Lactobacillus casei LC-HG35 with helicobacter pylori resistance and application thereof

Abstract

The invention discloses a lactobacillus casei LC-HG35 screened from naturally fermented yellow serofluid, which is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC No.M20221996, the classification name is lactobacillus casei LC-HG35, the preservation date is December 30, 2022, and the preservation address is Wuhan University, Wuhan, China. The traditional Chinese medicine composition has a strong anti-HP effect, is effective in live bacteria sterilization, also has an unexpected liver protection effect, and has a wide application prospect of medicinal or health-care products.

Description

Technical Field

[0001] The present application relates to the technical field of Lactobacillus casei, and in particular to a strain of Lactobacillus casei LC-HG35 having resistance to Helicobacter pylori and its application. Background Art

[0002] Lactobacillus casei belongs to the genus Lactobacillus, is a Gram-positive bacterium, does not produce spores, has no flagella, does not move, facultatively heteroferment lactose, and does not liquefy gelatin; the optimal growth temperature is 37°C, and the G+C content is 45.6% to 47.2%; the bacteria vary in length, with square ends and often in chains; the colonies are rough, off-white, and sometimes slightly yellow, and can ferment a variety of sugars. As a kind of probiotic, Lactobacillus casei can tolerate the defense mechanisms of organisms, including enzymes in the mouth, low pH values ​​in gastric juice, and bile acids in the small intestine. Therefore, after entering the human body, Lactobacillus casei can survive in large quantities in the intestine, playing a role in regulating the balance of intestinal flora and promoting human digestion and absorption. At the same time, Lactobacillus casei has the functions of effectively lowering blood pressure and cholesterol, promoting cell division, producing antibody immunity, enhancing human immunity, preventing cancer, and inhibiting tumor growth; it also has probiotic health care effects such as relieving lactose intolerance and allergies. Because Lactobacillus casei has significant beneficial effects on host nutrition, immunity, and disease prevention, it has become the focus of research, development, and production. With the increase in the number of people with hypertension, the research on Lactobacillus casei and the development of functional dairy products using it are of great significance.

[0003] Wastewater produced in the tofu preparation process is called yellow pulp water. At present, most soybean product enterprises in my country directly discharge yellow pulp water into the environment, which not only fails to fully develop and utilize its nutritional value, but also causes environmental pollution. Since the yellow pulp water of tofu contains a large amount of nutrients, microorganisms from the natural environment, especially probiotic lactic acid bacteria, will grow in large quantities therein, causing the pulp water to gradually acidify into acid pulp water and be used as a tofu coagulant. Therefore, the nutritional functional substances and microbial resources in the tofu pulp water have important development and utilization value. The present invention extracts a Lactobacillus casei LC-HG35 from the yellow pulp water.

[0004] The invention aims to provide a strain of Lactobacillus casei LC-HG35 having the function of resisting Helicobacter pylori and application thereof. Summary of the invention

[0005] In view of this, the purpose of the embodiments of the present application is to broaden the research and understanding of Lactobacillus casei, to provide its application in multiple fields, and even to provide a strain of Lactobacillus casei LC-HG35 with anti-Helicobacter pylori and its application, especially, even in an inactivated state, it has a certain effect.

[0006] In the first aspect, the embodiments of the present application disclose a strain of Lactobacillus casei LC-HG35, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No.M 20221996, classified as Lactobacillus casei LC-HG35, deposited on December 30, 2022, and deposited at Wuhan University, Wuhan, China.

[0007] In the second aspect, the examples of the present application disclose the application of the Lactobacillus casei LC-HG35 in the fight against Helicobacter pylori.

[0008] In a third aspect, the examples of the present application disclose the use of the Lactobacillus plantarum LC-HG35 in alleviating alcoholic liver damage and protecting the liver.

[0009] In a fourth aspect, the present application discloses a method for preparing the bacterial powder of Lactobacillus casei LC-HG35.

