A kind of plant lactobacillus J0334 and culture medium thereof

Abstract

The invention discloses a plant lactobacillus J0334, characterized in that the plant lactobacillus J0334 was deposited in Guangdong Province Microbiological Culture Collection Center on January 22, 2024, and the deposit number was GDMCC No: 64299; the plant lactobacillus J0334 provided by the invention has high ferrous ion chelating ability, and also has good DPPH free radical scavenging ability, hydroxyl free radical scavenging ability, superoxide anion scavenging ability and linoleic acid oxidation inhibition ability, and has good acid and bile salt resistance.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an antioxidant Lactobacillus plantarum J0334 with protein carbonyl scavenging ability and culture medium optimization thereof. Background Art

[0002] Oxidative stress refers to the level of endogenous and exogenous reactive oxygen free radicals (ROS) in biological organisms that exceeds the balance capacity of the body's own antioxidant system, resulting in excessive ROS attacking cell components, modifying cell proteins, lipids and DNA, and triggering a series of pathological reactions. Oxidative stress is closely related to many diseases, especially some senile and chronic diseases, such as diabetes, cardiovascular disease, inflammatory and immune diseases, hypertension, neurodegenerative diseases, etc. Studies have shown that the loss of NADPH oxidase genes (Nox1 and Nox2) or p47phox leads to a significant reduction in ROS generation and a reduced risk of atherosclerosis. Imbalance between intracellular reduction and oxidation activities can lead to β-cell dysfunction and mediate insulin resistance-related signaling pathways, which in turn leads to the occurrence and development of oxidative stress and diabetes and its related complications. In cancer, compared with normal cells, cancer cells have higher levels of ROS, which leads to changes in various signaling pathways of cell metabolism, resulting in an imbalance between ROS generation and elimination, thereby causing severe oxidative stress. Studies have shown that a moderate increase in ROS levels can accelerate the proliferation rate of cancer cells, but high concentrations of ROS can cause oxidative damage to proteins and nucleic acids, leading to the death of cancer cells. Therefore, regulating oxidative stress responses may be a potential method for cancer treatment. In alcoholic liver disease, ROS produced by alcohol metabolism in the liver induce oxidative stress responses. The accumulation of ROS increases the sensitivity of stem cells to lipid peroxidation, stimulates fatty degeneration of the liver, and leads to liver damage. Similarly, excessive ROS levels may cause pathological changes such as increased blood pressure, vascular remodeling, and vascular inflammation, promoting the occurrence and development of hypertension. Therefore, the risk of disease can be reduced by enhancing the body's antioxidant capacity and removing excessive ROS.

[0003] The chemically synthesized antioxidants commonly used at present have the advantages of low price and mature production process, but if the amount or method of use is improper, it will also cause potential harm to human health. For example, some chemical antioxidants such as butylated hydroxyphenyl ether and n-propyl gallate are reported to cause body damage. In addition, the antioxidants taken from the outside are easily digested and decomposed or directly discharged from the body, and cannot play an effective role. As people's pursuit of healthy consumption increases, the attention to natural antioxidants is also increasing. Natural or antioxidants from food resources (such as ingredients extracted from herbs, spices, seeds and fruits) are more favored by people for their safety, high efficiency, non-toxicity and other advantages, but VC, VE, etc. are extremely susceptible to the influence of factors such as temperature, oxygen, pH value and illumination, resulting in poor stability. More and more studies have shown that lactobacillus has an antioxidant effect, is rich in resources, grows fast, and has the characteristics of safety and non-toxicity as one of the common flora intestinal tract, and has great research and application value in antioxidant.

