Lactobacillus curvatus with antioxidant capacity and application thereof

Abstract

The application discloses a strain of lactobacillus curvatus with antioxidant capacity and an application, and the Latin name of the lactobacillus curvatus is Latilactobacillus curvatus, the independent number is FFZZH5L, and the preservation number is GDMCC NO: 63463. The lactobacillus curvatus is screened from household self-made pickled fermented foods in multiple regions in Anhui province, has good antioxidant capacity, and can effectively promote the increase of antioxidant enzyme activity in the body. Better gastrointestinal environment adaptation capacity increases the opportunity of the lactobacillus curvatus to be planted in the gastrointestinal tract of the human body and to play a probiotic role, and the acid, alkali and salt resistance is good in fermented pickles and yogurts, and the lactobacillus curvatus can be used as a fermentation starter of pickled foods and dairy products. The strain is sensitive to a plurality of antibiotics, has safety, and can also be used as a feed additive together with antibiotics not sensitive to the strain. The lactobacillus curvatus has no toxic side effects, can be used as health foods or medicines to improve the immunity of the body, resist aging, beautify, and regulate intestinal flora and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms and biotechnology, in particular to the field of development and application of functional lactic acid bacteria, and particularly to a Lactobacillus curvatus with antioxidant function. The present application also includes the application of the Lactobacillus curvatus. BACKGROUND

[0002] A large number of studies have shown that probiotics such as Lactobacillus and Bifidobacterium have good antioxidant capacity and can protect against oxidative stress to a certain extent. Lactic acid bacteria are one of the main sources of natural antioxidants and can exert antioxidant activity in various ways. Lactic acid bacteria can chelate metal ions to exert antioxidant effects. Lactic acid bacteria have the ability to synthesize antioxidant enzymes and can also produce various antioxidant substances such as folic acid, glutathione, and butyric acid. In addition, lactic acid bacteria can increase the production of host antioxidant activity substances, regulate the balance of gastrointestinal microecology and immune function, and regulate intestinal health by recombining the composition of intestinal flora. Detecting the free radical scavenging capacity of lactic acid bacteria, ferrous ion chelating capacity, and the influence of lactic acid bacteria on antioxidant-related enzymes and products of the host are the main methods for evaluating the antioxidant performance of probiotics.

[0003] Lactobacillus curvatus is a potential probiotic candidate strain that has attracted the attention of researchers due to its excellent fermentation characteristics and various health benefits. In 2012, it was included in the International Dairy Federation Bulletin and the Directory of Technically Necessary Microorganisms in Fermented Foods. In 2013, it was included in the European Food Safety Authority Qualification List as a recommended biological agent. In 2019, Lactobacillus curvatus was approved by the Chinese government as a new food additive strain for processed meat and dairy products and animal farm feed.

[0004] Recent studies have shown that Lactobacillus curvatus is beneficial to human health, such as reducing the effects of dextran sulfate sodium (DSS)-induced colitis in mice, alleviating obesity and hyperlipidemia, and effectively preventing dexamethasone-induced muscle atrophy, which is the first report of the inhibitory effect of lactic acid bacteria on muscle atrophy. However, the antioxidant performance and antioxidant mechanism of Lactobacillus curvatus are not fully understood.

[0005] The present application screens a strain of Lactobacillus curvatus from pickled food in some areas of Anhui Province, and names it Latilactobacillus curvatus FFZZH5L. It is proved by experiments that it has good probiotic quality, good tolerance to salt and acid-base, and adapts to the human gastrointestinal environment. At the same time, it can enhance the antioxidant capacity of nematodes, improve the tolerance of nematodes to heat stress, and enhance the movement ability of nematodes. Therefore, the Latilactobacillus curvatus FFZZH5L can improve the antioxidant level, so that the curvate can become a probiotic candidate strain with certain health value and commercial potential. SUMMARY

[0006] The present application aims to provide a foodborne Lactobacillus curvatus with antioxidant activity that can effectively improve the antioxidant capacity of organisms.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] The Lactobacillus curvatus with antioxidant capacity proposed by the present application is taxonomically named Lactobacillus curvatus, with the Latin name Latilactobacillus curvatus, and the independent number FFZZH5L. It has been preserved in the Guangdong Microbial Culture Collection Center, with the preservation number GDMCC NO: 63463, the preservation date May 14, 2023, and the address of the preservation unit 100, Martyrs Road, Guangzhou, China, 510070, Institute of Microbiology, Guangdong Academy of Sciences.

[0009] The 16S rDNA sequence of the Lactobacillus curvatus with antioxidant capacity proposed by the present application is shown in SEQ ID No. 1.

[0010] The 16S rDNA sequence of the Lactobacillus curvatus with antioxidant capacity proposed by the present application is shown in SEQ ID No. 1.

[0011] The Lactobacillus curvatus with antioxidant capacity proposed by the present application can be applied in the preparation of fermented food, fermented dairy products, health food, cosmetics, pharmaceuticals, feed and other fields.

[0012] The Lactobacillus curvatus with antioxidant capacity proposed by the present application is one of 18 strains of lactic acid bacteria isolated, screened and identified from hand-made pickled fermented dairy products in Anhui Province. By measuring the glutathione (GSH) content of the strain, a strain of Lactobacillus with antioxidant capacity and basic probiotic properties was selected, which can exert antioxidant and special probiotic functions in vivo.

[0013] The specific screening process steps are as follows:

[0014] (1) The microorganism in the hand-fermented food is cultured by the LBS culture medium, a characteristic strain is selected and purified, and a purified and separated strain is obtained.

[0015] (2) The strain is selected by the morphology and microscopic examination, 16S rDNA sequencing is carried out, Blast comparison is carried out in NCBI, 18 strains are identified and saved.

[0016] (3) The glutathione content of the lactobacillus identified in (2) is determined, and a lactobacillus with the highest glutathione content is screened out. After sequence comparison, it is determined as a lactobacillus curvatus.

[0017] (4) The antioxidant enzyme activity of the lactobacillus curvatus is determined, including CAT, SOD and GSH-px activity.

[0018] (5) The physiological and biochemical characteristics of the strain are determined, including acid and alkali resistance test, salt resistance test and unique carbon source utilization test.

[0019] (6) The gastrointestinal environment tolerance of the lactobacillus curvatus is evaluated through the body simulation gastrointestinal environment test.

[0020] (7) Through the animal experiment, the difference of antioxidant enzyme activity in the body of the Caenorhabditis elegans after eating the lactobacillus curvatus and the difference of the accumulation of oxidized substances in the body are detected and compared.

[0021] (8) It is determined that the lactobacillus curvatus FFZZH5L has antioxidant capacity in vivo and in vitro through steps (4) to (7), and can adapt to high salt environment and gastrointestinal environment.

[0022] The lactobacillus curvatus with high antioxidant activity provided by the application has high glutathione (GSH) content, and shows good probiotic characteristics in salt resistance test, acid and alkali resistance test and in vitro test simulating the gastrointestinal tract. It can be applied in the preparation of fermented dairy products and feed.

[0023] The lactobacillus curvatus with antioxidant activity provided by the application can effectively reduce the oxidative stress damage of Caenorhabditis elegans induced by juglone, improve the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), improve the total antioxidant capacity (T-AOC) in the body of Caenorhabditis elegans, and reduce the contents of malondialdehyde (MDA), reactive oxygen species (ROS) and lipofuscin in the body of Caenorhabditis elegans. Therefore, the lactobacillus curvatus can be widely applied in fermented dairy products, health products, medicines and feed, and has the ability of antioxidant and free radical scavenging.

[0024] Compared with the prior art, the beneficial effects of the application are as follows:

[0025] 1、The strain of Latilactobacillus curvatus FFZZH5L with antioxidant capacity can improve SOD, GSH-px enzyme activity and T-AOC total antioxidant capacity, reduce the synthesis of ROS, lipofuscin and MDA in the body, and has good antioxidant effect.

