Lactobacillus plantarum for inhibiting brown stain of soybean paste and application of lactobacillus plantarum

Abstract

The invention relates to the technical field of microorganisms and the field of food, in particular to lactobacillus plantarum for inhibiting brown stain of soybean paste and application of lactobacillus plantarum. The lactobacillus plantarum is screened from traditional naturally-fermented peasant soybean paste in northeast China, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on June 30, 2025, and the preservation number is CGMCC No.35048. The lactobacillus plantarum has an obvious browning inhibition effect and can be widely applied to fermented food production.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial applications and food production, and specifically relates to a plant lactobacillus that inhibits browning of soybean paste and its application. Background Technology

[0002] Soybean paste, with its unique aroma and flavor, is widely used as a distinctive condiment in Northeast China. Currently, there are many types of soybean paste products, and many exhibit instability in color. Color indicators provide consumers with immediate sensory information and serve as a basic visual standard for assessing soybean paste quality. Soybean paste with abnormal color can easily lead to negative consumer perceptions of product quality, thus affecting its market acceptance. The darkening of soybean paste's color is mainly due to browning caused by a series of biochemical reactions during fermentation. Undesirable browning can produce harmful substances, posing a carcinogenic risk to humans. The main causes of browning in soybean paste are the Maillard reaction and enzymatic browning. The Maillard reaction occurs when the proteins in soybeans, the raw material for soybean paste, are broken down by enzymes into compounds such as amino acids. These amino acids and reducing sugars undergo a carbonyl-amine condensation reaction under room temperature or heating conditions, forming unstable imine derivatives, which subsequently cyclize to N-glucosylamine. Through a series of reactions, hydroxymethylfurfural (HMF) is generated, and at the end of the reaction, aldehydes and amines also polymerize to form melanoidins, a black substance. Appropriate browning gives soybean paste its appealing color, while excessive browning reduces its sensory quality, thus affecting market acceptance. Enzymatic browning involves the conversion of colorless phenolic compounds in soybean paste raw materials into colored quinones under the action of various enzymes. Common enzymes involved in enzymatic browning include polyphenol oxidase, peroxidase, and phenylalanine ammonia-lyase. Polyphenol oxidase, as one of the key enzymes in the enzymatic browning process, possesses the activity of catalyzing the oxidation of phenolic compounds. When phenolic compounds in the raw materials are exposed to an aerobic environment, polyphenol oxidase drives the conversion of phenolic compounds into quinones. Peroxidase can also use hydrogen peroxide as an oxidant to catalyze the oxidation of phenolic compounds. In addition, phenylalanine ammonia-lyase participates in the catalytic process of the phenylpropane pathway, decomposing substances to produce phenolic compounds, which accumulate and become substrates for polyphenol oxidase. Phenylalanine ammonia-lyase indirectly promotes browning by regulating phenolic metabolism.

[0003] Methods for inhibiting browning in food range from refrigeration and modified atmosphere packaging to the use of chemical reagents such as citric acid, ascorbic acid, and sulfites. Each method has its drawbacks, including cost, potential off-flavors, and consumer acceptance. Therefore, there is a growing emphasis on safe, effective, and cost-effective solutions. Currently, physical processing technologies such as microwaves, irradiation, ozone, and cold plasma are gaining widespread attention due to their green and safe nature and significant browning inhibition effects. Although physical methods have proven effective in inhibiting browning, they often result in the loss of the original sensory and sensory characteristics of food during storage. In recent years, bio-strategies using natural extracts and their products have emerged as promising options. Fruit and vegetable extracts have recently been studied to explore their potential in anti-browning treatments and coatings. Furthermore, the extraction of anti-browning from animal-derived by-products is also a significant area. Animal-derived products contain bioactive peptides with structures similar to enzyme substrates, which are used to immobilize the enzyme substrates, thereby inhibiting the enzymatic browning process. Some animal materials, including egg white, sericin, fish skin, honey, mussels, and whey, can be considered as the next option for controlling food browning. While the above methods have garnered significant attention, limitations have also been identified with natural extracts such as coatings, which can affect the aroma and flavor of fruits and vegetables. Utilizing microorganisms to inhibit browning in food represents a multifunctional, green, safe, and sustainable new browning inhibition technology, holding significant importance for improving food quality and meeting consumer demands. Summary of the Invention

[0004] The purpose of this invention is to provide a *Lactobacillus plantarum* strain that can significantly inhibit browning in soybean paste. This *Lactobacillus plantarum* strain was screened from samples of traditionally naturally fermented soybean paste from farmers in Northeast China and exhibits a significant inhibitory effect on browning.

