Postbiotic of weizmannia coagulans, and preparation method therefor and use thereof

Abstract

Provided are a postbiotic of Weizmannia coagulans, and a preparation method therefor, and a use thereof. The postbiotic comprises inactivated bacteria and / or lipoteichoic acid. The postbiotic can enhance the phagocytosis activity of macrophages, the cytokine and NO secretion ability of macrophages, and the expression levels of immune-function-related genes and proteins, and has an effect of inducing human PBMC cells to kill cancer cells.

Description

A postbiotic of weissella coagulans and a preparation method and application thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202411096612.5 filed on August 12, 2024 in China, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the field of microbial technology, in particular to a postbiotic of weissella coagulans and a preparation method and application thereof. BACKGROUND

[0004] In May 2021, the International Scientific Association for Probiotics and Prebiotics (ISAPP) released a consensus statement on postbiotics: postbiotics are preparations of non-living microorganisms and / or their components that are beneficial to the health of the host, including non-living bacterial cells, probiotic metabolites and / or components after probiotic lysis. Among them, the metabolites mainly include exopolysaccharides, short-chain fatty acids, bacteriocins and organic acids, etc.; the cell components mainly include lipoteichoic acid, teichoic acid, peptidoglycan, derived polypeptide and cell surface protein, etc. A large number of researches in recent years have shown that postbiotics have important effects on human health, including regulating local or systemic immune response, regulating intestinal flora and enhancing intestinal epithelial cell barrier function, and regulating systemic metabolic activity, etc.

[0005] In the patent CN115068510A, a method for extracting lactobacillus lipoteichoic acid and its anti-inflammatory activity are disclosed, which has the advantages of fast and efficient extraction method, and it is proved that the lipoteichoic acid of various lactobacillus can reduce the inflammatory response of macrophage RAW264.7 caused by lipopolysaccharide (LPS). At present, there are few researches on postbiotics of weissella coagulans and its related applications. SUMMARY

[0006] OBJECTIVE

[0007] The purpose of the present application is to provide a postbiotic of weissella coagulans and a preparation method and application thereof. The postbiotic of weissella coagulans can enhance the phagocytic activity of macrophages, enhance the secretion capacity of cytokines and NO of macrophages, and enhance the expression level of immune function related genes and proteins, and has immune enhancing function; it can induce the killing effect of human peripheral blood mononuclear cells (PBMC) on cancer cells, and can be used as a drug for treating cancer.

[0008] TECHNICAL SCHEME

[0009] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:

[0010] In one aspect, the present application provides a probiotic of Weizmannia coagulans, which comprises inactivated bacteria and / or lipoteichoic acid.

[0011] Preferably, the inactivation condition of the inactivated bacteria comprises, but is not limited to, high-temperature inactivation at 121℃.

[0012] In one embodiment, the inactivated bacteria comprises heat-inactivated bacterial bodies and / or heat-inactivated spores.

[0013] In one embodiment, the lipoteichoic acid is extracted from Weizmannia coagulans HOM5301 strain.

[0014] The present application also provides a food or health food comprising the probiotic of Weizmannia coagulans as described above.

[0015] The present application also provides a method for preparing lipoteichoic acid of Weizmannia coagulans HOM5301 strain, which comprises the following steps:

[0016] (1) Bacterial body treatment: the Weizmannia coagulans HOM5301 strain is cultured in a liquid medium, the bacterial bodies are collected and dried; the bacterial bodies are resuspended in acetic acid buffer, n-butanol is added and shaken for incubation, the lower aqueous phase is collected by centrifugation, and after concentration, octyl sepharose column purification is performed,

[0017] characterized in that the concentration of the acetic acid buffer is 0.05-0.15M, the pH is 4.5-4.9, the incubation temperature is 35-40℃, the shaking speed is 150-250rpm, and the centrifugation speed is 9000-12000rpm;

[0018] (2) Sample purification: the concentrated aqueous phase liquid is purified by octyl sepharose column, the filtrate is collected after elution using n-propanol-ammonium acetate for washing and elution, and the lipoteichoic acid of Weizmannia coagulans HOM5301 is obtained after dialysis and drying,

[0019] characterized in that the volume of the sample introduced into the octyl sepharose column is 1 / 3-1 / 2 of the column volume, the speed of introducing the sample is 0.5-1.5mL / min, the speed of washing and eluting the sample is 1.5-3mL / min; the concentration of n-propanol in the n-propanol-0.1M ammonium acetate buffer used for washing is 12%-18%, the concentration of n-propanol in the n-propanol-0.1M ammonium acetate buffer used for elution is 30%-40%; and the pore size of the dialysis bag is 500-1000Da.

[0020] In another aspect, the present application also provides the use of the probiotic of Weizmannia coagulans as described above in the preparation of a medicament for enhancing the immune function of macrophages.

[0021] In an embodiment, the postbiotic is used for enhancing phagocytic activity of macrophages.

[0022] Preferably, the concentration of the inactivated bacteria is 5.0 x 10 5 TFU / mL to 5.0 x 10 7 TFU / mL, preferably 5.0 x 10 6 TFU / mL to 5.0 x 10 7 TFU / mL, more preferably 1.0 x 10 7 TFU / mL to 5.0 x 10 7 TFU / mL, most preferably 5.0 x 10 7 TFU / mL; and the concentration of the lipoteichoic acid is 25 μg / mL to 100 μg / mL, 25 μg / mL to 50 μg / mL, or 50 μg / mL to 100 μg / mL, preferably 100 μg / mL.

[0023] In an embodiment, the postbiotic is used for enhancing cytokine and NO secretion capacity of macrophages.

