Lactobacillus acidophilus BOAO001 and application of freeze-dried powder and metagen thereof in improvement or treatment of diarrhea

Abstract

The invention provides lactobacillus acidophilus BOAO001 as well as application of freeze-dried powder and postbiotics thereof in improvement or treatment of diarrhea, and belongs to the technical field of microorganisms. The invention provides lactobacillus acidophilus BOAO001, and the preservation number of the lactobacillus acidophilus BOAO001 is CGMCC (China General Microbiological Culture Collection Center) The invention further provides a method for separating the bacteriocin by utilizing the lactobacillus acidophilus BOAO001 and preparing the freeze-dried powder and the metabiotics by utilizing the lactobacillus acidophilus BOAO001. The lactobacillus acidophilus BOAO001 fermentation liquor and the bacteriocin of the lactobacillus acidophilus BOAO001 obtained by separation, which are disclosed by the invention, have a remarkable bacteriostatic effect. The lactobacillus acidophilus BOAO001 freeze-dried powder and the metagen prepared by the preparation method disclosed by the invention can be used for improving or treating diarrhea.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of Lactobacillus acidophilus BOAO001 and freeze-dried powder and postbiotics thereof in improving or treating diarrhea. Background Art

[0002] Diarrhea is a common disease, which is defined as more than 3 bowel movements per day, more than 200 grams of feces per day, thin stool texture, and water content >85%. Diarrhea is not an independent disease, but a common manifestation of many diseases. The causes of diarrhea can be divided into infectious diarrhea and non-infectious diarrhea. Infectious diarrhea is mostly caused by viral, bacterial, fungal and parasitic infections, and may be accompanied by symptoms such as abdominal pain, fever and leukocytosis; non-infectious diarrhea includes food, chemical poisoning, drug-induced diarrhea, bait diarrhea, symptomatic diarrhea, allergic diarrhea, and other innate metabolic disease diarrhea.

[0003] At present, the clinical treatment of diarrhea mainly relies on symptomatic drug treatment, such as montmorillonite powder, bismuth subcarbonate, tannic acid protein, lopendamide, aluminum hydroxide gel, etc. According to the duration of symptoms, it can be divided into acute diarrhea and chronic diarrhea, with symptoms lasting less than two weeks and at least two weeks, respectively. Antibiotics are the first choice for the treatment of infectious acute diarrhea for etiological treatment, but the use of antibiotics for treatment will also increase the patient's gastrointestinal side effects, and may even cause antibiotic-associated diarrhea and aggravate the condition. The treatment of chronic diarrhea is mostly long-term intermittent treatment, through dietary adjustment, supplemented with drugs such as montmorillonite powder and bismuth subcarbonate, or supplemented with microecological regulators such as live bifidobacteria and live Bacillus licheniformis, but the treatment effect is not obvious and the treatment period is long. Therefore, it is of great significance to find a safe and effective method to treat diarrhea.

[0004] Probiotics are a type of active microorganisms that are beneficial to the host. They can colonize in the human intestine, maintain the balance of intestinal flora, and play an important role in human health. Due to their relative safety and diverse efficacy, they are increasingly attracting widespread attention from researchers in this field. At present, some probiotics such as Bacteroides fragilis, Lactobacillus johnsonii, and Bifidobacterium breve have been proven to have an auxiliary therapeutic or alleviating effect on diarrhea, indicating that the use of probiotics to treat diarrhea has broad prospects and important clinical value. Therefore, continuing to screen probiotics with significant efficacy in treating diarrhea and developing related preparations remains the key to research in this field. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the object of the present invention is to provide a Lactobacillus acidophilus ( Lactobacillus acidophilus )BOAO001, the deposit number is CGMCC No.32195.

[0006] The present invention also aims to provide a bacteriocin separated from the fermentation culture of Lactobacillus acidophilus BOAO001, which has a significant antibacterial effect.

[0007] The present invention also aims to provide a freeze-dried powder, a postbiotic and a preparation method thereof, which have a good therapeutic effect on diarrhea.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a Lactobacillus acidophilus ( Lactobacillus acidophilus ) BOAO001, the preservation number of the Lactobacillus acidophilus BOAO001 is CGMCC No.32195.

[0009] The present invention also provides a method for preparing bacteriocin, comprising the following steps: fermenting and culturing the Lactobacillus acidophilus BOAO001, and separating and obtaining bacteriocin from the fermentation and culturing product.

[0010] Preferably, the fermentation culture medium is an MRS medium added with 0.4wt%~1.5wt% of plant powder, and the plant powder is selected from any one of aloe powder, tremella powder, sea buckthorn powder, Euryale powder and Poria powder; the fermentation culture temperature is 36~38°C; the fermentation culture time is 14~18h.

[0011] The present invention also provides the bacteriocin prepared by the above preparation method.

[0012] The present invention also provides the use of the Lactobacillus acidophilus BOAO001 or the bacteriocin in preparing a product for inhibiting pathogens, wherein the pathogens include Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, Klebsiella pneumoniae and Clostridium difficile.

