Lactobacillus paracasei K56 surface protein, extraction method thereof and application of lactobacillus paracasei K56 surface protein in regulating intestinal flora

Abstract

The invention relates to a lactobacillus paracasei K56 surface protein, an extraction method thereof and an application of the lactobacillus paracasei K56 surface protein in regulating intestinal flora. The extraction method comprises the following steps: (1) mixing the lactobacillus paracasei K56 with an extraction reagent acidic lithium chloride aqueous solution, and incubating; (2) centrifuging the mixture obtained in the step (1), and taking supernate which is a surface protein extracting solution of the lactobacillus paracasei K56; wherein the lactobacillus paracasei K56 is preserved in the German Colletion of Microorganisms and Cell Cultures, the preservation number of the lactobacillus paracasei K56 is DSM 27447, and the lactobacillus paracasei K56 is preserved in the German Microorganism Preservation Center (MCC), the preservation number of the lactobacillus paracasei K56 is DSM 27447, and the preservation number of the lactobacillus paracasei K56 is CGMCC NO. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the strain is CGMCC 15139, and the strain is also preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the strain is CGMCC 15139, and the preservation number of the strain is CGMCC 15139. And the ratio of the mass of the lactobacillus paracasei K56 to the volume of the extraction reagent acidic lithium chloride aqueous solution is 1g: 3mL. The method is high in surface protein extraction rate.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and food, and in particular to a Lactobacillus paracasei K56 surface protein, an extraction method thereof, and an application thereof in regulating intestinal flora. Background Art

[0002] Probiotics usually need to pass through the human digestive tract to enter the intestine and colonize before they can exert their corresponding probiotic effects. The pH of gastric juice in the human digestive tract is usually around 3.0, and the pH in the small intestine is about 7.6. At the same time, the bile salt concentration in the normal human small intestine is 0.3-3g / L. The bacterial strain not only needs to pass through the complex digestive tract environment of the human body, but also needs to be able to adhere to the digestive tract. Therefore, the bacterial strain needs to have a strong tolerance to the acid, alkali, and bile salts in the human digestive tract, and it also needs to have adhesion to the human digestive tract, especially the intestine, in order to exert its probiotic effects. Previous studies have shown that probiotics can use their surface proteins as mediating factors to adhere to and colonize intestinal cells, thereby achieving the characteristics of exerting a probiotic effect in the body.

[0003] Therefore, this study extracted and studied the surface proteins of Lactobacillus paracasei K56, which has far-reaching significance for explaining the mechanism of adhesion of Lactobacillus paracasei K56 and for explaining the probiotic properties of probiotics. Summary of the Invention

[0004] The Lacticaseibacillus paracasei K56 strain was deposited at the German Collection of Microorganisms and Cell Cultures on June 27, 2013, with the accession number DSM 27447. Furthermore, L. paracasei K56 was also deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 29, 2017, with the accession number CGMCC 15139.

[0005] After exploration and research, the inventors of the present application provide a method for extracting surface protein of Lactobacillus paracasei K56, which has a high extraction rate of surface protein.

[0006] In addition, after further research on the extracted surface protein, the inventors of the present application found that the surface protein can increase the abundance of beneficial intestinal bacteria norank_f_Ruminococcaceae, Lactobacillus and / or Coriobacteriaceae_UCG-002, especially the abundance of beneficial intestinal bacteria norank_f_Ruminococcaceae and / or Coriobacteriaceae_UCG-002, and has high application value.

[0007] To this end, in a first aspect of the present invention, the present invention provides a method for extracting surface protein of Lactobacillus paracasei K56, comprising:

[0008] (1) Lactobacillus paracasei K56 was mixed with an acidic lithium chloride aqueous solution as an extraction reagent and incubated;

[0009] (2) centrifuging the mixture obtained in step (1) and taking the supernatant, wherein the supernatant is a surface protein extract of Lactobacillus paracasei K56;

[0010] Wherein, the Lactobacillus paracasei K56 is deposited in the German Collection of Microorganisms and Cell Cultures with a deposit number of DSM 27447; it is also deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China National Committee for the Administration of Microbiological Culture Collection with a deposit number of CGMCC 15139;

[0011] The ratio of the mass of the Lactobacillus paracasei K56 to the volume of the extraction reagent acidic lithium chloride aqueous solution is 1g:(1-5mL), for example, 1g:1mL, 1g:2mL, 1g:3mL, 1g:4mL, 1g:5mL, preferably 1g:3mL.

[0012] In some embodiments, the concentration of lithium chloride in the acidic lithium chloride aqueous solution is 1-10 mol / L, preferably 4-6 mol / L, and more preferably 5 mol / L.

[0013] In some embodiments, the pH of the acidic lithium chloride aqueous solution is 1.0-3.0, preferably 2.0.

[0014] In some embodiments, the incubation time is at least 10 min, preferably 10-40 min, more preferably 30-40 min, and most preferably 30 min.

[0015] In some embodiments, the incubation is performed in an ice water bath.

[0016] In a second aspect of the present invention, the present invention provides an isolated surface protein of Lactobacillus paracasei K56.

[0017] In some embodiments, the Lactobacillus paracasei K56 surface protein comprises an LPXTG motif protein and a moonlighting protein.

[0018] In some embodiments, the LPXTG motif protein comprises KONBH9 (GenBank: CCK24055.1), KONA48 (GenBank: CCK23225.1), or KONAB9 (GenBank: CCK23405.1).

[0019] In some embodiments, the moonlighting proteins comprise A0A0K1KXQ2 (Gene ID: 57089615), K0N7K7 (GenBank: CCK23322.1), KOMU67 (GenBank: CCK22051.1), A0A0K1KYZ6 (Gene ID: 57090004), and K0N700 (GenBank: CCK23323.1).

[0020] In some embodiments, the surface protein of Lactobacillus paracasei K56 is as shown in Table A.

[0021] In some embodiments, the Lactobacillus paracasei K56 surface protein is obtained by any method of the first aspect.

