Lactic acid bacteria with preservative function and application thereof

Abstract

The invention relates to the technical field of biology, in particular to lactic acid bacteria with a preservative function and application of the lactic acid bacteria, the lactic acid bacteria are Lactobacillus plantarum GXSS-0004, the strain preservation number of the lactic acid bacteria is GDMCC No.65451, the lactic acid bacteria are preserved in Guangdong Microbiological Culture Collection Center on November 8, 2024, and the preservation unit address is Institute of Microbiology, Guangdong Academy of Sciences, Floor 59, No. 100 Courtyard, Xianlie Middle Road, Guangzhou. The Lactobacillus plantarum GXSS-0004 disclosed by the invention can be used for fermenting and producing acid by utilizing saccharides under a culture medium and a growth condition, so that the growth and reproduction of the Lactobacillus plantarum GXSS-0004 are promoted, and the Lactobacillus plantarum GXSS-0004 has the characteristics of antibacterial activity and drug resistance, also has the capability of degrading nitrite and generating biogenic amine, and can play a role in corrosion prevention in preparation of pickled leaf mustard, raphanic acid, pickled Chinese cabbage and pickled pawpaw.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a lactic acid bacterium with anti-corrosion function and its application. Background Art

[0002] Lactic acid bacteria, as a type of bacteria that can produce a large amount of lactic acid by utilizing fermentable carbohydrates, are unique in the food ecosystem. Compared with traditional chemical preservatives, they exhibit completely different characteristics in the field of pickled vegetable preservation.

[0003] During the production and storage of pickled vegetables, lactic acid bacteria play a key anti-corrosion role by virtue of their own metabolic advantages. During the growth and metabolism process, the lactic acid produced by lactic acid bacteria can rapidly reduce the pH value of the pickled vegetable environment. Common harmful microorganisms in pickled vegetables, such as Escherichia coli, molds, etc., are greatly inhibited in this acidic environment. Antibacterial active protein substances such as bacteriocins secreted by lactic acid bacteria can precisely act on the cell membranes and cell walls of harmful microorganisms, destroy their structural integrity, interfere with normal physiological functions, effectively prevent the erosion of pickled vegetables by harmful microorganisms, and effectively extend the shelf life of pickled vegetables. Screening lactic acid bacteria strains with strong anti-corrosion ability has become the core link in optimizing pickled vegetable anti-corrosion technology and innovating pickled vegetable preservation technology.

[0004] Nowadays, with consumers paying more and more attention to food safety, traditional chemical preservatives have been criticized for the potential health risks brought by long-term use, and biological anti-corrosion technology has thus emerged. However, there may be problems such as fluctuating effects and limited applicable scenarios when using some biological anti-corrosion methods alone. In the pickled vegetable industry, combining the application of lactic acid bacteria with physical preservation means such as low-temperature storage and vacuum packaging can play a synergistic effect. Low temperature can inhibit the growth rate of microorganisms, the vacuum environment reduces the oxidation effect of oxygen on pickled vegetables, and lactic acid bacteria inhibit the growth of harmful microorganisms from the source. By combining multiple measures, the anti-corrosion and preservation effect of pickled vegetables can be greatly enhanced, enabling pickled vegetables to always maintain excellent quality during the shelf life.

[0005] The use of lactic acid bacteria for pickled vegetable anti-corrosion has the outstanding characteristics of safety, naturalness, and greenness. This makes it highly favored in the pickled vegetable market and is widely used in various pickled vegetable products, whether traditional pickled cucumbers, pickled radishes, or emerging specialty pickled vegetable varieties. Lactic acid bacteria not only effectively inhibit the growth of harmful microorganisms but also can improve the flavor of pickled vegetables to a certain extent, providing consumers with healthier and more delicious pickled vegetable choices. At the same time, its successful application in pickled vegetable anti-corrosion also provides valuable reference for the expansion of food biological anti-corrosion technology in other pickled food fields, promoting the entire food industry to develop in a safer and greener direction. However, there is currently no lactic acid bacterium with anti-corrosion performance and its application in pickled vegetables. Therefore, a lactic acid bacterium with anti-corrosion function and its application are provided. Summary of the Invention

[0006] The object of the present invention is to address the deficiencies of the prior art by providing a lactic acid bacterium with anti-corrosion function and its application, so as to solve the problems raised in the above-mentioned background art.