[0004] The antioxidant research on Lactobacillus plantarum is divided into in vitro research and in vivo research. Among them, the main indicators measured in in vitro research include the scavenging rate of DPPH, the scavenging rate of hydroxyl radicals, the scavenging rate of superoxide anions, etc. For example, patent 202211499533.X discloses a strain of Lactobacillus plantarum SC75-2-2 with high antioxidant activity, whose DPPH scavenging rate is 73.25%, the scavenging rate of hydroxyl radicals is 6.65%, and the scavenging rate of superoxide anions is 35%. The in vivo antioxidant experiment mainly measures antioxidant enzymes such as superoxide dismutase (which parts), reduced glutathione and malondialdehyde, etc. For example, patent CN202211407098.3, a Lactobacillus plantarum KSFY01 and its application, after intervention with Lactobacillus plantarum KSFY01, the catalase activity and glutathione content increased significantly (p<0.05), and the MDA content was significantly reduced (p<0.05). Currently, there is no patent for the use of Lactobacillus plantarum to remove protein carbonyls from tissues and intestines in the body.

[0005] In addition, the demand for nutrients of Lactobacillus plantarum is strain-specific. For example, Luo Lirong et al. found that under the same culture conditions, Lactobacillus plantarum 5-1 can grow rapidly and enter the logarithmic growth phase during 4 to 16 hours of culture, while Lactobacillus plantarum 16-1 enters the logarithmic growth phase after 10 to 16 hours. Chen Cailing et al. showed that Lactobacillus plantarum P3 quickly enters the logarithmic growth phase after 5 to 10 hours, Lactobacillus plantarum P1 and P4 slowly enter the logarithmic growth phase after 5 to 15 hours, and Lactobacillus plantarum P2 slowly enters the logarithmic growth phase after 7 to 18 hours. The optimization of culture conditions for specific strains of Lactobacillus plantarum is also of great significance for biological culture efficiency and industrial fermentation. Summary of the invention

[0006] The purpose of the present invention is to provide an antioxidant Lactobacillus plantarum J0334 with protein carbonyl scavenging ability and culture medium optimization thereof.

[0007] The object of the present invention is achieved as follows: a plant lactobacillus J0334, wherein the plant lactobacillus J0334 is deposited in Guangdong Provincial Microbiological Culture Collection Center on January 22, 2024, and the deposit number is GDMCC No: 64299.

[0008] As a further preferred embodiment of the present invention, the 16s rRNA sequence number of the Lactobacillus plantarum J0334 is shown as SEQ ID NO: 1.

[0009] SEQ ID NO: 1:

[0010]

[0011] The culture medium components of plant lactobacillus J0334 are characterized in that they include the following components: Angel peptone FP10310g, beef extract 10g, Angel yeast extract FM802 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.05g, manganese sulfate 0.05g, agar 15g, distilled water 1000mL, pH7.0.

[0012] Compared with the prior art, the present invention has the following beneficial effects: in addition to high ferrous ion chelating ability, the present invention also has good DPPH free radical scavenging ability, hydroxyl free radical scavenging ability, superoxide anion scavenging ability and linoleic acid oxidation inhibition ability, and has good acid and bile salt resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the technical description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 This is the phylogenetic tree of Lactobacillus plantarum J0334.

[0015] Figure 2 To evaluate the DPPH radical scavenging ability of different Lactobacillus plantarum strains.

[0016] Figure 3 To evaluate the superoxide anion radical scavenging ability of different Lactobacillus plantarum strains.

[0017] Figure 4 To evaluate the Fe2+ ion chelating ability of different Lactobacillus plantarum strains.

[0018] Figure 5 To evaluate the hydroxyl radical scavenging ability of different Lactobacillus plantarum strains.

[0019] Figure 6 Evaluation of the linoleic acid antioxidant capacity of different Lactobacillus plantarum isolates.

[0020] Figure 7 The body weight changes of mice in different treatment groups.

[0021] Figure 8 Figure 2 shows the content of antioxidant enzymes and oxidative damage in the liver of mice in different treatment groups.

[0022] Fig. 9This is a graph showing the levels of antioxidant enzymes and oxidative damage in the intestines of mice in different treatment groups.