[0022] In a third aspect, the present invention provides a composition comprising the surface protein of Lactobacillus paracasei K56 obtained by any method of the first aspect or the surface protein of Lactobacillus paracasei K56 of the second aspect.

[0023] In some embodiments, the composition is a pharmaceutical composition, a food (regular food or health food), a food additive, a dietary supplement, or a feed.

[0024] In some embodiments, the composition is an oral dosage form. Preferably, the oral dosage form is selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, lozenges, tablets, chewing gums, and capsules.

[0025] In a fourth aspect of the present invention, the present invention provides a use of the surface protein of Lactobacillus paracasei K56 obtained by any method of the first aspect or the surface protein of Lactobacillus paracasei K56 of the second aspect in preparing a composition, wherein the composition has one or more uses selected from the following (1)-(2):

[0026] (1) Used to promote the proliferation of beneficial intestinal bacteria, wherein the beneficial intestinal bacteria are selected from one or more of norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002, preferably one or more of norank_f_Ruminococcaceae and Coriobacteriaceae_UCG-002;

[0027] (2) It helps regulate intestinal flora, wherein regulating intestinal flora is to promote the proliferation of beneficial intestinal bacteria, wherein the beneficial intestinal bacteria are selected from one or more of norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002, preferably one or more of norank_f_Ruminococcaceae and Coriobacteriaceae_UCG-002;

[0028] Preferably, the composition is a pharmaceutical composition, a food (regular food or health food), a food additive, a dietary supplement or a feed;

[0029] Preferably, the composition is in an oral dosage form, preferably, the oral dosage form is selected from the group consisting of solution, suspension, emulsion, powder, lozenge, pill, syrup, buccal lozenge, tablet, chewing gum, and capsule.

[0030] Beneficial effects

[0031] 1. The present invention provides a surface protein of Lactobacillus paracasei K56, an extraction method thereof, and an application thereof in regulating intestinal flora. The method has a high extraction rate of the surface protein.

[0032] 2, in the extracting method of Lactobacillus paracasei K56 surface protein proposed by the present invention, different bacterium liquid ratios (m:v, unit g / mL) have obvious impact on surface protein extraction rate, and do not have obvious rule.The inventor is surprised to find that, when bacterium liquid ratio is controlled at certain numerical value (such as 1:3), surface protein concentration is significantly higher than other scopes or other numerical values ​​(such as 4:1,3:1,2:1,1:1,1:4,1:5,1:6,1:7 etc.), and extraction rate is significantly more excellent.

[0033] 3. The present invention discovered the hydrophobicity and self-aggregation properties of Lactobacillus paracasei K56.

[0034] 4. The present invention discovered a strain of Lactobacillus paracasei K56 that has strong adhesion to collagen and bovine serum albumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : The surface protein concentration of Lactobacillus paracasei K56 under different treatment conditions in Example 1 of the present invention, wherein different lowercase letters represent significant differences (P<0.05) between the surface protein concentrations of Lactobacillus paracasei K56 under different treatment conditions.

[0036] Figure 2 This is the SDS-PAGE analysis gel electrophoresis diagram of the surface protein extracted using the acidic 5 mol / L LiCl method in Example 2 of the present invention, wherein M: protein marker; 1: surface protein of Lactobacillus paracasei K56.

[0037] Figure 3 This is the surface protein elution profile of Lactobacillus paracasei K56 in Example 2 of the present invention.

[0038] Figure 4 This is a gel electrophoresis diagram of SDS-PAGE analysis of the surface protein of Lactobacillus paracasei K56 after separation and purification in Example 2 of the present invention, wherein M: protein marker; 1: surface protein of Lactobacillus paracasei K56 treated with acidic 5M LiCl after elution.

[0039] Figure 5 The hydrophobicity of live, deproteinized, and killed Lactobacillus paracasei K56 bacteria in Example 3 of the present invention is shown in Table 1. Capital letters indicate significant differences between strains in the same group, while lowercase letters indicate significant differences between groups of the same strain (P < 0.05). LAL: live Lactobacillus paracasei K56 bacteria; LA: deproteinized Lactobacillus paracasei K56 bacteria; DLA: killed Lactobacillus paracasei K56 bacteria; 431: commercial strain 1; LGG: commercial strain 2.

[0040] Figure 6 This is the self-aggregation test of live, deproteinized, and killed Lactobacillus paracasei K56 cells from Example 4 of the present invention. Capital letters indicate significant differences between strains in the same group, and lowercase letters indicate significant differences between groups of the same strain (P < 0.05). LAL: live Lactobacillus paracasei K56 cells; LA: deproteinized Lactobacillus paracasei K56 cells; DLA: killed Lactobacillus paracasei K56 cells; 431: commercial strain 1; LGG: commercial strain 2.

[0041] Figure 7The adhesion rates of Lactobacillus paracasei K56 and commercial strain 3 to collagen, mucin, and BSA in Example 5 of the present invention are shown in Table 1. Capital letters indicate significant differences between strains in the same group, while lowercase letters indicate significant differences between groups of the same strain (P < 0.05). K56: Lactobacillus paracasei K56; SLT: commercial strain 3.

[0042] Figure 8 This is the effect of Lactobacillus paracasei K56 and its surface protein on the OTU species of the mouse intestinal flora in Example 6 of the present invention.