[0005] One aspect of this invention relates to a *Lactobacillus plantarum* strain that inhibits browning in soybean paste. This *Lactobacillus plantarum* strain is named *Lactobacillus plantarum* W19 and was deposited on June 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35048.

[0006] The 16S rDNA sequence of the *Lactobacillus plantarum* strain W19 is SEQ ID NO: 1.

[0007] This invention also relates to the application of the Lactobacillus plantarum strain W19 in the production of fermented foods.

[0008] Furthermore, the fermented food is soy sauce or bean paste.

[0009] The present invention also provides a probiotic fermentation agent comprising the Lactobacillus plantarum strain W19.

[0010] The probiotic starter also includes any one or more combinations of Bifidobacterium longum, Lactobacillus casei, Bacillus coagulans, Halophilic tetracoccus, Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Pediococcus lactis, and Leuconostoc mesenteroides.

[0011] The probiotic fermentation agent contains no less than 10 live bacteria of Lactobacillus plantarum W19 strain. 8 CFU / g.

[0012] The present invention also provides the application of the probiotic starter in food production.

[0013] The *Lactobacillus plantarum* W19 provided by this invention exhibits good biocompatibility, showing sensitivity to six common antibiotics, including amoxicillin, chloramphenicol, ceftazidime, tetracycline, gentamicin sulfate, and ampicillin. It also demonstrates strong antibacterial effects, with inhibition zones exceeding 15 mm in diameter against *Escherichia coli* and *Staphylococcus aureus*. Furthermore, it exhibits strong tolerance to the gastrointestinal environment, with a survival rate of 75.98% after 3 hours in artificial gastric fluid and 58.17% after 6 hours in intestinal fluid.

[0014] The *Lactobacillus plantarum* W19 strain effectively inhibits browning in fermented foods such as soybean paste and soy sauce. The activities of polyphenol oxidase, peroxidase, and peroxidase in the fermentation supernatant of this strain were 19.6 ± 0.02 U·mL. -1 155.57±0.15U·mL -1 6.05±0.02 U·mL -1 All exhibited low enzyme activity, demonstrating a significant browning inhibition effect. The *Lactobacillus plantarum* W19 provided by this invention can be widely used to inhibit food browning, meeting consumers' demands for safety and sustainability, and has broad prospects. Attached Figure Description

[0015] Figure 1 Growth curve of Lactobacillus plantarum W19;

[0016] Figure 2 Acid resistance test diagram of Lactobacillus plantarum W19;

[0017] Figure 3 Diagram of bile salt tolerance test for Lactobacillus plantarum W19. Detailed Implementation

[0018] The technical solutions of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are only used to explain and illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0019] All reagents used in the embodiments of this invention are domestically produced analytical grade reagents, and the culture media and formulations used are as follows:

[0020] MRS fermentation medium: 10 g / L peptone, 3 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.575 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate monohydrate, 20 g / L glucose, 2.42 g / L trisodium citrate, 4 g / L yeast extract, 8 g / L beef extract, 1 g / L Tween 80, add distilled water to a final volume of 1 L, and adjust the pH to 7.0.

[0021] Example 1: Isolation and identification of Lactobacillus plantarum W19

[0022] 1. Collection of soybean paste samples

[0023] Fifty-one samples of traditional homemade soybean paste were collected from seven regions in Northeast China. All samples were prepared using traditional natural fermentation techniques. After thorough mixing, the samples were aliquoted into 50 mL centrifuge tubes, quickly transported to the laboratory according to the sampling region number, and stored at -80°C for later use.

[0024] 2. Color determination of soybean paste samples

[0025] The brightness (L*), red / green (a*), yellow / blue (b*), and chromaticity (ΔE) values ​​of soybean paste samples from different regions were measured using a Chromameter CR-400 colorimeter. The soybean paste was sorted from lightest to darkest based on its chromaticity value; a smaller chromaticity value indicates a lighter color and a lower degree of browning.

[0026] Based on the color values ​​of the soybean paste samples, five light-colored soybean paste samples were selected, mixed, and used in the following experiments.

[0027] 3. Isolation of lactic acid bacteria

[0028] Accurately weigh 1.0 g of the light-colored soybean paste mixture into a 15 mL sterile centrifuge tube, bring the volume to 10 mL with sterile physiological saline, stir thoroughly, and let stand for 20–25 min. Transfer 1 mL of the supernatant to a new centrifuge tube and dilute with 9 mL of sterile physiological saline to 10⁻⁴, 10⁻⁵, and 10⁻⁶. Spread 100 μL of each dilution onto MRS solid medium and incubate at 36 °C for 2–3 days. After single colonies have grown, select healthy, suspected lactic acid bacteria colonies using the three-zone streak method, perform Gram staining, and isolate 72 Gram-positive strains. Confirm their lactic acid bacteria status through physiological and biochemical experiments. Incubate at 36 °C for 2 days, purify for three generations until no contamination is observed, and obtain pure colonies grown on MRS solid plates. Select high-quality single colonies and inoculate them into MRS liquid medium, place them on a shaker at 120 rpm and 37 °C for expansion culture to obtain single-cell suspensions. Finally, preserve the strains in glycerol for later use.