[0013] The present invention also provides a method for preparing freeze-dried powder, comprising the following steps: fermenting and culturing the Lactobacillus acidophilus BOAO001, centrifuging the fermentation culture product to obtain bacterial mud, mixing the bacterial mud with a protective agent and then freeze-drying to obtain freeze-dried powder of Lactobacillus acidophilus BOAO001.

[0014] The present invention also provides freeze-dried powder prepared by the above preparation method.

[0015] The present invention also provides a method for preparing a postbiotic, comprising the following steps: fermenting and culturing the Lactobacillus acidophilus BOAO001, and concentrating and spray-drying after the fermentation and culturing to obtain the postbiotic; the fermentation and culturing culture medium comprises: 40-60 g / L glucose, 25-35 g / L sucrose, 8-12 g / L aloe powder, 15-25 g / L yeast extract, 25-35 g / L Euryale powder, and 27-33 g / L Poria powder.

[0016] The present invention also provides postbiotics prepared by the above preparation method.

[0017] The present invention also provides use of the Lactobacillus acidophilus BOAO001 or the bacteriocin or the lyophilized powder or the postbiotic in preparing a medicament for improving or treating diarrhea.

[0018] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: The present invention obtains a strain of Lactobacillus acidophilus BOAO001 for the first time, and further separates and obtains Lactobacillus acidophilus BOAO001 bacteriocin based on Lactobacillus acidophilus BOAO001. The Lactobacillus acidophilus BOAO001 fermentation liquid and the bacteriocin separated therefrom of the present invention have good antibacterial effect, can significantly inhibit the growth of Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, Klebsiella pneumoniae and Clostridium difficile, and can be used to prevent or treat diarrhea caused by pathogenic bacteria.

[0019] The present invention prepares freeze-dried powder and postbiotics based on Lactobacillus acidophilus BOAO001, respectively. The obtained freeze-dried powder and postbiotics can significantly reduce the content of catalase and inflammatory factors in small intestinal tissue and serum, and relieve and treat diarrhea.

[0020] Biological Deposit Description Lactobacillus acidophilus BOAO001, classified as Lactobacillus acidophilus ( Lactobacillus acidophilus ), depository: China General Microbiology Center of Microbiological Culture Collection (CGMCC for short), depository address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No.32195, deposit date October 12, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :The macroscopic morphology of the colonies of Lactobacillus acidophilus BOAO001 in the plate; Figure 2 :Microscopic morphology of Lactobacillus acidophilus BOAO001 strain; Figure 3 :Experimental results on water content in mouse feces; Figure 4 :Experimental results of serum MOP, TNF-α and IL-6 levels in mice; Figure 5 :The experimental results of MOP, TNF-α and IL-6 contents in the small intestine of mice. DETAILED DESCRIPTION

[0022] The present invention provides a Lactobacillus acidophilus ( Lactobacillus acidophilus) BOAO001, the preservation number of the Lactobacillus acidophilus BOAO001 is CGMCC No.32195. The Lactobacillus acidophilus BOAO001 of the present invention is isolated and purified from traditional homemade yogurt in Kashgar, Xinjiang, and is proved to belong to Lactobacillus acidophilus , and was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on October 12, 2024. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] The fermentation broth of Lactobacillus acidophilus BOAO001 of the present invention can significantly inhibit the growth of Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, Klebsiella pneumoniae and Clostridium difficile. The fermentation medium of Lactobacillus acidophilus BOAO001 of the present invention is MRS liquid medium. As an optional embodiment, the present invention activates and cultures Lactobacillus acidophilus BOAO001 in MRS liquid medium, and after activation, it is inoculated into MRS liquid medium at an inoculation amount of 2-4% (v / v) for expansion culture and fermentation culture, and the inoculation amount is preferably 3%. The temperature of the fermentation culture of the present invention is 36-38°C, preferably 37°C; the fermentation culture time is 14-18h, preferably 15-16h.

[0024] Preferably, the fermentation medium of Lactobacillus acidophilus BOAO001 of the present invention is an optimized and improved MRS liquid medium, that is, 0.4wt%~1.5wt% of plant powder is added to the MRS liquid medium, and the plant powder is selected from any one of aloe powder, tremella powder, sea buckthorn powder, Euryale powder and Poria powder, and the addition amount is preferably 0.5~1wt%. The present invention can effectively promote the growth of Lactobacillus acidophilus BOAO001 and increase the viable count of Lactobacillus acidophilus BOAO001 by adding aloe powder, tremella powder, sea buckthorn powder, Euryale powder or Poria powder.

[0025] The present invention also provides a method for preparing bacteriocin, comprising the following steps: fermenting and culturing the Lactobacillus acidophilus BOAO001, and separating and obtaining bacteriocin from the fermentation and culturing product.