[0043] Figure 9 This is the Alpha diversity analysis of intestinal flora species of Lactobacillus paracasei K56 and surface protein treated mice in Example 6 of the present invention;

[0044] Figure 10 This is the species dilution curve of the intestinal flora of mice in the Lactobacillus paracasei K56 and surface protein treatment groups in Example 6 of the present invention;

[0045] Figure 11 This is the Beta diversity analysis of the intestinal flora of mice in the Lactobacillus paracasei K56 and surface protein treatment groups in Example 6 of the present invention;

[0046] Figure 12 This is a composition analysis of the intestinal flora of the mice in the Lactobacillus paracasei K56 and surface protein treatment groups at the phylum level in Example 6 of the present invention;

[0047] Figure 13 This is a composition analysis of the intestinal flora of mice in the Lactobacillus paracasei K56 and surface protein treatment groups at the genus level in Example 6 of the present invention;

[0048] Figure 14 The changes in beneficial bacteria at the genus level in the intestinal microorganisms of mice in the Lactobacillus paracasei K56 and surface protein treatment groups in Example 6 of the present invention;

[0049] Figure 15 This is the change of harmful bacteria at the genus level in the intestinal microorganisms of mice in the Lactobacillus paracasei K56 and surface protein treatment groups in Example 6 of the present invention. DETAILED DESCRIPTION

[0050] Below in conjunction with embodiment and accompanying drawing, embodiment of the present invention is described in detail, but those skilled in the art will understand that following embodiment and accompanying drawing are only used for illustrating the present invention, and are not limiting the scope of the present invention. According to the following detailed description of accompanying drawing and preferred embodiment, various purposes and advantages of the present invention will become apparent to those skilled in the art. Unless otherwise specified, each raw material and reagent all can be purchased commercially.

[0051] The Lactobacillus paracasei K56 used in the present invention was provided by Inner Mongolia Yili Industrial Group Co., Ltd., and the high-fat mice were provided by Nanjing Bioray Pharmaceutical Research Institute Co., Ltd.; various mouse ELISA kits were provided by Shanghai Zhuocai Biotechnology Co., Ltd.; and various enzyme activity assay kits were provided by Beijing Solebold Technology Co., Ltd.

[0052] The microscope, automatic biological tissue dehydrator, and slicer were provided by Leica Microsystems (Shanghai) Trading Co., Ltd.; the biological tissue freezing embedding machine, embedding machine freezing table, biological tissue spreader, and biological tissue baking machine were provided by Jinhua Yidi Medical Equipment Co., Ltd.

[0053] In the following Examples 1 and 2, the raw material, i.e., Lactobacillus paracasei K56 cells, can be obtained by the following method: activated Lactobacillus paracasei is inoculated into MRS broth at a ratio of 1% (v / v), cultured at 37° C. for 16 h, centrifuged at 3520×g and 4° C. for 10 min, the supernatant is discarded, and the cell pellet is collected.

[0054] Example 1: Effect of different extraction methods on the extraction rate of surface protein of Lactobacillus paracasei K56

[0055] 1. Effects of different reagents on surface protein extraction rate

[0056] Take 3.0g of bacteria and add different extraction reagents at a ratio of 1:4 (g / mL), namely acidic lithium chloride solution, guanidine hydrochloride solution, and lysozyme. The extraction methods are as follows:

[0057] (1) 5 mol / L acidic lithium chloride solution (pH = 2.0):

[0058] a. Prepare a 5 mol / L acidic lithium chloride solution (pH 2.0): Weigh 21.195 g of lithium chloride and dissolve it in 50 mL of sterile water. Adjust the pH to 2.0 with 0.1 mol / L hydrochloric acid solution and dilute to 100 mL with sterile water.

[0059] b. Extraction method: Take 3.0 g of Lactobacillus paracasei K56 cells, add 12 mL of 5 mol / L acidic lithium chloride solution (pH = 2.0), incubate in an ice water bath for 15 min, centrifuge (10,000 × g, 20 min) and collect the supernatant, which contains the surface protein.

[0060] (2) 4 mol / L guanidine hydrochloride solution:

[0061] a. Prepare 4 mol / L guanidine hydrochloride solution: Dissolve 38.212 g lithium chloride in 50 mL sterile water and dilute to 100 mL.

[0062] b. Extraction method: Take 3.0 g of Lactobacillus paracasei K56 cells, add 12 mL of 4 mol / L guanidine hydrochloride solution, incubate in an ice water bath for 15 min, centrifuge (10,000 × g, 20 min) and collect the supernatant, which contains the surface protein.

[0063] (3) Lysozyme: 15 mL of sterilized PBS (0.1 mol / L, pH = 7.4) was used to resuspend Lactobacillus paracasei K56 (1 mg / mL) cells, followed by the addition of 15 mg of lysozyme. The cells were incubated at 37°C for 1 h. The cell pellet was collected by centrifugation and then extracted with the previously prepared 5 mol / L acidic lithium chloride solution. The cell pellet and the extraction reagent, 5 mol / L acidic lithium chloride solution, were incubated in an ice-water bath for 15 min and centrifuged (10,000 × g, 20 min) to collect the supernatant.

[0064] The protein content in the supernatant collected by the above three extraction reagents was tested (for the test method, see GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food"). Figure 1 -As shown in A.

[0065] Depend on Figure 1 As can be seen from Figure 1, when different extraction reagents were used to extract surface proteins from Lactobacillus paracasei K56, the protein content extracted by lysozyme was significantly higher than that extracted using acidic lithium chloride solution and guanidine hydrochloride solution, reaching 14.45 μg / mL. This is because lysozyme extracts the entire protein of the bacteria, while acidic lithium chloride and guanidine hydrochloride extract the surface proteins of the bacteria. Therefore, the protein content extracted by lysozyme is higher.

[0066] In summary, by comparing the extraction rates of acidic lithium chloride and guanidine hydrochloride, acidic lithium chloride was selected as the best extraction reagent and subsequent experiments were continued.

[0067] 2. Effect of different bacterial liquid ratios on surface protein extraction rate

[0068] Surface proteins were extracted by adding 5 mol / L, pH 2.0, acidic lithium chloride solution (3.0 g) to different bacterial cell-to-liquid ratios (6 mL, 9 mL, and 12 mL, respectively). The cells were incubated with the acidic lithium chloride solution in an ice-water bath for 15 minutes, and the supernatant was collected by centrifugation (10,000 × g, 20 minutes).

[0069] The surface protein content in the supernatant collected at different bacterial liquid ratios was tested separately using the same method as above. Figure 1 -B.