[0029] 4. Identification of lactic acid bacteria

[0030] Physiological and biochemical characterization tests were conducted according to the "Classification, Identification and Test Methods of Lactic Acid Bacteria" and the "Handbook of Common Bacteria Identification". The obtained single-bacterial suspensions were subjected to molecular biological identification, and the PCR products were sequenced by Shanghai Sangon Biotech Co., Ltd. After amplification, 15 strains of lactic acid bacteria were accurately identified using 16S rDNA sequence homology comparison analysis. The results of the 16S rDNA sequence homology comparison of the strains are shown in Table 1.

[0031] Table 1. Homology comparison of 16S rDNA sequences of strains

[0032] strain number Test results homology H2 Halophilic tetracoccus (Tetragenococcus halophilus) 99.8% L6 Halophilic tetracoccus (Tetragenococcus halophilus) 98.8% T10 Halophilic tetracoccus (Tetragenococcus halophilus) 98.8% B11 Leuconostoc lactis 99.6% N13 Enterococcus faecium 99.8% M18 Leuconostoc mesenteroides 99.2% W19 Lactibacillus plantarum 99.90% P24 Pediococcus pentosaceus 99.86% L25 Lactibacillus plantarum 97.5% B26 Lactibacillus plantarum 94.5% F38 Lactibacillus plantarum 99.5% K21 Leuconostoc pseudomesenteroides 99.8% R27 Lactibacillus plantarum 97.6% G31 Lactibacillus plantarum 99.43% H41 Pediococcus acutissima (Lactococcus lacticis) 96.42%

[0033] 5. Secondary screening of lactic acid bacteria that inhibit browning

[0034] The 15 lactic acid bacteria strains obtained from the initial screening were activated and fermented. After centrifugation at 4℃ and 10000 r / min for 10 min, the bacterial precipitate was collected, lysozyme was added to break it up, and the supernatant was collected after centrifugation. The following experiments were conducted. By comparing the results of multi-index analysis, the dominant strains that could significantly inhibit browning of soybean paste were screened.

[0035] (1) Polyphenol oxidase (PPO) activity assay

[0036] The PPO activity of lactic acid bacteria was determined using a microbial polyphenol oxidase ELISA kit. Sample pretreatment and standard curve plotting were performed according to the manufacturer's instructions. OD450 values ​​were measured, and sample activity was calculated. The PPO enzyme activity results are shown in Table 2.

[0037] PPO is key to enzymatic browning. During enzymatic browning, PPO acts as a catalyst, promoting the conversion of phenolic substrates into quinone compounds. These quinones then undergo spontaneous polymerization to generate substances that cause browning. Therefore, PPO can be used as one of the screening criteria for strains that inhibit browning.

[0038] Table 2. Results of PPO enzyme activity

[0039] strain number <![CDATA[PPO enzyme activity (U·mL -1 )]]> strain number <![CDATA[PPO enzyme activity (U·mL -1 )]]> strain number <![CDATA[PPO enzyme activity (U·mL -1 )]] H2 <![CDATA[51.24±0.08 a ]]> W19 <![CDATA[19.6±0.02 yz ]]> L25 <![CDATA[34±0.09 l ]]> L6 <![CDATA[22.12±0.94 v ]]> H41 <![CDATA[43.96±0.26 d ]]> B26 <![CDATA[28.69±0.14 n ]]> T10 <![CDATA[24.85±0.03 qr ]]> M18 <![CDATA[21.16±0.03 x ]]> R27 <![CDATA[35.16±0.06 k ]]> B11 <![CDATA[20.09±0.03 y ]]> K21 <![CDATA[21.07±1.41 w ]]> G31 <![CDATA[41±0.39 g ]]> N13 <![CDATA[22.73±0.03 uv ]]> P24 <![CDATA[26.07±0.11 o ]]> F38 <![CDATA[42.88±0.02 f ]]>

[0040] Note: Data are presented as mean ± standard deviation; data with different letters in the same column indicate significant differences (P < 0.05).

[0041] As shown in Table 2, among the 15 lactic acid bacteria strains obtained in the initial screening of this invention, strain W19 had the lowest polyphenol oxidase (PPO) activity, effectively inhibiting PPO production and thus inhibiting enzymatic browning. Therefore, this strain has a certain browning-inhibiting effect.