[0026] 2、The strain of Latilactobacillus curvatus FFZZH5L with antioxidant capacity has good gastrointestinal environment adaptation ability, which increases the opportunity of colonization in human gastrointestinal tract and plays a probiotic role, and the strain is acid, alkali and salt resistant and performs well in fermented pickles and yogurt, and can be used as a fermentation starter for pickled food and dairy products. The strain is sensitive to a variety of antibiotics and has safety, and can also be used as a feed additive together with antibiotics not sensitive thereto. It has no toxic side effects and can be used as health food or medicine to improve immunity, anti-aging, beauty and regulation of intestinal flora. Therefore, the Latilactobacillus curvatus can be widely applied in fermented dairy products, health products, medicines and feed. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 For comparison of the GSH contents of the 18 strains screened in Example 2 of the present application, the GSH content of the strain FFZZH5L strain is the highest.

[0028] Figure 2 For the 16S rDNA phylogenetic tree of the lactobacillus FFZZH5L in Example 2 of the present application, it has the highest genetic relationship with a strain of Latilactobacillus FY1 and can be considered as a strain of Latilactobacillus.

[0029] Figure 3 For the gram staining microscopic examination of the Latilactobacillus FFZZH5L in Example 2 of the present application, the bacterial body is blue-purple after gram staining, and the Latilactobacillus FFZZH5L is a gram-positive bacterium.

[0030] Figure 4 For the litmus milk test chart in Example 4 of the present application (left is blank litmus milk medium, right is litmus milk inoculated with Latilactobacillus FFZZH5L), the Latilactobacillus FFZZH5L can utilize milk and ferment to produce acid to make litmus red.

[0031] Figure 5 For the growth status of the Latilactobacillus FFZZH5L in Example 4 of the present application in different salt content, different pH environment, gastrointestinal fluid and different bile salt concentration.

[0032] Figure 6 For the MTT method of inhibiting bacteria (the horizontal coordinate is the strain, and the vertical coordinate is the survival rate of E. coli).

[0033] Figure 7 For the antibacterial experiment, (A) the antibacterial activity of Lactobacillus curvature FFZZH5L was determined; (B) the self-agglutination ability of Lactobacillus curvature FFZZH5L was detected; and (C) the coagulation ability of Lactobacillus curvature FFZZH5L with the pathogenic bacterium Staphylococcus aureus was detected.

[0034] Figure 8 This is a comparison of the lifespan of nematodes fed with Lactobacillus curvature FFZZH5L and nematodes fed with OP50 in Example 5 of the present invention.

[0035] Figure 9 Example 5 of this invention compares the number of eggs laid by nematodes fed with Lactobacillus curvature FFZZH5L and nematodes fed with OP50. (a) is the negative control group, (b) is the mixed feeding group of 50% OP50 + 50% FFZZH5L, (c) is the Lactobacillus curvature FFZZH5L group, (d) is the positive control Lactobacillus rhamnosus LGG group, and (e) is a bar chart comparing the number of eggs laid by nematodes in each group after 3 days of feeding.

[0036] Figure 10 This is a comparison of the antioxidant enzyme content in nematodes fed with Lactobacillus FFZZH5L and nematodes fed with OP50 in Example 5 of the present invention. (a) GSH-px enzyme activity, (b) SOD enzyme activity, (c) T-AOC enzyme activity, and (d) CAT enzyme activity.

[0037] Figure 11 The comparison of oxidative substances in nematodes fed with Lactobacillus FFZZH5L and nematodes fed with OP50 in Example 5 of the present invention is shown in (a) MDA content, (b) average fluorescence intensity of lipofuscin, and (c) average fluorescence intensity of ROS.

[0038] Figure 12 This illustrates the effect of different foods on the survival rate of nematodes under the oxidative stress model in Example 5 of this invention.

[0039] Figure 13 The effects of different foods on the activity of antioxidant enzymes in nematodes under the oxidative stress model in Example 5 of this invention are shown in the figures: (a) GSH-px enzyme activity, (b) SOD enzyme activity, (c) T-AOC enzyme activity, and (d) CAT enzyme activity.

[0040] Figure 14 The effects of different foods on the content of oxidative products in nematodes under the oxidative stress model in Example 5 of this invention are shown in (a) MDA content, (b) average fluorescence intensity of lipofuscin, and (c) average fluorescence intensity of ROS.

[0041] Figure 15The images show a comparison of lipofuscin fluorescence under normal conditions in Example 5 of this invention. (a) shows nematodes in the negative control group, (b) shows nematodes fed with mixed feed, (c) shows nematodes fed with Lactobacillus curvularis FFZZH5L, and (d) shows nematodes fed with LGG.

[0042] Figure 16 The following are comparisons of ROS fluorescence images under normal conditions in Example 5 of this invention: (a) Nematodes in the negative control group, (b) Nematodes fed with mixed feeding, (c) Nematodes fed with Lactobacillus curvatureis FFZZH5L, and (d) Nematodes fed with LGG.

[0043] Figure 17 Comparison of lipofuscin fluorescence images under different conditions in Example 5 of the present invention: (a) OP50-fed nematodes without oxidative stress, (b) OP50-fed nematodes after oxidative stress, (c) Lactobacillus curvilinearis FFZZH5L-fed nematodes after oxidative stress, and (d) Lactobacillus curvilinearis FFZZH5L-fed nematodes without oxidative stress.

[0044] Figure 18 For comparison of ROS fluorescence images under different conditions in Example 5 of the present invention, (a) nematodes fed with OP50 without oxidative stress, (b) nematodes fed with OP50 after oxidative stress, (c) nematodes fed with Lactobacillus curvilinearis FFZZH5L after oxidative stress, and (d) nematodes fed with Lactobacillus curvilinearis FFZZH5L without oxidative stress.

[0045] Figure 19 This invention relates to the effect of Lactobacillus curvature FFZZH5L on the expression level of antioxidant-related mRNAs in nematodes in Example 5 of this invention. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings.

[0047] Related culture medium formulations and buffer formulations:

[0048] LBS Lactobacillus selection medium (per liter): 10g tryptone, 17g anhydrous sodium acetate, 5g yeast extract, 0.575g magnesium sulfate, 20g glucose, 0.12g manganese sulfate, 1mL Tween 80, 0.034g ferrous sulfate, 6g potassium dihydrogen phosphate, 2g ammonium citrate, pH 5.4±0.2. Add 15g agar powder when preparing the solid medium.

[0049] MRS liquid medium (per liter): 10.0 g peptone, 1.0 mL Tween-80, 10.0 g yeast extract, 20.0 g glucose, 5.0 g anhydrous sodium acetate, 2.0 g ammonium citrate, 5.0 g beef extract, 0.58 g MgSO4, 0.25 g MnSO4, 0.28 g CuSO4, 2.0 g K2HPO4·3H2O, add water to 1000.0 mL, adjust pH to 6.4, sterilize at 121°C for 20 min. When preparing solid medium, add 15.0 g agar powder.

[0050] Litmus milk culture medium: Mix 2.5% litmus aqueous solution with skim milk at a ratio of 4:100 (v / v) to make the milk turn lilac purple; dispense into test tubes and sterilize at 110℃ for 20 min.

[0051] Physiological saline (per liter): 0.85% NaCl solution, with the addition of 0.05% cysteine ​​hydrochloride, pH: 7.0; sterilized at 115℃ for 20 min.

[0052] NGM medium (per liter): 3g NaCl, 2.5g peptone, 20g agar, and double-distilled water to a final volume of 1000mL. Sterilize at 121℃ for 20min. Cool to 60℃, then add filtered sterilized 5mg / L cholesterol-ethanol solution, 1mM MgSO4, 1mM CaCl2, and 12.5mM KH2PO4-K2HPO4 solution.

[0053] Water agar medium (per liter): 2g agar powder, dissolved in 100mL ddH2O, sterilized at 121℃ for 20min.

[0054] LB medium (per liter): 5.0 g yeast extract, 10.0 g peptone, 5.0 g NaCl, ddH2O to a final volume of 1000 mL, sterilize at 121 °C for 20 min.

[0055] M9 Buffer (per liter): 3g KH2PO4, 6g Na2HPO4, 5g NaCl, add water to make up to 1L, sterilize at 121℃ for 20min, cool and then add 1mL MgSO4 solution (1mol / L).