[0070] Depend on Figure 1-B shows that by comparing the effects of different added amounts of acidic lithium chloride solution on the surface protein extraction efficiency, it was found that when the bacterial liquid ratio was 1:3 (m:v), the surface protein concentration was the highest and significantly higher than 1:4. Therefore, a bacterial liquid ratio of 1:3 was preferred as the optimal ratio for further experiments.

[0071] 3. Effect of different incubation times on surface protein extraction rate

[0072] Surface proteins were extracted by adding 5 mol / L, pH 2.0, acidic lithium chloride solution to 3.0 g of bacterial cells at a 1:3 (m:v) bacterial-liquid ratio. The cells were incubated in an ice-water bath with the acidic lithium chloride solution for 10, 20, 30, and 40 minutes, respectively. The supernatant was collected by centrifugation (10,000 × g, 20 minutes).

[0073] The surface protein content in the supernatant collected at different incubation times was detected using the same method as above. Figure 1 -C shown.

[0074] Depend on Figure 1 -C shows that when the incubation time is 30min and 40min, the extracted surface protein concentration is significantly higher than that of 10min and 20min, while there is no significant difference between 30min and 40min. Therefore, the 30min treatment time is the optimal time for surface protein extraction.

[0075] In summary, the optimal extraction conditions for the surface protein of Lactobacillus paracasei K56 are: 5 mol / L, pH = 2.0 acidic lithium chloride solution, bacteria: acidic lithium chloride = 1:3 (m:v), and treatment in an ice water bath for 30 min.

[0076] Example 2: Determination of surface protein of Lactobacillus paracasei K56

[0077] 1. Identification of surface proteins by SDS-PAGE gel electrophoresis

[0078] The surface protein of Lactobacillus paracasei K56 was extracted using the optimal method in Example 1. The collected supernatant was concentrated and then identified by SDS-PAGE. A 5% stacking gel and a 10% separation gel were prepared. The sample was added to a 5× protein loading buffer and boiled at 100°C for 3-5 minutes before spotting. The sample was stained with Coomassie brilliant blue or silver staining, and the electrophoresis pattern was analyzed using a gel imager. The results are shown in Figure 2. Figure 2 shown.

[0079] Depend on Figure 2 It can be found that the surface protein band of Lactobacillus paracasei K56 is higher in the range of 25-35 kDa, and the band at 45-60 kDa is lighter.

[0080] 2. Surface protein gel filtration chromatography

[0081] The following operations were performed on the surface protein of Lactobacillus paracasei K56 extracted by the optimal method in Example 1. Sephadex G-75 gel that had been swollen with boiling water was filled into the chromatography column according to the conventional column packing method, and 0.025 mol / LTris-HCl buffer (pH = 9.5) was prepared to balance the chromatography column. After the baseline was stable, the sample was loaded and eluted with 0.025 mol / LTris-HCl buffer (pH = 9.5) at an elution rate of 1 mL / min and a collection time of 1.5 min / tube. The ultraviolet detection wavelength was 280 nm, and the elution peak was collected and freeze-dried before SDS-PAGE analysis. The results are shown in FIG. Figure 3 and Figure 4 shown.

[0082] Depend on Figure 3 It can be seen that the elution spectrum shows an absorption peak with an absorbance value of about 0.28 at about 40 minutes, and an absorption peak with an absorbance value of about 0.2 at 210 minutes. The surface protein at peak A was then collected and freeze-dried, and re-dissolved to a concentration of 182.12 μg / mL. SDS-PAGE ( Figure 4 ) The molecular weight is concentrated between 25-35kDa.

[0083] 3. Mass spectrometry identification of surface proteins of Lactobacillus paracasei K56

[0084] Surface protein is secreted into the extracellular or is attached to cell surface after intracellular synthesis, performs various functions, as maintaining cell morphology, adhesion and regulating immunity, and surface protein mainly comprises S-layer protein, LPXTG motif protein and part-time protein etc.The present inventor, by the protein spectrum identification (Table A) to Lactobacillus paracasei K56 surface protein, finds that Lactobacillus paracasei K56 does not contain S-layer protein, mainly comprises LPXTG motif protein K0NBH9 (170kDa), KONA48 (30kDa), K0NAB9 (46kDa), and part-time protein A0A0K1KXQ2 (37kDa), K0N7K7 (58kDa), KOMU67 (47kDa), A0A0K1KYZ6 (44kDa), K0N700 (10kDa) etc.

[0085] Table A Surface protein spectrum identification results of Lactobacillus paracasei K56

[0086]

[0087] Example 3: Determination of surface hydrophobicity of different strains

[0088] First, resuspensions of live Lactobacillus paracasei K56, surface protein-depleted Lactobacillus paracasei K56, dead Lactobacillus paracasei K56 (i.e., inactivated bacteria) and other two commercial bacterial strains were prepared respectively.

[0089] (1) Live Lactobacillus paracasei

[0090] The activated Lactobacillus paracasei K56 or two other commercial strains were inoculated into MRS broth at a ratio of 1% (v / v) and cultured at 37°C for 16 hours. The cells were centrifuged at 3520×g and 4°C for 10 minutes, the supernatant was discarded, and the bacterial pellet was collected. The cells were washed with sterilized 0.9% saline solution, and the concentration of the bacterial solution was adjusted to 10% with 0.9% sterile saline solution. 8 CFU / mL is reserved.

[0091] (2) Heat-killed Lactobacillus paracasei K56

[0092] The activated Lactobacillus paracasei was inoculated into MRS broth at a ratio of 1% (v / v) and cultured at 37°C for 16 h. The bacterial precipitate was collected by centrifugation (3520×g, 10 min, 4°C), washed twice with sterile PBS, and resuspended in 0.9% sterile saline to adjust the bacterial solution concentration to 10 8 The suspension was sterilized by high pressure steam at 121°C for 15 min.