[0007] To achieve the above object, the present invention provides the following technical solution: A lactic acid bacterium Lactiplantibacillus plantarum GXSS-0004, with the preservation number of the strain: GDMCC No. 65451, which was deposited in the Guangdong Microbial Culture Collection Center on November 8, 2024. The address of the depositary institution: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences.

[0008] As a preferred technical solution of the present invention, the biological characteristics of this bacterium are as follows: The colonies of this bacterium are milky white, moist and smooth, with neat edges, the surface is relatively flat and slightly raised, and it is a Gram-positive bacillus; Through the 16S rDNA sequencing analysis of the strain, its homology with Lactobacillus plantarum reaches 100%; The optimal growth conditions of this bacterium are: in MRS medium, yeast extract 5 g / L, peptone 10 g / L, beef extract powder 10 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 mL / L, pH 6.2 - 6.4, cultured at 37 °C. Under this medium and growth conditions, it can utilize sugars to ferment and produce acid, thereby promoting its own growth and reproduction, forming round, smooth, moist colonies with neat edges, and the colony color is mostly white or off-white.

[0009] An application of the lactic acid bacterium Lactiplantibacillus plantarum GXSS-0004 as described above in the preparation of sauerkraut.

[0010] As a preferred technical solution of the present invention, the specific steps are as follows:

[0011] Step 1: Select fresh Chinese cabbages, remove the defective parts, wash, drain and cut them into pieces.

[0012] Step 2: After activating and culturing the lactic acid bacterium strain with strong acid resistance, prepare a bacterial solution with a concentration of 10 6 -10 8 CFU / mL.

[0013] Step 3: In the fermentation container, lay the Chinese cabbages in layers, evenly spray the bacterial solution on each layer, use 50 - 100 mL of the bacterial solution per kilogram of Chinese cabbage, and compact it to expel air.

[0014] Step 4: Ferment at 25-28°C for 3 days in the early stage to facilitate the rapid reproduction of lactic acid bacteria; then ferment at 18-22°C for 4-7 days;

[0015] Step 5: Regularly detect. When the pH value of the pickled Chinese cabbage reaches 3.5-4.0 and has a strong sour aroma, the fermentation is completed; the finished product is stored in a cool place at 5-10°C, vacuum-packed, and the product information is marked.

[0016] Application of Lactiplantibacillus plantarum GXSS-0004 as described above in the preparation of pickled papaya sauce.

[0017] As a preferred technical solution of the present invention, the specific steps are as follows:

[0018] Step 1: Take papayas with appropriate maturity, peel, remove the seeds, cut into pieces, soak in brine to remove astringency, and drain.

[0019] Step 2: Prepare the activated lactic acid bacteria into a bacterial solution.

[0020] Step 3: Add sugar and salt to the papaya pieces and stir. Pour in and mix evenly according to the amount of 80-120 mL of the bacterial solution per kilogram of papaya, and pack into a sealed container.

[0021] Step 4: Keep the temperature at 20-25°C throughout the fermentation process. After 5-7 days, when the pickled papaya sauce has a moderate sweet and sour taste, firm texture, and no peculiar smell, the fermentation is completed; store in a cool place, nitrogen-filled package, and mark the relevant product details.

[0022] Application of Lactiplantibacillus plantarum GXSS-0004 as described above in the preparation of pickled radish.

[0023] As a preferred technical solution of the present invention, the specific steps are as follows:

[0024] Step 1: After washing and cutting fresh radishes, marinate them with coarse salt for 1-2 hours, pour out the brine, rinse, and drain.

[0025] Step 2: Make the lactic acid bacteria into a bacterial solution, put the radishes into a jar, evenly pour and sprinkle the bacterial solution, use 80-150 mL of the bacterial solution per kilogram of radish, add cooled boiled water to cover the radishes, and then add 1%-2% white vinegar.