[0010] The preparation method is as follows:

[0011] S1, prepare seed suspension, melt the glycerol tube of Lactobacillus casei LC-HG35 preserved at -20 ℃, take 1mL and inoculate into 40~105mL containing 10g / L protein Chen, 10g / L beef extract, 5g / L yeast powder, 2g / L dipotassium hydrogen phosphate, 2g / L tricitrate, 5g / L sodium acetate, 1mL / L Tween-80, 0.2g / L magnesium sulfate, 0.05g / L sulfuric acid, 20g / L of glucose sterilized separately, pH=6.5, in MRS liquid medium sterilized for 30min at 121 ℃, stand and culture for 12~18h at 32~37 ℃, after culturing, centrifuge and wash 3 times with sterile physiological water, and then adjust the bacterial suspension to suitable concentration with sterile water for standby use;

[0012] S2, obtaining a fermentation broth, wherein the fermentation broth is obtained by transferring the seed suspension to the fermentation broth, wherein the fermentation broth is composed of 50 g / L skim milk powder, 8 g / L soy protein isolate, 20 g / L glucose, 8 g / L beef extract, 23 g / L NaCl, 2 g / LMgSO4·7H2O, 1 g / L Span S-80, adjusting pH=5.80, and sterilizing at 121°C for 20 min, transferring the seed suspension obtained in step S1 to the fermentation broth, and aerating and fermenting at 32-37°C, 100-120 rpm stirring conditions, and fermenting for 32-76 h to obtain the fermentation broth, wherein the ratio of air volume to tank volume is 1:0.5-1 (v / vm);

[0013] S3, centrifugal collection, collect the fermentation liquid 35ml and place it in a sterile 50ml centrifuge tube for centrifugation, the centrifugation conditions are 5000r / min, 10min, 4℃, 5000r / min, 20min, 4℃, 8000r / min, 10min, 4℃, 8000r / min, 20min, 4℃, after the centrifugation is completed, discard the supernatant and collect the bacterial mud;

[0014] S4. Preparation of freeze-dried bacterial suspension: collect centrifuged bacterial cells, add a bacterial mud protective agent prepared by 20% milk powder, 10% trehalose, 2% glycerol, 5% xylo-oligosaccharide and ultrapure water, vortex and mix well, the volume of the protective agent added is 1 / 5 of the culture solution before centrifugation, and obtain a freeze-dried bacterial suspension, which is placed at room temperature for 1 hour to allow the protective agent and bacterial cells to fully fuse;

[0015] S5. Vacuum freeze drying: pre-freeze in a -80°C refrigerator for 3 hours to freeze the freeze-dried bacterial suspension into a solid. Put it into a freeze dryer immediately after taking it out to avoid damage to the bacteria due to a sudden rise in temperature. Place the sample in the freeze dryer and continue freeze drying for 24 hours to obtain Lactobacillus casei LC-HG35 powder. Before starting freeze drying, the freeze dryer should be turned on in advance to reduce the cold trap temperature to -60°C and the vacuum degree to 1000Pa during operation.

[0016] The Lactobacillus casei LC-HG35 of the present invention is screened from naturally fermented yellow pulp water.

[0017] Compared with the prior art, this application has at least the following beneficial effects:

[0018] The present application screened a strain of Lactobacillus casei LC-HG35 against Helicobacter pylori from naturally fermented yellow pulp water, which can colonize in the stomach and has strong anti-Helicobacter pylori HP, both in live bacteria and inactivated states. In addition, it was unexpectedly found that the bacteria has a good liver protection effect and has a wide range of application prospects for medicinal or health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The colony was imaged by scanning electron microscope.

[0020] Figure 2 This is a graph of ALT activity in mouse serum.

[0021] Figure 3 This is a graph of mouse serum AST activity.

[0022] Figure 4 This is a graph of mouse serum γ-GT activity.

[0023] Figure 5 This is a graph showing the levels of inflammatory factors in mouse liver.

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 paying creative labor. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0026] Example 1 Screening and Identification of Lactobacillus casei LC-HG35

[0027] The applicant has conducted a large number of screenings from naturally fermented yellow pulp water and obtained several strains of bacteria with good health functions, among which the one involved in the present invention is named Lactobacillus casei LC-HG35. Identification of Lactobacillus casei LC-HG35

[0028] Physiological and biochemical identification

[0029] According to the Bergey's Bacterial Identification Manual and the Common Bacterial System Identification Manual, the strain LC-HG35 was tested for corresponding physiological and biochemical indicators. The specific results are shown in Table 1. Referring to the bacterial species identification manual, it can be known that the strain LC-HG35 meets the characteristics of Lactobacillus bacteria and can be preliminarily determined to be a Lactobacillus bacterium.

