Lactobacillus plantarum strain LT304 and application thereof
By screening and applying Lactobacillus plantarum strain LT304 and its fermentation broth to prepare antibacterial agents, combined with amino acid regulation of enzyme activity, the foodborne contamination and browning problems of pickled mustard greens were solved, and the safety and sensory quality were improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGXI ZHUANG AUTONOMOUS REGION ACAD OF AGRI SCI
- Filing Date
- 2026-04-15
- Publication Date
- 2026-05-12
AI Technical Summary
In the industrial production of pickled mustard greens, there is a risk of potential foodborne pathogenic microorganism contamination and enzymatic browning during fermentation, which affects product safety and sensory quality. Existing methods such as chemical preservatives and physical sterilization technologies have limitations.
Lactobacillus plantarum strain LT304 was screened out, and an antibacterial agent was prepared by salting out the NH4)2SO4 extract from its fermentation broth. Combined with the enhancers histidine and proline, the activity of key enzymes was regulated to prevent browning and improve the brightness of sauerkraut.
It effectively inhibits Salmonella enteritidis, Escherichia coli and Listeria monocytogenes, reduces the activity of enzymes such as polyphenol oxidase and peroxidase, improves the brightness of sauerkraut, prevents browning, and enhances product safety and sensory quality.
Smart Images
Figure CN122012355A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to Lactobacillus plantarum strain LT304 and its applications. Background Technology
[0002] Pickled mustard greens, as a traditional fermented vegetable product, are widely loved by consumers for their unique flavor. However, their industrial production and quality control still face two major challenges: first, the risk of contamination by potential foodborne pathogens during fermentation, which directly affects the product's food safety; and second, the enzymatic browning phenomenon that easily occurs during fermentation and storage, which leads to a darkening of the product's color and a decline in sensory quality, seriously affecting its commercial value.
[0003] Currently, common methods for controlling pathogenic bacteria and improving color in fermented foods include adding chemical preservatives, using physical sterilization techniques, or screening functional fermentation agents. However, chemical preservatives face limitations due to consumer acceptance and safety regulations; physical methods such as pasteurization may damage the texture and flavor of the product; and many traditional or commercial fermentation agents still lack sufficient comprehensive performance in effectively inhibiting specific pathogenic bacteria and simultaneously preventing browning. Therefore, developing novel microbial fermentation agents that can simultaneously improve the safety and sensory quality of fermented foods, especially those with excellent anti-browning functions, has become a key focus of industry and research.
[0004] Regarding browning prevention, our research group's extensive studies have confirmed that browning in fruits and vegetables is closely related to the activity of a series of enzymes, including polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine ammonia-lyase (PAL). Regulating the activity of these key enzymes through microbial fermentation is one effective strategy for controlling browning. Furthermore, browning in sauerkraut may also be related to some amino acid metabolic pathways. Based on these previous studies, we believe that developing anti-browning agents that utilize microorganisms to stabilize color and significantly improve the brightness of sauerkraut is worth considering.
[0005] On the other hand, fermentation broths or microbial metabolites with antibacterial activity are important resources for developing biopreservatives or functional peptides (such as antimicrobial peptides). Screening for lactic acid bacteria strains that can impart excellent sensory qualities to products and whose fermentation broths have the potential to inhibit common foodborne pathogens is of great significance for developing high-value-added fermented products and derived functional ingredients.
[0006] In conclusion, there is an urgent need to explore lactic acid bacteria resources that can be used for multiple purposes. These resources should not only ensure the safety of the fermentation process but also effectively control product browning, improve appearance quality, and provide new raw materials and ideas for the subsequent development of natural anti-browning agents or antibacterial active substances. Summary of the Invention
[0007] In view of the above, it is necessary to explore lactic acid bacteria resources that can be used for multiple purposes. These resources should not only ensure the safety of the fermentation process, but also effectively control product browning, improve appearance quality, and provide new raw materials and ideas for the subsequent development of natural anti-browning agents or antibacterial active substances.