[0026] Step 3: Ferment at 28-30°C for 2 days in the initial stage, then adjust to 20-25°C and ferment for 2-5 days; when the pickled radish has a crispy taste and obvious sourness, the fermentation is completed.

[0027] Application of Lactiplantibacillus plantarum GXSS-0004 as described above in the preparation of pickled mustard greens.

[0028] As a preferred technical solution of the present invention, the specific steps are as follows:

[0029] Step 1: Select fresh and tender mustard, remove the roots and yellow leaves, wash it, dry it until slightly wilted, and cut it into sections;

[0030] Step 2: Cultivate lactic acid bacteria into a bacterial liquid, lay the mustard in layers in a fermentation container, evenly sprinkle the bacterial liquid on each layer, use 60 - 100 mL of bacterial liquid per kilogram of mustard, add 5% - 8% brine to cover the mustard, and seal the container with water;

[0031] Step 3: Ferment at 22 - 25 °C for the first 3 days, and then ferment at 15 - 20 °C for 7 - 12 days; wait until the mustard turns yellow, has a pure sour taste, and good texture to complete fermentation. Store it in a cool place, and mark all product information after packaging

[0032] Compared with the prior art, the beneficial effects of the present invention are: The lactic acid bacteria Lactiplantibacillus plantarum GXSS - 0004 of the present invention can utilize sugars to ferment and produce acid under the culture medium and growth conditions, thereby promoting its own growth and reproduction, and can play an anti - corrosion role in the preparation of pickled mustard, pickled radish, pickled Chinese cabbage, and pickled papaya sauce.

[0033] The lactic acid bacteria Lactiplantibacillus plantarum GXSS - 0004 of the present invention has the ability to degrade nitrite and produce biogenic amines. The lactic acid bacteria Lactiplantibacillus plantarum GXSS - 0004 has the ability to utilize or transform nitrite during metabolism; the lactic acid bacteria Lactiplantibacillus plantarum GXSS - 0004 can also produce different biogenic amines, which is of great significance for evaluating the potential safety risks and other aspects in the application of lactic acid bacteria in food fermentation and other fields. Brief Description of the Drawings

[0034] Figure 1 It is a photograph of the Gram - staining of the strain of the present invention;

[0035] Figure 2 It is a diagram of the agarose gel electrophoresis result of the PCR amplification product of the present invention;

[0036] Figure 3 It is a phylogenetic tree diagram of the 16S rDNA of the strain of the present invention;

[0037] Figure 4 It is a growth ability diagram of the strain of the present invention;

[0038] Figure 5 It is an acid - producing ability diagram of the strain of the present invention;

[0039] Figure 6 This is the ability diagram of the strain of the present invention to degrade nitrite;

[0040] Figure 7 This is the antibacterial diagram of the strain of the present invention against Escherichia coli;

[0041] Figure 8 This is the antibacterial diagram of the strain of the present invention against Staphylococcus aureus;

[0042] Figure 9 This is the drug resistance diagram of the strain of the present invention. Detailed implementation manners

[0043] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making the protection scope of the present invention more clearly defined.

[0044] Example 1: The present invention provides a technical solution: a strain of Lactiplantibacillus plantarum GXSS - 0004, with the preservation number of the strain: GDMCC No. 65451, which was deposited in the Guangdong Provincial Culture Collection of Microorganisms on November 8, 2024. The address of the depository unit is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences.

[0045] The biological characteristics of this strain are as follows: The colonies of this strain are milky white, moist and smooth, with neat edges, relatively flat and slightly raised surfaces, and it is a Gram - positive bacillus. Through 16S rDNA sequencing analysis of the strain, its homology with Lactobacillus plantarum reaches 100%. Combining the morphological characteristics and molecular identification results, this strain is identified as Lactobacillus plantarum.

[0046] The optimal growth conditions are: in MRS medium, yeast extract 5 g / L, peptone 10 g / L, beef extract powder 10 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 mL / L, pH 6.2 - 6.4, cultured at 37 °C. Lactobacilli are Gram - positive bacteria, and most are anaerobic or facultative anaerobic bacteria. Under this medium and growth conditions, they can utilize sugars such as glucose for fermentation to produce acid, thereby promoting their own growth and reproduction, forming round, smooth, moist, and neatly - edged colonies, and the colony colors are mostly white or off - white.