[0042] (2) Peroxidase (POD) activity assay

[0043] The POD activity of lactic acid bacteria was determined using a microbial peroxidase ELISA kit. Sample pretreatment and standard curve plotting were performed according to the manufacturer's instructions. OD450 values ​​were measured, and sample activity was calculated. POD activity results are shown in Table 3.

[0044] During the catalytic oxidation process, POD itself can generate a variety of free radicals. These free radicals trigger membrane lipid peroxidation, which intensifies the degree of membrane lipid peroxidation and ultimately exacerbates the browning phenomenon in tissues. Therefore, it can be used as one of the key indicators for inhibiting enzymatic browning.

[0045] Table 3. Results of POD enzyme activity

[0046] strain number <![CDATA[POD enzyme activity (U·mL -1 )]]> strain number <![CDATA[POD enzyme activity (U·mL -1 ).]]> strain number <![CDATA[POD enzyme activity (U·mL -1 )]]> H2 <![CDATA[398.05±0.25 b ]]> M18 <![CDATA[167.32±0.18 j1 ]]> R27 <![CDATA[314.51±0.08 e ]]> L6 <![CDATA[184.99±0.27 b1 ]]> W19 <![CDATA[155.57±0.15 l1 ]]> G31 <![CDATA[257.32±0.17 l ]]> T10 <![CDATA[187.79±0.19 z1 ]]> P24 <![CDATA[185.74±0.06 a1 ]]> F38 <![CDATA[269.67±0.11 j ]]> B11 <![CDATA[167.7±0.2 i1 ]]> L25 <![CDATA[340.71±0.2 c ]]> H41 <![CDATA[271.4±0.1 i ]]> N13 <![CDATA[207.79±0.17 t ]]> B26 <![CDATA[311.51±0.42 f ]]> K21 <![CDATA[203.5±0.09 u ]]>

[0047] Note: Data are presented as mean ± standard deviation; within the same column, data labeled with different letters are statistically significant (P<0.05).

[0048] As shown in Table 3, and as shown in Table 2, among the 15 lactic acid bacteria strains obtained in the initial screening of this invention, strain W19 had the lowest peroxidase (POD) activity. This indicates that this strain has a certain inhibitory effect on browning.

[0049] (3) Assay of phenylalanine ammonia-lyase (PAL) activity

[0050] The PAL activity of lactic acid bacteria was determined using a microbial phenylalanine ammonia-lyase ELISA kit. Sample pretreatment and standard curve plotting were performed according to the manufacturer's instructions. OD450 values ​​were measured, and sample activity was calculated. PAL enzyme activity results are shown in Table 4.

[0051] PAL catalyzes the deamination of phenylalanine to produce trans-cinnamic acid, which in the subsequent phenylpropane metabolic pathway produces secondary metabolites such as phenols, flavonoids, and lignin, participating in Maillard and oxidation reactions. Therefore, it can be used as a key indicator for inhibiting enzymatic browning.

[0052] Table 4. Results of PAL enzyme activity

[0053] strain number <![CDATA[PAL enzyme activity (U·mL -1 )]]> strain number <![CDATA[PAL enzyme activity (U·mL -1 )]] strain number <![CDATA[PAL enzyme activity (U·mL -1 )]]> H2 <![CDATA[6.26±0.02 a1b1 ]]> M18 <![CDATA[8.41±0.04 p ]]> B26 <![CDATA[11.15±0.01 jk ]]> L6 <![CDATA[8.3±0.03 q ]]> W19 <![CDATA[6.05±0.02 c1 ]]> R27 <![CDATA[9.68±0.05 n ]]> T10 <![CDATA[17.38±0.05 a ]]> K21 <![CDATA[6.68±0.01 z ]]> G31 <![CDATA[14.11±0.02 c ]]> B11 <![CDATA[7.07±0.01 wx ]]> P24 <![CDATA[7.24±0.02 v ]]> F38 <![CDATA[12.81±0.03 h ]]> N13 <![CDATA[7.15±0.01 vw ]]> L25 <![CDATA[11.11±0.02 k ]]> H41 <![CDATA[14.18±0.05 c ]]>

[0054] Note: Data are presented as mean ± standard deviation; within the same column, data labeled with different letters are statistically significant (P<0.05).

[0055] As shown in Table 4, among the 15 lactic acid bacteria strains obtained in the initial screening of this invention, strain W19 had the lowest phenylalanine ammonia-lyase (PAL) activity, at only 6.05 ± 0.02 U·mL.-1 This demonstrates that *Lactobacillus plantarum* W19 can, to some extent, inhibit the phenylalanine reaction and suppress browning.