[0030] Table 1 Identification results of physiological and biochemical characteristics of strain LC-HG35

[0031] project result project result Sportiness - Gluconate + Catalase - Mannitol + Oxidase - Lactic acid production L(+) Lactic acid Amylase hydrolysis + lactose + L-arabinose - Galactose + Indole test - 10℃ Growth VP test + 45℃ Growth Methyl red + Gelatin liquid +

[0032] Lactic acid bacteria isolation medium (MRS medium): 10g beef extract, 10g protein, 5g acetic acid, 2g dipotassium hydrogen phosphate, 2g hydrogen citrate, 0.58g magnesium sulfate heptahydrate, 0.25g sulfuric acid tetrahydrate, 20g glucose, 1mL Tween-80, 1L distilled water, pH 6.2-6.4, sterilize at 115℃ for 20min for standby use, and add .5-2% agar to the solid culture medium.

[0033] Preparation of TE buffer: Take 5 ml of Tris-HCl Buffer solution with a pH of 8.0 and a concentration of 1 M, and then take 1 ml of EDTA solution with a pH of 8.0 and a concentration of 0.5 M, place them in a beaker, add about 400 ml of deionized water to the beaker and mix evenly, then transfer to a volumetric flask and make up the solution to 500 ml, sterilize it at high temperature and high pressure, and store it at room temperature.

[0034] Lactobacillus casei LC-HG35 strain was inoculated into MRS medium at a 1% inoculation rate, cultured overnight at 37°C for 12 hours, 5 ml of bacterial solution was centrifuged at 9000 pm for 2 minutes, and the supernatant was discarded to obtain the bacteria. After centrifugation and washing twice with TE buffer, the genome of the target strain was extracted using the bacterial genome extraction kit of OMEGA.

[0035] The PCR amplification and sequence determination of 16SrDNA gene used universal primers for prokaryotic 16SrDNA amplification, forward primer: fD1 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer: rP1 5'-GGTTACCTTGTTACGACTT-3'.

[0036] PCR reaction system includes: 10x PCR buffer, 5pL; 10mmol / L MgCl2, 3μL; 2.0μmol / L dNTP, 4μL; 10mol / L fD1, 3μL; 10mol / L rP1, 3μL; 1.0mol / L DNA template, 1μL, 5U / μL Tag TM DNA polymerase (TaKaRa), 1 μL; ddH2O, 30L; the total reaction system is 50 pL. The target fragment obtained by PCR amplification is connected to the T vector to transform Escherichia coli DH5a, and then the transformant is picked and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and the obtained 16SrDNA gene sequence is searched and compared for homology in the gene library. It was found that the Lactobacillus casei obtained by screening in the embodiment of the present application has 99.7% homology with Lactobacillus casei subsp.casei ATCC 393, so it is named Lactobacillus casei LC-HG35, which is preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC No.M20221996, and is classified as Lactobacillus casei LC-HG35. The preservation date is December 30, 2022, and the preservation address is Wuhan University, Wuhan, China.

[0037] Example 2 Preparation of bacterial powder

[0038] S1, prepare seed suspension, melt the glycerol tube of Lactobacillus casei LC-HG35 preserved at -20 ℃, take 1mL and inoculate into 40~105mL containing 10g / L protein Chen, 10g / L beef extract, 5g / L yeast powder, 2g / L dipotassium hydrogen phosphate, 2g / L tricitrate, 5g / L sodium acetate, 1mL / L Tween-80, 0.2g / L magnesium sulfate, 0.05g / L sulfuric acid, 20g / L of glucose sterilized separately, pH=6.5, in MRS liquid medium sterilized for 30min at 121 ℃, stand and culture for 12~18h at 32~37 ℃, after culturing, centrifuge and wash 3 times with sterile physiological water, and then adjust the bacterial suspension to suitable concentration with sterile water for standby use;

[0039] S2, obtaining a fermentation broth, wherein the fermentation broth is obtained by transferring the seed suspension to the fermentation broth, wherein the fermentation broth is composed of 50 g / L skim milk powder, 8 g / L soy protein isolate, 20 g / L glucose, 8 g / L beef extract, 23 g / L NaCl, 2 g / LMgSO4·7H2O, 1 g / L Span S-80, adjusting pH=5.80, and sterilizing at 121°C for 20 min, transferring the seed suspension obtained in step S1 to the fermentation broth, and aerating and fermenting at 32-37°C, 100-120 rpm stirring conditions, and fermenting for 32-76 h to obtain the fermentation broth, wherein the ratio of air volume to tank volume is 1:0.5-1 (v / vm);