[0047] Characteristics of bacteriostasis and drug resistance: According to the bacteriostatic and drug-resistant characteristics of lactic acid bacteria, the double-layer agar plate combined with the Oxford cup method was used to detect the bacteriostasis of lactic acid bacteria. Using Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922 as indicator bacteria, the overnight-precultured pathogenic bacteria were first added to the LB agar medium cooled to 55 °C at a ratio of 0.1% (volume ratio). After mixing, it was transferred to a sterile petri dish with three Oxford cups arranged in a triangle. After the medium solidified, the Oxford cups were removed, and 100 μL of the bacterial suspension of the isolated strain was added to the wells and allowed to stand and absorb. After culturing in an incubator at 28 °C for 24 hours, observations were made. If a clear zone appeared around the Oxford cup, it indicated that the isolated lactic acid bacteria strain had bacteriostatic ability against the pathogenic bacteria. The strength of the bacteriostatic ability was judged by the diameter of the inhibition zone. This was mainly due to the combined effects of bacteriostatic substances (such as bacteriocins, etc.) produced by lactic acid bacteria, organic acids produced by metabolism (lowering the pH of the surrounding environment), and competition with pathogenic bacteria for nutrients and space, etc.

[0048] In terms of drug resistance detection, according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) of the United States, the disk diffusion (K-B) method was used. The concentration of the fresh lactic acid bacteria cell suspension was adjusted to 107 colony-forming units / mL, spread on the MRS agar medium, and antibiotic susceptibility disks of different types (ampicillin, cefmetazole, amikacin, etc.) and concentrations were applied. After culturing at 37 °C for 24 hours, the diameter of the inhibition zone was measured. The sensitivity of lactic acid bacteria to different antibiotics was judged according to the diameter of the inhibition zone. When the diameter of the inhibition zone ≤ 15 mm, it was determined as drug-resistant; when the diameter was between 15 and 21 mm, it was intermediate; when the diameter ≥ 21 mm, it was sensitive. Thus, the drug-resistant or sensitive situation of lactic acid bacteria to a variety of commonly used antibiotics can be accurately evaluated, providing a scientific basis for the rational use of antibiotics and the application of lactic acid bacteria in related products.

[0049] Characteristics of nitrite degradation ability and biogenic amine production ability of lactic acid bacteria: When measuring the nitrite degradation ability of lactic acid bacteria, the isolated lactic acid bacteria strain was inoculated into 40 mL of sterile MRS broth containing 125 μg / mL nitrite at an inoculation amount of 2% (volume ratio), cultured at 30 °C, and the fermentation broth was collected at 0, 24, 36, 48, and 72 hours respectively to detect the nitrite concentration, so as to evaluate the degradation effect of lactic acid bacteria on nitrite, which reflected the ability of lactic acid bacteria to utilize or transform nitrite during metabolism.

[0050] In the detection of biogenic amine production ability, first, 1 mL of lactic acid bacteria isolate cultured overnight in MRS medium was added to 9 mL of MRS medium containing 0.1% of precursor amino acids such as histidine, tyrosine, or ornithine, and cultured at 25 °C for 24 hours. Subsequently, 10 μL of the culture broth sample was inoculated onto a decarboxylase medium plate with a specific formula (containing multiple components such as 0.5% tryptone, pH value of 5.3) and containing 1.0% (volume ratio) of precursor amino acids, and cultured at 25 °C for 48 hours. If purple appears around the colonies, it indicates that the lactic acid bacteria isolate can produce biogenic amines. Using Lactobacillus ATCC33222 TM and Enterococcus faecalis ATCC29212 TM as positive controls for the production of histamine, putrescine, and tyramine respectively, can accurately judge the ability of lactic acid bacteria to produce different biogenic amines, which is of great significance for evaluating the potential safety risks and other aspects in the application of lactic acid bacteria in food fermentation and so on.

