Screening of tomato-sourced plant lactobacillus and application of tomato-sourced plant lactobacillus in tomato enzyme

Abstract

The invention discloses screening of tomato-sourced plant lactobacillus and application of the tomato-sourced plant lactobacillus in tomato enzymes, and belongs to the technical field of microorganisms. According to the invention, a strain of lactobacillus plantarum A17 is obtained through screening, and the lactobacillus plantarum A17 has good physiological characteristics and probiotic characteristics, can efficiently utilize different carbon sources for fermentation, and has good tolerance to acid stress and gastrointestinal fluid tolerance, and the fermented tomato enzyme is fine and smooth in tissue state. The lactobacillus plantarum A17 provided by the invention has a huge application prospect in the fermentation of fruits and vegetables, and the lactobacillus plantarum A17 can be used for fermenting fruits and vegetables.

Description

TECHNICAL FIELD

[0001] The present application relates to a tomato-derived Lactiplantibacillus plantarum strain screening and its application in tomato enzyme, belonging to the field of microbial technology. BACKGROUND

[0002] Lactiplantibacillus plantarum is a probiotic bacteria widely distributed in plants, fermented foods and human intestines. It has attracted attention due to its significant role and benefits in acid tolerance, gastrointestinal tolerance, ability to utilize multiple carbon sources, and good fermentation performance. Therefore, it is widely used in fruit and vegetable enzyme fermentation process. Studies have shown that Lactiplantibacillus plantarum has a wide range of carbon source utilization ability, and can effectively utilize various monosaccharides, disaccharides and polysaccharides as carbon sources for growth and fermentation. This is of great significance to improve its application potential in the food industry.

[0003] Lactiplantibacillus plantarum can tolerate low pH, which makes it an important probiotic bacteria. In the production of fermented foods, Lactiplantibacillus plantarum can grow and ferment in acidic environments, producing organic acids such as lactic acid, not only giving the food a unique flavor and texture, but also reducing the pH of the food and inhibiting the growth of harmful microorganisms, prolonging the shelf life of the food. Therefore, applying Lactiplantibacillus plantarum to tomato enzyme fermentation not only improves the flavor and texture of tomato enzyme, but also gives it more health benefits.

[0004] In addition, Lactiplantibacillus plantarum can exert probiotic effects in the intestinal tract, which depends on its colonization ability in extreme environments such as gastric juice, digestive enzymes and small intestinal bile salts. Therefore, studying its tolerance to gastrointestinal juice is one of the important indicators for evaluating its colonization ability and potential probiotic function.

[0005] However, not all Lactiplantibacillus plantarum have excellent probiotic function and fermentation performance. In practical application, it is necessary to screen Lactiplantibacillus plantarum strains with good probiotic function and stable fermentation performance to ensure the quality and safety of enzyme products. Through traditional isolation, screening and identification methods, Lactiplantibacillus plantarum strains with good carbon source utilization, growth tolerance and gastrointestinal tolerance can be obtained. Summary of the Invention

[0006] This invention provides a strain of Lactiplantibacillus plantarum XJU-LP-A17 (abbreviated as Lactiplantibacillus plantarum A17) with good probiotic function and fermentation performance. It was deposited on May 6, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66259, and the deposit address is 5th Floor, Building 58, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In one embodiment, the *Lactobacillus plantarum* A17 has the following characteristics:

[0008] (1) The 16S rDNA sequence of the tomato was obtained from farmland in the 105th Regiment of Wujiaqu area, Xinjiang.

[0009] (2) Colonies on MRS medium are round, smooth, raised, relatively moist, milky white, and stain purple with Gram staining;

[0010] (3) It can make better use of different monosaccharides, disaccharides and polysaccharides (OD). 600 >0.8), and compared with other plant lactobacilli of the same genus, the utilization ability was significantly different (p<0.05);

[0011] (4) It has good tolerance to acid stress;

[0012] (5) It has good tolerance to simulated gastrointestinal tract.

[0013] The present invention also provides a microbial preparation containing the aforementioned Lactobacillus plantarum A17.

[0014] In one embodiment, the microbial preparation includes, but is not limited to, direct-inoculation fermentation agents.

