Lactobacillus plantarum strain LT400 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum strain LT400 and application thereof.The lactobacillus plantarum strain LT400 is separated from acid materials, can remarkably improve the aroma of sour mustard and increase the content of flavor substances, has a bacteriostatic effect on Escherichia coli, and can be used for preparing sour mustard. A fermentation product obtained by fermenting the strain and leaf mustard has an antibacterial effect on salmonella enteritidis, escherichia coli and staphylococcus aureus; the bacterial strain LT400 cooperates with alkaloids: mulberry alkaloid, passion fruit alkaloid and / or rosa roxburghii tratt fruit alkaloid, so that the brightness of the pickled Chinese cabbages can be effectively improved, and the pickled Chinese cabbages with better color and luster can be prepared; experiments prove that the alkaloid comes from fruits, the product source is green and healthy, the alkaloid is a healthy and effective pickled Chinese cabbage brightness improving agent, the alkaloid and the strain LT400 cooperate to provide a new thought for subsequent development of an anti-browning agent, the fermentation liquor has the value of researching and developing antibacterial peptides, and a new technical concept is provided for later improvement of the added value of the product.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Lactobacillus plantarum strain LT400 and its applications. Background Technology

[0002] Fermented vegetables, such as sauerkraut and kimchi, are traditional side dishes in my country and many East Asian countries, and their unique flavor, texture, and potential health benefits are highly favored by consumers. Sauerkraut, made from mustard greens and other similar vegetables, is particularly prized for its quality, which hinges on the characteristic flavor compounds formed during fermentation, its stable color, and its excellent hygiene and safety. Currently, traditional and modern sauerkraut production largely relies on the natural fermentation or guided fermentation of the raw materials themselves or by adding starter cultures. However, this production method often faces several challenges: First, the formation of flavor compounds is unstable; different vegetables have different characteristic aroma compounds, and insufficient characteristic aroma compounds can lead to a monotonous or unremarkable flavor. Second, during fermentation and storage, vegetables are prone to browning or discoloration, affecting the sensory quality and commercial value of the product. Third, the microbial safety of the fermentation system is crucial; effectively inhibiting the growth of common foodborne pathogens is key to ensuring product safety and extending shelf life.

[0003] To address the aforementioned issues, existing technologies typically employ various methods for improvement. These include screening specific functional microbial strains as fermentation agents to enhance flavor or inhibit bacterial growth, or adding chemical color-protecting agents and preservatives to improve color and shelf life. However, the use of chemical additives contradicts consumers' pursuit of "clean labels" and natural, healthy foods. Therefore, developing microbial fermentation agents derived from natural ingredients with multiple improvement functions, or exploring the synergistic effects of natural bioactive components and probiotic strains to simultaneously enhance the flavor, color, and safety of sauerkraut, has become an important research direction in this field. In particular, research on strains possessing aroma-producing, antibacterial, and color-protecting functions, as well as their synergistic effects with natural plant active ingredients, remains insufficient, indicating a vast space for exploration and application potential.

[0004] Therefore, in summary: to improve the quality of sauerkraut, this invention aims to provide a functional lactic acid bacteria strain isolated and screened from traditional fermented sauerkraut. This strain can not only significantly improve the content of characteristic aroma and flavor substances in fermented products, but also has the activity of inhibiting common pathogenic bacteria. Furthermore, it has adaptively developed a microbial fermentation technology that can synergize with natural alkaloids, effectively improve the color of sauerkraut, and enhance antibacterial properties, which has important application value. Summary of the Invention

[0005] In view of the above, in order to improve the quality of sauerkraut, a functional lactic acid bacteria strain isolated and screened from traditional fermentation materials was developed. This strain can not only significantly improve the content of characteristic aroma and flavor substances in fermented products, but also has the activity of inhibiting common pathogenic bacteria. It has also adaptively developed a microbial fermentation technology that can synergize with natural alkaloids, effectively improve the color of sauerkraut and enhance antibacterial properties. It can also effectively control product browning, improve appearance quality and enhance the flavor of pickled mustard greens. Furthermore, it provides new raw materials and ideas for the subsequent development of natural anti-browning agents, flavor enhancers or antibacterial active substances that can synergize with the strain.