[0051] An application of Lactiplantibacillus plantarum GXSS - 0004 as described above in the preparation of sauerkraut, the specific steps are as follows:

[0052] Step 1: Select fresh Chinese cabbages, remove the defective parts, wash, drain, and cut into pieces;

[0053] Step 2: After activating and culturing the lactic acid bacteria strain with strong acid resistance, prepare a bacterial suspension with a concentration of 10 6 -10 8 CFU / mL;

[0054] Step 3: In the fermentation container, lay the Chinese cabbages in layers, evenly spray the bacterial suspension on each layer, use 50 - 100 mL of bacterial suspension per kilogram of Chinese cabbage, and compact to expel air.

[0055] Step 4: Ferment at 25 - 28 °C for 3 days in the early stage to facilitate the rapid reproduction of lactic acid bacteria; then ferment at 18 - 22 °C for 4 - 7 days;

[0056] Step 5: Regularly detect. When the pH value of the sauerkraut reaches 3.5 - 4.0 and has a strong sour aroma, the fermentation is completed; the finished product is stored in a cool place at 5 - 10 °C, packaged in vacuum, and label the product information.

[0057] An application of Lactiplantibacillus plantarum GXSS - 0004 as described above in the preparation of pickled papaya sauce, the specific steps are as follows:

[0058] Step 1: Take papayas with appropriate maturity, peel, remove the seeds, cut into pieces, soak in brine to remove astringency, and drain;

[0059] Step 2: Prepare the activated and cultured lactic acid bacteria into a bacterial solution;

[0060] Step 3: Add sugar and salt to the papaya pieces, stir, pour in and mix evenly according to the amount of 80 - 120 mL of bacterial solution per kilogram of papaya, and pack into a sealed container;

[0061] Step 4: Keep the whole fermentation process at 20 - 25 °C. After 5 - 7 days, when the papaya jam has a moderate sour and sweet taste, a firm texture and no strange smell, the fermentation is completed; store it in a cool place, use nitrogen - filled packaging, and label the relevant product details.

[0062] Application of Lactiplantibacillus plantarum GXSS - 0004 as described above in the preparation of radish acid, the specific steps are as follows:

[0063] Step 1: After washing and cutting the fresh radish, marinate it with coarse salt for 1 - 2 hours, pour out the brine, rinse and drain;

[0064] Step 2: Make the lactic acid bacteria into a bacterial solution, put the radish into a jar, evenly pour and sprinkle the bacterial solution, use 80 - 150 mL of bacterial solution per kilogram of radish, add cooled boiled water to cover the radish, and then add 1% - 2% white vinegar;

[0065] Step 3: Ferment at 28 - 30 °C for 2 days in the initial stage, and then adjust to 20 - 25 °C for 2 - 5 days; when the radish acid has a crispy taste and an obvious sour taste, the fermentation is completed.

[0066] Application of Lactiplantibacillus plantarum GXSS - 0004 as described above in the preparation of pickled mustard, the specific steps are as follows:

[0067] Step 1: Select tender mustard, remove the roots and yellow leaves, wash it, dry it in the sun until slightly wilted and cut it into sections;

[0068] Step 2: Culture the lactic acid bacteria into a bacterial solution, lay the mustard in layers in the fermentation container, evenly sprinkle the bacterial solution on each layer, use 60 - 100 mL of bacterial solution per kilogram of mustard, add 5% - 8% brine to cover the mustard, and seal the container with water;

[0069] Step 3: Ferment at 22 - 25 °C for the first 3 days, and then ferment at 15 - 20 °C for 7 - 12 days; when the mustard turns yellow, has a pure sour taste and good texture, the fermentation is completed. Store it in a cool place, and label all the product information after packaging.

[0070] Example 2:

[0071] 1. Screening method of the strain of the present invention

[0072] 1.1 Culture media involved in the present invention:

[0073] Lactic acid bacteria screening medium: Prepared with 50 g / L of MRS broth, 20 g / L of glucose, 1 mL / L of Tween 80, and pure water, pH 6.2.