[0015] In one embodiment, the viable count of *Lactobacillus plantarum* A17 in the microbial preparation is ≥1×10⁻⁶. 7 CFU / mL.

[0016] The present invention also provides a method for preparing tomato enzyme by fermentation, which involves fermenting the Lactobacillus plantarum A17 in tomato pulp at 35-40°C.

[0017] In one embodiment, the tomato pulp is subjected to enzymatic hydrolysis.

[0018] In one embodiment, the enzymatic hydrolysis includes, but is not limited to, co-enzymatic hydrolysis by pectinase, cellulase, and hemicellulase.

[0019] In an embodiment, the enzymolysis is carried out at 50-60℃ for 2-4h.

[0020] In an embodiment, the tomato juice is prepared by crushing tomatoes and mixing with water at a mass ratio of 1:2-3, and adjusting the sugar content to 12°Brix.

[0021] In an embodiment, the method comprises the following steps:

[0022] (1) crushing tomatoes and mixing with water at a mass ratio of 1:2-3, and adjusting the sugar content to 12°Brix;

[0023] (2) subjecting the tomato juice after step (1) to enzymolysis treatment with pectinase, cellulase and hemicellulase;

[0024] (3) fermenting the tomato juice after step (2) with the Lactobacillus plantarum A17.

[0025] The application also provides a tomato ferment prepared by the method.

[0026] In an embodiment, the tomato ferment has an SOD enzyme activity of ≥150U / mL, an ABTS free radical scavenging rate of ≥60%, a solid content of 12-15°Brix, and a pH of 3.8-4.2.

[0027] The application also provides use of the Lactobacillus plantarum A17 or the microbial preparation in the field of food.

[0028] In an embodiment, the use includes but is not limited to fermented fruit and vegetable products.

[0029] Advantages:

[0030] 1. The application is directed to the suitability of the fermentation strain required for the production of Xinjiang characteristic forest fruit tomato ferment, and the Lactobacillus plantarum from tomatoes is subjected to directional breeding, and the Lactobacillus plantarum A17 with good physiological characteristics and probiotic properties is screened. The strain can tolerate the pH environment and has good gastrointestinal fluid tolerance. The strain is superior to three commercial strains CICC25125, CICC25124 and CICC6114 in the utilization ability of monosaccharides, disaccharides and polysaccharides, and shows its utilization potential on various sugar substrates, which helps the strain to utilize various sugars in tomatoes to prepare ferment products with antioxidant function.

[0031] 2. The application also provides a method for preparing tomato ferment using the Lactobacillus plantarum A17, which can improve the antioxidant capacity of the prepared tomato ferment, help to neutralize free radicals and regulate oxidative stress, and has multiple benefits to human health, which is embodied in:

[0032] (1) SOD enzyme activity: ≥ 150 U / mL

[0033] (2) ABTS free radical scavenging rate: ≥ 60% (1 mg / mL fermentation broth);

[0034] (3) TSS: 12-15 °Brix;

[0035] (4) pH: end point pH of fermentation 3.8-4.2.

[0036] Therefore, Lactiplantibacillus plantarum A17 has great application prospects in the application of tomato enzyme and other fruit and vegetable fermentation.

[0037] Biological material preservation

[0038] Lactiplantibacillus plantarum XJU-LP-A17 (abbreviated as Lactiplantibacillus plantarum A17) is classified as Lactiplantibacillus plantarum, which has been preserved in Guangdong Microbial Culture Collection Center on May 6, 2025, with the preservation number GDMCC No: 66259 and the preservation address being No. 58 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 It is a colony morphological characteristic diagram of Lactiplantibacillus plantarum A17.

[0040] Figure 2 It is the utilization ability of Lactiplantibacillus plantarum A17 to different carbon sources.

[0041] Figure 3 It is the acid stress tolerance ability of different Lactiplantibacillus.

[0042] Figure 4 It is the simulated gastrointestinal tolerance ability of Lactiplantibacillus plantarum A17.

[0043] Figure 5 It is the SOD enzyme activity of different Lactiplantibacillus plantarum fermented tomato enzyme.