[0006] To achieve the above objectives, this invention has screened out a new strain: Lactiplantibacillus plantarum LT400, its classification name is: Lactiplantibacillus plantarum The strain is classified and named as Lactobacillus plantarum in Chinese, with accession number GDMCC NO: 66637. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 3, 2025.

[0007] Furthermore, the *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The (NH4)2SO4 salting-out extract of sauerkraut fermentation broth of strain LT400 can be used to prepare antibacterial agents.

[0008] Furthermore, the pathogen inhibited by the antibacterial agent is: Salmonella enteritidis (…). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Staphylococcus aureus Staphylococcus aureus One or more of the following.

[0009] Furthermore, the mass concentration of (NH4)2SO4 during salting out is 20%.

[0010] The present invention also includes the Lactobacillus plantarum ( Lactiplantibacillus plantarum Application of strain LT400 in enhancing the aroma of sour mustard greens.

[0011] Furthermore, the flavor compounds of the aroma are isothiocyanate compounds.

[0012] The present invention also includes the Lactobacillus plantarum ( Lactiplantibacillus plantarum LT400 strain inhibits Escherichia coli ( Escherichia coli Applications during growth.

[0013] The present invention also includes the Lactobacillus plantarum ( Lactiplantibacillus plantarum Application of strain LT400 in enhancing the brightness of sauerkraut, the plant lactobacillus ( Lactiplantibacillus plantarumThe LT400 strain used an enhancer to improve the brightness of sauerkraut. The enhancer is one or more of mulberry alkaloids, passion fruit alkaloids, and prickly pear fruit alkaloids.

[0014] Furthermore, the *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The fermentation method for enhancing the brightness of sauerkraut using strain LT400 is as follows: Wash the whole mustard greens and place them in a fermentation tank for pickling. Add alkaloids at a rate of 3% of the mustard greens' weight, mix thoroughly, and then inoculate with *Lactobacillus plantarum* at a rate of 2% of the mustard greens' weight. Lactiplantibacillus plantarum The alkaloids are obtained by mixing and pickling LT400 strains, and the alkaloids are one or more of mulberry alkaloids, passion fruit alkaloids, and prickly pear fruit alkaloids.

[0015] Furthermore, the mass ratio of the mulberry alkaloids, passion fruit alkaloids, and prickly pear alkaloids is 2:0-3:4-5.

[0016] The present invention has the following beneficial effects: The LT400 strain of the present invention is isolated from pickled mustard greens. This strain can significantly improve the aroma of pickled mustard greens and increase the content of flavor substances, especially isothiocyanates. In addition, this strain also has a significant effect on Escherichia coli (Escherichia coli). Escherichia coli This strain has antibacterial effects, and the fermentation products of mustard fermentation are effective against Salmonella enteritidis (Salmonella enteritidis). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Staphylococcus aureus Staphylococcus aureus One or more pathogens in the strain LT400 exhibit antibacterial effects; the synergistic fermentation of this strain with alkaloids—mulberry alkaloids, passion fruit alkaloids, and / or prickly pear alkaloids—effectively enhances the brightness of sauerkraut, resulting in sauerkraut with better color. Our experiments have verified that these alkaloids originate from fruits. Due to their natural source, these fruit-derived alkaloids have potential green and healthy applications in food processing, making them a healthy and effective sauerkraut brightness enhancer. The synergistic effect of these alkaloids and strain 400 can provide new ideas for the subsequent development of anti-browning agents. The fermentation broth possesses the potential for developing antimicrobial peptides, providing new technical concepts for enhancing the added value of products in the future. Attached Figure Description

[0017] Figure 1 The image shows the plate growth of the *Lactobacillus plantarum* strains described in this application, where A represents strain LXC, B represents strain LT303, C represents strain LT304, and D represents strain LT400.