[0074] Antibacterial zone medium: Prepared with 3 g / L of beef extract, 10 g / L of peptone, 5 g / L of NaCl, 15 g / L of agar, and pure water, pH 7.2.

[0075] Drug resistance medium: Prepared with 30 g / L of ordinary nutrient broth, the target antibiotic (at the concentration set according to the experiment), and pure water, pH 7.0.

[0076] Biogenic amine degradation medium: Prepared with biogenic amine (such as 2 g / L of putrescine), 10 g / L of glucose, 0.2 g / L of MgSO4·7H2O, 2 g / L of KH2PO4, and pure water, pH 7.2.

[0077] Nitrite medium: Prepared with 10 g / L of peptone, 3 g / L of beef extract, 5 g / L of NaCl, 0.1 g / L of NaNO2, and pure water, pH 7.0.

[0078] 1.2 Screening method of strains

[0079] Pickled bamboo shoots from various regions of Guangxi. The pickled bamboo shoot brine was serially diluted in sterile 0.85% (mass / volume) sodium chloride solution, and then inoculated onto MRS (produced by Qingdao Haibo Biotechnology Co., Ltd.) agar medium and cultured at 30 °C for 48 hours. Typical colonies with different morphologies were picked from the plates of the final dilution and purified. Gram-positive, catalase-negative, non-motile cocci and decarboxylase-negative isolates were regarded as suspected lactic acid bacteria isolates and further purified by continuous subculture.

[0080] 2. Morphological characteristics of the strains of the present invention

[0081] 2.1 Colony morphological characteristics:

[0082] After Gram staining, lactic acid bacteria showed typical Gram-positive characteristics under the microscope, presenting an overall purple color. Morphologically, lactic acid bacteria are rod-shaped, with their rod shapes varying in length, relatively uniform in width, generally straight, blunt at both ends, often existing individually or arranged in chains, clearly distinguishable against the purple background. This rod shape and the purple appearance after staining are significant identifiers of lactic acid bacteria at the microscopic level ( Figure 1 ).

[0083] 2.2 Molecular biological identification of strains

[0084] According to the manufacturer's operating instructions, the genomic DNA of the isolated strain was extracted from the overnight culture using the MiniBEST Bacterial Genomic DNA Extraction Kit Ver. 3.0. The 16S rRNA gene was amplified using the primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. in China, and then the obtained sequences were aligned with the sequence database of the National Center for Biotechnology Information (NCBI) in the United States. The strains were identified at the species level through the Basic Local Alignment Search Tool (BLAST). Strains with a homology of at least 97% were considered to be of the same species. The cultures were stored at -80 °C with 20% (v / v) glycerol as the preservative solution and were routinely subcultured by culturing in MRS broth at 30 °C for 24 hours. The sequencing results were submitted to the National Center for Biotechnology Information (NCBI) database for alignment, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA7.0 software. The PCR amplification products of the 16S rDNA sequence of strain 2 were detected by 2% agarose gel electrophoresis, and the results are shown in Figure 2 . After detection, the base length of the 16S rDNA was 1465 bp. The sequence was analyzed by BLAST on NCBI. The homology of the 16S rDNA of the strain with Bacillus nitratireducens was 100%. Considering the comprehensive morphological characteristics, the strain was identified as Bacillus sp. A phylogenetic tree was constructed using the NJ method in MEGA7.0 software, and the results are shown in Figure 3 .

[0085] 3. Growth and acidification ability of the strain of the present invention

[0086] The activated culture of the isolated strain (cultured for 18 hours) was inoculated into fresh sterile MRS broth at an inoculum size of 2% (v / v) and cultured at 30 °C for 48 hours. Then, the cells were collected by centrifugation at 12,000 × g for 20 minutes at 4 °C and washed twice with sterile 0.85% (w / v) saline solution. Finally, the cells were resuspended in the same solution and inoculated into sterile MRS broth as an inoculum to evaluate their growth rate (absorbance at 600 nm, OD600) and acidification ability (by measuring pH value and titratable acidity (TA)). The OD600 of the cell suspension was measured and adjusted to obtain a microbial culture with an initial OD600 of approximately 0.1. Within 48 hours after inoculation, the fermentation broth was taken out every 2 hours, stirred before measuring the OD600 value, and the OD600 value was measured at 0, 2, 4, 6, 8, 10, 12, and 24 hours to determine the pH value; the titratable acidity (TA) was detected at 0, 24, 36, 48, and 72 hours, respectively. Acidification and growth abilities of the strain and other bacteria Figure 4 and Figure 5 。