[0044] Figure 6 It is the ABTS free radical scavenging rate of different Lactiplantibacillus plantarum fermented tomato enzyme. DETAILED DESCRIPTION

[0045] The present application will be further described below in combination with specific examples.

[0046] The glucose involved in the following examples was purchased from Urumqi Keweiye Biological Technology Co., Ltd., and the yeast powder was purchased from Shaanxi Tongshengchang Biological Technology Co., Ltd.

[0047] Culture medium:

[0048] MRS solid medium (g / L): glucose 20 g / L, peptone 10 g / L, yeast powder 5 g / L, beef infusion powder 10 g / L, anhydrous sodium acetate 2 g / L, diamine citric acid 2 g / L, dipotassium hydrogen phosphate 2.6 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween 80 1 g / L, agar 20 g / L, distilled water 1 L.

[0049] MRS liquid medium (g / L): glucose 20 g / L, peptone 10 g / L, yeast powder 5 g / L, beef infusion powder 10 g / L, anhydrous sodium acetate 2 g / L, diamine citric acid 2 g / L, dipotassium hydrogen phosphate 2.6 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween 80 1 g / L, distilled water 1 L.

[0050] Preparation method of simulated gastric juice and intestinal juice:

[0051] Preparation of simulated gastric juice and intestinal juice:

[0052] ① Simulated gastric juice: 0.9% w / v, physiological saline adjusted to pH 3.0 with hydrochloric acid; ② pepsin was dissolved in sterilized physiological saline (0.9% w / v, adjusted to pH 3.0 with hydrochloric acid) to a final concentration of 3 g / L. Filtered with 0.22 μm sterile filter membrane, and used immediately.

[0053] ① Simulated intestinal juice: 0.9% w / v, physiological saline adjusted to pH 8.0 with NaOH; ② trypsin was dissolved in sterilized physiological saline (0.9% w / v, adjusted to pH 8.0 with NaOH) to a final concentration of 1 g / L, and bile salts were added to a final concentration of 0.3%. Filtered with 0.22 μm sterile filter membrane, and used immediately.

[0054] Detection method:

[0055] SOD enzyme activity:

[0056] The activity of tomato enzyme superoxide dismutase (Superoxide Dismutase, SOD) was determined according to the kit instructions. Before determining the SOD activity, 2 mL of sample was centrifuged at 3000 g / min for 5 min at 4℃, and the supernatant was diluted twice. OD 550nm SOD activity was detected. According to the formula:

[0057]

[0058] ABTS radical scavenging rate:

[0059] Prepare a standard mother liquor (0.5 mg / mL) by adding 2 mL of methanol before use, and mix well. Dilute the mother liquor with methanol to prepare standard solutions with the following concentration gradient: 0, 20, 40, 60, 80, 100 μg / mL. Measure the absorbance at 734 nm, draw a standard curve, and determine the ABTS scavenging rate according to the method in the literature.

[0060] Soluble solids content:

[0061] Determine using a handheld refractometer, and take 1 mL of sample and drop it into the refractometer sample inlet for reading.

[0062] Example 1: Isolation and screening of Lactobacillus plantarum and strain identification

[0063] (1) Isolation and purification of lactic acid bacteria

[0064] Tomatoes grown in the farmland of the 105th Regiment in Wujiaqu, Xinjiang were washed and crushed, and the sugar content was determined at room temperature (the initial sugar content of tomato juice was 4.3 °Brix). 4% (w / v) white sugar was added to adjust the sugar content to 12 °Brix. Then, enzymatic hydrolysis was performed at 55 °C for 3 h (pectinase 50000 U / g, cellulase 50 U / mg, hemicellulase 20000 U / g, addition ratio 2:1:1).

[0065] Tomato enzyme was screened and enriched by 6 methods under sterile conditions. 100 μL of the dilution was taken from test tubes with dilutions of 10 -4 , 10 -5 , and 10 -6 , and evenly coated on MRS agar plates. After incubation at 37 °C for 48 h, typical colonies were picked and purified on MRS solid plates to obtain single pure bacteria. Figure 1 Through observation of colony morphological characteristics and examination under a microscope, further gram staining test and hydrogen peroxide enzyme test were performed to preliminarily screen lactic acid bacteria strains. The identified lactic acid bacteria were numbered and stored in 40% glycerol, and were stored in a 4 °C refrigerator and a -80 °C refrigerator for standby use.