[0093] (3) Lactobacillus paracasei K56 surface protein-removing bacteria

[0094] The activated Lactobacillus paracasei was inoculated into MRS broth at a ratio of 1% (v / v) and cultured at 37°C for 16h. The cells were centrifuged at 3520 × g for 10min at 4°C, and the supernatant was discarded to collect the bacterial precipitate. 9mL of 5mol / L, pH 2.0 acidic lithium chloride solution was added to every 3.0g of cells. The cells and the acidic lithium chloride solution were incubated in an ice-water bath for 30min, centrifuged at 10000 × g for 20min, and the bacterial precipitate was collected. The cells were washed with sterilized 0.9% saline solution, and the concentration of the bacterial solution was adjusted to 10% with 0.9% sterile saline after three repetitions. 8 CFU / mL is reserved.

[0095] Then, the OD of each resuspended bacterial suspension was measured. 600 Then, 1 mL of xylene (non-polar solvent), ethyl acetate (electron donor), and chloroform (electron acceptor) were mixed with 3 mL of bacterial suspension, placed at room temperature for 10 minutes, mixed again, and allowed to stand for 20 minutes to measure the OD value of the aqueous phase. 600The hydrophobicity of live Lactobacillus paracasei K56, protein-depleted Lactobacillus paracasei K56, dead Lactobacillus paracasei K56 and two other commercial strains was calculated using the following value:

[0096]

[0097] The results are as follows Figure 5 As shown in the results, it can be seen that the hydrophobicity of the live bacteria of Lactobacillus paracasei K56 is significantly higher than that of the commercial strain 2.

[0098] Example 4: Determination of self-aggregation of different strains

[0099] Resuspended solutions of live Lactobacillus paracasei K56, Lactobacillus paracasei K56 surface protein-free bacteria, dead Lactobacillus paracasei K56 and two other commercial strains were prepared (the specific method is as in Example 3), and the OD of the resuspended bacterial suspension was measured. 600 Then add 4 mL of bacterial suspension to each test tube and let it stand at room temperature for 1, 2, and 3 hours respectively. Then take 1 mL of the upper solution and measure its OD 600 The values ​​were repeated three times, and the average value was recorded as A2. The self-agglutination ability of live Lactobacillus paracasei K56, Lactobacillus paracasei K56 with surface protein removed, dead Lactobacillus paracasei K56 and other two commercial strains was calculated:

[0100]

[0101] The results are as follows Figure 6 The results show that the self-aggregation of K56 increases with time, reaching 28.47% after 5 hours, which is significantly higher than that of commercial strain 1.

[0102] Example 5: Determination of the adhesion ability of Lactobacillus paracasei K56 to mucin and collagen

[0103] Under sterile conditions, the adhesion of Lactobacillus paracasei K56 and commercial strain 3 to mucin and collagen was determined in 12-well polystyrene plates. A 5 mg / mL concentration of bovine serum albumin, mucin, and collagen was prepared in sterile PBS (0.1 mol / L, pH = 7.4). 2 ml of the solution was added to the 12-well plate and allowed to fix overnight at 4°C. The wells were washed twice with sterile PBS to remove unbound matrix. The bacterial concentration was adjusted to 10 6cfu / mL, and perform viable bacteria count. After adding 2mL of bacterial solution, incubate at 37℃ for 2h, and wash the wells 5 times with sterile PBS to remove unbound bacteria. Add 1mL of 0.05% (v / v) Triton X-100 and treat at 37℃ for 20min to release the adhered Lactobacillus paracasei K56 and commercial strain 3, and then wash once with 1mL of sterile PBS. Use according to GB 4789.35-2016 to count viable bacteria. The adhesion rate calculation formula is as follows. 0.05% (v / v) Triton X-100 treatment of Lactobacillus paracasei K56 at 37℃ for 30 minutes does not affect its viability.

[0104] Adhesion rate

[0105] Where: N1 represents the number of viable bacteria adhering to the substrate; N0 represents the number of viable bacteria in the bacterial solution before adhesion.

[0106] The results are as follows Figure 7 The results show that Lactobacillus paracasei K56 and commercial strain 3 have adhesion to mucin, collagen, and bovine serum albumin, and K56 has a stronger adhesion ability.

[0107] Example 6: Lactobacillus paracasei K56 surface protein regulates intestinal flora imbalance caused by high-fat diet

[0108] 1. Establishment of high-fat diet model

[0109] C57BL / 6 male mice, 6 weeks old, weighing 15-20g. The temperature of the animal laboratory was 22±2℃, the humidity was 40-60%, and the light-dark cycle was 12h (lighting time 8:00A.M~8:00P.M). The mice were free to drink water and eat SPF bar feed, and were pre-fed for one week to adapt to the environment. All mice were divided into two groups, a normal group (15 mice) and a model group (65 mice). The normal group was fed with normal feed, and the model group was fed with high-fat feed. The mice were kept in separate cages, 5 mice per cage, and the weight of the mice was measured once a week. After 9 weeks of feeding, 10 normal mice (blank group (Con)) and 60 successfully modeled high-fat mice were taken.

[0110] Sixty high-fat mice were randomly divided into six groups (n=10): a model group (Mod), a live Lactobacillus paracasei K56 group (LAL), a Lactobacillus paracasei K56 surface protein group (SLA), a Lactobacillus paracasei K56 surface protein-deficient group (LA), a killed Lactobacillus paracasei K56 group (DLA), and a drug-positive control group (ORT). The blank group continued to be fed a normal diet, while the remaining groups continued to be fed a high-fat diet. After six weeks of intervention, intestinal microbiota was assessed in each group. The specific gavage schedule for each group is shown in Table 1. Daily before treatment, the mice's growth status was recorded, and observations were made for mortality, mental status, hair gloss, and activity. Body weight was measured once a week at a fixed time before gavage.

[0111] Table 1 Grouping of mice and daily gavage

[0112]

[0113]

[0114] The preparation methods of the resuspensions of live Lactobacillus paracasei K56 in the LAL group, protein-depleted Lactobacillus paracasei K56 in the LA group, and inactivated Lactobacillus paracasei K56 in the DLA group are shown in Example 3.