[0008] To achieve the above objectives, this invention has screened out a new strain: Lactiplantibacillus plantarum LT304, its classification name is: Lactiplantibacillus plantarum The strain is classified and named *Lactobacillus plantarum* in Chinese, with accession number GDMCC NO: 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.
[0009] Furthermore, the *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The (NH4)2SO4 salt-out extract of sauerkraut fermentation broth of strain LT304 can be used to prepare antibacterial agents.
[0010] Furthermore, the pathogen inhibited by the antibacterial agent is: Salmonella enteritidis (…). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Listeria monocytogenes ( ) Listeria monocytogenes One or more of the following.
[0011] Furthermore, the mass concentration of (NH4)2SO4 during salting out is 45%.
[0012] Furthermore, the preparation method of the sauerkraut fermentation broth (NH4)2SO4 salting-out extract is as follows: Measure the sauerkraut fermentation broth, centrifuge it at room temperature, discard the lower layer of impurities and the bacterial sludge mixture, retain the upper clear liquid, and then concentrate it under vacuum and reduced pressure in a vacuum water bath rotary evaporator under a series of temperature gradients until the volume is 1 / 10 of the original liquid to obtain a concentrated liquid. Then, add a 45% (NH4)2SO4 solution to the concentrated liquid for salting out. After salting out, centrifuge it in a centrifuge tube, take the supernatant for secondary salting out, and centrifuge until no more precipitation occurs.
[0013] The present invention also includes the Lactobacillus plantarum ( Lactiplantibacillus plantarum Application of strain LT304 in enhancing the brightness of sauerkraut.
[0014] Furthermore, the *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The LT304 strain also used an enhancer to improve the brightness of sauerkraut, which is histidine and / or proline.
[0015] Furthermore, the *Lactobacillus plantarum* (Lactiplantibacillus plantarum The fermentation method for improving the brightness of sauerkraut using strain LT304 is as follows.
[0016] After washing the whole mustard greens, place them in a fermentation tank for pickling sauerkraut, and inoculate with *Lactobacillus plantarum* at a rate of 2% by weight. Lactiplantibacillus plantarum It is obtained by pickling and fermenting strain LT304.
[0017] Alternatively, wash the whole mustard greens and place them in a fermentation tank for pickling sauerkraut, then inoculate with the aforementioned *Lactobacillus plantarum* at a rate of 2% by weight. Lactiplantibacillus plantarum The product is obtained by mixing and fermenting strain LT304 with amino acids, wherein the amino acids are one or more of histidine and proline.
[0018] The present invention also includes the Lactobacillus plantarum ( Lactiplantibacillus plantarum Application of strain LT304 in the preparation of anti-browning agents.
[0019] The present invention also includes the Lactobacillus plantarum ( Lactiplantibacillus plantarum Application of strain LT304 in reducing the activity of polyphenol oxidase, peroxidase, phenylalanine ammonia-lyase, ascorbic acid peroxidase, catalase and / or increasing lipoxygenase in sauerkraut.
[0020] The present invention has the following beneficial effects: The LT304 strain of the present invention is isolated from acidified material. This strain has no significant antibacterial effect on pathogens, but the fermentation product of this strain and mustard fermentation has an effect on Salmonella enteritidis (…). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Listeria monocytogenes ( ) Listeria monocytogenes One or more of the following have antibacterial effects: After fermentation by strain LT304, compared with fermentation by other strains of the same species, the enzyme activities of PAL, POD, PPO, APX and CAT in sauerkraut are reduced, while the enzyme activity of LOX is increased, and the sauerkraut has the brightest color. This indicates that Lactobacillus plantarum strain LT304 has a significant effect on preventing browning in mustard sauerkraut. Moreover, after fermentation with the addition of histidine (His) and proline (Pro), the brightness is further improved, and the color of sauerkraut is better. Therefore, the LT304 strain of this application can produce sauerkraut with better color and can effectively prevent browning, providing a new idea for the subsequent development of anti-browning agents. The fermentation broth has the value of developing antimicrobial peptides, providing a new technical concept for improving the added value of products in the future. Attached Figure Description
[0021] 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.