[0023] Fig.10 Evaluation of yeast extract preference for Lactobacillus plantarum J0334.

[0024] Fig.11 Evaluation of peptone preference for Lactobacillus plantarum J0334.

[0025] Fig.12 To evaluate the trace element supplementation preference of Lactobacillus plantarum J0334.

[0026] Fig.13 Evaluation of pH preference for Lactobacillus plantarum J0334.

[0027] Fig.14 To evaluate the growth of different Lactobacillus plantarum cultures before and after optimization. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention optimizes the cultivation of Lactobacillus plantarum J0334, and the culture medium formula is (based on 1L): 40g of glucose, 25g of yeast extract powder, 9g of peptone, 1g of Tween 80, pH 7.0, 0.25g of MnSO4, and 0.25g of MgSO4.

[0030] The culture medium formula involved in the present invention is:

[0031] MRS agar medium: peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g, agar 15g, distilled water 1000mL, pH 6.2-6.6.

[0032] MRS optimized agar medium: Angel peptone FP103 (25g / L) 10g, beef extract 10g, Angel yeast extract FM802 (9g / L) 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.05g, manganese sulfate 0.05g, agar 15g, distilled water 1000mL, pH 7.0.

[0033] 1. Isolation, purification, identification and storage of Lactobacillus plantarum J0334

[0034] Fecal samples were collected from healthy people, and the collected samples were immediately placed in an insulated box with ice packs and taken back for the experiment. After the samples were brought back to the laboratory, part of the samples were placed in a glass test tube with a corresponding label (indicating the source and time of the sample) containing 2mL of 0.85% sterile saline, and mixed at a low speed with an oscillator until uniform, so that the microorganisms in the intestine were released as much as possible to obtain a resuspension. The Lactobacillus selective culture medium LAMVAB (peptone 10g, beef extract 10g, yeast powder 5g, dipotassium hydrogen phosphate 2.6g, sodium acetate 2g, diammonium hydrogen citrate 2g, magnesium sulfate heptahydrate 0.5g, manganese sulfate monohydrate 0.25g, arabinose 20g, L-cysteine ​​0.5g, vancomycin 20×10 -3 g, pH 5.0±0.1) and screened lactobacilli according to the following steps: (a) enrichment culture: take 0.5 mL of the above suspension and add it to 5 mL of LFMVAB liquid culture medium under sterile conditions, and enrich and culture it under anaerobic conditions at 35-37°C for 18-24 h; (b) gradient dilution: in step (1), 1 mL of the suspension was oscillated on an oscillator in a liquid tube and placed in 9 mL of physiological saline, and mixed thoroughly to obtain a 10-1 dilution. Repeat the above dilution steps to obtain 10-1 dilutions. -2 , 10 -3 , 10 -4 , 10 -5 (c) Spreading culture: 100 μL of the above 10 -3 , 10 -4 , 10 -5Three gradient dilutions were evenly spread on LFMVAB solid medium with a spreader rod, and cultured at 37°C under anaerobic conditions for 48 hours; (d) Primary purification culture: Take the dilution spread plate with a colony count in the range of 30-300, randomly select 5 single colonies of different shapes and sizes for each sample and streak them on the MRS solid plate, and culture them at 37°C under anaerobic conditions for 48 hours. (e) Secondary purification culture: Take the single colony on the streaked plate in step (4) and connect it to a liquid MRS tube, and culture it at 37°C under anaerobic conditions for 20 hours. (f) Strain preservation: Mix the secondary purification culture suspension with sterilized 60% glycerol in a 1:1 ratio, and the final glycerol concentration is 30%. The mixed solution is divided into cryopreservation tubes and stored in a -80°C ultra-low temperature refrigerator. (g) Strain identification: Centrifuge the bacterial suspension to obtain bacterial sludge, resuspend and wash the bacterial sludge with 0.85% sterile saline, and then amplify 16S DNA PCR. The upstream and downstream primers of 16SDNA were 27F: 5'-AGAGTTTGATCCTGGCCTCA-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR conditions were: 94℃, 5min; 94℃, 30s; 55℃, 30s; 72℃, 2min; 72℃, 10min; 30 cycles of 2-4. The PCR product was confirmed by nucleic acid electrophoresis analysis and sent for sequencing. The obtained spliced ​​sequence was confirmed by BLAST of NCBI database for species confirmation. The sequence analysis was performed using software MEGA7.0, and the phylogenetic tree was constructed. Figure 1 The lactic acid bacteria isolated by the present invention have the closest relationship with Lactobacillus plantarum, with a homology of 99%, and both belong to the genus Lactobacillus plantarum. Therefore, the strain is identified as Lactobacillus plantarum and named Lactobacillus plantarum J0334.