[0115] The preparation method of the surface protein of the SLA group is the optimal method for extracting the surface protein of Lactobacillus paracasei K56 in Example 1, and the specific method is as follows:

[0116] The activated Lactobacillus paracasei was inoculated into MRS broth at a 1% (v / v) ratio and cultured at 37°C for 16 hours. The mixture was centrifuged at 3520×g for 10 minutes at 4°C, the supernatant discarded, and the bacterial precipitate collected. A 5 mol / L, pH 2.0 acidic lithium chloride solution was added at a ratio of 9 mL per 3.0 g of bacterial cells. The cells and the acidic lithium chloride solution were incubated in an ice-water bath for 30 minutes, centrifuged at 10,000×g for 20 minutes, and the supernatant collected. The supernatant contained the surface protein.

[0117] 2. Specific experimental plan

[0118] Total bacterial DNA was extracted from mouse intestinal contents according to the QIAamp DNA Stool Mini Kit instructions. DNA was quantified using Nanodrop, and the quality of the extracted DNA was verified by 1.2% agarose gel electrophoresis. Primers were designed to amplify conserved regions of bacterial 16S rDNA using polymerase chain reaction (PCR), and sample-specific barcode sequences were added.

[0119] PCR conditions were: 95°C, 3 min; 24 cycles of 95°C, 5 s, 57°C, 90 s, 72°C, 10 s; and 72°C, 5 min. A 20 μL reaction system included: 2.5 μL TransStart buffer, 2 μL dNTPs, 1 μL forward primer, 1 μL reverse primer, 0.5 μL Pfu polymerase (all-gold), and 20 ng template DNA. The PCR amplification products were quantified using the Quant-iTPicoGreen dsDNA Assay Kit using a Microplate reader (BioTek, FLx800). Based on the fluorescence quantification results and the sequencing requirements for each sample, the samples were mixed in the appropriate proportions.

[0120] Sequencing libraries were then prepared using the TruSeq Nano DNA LT Library Prep Kit (Illumina). The positive and negative reads obtained from the double-end sequencing were first spliced ​​in pairs. Sequences containing N in the spliced ​​results were filtered, and sequences greater than 200 bp were retained. After quality filtering and purification of chimeric sequences, the resulting sequences were clustered into operational taxonomic units (OTUs) at 97% sequence homology using the QIIME2 dada2 analysis pipeline. These OTUs were aligned with the Greengenes database using PyNAST software and taxonomic information was annotated. α-diversity and β-diversity were analyzed using QIIME2 software (2019.4) and R software (V3.4.1). Principal coordinates analysis (PCoA) was performed on each group of samples using the Jaccard method, and distance box plots of the samples were drawn. Using the Galaxy online analysis platform, the LEfSe analysis method simultaneously performed differential analysis on all classification levels, drew LDA value distribution bar charts and species taxonomy branch diagrams of significantly different species, and searched for robust differential species between groups.

[0121] 2.1. Mouse intestinal flora species out

[0122] The Venn diagram illustrates the effects of Lactobacillus paracasei K56 and its postbiotics on the OTUs contained in high-fat diet-induced obese mice, indicating the similarity of the intestinal flora contained in different samples. OTU clustering was performed on non-repeated sequences (excluding single sequences) at 97% similarity, and chimeras were removed during the clustering process to obtain representative sequences of OTUs. Figure 8As shown, a Venn diagram illustrates the relationship between the microbial composition diversity of the control, model, orlistat, LAL, SLA, DLA, and LA groups. A total of 1152 OTUs were identified, including 483 unique OTUs in the control group, 103 of which were unique; 96 unique OTUs in the model group; 105 unique OTUs in the orlistat group; 171 unique OTUs in the LAL group; 152 unique OTUs in the LA group; 79 unique OTUs in the SLA group; and 66 unique OTUs in the DLA group. A total of 380 OTUs were shared among the seven groups, accounting for 33% of the total OTUs. Lactobacillus paracasei K56 and its postbiotic intervention influenced the OTU composition of the mouse gut microbiota to a certain extent, with the LAL group having the most unique OTUs and the DLA group having the least unique OTUs.

[0123] 2.2 Alpha diversity analysis of mouse intestinal flora species

[0124] Alpha diversity can reflect the abundance and diversity of microbial communities, including a series of statistical analysis indices to estimate the species abundance and diversity of environmental communities. It is mainly related to two factors. One is the number of species in the sample, that is, richness, which is measured by the Chao1 index. The higher the Chao1 index value, the richer the species in the community; the second is the relative density of each species in the sample, that is, the uniformity of individual distribution in the community, which is represented by the Shannon index and the Simpson index. The larger the Shannon index value, the higher the community diversity; the larger the Simpson index value, the lower the community diversity. The Goodscoverage indicator reflects the sequencing depth and coverage of the sample. The changes in the Alpha diversity of the intestinal flora after treatment in different groups are as follows. Figure 9 As shown. Compared with the Control group, the Chao1 index in the LAL group was significantly increased (P < 0.05), indicating that the LAL group had a higher species richness. Compared with the Control and Model groups, the Orlistat group had a lower Simpson index, while the Shannon index in the LA and Orlistat groups was significantly increased (P < 0.05), indicating higher community diversity. The Goodscoverage values ​​of all samples were greater than 0.995, indicating that the sequencing coverage of the samples was high.

[0125] 2.3. Species dilution curve of mouse intestinal flora

[0126] The rarefaction curve mainly uses the microbial alpha diversity index of each sample at different sequencing depths to construct a curve to reflect the microbial diversity of each sample at different sequencing quantities. If the diversity index is sobs (representing the number of species actually observed), when the curve tends to be flat, it means that the amount of sequencing data is reasonable, and more data will only produce a small number of new species. Figure 10 This shows that the sequencing depth of this experiment covers most species diversity.

[0127] The Shannon curve is constructed based on the sequence of each sample and the microbial diversity index at different sequencing depths. A flat curve indicates that the sequencing data volume is sufficient to reflect the vast majority of microbial diversity in the sample. The figure below shows that this experiment meets the requirements for further analysis.