[0087] 4. Ability of the strain to degrade nitrite

[0088] The isolated lactic acid bacteria strains were inoculated into 40 mL of sterile MRS broth containing 125 μg / mL nitrite at an inoculum size of 2% (v / v) to determine their nitrite removal ability. All experimental groups were cultured at 30 °C, and the fermentation broth was collected at 0, 24, 36, 48, and 72 hours, respectively, for the determination of nitrite concentration. Figure 6 is the ability to degrade nitrite.

[0089] 5. Antibacterial property of the strain

[0090] The double-layer agar plate method (Kang et al., 2022) was used to screen lactic acid bacteria with antibacterial activity. Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922 were used as indicator bacteria. The specific operation was as follows: The pathogen pre-cultured overnight was added to LB agar cooled to 55 °C at a ratio of 0.1% (v / v). Then, the mixture was poured into sterile plates with three Oxford cups placed in a triangular pattern. After solidification, the Oxford cups were removed, and 100 μL of the cell suspension of the isolated strain was added to the wells and allowed to adsorb statically. Finally, the plates were placed in an incubator at 28 °C for 24 hours, and then the inhibition zone was measured. If a clear circle appeared around the Oxford cup, it indicated that the isolated strain had antibacterial ability against the pathogen. The strength of the antibacterial ability was judged by the diameter of the circle. Figure 7 is the antibacterial property of the strain against Escherichia coli, Figure 8 is the antibacterial property of the strain against Staphylococcus aureus.

[0091] 6. Drug resistance of bacteria

[0092] The disk diffusion method was used to determine the susceptibility of lactic acid bacteria to different antibiotics according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2016). First, the concentration of the fresh lactic acid bacteria cell suspension was adjusted to 10 7 colony forming units / mL, and then it was evenly spread on MRS agar medium. Then, antibiotic susceptibility disks with different concentrations (provided by a microbial reagent company in Hangzhou, China) were placed on the medium. According to the recommendations of CLSI, the antibiotic disks used included: ampicillin (10 μg), cefazolin (30 μg), amikacin (30 μg), gentamicin (10 μg), norfloxacin (10 μg), ciprofloxacin (5 μg), compound sulfamethoxazole (25 μg), chloramphenicol (30 μg), erythromycin (15 μg), and penicillin (10 μg). The inoculated medium was incubated at 37 °C for 24 hours. After the incubation, the diameter of the inhibition zone was measured, and the susceptibility of lactic acid bacteria to the corresponding antibiotics was judged according to the size of the inhibition zone diameter. The results were divided into resistant (inhibition zone diameter ≤ 15 mm), intermediate (inhibition zone diameter 15 - 21 mm), and sensitive (inhibition zone diameter ≥ 21 mm). As Figure 9 shown.

[0093] The above embodiments only express the implementation modes of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims

1. A Lactiplantibacillus plantarum GXSS-0004, characterized in that, Its strain preservation number: GDMCC No. 65451, which was preserved in the Guangdong Provincial Culture Collection of Microorganisms on November 8, 2024. The address of the preservation unit is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences.

2. A Lactiplantibacillus plantarum GXSS-0004 as claimed in claim 1, wherein: The biological characteristics of this bacterium are as follows: The colony of this bacterium is milky white, moist and smooth, with neat edges, relatively flat and slightly raised surface, and it is a Gram-positive bacillus; Through 16S rDNA sequencing analysis of the strain, its homology with Lactobacillus plantarum reaches 100%; The optimal growth conditions of this bacterium are: in MRS medium, yeast extract 5 g / L, peptone 10 g / L, beef extract powder 10 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 mL / L, pH 6.2 - 6.4, cultured at 37 °C. Under this medium and growth conditions, it can utilize sugars to ferment and produce acid, thereby promoting its own growth and reproduction, forming round, smooth, moist and neatly edged colonies, and the colony color is mostly white or grayish white.