[0024] Preferably, the cytokine is selected from IL-6 and TNF-α.

[0025] In an embodiment, the postbiotic is used for increasing expression level of immune function related genes and proteins in macrophages.

[0026] Preferably, the postbiotic is used for increasing expression level of MyD88 and IL-1β.

[0027] Preferably, the lipoteichoic acid is used for increasing expression level of TNF, CXCL2, TLR2 and CD14.

[0028] In another aspect, the present application also provides use of the postbiotic of the W. coagulans in the preparation of a medicament for treating cancer.

[0029] In an embodiment, the postbiotic is used for inducing killing effect of peripheral blood mononuclear cells (PBMCs) on cancer cells.

[0030] Preferably, the cancer is selected from one or more of intestinal cancer, liver cancer and lung cancer.

[0031] Preferably, the cancer is selected from one or more of intestinal cancer and lung cancer.

[0032] In addition, the present application also provides use of the W. coagulans HOM5301 strain in the preparation of a medicament for treating cancer. The cancer is selected from one or more of intestinal cancer, liver cancer and lung cancer. Preferably, the cancer is selected from one or more of intestinal cancer and lung cancer. Beneficial effects

[0033] (1) The postbiotic of the Weizmannia coagulans of the present application can enhance the phagocytic ability of mouse macrophage RAW264.7; promote the ability of mouse macrophage RAW264.7 to produce NO, TNF-a and IL-6 cytokines; and can enhance the expression level of immune function related genes and proteins in mouse macrophage RAW264.7, thus having immune enhancing function.

[0034] (2) The Weizmannia coagulans and its postbiotic of the present application have the effect of inducing human PBMC cells to kill cancer cells, thus having the effect of treating cancer.

[0035] (5) The postbiotic of the Weizmannia coagulans of the present application has wide application prospects in enhancing immune drugs, food or health food. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 shows the elution curve of octyl sepharose gel column and the corresponding phosphorus content.

[0037] Figure 2 shows the infrared spectrum of the lipoteichoic acid of Staphylococcus aureus.

[0038] Figure 3 shows the infrared spectrum of the lipoteichoic acid of Weizmannia coagulans HOM5301 strain.

[0039] Figure 4 shows the nuclear magnetic resonance hydrogen spectrum of the lipoteichoic acid of Staphylococcus aureus.

[0040] Figure 5 shows the nuclear magnetic resonance hydrogen spectrum of the lipoteichoic acid of Weizmannia coagulans HOM5301 strain.

[0041] Figure 6 shows the mass spectrum (MALDI-TOF, molecular weight <10k) of the lipoteichoic acid of Weizmannia coagulans HOM5301 strain.

[0042] Figure 7 shows the mass spectrum (MALDI-TOF, molecular weight 10k-100k) of the lipoteichoic acid of Weizmannia coagulans HOM5301 strain.

[0043] Figure 8 shows the liquid chromatogram of the lipoteichoic acid of Weizmannia coagulans HOM5301 strain.

[0044] Figure 9 shows the effect of Weizmannia coagulans HOM5301 strain and its postbiotic lipoteichoic acid on the cell viability of mouse macrophage RAW264.7.

[0045] Figure 10 shows the volcano plot of differentially expressed genes of the live bacteria group of Weizmannia coagulans HOM5301 strain compared with the control group, the heat-killed bacteria group compared with the control group, the lipoteichoic acid (LTA) group compared with the control group, and the live bacteria group compared with the heat-killed bacteria group.

[0046] Figure 11 shows the KEGG enrichment analysis bubble chart of differentially expressed genes of the live bacteria group of the Weizmannia coagulans HOM5301 strain compared to the control group, the heat-killed bacteria group compared to the control group, the lipoteichoic acid (LTA) group compared to the control group, and the live bacteria group compared to the heat-killed bacteria group.

[0047] Figure 12 shows the average expression amount of differentially expressed proteins of the Weizmannia coagulans HOM5301 strain and its postbiotic lipoteichoic acid (LTA) on mouse macrophage RAW264.7. Note: *0.01 < p < 0.05, **0.001 < p < 0.01, ***p < 0.001.

[0048] Figure 13 shows the killing rate (%) of Caco-2 cells by human PBMC induced by the Weizmannia coagulans HOM5301 strain and its postbiotic lipoteichoic acid (LTA). Note: *0.01 < p < 0.05, **0.001 < p < 0.01.

[0049] Figure 14 shows the killing rate (%) of HepG2 cells by human PBMC induced by the Weizmannia coagulans HOM5301 strain and its postbiotic lipoteichoic acid (LTA). Note: *0.01 < p < 0.05, **0.001 < p < 0.01.

[0050] Figure 15 shows the killing rate (%) of A549 cells by human PBMC induced by the Weizmannia coagulans HOM5301 strain and its postbiotic lipoteichoic acid (LTA). Note: *0.01 < p < 0.05, **0.001 < p < 0.01. DETAILED DESCRIPTION

[0051] The strain, characteristics and applications are disclosed, and those skilled in the art can refer to the content herein and appropriately improve the process parameters for implementation. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0052] Postbiotics are collectively referred to as the components of probiotic metabolites after processing, including bacterial cells and metabolites. The main components of bacterial cells include lipoteichoic acid, teichoic acid, peptidoglycan, derived polypeptide and cell surface protein, etc. The main metabolites include exopolysaccharide, short-chain fatty acid, bacteriocin and organic acid. Some in vitro and animal experiments have confirmed that the screened postbiotics have better immune enhancing ability than the original live bacteria, and still have high physiological activity even after being treated by high temperature or gastrointestinal juice. It is known that postbiotics have many health benefits for the human body.