[0056] Example 1: Isolation and identification of Lactobacillus from homemade fermented and pickled foods in multiple regions of Anhui Province

[0057] I. Sample Source

[0058] These are homemade pickled and fermented foods originating from Hefei, Xuancheng, and Suzhou cities in Anhui Province, my country. Specific products include pear syrup, pickled flounder, pickled cowpeas, yogurt, pickled mustard greens, broad bean paste, pickled ginger, pickled garlic, sauerkraut, pickled greens, and pickled peppers. Solid products are stored at -80°C using 30% sterile glycerin, and liquid products are stored at -80°C using 60% sterile glycerin.

[0059] II. Isolation of Lactobacillus from Traditional Fermented Foods

[0060] Take 1 mL of the melted fermented food and crush it. Dilute the homogenate with sterile physiological saline at a 1:1 ratio. This is 10. -1 Dilution. Then take 1 mL of the above diluted solution and dilute it 10 times to obtain a 10:10 solution. -2 Dilution, and so on. Select 10. 0 10 -1 10 -2 10 -3 10 -4 Five dilution gradients were used, and 100 μL of each dilution was evenly spread on LBS plates. The plates were incubated at 37°C for 48 h. Characteristic colonies were picked and further purified to obtain purified and isolated Lactobacillus strains.

[0061] III. Identification of the strains

[0062] Single colonies were picked and inoculated into MRS liquid medium and incubated at 37°C and 160 rpm for 12 h. The bacterial culture was centrifuged at 4500 rpm for 5 min, and the bacterial pellet was collected. DNA was extracted from the pellet according to the instructions of the Gram-positive bacterial DNA extraction kit. Using the extracted DNA as a template, 16S rDNA PCR was performed on the bacterial DNA using universal primers 27F (sequence AGAGTTTGATCMTGGCTCAG) and 1492R (sequence TACGGYTACCTTGTTACGACTT). The PCR system and template are as follows:

[0063] Table 1 PCR system settings

[0064]

[0065] Table 2 PCR reaction procedures

[0066]

[0067] The PCR amplification products were detected by 1% agarose gel electrophoresis, with the expected PCR product length being approximately 1500 bases. The obtained PCR products were sent to Hefei General Biotechnology Co., Ltd. for sequencing. Using NCBI, the sequencing results were compared using the basic local alignment search tool (BLAST). Based on the alignment results, homology analysis was performed, and Lactobacillus sequences with high homology to the isolated Lactobacillus 16S rDNA gene sequence were selected as reference strains.

[0068] A total of 18 strains were identified in this invention (as shown in Table 3), and all of them were used for screening of lactobacilli with antioxidant activity.

[0069] Table 3. Results of Lactobacillus 16S rDNA identification.

[0070]

[0071]

[0072] Example 2: Screening of Lactobacilli with antioxidant activity

[0073] Reduced glutathione (GSH) is present in some Gram-positive bacteria and is an abundant small sulfhydryl peptide. GSH participates in redox reactions within bacteria, maintaining systemic balance in enzymatic reactions. This allows bacteria to resist the high oxygen and high osmotic pressure caused by the environment and their own metabolism, and can inhibit the oxidation of proteins and lipids. The GSH content in bacteria is one of the indicators of their antioxidant activity. The GSH content of 18 bacterial strains isolated from fermented foods was determined.

[0074] Determination of glutathione content in Lactobacillus: Eighteen purified strains of Lactobacillus were cultured in MRS liquid medium for 12 h, and the cells were collected by centrifugation at 4500 rpm for 5 min. The cells were then sonicated and the glutathione (GSH) content of the homogenate was determined according to the kit instructions.

[0075] like Figure 1 As shown, the comparison of GSH content revealed that Lactobacillus FFZZH5L had the highest glutathione (GSH) content (***P<0.01).

[0076] The 16S rDNA sequence of *Lactobacillus FFZZH5L* was obtained by sequencing, as shown in SEQ ID No. 1. BLAST analysis showed that this isolated strain had the highest homology with *Latilactobacillus curvatus* FY1 (GenBank accession number ON758920.1), with 100% coverage and 99.83% homology. (16S rDNA phylogenetic tree) Figure 2 The diagram shows the evolutionary relationship of this strain within the genus *Lactobacillus*. Based on the above data, the isolated strain was identified as *Latilactobacillus curvatus*, and self-named *Latilactobacillus curvatus* FFZZH5L.

[0077] The *Lactobacillus curvatureii* FFZZH5L strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC NO: 63463), dated May 14, 2023. The address of the institution is Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode 510070. The 16S rDNA sequence of *Lactobacillus curvatureii* FFZZH5L has also been registered in GenBank, obtaining the strain sequence number OQ748066.

[0078] The selected *Lactobacillus curvilinearis* FFZZH5L was processed using a Gram staining kit, and its morphology was observed under a microscope. Figure 3 As shown, this strain is short rod-shaped and usually consists of four or more cells linked together. Generally, blue-purple indicates Gram-positive bacteria, and red indicates Gram-negative bacteria. The *Lactobacillus curvilinearis* FFZZH5L purified and isolated in this invention is a Gram-positive bacterium.

[0079] Example 3: Evaluation of the in vitro antioxidant capacity of Lactobacillus curvature FFZZH5L

[0080] I. Preparation of intact cell and cell-free extracts and fermentation supernatant of the bacterial strain

[0081] The bacterial strain was activated three times. The third activated bacterial solution was centrifuged at 6000 rpm for 10 min at low temperature, and the supernatant and bacterial cells were collected separately. The supernatant was filtered through a 0.22 μm sterile filter membrane to obtain the fermented supernatant (FS). The bacterial cells were washed three times with sterile double-distilled water, and a portion of the cells was stored at 4℃; this portion was the intracellular cells (IC). The remaining bacterial cells were resuspended in sterile physiological saline, and the cell count was adjusted to 10⁹ CFU / mL. The cells were then sonicated in an ice bath (800 W, 2 s, pause 2 s, total 10 min). The lysate was centrifuged at 12000 rpm for 15 min, and the supernatant was collected. A sample was examined under a microscope. If cells were still present, centrifugation at 12000 rpm was continued for 5 min until no cells were observed in the supernatant under a microscope. This supernatant was then the cell-free extract (CFE).

[0082] II. Evaluation of the antioxidant capacity of each component of the strain:

[0083] (1) Determination of the free radical scavenging ability of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH)

[0084] Weigh 8 mg of DPPH and dissolve it in anhydrous ethanol, then dilute to 100 mL to prepare a 0.2 mmol / L DPPH solution. Pipette 1 mL of bacterial suspension (1 × 10⁻⁶) 8 Add 2 mL of 0.2 mmol / L DPPH anhydrous ethanol solution to CFU / mL, react at room temperature (20-25℃) in the dark for 30 min, centrifuge at 8000g, 4℃ for 10 min, take the supernatant, measure the absorbance of the supernatant at 517 nm, and zero with deionized water.

[0085] DPPH clearance rate (%) = [1 - (Ai - Aj) / Ac] × 100

[0086] Ai: 1 mL DPPH + 1 mL sample; Aj: 1 mL ethanol + 1 mL sample; Ac: 1 mL DPPH + 1 mL PBS.

[0087] (2) Determination of ferrous ion chelating ability

[0088] Add 25 μL of 2 mmol / L FeCl2 solution to 500 μL of the sample to be tested, vortex for 3 min, add 100 μL of 5 mmol / L phenoxyazine solution, vortex for 3 min, and incubate at 25℃ for 10 min. Measure the OD value of the solution at 562 nm and record it as A1. Use sterile double-distilled water instead of the sample in the control tube, and record the absorbance as A2. The ferrous ion chelation rate is calculated using the following formula:

[0089]

[0090] (3) According to the instructions of the test kit, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT) were tested on the extracted fermentation supernatant, intracellular extract and cell precipitate respectively.