[0026] The culture medium for fermentation culture of the present invention is an MRS culture medium added with 0.4wt%~1.5wt% of plant powder, the addition amount of the plant powder is preferably 0.5~1wt%, and the plant powder is selected from any one of aloe powder, tremella powder, seabuckthorn powder, Euryale powder and Poria powder. The present invention can effectively promote the growth of Lactobacillus acidophilus BOAO001 and increase the content of bacteriocin in the fermentation liquid by adding aloe powder, tremella powder, seabuckthorn powder, Euryale powder or Poria powder.

[0027] The fermentation culture temperature of the present invention is 36-38° C., preferably 37° C.; the fermentation culture time is 14-18 h, preferably 15-16 h.

[0028] The separation method of the present invention is preferably an ammonium sulfate salting-out method. The present invention centrifuges the fermentation culture product (fermentation broth) to obtain the supernatant, and the centrifugation conditions are preferably centrifuged at 4°C and 12000r / min for 10min. The present invention adds ammonium sulfate to the centrifuged supernatant to make its saturation reach 80%, and salts out at 4°C overnight. After salting out, the present invention collects the precipitate by centrifugation to obtain bacteriocin, and the centrifugation conditions are preferably centrifuged at 4°C and 12000r / min for 10min.

[0029] The present invention also provides a bacteriocin prepared by the above preparation method, wherein the bacteriocin can significantly inhibit the growth of Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, Klebsiella pneumoniae and Clostridium difficile.

[0030] The present invention also provides the use of the Lactobacillus acidophilus BOAO001 or the bacteriocin in preparing a product for inhibiting pathogens, wherein the pathogens include Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, Klebsiella pneumoniae and Clostridium difficile.

[0031] The present invention also provides a method for preparing a freeze-dried powder, comprising the following steps: fermenting and culturing the above-mentioned Lactobacillus acidophilus BOAO001, centrifuging the fermentation culture product to obtain bacterial mud, mixing the bacterial mud with a protective agent and freeze-drying to obtain freeze-dried powder of Lactobacillus acidophilus BOAO001. The fermentation medium of Lactobacillus acidophilus BOAO001 of the present invention is preferably an MRS medium added with 0.4wt%~1.5wt% plant powder, more preferably an MRS liquid medium added with 0.4wt%~1.5wt% aloe powder. As an optional embodiment, the present invention activates and cultures Lactobacillus acidophilus BOAO001 in an MRS liquid medium, and after activation, the inoculation amount of 2~4% (v / v) is inoculated into the fermentation medium for expansion culture and fermentation culture. The temperature of the fermentation culture of the present invention is 36~38°C, preferably 37°C; the fermentation culture time is 14~18h, preferably 15~16h. The present invention centrifuges the culture product, and the centrifugation conditions are preferably 4°C, 8000r / min, and centrifugation for 20min. After centrifugation, the supernatant is discarded to obtain bacterial sludge, and the bacterial sludge is fully mixed with a protective agent in a mass ratio of 1:2. The protective agent includes 10g skim milk, 8g trehalose, 4g sucrose, 3g lactose, 0.1g vitamin C, and 100g water. The protective agent is sterilized at 110°C for 20min and cooled to 4°C for standby use. The freeze drying of the present invention is preferably carried out at a cold trap temperature of -70°C and freeze drying for 72h to obtain freeze-dried bacterial powder. The number of viable bacteria of the freeze-dried powder provided by the present invention is 1×1011 ~9×10 11 cfu / g.

[0032] The present invention also provides a method for preparing postbiotics, comprising the following steps: fermenting and culturing the above-mentioned Lactobacillus acidophilus BOAO001, concentrating and spray drying after the fermentation and culturing to obtain postbiotics; the fermentation medium of the Lactobacillus acidophilus BOAO001 of the present invention comprises: 40-60 g / L glucose, 25-35 g / L sucrose, 8-12 g / L aloe powder, 15-25 g / L yeast extract, 25-35 g / L Euryale powder, 27-33 g / L Poria powder; preferably comprises: 50 g / L glucose, 30 g / L sucrose, 10 g / L aloe powder, 20 g / L yeast extract, 30 g / L Euryale powder, 30 g / L Poria powder. As an optional embodiment, the present invention activates and cultures Lactobacillus acidophilus BOAO001 in MRS liquid culture medium, and after activation, inoculates it into the fermentation medium at an inoculation amount of 2-4% (v / v) for expansion and fermentation. The temperature of the fermentation culture of the present invention is 36-38°C, preferably 37°C; the fermentation culture ends when the pH value no longer decreases. The fermentation broth of the present invention is concentrated under reduced pressure, preferably by evaporation under 0.09 MPa and 45°C, and concentrated to half the volume of the fermentation broth. After the reduced pressure concentration of the present invention, spray drying is performed to obtain postbiotics, and the conditions of the spray drying are that the inlet temperature is 170°C and the feed rate is 10.4 mL / min.

[0033] The freeze-dried powder and postbiotics obtained by the present invention can significantly reduce the content of catalase and inflammatory factors (TNF-α and IL-6) in small intestinal tissue and serum, and relieve and treat diarrhea.

[0034] The present invention also provides use of the Lactobacillus acidophilus BOAO001 or the bacteriocin or the lyophilized powder or the postbiotic in preparing a medicament for improving or treating diarrhea.