[0053] Teichoic acid (TA) is a negatively charged polymer on the surface of gram-positive bacteria. According to its anchoring position, it can be divided into lipoteichoic acid (LTA) anchored on the cell membrane and wall teichoic acid (WTA) rich in phosphate and covalently bound to the cell wall peptidoglycan. Lipoteichoic acid was originally called membrane teichoic acid and belongs to amphiphilic molecules across the peptidoglycan layer of the bacterial surface, which has both hydrophilic (polyphosphoglycerol chain) and hydrophobic (fatty acid chain of glycolipid) properties. As a specific antigen component of gram-positive bacteria, lipoteichoic acid can stimulate the body to produce an immune response and has certain value for vaccine development and immunotherapy. There is evidence that representative components of postbiotics (such as lipoteichoic acid) are the key to determining the acid, alkali and heat resistance of postbiotics.

[0054] Lipoteichoic acid is an amphiphilic molecule with hydrophobic properties, while nucleic acids, most proteins, peptidoglycans, polysaccharides and wall teichoic acids are polar substances without hydrophobicity. Therefore, octyl sepharose hydrophobic interaction chromatography is used, and an organic solvent n-propanol with both amphiphilic and detergent-like properties is used for elution.

[0055] Macrophages are important immune effector cells differentiated from peripheral blood mononuclear cells, and as a component of the body's innate immunity, they play an important role in regulating immunity. Macrophages in the body are generally in a relatively static state and only have certain non-specific phagocytic and chemotactic abilities. Studies have found that stimulation of lipopolysaccharide or other immunomodulators can effectively activate macrophages to differentiate into M1 type macrophages. The typical characteristics of M1 type macrophages are enhanced phagocytic ability, significantly increased secretion of NO and pro-inflammatory factors, and they are mainly involved in pathogen clearance and immune initiation.

[0056] Biological material description

[0057] The detailed information of the Weizmannia coagulans HOM5301 strain used in the present application is described in the granted Chinese patent CN112522134B, which was deposited at the China General Microbiological Culture Collection Center on July 16, 2020, at the address of the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Haidian District, Beijing; the preservation number is CGMCC No. 20383. In addition, other information about the Weizmannia coagulans HOM5301 strain, including isolation, identification, preservation, fermentation, etc., can be referred to the Chinese patent (application date: November 16, 2020, application number: 202011279981.X, invention title: "A strain of Bacillus coagulans and its application") with the granted publication number CN112522134B.

[0058] The Weizmannia coagulans HOM5301 strain of the present application, also known as Bacillus coagulans HOM5301 strain, was sent to the Institute of Microbiology, Chinese Academy of Sciences for identification in June 2023.

[0059] The detection and identification conclusion is as follows:

[0060] Under the conditions of the laboratory, according to the experimental data of cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, idh gene sequence, etc. of the sent strain, referring to the "Berger's Systematic Bacteriology Manual" and the relevant research papers of International Journal of Systematic and Evolutionary Microbiology, the identification result of the sent strain (strain number: HOM5301 strain) is: Weizmannia coagulans (Weizmannia coagulans). Synonym: Bacillus coagulans (Bacillus coagulans).

[0061] The cell morphology of the strain is:

[0062] Rod-shaped, gram-positive, spore-forming, contact enzyme positive (+), and oxidase negative (-); the 16S rRNA gene sequence is shown in SEQ ID NO: 1, and the idh gene sequence is shown in SEQ ID NO: 2.

[0063] In the present application, Weizmannia coagulans and Bacillus coagulans can be used interchangeably.

[0064] The coagulans weizmannii HOM5301 strain and the postbiotic lipoteichoic acid thereof in the application can enhance the phagocytic activity of macrophages, enhance the ability of macrophages to secrete cytokines, induce the killing effect of human PBMC on cancer cells, and have the potential to become an immune enhancer, food or health food.

[0065] In order to further understand the application, the coagulans weizmannii, the postbiotic and the preparation method and application thereof provided by the application are described in detail below in combination with examples, and the protection scope of the application is not limited by the following examples.

[0066] It should be noted that the experimental methods used in the application are conventional methods unless otherwise specified.

[0067] The control strain coagulans weizmannii (Weizmannia coagulans) BC-XX used in the application is isolated from a commercially available product.

[0068] The reagents and materials used in the application are prepared by conventional methods or obtained by commercial means unless otherwise specified.

[0069] Example 1 Preparation of coagulans weizmannii HOM5301 strain and postbiotic thereof

[0070] 1.1 Preparation of live bacterial sample and heat-inactivated bacterial sample of coagulans weizmannii HOM5301 strain:

[0071] The coagulans weizmannii HOM5301 strain was inoculated into a sterilized TSB culture medium at an inoculation amount of 1% of the total amount of the culture medium, and cultured in a shaking bed at 45℃ and 280rpm for 24h, and the culture was activated for 3 generations. The bacterial fermentation broth was centrifuged at 6500rpm for 10min, and washed with sterile physiological saline for 3 times. The bacterial body was resuspended in DPBS to obtain a live bacterial sample, which was stored at 2-8℃ for standby; the live bacterial sample was sterilized at 121℃ for 15min to obtain a heat-inactivated bacterial sample, which was stored at 2-8℃ for standby.