[0091] Table 4. Antioxidant capacity of each component of Lactobacillus curvature FFZZH5L

[0092]

[0093] As shown in Table 4, the ferrous ion chelation rate and DPPH free radical scavenging ability of different components of *Lactobacillus curvatureis* FFZZH5L were tested. Significant differences in the ferrous ion chelation ability were observed among the components. The chelation rate of the fermentation supernatant (FS) was 21.46%, and the chelation rate of the intracellular extract (CFE) was 20.32%, lower than the ferrous ion chelation rate of the cell line (IC) at 37.64%. This result indicates that the functional active substances of *Lactobacillus curvatureis* FFZZH5L capable of chelating ferrous ions are mainly present on the cell surface, with a small portion in the cell contents and relatively low levels in extracellular secretions.

[0094] Different components of Lactobacillus curvature FFZZH5L exhibited varying DPPH radical scavenging abilities. Specifically, the fermentation supernatant showed the highest DPPH radical scavenging rate at 64.27%, while the intracellular extract and bacterial cells showed significantly lower DPPH radical scavenging rates of only 24.69% and 15.37%, respectively (P < 0.05).

[0095] The SOD enzyme activity in the fermentation supernatant of *Lactobacillus curvatureis* FFZZH5L was significantly higher than that in the intracellular extract and cell culture, by 76.34% and 83.68% respectively (P < 0.05). CAT enzyme activity was detected in the fermentation supernatant and cell culture, at 5.09 U / mL and 1.16 U / mL respectively, but not in the intracellular extract. Similarly, GSH-px enzyme activity was detected only in the fermentation supernatant and intracellular extract, at 9.04 U / mL and 15.95 U / mL respectively, but not on the cell surface. These results indicate that SOD enzymes are present in both extracellular secretions and intracellular and extracellular environments, with lower activity in the latter. CAT enzymes are mostly found in extracellular secretions, with a small portion on the cell surface and very low activity, while GSH-px enzymes are mainly found intracellularly, with some in the fermentation supernatant.

[0096] Example 4: Evaluation of the physiological and biochemical characteristics of Lactobacillus curvature FFZZH5L

[0097] I. Experiment simulating the digestive tract of bacterial strains

[0098] (1) Evaluation of tolerance to artificial gastric and intestinal fluids

[0099] Preparation of artificial gastric juice: NaCl 0.2%, pepsin 0.3%, pH adjusted to 2.5 with HCl, filtered and sterilized for later use.

[0100] Preparation of pancreatic juice: Add 0.1% trypsin, adjust the pH to 8.0, filter and sterilize for later use. Preparation of bile: Add 1.80% bile salts, adjust the pH to 8.0, filter and sterilize for later use. Mix pancreatic juice and bile in a 2:1 ratio to obtain artificial intestinal fluid.

[0101] The method of Zhao Fang et al. (Zhao Fang, Li Yanqin, Li Binchun. Screening of probiotic Lactobacillus by simulating the human gastrointestinal environment [J]. Bulletin of Microbiology, 2016, 43(6):1396-1403.) was adopted. First, Lactobacillus curvature FFZZH5L strain was activated and cultured in MRS liquid medium for 12 h. 1 mL of the bacterial suspension was mixed with 9 mL of artificial gastric fluid and cultured in a 37℃ constant temperature shaker at 160 rpm. Samples were taken at 0 h and 2 h, and the viable count was determined by plating. Then, 1 mL of the mixture was taken from the 2 h-treated artificial gastric fluid containing bacteria and mixed with 9 mL of pH 8.0 artificial intestinal fluid. The mixture was cultured in a 37℃ constant temperature shaker at 160 rpm, and the viable count (CFU / mL) was counted at 6, 10, 14, and 26 h to calculate the survival rate.

[0102] (2) Evaluation of bile salt tolerance

[0103] Lactobacillus activated in liquid MRS was inoculated at a 1% inoculum into bile salt liquid medium (bile salt concentrations of 0%, 0.05%, 0.1%, 0.15%, 0.2%, and 0.25%) and cultured at 37°C in a shaker at 160 rpm. Samples were taken every 1 hour in a sterile laminar flow hood, and the OD value at 620 nm was measured until the OD value increased by more than 0.3 units or the time reached 24 hours. The lag phase of the strain was calculated based on the fitted curve.

[0104] Lag period (LT) = OD of the strain in a culture medium containing bile salts 620 Time required for the value to increase by 0.3 units - OD of the strain in a bile-free culture medium 620 The time required for the value to increase by 0.3 units.

[0105] Table 5. Tolerance of Lactobacillus curvature FFZZH5L to gastric and intestinal fluids

[0106]

[0107] Table 6 Results of bile salt tolerance test for Lactobacillus curvature FFZZH5L

[0108]

[0109] The ability to withstand gastric acid and bile salts is one of the important indicators for lactobacilli as a food supplement. The strength of tolerance to bile salts determines whether lactobacilli can survive normally and successfully colonize the intestines after entering them. Gastric acid and bile salts also have antibacterial effects. After being ingested by humans, lactobacilli enter the stomach and small intestine through the mouth and eventually reach the terminal small intestine. Therefore, to determine whether a lactobacillus is qualified to be a probiotic, it is necessary to conduct a test to simulate the tolerance of the gastrointestinal environment. In in vitro tests, the study of the bile salt tolerance of probiotics is mainly conducted through two methods: survival and growth.

[0110] Tolerance tests were conducted on *Lactobacillus curvatureii* FFZZH5L in a simulated gastrointestinal environment. The results showed that, in the simulated gastric environment test, the survival rate of *Lactobacillus curvatureii* FFZZH5L was 69.19% after 2 hours of gastric juice treatment, and stabilized at 45% after 26 hours. Figure 5 (See Table 5 for reference).

[0111] After testing for bile salt concentration, the lag phase of *Lactobacillus curvaturelis* FFZZH5L was 2.63 h at a bile salt concentration of 0.05%, and 7.81 h and 13.54 h at bile salt concentrations of 0.1% and 0.15%, respectively. Figure 5 (d as shown in Table 6).

[0112] II. Evaluation of the strain's sole carbon source utilization

[0113] Preparation of PY basal medium: 0.5 g peptone, 1.0 g yeast extract, 0.5 g trypsin-hydrolyzed casein, 4.0 mL salt solution II, 1000 mL distilled water. Sterilize at 121℃ for 20 min.

[0114] Preparation of salt solution II: CaCl2 0.2g, MgSO4·7H2O 0.48g, K2HPO4 1.0g, KH2PO4 1.0g, NaHCO3 10.0g, NaCl 2.0g, distilled water 1000mL.

[0115] BTB-MR detection solution: 0.2 g bromothymol blue, 0.1 g methyl red, 300 mL 95% ethanol, 200 mL distilled water.

[0116] In the experiment, mannitol, D-sorbitol, maltose, D-galactose, glucose, glycerol, fructose, and sucrose were used as the sole carbon sources and added to PY basal medium at a concentration of 1%. The results were detected using BTB-MR indicator.

[0117] Table 7 Results of Utilization of the Sole Carbon Source

[0118]

[0119] The experimental results are shown in Table 7: Except for the inability to utilize glycerol, Lactobacillus FFZZH5L can utilize mannitol, D-sorbitol, maltose, D-galactose, fructose, sucrose, and glucose as the sole carbon source for growth.

[0120] III. Antibiotic Susceptibility Evaluation

[0121] MH medium: 10g beef extract, 1.5g starch, 17.5g hydrolyzed casein, 17g agar, final pH 7.3, autoclaved at 121℃ for 20min.

[0122] According to Bergey's Manual of Bacteria, the KB method is used to test the antibiotic susceptibility of bacterial strains.

[0123] Antibiotic susceptibility was evaluated using MH medium.

[0124] Using a sterile cotton swab, apply a calibrated 0.5 McFarland turbidity concentration bacterial suspension to the surface of MH medium. Rotate the plate 60° each time, applying the suspension three times, and finally wipe around the perimeter of the petri dish twice. After the moisture on the plate has been completely absorbed by the agar, attach the drug-sensitive paper discs. Use tweezers to pick up the drug sensitivity paper discs and attach them to the surface of the plate. Incubate at 37°C inverted for 24 hours and record the results.

[0125] Eleven antibiotics were selected (penicillin, cefotaxime, ampicillin, streptomycin, kanamycin, tetracycline, ciprofloxacin, norfloxacin, vancomycin, clindamycin, and erythromycin), and the test results were determined according to the EU drug susceptibility testing standards (2016).

