A kind of fermented mucus lactobacillus and its application

Abstract

The present invention discloses a fermented mucus lactobacillus and its application. The present invention provides a fermented mucus lactobacillus A10 with high extracellular polysaccharide production, and its deposit number is CCTCC NO: M20242082. The fermented mucus lactobacillus A10 has strong bile salt and gastrointestinal digestion tolerance, and has obvious antibacterial effects on Escherichia coli and Staphylococcus aureus. The extracellular polysaccharide synthesized by the fermented mucus lactobacillus A10 is a Wzy type extracellular polysaccharide, which is a heteropolysaccharide composed of rhamnose, galactose, glucose, mannose, and glucuronic acid. It has good thermal stability and antioxidant activity, and can significantly scavenge free radicals. The fermented mucus lactobacillus A10 extracellular polysaccharide provided by the present invention can exert antioxidant activity by activating the Keap1-Nrf2 / ARE signaling pathway, and is expected to be developed into a natural antioxidant that interacts with Keap1-Nrf2 in a targeted manner.

Description

Technical Field

[0001] The invention relates to the field of lactic acid bacteria, in particular to a fermented mucus lactobacillus and an application thereof. Background Art

[0002] Reactive oxygen species (ROS) are metabolic products produced by cells. Excessive accumulation of ROS can lead to lipid peroxidation, protein and DNA damage, and cause inflammation, diabetes, atherosclerosis and neurodegenerative diseases. In order to resist the damage caused by ROS, the body has established a complex oxidative stress response system (Keap1-Nrf2 / ARE signaling pathway) to reduce the damage to cells. Nrf2 transcription factors interact with antioxidant response elements (AREs) in the cell nucleus to activate the activities of most antioxidant enzymes [heme oxygenase (HO-1), glutamylcysteine ​​ligase (GCLC), cysteine ​​ligase (GCLM) and thioreductase 1 (TXNRD1)], thereby scavenging free radicals in the body and reducing the effects of oxidative stress on cells and tissues.

[0003] Exopoly Saccharides (EPS) are secondary metabolites secreted outside the cell wall by lactic acid bacteria during growth and metabolism. Exopolysaccharides are widely used in the food industry due to their unique health benefits and physicochemical properties. Exopolysaccharides can be used as natural thickeners to improve the viscosity, texture and sensory properties of food; in addition, EPS has a variety of physiological activities, including anti-tumor, antioxidant, antibacterial, anti-inflammatory and hypoglycemic activities, among which the antioxidant function is the basis of other functions and is the most important and the one that needs to be studied in depth. The physicochemical properties and functional activities of exopolysaccharides are closely related to their structural characteristics (such as monosaccharide composition, molecular weight, functional groups, potential, glycosidic configuration and degree of branching). Therefore, characterizing the structural characteristics of exopolysaccharides is crucial to exploring their functional activities.

[0004] Extracellular polysaccharides are safe, non-toxic, and have good biocompatibility, and are expected to be developed into a natural antioxidant that interacts with Keap1-Nrf2 in a targeted manner. Summary of the invention

[0005] The present invention provides a fermented Lactobacillus mucoides A10 extracellular polysaccharide with antioxidant activity and its application. The present invention separates a fermented Lactobacillus mucoides strain from a meat product sample collected from Shangri-La, Yunnan, the strain has a high production of extracellular polysaccharide, and the extracellular polysaccharide has a high antioxidant activity.

[0006] The technical solution of the present invention is as follows: a fermented mucus lactobacillus, the fermented mucus lactobacillus ( Limosilactobacillus fermentum) was named: Fermented Lactobacillus mucus A10; it was deposited in the China Center for Type Culture Collection on September 25, 2024, with the deposit number CCTCC NO: M20242082, and the deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The invention also provides a microbial agent containing the fermented mucus lactobacillus.

[0008] The present invention also provides the use of the fermented mucus lactobacillus or the microbial agent in antibacterial application, wherein the antibacterial application is used for non-therapeutic purposes.

[0009] As a further description of the above scheme: the antibacterial effect refers to the antibacterial effect on Escherichia coli or Staphylococcus aureus.