[0035] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. 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.

[0036] In the examples of the present invention, Escherichia coli CMCC (B) 44102, Salmonella paratyphi B CMCC (B) 50094, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae ATCC 700603, and Clostridium difficile ATCC 9689 were all purchased from Shanghai Beinuo Biotechnology Co., Ltd. Aloe vera powder (Aloe vera gel powder) was purchased from Shaanxi Yilin Biotechnology Co., Ltd.; Tremella fuciformis powder was purchased from Shaanxi Qinglan Biotechnology Co., Ltd.; Sea buckthorn powder was purchased from Tianjin Jincheng Biotechnology Co., Ltd.; Euryale ferox powder was purchased from Shaanxi Qinglan Biotechnology Co., Ltd.; and Poria cocos powder was purchased from Shaanxi Qinglan Biotechnology Co., Ltd.

[0037] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] Example 1 Isolation and identification of Lactobacillus acidophilus BOAO001: Traditional homemade yogurt from Kashgar, Xinjiang was refrigerated and sent to the strain isolation laboratory. The homemade yogurt was diluted in multiple gradients using a commercial modified MRS solid culture medium and then anaerobically cultured for 72 hours for strain isolation. Single colonies were picked and cultured until the strain was purified. A single strain was screened out, namely Lactobacillus acidophilus BOAO001.

[0040] Lactobacillus acidophilus BOAO001 was inoculated on a modified MRS solid medium and cultured at 37°C for 72 hours. The colonies were found to be white, round, moist, opaque, and with neat edges. Figure 1 shown.

[0041] Lactobacillus acidophilus BOAO001 was inoculated into a modified MRS liquid medium and cultured at 37°C for 18 h. The bacteria were observed and photographed under an ordinary optical microscope. The bacteria were found to be rod-shaped, 0.5-0.7 μm × 1.8-4.5 μm, arranged singly or in pairs, and were Gram-positive bacteria. Figure 2 As shown, the scale bar in the figure is 5 μm.

[0042] Physiological, biochemical and glycolytic experiments were conducted on Lactobacillus acidophilus BOAO001. The results are shown in Table 1.

[0043] Table 1 Results of physiological and biochemical and glycolysis experiments of Lactobacillus acidophilus BOAO001

[0044] Note: “+” indicates positive, “-” indicates negative.

[0045] According to the results in Table 1, the isolated single strain can be preliminarily judged as lactic acid bacteria in the above experimental process. The strain was sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA sequence sequencing. The 16S rDNA sequence is shown in SEQ ID No. 1. The identification result is Lactobacillus acidophilus. According to the strain preservation of this experiment, it is numbered and named Lactobacillus acidophilus BOAO001.

[0046] Lactobacillus acidophilus BOAO001 is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short). The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number: CGMCC No.32195 and the deposit date of October 12, 2024.

[0047] Example 2 Antibacterial experiment of Lactobacillus acidophilus BOAO001: BHIs liquid culture medium: Accurately weigh 18.5g of brain heart infusion medium and 2.5g of yeast extract, and place them in a 1L beaker. Use a measuring cylinder to accurately measure 500mL of ultrapure water and add it to the beaker, and stir it thoroughly with a glass rod to make BHI liquid. Pour the mixed liquid into a 500mL blue-mouth bottle, loosen the bottle cap, use a high-pressure steam sterilizer, sterilize at 121℃ for 20min, take it out and store it in a sealed container at room temperature. When using, add a sterile 10% Cys solution to the BHI liquid culture medium to a final concentration of 0.1% of the total concentration, and make it into BHIs liquid culture medium for use.

[0048] After Lactobacillus acidophilus BOAO001 was activated for two generations in MRS liquid medium, it was inoculated into MRS liquid medium at a 3% (v / v) inoculum and cultured at 37°C for 16-18 h. 600 is 5.00 to obtain the culture solution of Lactobacillus acidophilus BOAO001 for use.

[0049] Clostridium difficile ATCC9689 was revived by taking out glycerol frozen bacteria from the ultra-low temperature refrigerator and transferring them to 10 mL BHIs liquid medium. The culture was incubated in an anaerobic incubator at 37°C for 16 h overnight. The bacterial solution after overnight culture was transferred to fresh 10 mL BHIs liquid medium at a 3% (v / v) inoculation volume and incubated in an anaerobic incubator at 37°C for 16 h overnight. The OD 600 1.2~1.6, for spare use.

[0050] Escherichia coli CMCC (B) 44102, Salmonella paratyphi B CMCC (B) 50094, Staphylococcus aureus ATCC6538, and Klebsiella pneumoniae ATCC700603 were inoculated into nutrient broth medium and cultured in a shaker at 37°C and 180 rpm for 24 hours for activation. 1 mL of bacterial suspension of Escherichia coli CMCC (B) 44102, Salmonella paratyphi B CMCC (B) 50094, Staphylococcus aureus ATCC6538, and Klebsiella pneumoniae ATCC700603 were added to 55°C nutrient broth solid medium and shaken evenly to avoid bubbles. The number of viable bacteria was kept at 10 7 CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium has cooled and solidified, take out the Oxford cup, inject 200μL of Lactobacillus acidophilus BOAO001 culture solution into each well, cover the plate lightly and place it upright in a 37℃ incubator for incubation for 3 days, and measure the diameter of the inhibition zone with a vernier caliper, and make three parallels.