[0126] Table 8 Results of Antimicrobial Susceptibility Testing

[0127]

[0128] The experimental results are shown in Table 8: *Lactobacillus curvatureii* FFZZH5L exhibited resistance to vancomycin, streptomycin, kanamycin, ciprofloxacin, and norfloxacin, but showed sensitivity to tetracycline, penicillin, cefotaxime, ampicillin, clindamycin, and erythromycin. In contrast to conventional understanding, *Lactobacillus curvatureii* is generally insensitive to tetracycline, and tetracycline cannot inhibit its growth. The *Lactobacillus curvatureii* FFZZH5L isolated in this invention can have its growth inhibited by tetracycline and is sensitive to tetracycline, indicating that this strain has high safety.

[0129] IV. Litmus Milk Experiment:

[0130] Lactobacillus curvature FFZZH5L was tested using litmus milk culture medium.

[0131] Inoculate with activated Lactobacillus curvatureus FFZZH5L, incubate at 37°C, and observe continuously for 2 days.

[0132] like Figure 4 As shown, the color of litmus milk changes from lilac purple to pink, indicating that it contains casein hydrolase, which can utilize milk for growth and metabolism. Furthermore, the fermentation metabolites are acidic, turning the indicator litmus red.

[0133] V. Salt Tolerance Evaluation

[0134] The activated Lactobacillus curvature FFZZH5L strain was inoculated at a rate of 1% into MRS liquid medium with NaCl concentrations of 3%, 4%, 6.5%, 8%, 12%, and 15%, and incubated at 37°C. Oddi concentrations were measured every two hours. 600 Growth curves were plotted, with MRS liquid medium without NaCl serving as a control.

[0135] Probiotics used as additives in fermented and pickled foods need to be tolerant of salt and low pH. Salt and acid / alkali tolerance tests were conducted on *Lactobacillus curvatureii* FFZZH5L, and it was found that *Lactobacillus curvatureii* FFZZH5L could still grow normally at a salt content of 4% (e.g., ...). Figure 5 (as shown in a), but its growth is inhibited when the salt content reaches 6.5%.

[0136] VI. Evaluation of acid and alkali resistance

[0137] After sterilizing the MRS liquid medium, the pH of the medium was adjusted to pH 3.0, pH 4.0, pH 4.5, pH 9.2, and pH 9.6 using sterile HCl and NaOH. Activated *Lactobacillus curvatureis* strain FFZZH5L was inoculated at a rate of 1% into the MRS medium at different pH values. Odulocyte count (OD) was measured every two hours. 600 Draw the growth curve.

[0138] When *Lactobacillus curvatureii* FFZZH5L was placed in different culture media ranging from pH 3.0 to pH 9.6, it was found that it could tolerate environments ranging from pH 4.5 to pH 9.6, and within this range, *Lactobacillus curvatureii* FFZZH5L could achieve normal growth levels (e.g., ...). Figure 5 (as shown in b), but the growth of Lactobacillus curvature FFZZH5L was inhibited when the pH was below 4.

[0139] VII. Antibacterial Test

[0140] (1) MTT assay for antibacterial activity

[0141] Escherichia coli was cultured in liquid LB medium as an indicator bacterium for inhibition, and lactic acid bacteria screened in liquid LBS medium were used as the test bacteria. The cultures were incubated in a shaker at 37°C for 12 hours.

[0142] Mix 50 μL of *E. coli* fermentation broth with 50 μL of the test strain fermentation broth and let stand for 2 h. Add 50 μL of liquid LB medium and incubate at 37℃ for 12 h, then transfer to a 96-well plate. A control group was also set up, containing sterile water and uncultured liquid medium. Add 5 μL of MTT and 120 μL of DMSO, and measure the OD value at 570 mmHg. The final OD value was then measured. 570 The smaller the value, the weaker the activity of E. coli, reflecting a stronger antibacterial ability of the screened strain. Results are as follows... Figure 6 As shown, Lactobacillus curvature FFZZH5L has the strongest antibacterial ability.

[0143] (2) Determination of the minimum inhibitory concentration of Lactobacillus curvature FFZZH5L

[0144] Centrifuge the probiotic culture at 6000 rpm for 3 min, collect the supernatant, and dilute using the two-fold dilution method. Mix the Lactobacillus supernatant with the Staphylococcus aureus culture at a 1:1 ratio, incubate at 37°C for 24 h, then repeatedly pipette to disperse the biofilm. Perform a ten-fold serial dilution with PBS buffer, and evenly spread an appropriate concentration of the diluted solution onto LB agar plates. Incubate at 37°C for 24 h. Use LB agar plates containing the bacterial culture without supernatant as a blank control, and count the colonies on the plates. The minimum effective concentration of probiotics with fewer colonies than the blank control is the minimum inhibitory concentration (MIC).

[0145] Table 9 Minimum Inhibitory Concentration

[0146]

[0147] The results are shown in Table 9. The supernatant of Lactobacillus curvature effectively inhibited the growth of Staphylococcus aureus, with a minimum inhibitory concentration of 25%.

[0148] (3) Determination of antibacterial activity of Lactobacillus curvature FFZZH5L

[0149] Escherichia coli was evenly spread on the surface of solid MRS medium. A 15 mm diameter circular sterile paper was dipped into a small amount of lactic acid bacteria culture and placed in the center of the medium. The plate was sealed and inverted in an incubator at 37°C for 24 hours. The diameter of the inhibition zone was measured. MRS liquid medium was used as a negative control. The inhibition zone diameter (DIZ) was used to determine the inhibitory activity of the lactobacillus against pathogenic bacteria.

[0150] The antibacterial assay results for Lactobacillus against Escherichia coli showed that the supernatant of Lactobacillus bacterial culture exhibited antibacterial activity. The inhibition zone diameter (DIZ) was used as the standard for inhibitory activity against pathogenic bacteria: DIZ ≥ 25 mm, high inhibition; 25 mm > DIZ ≥ 20 mm, moderate inhibition; 20 mm > DIZ ≥ 15 mm, low inhibition; 15 mm > DIZ, no inhibition. Results are as follows: Figure 7 As shown in (A), the inhibition zone diameter of Lactobacillus curvature FFZZH5L is 22 mm, and it has a moderate inhibitory effect on Escherichia coli.

[0151] (4) Detection of the self-agglutination ability of probiotic strains

[0152] The self-agglutination ability of probiotic strains is one of the important indicators for evaluating the potential probiotic properties. The specific operation steps are as follows: vortex 10 mL of probiotic suspension for 10 seconds, incubate at 37℃ for 2 hours and 4 hours, and then test the supernatant.

[0153]

[0154] The absorbance of the clear liquid. The autoagglutination rate (Ac,%) of the probiotics was calculated using the formula.

[0155] A0 represents the original OD measured 0h after vortexing the probiotic suspension. 600 Numerical value; A ih This refers to the supernatant OD after IH has been left to stand. 600 Numerical value.

[0156] The results are as follows Figure 7 As shown in (B), Lactobacillus curvature FFZZH5L has a strong autoagglutination ability, reaching 6.0% at 2 h and 34.8% at 4 h.

[0157] (5) Detection of the coagulation ability between probiotic strains and pathogenic bacteria

[0158] Probiotics prevent the adhesion and colonization of pathogenic bacteria to intestinal epithelial cells by co-aggregating with them. The specific procedure is as follows: Take 5 mL of a well-vortexed probiotic suspension and mix it with an equal volume of 5 mL of Staphylococcus aureus, then vortex for 10 seconds. Incubate the 10 mL mixture at 37°C for 2 hours and 4 hours, then immediately measure the absorbance of the supernatant. Finally, calculate the co-aggregation rate (Cc,%) between the probiotics and pathogenic bacteria using the formula.

[0159] C c (%) = [1-C 0mix / (C 0strain +C 0pathogen ) / 2]×100

[0160] C 0mix This indicates the OD measured after co-culturing the mixed bacterial culture for 2 hours / 4 hours. 600 Numerical value; C 0strain This indicates the OD value measured before mixing probiotics and pathogenic bacteria. 600 Numerical value; C 0pathogen This represents the OD value before mixing Staphylococcus aureus and probiotic culture. 600 Numerical value.