[0022] Figure 2 The 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.
[0023] Information on the preservation of biological materials.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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
[0028] 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.
[0029] Unless otherwise stated, each feature disclosed in this specification is merely one example of a series of equivalent or similar features.
[0030] Example 1
[0031] This example uses *Lactobacillus plantarum* (… Lactiplantibacillus plantarum Isolation and identification of strains.
[0032] 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 ℃.
[0033] 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.
[0034]
[0035] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.
[0036] 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.
[0037] 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 represents 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.
[0038] 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 .
[0039] Example 2
[0040] This embodiment is a performance test, as detailed below.
[0041] 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.
[0042]
[0043] 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.
[0044] Table 2 shows that 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.
[0045] Example 3
[0046] This example describes a sauerkraut fermentation experiment using strains LXC, LT303, LT304, and LT400, as detailed below.
[0047] 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.
[0048]
[0049] Table 3 shows that different strains have different effects on... The value had no effect (p>0.05); 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.
[0050]
[0051] 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.
[0052] Example 4
[0053] This example demonstrates the testing of color-related enzyme activity after sauerkraut fermentation.
[0054] 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 enzyme activities of PPO (polyphenol oxidase), POD (peroxidase), PAL (phenylalanine ammonia-lyase), APX (ascorbic acid peroxidase), CAT (catalase), and LOX (lipoxygenase) were measured using corresponding activity assay kits manufactured by Beijing Solarbio®. Our previous studies found that color changes during mustard fermentation are closely related to these enzymes. In plant tissues, PPO, POD, and PAL are three key defense-related enzymes. PAL can catalyze the synthesis of phenolic compounds and convert L-phenylalanine into trans-cinnamic acid. In the plant enzyme-mediated antioxidant system, CAT and APX are two of the most important free radical scavenging enzymes, capable of breaking down H2O2 into H2O. LOX is an integrase located on the cell membrane. Under normal circumstances, the enzyme and substrate are separated in different cell compartments, thus preventing them from interacting directly.
[0055] In our previous study, we found that as sauerkraut fermented and browned, PAL activity decreased significantly from day 0 to day 8, but not significantly in the later stages, reaching its lowest point and stabilizing after day 25. POD activity decreased significantly, showing a similar trend to PAL activity, reaching its lowest point and stabilizing after day 25. However, other studies have found that POD in lotus root was almost completely inactivated at pH 3.0–4.0, indicating that the enzyme activity trends of different fermentation substrates are completely different. In addition, PPO activity increased slightly from day 0 to day 3, then decreased at a faster rate between day 3 and day 12, reaching its lowest point after day 25. The enzyme activity reached its lowest level and remained stable on day 4. The changes in APX and CAT activities were similar, both decreasing significantly during fermentation: APX activity increased to some extent from day 0 to day 1, then decreased rapidly and almost reached inactivation; CAT activity showed a slight upward trend and reached its peak on day 6, then continued to decrease, reaching its lowest level and remaining stable after day 25; while LOX activity continued to increase, reaching its peak on day 24 and then stabilizing. Therefore, combined with the aforementioned color indicators, we believe that testing the above enzyme activities can verify the changes in sauerkraut color. The enzyme activity test results for different experimental groups are shown in Table 5.
[0056]
[0057] As shown in Table 5, the activities of PAL, POD, PPO, APX, and CAT in the sauerkraut fermentation group inoculated with LT304 strain were significantly lower than those in other experimental groups, while the activity of LOX was significantly higher than that in other experimental groups. p<0.05 The activities of PAL, POD, PPO, APX, CAT, and LOX in fermented sauerkraut from other strains (LXC, LT303, and LT400) were not significantly different from the control (CK). Based on the color data, we can infer that the activities of PAL, POD, PPO, APX, CAT, and LOX are closely related to the color of the sauerkraut. Better sauerkraut color leads to decreased activities of PAL, POD, PPO, APX, and CAT, while increasing the activity of LOX. Based on the difference in enzyme activity and sauerkraut brightness, we selected strain LT304 for further experiments.