[0010] The present invention also provides the use of the fermented Lactobacillus mucilaginosus or the microbial agent in producing extracellular polysaccharides. The fermented Lactobacillus mucilaginosus A10 can synthesize extracellular polysaccharides during the fermentation process, with a yield of more than 309 mg / L.

[0011] The present invention also provides an extracellular polysaccharide, which is obtained by fermenting the fermented mucus lactobacillus or the microbial agent.

[0012] As a further description of the above scheme: the extracellular polysaccharide is a heteropolysaccharide composed of rhamnose, galactose, glucose, mannose and glucuronic acid; the molar ratio of rhamnose, galactose, glucose, mannose and glucuronic acid is 0.13-0.16: 1.80-1.97: 1.09-1.11: 0.88-1.00: 0.26-0.27. FTIR and XRD show that the extracellular polysaccharide has a typical characteristic absorption peak and crystal structure; and has good thermal stability.

[0013] As a further description of the above scheme: the extracellular polysaccharide synthesis pathway is a Wzx / Wzy dependent pathway, and the synthesized extracellular polysaccharide is a Wzy type extracellular polysaccharide.

[0014] The present invention also provides the use of the exopolysaccharide as a natural antioxidant. The exopolysaccharide has good ability to remove DPPH free radicals, ABTS free radicals and hydroxyl free radicals. The exopolysaccharide can be used to protect HepG2 cells from oxidative damage induced by H2O2.

[0015] The present application screened a strain of lactic acid bacteria with high extracellular polysaccharide production from meat product samples in Shangri-La, Yunnan. The strain was identified as fermentative mucus lactobacillus A10 through morphological and molecular biological identification. The colony morphology of the fermentative mucus lactobacillus A10 on MRS solid culture medium is as follows: Figure 1As shown in A, the colony is round, milky white, translucent, with neat edges and obvious protrusions, and the Gram staining result is purple positive bacteria; scanning electron microscopy observed that the strain is round and long rod-shaped, and each strain adheres to a mass. The fermented mucus Lactobacillus A10 has strong bile salt and gastrointestinal digestion tolerance, and has obvious antibacterial effects on Escherichia coli and Staphylococcus aureus.

[0016] The fermented mucus lactobacillus A10 is cultured in MRS liquid medium, preferably at 37°C for 30 hours, and centrifuged to obtain a cell-free fermentation supernatant. After the supernatant is deproteinized, the exopolysaccharide is extracted by precooled ethanol precipitation, and the yield is as high as 309.3 mg / L or more. FTIR and XRD show that the exopolysaccharide has a typical characteristic absorption peak and a crystal structure. The exopolysaccharide has good thermal stability.

[0017] The extracellular polysaccharide has the ability to remove DPPH free radicals, ABTS free radicals and hydroxyl free radicals. The extracellular polysaccharide can be used for protecting HepG2 cells from oxidative damage induced by H2O2.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention screened a strain of lactic acid bacteria A10 with high extracellular polysaccharide production from meat product samples in Shangri-La, Yunnan. The lactic acid bacteria were identified by morphological identification, molecular biological identification and whole genome sequencing. Limosilactobacillus fermentum , which is fermented mucus Lactobacillus, and was named fermented mucus Lactobacillus A10.

[0020] (2) The fermented mucus Lactobacillus A10 provided by the present invention has strong bile salt and gastrointestinal digestion tolerance and antibacterial activity.

[0021] (3) The fermented Lactobacillus mucilaginosus A10 provided by the present invention produces a high amount of extracellular polysaccharide, wherein the extracellular polysaccharide is a heteropolysaccharide composed of rhamnose, galactose, glucose, mannose and glucuronic acid.

[0022] (4) The extracellular polysaccharide of the fermented Lactobacillus mucilaginosus A10 provided by the present invention has a typical characteristic absorption peak and crystal structure, and has good thermal stability.

[0023] (5) The extracellular polysaccharide of fermented Lactobacillus mucilaginosus A10 provided by the present invention has good in vitro antioxidant activity and cellular antioxidant activity.