[0161] The results are as follows Figure 7 As shown in (C), this Lactobacillus has a strong coagulation ability with Staphylococcus aureus, reaching 69.1% in 2 hours and 82.2% in 4 hours.

[0162] Example 5: Animal Model Experiment

[0163] The antioxidant activity of *Lactobacillus curvatureii* strain FFZZH5L against *Caenorhabditis elegans* was investigated using the standard strain *Lactobacillus rhamnosus* LGG (ATCC 53103) as a positive control. The experimental animals used in this example—wild-type *Caenorhabditis elegans* N2—were obtained from the School of Life Sciences, University of Science and Technology of China. *Escherichia coli* (E. coli) OP50 was preserved in our laboratory; *Lactobacillus rhamnosus* (LGG) ATCC 53101 was purchased from the ATCC Bioresource Center in the United States.

[0164] (1) Culture of Caenorhabditis elegans

[0165] Escherichia coli OP50 was spread on NGM medium as food for nematodes. Wild-type Caenorhabditis elegans N2 was picked and placed on the surface of the NGM medium and incubated in a 20°C biochemical incubator. During the incubation period, the nematodes were transferred to fresh NGM plates coated with E. coli OP50 every two days. If a single nematode was to be passaged, the picking needle was flammed with an alcohol lamp, cooled, and then a single nematode was picked and transferred to a new NGM medium. If a large number of nematodes were to be passaged, the nematodes in the medium were washed off with M9 buffer, allowed to settle, the supernatant was discarded, and the nematodes were transferred to a new NGM medium.

[0166] (2) Synchronization of Caenorhabditis elegans

[0167] Add 1 mL of M9 buffer to Caenorhabditis elegans to wash away excess E. coli OP50. After standing and allowing the pellet to settle, remove the supernatant and repeat the washing process three times. Then add 1 mL of lysis buffer and shake thoroughly for 1 min. Remove the supernatant and then centrifuge with M9 buffer (3000 rpm, 1 min) and wash three times to remove excess supernatant and retain the eggs. Transfer the eggs to a new NGM plate and incubate them in a 20°C biochemical incubator. After about 48 hours, they will grow into L4 stage larvae.

[0168] (3) Determination of the characterization of Caenorhabditis elegans

[0169] Lactobacillus curvatureis FFZZH5L, Escherichia coli OP50, and LGG were cultured in liquid medium to the logarithmic growth phase. The mixtures were adjusted to the same OD value with sterile water. The three bacterial solutions were then mixed with OP50 in a 1:1 ratio and spread onto NGM medium. The samples were then fed to Caenorhabditis elegans N2 and their lifespan, body length, and body wiggling frequency were measured.

[0170] Ninety wild-type *C. elegans* N2 nematodes in stage L4, synchronized as described above, were selected and placed on NGM plates containing *Lactobacillus curvatureis* FFZZH5L and *Escherichia coli* OP50, respectively, and incubated at 20°C in a biochemical incubator. Every 24 hours, all surviving nematodes were transferred to freshly coated NGM plates to prevent newly hatched nematodes from affecting the experimental results. After the nematodes finished oviposition, surviving nematodes were transferred to corresponding fresh food NGM plates every two days. The experiment continued until all nematodes died, and nematode lifespan curves were plotted.

[0171] Ten 7-day-old nematodes were randomly selected from four groups, placed on agarose plates, and their body length and number of body swings within 10 seconds were observed and measured using an optical microscope.

[0172] One L4 nematode was selected and plated onto NGM plates containing *Lactobacillus curvatureis* FFZZH5L, *Escherichia coli* OP50, a mixture of both, and LGG, with three replicates per group. During the nematode oviposition period, adult nematodes that had laid eggs were transferred to new plates every day, while the old plates were kept in a 20°C biochemical incubator until the eggs hatched. The total number of larvae hatched from the fresh plates was counted until the nematode oviposition ended.

[0173] (4) Determination of antioxidant capacity of Caenorhabditis elegans

[0174] *Caenorhabditis elegans* was cultured using *Lactobacillus curvatureis* strain FFZZH5L. Fifty nematodes were fed *Escherichia coli* OP50 as a control group. After culturing for 7 days in the L4 stage, the nematodes were collected, washed three times with M9 buffer, and allowed to settle naturally for 5 minutes. The nematodes were then homogenized in a homogenizer on ice for 5 minutes, centrifuged at 4000 rpm at 4°C, and the supernatant was collected as the protein homogenate. The contents of lipofuscin, malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-px) activity, catalase (CAT) activity, and total antioxidant capacity (T-AOC) were measured according to the kit instructions.

[0175] MDA content determination: Collect all nematodes and rinse with M9 Buffer. Perform the procedure according to the MDA content detection kit instructions and calculate the MDA content.

[0176] Lipofuscin content determination: Nematodes were placed on 2% agarose plates and anesthetized with 2mM levamisole for 1 min. The fluorescence microscope was adjusted to an excitation wavelength of 340-380 nm and an emission wavelength of 430 nm for photographing and recording lipofuscin fluorescence images. The images were then analyzed quantitatively using ImageJ software.

[0177] ROS content determination: Ten nematodes were transferred to a 96-well plate, and 50 μL of M9 Buffer and 50 μL of LCFH-DA probe were added to achieve a final concentration of 50 μM. The plate was incubated at 25°C in the dark for 30 min. The nematodes were then transferred to a glass slide, and anesthetized with 2 mM levamisole for 1 min. The fluorescence microscope was adjusted to an excitation wavelength of 460-550 nm and an emission wavelength of 590 nm to capture ROS fluorescence images. The images were then analyzed quantitatively using ImageJ software.

[0178] Superoxide dismutase activity, glutathione peroxidase activity, catalase activity, and total antioxidant capacity were measured according to the kit instructions.

[0179] (5) Protective effect of Lactobacillus curvature FFZZH5L against juglone-induced nematodes

[0180] After synchronization treatment, L4-stage nematodes were cultured for 7 days on NGM containing OP50 and NGM containing Lactobacillus curvatureis FFZZH5L. They were then transferred to plates containing juglone at a final concentration of 0.5 mM. Nematode survival was recorded hourly, and dead nematodes were promptly removed until all nematodes died. Survival rate curves were plotted.

[0181] After synchronization treatment, L4 stage nematodes were cultured for 7 days on NGM containing OP50 and NGM containing Lactobacillus curvature FFZZH5L. They were then transferred to plates containing juglone at a final concentration of 0.5 mM for oxidative stress. The nematodes were collected, and the antioxidant enzyme activity and oxidative substance content were determined in the same way as in (4).

[0182] (6) Effects of Lactobacillus curvature on the expression levels of antioxidant-related mRNAs in nematodes

[0183] After synchronization, nematodes were transferred to NGM plates coated with OP50 (control group) and NGM plates coated with Lactobacillus curvature FFZZH5L (experimental group). They were transferred to new plates daily for 7 days. Afterward, nematodes were collected, washed, and lysed. The nematodes were then centrifuged at 8000g, and the supernatant was collected. The concentration of extracted RNA was determined, and the RNA concentrations of the two groups were recorded. The extracted RNA was processed using the reverse transcription kit instructions. The extracted cDNA was quantified using the SYBR reagent according to the instructions. The target gene was detected by qPCR using the Takara Bio Chimeric Fluorescence Detection Kit and a Thermo Fisher Scientific Real-Time PCR instrument. The experimental results were analyzed using the Design & Analysis 2.5 Real-Time PCR System. The relative transcription levels of the akt-1, daf-16, skn-1, gst-1, gst-4, gst-10, sod-1, sod-3, and sod-5 genes in the negative control group and the experimental group were calculated using the relative quantification method (2-ΔΔCt). Tba-1 was used as an internal reference gene.