[0035] 2 In vitro experiments

[0036] 2.1 Evaluation of DPPH free radical scavenging rate

[0037] Plant lactobacillus J0334 was activated 3 times at 1% inoculum, and each culture was 18 hours. Take 3 ml of the activated fermentation liquid and centrifuge at 3500 r / min for 10 minutes, discard the precipitate, retain the supernatant, and filter through a 0.22 μm sterile filter membrane to obtain the fermentation supernatant. 1 mL of the fermentation supernatant and 1 mL of 0.2 mmol / L DPPH-anhydrous ethanol solution were reacted at room temperature in the dark for 30 minutes, then centrifuged at 3500 r / min for 10 minutes, and the absorbance of the supernatant at 517 nm was measured in triplicate. Rhamnosus lactobacillus LGG was used as a positive control.

[0038]

[0039] Among them, Ai: DPPH solution + sample, Aj: anhydrous ethanol + sample, A0: DPPH + MRS.

[0040] Depend on Figure 2 It can be seen that with Lactobacillus rhamnosus LGG as the positive control strain, only Lactobacillus plantarum J0334 had a better and significantly different DPPH free radical scavenging rate than Lactobacillus rhamnosus LGG, with a DPPH free radical scavenging rate of 96.26%. The other 8 strains had no significant differences compared with Lactobacillus rhamnosus LGG.

[0041] Evaluation of superoxide anion radical scavenging rate

[0042] Take 3 ml of the activated fermentation liquid and centrifuge it at 3500 r / min for 10 min, discard the precipitate, retain the supernatant, and filter it through a 0.22 μm sterile filter membrane to obtain the fermentation supernatant. The superoxide anion free radical scavenging ability experiment is divided into 4 groups:

[0043] A 11 Group A: 0.6 mL of sample was added to 2 mL of Tris-HCl buffer (150 mmol / L, pH 8.0), and then 0.4 mL of pyrogallol solution preheated to 25°C was added to start the reaction; 10 Group A: 0.4 mL MRS was used to replace the pyrogallol solution; 01 Group A: Replace the sample with 0.6 mL MRS; 00 Group: 1 mL MRS was used to replace 0.4 mL pyrogallol and 0.6 mL sample; the total reaction volume was 3 mL. After 20 min of reaction in a 25°C water bath, the absorbance was measured in triplicate at 325 nm. Lactobacillus rhamnosus LGG was used as a positive control.

[0044] Superoxide anion free radical scavenging rate (%) = (A 11 -A 10 ) / (A 01 -A 00 )×100%

[0045] Depend on Figure 3 It can be seen that with Lactobacillus rhamnosus LGG as the positive control strain, there are two strains that are better than Lactobacillus rhamnosus LGG, among which Lactobacillus plantarum J0334 has the highest superoxide anion radical scavenging ability of 94.3%. In addition, J0232 also has significant differences compared with Lactobacillus rhamnosus, and its superoxide anion radical scavenging ability is 89.29%.

