 2-3 days (until it wilts and some moisture is removed). Rotten and withered leaves are removed, the roots are cut off and the cabbage is washed clean. The whole cabbage is then cut into quarters along its long axis.

[0110] 2. Pack approximately 4.5 kg of pretreated Chinese cabbage into a fermentation tank, compact it, and remove the air. Add approximately 2.9 L of water and 2.0% salt by weight. Inoculate the compound fermentation agents 1-7 obtained in Examples 3-9, the culture solutions 1-3 obtained in Example 3, and the compound fermentation agents 8-10 obtained in Comparative Examples 1-3 at an inoculation rate of 5 wt.% of the Chinese cabbage weight, and seal and ferment at 17°C for 28 days. Three parallel groups were set up for each treatment method.

[0111] 3. Referring to the literature (Tian Jiaxue. Preparation of compound fermentation agent for Northeast sauerkraut and its influence on sauerkraut quality [D]. Jilin University. 2024.), the main flavor components of the fermented sauerkraut prepared above were detected, and the results are shown in Table 6.

[0112] Table 6. Quantities of main flavor components in sauerkraut fermented using different methods

[0113]

[0114] As shown in Table 6, compound fermentation agent 7 exhibits excellent performance in terms of the quantity of major flavor components such as alcohols, esters, ketones, sulfur-containing compounds, nitrogen-containing compounds, and amino acids, especially showing significant advantages in esters, ketones, sulfur-containing compounds, nitrogen-containing compounds, and amino acids. This indicates that compound fermentation agent 7 can effectively promote the generation of multiple flavor components, thereby improving the overall flavor and quality of fermented sauerkraut.

[0115] 4. Referring to the literature (Tian Jiaxue. Preparation of compound fermentation agent for Northeast sauerkraut 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 Northeast 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 fermented sauerkraut prepared using culture medium 1 and compound fermentation agent 7 were detected. Natural fermentation treatment without inoculation of fermentation agent was used as a control. The results are shown in Table 7.

[0116] Table 7 Evaluation results of crispness and safety of sauerkraut fermented by different methods

[0117]

[0118] As shown in Table 7, compared to natural fermentation and fermentation using only Leuconostoc mesenteroides, the sauerkraut fermented using compound fermentation agent 7 exhibited the best crispness, reaching 21N, indicating a tender and crisp texture. Furthermore, the levels of nitrite, sulfur dioxide residue, and E. coli all met or exceeded food safety standards, demonstrating excellent safety performance. Compound fermentation agent 7 promotes the esterification reaction of lactic acid and ethanol, regulating the acidity of the sauerkraut and improving its taste, resulting in a moderately acidic sauerkraut with a more palatable flavor. Leuconostoc mesenteroides alone caused the sauerkraut to become overly acidic, and natural fermentation is difficult to control in terms of acid production. Overall, the sauerkraut fermented using compound fermentation agent 7 not only possesses excellent crispness but also meets high safety standards, making it a high-quality fermented sauerkraut product.

[0119] As can be seen from the above, the compound microbial agent provided by the present invention can improve the flavor quality of fermented sauerkraut, ensure the consistency and safety of sauerkraut, and guarantee the taste and crispness of sauerkraut.

[0120] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A compound microbial agent, characterized in that, Including Leuconostoc mesenteroides ( Leuconostoc citreum ), Lactobacillus putrefactiveis ( Loigolactobacillus coryniformis Z5-1 and lipolytic yeast ( Yarrowia lipolytica Z6-2; The Lactobacillus putrefactivee Z5-1 has the accession number CGMCC No.31547 and was deposited at the China General Microbiological Culture Collection Center on August 5, 2024. The Yerovia lipolytica Z6-2 has the accession number CGMCC No.31548 and was deposited at the China General Microbiological Culture Collection Center on August 5, 2024.

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

3. The compound microbial agent according to claim 2, characterized in that, The ratio of viable bacteria in the compound microbial agent is (1~3):(1~3):(1~2).

4. The compound microbial agent according to any one of claims 1 to 3, characterized in that, The compound microbial agent includes a biodegradable adsorbent carrier.

5. The method for preparing the compound microbial agent according to any one of claims 1 to 4, characterized in that, Includes the following steps: Leuconostoc melanocytoxin, Lactobacillus putrefactivee Z5-1 and Jeroensis lipolytica Z6-2 were cultured separately to obtain Leuconostoc melanocytoxin culture medium, Lactobacillus putrefactivee Z5-1 culture medium and Jeroensis lipolytica Z6-2 culture medium; The compound bacterial agent is obtained by mixing the culture medium of Leuconostoc mesenteroides, the culture medium of Lactobacillus putrefactiveus Z5-1, and the culture medium of Yersinia lipolytica Z6-2.

6. The preparation method according to claim 5, characterized in that, After mixing the Leuconostoc mesenteroides culture medium, Lactobacillus putrefactive strain Z5-1 culture medium, and Yersinia lipolyticis Z6-2 culture medium, the mixture further includes: adsorbing the mixture using a biodegradable adsorption carrier to obtain the composite microbial agent.

7. The application of the compound microbial agent according to any one of claims 1 to 4 or the compound microbial agent obtained by the preparation method according to claim 5 or 6 in fermented sauerkraut.

8. A method for preparing fermented sauerkraut, characterized in that, The process includes the following steps: fermenting the raw materials for preparing sauerkraut using the compound microbial agent described in any one of claims 1 to 4 or the compound microbial agent obtained by the preparation method described in claim 5 or 6, thereby obtaining fermented sauerkraut.

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

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

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