[0072] 1.2 Preparation of lipoteichoic acid (LTA) sample of coagulans weizmannii HOM5301 strain:

[0073] 1.2.1 Sample extraction: The fermentation of Weizmannia coagulans HOM5301 strain was carried out according to the Chinese patent with publication number CN112522134A. The fermentation broth was centrifuged at 6500 rpm for 10 min, and the bacterial cells were collected and freeze-dried. 5 g of freeze-dried bacterial cell powder was added to 0.1 M Tris-HCl buffer (pH = 8) and washed three times. The bacterial cells were resuspended in 20 mL of 0.1 M ammonium acetate buffer (pH = 4.7), and an equal volume of n-butanol was added. The mixture was incubated at 37°C on a shaker at 200 rpm for 1 h. The mixture was centrifuged at 10,000 rpm for 15 min at 4°C, and the middle layer of the water phase (the order of the three layers is n-butanol, water phase, and bacterial cells) was collected. The lower layer of bacterial cells was extracted again. The two water phases were combined and centrifuged at 10,000 rpm for 15 min at 4°C to remove residual bacterial cells and n-butanol. The water phase was filtered using a 0.45 μm filter membrane, and concentrated to 10 mL using nitrogen blowing to obtain a crude LTA extract.

[0074] 1.2.2 Sample purification: An octyl sepharose gel column (column volume 23 mL) was equilibrated with 70 mL of 0.1 M ammonium acetate buffer (pH = 4.7) until the pH of the effluent was 4.7. The crude LTA extract was loaded onto the column at a flow rate of 1 mL / min. After the crude extract was completely loaded into the gel column, 90 mL of 15% n-propanol-0.1 M ammonium acetate buffer (pH = 4.7) was added for elution, followed by 90 mL of 35% n-propanol-0.1 M ammonium acetate buffer (pH = 4.7) for elution at a flow rate of 2 mL / min. A total of 18 fractions of 10 mL each were collected. The phosphorus content in each fraction was determined, and the results are shown in Figure 1. The phosphorus-containing substances in fractions 1-9 were teichoic acids, and the phosphorus-containing substances in fractions 9-18 were LTA. Fractions 11 and 12, which had higher phosphorus content, were combined and concentrated to 10 mL using nitrogen blowing. The concentrated solution was transferred to a 500 Da dialysis bag, and the concentrated solution accounted for 1 / 3 to 1 / 2 of the volume of the dialysis bag. The dialysis bag was sealed and placed in 1 L of deionized water at 4°C in the refrigerator for dialysis. The deionized water was changed every 24 h, and the pH of the deionized water was measured each time. The dialysis was completed when the pH of the deionized water was consistent with that of pure deionized water, and the dialysis lasted for 5 days and 6 nights. The concentrated solution in the dialysis bag was removed and concentrated to a volume of about 3 mL using nitrogen blowing. The concentrated solution was transferred to a 20 mL vial and freeze-dried to obtain LTA freeze-dried powder.

[0075] 1.2.3 Structure identification:

[0076] ① Infrared spectroscopy structure identification: The infrared spectrum of the LTA of Staphylococcus aureus is shown in Figure 2, and the infrared spectrum of the LTA of Weizmannia coagulans HOM5301 strain is shown in Figure 3. The results showed that the LTA of Weizmannia coagulans HOM5301 strain was similar in structure to the LTA of Staphylococcus aureus. As shown in Figure 3, the band at 1202.17 cm-1 vibration of P=0, band 2920.60 cm -1 vibration of OH, band 1654.15 cm -1 vibration of amido, band 1023.57 cm -1 vibration of amino acid, sugar and other residues, the spectrum shows that the sample has glycerophosphate structure, amido, amino acid, sugar residues and other groups, which proves that the sample prepared in Example 1 is LTA.

[0077] ②NMR structure identification: the NMR spectrum of LTA of S. aureus is shown in Figure 4, and the NMR spectrum of LTA of W. coagulans HOM5301 strain is shown in Figure 5. It is found that the main peak position of LTA of W. coagulans HOM5301 strain is close to that of S. aureus, indicating that the structures of the two are similar. As shown in Figure 5, 5.1 ppm represents the vibration of OH, 4.2 ppm represents the vibration of CH, 2.0-2.1 ppm represents the vibration of CH2, 1.5 ppm represents the vibration of CH3, 4.0 ppm represents the vibration of H in sugar residues, and there is no peak at 7.0 ppm. It is shown that the LTA of W. coagulans HOM5301 strain has no nucleic acid contamination, and the sample is relatively pure.

[0078] ③Molecular weight identification: the mass spectrum of LTA of W. coagulans HOM5301 strain is shown in Figure 6 and Figure 7. There is no obvious signal of LTA of W. coagulans HOM5301 strain less than 10 kDa except the solvent response peak, and there is a strong response signal at 10-16 kDa, indicating that the molecular weight of LTA of W. coagulans HOM5301 strain is 10 k-16 kDa.

[0079] ④Purity identification: the purity of LTA of W. coagulans HOM5301 strain was identified by liquid chromatography with a differential refractometer detector. The chromatographic column was Bio Core SEC-300, 300 mm x 7.8 mm, 5 μm; the mobile phase was 0.15 mol / L sodium chloride-0.02 mol / L phosphate buffer solution with pH=7; the flow rate was 0.5 mL / min; the detector temperature was 36℃; the column temperature was 30℃; and the injection volume was 10 μL. As shown in Figure 8, the LTA of W. coagulans HOM5301 strain was monitored by the differential refractometer detector after passing through the size exclusion chromatographic column, and a sharp chromatographic peak was obtained. It is shown that the molecular weight of the extracted LTA of W. coagulans HOM5301 strain is concentrated and the purity is high, which is consistent with the conclusion of molecular weight identification.