[0018] Figure 2The images show microscopic examination of the strains of *Lactobacillus plantarum* from this application, where A is strain LXC, B is strain LT303, C is strain LT304, and D is strain LT400.

[0019] Information on the preservation of biological materials.

[0020] The strain information deposited in this application is as follows: Lactiplantibacillus plantarum LXC, its classification name is: Lactiplantibacillus plantarum The strain is classified and named *Lactobacillus plantarum* in Chinese, with accession number GDMCCNO: 66634. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 3, 2025.

[0021] The strain information deposited in this application is as follows: Lactiplantibacillus plantarum LT303, its classification name is: Lactiplantibacillus plantarum The strain is classified and named *Lactobacillus plantarum* in Chinese, with accession number GDMCCNO: 66635. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 3, 2025.

[0022] The strain information deposited in this application is as follows: Lactiplantibacillus plantarum LT304, its classification name is: Lactiplantibacillus plantarum The strain is classified and named *Lactobacillus plantarum* in Chinese, with accession number GDMCCNO: 66636. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 3, 2025.

[0023] The strain information deposited in this application is as follows: Lactiplantibacillus plantarum LT400, its classification name is: Lactiplantibacillus plantarum The strain is classified and named *Lactobacillus plantarum* in Chinese, with accession number GDMCCNO: 66637. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 3, 2025. Detailed Implementation

[0024] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0025] Unless otherwise stated, each feature disclosed in this specification is merely one example of a series of equivalent or similar features.

[0026] Example 1.

[0027] This example uses *Lactobacillus plantarum* (… Lactiplantibacillus plantarum Isolation and identification of strains.

[0028] 1. Strain screening and isolation: Mother brine from market-prepared pickled cabbage, pickled cowpeas, and pickled bamboo shoots was added to MRS broth medium and incubated at 37 ℃ for 24 h for enrichment. 10 mL of the enriched strain was transferred to a sterile Erlenmeyer flask, 90 mL of physiological saline was added, and the mixture was shaken to mix thoroughly. The mixture was then serially diluted, and 100 µL was plated onto MRS agar plates containing calcium carbonate and incubated at 37 ℃ for 48 h. Stranding was performed multiple times on strains exhibiting calcium dissolution zones for purification. The strains were classified according to colony morphology, and after three generations of streaking isolation, pure strains were obtained and stored at -80 ℃.

[0029] 2. The strains in the enrichment solution were initially screened using MRS solid medium containing calcium carbonate. Stranding of strains exhibiting calcium dissolution zones was performed multiple times for purification, identifying suspected lactic acid bacteria. Morphological classification and Gram staining were then performed on the suspected strains. The results are shown in Table 1 and... Figure 1 , Figure 2 As shown.

[0030]

[0031] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.

[0032] From Table 1 and Figure 1 visible: Figure 1 This is a plate image showing the growth of the *Lactobacillus plantarum* strain of this application. Figure 1 In the text, A represents strain LXC, whose colonies are round and raised, milky white, small, with neat edges, moist, and opaque. Figure 1 In the text, B represents strain LT303, whose colonies are round, milky white, with neat edges, and opaque. Figure 1 C in the text refers to strain LT304, whose colonies are round, milky white, with neat edges, moist, and opaque. Figure 1 In Table 1, D represents strain LT400, whose colonies are round, milky white, with neat edges, and opaque. As can be seen from Table 1, the colonies of strain XD are round, milky white, with neat edges, moist, and opaque; the colonies of strain XC2 are round, milky white, and opaque; and the colonies of strain SD are round, milky white, with neat edges, moist, and opaque.