[0040] S3, centrifugal collection, collect the fermentation liquid 35ml and place it in a sterile 50ml centrifuge tube for centrifugation, the centrifugation conditions are 5000r / min, 10min, 4℃, 5000r / min, 20min, 4℃, 8000r / min, 10min, 4℃, 8000r / min, 20min, 4℃, after the centrifugation is completed, discard the supernatant and collect the bacterial mud;

[0041] S4. Preparation of freeze-dried bacterial suspension: collect centrifuged bacterial cells, add a bacterial mud protective agent prepared by 20% milk powder, 10% trehalose, 2% glycerol, 5% xylo-oligosaccharide and ultrapure water, vortex and mix well, the volume of the protective agent added is 1 / 5 of the culture solution before centrifugation, and obtain a freeze-dried bacterial suspension, which is placed at room temperature for 1 hour to allow the protective agent and bacterial cells to fully fuse;

[0042] S5. Vacuum freeze drying: pre-freeze in a -80°C refrigerator for 3 hours to freeze the freeze-dried bacterial suspension into a solid. Put it into a freeze dryer immediately after taking it out to avoid damage to the bacteria due to a sudden rise in temperature. Place the sample in the freeze dryer and continue freeze drying for 24 hours to obtain Lactobacillus casei LC-HG35 powder. Before starting freeze drying, the freeze dryer should be turned on in advance to reduce the cold trap temperature to -60°C and the vacuum degree to 1000Pa during operation.

[0043] Preparation of inactivated bacteria powder: except that the bacterial sludge obtained in the above step S3 is treated at 121° C. for 30 minutes to obtain inactivated bacteria, the remaining steps are the same as the above steps.

[0044] Comparative Example 1: To this end, the present application also provides a method of using existing plant lactobacillus as comparative example 1. The steps are the same as those of the above-mentioned Example 1, except that the strain used for fermentation is Lactobacillus casei (LC for short, product number BMZ134814, Mingzhou Biotechnology), and LC bacterial powder is also obtained through the above steps.

[0045] Example 3 HP inhibition test

[0046] Preparation of Helicobacter pylori culture:

[0047] Helicobacter pylori (HP, catalog number B84182) was purchased from Mingzhou Biotechnology.

[0048] HP recovery and activation: Use the direct streak inoculation method to pick HP with an inoculation loop and smear it on Columbia blood plate culture medium. Place the coated culture dish at 37°C, humidity>90%, and culture it in a microaerobic environment (5% O2, 10% CO2, 85% N2) for 72 hours, and pass it twice in a row. The activated HP is inoculated into BHI culture medium containing 10% fetal bovine serum and cultured at 37°C in a microaerobic environment (5% O2, 10% CO2, 85% N2) for 96 hours. After washing with PBS, adjust the bacterial concentration to 10 9 The concentration of cfu / mL was divided into eppdorf tubes for later use to obtain HP bacterial solution.

[0049] Preparation of Lactobacillus casei LC-HG35 bacterial liquid: inoculate Lactobacillus casei LC-HG35 fermentation liquid into 50-100 mL of MRS liquid medium containing 10 g / L protein Chen, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L dipotassium hydrogen phosphate, 2 g / L tricitrate, 5 g / L sodium acetate, 1 mL / L Tween-80, 0.2 g / L magnesium sulfate, 0.05 g / L sulfuric acid, 20 / L of separately packaged sterilized glucose, pH=6.5, sterilized at 121°C for 20 min, and culture at 30-35°C until the bacterial liquid concentration is 10 9 cfu / mL, the Lactobacillus casei LC-HG35 bacterial liquid can be obtained.

[0050] Preparation of inactivated bacterial solution of Lactobacillus casei LC-HG35: The prepared Lactobacillus casei LC-HG35 bacterial solution was heated at 60°C for 1 h to inactivate the solution and obtain a concentration of 10 9 cfu / mL of inactivated Lactobacillus casei LC-HG35 solution.