[0046] 2.2 Evaluation of ferrous ion chelating ability

[0047] Take 3ml of activated fermentation broth and centrifuge at 3500r / min for 10min, discard the precipitate, retain the supernatant, and filter through a 0.22μm sterile filter membrane to obtain the fermentation supernatant. The Fe2+ chelating ability experiment is divided into 3 groups: A1: 0.1mL 10g / L ascorbic acid solution, 0.1mL 4g / L ferrous sulfate solution and 1mL 0.2M sodium hydroxide solution are mixed with 0.5mL fermentation supernatant, and then placed in a 37℃ water bath for 20min, and then 0.2mL 10% trichloroacetic acid solution is added. After centrifugation at 3000r / min for 10min, the supernatant is obtained, and then 0.2mL of the supernatant is added to 2mL 0.1% o-phenanthroline solution for 10min. The absorbance is measured in triplicate at 510nm, and Lactobacillus rhamnosus LGG is used as a positive control. The fermentation supernatant of group A0 is replaced with MRS medium, and the ferrous sulfate solution of group A2 is replaced with MRS medium.

[0048] Fe 2+ Chelating ability (%) = [1-(A1-A2) / A0] × 100%

[0049] Depend on Figure 4 It can be seen that, with Lactobacillus rhamnosus LGG as the positive control strain, 6 strains have better Fe2+ ion chelating ability than Lactobacillus rhamnosus LGG, among which Lactobacillus plantarum J0344, Lactobacillus plantarum J0403, Lactobacillus plantarum J0232, and Lactobacillus plantarum J0334 are the most significant, with clearance rates of 88.23%, 94.71%, 96.6%, and 97.31%, respectively. In addition, Lactobacillus plantarum J059 and Lactobacillus plantarum J052 also have significant differences compared with Lactobacillus rhamnosus LGG, with clearance rates of 84.83% and 84.60%, respectively. The effects of the other three strains have no significant differences from Lactobacillus rhamnosus LGG.

[0050] 2.3 Evaluation of hydroxyl radical scavenging rate

[0051] Take 3ml of activated fermentation liquid and centrifuge at 3500r / min for 10min, discard the precipitate, retain the supernatant, and filter through a 0.22μm sterile filter membrane to obtain the fermentation supernatant. The hydroxyl radical scavenging ability experiment is divided into 3 groups. Sample group: 1mL 0.02mol / L PBS (pH 7.4) solution is added with 0.5mL 2.5mmol / L o-phenanthroline solution, 0.5mL 2.5mmol / L FeSO4 solution and 0.5mL 20mmol / L H2O2 solution in sequence, and then 0.5mL sample is added after sufficient mixing, as sample group As; control group: 0.5mL sample is replaced with 0.5mL distilled water, as control group Ac; blank group: 1mL distilled water replaces sample and H2O2, as blank group Ab. 37℃ constant temperature water bath for 1h, and the absorbance value is measured in triplicate at 536nm. Lactobacillus rhamnosus LGG is used as the positive control.

[0052] Hydroxyl radical scavenging rate (%) = (As-Ab) / (Ac-Ab) × 100%

[0053] Depend on Figure 5 It can be seen that with Lactobacillus rhamnosus LGG as the positive control strain, 4 strains have significant differences in hydroxyl radical scavenging ability compared with Lactobacillus rhamnosus LGG, but only Lactobacillus plantarum J0334 has an effect higher than Lactobacillus rhamnosus LGG, with a hydroxyl radical scavenging rate of 78.50%.

[0054] 2.4 Evaluation of linoleic acid oxidation inhibition ability

[0055] Take 3ml of activated fermentation liquid and centrifuge at 3500r / min for 10min, discard the precipitate, retain the supernatant, and filter through a 0.22μm sterile filter membrane to obtain the fermentation supernatant. Add 1mL of linoleic acid emulsion to 0.5mL of phosphate buffer solution (pH=7.4), add 1mL of ferrous sulfate (FeSO4 mass fraction 1%), and then add 0.5mL of fermentation supernatant, and react in a water bath at 37℃ for 1.5h. Add 0.2mL of trichloroacetic acid (TAC mass fraction 4%) and 2mL of thiobarbituric acid (TBA mass fraction 0.8%) to the mixture, mix well, and react in a water bath at 100℃ for 30min, take out and cool quickly, centrifuge at 5000r / min for 10min, collect the supernatant, measure the absorbance at 532nm, and use phosphate buffer (PBS) as a blank control. Use Lactobacillus rhamnosus LGG as a positive control.