[0051] Inhibition of Clostridium difficile ATCC9689 experiment: Cool the BHIs solid medium to 55°C, take 1 mL of Clostridium difficile ATCC9689 suspension and add it to the above mixed medium, shake it evenly to avoid bubbles, and make the number of viable pathogens in the system reach 10 7 CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium cooled and solidified, take out the Oxford cup, inject 200μL of Lactobacillus acidophilus BOAO001 culture solution into each well, cover the plate lightly and place it upright at 37℃ for anaerobic culture for 3 days, and measure the diameter of the inhibition zone with a vernier caliper, and make three parallels.

[0052] The test results are as follows: Table 2 Antibacterial test results of Lactobacillus acidophilus BOAO001

[0053] It can be seen from the above table that Lactobacillus acidophilus BOAO001 has an inhibitory effect on 5 pathogens, among which the inhibitory effect on Gram-negative bacteria is strong, especially the strongest inhibitory effect on Escherichia coli CMCC(B)44102; it has an inhibitory effect on Gram-positive bacteria, which is slightly weaker than that on Gram-negative bacteria.

[0054] Example 3 Bacteriocin isolation and antibacterial experiment of Lactobacillus acidophilus BOAO001: After Lactobacillus acidophilus BOAO001 was activated for two generations in MRS liquid medium, it was inoculated into MRS liquid medium at a 3% (v / v) inoculum and cultured at 37°C for 16-18 h. 600 is 5.00 to obtain the culture solution of Lactobacillus acidophilus BOAO001 for use.

[0055] The culture of Lactobacillus acidophilus BOAO001 was inoculated into 500 mL MRS broth at 3% (v / v) and cultured at 37°C for 40 hours. The fermentation broth was centrifuged at 4°C and 12000 r / min for 10 minutes, and the supernatant was stored in a refrigerator at 4°C for later use. The ammonium sulfate salting-out method was adopted. Ammonium sulfate was added to 100 mL of the fermentation supernatant to make its saturation reach 80%, and the salting-out precipitate was precipitated at 4°C overnight (18 hours). The precipitate was centrifuged at 4°C and 12000 r / min for 10 minutes, and the precipitate (1.2 g) was collected and dissolved with 100 mL of distilled water to obtain the bacteriocin solution of Lactobacillus acidophilus BOAO001, which was stored in a refrigerator at -20°C.

[0056] Five pathogenic bacteria, namely, Escherichia coli CMCC (B) 44102, Salmonella paratyphi B CMCC (B) 50094, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae ATCC 700603, and Clostridium difficile ATCC 9689, were used to detect the antibacterial effect of Lactobacillus acidophilus BOAO001 bacteriocin using the antibacterial zone experimental method of Example 2 (the only difference is that 200 μL of Lactobacillus acidophilus BOAO001 culture solution was injected into each well instead of 200 μL of Lactobacillus acidophilus BOAO001 bacteriocin solution). The results are shown in the following table: Table 3 Inhibition test results of bacteriocin of Lactobacillus acidophilus BOAO001

[0057] It can be seen from the above table that the bacteriocin isolated from the supernatant of Lactobacillus acidophilus BOAO001 has an inhibitory effect on five pathogens, and there is no significant difference compared with the unpurified fermentation broth, indicating that the bacteriocin produced by Lactobacillus acidophilus BOAO001 has a good inhibitory effect on pathogens.

[0058] Example 4 The inhibitory effect of different strains on pathogens: Lactobacillus acidophilus BOAO001, Lactobacillus acidophilus LA-5 (purchased from Chr. Hansen, Denmark), Lactobacillus acidophilus NCFM (purchased from DuPont Danisco), Lactobacillus acidophilus La28 (purchased from Hebei Yiran Biological), and Lactobacillus acidophilus LS001 (CGMCC23649) were activated in MRS liquid medium for two generations, inoculated with 3% (v / v) inoculation volume into MRS liquid medium, and cultured at 37°C for 16-18 h. OD 600 is 5.00 to prepare culture solution for later use.

[0059] Take 1 mL of Escherichia coli CMCC (B) 44102, Salmonella paratyphi B CMCC (B) 50094, Staphylococcus aureus ATCC 6538, and Klebsiella pneumoniae ATCC 700603 bacterial suspensions respectively and add them to 55°C nutrient broth solid culture medium, shake evenly to avoid bubbles, and make the number of viable bacteria reach 10 7 CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium cooled and solidified, take out the Oxford cup, inject 200μL of culture solution of Lactobacillus acidophilus BOAO001, Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus La28, and Lactobacillus acidophilus LS001 respectively, cover the plate lightly and place it upright in a 37℃ incubator for 3d, and measure the diameter of the inhibition zone with a vernier caliper, and make three parallels.