[0080] 1.3 Preparation of live and heat-killed samples of W. coagulans BC-XX control strain

[0081] Example 2 In vitro immune-enhancing experiment of W. coagulans HOM5301 strain and its postbiotic

[0082] 2.1 Preparation of cell culture medium

[0083] Complete medium: Dulbecco's modified Eagle's medium (DMEM) with high glucose, supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin), and stored at 2-8 °C after mixing.

[0084] Incomplete medium: DMEM medium with high glucose, supplemented with 10% (v / v) fetal bovine serum (FBS) and stored at 2-8 °C after mixing.

[0085] 2.2 Culture of mouse macrophage RAW264.7 cells

[0086] Mouse macrophage RAW264.7 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were inoculated in complete medium and subcultured at a ratio of 1:15 (v / v). Cells from passages 3-10 were selected for experiments.

[0087] 2.3 Cell viability experiment

[0088] 2.3.1 Sample preparation

[0089] W. coagulans HOM5301 live bacteria group: The concentration of live bacteria of W. coagulans HOM5301 strain prepared in Example 1 was adjusted to 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , and 5 x 10 8 CFU / mL using incomplete medium.

[0090] W. coagulans HOM5301 heat-killed bacteria group: The concentration of heat-killed bacteria of W. coagulans HOM5301 strain prepared in Example 1 was adjusted to 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , and 5 x 10 8 TFU / mL using incomplete medium.

[0091] W. coagulans BC-XX live bacteria group (control group): The concentration of live bacteria of W. coagulans BC-XX strain prepared in Example 1 was adjusted to 1 x 10 7 , 5 x 107 1 x 10 8 8 CFU / mL.

[0092] Heat-killed W. coagulans BC-XX group (control group): the concentration of heat-killed W. coagulans BC-XX strain prepared in Example 1 was adjusted to 1 x 10 7 5 x 10 7 8 CFU / mL and 5 x 10 8 TFU / mL using incomplete medium.

[0093] LTA group: the concentration of LTA of W. coagulans HOM5301 strain prepared in Example 1 was adjusted to 20, 50, 100 and 250 μg / mL using incomplete medium.

[0094] 2.3.2 Cell viability detection

[0095] The cell number was adjusted to 5 x 10 5 CFU / mL with incomplete medium, inoculated in a 96-well culture plate, 0.1 mL per well, and cultured at 37°C in a 5% CO2 incubator for 4 hours until the cells adhered. The cultured 96-well plate cells were taken, the cell culture solution was aspirated, 0.1 mL of different concentrations of sample-containing medium prepared in step 2.3.1 was added to each well, 0.1 mL of incomplete medium was added to each well in the negative control group, each treatment was repeated 3 times, and cultured at 37°C in a 5% CO2 incubator for 24 hours. The cell viability was detected according to the cell proliferation and cytotoxicity detection reagent MTS produced by Promega. 20 μL of MTS reagent was added to each well, and incubated in the incubator for 4 hours in the dark. The absorbance value was read at 490 nm using a microplate reader. The relative viability of the cells was calculated with the negative control group as the control. The detection results are shown in Figure 9. As can be seen from the results of Figure 9, when the concentration of live W. coagulans HOM5301 was not more than 1 x 10 7 CFU / mL, the concentration of heat-killed W. coagulans HOM5301 was not more than 1 x 10 8 TFU / mL, the concentration of live W. coagulans BC-XX was not more than 1 x 10 7 CFU / mL, the concentration of heat-killed W. coagulans BC-XX was not more than 5 x 10 7 TFU / mL, and the concentration of LTA was not more than 100 μg / mL, the viability of macrophage RAW264.7 was higher than 100%.

[0096] 2.4 Macrophage cytokine and NO secretion experiment

[0097] 2.4.1 Sample preparation ​​

[0098] Live Weizmannia congelensis HOM5301 group: The concentration of live bacteria of Weizmannia congelensis HOM5301 strain prepared in Example 1 was adjusted to 1.0 x 10 5 , 5.0 x 10 5 , 1.0 x 10 6 , 5.0 x 10 6 and 1.0 x 10 7 CFU / mL using incomplete medium.

[0099] Heat-inactivated Weizmannia congelensis HOM5301 group: The concentration of heat-inactivated bacteria of Weizmannia congelensis HOM5301 strain prepared in Example 1 was adjusted to 5.0 x 10 5 , 1.0 x 10 6 , 5.0 x 10 6 , 1.0 x 10 7 and 5.0 x 10 7 TFU / mL using incomplete medium.

[0100] Live Weizmannia congelensis BC-XX group (control group): The concentration of live bacteria of Weizmannia congelensis BC-XX strain prepared in Example 1 was adjusted to 1.0 x 10 5 , 5.0 x 10 5 , 1.0 x 10 6 , 5.0 x 10 6 and 1.0 x 10 7 CFU / mL using incomplete medium.

[0101] Heat-inactivated Weizmannia congelensis BC-XX group (control group): The concentration of heat-inactivated bacteria of Weizmannia congelensis BC-XX strain prepared in Example 1 was adjusted to 5.0 x 10 5 , 1.0 x 10 6 , 5.0 x 10 6 , 1.0 x 10 7 and 5.0 x 10 7 TFU / mL using incomplete medium.

[0102] LTA group: The concentration of LTA of Weizmannia congelensis HOM5301 strain prepared in Example 1 was adjusted to 20, 50 and 100 μg / mL using incomplete medium.