[0056] Total reducing power (%) = (1-A 样品 ) / A 空白 ×100%

[0057] Depend on Figure 6It can be seen that, with Lactobacillus rhamnosus LGG as the positive control strain, there are 3 strains with significant differences in the ability to inhibit linoleic acid oxidation compared with Lactobacillus rhamnosus LGG, among which only Lactobacillus plantarum J0334 has a higher ability to inhibit oleic acid oxidation than Lactobacillus rhamnosus LGG, and its clearance rate is 44.95%.

[0058] 3 In vivo experiments

[0059] Forty male C57BL / 6J mice aged 6 weeks were fed with normal diet for 1 week, with 12h interval between light and dark, and the mice were free to forage for food and drink water. The mice were randomly divided into five groups (n=10), namely: positive control group, negative control group, Lactobacillus plantarum J0334 group, and model bacteria LGG group. The mice in the negative control group were fed with normal diet, while the other three groups were fed with high-fat diet. (1) Negative control group: normal fat diet, gavage with 0.2mL normal saline once a day. (2) Positive control group: high-fat diet, gavage with 0.2mL normal saline once a day. (3) Lactobacillus plantarum J0334 group: high-fat diet, gavage with 0.2mL 1×109CFUmL-1 Lactobacillus plantarum J0334 once a day. (4) Model bacteria LGG group: High-fat diet, oral gavage of 0.2 mL 1×109 CFU mL-1 Lactobacillus rhamnosus LGG, once a day, the experiment lasted for 10 weeks, and body weight was measured once a week. After the last oral gavage, all mice were fasted and allowed to drink water freely for 12 hours, and then killed by cervical dislocation.

[0060] 3.1 Effects of different treatments on mouse body weight

[0061] like Figure 7 It can be seen that the weight of mice in the model group gradually increased over time. After 10 weeks, the average weight of mice in the positive control group increased by 20.66g, while the average weight of mice in the negative control group increased by 9.36g. The experimental results showed that the weight gain induced by high-fat feeding was significant, and the positive control group was established. Intragastric administration of Lactobacillus plantarum J0334 and LGG significantly inhibited the weight gain induced by high fat. The weight of mice in the Lactobacillus plantarum J0334 group increased by 5.71g, and the weight of mice in the model bacteria LGG group increased by 8.31g. Compared with the positive control group, the maximum weight difference of mice in the Lactobacillus plantarum J0334 group was 14.95g, indicating that the intake of Lactobacillus plantarum J0334 can reduce the weight gain of mice fed with high fat.

[0062] 3.2 Effects of different treatments on antioxidant enzymes and oxidative stress indicators in mouse liver