[0024] (6) The fermented Lactobacillus mucilaginosus A10 extracellular polysaccharide provided by the present invention is a promising natural antioxidant and can be used in functional foods.

[0025] Microbiological deposit information

[0026] Classification and naming: Fermented Lactobacillus mucilaginosus A10 Limosilactobacillus fermentum A10;

[0027] Deposit number: CCTCC NO: M20242082;

[0028] Deposit date: September 25, 2024;

[0029] Depository: China Center for Type Culture Collection;

[0030] Storage address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Figure 2 is the colony morphology of fermented Lactobacillus mucilaginosus A10. A is the colony morphology of the strain grown on MRS solid medium for 24 h. B is the colony morphology of the strain after Gram staining. CE is the microstructure of fermented Lactobacillus mucilaginosus A10 at 2,000× (C), 5,000× (D), and 20,000× (E).

[0032] Figure 2 is the phylogenetic tree of Lactobacillus mucilaginosus A10;

[0033] Figure 3 is the growth curve of fermenting Lactobacillus mucilaginosus A10;

[0034] Figure 4 The acid production performance of fermented Lactobacillus mucilaginosus A10;

[0035] Figure 5 The exopolysaccharide synthesis pathway of Lactobacillus mucilaginosus A10 based on the Wzx / wzy-dependent pathway;

[0036] Figure 6 The monosaccharide composition of the exopolysaccharide of fermented Lactobacillus mucilaginosus A10;

[0037] Figure 7 is the microstructure of exopolysaccharide fermented by Lactobacillus mucilaginosus A10, AD is the microstructure of exopolysaccharide at 200×, 500×, 1,000×, and 5,000×, respectively;

[0038] Figure 8 FTIR spectrum of exopolysaccharide of fermented Lactobacillus mucilaginosus A10;

[0039] Fig. 9 Zeta potential (A) and particle size (B) of exopolysaccharide fermented by Lactobacillus mucilaginosus A10;

[0040] Fig.10is the thermal stability of the extracellular polysaccharide fermented by Lactobacillus mucilaginosus A10, A is the TGA-DTG curve of the extracellular polysaccharide,

[0041] B is the DSC curve;

[0042] Fig.11 is the XDR spectrum of the exopolysaccharide of fermented Lactobacillus mucilaginosus A10;

[0043] Fig.12 is the in vitro antioxidant activity of the exopolysaccharide of fermented Lactobacillus mucilaginosus A10, A is the DPPH free radical scavenging activity, B is the ABTS free radical scavenging activity, and C is the hydroxyl free radical scavenging activity;

[0044] Fig.13 The antioxidant activity of the cells fermenting the exopolysaccharide of Lactobacillus mucilaginosus A10;

[0045] Fig.14 To investigate the docking structure-activity relationship between the monosaccharide components of the extracellular polysaccharide of fermented Lactobacillus mucilaginosus A10 and the Keap1 protein. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific embodiments. The description in this section is only exemplary and explanatory and should not have any limiting effect on the scope of protection of the present invention.

[0047] Example 1 Isolation and purification of bacterial strains

[0048] 10 g of meat sample collected from Deqin County, Shangri-La, Yunnan was added to 90 mL of MRS liquid culture medium. After enrichment culture at 37 °C for 48 h, 1 mL of culture medium was added to 9 mL of normal saline and fully shaken for 30 s. Gradual dilution was performed in sequence, with the highest dilution being 10 8 . Take 200 μL of the dilution and spread it on MRS solid culture medium and culture it at 37℃ for 48h. Preliminarily, according to the colony morphology, size, size of calcium dissolution circle, color, and growth position in the culture medium (surface, inside and bottom), select colonies with different appearance characteristics and larger calcium dissolution circle for streak culture and purification. Repeat the streak purification several times with the same method until the final plate has colonies of the same morphology. Pick a single colony and inoculate it in MRS liquid culture medium at 37℃ for 24h for preservation and identification.