[0058] Example 5
[0059] This embodiment tests the antibacterial effect of strain LT304 against common pathogenic bacteria.
[0060] 1. Determination of the inhibitory effect of the strain on pathogens: The inhibitory effect of strain LT304 on Salmonella enteritidis (Salmonella enteritidis) was determined using the perforated agar diffusion method. Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Staphylococcus aureus Staphylococcus aureus ) and Listeria monocytogenes ( Listeria monocytogenesThe 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 6.
[0061]
[0062] Note: “—” in the table indicates no inhibitory effect.
[0063] As shown in Table 6, strain LT304 had no inhibitory effect on the above four pathogens: Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes, and had no antibacterial effect on other pathogens.
[0064] 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 7. 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 8.
[0065]
[0066] As shown in Table 7, precipitate can only be precipitated in the sauerkraut fermentation broth at a concentration of 45%, while no precipitate can be precipitated at other concentrations.
[0067]
[0068] Note: The significance of different bacteria and different salting-out concentrations in the table was analyzed by grouping.
[0069] Table 8 shows that at a salting-out concentration of 45%, Salmonella enteritidis (… Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli The supernatant and the inhibition zone of the precipitate were significantly different from those of the blank. p<0.05 Listeria monocytogenes ( ); Listeria monocytogenes ( Listeria monocytogenes The inhibition zone of the supernatant was not significantly different from that of the blank. p>0.05 ).
[0070] Furthermore, at this salting-out concentration, the supernatant and precipitate showed good resistance to Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus The difference was not significant compared to the blank. p>0.05 This indicates that the fermentation broth of sauerkraut at two concentrations has an effect on Escherichia coli (Escherichia coli). Escherichia coli It has no antibacterial effect.
[0071] Therefore, in this embodiment, strain LT304 has no inhibitory effect on the above four pathogens, but after fermentation of sauerkraut, the fermentation broth has an inhibitory effect on Salmonella enteritidis (Salmonella enteritidis). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Listeria monocytogenes ( ) Listeria monocytogenes One or more of these bacteria have antibacterial effects, and they are effective against Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus It has no antibacterial effect.
[0072] Example 6
[0073] This embodiment studies the changes in the color of sauerkraut after adding amino acids to assist fermentation.
[0074] Our previous research showed that the amino acid content of mustard greens decreases sharply after fermentation, which may be the reason for browning in sauerkraut. Therefore, we considered adding some amino acid solution to the substrate during fermentation to inhibit browning and improve the color of sauerkraut. In our previous research, we found that proline (Pro) and γ-aminobutyric acid (GABA) may inhibit PPO enzyme activity, thereby inhibiting browning in sauerkraut. Histidine (His) also showed high reactivity in the Maillard reaction and may also inhibit browning in sauerkraut. However, we did not clearly verify the relationship between these amino acids and the enzyme activities of PPO, POD, PAL, APX, CAT, and LOX in sauerkraut, nor did we verify the effect of these amino acids on the color of sauerkraut. Therefore, we chose to add these amino acids to sauerkraut during fermentation and measure their effects on enzyme activity and color of sauerkraut tissue, as detailed below.
[0075] After washing the whole mustard greens, they were placed in a fermentation tank for pickling sauerkraut. Then, 2g / 100mL of the above-mentioned amino acid aqueous solution was added, with the amount of aqueous solution added being 5% of the mass of the base material. The mixture was thoroughly mixed, and then LT304 strain was inoculated at a mass ratio of 2%. After pickling and fermenting for 18 days, the experimental group was obtained. The sauerkraut fermentation without adding the strain but only adding the amino acid aqueous solution was used as the control group, and the sauerkraut fermentation group without adding the strain and without using the amino acid aqueous solution was used as the blank group (only one group was done, 3 replicates). Each experimental group was done in 3 replicates. The enzyme activities of PPO, POD, PAL, APX, CAT and LOX in each group of sauerkraut were measured and the results are shown in Table 9. The color content was also measured and the results are shown in Table 10.