[0063] like Figure 8As shown, the total superoxide dismutase, glutathione peroxidase and catalase activities of mice in the positive control group were significantly lower than those in the negative control group (p<0.05), indicating that a high-fat diet can cause a decrease in the antioxidant enzyme activity in the mouse liver. After oral administration of Lactobacillus plantarum J0334, the total superoxide dismutase, glutathione peroxidase and catalase activities in the mouse liver increased. The antioxidant enzyme activity of mice in the J0334 group was higher than that in the Lactobacillus rhamnosus LGG group. Malondialdehyde is a product of lipid peroxidation, and protein carbonyl is the main sign of protein oxidative damage, and can be used as an important biomarker of oxidative damage. Fig. 9 As shown in the figure, compared with the negative control group, the malondialdehyde and protein carbonyl contents in the liver tissue of mice in the positive control group were significantly increased (p<0.05), indicating that a high-fat diet can cause lipid peroxidation and protein oxidative damage in the liver of mice. Compared with the positive control group, the malondialdehyde and protein carbonyl contents in the intestinal tissue of mice were significantly reduced after intervention with Lactobacillus plantarum J0334 (p<0.05). Compared with the positive control, the total antioxidant capacity and reduced glutathione content of mice were significantly increased after oral administration of Lactobacillus plantarum J0334 (p<0.05). The results showed that Lactobacillus plantarum J0334 intervention can increase the total superoxide dismutase, glutathione peroxidase and catalase activities of mice fed a high-fat diet, improve the total antioxidant capacity and reduced glutathione content, reduce the malondialdehyde and protein carbonyl contents, and alleviate the intestinal oxidative damage of mice caused by a high-fat diet.

[0064] 3.3 Effects of different treatments on intestinal antioxidant enzymes and oxidative stress indicators in mice

[0065] like Fig. 9As shown in the figure, compared with the positive control, the total antioxidant capacity of mice was significantly improved after oral administration of Lactobacillus plantarum J0334 (p<0.05), and the reduced glutathione content was not significantly different from the positive control. The total superoxide dismutase, glutathione peroxidase and catalase activities of mice in the positive control group were significantly reduced compared with those in the negative control group (p<0.05), indicating that a high-fat diet can cause a decrease in the activity of antioxidant enzymes in the intestine of mice. After oral administration of Lactobacillus plantarum J0334, the activities of total superoxide dismutase, glutathione peroxidase and catalase in the intestine of mice increased. Compared with the negative control group, the malondialdehyde and protein carbonyl contents in the intestinal tissue of mice in the positive control group were significantly increased (p<0.05), indicating that a high-fat diet can cause lipid peroxidation and protein oxidative damage in the intestine of mice. Compared with the positive control group, the malondialdehyde and protein carbonyl contents in the intestinal tissue of mice were significantly reduced (p<0.05) after intervention with Lactobacillus plantarum J0334. The results showed that Lactobacillus plantarum J0334 intervention could increase the total superoxide dismutase, glutathione peroxidase and catalase activities of mice fed a high-fat diet, improve the total antioxidant capacity and reduced glutathione content, reduce the malondialdehyde and protein carbonyl content, and alleviate the intestinal oxidative damage caused by a high-fat diet in mice.

[0066] 4 Optimization of culture medium for Lactobacillus plantarum J0334

[0067] Based on the traditional MRS culture medium components (10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium hydrogen citrate, 20 g glucose, 1 mL Tween 80, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 1000 mL distilled water, pH 6.2-6.6), the yeast extract, peptone, magnesium and manganese trace element additions and pH index of the culture medium were adjusted.

[0068] 4.1 Angel Yeast Yeast Extract Preference Analysis

[0069] Angel peptone FP401 is fixed peptone (9g / L), glucose 40g / L, Tween 80 1g / L, MnSO4 0.05g / L, MgSO4 0.05g / L, pH 7.0; Angel yeast yeast extract FM985, Angel yeast yeast extract FM503, Angel yeast yeast extract FM802, Angel yeast yeast extract FM601 (25g / L) are variable yeast extracts. The above culture medium is divided into 50mL conical flasks, inoculated into conical flasks at a 1% inoculation amount, and OD600 is measured at 0h, 12h, 16h, 20h, and 24h to check its yeast extract preference.

[0070] Depend on Fig.10It can be seen that Lactobacillus plantarum J0334 used Angel Yeast Yeast Extract FM802 as yeast extract, and the growth effect was the best at OD 600 It can grow to 1.86, followed by Angel Yeast Yeast Extract FM985, whose OD of 24h 600 It can grow to 1.81, Angel Yeast Yeast Extract FM503, its 24h OD 600 It can grow to 1.78, Angel Yeast Yeast Extract FM601, its 24h OD 600 Can grow up to 1.60.