[0056] (4) Determination of total browning

[0057] Browning degree is a quantitative indicator that measures the depth of color. It can usually reflect the color change caused by a series of chemical reactions, mainly related to processes such as Maillard reaction and enzymatic browning.

[0058] Simulated fermentation medium for soybean paste: 20 g soy protein isolate, 8 g wheat flour, 0.028 g calcium chloride, and 28 g deionized water were mixed and sterilized at 121 ℃ for 30 min. After cooling to 38 ℃, *Aspergillus oryzae* strain 3.042 was inoculated. After 3 days of fermentation, 100 mL of phosphate buffer solution was added, and the mixture was stirred vigorously for 1 h. After centrifugation at 10000 g / min for 15 min at 4 ℃, the supernatant was collected, 10% sodium chloride was added, and the mixture was sterilized at 121 ℃ for 15 min. Inoculation was performed at a 1% inoculum size. After two weeks of fermentation, the absorbance was measured at 420 nm using a microplate reader to evaluate the browning degree of fermented soybean paste from different strains. The results are shown in Table 5.

[0059] Table 5 Results of Browning Degree Measurement

[0060] strain number <![CDATA[Degree of Browning OD 420 > strain number <![CDATA[Browning degree OD 420 > strain number <![CDATA[Browning degree OD 420 > H2 <![CDATA[0.4174±0.002 cd ]]> M18 <![CDATA[0.3953±0.002 ghij ]]> B26 <![CDATA[0.3923±0 hijk ]]> L6 <![CDATA[0.382±0.004 mnopq ]]> W19 <![CDATA[0.3111±0.001 s ]]> R27 <![CDATA[0.3747±0.005 qrs ]]> T10 <![CDATA[0.3689±0.004 s ]]> K21 <![CDATA[0.398±0.002 fgh ]]> G31 <![CDATA[0.3873±0.001 jklmn ]]> B11 <![CDATA[0.4198±0.003 c ]]> P24 <![CDATA[0.3763±0.001 pqrs ]]> F38 <![CDATA[0.4113±0 de ]]> N13 <![CDATA[0.3865±0.019 klmno ]]> L25 <![CDATA[0.3366±0.004 uv ]]> H41 <![CDATA[0.3491±0.004 w ]]> null <![CDATA[0.3429±0.002 u ]]>

[0061] Note: Data are presented as mean ± standard deviation; within the same column, data labeled with different letters are statistically significant (P<0.05).

[0062] As shown in Table 5, compared with the blank control group, among the 15 lactic acid bacteria strains obtained in the initial screening of this invention, only strain W19 showed a significant decrease in browning degree, which was only 0.311±0.001. The browning degree of the other strains was aggravated to varying degrees.

[0063] (5) Determination of 5-hydroxymethylfurfural (HMF)

[0064] 5-Hydroxymethylfurfural (HMF) is a yellow to brown compound produced by a complex reaction between amino acids and reducing sugars in the Maillard reaction, and therefore can be used as one of the screening criteria for inhibiting browning strains.

[0065] A simulated HMF medium was used, with HMF standards inoculated into the medium to a final concentration of 1%. The inoculated strain was then fermented. After two weeks of fermentation, the absorbance was measured at 420 nm using a microplate reader. The results are shown in Table 6.

[0066] Table 6 HMF Measurement Results

[0067] serial number <![CDATA[HMF value OD 420 > serial number <![CDATA[HMF value OD 420 > strain number <![CDATA[HMF value OD 420 > H2 <![CDATA[0.4982±0.001 mn ]]> M18 <![CDATA[0.421±0.001 r ]]> B26 <![CDATA[0.3859±0 x ]]> L6 <![CDATA[0.4709±0.001 q ]]> W19 <![CDATA[0.1258±0.001 s ]]> R27 <![CDATA[0.3989±0.001 u ]]> T10 <![CDATA[0.5212±0.001 hi ]]> K21 <![CDATA[0.6008±0.001 b ]]> G31 <![CDATA[0.5372±0 e ]]> B11 <![CDATA[0.5212±0.002 hi ]]> P24 <![CDATA[0.41±0.001 t ]]> F38 <![CDATA[0.6136±0 a ]]> N13 <![CDATA[0.5294±0.001 f ]]> L25 <![CDATA[0.3891±0.002 w ]]> H41 <![CDATA[0.3971±0.001 uv ]]> null <![CDATA[0.492±0.001 o ]]>

[0068] Note: Data are presented as mean ± standard deviation; within the same column, data labeled with different letters are statistically significant (P<0.05).