[0184] (7) Experimental Results

[0185] ① Effects of Lactobacillus curvature FFZZH5L on the phenotype of Caenorhabditis elegans under natural conditions

[0186] After statistically analyzing the lifespan of different groups of *Caenorhabditis elegans*, Kplan-Merie survival curves were plotted as follows: Figure 8 As shown in Table 10, the statistical results indicate that the nematodes fed with Lactobacillus curvature FFZZH5L had a 9% longer average lifespan compared to the negative control group fed with OP50 (**p<0.05, log-rank test), while there was no significant difference between the mixed-fed nematodes and the negative control group.

[0187] Table 10. Effects of different diets on the lifespan of nematodes.

[0188]

[0189] The body length of nematodes reflects their growth status and growth rate. The body lengths of different groups of *C. elegans* are shown in Table 11. The results showed that the average body length of nematodes in the negative control group was the longest among the four groups. The body length of nematodes fed with mixed diets was shortened by 2.7%, which was not statistically significant. The body length of nematodes fed exclusively with *Lactobacillus curvatureis* FFZZH5L was shortened by 7.4% (**P<0.05). The body length of nematodes in the positive control group fed with *Lactobacillus rhamnosus* was the shortest, 7.7% shorter than that of the negative control group (**P<0.05).

[0190] Table 11 Effects of different foods on nematode body length

[0191]

[0192] Ten 7-day-old nematodes were randomly selected and placed on agar plates. The number of times the body bent within 10 seconds was used as an indicator to test the movement ability of different groups of Caenorhabditis elegans. The results are shown in Table 12. It was found that the number of movements of nematodes fed with mixed feeding in the negative control group increased by 12.5%, the number of movements of nematodes fed with Lactobacillus curvature FFZZH5L increased significantly by 20.7% (*P<0.05), and the number of movements of nematodes fed with Lactobacillus curvature FFZZH5L increased by 26.1% in the positive control group.

[0193] Table 12 Effects of different foods on the number of body bends of nematodes within 10 seconds

[0194]

[0195] The reproductive capacity of nematodes, i.e., the number of eggs laid, is an important reference for their survival status and directly affects their sustainability. To test the reproductive capacity of four groups of nematodes, the number of eggs laid by synchronized *C. elegans* on a 60 mm plate from day 1 to day 3 was statistically analyzed. The results are as follows: Figure 9 As shown, in terms of total number of eggs laid, the negative control group had the most eggs laid, followed by the mixed-fed nematodes. The nematodes fed with Lactobacillus curvatureis FFZZH5L had the fewest eggs laid, which was 11.5% lower than the negative control group, but there was no significant difference. It can be concluded that Lactobacillus curvatureis FFZZH5L has no reproductive toxicity to nematodes.

[0196] ② Effects of Lactobacillus curvature FFZZH5L on antioxidant enzymes in Caenorhabditis elegans

[0197] The antioxidant enzyme activity of four groups of nematodes was measured, and the results are as follows: Figure 10 As shown, the GSH-px activity of nematodes fed a mixed diet was 26.0% higher than that of the negative control group fed with OP50, while the GSH-px enzyme activity of nematodes fed with Lactobacillus curvularia FFZZH5L was 43.4% higher than that of the negative control group (*P<0.01), and also higher than that of the positive control group fed with LGG. However, the SOD enzyme activity of nematodes fed a mixed diet only increased by 6%, which was not significant, while the SOD enzyme activity of nematodes fed exclusively with Lactobacillus curvularia FFZZH5L increased by 33.6% (*P<0.05), and the SOD activity of the positive control group increased by 61.8%. Enzyme activity: There was no significant difference in CAT enzyme activity between the negative control group and the mixed-feed nematodes. The CAT enzyme activity of nematodes fed only with FFZZH5L was 16.79% higher than that of the negative control group. After measuring the total antioxidant capacity of T-AOC, the results showed that the T-AOC enzyme activity of nematodes fed with mixed food was 39.5% higher than that of the negative control group fed only with OP50. The total antioxidant capacity of nematodes fed entirely with Lactobacillus curvularia FFZZH5L was 58.3% higher than that of the negative control group, which was significantly different (*P<0.05). The total antioxidant capacity of nematodes fed with LGG in the positive control group was 75% higher than that of the negative control group.

[0198] The results showed that Lactobacillus curvature FFZZH5L significantly enhanced the GSH-px enzyme activity in nematodes, similar to its effect on the total antioxidant capacity of T-AOC, and the degree of enhancement was positively correlated with the proportion of Lactobacillus curvature FFZZH5L in the diet. Similarly, Lactobacillus curvature FFZZH5L also enhanced the SOD enzyme activity in nematodes.

[0199] ③ Effects of Lactobacillus curvaturei FFZZH5L on the content of oxidative stress in Caenorhabditis elegans

[0200] Results of oxidative stress testing on nematodes fed different diets are as follows: Figure 11 As shown. Free radicals can participate in the peroxidation of various substances in organisms, but excessive free radicals beyond a certain range will lead to the overproduction of MDA (malondialdehyde). The level of MDA is generally considered a marker of oxidative stress and antioxidant status. Compared with the MDA content of the negative control group, the MDA content in nematodes fed with mixed diets decreased by 8%, and the MDA content in nematodes fed exclusively with Lactobacillus curvatureis FFZZH5L decreased by 9%.

[0201] Lipofuscin (LPF) is widely distributed near the cell nucleus of organisms and is composed of fat remnants and some lysosomes, gradually increasing in amount with age. The LPF content of four groups of nematodes was measured, and the results are as follows: Figure 15 As shown, the lipofuscin content in nematodes fed with mixed food was reduced by 19.0% compared to the negative control group, and the lipofuscin content in nematodes fed exclusively with Lactobacillus curvularis FFZZH5L decreased by 36.4%, which was statistically significant (*P<0.05). The lipofuscin content in the positive control LGG group decreased by 61.5%.

[0202] ROS accumulation due to cellular damage is one of the causes of oxidative damage. The imbalance between ROS and the body's cellular stress defense mechanisms leads to mitochondrial dysfunction, resulting in the production of more ROS and further oxidative damage, causing systemic imbalance. Compared to the negative control group of nematodes fed on OP50, the results of ROS content measurement using the DCFH-DA probe are as follows: Figure 16 As shown, Lactobacillus curvature FFZZH5L reduced the ROS content in nematodes by 9%.

[0203] ④ Effects of Lactobacillus curvature FFZZH5L on the lifespan of Caenorhabditis elegans under oxidative stress

[0204] After feeding *C. elegans* with *Lactobacillus curvatureensis* as the sole food source for 7 days, they were transferred to a medium containing 0.5 mM juglone NGM for oxidative stress injury. Kplan-Merie survival curves are shown in Table 13. Compared with the control group fed only OP50, nematodes fed *Lactobacillus curvatureensis* FFZZH5L had a significantly longer average lifespan (13.82%) under juglone oxidative stress injury (**p<0.05, log-rank test). This indicates that *Lactobacillus curvatureensis* FFZZH5L has a protective effect against mortality caused by juglone oxidative stress injury, producing a pre-protective effect (e.g., *Lactobacillus curvatureensis* FFZZH5L). Figure 12 (As shown).

[0205] Table 13 Effects of different diets on the survival time of nematodes under oxidative stress model.

[0206]

[0207]

[0208] ⑤ Effects of Lactobacillus curvature FFZZH5L on antioxidant enzymes in nematodes induced by juglone oxidative damage

[0209] After feeding *C. elegans* with *Lactobacillus curvularis* as the sole food source for 7 days, the nematodes were transferred to a medium containing juglone NGM at a final concentration of 0.5 mM for oxidative stress injury. Compared with the control group fed with OP50 as the sole food source, the experimental group fed with *Lactobacillus curvularis* FFZZH5L showed the following results regarding the antioxidant enzyme activity in nematodes under juglone oxidative stress injury: Figure 13 As shown, the GSH-px enzyme activity in the experimental group decreased by 51.2%, while the GSH-px enzyme activity in the control group decreased by 66.5%. The decrease in GSH-px enzyme activity in the experimental group was significantly lower than that in the control group (***p<0.01). The SOD enzyme activity in the experimental group decreased by 30.57%, while the SOD enzyme activity in the control group decreased by 50.32%. The decrease in SOD enzyme activity in the experimental group was significantly lower than that in the control group (****p<0.01). Similarly, the T-AOC enzyme activity in the experimental group decreased by 28.03%, while the T-AOC enzyme activity in the control group decreased by 35.04%, showing a significant difference between the two groups (***p<0.01). Lactobacillus FFZZH5L has a protective effect against the accumulation of juglone-induced oxidative stress damage in terms of its influence on SOD enzyme activity and the total antioxidant capacity of T-AOC, and can slow down the downward trend of GSH-px enzyme, SOD enzyme, and the total antioxidant capacity of T-AOC.