[0060] Inhibition of Clostridium difficile ATCC9689 experiment: Cool the BHIs solid medium to 55°C, take 1 mL of Clostridium difficile ATCC9689 suspension and add it to the above mixed medium, shake it evenly to avoid bubbles, and make the number of viable pathogens in the system reach 10 7 CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium is cooled and solidified, take out the Oxford cup, inject 200μL of Lactobacillus acidophilus BOAO001, Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus La28, and Lactobacillus acidophilus LS001 culture solution into each well, cover the plate lightly and place it upright at 37℃ for anaerobic culture for 3d, and measure the diameter of the inhibition zone with a vernier caliper, and make three parallels.

[0061] The test results are as follows: Table 4 Antibacterial test results of different strains

[0062] Note: Lowercase letters in the table indicate differences among groups.

[0063] From the above table, we can see that Lactobacillus acidophilus BOAO001 has obvious inhibitory effect on five pathogens, among which only Lactobacillus acidophilus NCFM has a slightly higher inhibitory effect on Clostridium difficile ATCC9689 than Lactobacillus acidophilus BOAO001, but has no significant inhibitory effect on Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, and Klebsiella pneumoniae; Lactobacillus acidophilus LS001 has an inhibitory effect on Escherichia coli CMCC(B)44102 close to that of Lactobacillus acidophilus BOAO001. Overall, Lactobacillus acidophilus BOAO001 has a good inhibitory effect on pathogens.

[0064] Example 5 1. Lactobacillus acidophilus BOAO001 enrichment experiment: MRS medium: peptone 10g, beef extract powder 10g, yeast extract powder 5g, glucose 20g, magnesium sulfate 0.1g, sodium acetate 5g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, manganese sulfate 0.05g, Tween 80 1.0g, water 1000g, pH 6.8, sterilization at 121℃ for 15min.

[0065] MRS-1 medium: Add 5 g of aloe powder to MRS medium, sterilize at 121℃ for 15 min, and set aside.

[0066] MRS-2 medium: Add 5 g Tremella powder to MRS medium, sterilize at 121℃ for 15 min, and set aside.

[0067] MRS-3 medium: Add 5 g seabuckthorn powder to MRS medium, sterilize at 121℃ for 15 min, and set aside.

[0068] MRS-4 medium: Add 5 g of Euryale ferox powder to MRS medium, sterilize at 121℃ for 15 min, and set aside.

[0069] MRS-5 medium: Add 5 g of Poria powder to MRS medium, sterilize at 121℃ for 15 min, and set aside.

[0070] Lactobacillus acidophilus BOAO001 was activated using MRS medium, inoculated into the MRS medium at 3% (v / v), and cultured at 37°C for 16 hours before use.

[0071] The activated Lactobacillus acidophilus BOAO001 was inoculated into MRS, MRS-1, MRS-2, MRS-3, MRS-4, and MRS-5 culture media at a 3% (v / v) inoculation rate. After static culture at 37°C for 16 hours, the number of viable bacteria was detected. 100 mL of fermentation broth was taken from each group and centrifuged at 4°C and 12000 r / min for 10 minutes, and the supernatant was stored in a refrigerator at 4°C for later use. The ammonium sulfate salting-out method was adopted, and ammonium sulfate was added to 100 mL of fermentation supernatant to a saturation of 80%. Salting-out precipitation was carried out at 4°C overnight (18 hours), and centrifuged at 4°C and 12000 r / min for 10 minutes. The precipitate was collected and weighed. The results are as follows: Table 5 Results of the enrichment experiment of Lactobacillus acidophilus BOAO001

[0072] Note: Compared with MRS,*: P <0.05.

[0073] As shown in the table above, when aloe powder, tremella powder, and sea buckthorn powder were added to the MRS medium, the bacterial cell and bacteriocin of Lactobacillus acidophilus BOAO001 increased. The number of live bacteria increased the most after adding aloe powder, and the bacteriocin metabolism was the most. The effect of adding sea buckthorn powder was not significant compared with the MRS medium without adding sea buckthorn powder. The effects of tremella powder, Poria powder, and Euryale powder were in the middle. This experiment shows that aloe powder promotes the growth of Lactobacillus acidophilus BOAO001 and the metabolism of bacteriocin. Subsequent experiments use aloe powder to study Lactobacillus acidophilus BOAO001.

[0074] 2. Experiment on the bacterial growth of aloe vera powder on Lactobacillus acidophilus BOAO001: Lactobacillus acidophilus BOAO001 was activated using MRS medium, inoculated into the MRS medium at 3% (v / v), and cultured at 37°C for 16 hours before use.