[0128] The Rank-Abundance curve can be used to explain two aspects of diversity, namely species richness and community evenness. Figure 10 As shown in the figure, in the horizontal direction, the species richness is reflected by the width of the curve. The larger the range of the curve on the horizontal axis, the higher the species richness. The shape of the curve (flatness) reflects the uniformity of the community in the sample. The flatter the curve, the more uniform the species distribution.

[0129] 2.4 Beta diversity analysis of mouse intestinal flora

[0130] Beta diversity refers to the differences in species composition between different environmental communities. Sample hierarchical cluster analysis, principal component analysis (PCA), principal coordinates analysis (PCoA), and non-metric multidimensional scaling (NMDS) are commonly used to reflect beta diversity between samples. To better reflect the diversity of microbial colony results between groups, PCoA analysis is used. When samples are close together, it indicates a similar species structure, so samples with high similarity are more likely to cluster together. The PCoA analysis method is similar to PCA analysis, but PCA is based on Euclidean distance, while PCoA is not restricted by distance algorithms.

[0131] Jaccard analysis was used to analyze the beta diversity of the intestinal flora of each group of mice. The profiles of the intestinal flora of the seven groups of samples were different, indicating that there were differences in the flora structure of the seven groups. Figure 11A reflects the effects of ordinary feed and high-fat feed on the changes in intestinal flora structure. The sample composition between the Model group and the Control group was significantly different. Figure 11 B reflects the differences in sample composition between each treatment group and the Control group. The differences in sample composition between the LAL group, LA group, SLA group, Orlistat group and the Control group were significant, and there was some overlap in sample composition between the DLA group and the Control group. Figure 11 C reflects the differences in sample composition between the treatment groups and the Model group. The sample composition between the LAL group, LA group, SLA group and the Model group is similar, which may be because the basic diets of the treatment group and the Model group are all high-fat feed. The microbial aggregation of the SLA group is tighter than that of the Model group, and the parallelism is better. Figure 11 As shown in D, high-fat diet intervention caused significant changes in the structure of the microbial community. The microbial community also changed after intervention with Lactobacillus paracasei K56 and its postbiotics. The microorganisms in the LAL group, DLA group, and SLA group clustered more closely.

[0132] 2.5 Compositional analysis of mouse intestinal flora at the phylum level

[0133] The intestinal flora of mice in the control group, model group, Lactobacillus paracasei K56 and its metabiotic group, Lactobacillus paracasei K56 surface protein-depleted group, and orlistat group were compared at the phylum and genus levels to analyze the differences in the flora. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the dominant phyla in the cecal microbial community. The results of the phylum-level analysis are as follows: Figure 12As shown, the intestinal microbiota of experimental mice was mainly composed of Firmicutes, Bacteroidetes, Actionobacteria, Desulfobacterota, Verrucomicrobiota, Patescibacteria, and Proteobacteria at the phylum level. Firmicutes and Bacteroidetes dominated the intestinal microbiota of mice, accounting for over 70%. Compared with the control group, the relative abundance of Firmicutes and Bacteroidetes in the model group was not significantly different, while the relative abundance of Actionobacteria was significantly increased (P < 0.05). Compared with the model group, the relative abundance of Firmicutes was significantly decreased in the DLA and orlistat groups (P < 0.05). The relative abundance of Bacteroidetes and Actionobacteria did not differ significantly among the treatment groups.

[0134] The Firmicutes / Bacteroidetes ratio is positively correlated with host fat storage, body weight, visceral fat, and subcutaneous fat. Probiotics and prebiotics can reduce the Firmicutes / Bacteroidetes (F / B) ratio. Compared with the control group, the F / B ratio was significantly decreased in the LAL, DLA, LA, SLA, and ORT groups (P < 0.05), indicating that Lactobacillus paracasei K56 and its postbiotics can prevent and improve obesity by reducing the Firmicutes-to-Bacteroidetes ratio.

[0135] 2.6 Composition Analysis of Mouse Gut Microbiota at the Genus Level

[0136] The changes in intestinal flora were further analyzed at the genus level. Figure 13-15It can be seen that the intestinal flora of the seven groups of mice at the genus level mainly include beneficial bacteria such as norank_f_Muribaculaceae, Dubosiella, Lachnospiraceae_NK4A136 (Lachnospiraceae_NK4A136_group), norank_f_Ruminococcaceae, Lactobacillus, Enterobacter, Coriobacteriaceae_UCG-002, Akkermansia, and Bifidobacterium; and harmful bacteria such as Ileibacterium, Desulfovibrio, Clostridia UCG-014_norank, norank_f__Erysipelotrichaceae, and norank_f__Oscillospiraceae.

[0137] norank_f_Muribaculaceae can inhibit intestinal barrier dysfunction, lipid metabolism disorders, suppress harmful bacteria and oxidative stress, and improve intestinal mucosal inflammation. The abundance of Dubosiella is negatively correlated with body weight; Lachnospiraceae_NK4A136_group is negatively correlated with a variety of metabolic diseases and chronic inflammation; Ruminococcaceae is positively correlated with the proportion of carbohydrates in high-fat feeds, especially plant polysaccharides. Lachnospiraceae_NK4A136_group and Ruminococcaceae are bacteria rich in the production of short-chain fatty acids (SCFAs) in healthy intestines. SCFAs are beneficial to maintaining the anaerobic environment in the intestine. Lactobacillus can prevent pathogens from invading and colonizing the intestine, inhibit the production of pathogens and endotoxins, and can also produce lactic acid, acetic acid and propionic acid to promote intestinal health; Enterorhabdus is negatively correlated with the concentration of pro-inflammatory cytokines and plays a positive role in intestinal homeostasis. Coriobacteriaceae_UCG-002 can improve the steatosis of non-alcoholic fatty liver disease, and may have the function of improving steatosis by affecting the sphingolipid metabolic pathway, thereby reducing the steatosis of liver cells. Studies have shown that Coriobacteriaceae can alleviate insulin resistance and improve liver steatosis. Akkermansia is a microorganism that inhibits the intestinal mucus layer. Because Akkermansia can increase the amount of mucin and reduce colonic inflammation, Akkermania is believed to have a positive effect on maintaining intestinal barrier homeostasis. The abundance of beneficial bacteria such as norank_f_Muribaculaceae, Lachnospiraceae_NK4A136_group, and Akkermansia did not change significantly at the genus level. Compared with the Control group, the Model group significantly reduced the relative abundance of beneficial bacteria such as Bifidobacterium (P < 0.05). Compared with the Model group, the SLA group significantly increased the relative abundance of norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002; the LA group significantly increased the relative abundance of Lactobacillus (P<0.05).