[0069] As shown in Table 6, compared with the blank control group, the HMF value of the W19 strain provided by this invention decreased the most, by only 0.1258 ± 0.001. This indicates that the strain has a certain effect on inhibiting HMF production.

[0070] 6. Factor analysis

[0071] Based on the above experimental results, different lactic acid bacteria strains showed different browning inhibition indices. To comprehensively evaluate their impact on color, the strains were classified according to variable correlation, and factor scores were used to complete a comprehensive evaluation of 15 lactic acid bacteria strains.

[0072] The results showed that strain W19 had the highest overall factor score, indicating that it had the strongest ability to inhibit browning.

[0073] 7. Identification of W19 strain

[0074] The 16S rDNA sequence of strain W19 is shown in SEQ ID NO: 1.

[0075]

[0076] The taxonomic position of strain W19 was analyzed using 16S rDNA sequencing technology. The sequencing results were compared with sequences in the NCBI database using the BLAST algorithm. A phylogenetic tree was constructed using MEGA10 software to determine the phylogenetic relationships among the strains.

[0077] Based on homology retrieval analysis and the taxonomic position of the strain in the phylogenetic tree, the W19 strain obtained by screening in this invention was identified as Lactibacillus plantarum.

[0078] The applicant deposited Lactobacillus plantarum strain W19 at the China General Microbiological Culture Collection Center (CGMCC) on June 30, 2025. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35048.

[0079] Example 2: Growth curve determination of Lactobacillus plantarum W19

[0080] Lactobacillus plantarum W19 was inoculated into MRS liquid medium, and the OD600nm was measured and recorded every 2 hours.

[0081] Figure 1 The graph shows the growth curve of Lactobacillus plantarum W19. It can be seen from the graph that Lactobacillus plantarum W19 grows most vigorously at 16 hours and then begins to decline.

[0082] Example 3 Safety evaluation of Lactobacillus plantarum W19

[0083] The susceptibility of the tablets to amoxicillin (25 μg / tablet), chloramphenicol (30 μg / tablet), ceftazidime (30 μg / tablet), tetracycline (30 μg / tablet), gentamicin sulfate (10 μg / tablet), and ampicillin (10 μg / tablet) was tested according to the Kirby-Bauer disk diffusion method recommended by the World Health Organization.

[0084] The glycerol-preserved strain was activated in sterilized MRS liquid tubes, passaged, and cultured in a fermenter for 24 hours to obtain the second-generation strain. A culture of the second-generation *Lactobacillus plantarum* W19 strain was spread onto MRS solid medium using a sterile cotton swab. Antibiotic discs were placed on the surface of the solid medium using sterile forceps. The medium was incubated upside down at 38°C for 48 hours, and the diameter of the inhibition zone (mm) was measured and recorded. The susceptibility of *Lactobacillus plantarum* to six common antibiotics was assessed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Specific results are shown in Table 7.

[0085] Table 7 Results of antibiotic susceptibility testing of Lactobacillus plantarum W19

[0086] antibiotic Sensitive S Moderately sensitive M Drug-resistant R Diameter of the inhibition zone W19 Sensitivity Amoxicillin 17–24 9–16 ≤8 18mm S Chloramphenicol ≥18 13–17 ≤12 30mm S Ceftazidime 17–24 9–16 ≤8 20mm S tetracycline ≥15 12–14 ≤11 19mm S Gentamicin sulfate ≥15 13–14 ≤12 20mm S Ampicillin ≥17 9–16 ≤8 25mm S

[0087] As shown in Table 7, Lactobacillus plantarum W19 is sensitive to six common antibiotics, including amoxicillin, chloramphenicol, ceftazidime, tetracycline, gentamicin sulfate, and ampicillin.

[0088] Example 4: In vitro antibacterial analysis of Lactobacillus plantarum W19

[0089] Two indicator bacteria (Staphylococcus aureus and Escherichia coli) were used to determine the antibacterial ability of Lactobacillus plantarum W19.

[0090] Glyceryl-preserved *E. coli* and *Staphylococcus aureus* were cultured in LB liquid medium for 24 hours. The bacterial suspensions of *E. coli* and *S. aureus* were then spread onto LB solid medium using sterile cotton swabs. *Lactobacillus plantarum* W19, also preserved in glycerol, was cultured in MRS liquid medium for 24 hours. A filter paper disc was then used to pick up a suspension of *Lactobacillus plantarum* W19 and place it onto LB solid medium. The LB solid medium was placed in a 37°C incubator and incubated for 24 hours. Afterward, the presence and diameter of inhibition zones around the colonies were observed and measured.