[0103] 2.4.2 Macrophage cytokine and NO secretion experiment

[0104] The number of cells was adjusted to 5 x 10 5The RAW264.7 cells were inoculated in a 24-well culture plate at 1 mL per well, and cultured at 37°C in a 5% CO2 incubator for 4 hours until the cells adhered. The cultured 24-well plate cells were taken, and the cell culture solution was removed. Different concentrations of sample-containing medium prepared in step 2.5 were added to each well at 1 mL, the negative control group was added with 1 mL of incomplete medium per well, and the positive control group was added with 1 mL of incomplete medium containing 100 μg / mL of LTA of S. aureus per well. Each treatment was repeated three times, and the cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. After the culture ended, the cell culture solution was taken into a 1.5 mL EP tube and centrifuged at 3500 rpm for 10 minutes. The supernatant was taken into a clean EP tube, and the NO content was determined using the NO detection reagent produced by the Plomegas company. The TNF-α and IL-6 contents were determined using the mouse TNF-α and IL-6 enzyme-linked immunosorbent assay kit produced by the Dako company. The results were analyzed by one-way ANOVA (One Way ANOVA) using SPSS 26, and the post-hoc multiple comparisons were performed using Duncan's Multiple Range Test. The results are shown in Table 1.

[0105] As can be seen from the results in Table 1, the strain of W. coagulans HOM5301 and its postbiotic can significantly (p<0.01) increase the secretion of TNF-α and NO by RAW264.7 cells compared with the negative control group and the positive control group. High concentrations of live bacteria, heat-killed bacteria, and lipoteichoic acid (LTA) can significantly (p<0.01) increase the secretion of IL-6 compared with the negative control group and the positive control group, and the live bacteria group has a stronger ability to stimulate macrophages to secrete IL-6 and is better than the control strain BC-XX. It can be seen that the strain of W. coagulans HOM5301 and its postbiotic activate RAW264.7 cells to differentiate into M1 type macrophages, promote the secretion of NO and cytokines, and thus activate the immune response.

[0106] Table 1: W. coagulans HOM5301 strain and its postbiotic promote the secretion of cytokines and NO by mouse macrophages

[0107] Note: Different letters in the same column indicate significant differences (p<0.05)

[0108] The remaining adherent cells were washed with DPBS for 3 times, 300 μL of 0.075% neutral red solution was added to each well, and then incubated at 37°C for 1 h. After that, the supernatant was discarded, and the cells were washed with DPBS for 3 times. Then, 500 μL of lysis solution (50% ethanol + 1% acetic acid + 49% pure water) was added to each well, and the cells were incubated at room temperature for 20 min until they were fully lysed. After shaking, 100 μL of the lysis solution was transferred to a clean 96-well plate, and 100 μL of blank lysis solution was used as a blank control. The absorbance value was measured at 540 nm. The calculation formula was: phagocytic activity (%) = (A 样品 -A 空白 ) / (A 阴性对照 -A 空白 ) x 100%. The results were analyzed by One Way ANOVA and Duncan's Multiple Range Test using SPSS 26. The results are shown in Table 2. The 100 μg / mL LTA had the strongest ability to activate the phagocytic activity of RAW264.7 cells, which was significantly higher than the negative control group and the positive control group (p < 0.05). The live bacteria of the Weizmannia coagulans HOM5301 strain and the heat-inactivated bacteria also enhanced the phagocytic activity of RAW264.7 cells compared with the negative control group. The phagocytic activity of macrophages stimulated by heat-inactivated bacteria increased with increasing bacterial concentration and was better than BC-ⅩⅩ.

[0109] Table 2 Phagocytic activity of mouse macrophages promoted by Weizmannia coagulans HOM5301 strain and its postbiotic

[0110] Note: Different letters in the same column indicate significant differences (p < 0.05)

[0111] 2.5 Transcriptome and proteome analysis of macrophages

[0112] 2.5.1 Sample preparation

[0113] The live bacteria of the Weizmannia coagulans HOM5301 strain were prepared in Example 1. The concentration of the live bacteria was adjusted to 1.0 x 10 7 CFU / mL using incomplete culture medium.

[0114] The heat-inactivated bacteria of the Weizmannia coagulans HOM5301 strain were prepared in Example 1. The concentration of the heat-inactivated bacteria was adjusted to 1.0 x 10 7 TFU / mL using incomplete culture medium.

[0115] LTA group: The concentration of LTA of the *Weizmannii coagulans* strain HOM5301 prepared in Example 1 was adjusted to 100 μg / mL using incomplete culture medium.

[0116] 2.5.2 Macrophage Transcriptome and Proteome Analysis Experiments

[0117] The cell number was adjusted to 5 × 10⁶ using incomplete culture medium. 5 Cells were seeded at a density of 5 mL / mL into 6-well plates and incubated at 37°C with 5% CO2 for 4 hours until cell adhesion. After incubation, the culture medium was removed from each well, and 5 mL of the different concentrations of culture medium prepared in step 2.7 (containing the sample) was added to each well. For the negative control group, 5 mL of incomplete culture medium was added to each well. Each treatment was repeated in triplicate, and the cells were incubated at 37°C with 5% CO2 for 24 hours. After incubation, cells were scraped off using a cell scraper, and the cell suspension was collected in 50 mL centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, and the culture medium was discarded. The cells were resuspended in DPBS, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. This washing process was repeated three times. The cell pellet was then evenly transferred to two clean 1.5 mL EP tubes, and centrifuged to remove residual DPBS (Duboré phosphate buffer). One tube was used for transcriptome analysis. 1 mL of TRIzol reagent (produced by Tiangen Biotech) was added and mixed thoroughly by pipetting. The tube was then flash-frozen in liquid nitrogen for 0.5 h and stored at -80°C. The other tube was used for proteome analysis. It was flash-frozen in liquid nitrogen for 0.5 h and stored at -80°C. Both tubes were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for transcriptome and proteome analysis.