[0033] From Table 1 and Figure 2 visible: Figure 2This is a microscopic image of the *Lactobacillus plantarum* strain described in this application. Figure 2 In this context, A represents strain LXC. Figure 2 B is strain LT303. Figure 2 C is strain LT304, Figure 2 D is strain LT400; from Figure 2 As can be seen in Table 1, strains LXC, LT303, LT304, and LT400 showed a purple Gram staining, indicating a positive result. Under the microscope, the strains were observed to be in pairs and rod-shaped. In addition, as shown in Table 1, strains XD, XC2, and SD also showed a Gram staining, and under the microscope, the strains were observed to be in pairs and rod-shaped.

[0034] 3. Molecular identification: The 16S rDNA sequences of strains LXC, LT303, LT304, LT400, XD, XC2, and SD listed in Table 1 were sequenced. The sequences were submitted to the National Center for Biotechnology Information (NCBI) database for BLAS homology comparison. The comparisons showed that these strains were homologous with... Lactiplantibacillus plantarum The homology reached 100%. Based on the morphological characteristics in Table 1, strains LXC, LT303, LT304, LT400, XD, XC2, and SD were identified as... Lactiplantibacillus plantarum .

[0035] Example 2.

[0036] This embodiment is a performance test, as detailed below.

[0037] 1. The activated bacterial strain was inoculated into MRS broth at a volume fraction of 2% and cultured at 37 ℃ for 24 h. The pH value of the fermentation broth was measured at regular intervals using a pH meter. The total acid content was determined according to GB 12456—2021 "National Food Safety Standard - Determination of Total Acid in Food". The results are shown in Table 2.

[0038]

[0039] Note: Different lowercase letters in the table indicate significant differences in the data within the same column. p<0.05 The same letter indicates that the differences in the same column are not significant. p>0.05 ), the same as in the table below.

[0040] As shown in Table 2, among the seven *Lactobacillus plantarum* strains, strains LXC and LT304 had higher total acid content and stronger acid production capacity, followed by strain LT400. Strains XD and SD had the lowest total acid content, while strains LT303 and XC2 had relatively weak total acid content. In terms of pH, strain XD had the highest total acid content, followed by strain SD. After screening, the four strains with the highest total acid content—strains LXC, LT303, LT304, and LT400—were selected for sauerkraut fermentation experiments.

[0041] Example 3.

[0042] This example describes a sauerkraut fermentation experiment using strains LXC, LT303, LT304, and LT400, as detailed below.

[0043] Four bacterial strains were inoculated into mustard greens for sauerkraut fermentation experiment and compared with CK (fermentation group without any bacterial strains) as control. After 20 days of fermentation, the total acid, color difference and texture index of the finished vegetables were measured. The specific results are shown in Tables 3 and 4.

[0044]

[0045] Table 3 shows that different strains have different effects on... The value has no effect. p>0.05 );exist Regarding values, there were no significant differences between CK, strain LXC, strain LT400, strain LT304, and strain LT303. p>0.05 However, strains LT304 and LT303 showed significant differences. p<0.05 );exist Regarding values, there were no significant differences between CK, strain LXC, strain LT303 and strain LT304, strain LT400. p>0.05 However, strains LT304 and LT400 showed significant differences. p<0.05 This indicates that strain LT303 reduces the brightness of sauerkraut, while strain LT304 increases the brightness of sauerkraut and alleviates the increase in yellowness.

[0046]

[0047] The effects of different bacterial strains on the texture of sauerkraut fermented with different strains on its quality were investigated in five aspects: hardness, elasticity, adhesiveness, cohesiveness, and chewiness. Table 4 shows that, in terms of hardness and chewiness, strains LXC and LT400 showed no significant differences compared to strains CK, LT303, and LT304. p>0.05 However, strains LXC and LT400 showed significant differences. p< 0.05In terms of hardness and chewiness, the order was LXC > CK > LT303 > LT304 > LT400. Strain LXC had a hardness of 535.60 g and a chewiness of 3.52 mJ, while strain LT400 had a hardness of 377.75 g and a chewiness of 2.30 mJ. Regarding elasticity, there was no significant difference among the five strains. p>0.05 In terms of adhesiveness, CK showed the highest level, with strains LT304 and LT400 showing significant differences from CK and LXC. Regarding cohesion, strain LT304 showed the highest level, with a significant difference from CK.