[0138] norank_f__Erysipelotrichaceae and Ileibacterium both belong to the Erysipelotrichaceae family and are closely associated with metabolic disorders. Studies have shown that the abundance of Ileibacterium can increase tenfold in models of obesity and high-fat diets. Ileibacterium is likely associated with metabolic diseases, and its specific role requires further investigation. Desulfovibrio is a bacterium associated with intestinal inflammation and is associated with LPS production in mice with high-fat diet-induced obesity. Reducing the relative abundance of Desulfovibrio may help improve intestinal integrity and reduce intestinal inflammation. ClostridiaUCG-014_norank, Clostridium, is highly abundant in the intestine of early-stage liver cancer. Increased abundance of some pro-inflammatory bacteria, such as ClostridiaUCG-014, can induce inflammatory responses and upregulate pro-inflammatory lipid metabolites. norank_f__Oscillospiraceae is a pathogenic intestinal bacterium associated with inflammation that may participate in mucin degradation, triggering intestinal barrier damage and systemic inflammatory responses. The relative abundance of norank_f__Oscillospiraceae did not change significantly in any of the groups. Compared with the Model group, the relative abundance of Ileibacterium was significantly decreased in the DLA and ORT groups, while the relative abundance of Desulfovibrio increased in the DLA group, the relative abundance of norank_o__Clostridia_UCG-014 increased in the LA group, and the relative abundance of norank_f__Erysipelotrichaceae increased significantly in the ORT group (P < 0.05).

[0139] In summary, our results suggest that a high-fat diet can, to a certain extent, cause intestinal microbial disturbances in mice. Lactobacillus paracasei K56 and its postbiotic intervention can modulate some of these microbiota, with differences observed between different groups. In particular, Lactobacillus paracasei K56 surface proteins significantly increased the relative abundance of the beneficial bacteria norank_f_Ruminococcaceae, Lactobacillus, and Coriobacteriaceae_UCG-002. In particular, the Lactobacillus paracasei K56 surface proteins significantly increased the relative abundance of the beneficial bacteria norank_f_Ruminococcaceae and Coriobacteriaceae_UCG-002 compared to the live LAL group, achieving unexpected results.

[0140] It should be understood that the invention described herein is not limited to specific methodology, experimental protocols or reagents, as these may vary. The discussion and examples provided herein are presented only to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the claims.

Claims

1. A method for extracting the surface protein of Lactobacillus paracasei K56, comprising: (1) Lactobacillus paracasei K56 was mixed with an acidic lithium chloride aqueous solution as an extraction reagent and incubated; (2) centrifuging the mixture obtained in step (1) and taking the supernatant, wherein the supernatant is a surface protein extract of Lactobacillus paracasei K56; Wherein, the Lactobacillus paracasei K56 is deposited in the German Collection of Microorganisms and Cell Cultures with a deposit number of DSM 27447; it is also deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China National Committee for the Administration of Microbiological Culture Collection with a deposit number of CGMCC 15139; The ratio of the mass of the Lactobacillus paracasei K56 to the volume of the extraction reagent acidic lithium chloride aqueous solution is 1g:(1-5mL).

2. The method according to claim 1, wherein In the acidic lithium chloride aqueous solution, the concentration of lithium chloride is 1-10 mol / L, preferably 4-6 mol / L, more preferably 5 mol / L.

3. The method according to any one of claims 1 to 2, wherein: The pH of the acidic lithium chloride aqueous solution is 1.0-3.0, preferably 2.

0.

4. The method according to any one of claims 1 to 3, wherein: The incubation time is at least 10 min, preferably 10-40 min, more preferably 30-40 min, and most preferably 30 min.

5. The method according to any one of claims 1 to 4, wherein: The incubation was performed in an ice water bath.

6. An isolated Lactobacillus paracasei K56 surface protein, wherein the Lactobacillus paracasei K56 surface protein comprises an LPXTG motif protein and a moonlighting protein; Preferably, the LPXTG motif protein comprises K0NBH9 (GenBank: CCK24055.1), KONA48 (GenBank: CCK23225.1), and K0NAB9 (GenBank: CCK23405.1); Preferably, the part-time protein comprises A0A0K1KXQ2 (GeneID: 57089615), K0N7K7 (GenBank: CCK23322.1), KOMU67 (GenBank: CCK22051.1), A0A0K1KYZ6 (Gene ID: 57090004), and K0N700 (GenBank: CCK23323.1); Preferably, the surface protein of Lactobacillus paracasei K56 is as shown in Table A.

7. Lactobacillus paracasei K56 surface protein according to claim 6, which is obtained by the method described in any one of claims 1-5.

8. A composition comprising the surface protein of Lactobacillus paracasei K56 obtained by the method according to any one of claims 1 to 5 or the surface protein of Lactobacillus paracasei K56 according to any one of claims 6 to 7.

9. The composition according to claim 8, wherein The composition is a pharmaceutical composition, a food (ordinary food or health food), a food additive, a dietary supplement or a feed.

10. The composition according to any one of claims 8 to 9, wherein The composition is in an oral dosage form. Preferably, the oral dosage form is selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, buccal lozenges, tablets, chewing gums, and capsules.

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