[0091] Table 8 Results of antibacterial experiment with Lactobacillus plantarum W19

[0092] indicator bacteria Diameter of the inhibition zone (mm) E. coli 15.96 Staphylococcus aureus 17.35

[0093] The results are shown in Table 8. Lactobacillus plantarum W19 has a strong inhibitory effect on Escherichia coli and Staphylococcus aureus.

[0094] Example 5: In vitro evaluation of the probiotic function of Lactobacillus plantarum W19

[0095] 1. Acid resistance test

[0096] The pH of MRS medium was adjusted to 2.0, 3.0, and 4.0 using 0.1% hydrochloric acid, and then aliquoted into sterile test tubes. A blank control group was also prepared using untreated MRS medium, which was then sterilized. Activated *Lactobacillus plantarum* W19 was cultured at 37°C for 24 hours, then centrifuged at 5000 rpm for 10 min, and the OD600nm was adjusted to 0.5 with sterile PBS buffer. Then, *Lactobacillus plantarum* W19 was inoculated at 1% into MRS medium at different pH values ​​and into the blank control group medium. OD600nm was measured, and the culture was incubated at room temperature for 4 hours before measuring OD600nm again. Each strain was measured three times. The survival rate was calculated using the following formula:

[0097] Survival rate (%) = 100% × OD value of experimental group / OD value of control group.

[0098] The results are as follows Figure 2 As shown, the survival rate of Lactobacillus plantarum W19 was 25.30% after culturing at pH 2.0 for 4 hours.

[0099] 2. Bile salt tolerance test

[0100] MRS culture media were prepared by adding 0.1%, 0.2%, and 0.3% bile salts, and then aliquoted into sterile test tubes. Activated *Lactobacillus plantarum* W19 was cultured at 37°C for 24 hours, then centrifuged at 5000 rpm for 10 min, and the OD600nm value was adjusted to 0.5 with sterile PBS buffer. Then, *Lactobacillus plantarum* W19 was inoculated at 1% into MRS culture media containing different concentrations of bile salts and a blank control group, and the OD600nm value was measured. After culturing at room temperature for 4 hours, the OD600nm value was measured again. The survival rate was calculated using the following formula:

[0101] Survival rate (%) = 100% × OD value of experimental group / OD value of control group.

[0102] The results are as follows Figure 3 As shown, the survival rate of Lactobacillus plantarum W19 after culturing in 0.1% bile salt environment for 4 hours was 38.46%.

[0103] 3. Simulated gastrointestinal tolerance experiment

[0104] 200 μL of activated *Lactobacillus plantarum* W19 bacterial suspension was placed in a 10 mL LMR Strain solution and incubated at 37 °C for 18 h. The suspension was then centrifuged at 8000 rpm for 10 min to collect the bacterial cells. The bacterial suspension was washed with PBS to adjust the OD600 nm to 1. The PBS solution was adjusted to pH 3.0, sterilized, and then 0.6 g of pepsin was added for membrane filtration to simulate artificial gastric fluid. The PBS solution was then adjusted to pH 8.0, sterilized, and then 0.2 g of trypsin and 0.3% bile salts were added for membrane filtration to simulate artificial intestinal fluid. 1 mL of bacterial suspension and 9 mL of artificial gastric fluid were mixed and the OD600 nm was measured. After 3 hours, 1 mL of the mixture of *Lactobacillus plantarum* W19 bacterial suspension and artificial gastric fluid was added to 9 mL of artificial intestinal fluid, mixed, and the OD600 nm was measured. After 6 hours, the OD600 nm was measured again. The survival rate was calculated using the following formula:

[0105] Survival rate (%) = 100% × OD value of experimental group / OD value of control group.

[0106] The results showed that Lactobacillus plantarum W19 had strong tolerance to the gastrointestinal environment, with a survival rate of 75.98% after 3 hours in artificial gastric fluid and 58.17% after 6 hours in intestinal fluid.

[0107] 4. Adhesion ability evaluation

[0108] (1) Surface hydrophobicity determination

[0109] Centrifuge the activated *Lactobacillus plantarum* W19 bacterial suspension at 1000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Wash the suspension 2-3 times with 0.85% NaCl physiological saline to ensure the OD600nm of the bacterial suspension is between 0.5 ± 0.05, and record the initial absorbance A0 at 600nm. Mix 3 mL of the bacterial suspension and 3 mL of xylene in a 10 mL centrifuge tube, vortex for 30 s to mix thoroughly, and let the mixture stand at 37°C for 30 min. Then, collect the supernatant and measure the OD600nm (A1).

[0110] Hydrophobicity formula: Hydrophobicity (%) = (A0-A1) / A0×100%.

[0111] The results showed that the hydrophobicity of Lactobacillus plantarum W19 was 35.42%.