[0048] Example 4.

[0049] This example illustrates the effect of strain LT400 on the browning effect of fermented sauerkraut after substrate optimization, as detailed below.

[0050] Our research group conducted preliminary metabolomics studies on the browning of sauerkraut. During the study, we discovered that the content of two phenolic compounds—icariin and benzyl-β-gentiobiglycoside—among the alkaloids in sauerkraut showed a significant negative correlation with color parameters. These two phenolic compounds can serve as markers of sauerkraut browning. Furthermore, we found that in sauerkraut fermented with four strains—LXC, LT303, LT304, and LT400—LT400 showed the highest levels of icariin in the browning process. The content of gentiopicroside and benzyl-β-gentiopicroside was the lowest, followed by LT304, with LXC and LT303 being the highest. However, the color test results of sauerkraut fermented by strain LT400 showed no significant difference in color compared to the control. Nevertheless, we believe that the color of strain LT400 might be improved by optimizing the fermentation substrate. To verify this hypothesis, we considered adding alkaloids to verify their potential inhibitory effect on browning of sauerkraut by the metabolism of strain LT400, as detailed below:

[0051] 1. Preparation of the corresponding alkaloids: The total alkaloids were extracted and purified using a combination of heating reflux and macroporous adsorption resin as follows:

[0052] Mulberry alkaloids: Dried mulberries, dried passion fruit peels, dried dragon fruit peels, dried whole prickly pear fruits, and dried mulberry leaves were crushed separately to obtain corresponding coarse powders. Then, 20 times the volume of 75% ethanol was added, and the mixture was heated under reflux twice, 120 min each time. After concentration under reduced pressure, centrifugation, and filtration under reduced pressure, the filtrate was diluted to 500 mL and used as the loading solution. Elution was then performed using HPD100 macroporous resin. The resulting eluent was concentrated, freeze-dried, and ground to obtain the corresponding alkaloid powders from mulberries, passion fruit, dragon fruit, prickly pear fruits, and mulberry leaves.

[0053] 2. Optimize the substrate fermentation of mustard greens using the alkaloids prepared in step 1: Wash the whole mustard greens and put them into a fermentation tank for pickling. Then, add the above-mentioned alkaloids at a rate of 3% of the mustard greens' mass, mix thoroughly, and then inoculate with strain LT400 at a rate of 2% by mass. After pickling and fermenting for 18 days, the experimental group was obtained. The control group was the sauerkraut fermentation group with only alkaloids added and no strain added, and the blank group was the sauerkraut fermentation group without strains or alkaloids (only one group was done, 3 in parallel). Each experimental group was done in 3 parallels. The color content of the sauerkraut in each group was measured and the results are shown in Table 5.

[0054]

[0055] As shown in Table 5, there were no significant differences in redness and yellowness among the experimental groups. p>0.05 Significant differences in brightness were observed among the experimental groups, specifically: the brightness of the passion fruit alkaloid-experimental group and the prickly pear alkaloid-experimental group was significantly higher than that of the blank group. p< 0.05 The mulberry alkaloids experimental group had a higher brightness than the blank group, but the difference was not significant. p>0.05 The brightness of the mulberry alkaloids-control group, passion fruit alkaloids-control group, dragon fruit alkaloids-experimental group, and dragon fruit alkaloids-control group was significantly lower than that of the blank group. p<0.05 The brightness of the mulberry leaf alkaloids experimental group and the mulberry leaf alkaloids control group was not significantly different from that of the blank group. p< 0.05 This indicates that in the experiments of this application, the addition of plant alkaloids alone had little effect on the color of fermented mustard greens. However, the addition of a mixture of alkaloids—mulberry alkaloids, passion fruit alkaloids, and prickly pear alkaloids—fermented with a specific strain LT400 effectively improved the color of the mustard greens. In contrast, the addition of dragon fruit alkaloids and mulberry leaf alkaloids did not significantly improve the color of the mustard greens fermented with strain LT400. This suggests that the reason why the above-mentioned alkaloids can improve the color of the mustard greens is more due to their influence on the metabolic pathway of strain LT400, thereby affecting the color of the mustard greens, rather than the fact that the alkaloids themselves have an anti-browning effect.