[0066] (2) Strain identification

[0067] The purified single colonies were inoculated into 5 mL of MRS liquid medium and cultured for 24 h. The bacterial suspension was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the bacterial sludge was washed twice with sterile water. The suspension was then resuspended in 50 μL of sterile water as a template. Primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used. The PCR reaction system consisted of: 12.5 μL TaqMaster Mix (2×); 10.5 μL double-distilled water; 1 μL template; and 0.5 μL each of forward and reverse primers. The amplification program was: 94℃ for 5 min; 94℃ for 30 s; 55℃ for 30 s; 72℃ for 1 min, 35 cycles, followed by 72℃ for 10 min. After PCR amplification products were detected by 1.8% agarose gel electrophoresis, the PCR products with correct electrophoretic bands (shown in SEQ ID NO.1) were sent to Xinjiang Youkang Biotechnology Co., Ltd. for sequencing. The obtained sequences were compared online at the National Center for Biotechnology Information (NCBI), and those with a similarity of 98% or higher were defined as belonging to the same bacterial species.

[0068] Example 2: Analysis of the ability of Lactobacillus plantarum to utilize different carbon sources

[0069] The growth of the strain was determined in media containing fructose, sucrose, trehalose, cellobiose, mannitol, and sorbitol as carbon sources. Carbon-free MRS liquid medium was sterilized at 121°C for 15 min. The sugars to be measured were then filtered through a sterile 0.22 μm aqueous microfiltration filter and added to the carbon-free MRS liquid medium to achieve a sugar concentration of 5 g / L. The strain preserved in glycerol was inoculated into MRS liquid medium at a 2% inoculum and cultured at 37°C for 24 h for activation. This process was repeated for three generations. After three generations, the bacterial cells were collected by centrifugation at 8000 rpm at 4°C for 10 min, washed twice with sterile PBS (7.2-7.4) buffer, and then resuspended in PBS buffer. The prepared media with different carbon sources were added to 96-well plates, with three replicates. The bacterial suspension was inoculated into culture media with different carbon sources at an inoculum volume of 1%, and incubated at 37°C for 24 hours. The OD was then measured using a microplate reader. 600 nm value. Results are as follows: Figure 2 As shown, the Lactobacillus plantarum A17 screened in Example 1 can efficiently utilize different monosaccharides, disaccharides and polysaccharides.

[0070] Example 3: Analysis of acid stress tolerance of Lactobacillus plantarum

[0071] MRS liquid medium was adjusted to pH 2.0, 3.0, 4.0, 5.0 by 1 mol / L hydrochloric acid, and then sterilized at 115°C for 20 min. The activated Lactobacillus plantarum A17 was inoculated into the MRS liquid medium with different pH at 4% (v / v), and cultured at 37°C. The samples were taken at 0h, 16h, 22h, and the MRS medium was used as a blank control. Three commercial strains Lactobacillus plantarum CICC25125, Lactobacillus fermentum CICC25124, and Lactobacillus casei CICC6114 were used as control groups, and cultured under the same conditions. The results showed that the A17 strain had stronger growth ability and adaptability under different pH conditions, especially at pH 3.0, which was better than the three commercial strains. Figure 3 )。

[0072] Example 4: Analysis of the simulated gastrointestinal tolerance of Lactobacillus plantarum

[0073] The strain preserved in glycerol was inoculated into MRS liquid medium at 2% inoculation, and cultured at 37°C for 24h for activation. After continuous culture for 3 generations, the bacterial cells were collected by centrifugation at 8000r / min for 10min, the supernatant was discarded, and the bacterial cells were washed twice with 0.9% sterile physiological saline, and resuspended in an equal volume of simulated gastric juice. The mixture was mixed uniformly with a vortex mixer, and cultured at 37°C for 3h, and then the viable cell count was performed. Similarly, the bacterial suspension was resuspended in simulated intestinal juice, mixed uniformly with a vortex mixer, and cultured at 37°C for 4h, and then the viable cell count was performed. The results showed that the survival rates of strain A17 after gastric and intestinal juice culture for 4h were 65.44% and 71.78%, respectively.