[0112] (2) Determination of self-agglomeration rate

[0113] Centrifuge the activated *Lactobacillus plantarum* W19 bacterial suspension at 1000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Wash the suspension 2-3 times with 0.85% NaCl physiological saline to ensure the OD600nm of the bacterial suspension is between 0.5 ± 0.05, and record the initial absorbance A0 at 600nm. Incubate the bacterial suspension at 37℃ for 5 hours, and measure the absorbance (A1) of the supernatant at 600nm.

[0114] Self-agglomeration formula: Self-agglomeration rate (%) = (A0-A1) / A0×100%.

[0115] The results showed that the autoagglutination rate of Lactobacillus plantarum W19 was 33.66%.

[0116] Example 6: Application of Lactobacillus plantarum W19 in the fermentation preparation of soybean paste

[0117] 1. Preparation method of fermented soybean paste:

[0118] (1) Place the soaked soybeans on a steamer and steam for 45 minutes until the soybeans are cooked through and can be easily crushed with your fingers;

[0119] (2) Place the flour on a steamer rack and steam for 10 minutes;

[0120] (3) Mix Aspergillus oryzae with cooked flour cooled to room temperature at a mass ratio of 1:2500, and mix with wet soybeans cooled to room temperature at a ratio of 4:1 to obtain soybean meal;

[0121] (4) Place the soybean meal in a constant temperature incubator, cover the surface with a layer of damp gauze, and make koji for 48 hours. The temperature is controlled at 34-36℃ for the first 24 hours and adjusted to 28℃ for the last 24 hours. Turn the koji over once every 12 hours to obtain soy sauce koji.

[0122] (5) Add the prepared koji and a sterilized salt solution with a mass-volume ratio of 15% to a sterilized fermentation tank at a mass ratio of 1:2, and stir evenly;

[0123] (6) On day 0, inoculate activated Lactobacillus plantarum W19 bacterial suspension at an inoculation rate of 15 mL / kg (10 9 (CFU / mL), fermentation temperature 30℃, daily stirring and raking of the koji, fermentation for 49 days to obtain fermented soybean paste.

[0124] Samples were taken every 7 days during the fermentation process of the soybean paste. Meanwhile, following the same preparation method as described above, a control group of fermented soybean paste was prepared without inoculation with *Lactobacillus plantarum*.

[0125] 2. Measurement of color of soybean paste

[0126] The brightness (L*), red / green (a*), and yellow / blue (b*) of the soybean paste samples were measured using a Chromameter CR-400 colorimeter. The results are shown in Table 9.

[0127] Table 9 Color changes during soybean paste fermentation

[0128] Note: Data are presented as mean ± standard deviation; within the same column, data labeled with different letters are statistically significant (P<0.05).

[0129] As shown in Table 9, the L value of the soybean paste gradually increased in the early stage of fermentation, and began to decrease after 21 days; the a and b values ​​also first increased and then decreased. At the end of fermentation, the L, a, and b values ​​of the fermented soybean paste inoculated with *Lactobacillus plantarum* W19 were significantly higher than those of the control group, indicating that *Lactobacillus plantarum* W19 can improve the brightness, redness, and yellowness of the soybean paste to a certain extent. The color change of the fermented soybean paste was evaluated by calculating ΔE. At the end of fermentation, the color value of the fermented soybean paste inoculated with *Lactobacillus plantarum* W19 was higher than that of the control group. This demonstrates that the *Lactobacillus plantarum* W19 provided by this invention can significantly inhibit browning of soybean paste, achieving unexpected technical effects.

Claims

1. A type of Lactobacillus plantarum ( Lactobacillus plantarum ), characterized in that, The preservation number of the Lactobacillus plantarum is CGMCC No. 35048.

2. The *Lactobacillus plantarum* as described in claim 1, characterized in that, The 16S rDNA sequence of the *Lactobacillus plantarum* is SEQ ID NO:

1.

3. The application of Lactobacillus plantarum as described in claim 1 in the production of fermented foods.

4. The application as described in claim 3, characterized in that, The fermented food is soy sauce or bean paste.

5. A probiotic starter, characterized in that, The probiotic fermentation agent comprises the *Lactobacillus plantarum* as described in claim 1.

6. The probiotic starter culture as described in claim 5, characterized in that, The probiotic starter also includes any one or more combinations of Bifidobacterium longum, Lactobacillus casei, Bacillus coagulans, Halophilic tetracoccus, Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Pediococcus lactis, and Leuconostoc mesenteroides.

7. The probiotic starter culture as described in claim 5 or 6, characterized in that, The probiotic starter contains no less than 10 live Lactobacillus plantarum bacteria. 8 CFU / g.

8. The use of the probiotic starter culture according to any one of claims 5-7 in food production.

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