[0056] This indicates that mulberry alkaloids, passion fruit alkaloids, and prickly pear alkaloids can increase the brightness of mustard greens fermented with strain LT400 to varying degrees. Therefore, we chose to mix these alkaloids with strain LT400 to prepare an anti-browning agent. Specifically, mulberry alkaloids, passion fruit alkaloids, and prickly pear alkaloids were mixed in different mass ratios and added to the mustard greens at a rate of 3% of the mustard greens' mass. After thorough mixing, the mixture was inoculated with strain LT400 for fermentation to obtain fermented sauerkraut. The color of the sauerkraut was then tested, with brightness as the primary indicator. The results obtained by selecting the criteria are shown in Tables 6 and 7.

[0057]

[0058] The orthogonal analysis of the above proportions and the results are shown in Table 7:

[0059]

[0060] Table 7 shows that when mulberry alkaloids, passion fruit alkaloids, and prickly pear alkaloids were mixed and fermented with strain LT400, the brightness of the pickled vegetables varied greatly. Specifically, the brightness of experiments 4 and 5 was significantly higher than that of experiment 1 (control) without added alkaloids. p<0.05 The brightness of experiments 3, 6, and 7 was not significantly different from that of experiment 1 (control) without added alkaloids. p>0.05 The brightness of tests 2, 8, and 9 was significantly lower than that of test 1 (control) without the addition of alkaloids. p<0.05 This indicates that when alkaloids are mixed, different proportions of alkaloids can have a synergistic effect on the brightness of pickled mustard greens, while other proportions can reduce the brightness. This suggests that the different metabolic effects of alkaloid mixtures on the bacterial strains may lead to significant differences in the final brightness of the pickled mustard greens. Based on this data, we believe that when mulberry alkaloids, passion fruit alkaloids, and prickly pear alkaloids are mixed in a mass ratio of 2:0-3:4-5, they can effectively synergistically enhance the brightness of pickled mustard greens by bacterial strain LT400.

[0061] In terms of range: mulberry alkaloids > passion fruit alkaloids > prickly pear alkaloids. Therefore, in terms of the brightness of sauerkraut, mulberry alkaloids have the greatest impact, followed by passion fruit alkaloids, and prickly pear alkaloids have the least impact.

[0062] In summary, for this application, the optimal preparation method for improving the brightness of pickled mustard greens is as follows: after washing the whole mustard greens, put them into a fermentation tank for pickling mustard greens, add alkaloids at a rate of 3% of the mustard greens' weight, mix thoroughly, and then inoculate with strain LT400 at a rate of 2% by weight and pickle and ferment. The alkaloids are mulberry alkaloids, passion fruit alkaloids, and prickly pear fruit alkaloids mixed in a weight ratio of 2:0-3:4-5.

[0063] Example 5.

[0064] This embodiment tests the antibacterial effect of strain LT400 against common pathogenic bacteria.

[0065] 1. Determination of the inhibitory effect of the strain on pathogens: The inhibitory effect of strain LT400 on Salmonella enteritidis (Salmonella enteritidis) was determined using the perforated agar diffusion method. Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Staphylococcus aureusStaphylococcus aureus ) and Listeria monocytogenes ( Listeria monocytogenes The antibacterial activity of the inhibition zone was measured by measuring the diameter of the transparent zone (diameter of inhibition zone = measurement diameter - pore diameter), and the results are shown in Table 8.