[0051] Preparation of Lactobacillus casei LC bacterial liquid: inoculate Lactobacillus casei LC fermentation liquid into 50-100 mL of MRS liquid medium containing 10 g / L protein Chen, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L dipotassium hydrogen phosphate, 2 g / L tricitrate, 5 g / L sodium acetate, 1 mL / L Tween-80, 0.2 g / L magnesium sulfate, 0.05 g / L sulfuric acid, 20 / L of separately sterilized glucose, pH=6.5, sterilized at 121°C for 20 min, and culture at 30-35°C until the bacterial liquid concentration is 10 9 cfu / mL, then Lactobacillus casei LC bacterial liquid can be obtained.

[0052] Lactobacillus casei LC-HG35 inhibition HP test

[0053] Take 5 portions of 0.1 ml of Helicobacter pylori bacterial liquid and evenly spread them on 5 Columbia blood agar culture plates respectively. Use a 6 mm diameter puncher to punch four holes in each plate. After punching, number the Columbia blood agar culture plates as A, B, C, D, and E.

[0054] No bacterial solution was injected into the wells in the Columbia blood agar medium plate numbered A. As a negative control, the blood agar plate was lightly covered and placed upright at 37°C incubator (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 48 to 72 hours, and then observed and the diameter of the inhibition zone was measured with a vernier caliper.

[0055] Inject 200 μL of Lactobacillus casei LC-HG35 bacterial solution into each well of the Columbia blood agar plate numbered B, cover the plate lightly and place it upright at 37°C incubator (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 48 to 72 hours, then observe and measure the diameter of the inhibition zone with a vernier caliper.

[0056] Inject 200 μL of inactivated Lactobacillus casei LC-HG35 solution into each well of the Columbia blood agar plate numbered C, cover the plate lightly and place it upright at 37°C incubator (5% oxygen, 10% carbon dioxide, 85% nitrogen), incubate for 48 to 72 hours, observe and measure the diameter of the inhibition zone with a vernier caliper.

[0057] Inject 200 μL of Lactobacillus casei LC solution into each well of the Columbia blood agar plate numbered D, cover the plate lightly and place it upright at 37°C incubator (5% oxygen, 10% carbon dioxide, 85% nitrogen), incubate for 48 to 72 hours, observe, and measure the diameter of the inhibition zone with a vernier caliper.

[0058] Inject 200 μL of 0.05 mg / mL amoxicillin solution into each well of the Columbia blood agar plate numbered E as a positive control. Cover the plate lightly and place it upright at 37°C incubator (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 48 to 72 hours, then observe and measure the diameter of the inhibition zone with a vernier caliper.

[0059] Table 1

[0060]

[0061]

[0062] According to the antibacterial experiment, the inhibition zone produced by Lactobacillus casei LC-HG35 can reach a maximum of 20.3±0.4 (mm). At the same time, the Lactobacillus casei LC-HG35 after fire extinguishing also has a certain inhibitory effect on Helicobacter pylori, and the inhibitory effect is better than that of Lactobacillus casei LC bacterial solution and amoxicillin, indicating that Lactobacillus casei LC-HG35 can effectively inhibit the growth of Helicobacter pylori and prevent Helicobacter pylori infection.

[0063] Example 4 Experiment on Alleviating Alcoholic Liver Damage and Protecting the Liver

[0064] 50 SPF level male C57BL / 6 mice, weighing 26~28g, were randomly divided into normal group, model group, embodiment group, comparative example group and inactivation group after three weeks of adaptive feeding in SPF level animal room. Feeding conditions: animals were free to eat and drink water, the temperature was maintained at 23 ℃~25 ℃, humidity was 55%±5%, and the daily 12h day and night cycle was continued. Except that the normal group continued normal feeding, the feed was replaced by alcoholic Lieber DeCarli alcohol liquid feed in all other groups, and after one week of alcohol adaptive feeding, the alcohol content was increased, and the Lieber DeCarli alcohol liquid feed containing 28% caloric alcohol was continuously fed for 8 weeks. Wherein the embodiment group, comparative example group and inactivation group, in the last four weeks, every mouse was fed respectively with the lactobacillus casei LC-HG35 powder obtained in the embodiment 2, the lactobacillus casei LC powder obtained in the comparative example 1 and the inactivated lactobacillus casei LC-HG35 powder according to the dosage of 0.2g / per day.