3. Use of the lactic acid bacterium Lactiplantibacillus plantarum GXSS - 0004 as claimed in claim 1 or 2 in the preparation of sauerkraut.

4. The application according to claim 3, wherein: The specific steps are as follows: Step 1: Select fresh Chinese cabbages, remove the defective parts, wash, drain and cut into pieces. Step 2: After activating and culturing the lactic acid bacteria strains with strong acid resistance, prepare a bacterial liquid with a concentration of 10 6 -10 8 CFU / mL; Step 3: In the fermentation container, lay the Chinese cabbages in layers, evenly spray the bacterial liquid on each layer, use 50 - 100 mL of bacterial liquid per kilogram of Chinese cabbage, and compact to expel air. Step 4: Ferment at 25 - 28 °C for 3 days in the early stage to facilitate the rapid reproduction of lactic acid bacteria; then ferment at 18 - 22 °C for 4 - 7 days. Step 5: Regularly detect. When the pH value of the sauerkraut reaches 3.5 - 4.0 and has a strong sour aroma, the fermentation is completed; The finished product is stored in a cool place at 5 - 10 °C, packed in vacuum, and label the product information.

5. Use of the lactic acid bacterium Lactiplantibacillus plantarum GXSS - 0004 as claimed in claim 1 or 2 in the preparation of pickled papaya with sauce.

6. The application according to claim 5, wherein: The specific steps are as follows: Step 1: Take papayas with appropriate maturity, peel, remove the seeds and cut into pieces, soak in brine to remove astringency and drain. Step 2: Prepare the bacterial liquid by activating and culturing the lactic acid bacteria. Step 3: Add sugar and salt to the papaya pieces and stir, pour in and mix according to the amount of 80 - 120 mL of bacterial liquid per kilogram of papaya, and put into a sealed container. Step 4: Keep the whole fermentation process at 20 - 25 °C. After 5 - 7 days, when the pickled papaya with sauce is moderately sour and sweet, firm in texture and odorless, the fermentation is completed; Store in a cool place, packed with nitrogen filling, and label the relevant product details.

7. Use of the lactic acid bacterium Lactiplantibacillus plantarum GXSS - 0004 as claimed in claim 1 or 2 in the preparation of pickled radish.

8. The application according to claim 7, wherein: The specific steps are as follows: Step 1: After washing and cutting fresh radishes, marinate them with coarse salt for 1 - 2 hours, pour out the brine, rinse, and drain. Step 2: Prepare the lactic acid bacteria into a bacterial solution. Put the radishes into a jar, evenly pour and sprinkle the bacterial solution. Use 80 - 150 mL of the bacterial solution per kilogram of radishes, add cooled boiled water to cover the radishes, and then add 1% - 2% white vinegar. Step 3: Ferment at 28 - 30 °C for 2 days initially, and then adjust the temperature to 20 - 25 °C for 2 - 5 days; the fermentation is completed when the radishes are sour, crispy, and have an obvious sour taste.

9. Use of Lactiplantibacillus plantarum GXSS - 0004 as claimed in claim 1 or 2 in the preparation of pickled mustard greens.

10. The application according to claim 9, characterized in that: The specific steps are as follows: Step 1: Select tender mustard greens, remove the roots and yellow leaves, wash them, dry them in the sun until slightly wilted, and cut them into sections. Step 2: Culture the lactic acid bacteria into a bacterial solution. Layer the mustard greens in the fermentation container, evenly sprinkle the bacterial solution on each layer. Use 60 - 100 mL of the bacterial solution per kilogram of mustard greens, add 5% - 8% brine to cover the mustard greens, and seal the container with water. Step 3: Ferment at 22 - 25 °C for the first 3 days, and then ferment at 15 - 20 °C for 7 - 12 days; the fermentation is completed when the mustard greens turn yellow, have a pure sour taste, and good texture. Store in a cool place, and label all product information after packaging.

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