[0066]

[0067] Note: “—” in the table indicates no inhibitory effect.

[0068] As shown in Table 8, strain LT400 has an inhibitory effect on the pathogen Escherichia coli, but no inhibitory effect on Salmonella enteritidis, Staphylococcus aureus and Listeria monocytogenes.

[0069] 2. Determination of the inhibitory effect of fermented sauerkraut broth on pathogens: A sample of sauerkraut fermentation broth was centrifuged at room temperature to remove the lower layer of impurities and bacterial sludge, retaining the supernatant. The broth was then concentrated using a vacuum water bath rotary evaporator at a series of temperature gradients until the volume was 1 / 10 of the original broth. (NH4)2SO4 solution was added to the concentrate for gradient salting out (salting out concentration gradients: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%). After salting out, the centrifuge tubes were centrifuged, and the supernatant was subjected to a second salting out and centrifugation until no more precipitate appeared. The amount of precipitate from the gradient concentration salting out is shown in Table 9. Then, the precipitate samples were used for a filter paper disc antibacterial test. The specific method was as follows: a centrifuge tube with precipitate was taken, and the supernatant and precipitate were used as test samples. (NH4)2SO4 of the same concentration was used as a blank for the antibacterial test, and the size of the inhibition zone was observed. The results are shown in Table 10.

[0070]

[0071] As shown in Table 9, precipitate can only be precipitated in the sauerkraut fermentation liquid at a concentration of 20%, while no precipitate can be precipitated at other concentrations.

[0072]

[0073] Note: The significance of different bacteria and different salting-out concentrations in the table was analyzed by grouping.

[0074] Table 10 shows that at a salting-out concentration of 20%, both the supernatant and the precipitate showed resistance to Salmonella enteritidis (Salmonella enteritidis). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Staphylococcus aureus Staphylococcus aureus The inhibition zone diameter of one or more pathogens in the sample was significantly higher than that of the control group. p<0.05This indicates an antibacterial effect, while it is effective against Listeria monocytogenes (…). Listeria monocytogenes The diameter of the inhibition zone was not significantly different from that of the control group. p>0.05 This indicates that there is no antibacterial effect.

[0075] Example 6.

[0076] This example demonstrates a test of the aroma of fermented sauerkraut.

[0077] Strains LXC, LT303, LT304, and LT400 were inoculated into mustard greens as experimental groups, while mustard greens without any added strains served as the control (CK) group. After fermentation in direct-inoculation fermenters for 25 days, the volatile components in the fermentation broth were tested using GC-MS. Three parallel levels were measured for each fermentation broth. Analysis revealed significant differences in volatile components among different strains. Isothiocyanates, a hallmark flavor compound of pickled mustard greens, exhibited significant differences in their pungent and mustard-like flavor across different strains. Furthermore, the analysis of volatile components revealed that pickled mustard greens fermented by strain LT400 possessed a unique sweet flavor, with ethyl formate and ethyl palmitate, representing the sweet flavor, being the most abundant. Therefore, we compared the total isothiocyanates and the higher-content isothiocyanates with the contents of ethyl benzoate and ethyl palmitate. The results are shown in Table 11.

[0078]

[0079] Note: “—” in the table indicates that it was not detected or the concentration was too low.

[0080] Table 11 shows that the total isothiocyanate content of fermented mustard greens by strain LT400 was significantly higher than that of fermented mustard greens by strains LXC, LT303, and LT304, and the control group (CK) without lactic acid bacteria inoculation. p<0.05 Among the total isothiocyanate compounds, the top three isothiocyanate compounds with the highest content were allyl isothiocyanate, 2-phenylethyl isothiocyanate, and 3-butenyl isothiocyanate. The test results showed that the contents of allyl isothiocyanate, 2-phenylethyl isothiocyanate, and 3-butenyl isothiocyanate fermented by strain LT400 were significantly higher than those fermented by strains LXC, LT303, and LT304, and in uninoculated sour mustard greens. p<0.05 The total isothiocyanate content in fermented mustard greens by strains LXC, LT303, and LT304 was not significantly different from that in the control group (CK). p> 0.05 ).