[0210] After measuring the CAT enzyme activity of the two groups of nematodes, it was found that the CAT enzyme activity in the experimental group decreased by 49.2%, while the CAT enzyme activity in the control group fed with OP50 decreased by 47.4%. There was no significant difference between the two groups, indicating that Lactobacillus curvularis FFZZH5L did not have a protective effect against the decrease in CAT enzyme activity in nematodes under oxidative stress.

[0211] ⑥ Effect of Lactobacillus curvature FFZZH5L on the content of oxides in nematodes induced by juglone oxidation damage

[0212] After feeding *C. elegans* with *Lactobacillus curvatureensis* as the sole food source for 7 days, they were transferred to a medium containing 0.5 mM juglone NGM for oxidative stress injury. The results of the determination of oxidative stress levels in *C. elegans* were compared with those of the control group fed only OP50. Figure 14 In the experimental group of nematodes, the MDA content increased by 29.4% compared to the control group under juglone oxidative stress, while the MDA content of nematodes fed OP50 increased by 50.2%. This indicates that the increase in MDA in the experimental group was smaller than that in the control group (**p<0.05). Similarly, the lipofuscin content in nematodes fed OP50 increased by 69.9% after oxidative stress. Figure 17As shown in the figure, the lipofuscin content in nematodes fed with *Lactobacillus curvatureis* FFZZH5L increased by 55.6%, and in the juglone-induced oxidative stress model, the lipofuscin content in the experimental group was reduced by 48.9% compared with the control group; after measuring the ROS content, the results showed that the ROS content in the nematodes of the control group increased by 80.7% after oxidative stress. Figure 18 As shown in the figure, the ROS level in the nematodes of the experimental group increased by 72.9%, which was lower than that of the control group (**p<0.05).

[0213] The content of oxidative stress products in nematodes indicates that Lactobacillus curvatureis FFZZH5L has a mitigating effect on the accumulation of oxidative stress in nematodes caused by oxidative stress damage.

[0214] ⑦ Effects of Lactobacillus curvature FFZZH5L on the expression of antioxidant genes in nematodes

[0215] After nematode synchronization, the nematodes were fed *Escherichia coli* OP50 (control group) and *Lactobacillus curvatureis* FFZZH5L (experimental group) as their sole food source for 7 days. The expression of various antioxidant-related genes (akt-1, daf-16, skn-1, gst-1, gst-4, gst-10, sod-1, sod-3, and sod-5) was monitored using quantitative real-time PCR (qPCR) to explore which molecular mechanisms are linked to the antioxidant mechanism of *Lactobacillus curvatureis* FFZZH5L. The results are as follows: Figure 19 As shown, when nematodes were fed *Lactobacillus curvatureis* FFZZH5L as their sole food source, the expression level of the akt-1 gene decreased. There was no significant difference in daf-16 and skn-1 expression, with some even showing a downregulation trend. However, the gst-4 gene was significantly upregulated, and gst-1, gst-10, sod-3, and sod-5 were also upregulated to some extent. Nevertheless, the expression of sod-1 showed a tendency to be suppressed. The antioxidant mechanism of *Lactobacillus curvatureis* FFZZH5L may be achieved through the upregulation of the gst-4 gene expression. Gst-4 encodes the antioxidant enzyme glutathione S-transferase, which participates in inhibiting ROS formation and conferring longer survival time to nematodes.

[0216] In summary, the *Latilactobacillus curvatus* FFZZH5L strain provided by this invention can prolong the lifespan of nematodes, increase the activity of SOD, GSH-px enzymes and the total antioxidant capacity of T-AOC, and reduce the synthesis of ROS, lipofuscin and MDA in the body, thus exhibiting antioxidant effects.

[0217] Example 6: Application of Lactobacillus curvaturei FFZZH5L in kimchi

[0218] Lactobacillus curvature FFZZH5L was used at 107 CFU / mL was inoculated into homemade kimchi, with three parallel replicates. Homemade kimchi without inoculation served as a blank. The kimchi was incubated at 10℃ and 25℃, and samples were taken at 1, 3, 5, 7, 10, 15, and 20 days to determine the pH, total acid, nitrite, and biogenic amine content of the kimchi.

[0219] strain FFZZH5L was used at 10 7 CFU / mL was inoculated into homemade kimchi, and the kimchi was replaced every 2 days. The kimchi was tested to ensure stability during multiple fermentation cycles. After 20 generations of fermentation, the kimchi liquid did not become viscous or develop an off-flavor, indicating that the fermentation was relatively stable.

[0220] Example 7: Application of Lactobacillus curvature FFZZH5L in fermented milk

[0221] 1) Ingredients: Preheat water to 45-60℃, add skim milk powder and white sugar, disperse until dissolved, and continue stirring for 25-30 minutes after dissolution to obtain milk liquid.

[0222] 2) Hydration: After stopping stirring, keep it still for 25-35 minutes to allow all substances in the milk to be fully hydrated.

[0223] 3) Homogenization: Preheat and homogenize the hydrated milk. The preheating conditions are: temperature 65℃; the homogenization conditions are: first-stage pressure 25MPa and second-stage pressure 5MPa.

[0224] 4) Sterilization: After sterilization and homogenization, the milk is sterilized under the following conditions: temperature 90-96℃, time 5-10min, and then cooled to 42℃.

[0225] 5) Inoculation and fermentation: Add Lactobacillus curvatureis FFZZH5L to the sterilized and cooled liquid, stir and mix well, and place it in a constant temperature environment of 37-42℃ for static fermentation for 14-20 hours to obtain yogurt base.

[0226] 6) Demulsification and homogenization: The fermented yogurt base is stirred to demulsify and then homogenized using a homogenizer. The homogenization conditions are: first stage pressure 0-5MPa, second stage pressure 0MPa.

[0227] 7) Post-filling ripening: Filling is carried out at 15-25℃ to ensure aseptic operation. After filling, the sample is placed in an environment of 4℃ for ripening for 12-36 hours to obtain drinking-type fermented probiotic yogurt.

[0228] Experiments show that the *Lactobacillus curvatureii* FFZZH5L strain provided by this invention possesses good antioxidant capacity in both in vivo and in vitro tests, effectively promoting the increase of antioxidant enzyme activity in vivo. This strain exhibits a favorable gastrointestinal environment and bile salt tolerance. This product has no toxic side effects and can be used as a health food or medicine to improve immunity, combat aging, enhance beauty, and regulate intestinal flora.

[0229] The above description is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the concept of the invention or exceed the scope defined in the claims, they should all fall within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus curvilinearus* with antioxidant capabilities, taxonomically named *Lactobacillus curvilinearus*, with the Latin name... Latilactobacillus curvatus The sample, with the accession number FFZZH5L, has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO: 63463, on May 14, 2023. The address of the depository is Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, postal code 510070.

2. The application of Lactobacillus curvature as described in claim 1 in the preparation of fermented dairy products.

3. The application of Lactobacillus curvature as described in claim 1 in the preparation of health food products.

4. The use of Lactobacillus curvature as described in claim 1 in the preparation of cosmetics.

5. The application of Lactobacillus curvature as described in claim 1 in the preparation of feed.

6. The application of Lactobacillus curvature as described in claim 1 in the preparation of fermented foods.

7. Fermented food prepared by fermentation using Lactobacillus curvature as described in claim 1.

8. Fermented dairy products prepared by fermentation using Lactobacillus curvature as described in claim 1.

9. A health food prepared by fermentation using Lactobacillus curvature as described in claim 1.

10. A cosmetic prepared by fermentation of Lactobacillus curvature as described in claim 1.

11. Feed prepared by fermentation using Lactobacillus curvature as described in claim 1.

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