[0075] Different concentrations of aloe powder were added to the MRS medium, and the final concentrations of each group were: 0.5%, 1%, and 1.5%, respectively. The pH was 6.8 and sterilized at 121℃ for 15min. The activated Lactobacillus acidophilus BOAO001 was inoculated into the aloe powder medium with different concentrations at a 3% (v / v) inoculation rate. After static culture at 37℃ for 16h, the number of viable bacteria was detected, and the bacteriocin was separated by ammonium sulfate precipitation method (the specific scheme is the same as step 1). The results are as follows: Table 6 Results of bacterial enrichment experiments with different concentrations of aloe vera powder

[0076] Note: Compared with MRS,*: P <0.05;**: P <0.01.

[0077] From the table above, we can see that as the amount of aloe powder added increases, the number of live bacteria and bacteriocin metabolism of Lactobacillus acidophilus BOAO001 gradually increases. When the amount of aloe powder added is 1% and 1.5%, the difference in the number of live bacteria and bacteriocin increment of Lactobacillus acidophilus BOAO001 is not large. Therefore, the addition of aloe powder at 1% is more appropriate.

[0078] Example 6 1. Preparation of freeze-dried powder of Lactobacillus acidophilus BOAO001: Lactobacillus acidophilus BOAO001 was activated using MRS medium: 3% (v / v) was inoculated into MRS medium, and cultured at 37°C for 16 hours before use. The activated Lactobacillus acidophilus BOAO001 was inoculated into MRS medium supplemented with 1% aloe powder at a 3% (v / v) inoculation rate. After cultured at 37°C for 16 hours, it was centrifuged at 4°C and 8000r / min for 20 minutes, and the supernatant was discarded to obtain bacterial sludge. The bacterial sludge and the protective agent were thoroughly mixed in a ratio of 1:2. After mixing evenly, freeze-dried for 72 hours at a cold trap temperature of -70°C to obtain freeze-dried bacterial powder. The number of live bacteria in the freeze-dried bacterial powder was measured to be 2.3×10 11 cfu / g.

[0079] Protective agent: skim milk 10g, trehalose 8g, sucrose 4g, lactose 3g, vitamin C 0.1g, water 100g, sterilize at 110℃ for 20min, cool to 4℃ for use.

[0080] 2. Preparation of postbiotics from Lactobacillus acidophilus BOAO001 Liquid culture medium: glucose 50g / L, sucrose 30g / L, aloe powder 10g / L, yeast extract 20g / L, Euryale powder 30g / L, Poria powder 30g / L.

[0081] Lactobacillus acidophilus BOAO001 was activated using MRS medium: 3% (v / v) was inoculated into MRS medium, and cultured at 37°C for 16 hours before use. The activated Lactobacillus acidophilus BOAO001 was inoculated into the liquid medium at a 3% (v / v) inoculation rate for fermentation culture. The culture temperature was 37°C. The pH value of the fermentation liquid no longer decreased after 16 hours, and the fermentation was terminated. The fermentation liquid was concentrated by vacuum concentration, evaporated and concentrated at a vacuum degree of 0.09mpa and a temperature of 45°C, and spray dried when it was concentrated to one-half of the fermentation liquid. The spray drying conditions were an inlet air temperature of 170°C and a feed rate of 10.4mL / min to obtain Lactobacillus acidophilus BOAO001 postbiotics.

[0082] Example 7 The freeze-dried powder and postbiotics prepared in Example 6 were used to carry out an experiment on Lactobacillus acidophilus BOAO001 to relieve diarrhea in mice: Ninety SPF 6-week-old Balb / c male mice with a body weight of (20±2) g were randomly divided into 9 groups after 1 week of adaptive feeding, namely, control group, model group, positive control group, low-, medium- and high-dose groups of lyophilized powder, and low-, medium- and high-dose groups of postbiotics, with 10 mice in each group. The mice in the model group, positive control group, low-, medium- and high-dose groups of lyophilized powder, and low-, medium- and high-dose groups of postbiotics were modeled. The modeled mice were gavaged daily (Escherichia coli CMCC(B)44102, Salmonella paratyphi B CMCC(B)50094, Staphylococcus aureus ATCC6538, Klebsiella pneumoniae ATCC700603, Clostridium difficile ATCC9689, the five strains were mixed at a ratio of 1:1:1:1:1, and the dose was 1×10 7 CFU / kg), and the mice in the control group were intragastrically administered with the same volume of normal saline.

[0083] After modeling, the low, medium and high dose groups of freeze-dried powder and the low, medium and high dose groups of postbiotics were given corresponding different doses, the control group and the model group were given sterile saline, and the positive control group was given commercial products, with an intragastric volume of 0.1mL / 10g, once a day, for 7 consecutive days. The dosage is as follows: Table 7 Dosage

[0084] 12 hours after the last administration, blood was collected from the eyeballs of mice in each group and the serum was separated. Anticoagulant blood was also collected. The mice were killed and the small intestine was removed and frozen in liquid nitrogen for later use.

[0085] Experimental tests and results: Inspection standards: observe the general physiological state of mice (mentality, hair, diet, feces shape, etc.), and the diarrhea condition is evaluated according to the water content of feces. Criteria for successful modeling: mice have rough hair, listlessness, dirty perianal area, and obvious stains on buttocks. Collect fresh feces of mice within 2 hours in EP tubes by laying paper in the cage, weigh them, and bake them at 100℃ for 2 hours until they are completely dry, weigh them again, and calculate the water content of feces.