[0071] 4.2 Angel Peptone Preference Analysis

[0072] Angel Yeast Extract FM503 is a fixed yeast extract (25g / L), glucose 40g / L, Tween 80 1g / L, MnSO40.05g / L, MgSO4 0.05g / L, pH 7.0; Angel Peptone FP401, Angel Peptone FP103, Angel Peptone FP101 (9g / L) are variable peptones. The above culture medium is divided into 50mL conical flasks, inoculated into conical flasks at a 1% inoculation amount, and OD600 is measured at 0h, 12h, 16h, 20h, and 24h to check its peptone preference.

[0073] Depend on Fig.11 It can be seen that the growth effect of Lactobacillus plantarum J0334 using Angel Peptone FP103 is the best, and its OD 600 It can grow to 1.76, followed by Angel Peptone FP103 Angel Peptone FP101, and its OD of 24h 600 It can grow to 1.74, followed by Angel Peptone FP103, whose OD of 24h 600 Can grow to 1.67.

[0074] 4.3 Preference for trace element addition (MnSO4, MgSO4)

[0075] FP401 is fixed peptone (9g / L), FM503 is fixed yeast extract (25g / L), glucose 40g / L, Tween 80 1g / L, pH 7.0; MnSO4 and MgSO4 are 0.05g / L, 0.25g / L, and 0.45g / L, respectively. OD600 is measured at 0h, 8h, 12h, 16h, 20h, and 24h to check its trace element content preference.

[0076] When Lactobacillus plantarum J0334 grew to the 18h stable period, the best growth effect was achieved when the addition amount of MnSO4 and MgSO4 was 0.05g / L. 600It can grow to 1.52. When the addition amount of MnSO4 and MgSO4 is 0.15g / L and 0.25g / L, its OD 600 Can grow to 1.51.

[0077] 4.4 pH preference

[0078] FP401 is fixed peptone (9g / L), FM503 is fixed yeast extract (25g / L), glucose 40g / L, Tween 80 1g / L, MnSO4 0.05g / L, MgSO4 0.05g / L; pH is 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0 respectively. The above culture medium is divided into 50mL conical flasks, inoculated into conical flasks at 1% inoculation volume, and OD is measured at 0h, 8h, 12h, 16h, and 20h respectively. 600 to see its pH preference.

[0079] Depend on Fig.13 It can be seen that when Lactobacillus plantarum J0334 grew to 20h, pH 7.0 had the best growth effect, and its OD 600 It can grow to 1.81, followed by pH 6.5, pH 6.0, and pH 5.5, and its OD 600 They are 1.77, 1.57, 1.55 and 1.35 respectively.

[0080] 4.5 Growth evaluation of Lactobacillus plantarum J0334 before and after medium optimization

[0081] Angel peptone FP103 (25g / L) 10g, beef extract 10g, Angel yeast extract FM802 (9g / L) 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.05g, manganese sulfate 0.05g, agar 15g, distilled water 1000mL, pH 7.0. The above culture medium was divided into 50mL conical flasks, inoculated into the conical flasks at 1% inoculation, and OD was measured at 0h, 8h, 12h, 16h, 20h, and 24h respectively. 600 , check its growth.

[0082] Depend on Fig.14 It can be seen that the OD of Lactobacillus plantarum J0334 medium was 24 hours before optimization. 600 1.5, and the growth efficiency increased significantly after optimization. 600 Can reach 1.98.

[0083] The above description is only a specific implementation mode of the present invention, but the protection scope of the invention is not limited thereto. Any changes or substitutions that can be easily thought of by a technician familiar with the field within the technical scope disclosed by the present invention should be covered within the protection scope of the invention.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ) J0334, characterized in that, The plant lactobacillus J0334 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 22, 2024, with the collection number GDMCC No: 64299.

2. Use of plant lactobacillus J0334 according to claim 1 in the preparation of antioxidants.

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