[0118] 2.5.2.1 Differentially expressed gene analysis

[0119] Differentially expressed genes were analyzed using DESeq2 software with |log2(Fold change)|≥1 and Padjust≤0.05 as the criteria. The volcano plot results are shown in Figure 10. Figure 10 shows that after treatment with live *Weizmannii* strain HOM5301, there were 3022 differentially expressed genes compared to the control group, including 2024 upregulated genes and 998 downregulated genes; compared to the control group, there were 2491 differentially expressed genes in the heat-inactivated group, including 1735 upregulated genes and 756 downregulated genes; compared to the control group, there were 4750 differentially expressed genes in the LTA group, including 2924 upregulated genes and 1826 downregulated genes; and compared to the heat-inactivated group, there were 10 differentially expressed genes in the live group, including 9 upregulated genes and 1 downregulated gene. The number of differentially expressed genes between the live and heat-inactivated strain HOM5301 of Weizmannii coagulation bacteria was relatively small, indicating that the live and heat-inactivated bacteria had similar effects on the transcriptional level of RAW264.7 macrophages.

[0120] 2.5.2.2 KEGG pathway analysis of differentially expressed genes

[0121] KEGG (Kyoto encyclopedia of genes and genomes) enrichment analysis is mainly used to identify the biological pathways enriched in the gene set. KEGG analysis uses the annotation information in the KEGG database to associate the differentially expressed genes or proteins found in the study with known metabolic pathways and functions, further explaining which pathways or biological processes have changed under specific biological conditions. KEGG enrichment analysis was performed using the Python scipy package, Fisher's exact test was used for calculation, and Padjust < 0.05 was used as the threshold, multiple testing was performed using the BH (FDR) method, and the bubble chart of KEGG pathway enrichment analysis of differentially expressed genes is shown in Figure 11. In Figure 11, the vertical axis represents the pathway name, and the horizontal axis represents the ratio of the number of differentially expressed genes annotated to the pathway to the number of genes annotated to the pathway, the larger the enrichment coefficient, the greater the degree of enrichment, and the size of the point represents the number of differentially expressed genes in the pathway. The top 20 most significant pathways were selected for display. The differentially expressed genes in the live bacteria group compared with the control group and the differentially expressed genes in the heat-killed bacteria group compared with the control group were mainly enriched in similar signal pathways. The main enrichment was in the TNF signaling pathway, NF-κB signaling pathway, cytokine and receptor interaction, apoptosis and other pathways, among which the TNF signaling pathway was the most significant. The differentially expressed genes in the LTA group compared with the control group were mainly enriched in the TNF signaling pathway, P53 signaling pathway, NF-κB signaling pathway, among which the TNF signaling pathway was the most significant. These pathways are all directed to intracellular immune-related pathways. It shows that the Weizmannia coagulans HOM5301 strain and its postbiotic mainly act on the immune function direction of macrophages.

[0122] 2.5.2.3 Analysis of differentially expressed proteins among multiple groups

[0123] The R software was used for single factor variance analysis of the protein expression amount related to immune function between groups, and Tukey test was used for post hoc multiple comparisons. The differential proteins included tumor necrosis factor (TNF, involved in inflammatory response, anti-tumor, and immune regulation), chemokine ligand 2 (CXCL2, capable of stimulating immune cells to release inflammatory mediators, regulating immune cell activation, and enhancing immune cell antibacterial ability), Toll-like receptor 2 (TLR2, a pattern recognition receptor, capable of inducing T cell differentiation and playing an anti-pathogen and anti-tumor role after activation), interleukin-1β (IL-1β, regulating immune response and participating in nerve protection and tissue repair), myeloid differentiation factor 88 (MyD88, activating immune cells through Toll-like receptors and inducing NF-κB activation), leukocyte differentiation antigen (CD14, inducing NF-κB activation and cytokine secretion), and the like. The average expression amount of the differential expression proteins is shown in FIG. 12. As can be seen from FIG. 12, the coagulovum Weizmann strain HOM5301 and its postbiotic can significantly increase the expression amount of each protein (p<0.05), indicating that the coagulovum Weizmann strain HOM5301 and its postbiotic can promote the activation of the immune function of macrophages. Compared with the live bacteria group and the heat-killed bacteria group, the LTA group can more strongly increase the expression amount of TNF, CXCL2, TLR2 and CD14. Compared with the control group, the LTA group can also significantly increase the expression amount of MyD88 and IL-1β (p<0.05), indicating that LTA can activate the TLR2 receptor, depend on the MyD88 pathway, start the expression of nuclear-related genes through the NF-κB pathway, enhance the phagocytic ability of macrophages, promote the synthesis and release of NO, TNF-α, IL-6, IL-1β and other cytokines to the extracellular, and participate in the early immune response of the body, thereby enhancing the immune function of the body.

[0124] Example 3 Killing effect of coagulovum Weizmann strain HOM5301 and its postbiotic on cancer cells induced by PBMC

[0125] Human peripheral blood mononuclear cells (PBMCs) mainly include lymphocytes and monocytes, among which T cells account for about 50%-70%, B cells account for about 10%-15%, monocytes account for about 10%-25%, and there are also some NK cells and other cells. The conventional mitogen-induced T cell proliferation experiment and specific antigen-induced T cell proliferation experiment can directly use PBMCs as target cells, and the mixed mononuclear cells can act as antigen presenting cells to promote cell proliferation.