[0049] Morphological characteristics of strains

[0050] like Figure 1 As shown in A, after fermentation of Lactobacillus mucilaginosus A10 on MRS agar medium for 24 hours, the colony morphology was milky white, translucent, relatively moist, smooth, with neat edges and obvious protrusions; Figure 1As shown in B, the microscopic examination results of fermented mucus Lactobacillus A10 are: no spores, scattered rod-shaped cells, purple Gram-stained positive bacteria; scanning electron microscopy observed that the strain was round and long rod-shaped, and each strain adhered to a mass ( Figure 1 C. Figure 1 D and Figure 1 E).

[0051] Molecular biological identification of bacterial species

[0052] 16S rDNA gene sequencing

[0053] The bacterial genome was extracted using the TSINGKE plant DNA extraction kit (universal type). The genome of the extracted strain was used as a template, and the bacterial universal primers: 27F (5'-AGAGTTTGATCMTGGCTCAG -3', SEQ ID NO.2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.3) were used to perform a PCR experiment on 16S rDNA. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared and analyzed using the BLAST tool in the NCBI database (www.ncbi.nlm.gov / blast / ) with the existing sequences in the GenBank database. The homology of the strain to be tested and the corresponding sequences of the known strains was analyzed to determine the species of the sugar-producing strains screened out. The strain was sequenced by 16S rDNA gene, and the 16SrDNA sequence is shown in SEQ ID NO.1. The results of gene sequencing showed that the sequence had a homology of more than 99% with the 16S rDNA sequence of fermented mucus Lactobacillus, and the screened lactic acid bacteria were determined to be fermented mucus Lactobacillus ( Figure 2 ). Finally, the strain screened was determined to be fermentative mucoid Lactobacillus through morphology, 16S rDNA sequencing, whole genome sequencing and phylogenetic tree, and was named fermentative mucoid Lactobacillus 10, which was deposited in the China Center for Type Culture Collection with the deposit number: CCTCCNO: M20242082.

[0054] Example 2 Acid production and antibacterial activity of fermented Lactobacillus mucilaginosus A10

[0055] Growth curve

[0056] The lactic acid bacteria with high extracellular polysaccharide production were activated to the third generation, and 200 μL of the lactic acid bacteria activated to the third generation was placed in a 9-well plate, and its absorbance at 600 nm was measured every 2 hours for a total of 24 hours. The growth curve was drawn with time (h) as the horizontal axis and absorbance as the vertical axis. The results are shown in the figure. Figure 3As shown in the figure, with the extension of fermentation time, the OD600 nm value of the fermented mucus Lactobacillus A10 showed an upward trend. From 0 to 4 h, it was in the lag phase of growth, and the growth rate of the strain was relatively slow; from 4 to 10 h, it was in the logarithmic phase of growth, and the growth rate of the strain increased sharply, and the OD value increased exponentially; after 10 h, it entered the stable growth phase, and the OD600 nm value of the strain was not much different, and basically tended to be stable.

[0057] Acid production performance

[0058] The activated strain producing extracellular polysaccharides was added to the MRS liquid culture medium at a 3% inoculation rate, and the pH value during the fermentation process was measured. The measurement was stopped every 2 hours until the pH stabilized. The activated strain producing extracellular polysaccharides was inoculated into the MRS liquid culture medium at a 3% inoculation rate, and placed in a 37°C incubator for anaerobic culture. Every 2 hours, 5 mL of fermentation liquid was added to the flask, and then three drops of phenolphthalein and 10 mL of distilled water were added. Then, 0.1 mol of NaOH was used for titration, and the changes in titrated acidity were measured and recorded. The acid production performance of Lactobacillus mucilaginosus A10 during fermentation is shown in Figure 2. Figure 4 As shown in the figure, the pH value gradually decreased with the extension of fermentation time. When the fermentation time reached 14h, the pH value reached the lowest value and then stabilized. The trend of titrated acidity was opposite to that of pH value. At the end of fermentation, the titrated acidity of the fermentation liquid was 72, indicating that the fermented Lactobacillus mucilaginosus A10 had good fermentation performance.