[0065] Mouse sample collection and processing:

[0066] After the mice were fed for 8 weeks, fresh fecal samples were collected from the mice. After fasting for 12 hours, the eyeballs were quickly removed after anesthesia to collect blood, and the mice were killed by cervical dislocation. The blood samples were collected, left to stand at 4°C for 1 hour, centrifuged at 3000r / min for 15 minutes, and the upper serum was carefully collected. The mouse liver after dissection was rinsed and wiped dry in ice-cold saline, weighed after taking photos and observation, and the left lobe was sliced ​​for complete use; another 0.1g of liver tissue was taken for homogenate, and 9 times the weight of the tissue block was added to pre-cooled saline to fully grind the liver tissue, and centrifuged at 6000r / min for 15 minutes at 4°C, and the fat and precipitate were discarded. The supernatant was carefully collected as the liver homogenate. The mouse ileum tissue after dissection was rinsed and wiped dry in ice-cold saline, weighed, and 0.1g of intestinal segment was taken for RNA extraction. All samples were stored at -80°C.

[0067] Determination of mouse body weight, liver weight and liver index:

[0068] During the experiment, the body weight of mice was measured once a week. The fasting body weight of mice was measured before being killed, recorded as V1; after the mice were dissected, the complete liver was rinsed and dried in ice-cold saline, weighed, and recorded as V2; the liver index was the mass ratio of liver weight to body weight, recorded as V2 / V1.

[0069] Determination of mouse serum biochemical indicators:

[0070] The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transpeptidase (γ-GT) in mouse serum, as well as the levels of TG and total cholesterol (TC) in serum were measured using a fully automatic serum biochemical analyzer.

[0071] Determination of inflammatory factor levels in mouse liver:

[0072] The expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in mouse liver were detected by enzyme-linked immunosorbent assay according to the relevant operating instructions of the corresponding ELISA kit of R&D Company.

[0073] Table 2

[0074] Group Weight / g Liver weight / g Liver index / % Normal group 27.57±1.33 1.26±0.21 4.57±0.76 Model Group 26.31±1.46 1.42±0.16 5.40±0.60 Example Group 27.13±1.51 1.27±0.18 4.68±0.66 Comparative group 26.26±1.22 1.37±0.12 5.22±0.45 Inactivation group 27.07±1.63 1.30±0.15 4.80±0.55

[0075] According to the experimental result in Table 2, it can be seen that the weight of mice in each group changes little.But, compared with the normal group, the model group mice liver weight increases, and the liver index significantly increases.After the intervention of lactobacillus casei LC-HG35 powder in the embodiment group, the weight of mice liver and the liver index are significantly reduced, and it can be seen that the liver index also has a certain decline under the inactivated state, while the intervention in the comparative example group shows that the improvement effect is not significant.Show that lactobacillus casei LC-HG35 is under the viable bacteria or inactivated state, and can effectively suppress the rising of the liver weight and liver index of mice caused by chronic alcohol exposure.

[0076] according to Figures 2 to 4 The results in the study analyzed the effects of Lactobacillus casei LC-HG35 on liver function indicators in the serum of mice exposed to chronic alcohol. Compared with the normal group, the levels of ALT, AST, and γ-GT in the serum of mice in the alcohol model group were significantly increased, and the liver function returned to normal levels after intervention with Lactobacillus casei LC-HG35. In the serum of mice in the Lactobacillus casei LC intervention group, the liver function recovery effect was poor.

[0077] Example 5 LC-HG35 safety test

[0078] Acute toxicity test

[0079] The clean-grade Kunming male mice that had undergone adaptive feeding were randomly divided into 3 groups, 10 in each group. They were numbered as normal group, LC-HG35 group and LC-HG35 inactivated group. The prepared bacterial powder was fed at a dose of 0.2g / 10g body weight per mouse, 3 times a day, with an interval of 3h each time. The normal group was fed the same amount of feed. The activity status of the mice was observed within 7 days. After feeding, the mice were weighed and killed by cervical dislocation, and the liver coefficient and kidney coefficient of the mice were calculated.

[0080] Table 3

[0081] Group Weight / g Liver organ index / % Renal organ index / % Normal group 28.17±1.37 4.61±0.35 1.34±0.05 LC-HG35 Group 28.23±1.29 4.61±0.41 1.33±0.03 LC-HG35 inactivated group 28.11±1.51 4.60±0.39 1.34±0.02

[0082] Within 7 days of feeding, the mice's eating, drinking and activities were normal, with no abnormal behavior and normal fur. According to Table 3, the weight results showed that the mice in the LC-HG35 group and the LC-HG35 inactivated group had normal weights, with no significant difference from the normal group. The liver organ coefficient and kidney organ coefficient of the mice in the LC-HG35 group and the LC-HG35 inactivated group were not significantly different from those in the normal group, indicating that Lactobacillus casei LC-HG35 is safe and non-toxic.