[0126] Inoculate human colon cancer cells Caco-2, human hepatoma cells HepG2, and human lung cancer cells A549 in 96-well culture plates, respectively, and incubate overnight until the cells adhere to the growth. Human PBMCs are effector cells, and Caco-2, HepG2, and A549 cells are target cells. Add PBMCs to each cancer cell well at an effector-to-target ratio (E:T) of 10:1, and add 1x10 7 CFU / mL (E:T = 20:1) of live HOM5301 strain to the live bacteria group, and add 1x10 7 CFU / mL (E:T = 20:1) of heat-killed HOM5301 strain to the heat-killed bacteria group, and add 100 μg / mL of HOM5301 strain lipoteichoic acid to the LTA group. Set up separate target cell and effector cell groups, and set up 3 replicate wells for each group. Continue to incubate for 48 h, then add 20 μL of MTS reagent to each well, and continue to incubate for another 4 h. Detect the absorbance (A) value of each well at 490 nm on a microplate reader, and calculate the killing rate. The calculation formula is: killing rate = [1-(A 实验孔 -A 效应孔 ) / A 靶细胞孔 ]x100%. The obtained results are analyzed by one-way ANOVA using GraphPad prism 9.0, and the groups are compared by Tukey post-test. The results are shown in FIGS. 13, 14, and 15. The results show that 100 μg / mL of HOM5301 strain lipoteichoic acid has the strongest tumor cell killing effect, which is significantly higher than that of the control group and the heat-killed bacteria group (p<0.05). The live bacteria and the heat-killed bacteria also have strong tumor cell killing effects, which are significantly different from those of the control group (p<0.05). The killing effects of the live bacteria and the heat-killed bacteria on tumor cells are similar.

[0127] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

[0128] Although the embodiments of the present application have been disclosed as above, they are not limited to the applications and embodiments listed in the specification, and can be fully applied to various fields suitable for the present application. Those skilled in the art can easily make additional modifications, and therefore, the present application is not limited to specific details and the details shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. A postbiotic of Weizmannia coagulans, characterized in that, The postbiotic comprises inactivated bacteria and / or lipoteichoic acid.

2. The postbiotic of claim 1, wherein, The inactivated bacteria comprise heat-inactivated bacterial bodies and / or heat-inactivated spores.

3. The postbiotic of claim 1 or 2, wherein, The lipoteichoic acid is extracted from the Weizmannia conglobata HOM5301 strain.

4. A food or health food comprising the postbiotic of the Weizmannia conglobata according to any one of claims 1 to 3.

5. A method for preparing lipoteichoic acid of the Weizmannia conglobata HOM5301 strain, the method comprising the following steps: (1) Bacterial body treatment: the Weizmannia conglobata HOM5301 strain is cultured in a liquid medium, the bacterial bodies are collected and dried; the bacterial bodies are resuspended in an acetic acid buffer, n-butanol is added and shaken for incubation, the lower aqueous phase is collected by centrifugation, and after concentration, octyl sepharose gel column purification is performed, characterized in that the concentration of the acetic acid buffer is 0.05-0.15M, the pH is 4.5-4.9, the incubation temperature is 35-40℃, the shaking speed is 150-250rpm, and the centrifugal speed is 9000-12000rpm; (2) Sample purification: the concentrated aqueous phase liquid is purified by octyl sepharose gel column, the filtrate is collected after elution and washing with n-propanol-ammonium acetate, and the lipoteichoic acid of the Weizmannia conglobata HOM5301 is obtained after dialysis and drying, characterized in that the volume of the sample introduced into the octyl sepharose gel column is 1 / 3-1 / 2 of the column volume, the speed of introducing the sample is 0.5-1.5mL / min, the speed of eluting and washing the sample is 1.5-3mL / min; the concentration of n-propanol in the n-propanol-0.1M ammonium acetate buffer used for washing is 12%-18%, and the concentration of n-propanol in the n-propanol-0.1M ammonium acetate buffer used for elution is 30%-40%; the pore size of the dialysis bag is 500-1000Da.

6. Use of the postbiotic of the Weizmannia conglobata according to any one of claims 1 to 3 in the preparation of a medicament for enhancing the immune function of macrophages.

7. Use according to claim 6, characterized in that, The postbiotic is used to enhance the phagocytic activity of macrophages.

8. Use according to claim 6, characterized in that, The postbiotic is used to enhance the cytokine and NO secretion capacity of macrophages, preferably the cytokine is selected from IL-6 or TNF-α.

9. Use according to any one of claims 6 to 8, characterized in that, The postbiotic is used to increase the expression level of immune function-related genes and proteins in macrophages.

10. Use according to claim 9, characterized in that, The postbiotic is used to increase the expression of MyD88 and IL-1β; and / or the lipoteichoic acid is used to increase the expression of TNF, CXCL2, TLR2 and CD14.

11. Use of the postbiotic of the Weizmannia conglobata according to any one of claims 1 to 3 in the preparation of a medicament for treating cancer.

12. Use according to claim 11, characterized in that, The postbiotic is used to induce the killing effect of peripheral blood mononuclear cells (PBMCs) on cancer cells.

13. Use according to claim 11 or 12, characterized in that, The cancer is selected from one or more of intestinal cancer, liver cancer and lung cancer.

14. Use of a Weizmannia conglobata HOM5301 strain in the preparation of a medicament for treating cancer.

15. Use according to claim 14, characterized in that, The cancer is selected from one or more of intestinal cancer, liver cancer and lung cancer.

16. The use according to claim 15, characterized in that, The cancer is selected from one or more of intestinal cancer and lung cancer.

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