[0076]
[0077] As shown in Table 9, compared with the control group, the enzyme activities of PAL, POD, PPO, APX, and CAT in the experimental group with added proline (Pro) and fermented using strain LT304 were significantly reduced. p<0.05 LOX enzyme activity was significantly increased. p<0.05 The control group, which did not use strain LT304 for pickling and fermentation, showed a significant decrease in the enzyme activities of POD, PPO, APX, and CAT compared to the blank group after the addition of proline (Pro). p<0.05 LOX enzyme activity was significantly increased. p<0.05 PAL enzyme activity was not significantly different from that of the control group. p>0.05 ).
[0078] Compared with the control group, the experimental group fermented with γ-aminobutyric acid (GABA) using strain LT304 showed no significant difference in the enzyme activities of PAL, POD, PPO, APX, CAT, and LOX. p>0.05 In the control group, which was fermented with γ-aminobutyric acid (GABA) but not with strain LT304, the enzyme activities of PAL, POD, PPO, APX, and CAT were significantly decreased compared to the blank group. p<0.05 LOX enzyme activity was significantly increased. p<0.05 ).
[0079] Compared to the control group, the experimental group fermented with histidine (His) using strain LT304 showed a significant decrease in the enzyme activities of PAL, POD, PPO, APX, and CAT. p<0.05 LOX enzyme activity was significantly increased. p<0.05 The control group, which was fermented with histidine (His) but not using strain LT304, showed no significant difference in the enzyme activities of PAL, POD, PPO, APX, CAT, and LOX compared to the blank control group. p>0.05 ).
[0080] This indicates that, without the use of strain LT304 for pickling, the addition of proline (Pro) and γ-aminobutyric acid (GABA) has varying degrees of effect on the enzyme activities of PAL, POD, PPO, APX, CAT, and LOX. Generally, the addition of these two amino acids delays the browning of pickled mustard greens. However, the addition of histidine (His) has little effect on the enzyme activities of PAL, POD, PPO, APX, CAT, and LOX, indicating that the addition of histidine (His) has little effect on the browning of pickled mustard greens. In contrast, after mixed fermentation with strain LT304 and the corresponding added amino acids, the effects on the enzyme activities of PAL, POD, PPO, APX, CAT, and LOX were significantly reduced. The effects of the enzyme activities of PAL, CAT, and LOX on the control group without the strain were significantly different from those on the control group without the strain. This suggests that the addition of strain LT304 may have altered the metabolites in the fermentation products, thereby affecting the enzyme activities of PAL, POD, PPO, APX, CAT, and LOX. Based on the above experimental results, the experimental groups that may delay the browning of pickled mustard greens are: the Pro- experimental group (pickled with strain LT304 and simultaneously added proline (Pro), the His- experimental group (simultaneously added histidine (His), the Pro- control group (pickled without strain LT304), and the GABA- control group (pickled with γ-aminobutyric acid (GABA)).
[0081]
[0082] As shown in Table 10, the yellowness of the blank group was the highest among the results of colorimetric effects, but the difference was not significant compared with other experimental groups. p>0.05 The brightness showed significant differences, specifically: the brightness of the Pro-experimental group and the His-experimental group was significantly higher than that of the blank group. p<0.05 The brightness of the Pro-control group and the GABA-control group was higher than that of the control group, but the difference was not significant. p>0.05 The experimental results are basically consistent with the trend of enzyme activity changes of PAL, POD, PPO, APX, CAT and LOX, indicating that the Pro- and His- experimental groups can significantly improve the color of sauerkraut. That is, when using strain LT304 to pickle sauerkraut, the addition of proline (Pro) and histidine (His) for mixed fermentation can effectively improve the brightness of sauerkraut, delay browning and change the color of sauerkraut.