[0083] In summary, the present application screened a strain of Lactobacillus casei LC-HG35 from naturally fermented yellow pulp, which has a strong anti-HP effect and is effective in sterilizing live bacteria. In addition, it also has a good liver-protecting effect and has a wide range of application prospects in medicinal or health-care products.

[0084] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A strain of Lactobacillus casei LC-HG35, deposited in China Center for Type Culture Collection, with a deposit number of CCTCC No.M20221996, classified as Lactobacillus casei LC-HG35, with a deposit date of December 30, 2022, and a deposit address of Wuhan University, Wuhan, China.

2. Application of Lactobacillus casei LC-HG35 according to claim 1 in fermenting black ginseng to prepare anti-HP preparation.

3. the bacterium powder that Lactobacillus casei LC-HG35 described in claim 1 prepares. The bacterial powder according to claim 3, which is in a live bacteria or inactivated state.

5. The method for preparing the bacterial powder according to claim 3, wherein the steps are as follows: S1, prepare seed suspension, melt the glycerol tube of Lactobacillus casei LC-HG35 preserved at -20 ℃, take 1mL and inoculate into 40~105mL containing 10g / L protein Chen, 10g / L beef extract, 5g / L yeast powder, 2g / L dipotassium hydrogen phosphate, 2g / L tricitrate, 5g / L sodium acetate, 1mL / L Tween-80, 0.2g / L magnesium sulfate, 0.05g / L sulfuric acid, 20g / L of glucose sterilized separately, pH=6.5, in MRS liquid medium sterilized for 30min at 121 ℃, stand and culture for 12~18h at 32~37 ℃, after culturing, centrifuge and wash 3 times with sterile physiological water, and then adjust the bacterial suspension to suitable concentration with sterile water for standby use; S2, obtaining a fermentation broth, wherein the fermentation broth is obtained by transferring the seed suspension to the fermentation broth, wherein the fermentation broth is composed of 50 g / L skim milk powder, 8 g / L soy protein isolate, 20 g / L glucose, 8 g / L beef extract, 23 g / L NaCl, 2 g / LMgSO4·7H2O, 1 g / L Span S-80, adjusting pH=5.80, and sterilizing at 121°C for 20 min, transferring the seed suspension obtained in step S1 to the fermentation broth, and aerating and fermenting at 32-37°C, 100-120 rpm stirring conditions, and fermenting for 32-76 h to obtain the fermentation broth, wherein the ratio of air volume to tank volume is 1:0.5-1 (v / vm); S3, centrifugal collection, collect the fermentation liquid 35ml and place it in a sterile 50ml centrifuge tube for centrifugation, the centrifugation conditions are 5000r / min, 10min, 4℃, 5000r / min, 20min, 4℃, 8000r / min, 10min, 4℃, 8000r / min, 20min, 4℃, after the centrifugation is completed, discard the supernatant and collect the bacterial mud; S4. Preparation of freeze-dried bacterial suspension: collect centrifuged bacterial cells, add a bacterial mud protective agent prepared by 20% milk powder, 10% trehalose, 2% glycerol, 5% xylo-oligosaccharide and ultrapure water, vortex and mix well, the volume of the protective agent added is 1 / 5 of the culture solution before centrifugation, and obtain a freeze-dried bacterial suspension, which is placed at room temperature for 1 hour to allow the protective agent and bacterial cells to fully fuse; S5. Vacuum freeze drying: pre-freeze in a -80°C refrigerator for 3 hours to freeze the freeze-dried bacterial suspension into a solid. Put it into a freeze dryer immediately after taking it out to avoid damage to the bacteria due to a sudden rise in temperature. Place the sample in the freeze dryer and continue freeze drying for 24 hours to obtain Lactobacillus casei LC-HG35 powder. Before starting freeze drying, the freeze dryer should be turned on in advance to reduce the cold trap temperature to -60°C and the vacuum degree to 1000Pa during operation.

6. Application of Lactobacillus casei LC-HG35 according to claim 1 in preparing probiotic powder tablets.

7. The use according to claim 6, wherein: The bacterial powder contains a protective agent.

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