[0074] ( Figure 4 )。

[0075] Survival rate of strain = lg(N1-N0) x 100%;

[0076] In the formula: N1 is the number of surviving bacteria after treatment with simulated artificial gastric juice or intestinal juice, CFU / mL;

[0077] N0 is the viable cell count of the strain before treatment, CFU / mL.

[0078] Example 5: Analysis of the characteristics of tomato enzyme fermented by Lactobacillus plantarum

[0079] Tomatoes were thawed, washed and crushed. The tomato juice was mixed with water at a ratio of 1:2.6 by weight, and the sugar content was measured at room temperature. 4% (w / v) white sugar was added to adjust the sugar content to 12 °Brix, and the initial soluble solid content (TSS) was 4.3 °Brix at this time. 15 g / L pectinase (50000 U / g), 10 g / L cellulase (50 U / mg) and 1% (w / v) hemicellulase (20000 U / g) were added to the tomato juice, and then enzymolysis was carried out at 55°C for 3h. After the enzymolysis was completed, the sample was sterilized at 90°C for 20 min. After the temperature was cooled to 37°C, the plant lactobacillus A17 activated for 3 generations was inoculated into the enzymolyzed tomato juice for single bacterial fermentation, so that the initial bacterial concentration after inoculation was 4x10 7 CFU / mL, and the fermentation was carried out at 37°C for 22h. The SOD enzyme activity, ABTS free radical scavenging capacity, pH and soluble solid content (TSS) of the tomato juice were detected. The results showed that the SOD enzyme activity at the end of fermentation was 310.86 U / mL, the ABTS free radical scavenging rate was 66.26% (1 mg / mL of the fermentation broth), the TSS was 14.1 °Brix, and the pH at the end of fermentation was 3.5.

[0080] Comparative Example 1

[0081] Strains CICC25125, CICC25124 and CICC6114 were respectively fermented according to the same method as in Example 5 to prepare tomato juice, and the SOD enzyme activity and ABTS free radical scavenging rate of the samples at the end of fermentation were detected. The results are shown in Table 1. Figures 5-6 As shown in Table 1, the strain A17 was superior to the three commercial strains in terms of SOD enzyme activity and ABTS free radical scavenging rate.

[0082] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. Lactiplantibacillus plantarum, which has been preserved in Guangdong Microbial Culture Collection Center on May 6, 2025, with the preservation number of GDMCC No: 66259.

2. A microbial preparation containing the Lactiplantibacillus plantarum A17 of claim 1.

3. The microbial preparation according to claim 2, characterized in that, The viable cell count of the plant Lactobacillus A17 is ≥ 1 x 10 7 CFU / mL.

4. A method of preparing tomato ferment, characterized by, The Lactiplantibacillus plantarum A17 of claim 1 is fermented in tomato juice at 35-40℃.

5. The method of claim 4, wherein, The tomato juice is subjected to enzymatic hydrolysis.

6. The method of claim 5, wherein, The enzymatic hydrolysis includes but is not limited to the combined enzymatic hydrolysis of pectinase, cellulase and hemicellulase.

7. The method according to any one of claims 3 to 6, characterized in that, The method comprises the following steps: (1) After crushing the tomatoes, mix them with water at a mass ratio of 1:2-3, and adjust the sugar content to 12°Brix; (2) Subject the tomato juice treated in step (1) to enzymatic hydrolysis with pectinase, cellulase and hemicellulase; (3) Ferment the Lactiplantibacillus plantarum A17 in the tomato juice subjected to enzymatic hydrolysis in step (2).

8. The method of any one of claims 5-7, wherein, The enzymatic hydrolysis is carried out at 50-60℃ for 2-4h.

9. Tomato juice prepared by the method of any one of claims 4-8.

10. Use of the Lactiplantibacillus plantarum A17 of claim 1 or the microbial preparation of any one of claims 2-3 in the field of food.

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