[0083] In summary, the *Lactobacillus plantarum* strain LT304 of this application has no significant inhibitory effect on pathogens, but this strain, together with the fermentation products of mustard fermentation, has an effect on *Salmonella enteritidis* (…). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Listeria monocytogenes ( ) Listeria monocytogenesOne or more of the following have antibacterial effects; after fermentation by strain LT304, compared with fermentation by other strains of the same species, the enzyme activities of PAL, POD, PPO, APX and CAT in sauerkraut will decrease, while the enzyme activity of LOX will increase, and the sauerkraut will be the brightest; indicating that Lactobacillus plantarum strain LT304 has a significant effect on preventing browning of mustard sauerkraut, and after fermentation with the addition of histidine (His) and proline (Pro), the brightness will be further improved, and the color of sauerkraut will be better.
[0084] 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 LT304, with accession number GDMCC NO: 66636, 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 LT304, characterized in that, The plant lactobacillus ( Lactiplantibacillus plantarum The (NH4)2SO4 salt-out extract of sauerkraut fermentation broth of strain LT304 can be used to prepare antibacterial agents.
3. The *Lactobacillus plantarum* according to claim 2 ( Lactiplantibacillus plantarum strain LT304, characterized in that, The pathogen inhibited by the antibacterial agent is: Salmonella enteritidis (…). Salmonella enterica subsp. enterica serovar Enteritidis Escherichia coli ( Escherichia coli Listeria monocytogenes ( ) Listeria monocytogenes One or more of the following.
4. The *Lactobacillus plantarum* according to claim 2 ( Lactiplantibacillus plantarum strain LT304, characterized in that, The mass concentration of (NH4)2SO4 during salting out was 45%.
5. The *Lactobacillus plantarum* according to claim 2 ( Lactiplantibacillus plantarum strain LT304, characterized in that, The preparation method of the sauerkraut fermentation broth (NH4)2SO4 salting-out extract is as follows: Measure the sauerkraut fermentation broth, centrifuge it at room temperature, discard the lower layer of impurities and the bacterial sludge mixture, retain the upper clear liquid, and then concentrate it to 1 / 10 of the original volume using a vacuum water bath rotary evaporator under a series of temperature gradients to obtain a concentrated liquid. Then, add a 45% (NH4)2SO4 solution to the concentrated liquid for salting out. After salting out, centrifuge it in a centrifuge tube, take the supernatant for secondary salting out, and centrifuge until no more precipitate appears.
6. The *Lactobacillus plantarum* as described in claim 1 ( Lactiplantibacillus plantarum Application of strain LT304 in enhancing the brightness of sauerkraut.
7. The application according to claim 6, characterized in that, The plant lactobacillus ( Lactiplantibacillus plantarum The LT304 strain also used an enhancer to improve the brightness of sauerkraut, which is histidine and / or proline.
8. The application according to claim 6, characterized in that, The plant lactobacillus ( Lactiplantibacillus plantarum The fermentation method for improving the brightness of sauerkraut using strain LT304 is as follows: After washing the whole mustard greens, place them in a fermentation tank for pickling sauerkraut, and inoculate with *Lactobacillus plantarum* as described in claim 1 at a mass ratio of 2%. Lactiplantibacillus plantarum It is obtained by pickling and fermenting strain LT304; Alternatively, wash the whole mustard greens and place them in a fermentation tank for pickling sauerkraut, then inoculate with *Lactobacillus plantarum* as described in claim 1 at a mass ratio of 2%. Lactiplantibacillus plantarum The product is obtained by mixing and fermenting strain LT304 with amino acids, wherein the amino acids are one or more of histidine and proline.
9. The *Lactobacillus plantarum* as described in claim 1 ( Lactiplantibacillus plantarum Application of strain LT304 in the preparation of anti-browning agents.
10. The *Lactobacillus plantarum* as described in claim 1 ( Lactiplantibacillus plantarum Application of strain LT304 in reducing the activity of polyphenol oxidase, peroxidase, phenylalanine ammonia-lyase, ascorbic acid peroxidase, catalase and / or increasing lipoxygenase in sauerkraut.