[0086]

[0087] After mice were gavaged with clindamycin, ampicillin sodium, and streptomycin, they showed rough hair, slow and weak movements, and some mice had moist hair around the anus and some feces, indicating that the model was successfully established. Figure 3 Compared with the control group, # indicates P <0.05, ## indicates P <0.01; compared with the model group, * indicates P <0.05,** indicatesP <0.01. As shown in the figure: after the mouse model was established, the feces water content increased, and the stool was loose ( P <0.01), the model was successfully established; after gavage with lactobacillus tablets, Lactobacillus acidophilus BOAO001 freeze-dried powder and postbiotics, the effect was significant except for the low-dose freeze-dried powder group ( P <0.05), and the effects of other groups were extremely significant ( P <0.01), there was no significant difference between the positive control group and the experimental groups, indicating that the freeze-dried powder of Lactobacillus acidophilus BOAO001 and postbiotics had a significant effect on diarrhea in mice.

[0088] The serum of mice in each group was collected and small intestinal homogenate was prepared. The levels of inflammatory cytokines TNF-α and IL-6 in serum and small intestinal tissue homogenate supernatant were determined respectively. The operation method was carried out according to the instructions of ELISA kit. Mouse catalase (MOP) activity assay kit, mouse tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) enzyme-linked immunosorbent assay kit were purchased from Biolegend. The experimental results of mouse serum MOP, TNF-α and IL-6 content are shown in Figure 2. Figure 4 The experimental results of the content of MOP, TNF-α and IL-6 in the small intestine of mice are shown in Figure 5 shown. Figure 4~Figure 5 Compared with the control group, # indicates P <0.05, ## indicates P <0.01; compared with the model group, * indicates P <0.05,** indicates P <0.01. As shown in the figure: compared with the control group, the levels of catalase, TNF-α and IL-6 in the small intestinal tissue and serum of the model group were significantly increased ( P <0.01). Compared with the model group, the contents of the two groups of freeze-dried powder, high-dose and the three groups of postbiotics, low-dose, medium-dose and high-dose decreased significantly ( P <0.01); there was no significant difference between the positive control group and the freeze-dried powder and postbiotic experimental groups. Only the low-dose freeze-dried powder group showed significant differences in small intestinal TNF-α and serum catalase detection ( P <0.05). It is known that the freeze-dried powder of Lactobacillus acidophilus BOAO001 and postbiotics have a good effect in relieving and treating intestinal diarrhea in mice.

[0089] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Lactobacillus acidophilus ( Lactobacillus acidophilus ) BOAO001, characterized in that, The deposit number of the Lactobacillus acidophilus BOAO001 is CGMCC No.32195.

2. A method for preparing bacteriocin, characterized in that: The method comprises the following steps: fermenting and culturing the Lactobacillus acidophilus BOAO001 according to claim 1, and separating and obtaining bacteriocin from the fermentation and culturing product.

3. The preparation method according to claim 2, characterized in that: The fermentation culture medium is an MRS medium added with 0.4wt%~1.5wt% of plant powder, and the plant powder is selected from any one of aloe powder, tremella powder, sea buckthorn powder, Euryale powder and Poria powder; the fermentation culture temperature is 36~38°C; the fermentation culture time is 14~18h.

4. The bacteriocin prepared by the preparation method according to any one of claims 2 to 3.

5. Use of the Lactobacillus acidophilus BOAO001 according to claim 1 or the bacteriocin according to claim 4 in preparing a product for inhibiting pathogens, characterized in that: The pathogens include Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, Klebsiella pneumoniae and Clostridium difficile.

6. A method for preparing a freeze-dried powder, characterized in that: The method comprises the following steps: fermenting and culturing the Lactobacillus acidophilus BOAO001 according to claim 1, centrifuging the fermentation and culturing product to obtain bacterial mud, mixing the bacterial mud with a protective agent and then freeze-drying to obtain freeze-dried powder of Lactobacillus acidophilus BOAO001.

7. The freeze-dried powder prepared by the preparation method according to claim 6.

8. A method for preparing postbiotics, characterized in that: The method comprises the following steps: fermenting and culturing the Lactobacillus acidophilus BOAO001 described in claim 1, and concentrating and spray-drying after the fermentation and culturing to obtain postbiotics; the fermentation and culturing culture medium comprises: 40-60 g / L glucose, 25-35 g / L sucrose, 8-12 g / L aloe powder, 15-25 g / L yeast extract, 25-35 g / L Euryale powder, and 27-33 g / L Poria powder.

9. The postbiotics prepared by the preparation method according to claim 8.

10. Use of the Lactobacillus acidophilus BOAO001 according to claim 1, the bacteriocin according to claim 4, the lyophilized powder according to claim 6, or the postbiotic according to claim 9 in the preparation of a medicament for improving or treating diarrhea.

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