[0081] Furthermore, the mustard greens fermented by strain LT400 have unique sweet flavors due to the presence of ethyl benzoate and ethyl palmitate, while the mustard greens fermented by strains LXC, LT303, and LT304, as well as the mustard greens without lactic acid bacteria, do not contain ethyl benzoate and ethyl palmitate. Thus, we can see that strain LT400 can significantly enhance the aroma of mustard greens compared to other experimental groups, and also imparts a unique sweet flavor.

[0082] In summary, the *Lactobacillus plantarum* LT400 strain of this application can significantly enhance the aroma of pickled mustard greens, increase the content of flavor compounds, and inhibit the pathogenic bacteria *Escherichia coli* (…). Escherichia coli This strain has antibacterial effects, and the fermentation products of mustard fermentation are effective against Salmonella enteritidis (Salmonella enteritidis). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Staphylococcus aureus ( Staphylococcus aureus One or more of these substances have antibacterial effects; when this strain is used to prepare pickled mustard greens, the addition of mulberry alkaloids, passion fruit alkaloids and / or prickly pear fruit alkaloids can effectively improve the brightness of the pickled greens.

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum The strain LT400, with accession number GDMCC NO: 66637, is deposited at Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 3, 2025.

2. The *Lactobacillus plantarum* according to claim 1 ( Lactiplantibacillus plantarum strain LT400, characterized in that, The plant lactobacillus ( Lactiplantibacillus plantarum The (NH4)2SO4 salting-out extract of sauerkraut fermentation broth of strain LT400 can be used to prepare antibacterial agents.

3. The *Lactobacillus plantarum* according to claim 2 ( Lactiplantibacillus plantarum strain LT400, characterized in that, The pathogen inhibited by the antibacterial agent is: Salmonella enteritidis (…). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Staphylococcus aureus Staphylococcus aureus One or more of the following.

4. The *Lactobacillus plantarum* according to claim 2 ( Lactiplantibacillus plantarum strain LT400, characterized in that, The mass concentration of (NH4)2SO4 during salting out is 20%.

5. The *Lactobacillus plantarum* as described in claim 1 ( Lactiplantibacillus plantarum Application of strain LT400 in enhancing the aroma of sour mustard greens.

6. The application according to claim 5, characterized in that, The aroma's flavor compounds are isothiocyanate compounds.

7. The *Lactobacillus plantarum* as described in claim 1 ( Lactiplantibacillus plantarum LT400 strain inhibits Escherichia coli ( Escherichia coli Applications during growth.

8. The *Lactobacillus plantarum* as described in claim 1 ( Lactiplantibacillus plantarum The application of strain LT400 in enhancing the brightness of sauerkraut is characterized by, The plant lactobacillus ( Lactiplantibacillus plantarum The LT400 strain used an enhancer to improve the brightness of sauerkraut. The enhancer is one or more of mulberry alkaloids, passion fruit alkaloids, and prickly pear fruit alkaloids.

9. The application according to claim 8, characterized in that, The plant lactobacillus ( Lactiplantibacillus plantarum The fermentation method for enhancing the brightness of sauerkraut using strain LT400 is as follows: Wash the whole mustard greens and place them in a fermentation tank for pickling sauerkraut. Add the enhancer at a rate of 3% of the mustard greens' weight, mix thoroughly, and then inoculate with *Lactobacillus plantarum* as described in claim 1 at a rate of 2% by weight. Lactiplantibacillus plantarum It is obtained by mixing and pickling LT400 strains.

10. The application according to claim 8, characterized in that, The mass ratio of mulberry alkaloids, passion fruit alkaloids, and prickly pear alkaloids is 2:0-3:4-5.

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