[0059] Antibacterial properties

[0060] The antibacterial activity was determined by the Oxford cup punching method. Under sterile operation, 4 Oxford cups were placed on a clean plate, 2 mL of Escherichia coli or Staphylococcus aureus was injected into the plate, agar was added to make the volume 20-25 mL, and after the agar was completely solidified, the Oxford cup was gently pulled out with tweezers, and then 180 μL of culture medium was injected into the cup holes respectively. 20 μL of sterile fermentation supernatant was added to the experimental group, and 20 μL of sterile saline was used as a control. The plates were placed at 4°C for 4 h, and then moved to 37°C for 12 h to observe whether an inhibition zone was produced. The diameter of the inhibition zone was measured and recorded. The results are shown in Table 1. The diameter of the inhibition zone of fermented mucus lactobacillus A10 against Escherichia coli was 14.1 mm, and the diameter of the inhibition zone against Staphylococcus aureus was 15.7 mm, which was significantly higher than the antibacterial activity of fermented mucus lactobacillus A07 and B11. Fermentative mucus Lactobacillus A07 was isolated and purified from meat product samples in Deqin County, Shangri-La, Yunnan, and fermentative mucus Lactobacillus B11 was isolated from Dengchuan milk in Eryuan County, Dali. 16sDNA gene sequencing identified both strains as fermentative mucus Lactobacillus.

[0061] Table 1 Antibacterial activity of fermented Lactobacillus mucilaginosus A10

[0062]

[0063] Note: Different letters in the same column indicate significant differences (P<0.05).

[0064] Example 3 Simulated gastrointestinal digestion tolerance and bile salt tolerance of fermented Lactobacillus mucilaginosus A10

[0065] The activated bacterial solution was inoculated into 10 mL of sterilized MRS broth medium with a final bile salt concentration of 0.2%, 0.3% and 0.4% at a volume fraction of 1.0%. After incubation at 37°C for 4 hours, 100 μL of the fermentation solution was taken and appropriately diluted on MRS agar plates to count the number of viable bacteria. The culture medium without bile salt was used as a control to calculate the survival rate. The results are shown in Table 2. The number of viable bacteria of fermented Lactobacillus mucosa A10 after 4 hours of treatment with 0.4% bile salt reached 9.5×10 7 CFU / mL, which was higher than that of fermentative Lactobacillus mucilaginosus A07 and B11, indicating that strain A10 had a strong bile salt tolerance.

[0066] Table 2 Bile salt tolerance of fermented Lactobacillus mucilaginosus A10

[0067]

[0068] Note: Different superscript letters in the same column indicate significant differences (P < 0.05).

[0069] The activated bacterial solution was inoculated into the pre-set simulated gastric fluid (pH=2.0) at a 10% inoculation volume, mixed evenly, and placed under 37°C for anaerobic culture. Then, samples were taken at 0h and 3h for plate counts. After 3h of simulated gastric fluid, 3mL of the culture solution was extracted into 27mL of simulated intestinal fluid (pH=8.0) for mixing. After 24h of anoxic culture at 37°C, plate counts were performed and the survival rate was calculated according to the formula. The results are shown in Table 3. The number of viable bacteria of fermented mucus Lactobacillus A10 after digestion in simulated gastric fluid and intestinal fluid was 3.4×10 8 CFU / mL and 3.1×10 8 CFU / mL, which was higher than that of Lactobacillus muciflorus A07 and B11, indicating that strain A10 had a strong tolerance to simulated gastrointestinal digestion.

[0070] Table 3 Simulated gastrointestinal digestion tolerance of fermented Lactobacillus mucilaginosus A10

[0071]

[0072] Note: Different superscript letters in the same column indicate significant differences (P < 0.05).

[0073] Example 4 Whole genome sequencing of fermenting Lactobacillus mucilaginosus A10 and exopolysaccharide synthesis pathway

[0074] Whole genome sequencing

[0075] DNA was extracted using the DNA extraction kit of TGUIDE S96 magnetic bead technology. The content was determined by the Qubit method, the DNA integrity was determined by the Nanodrop method, and the DNA integrity was determined by agarose electrophoresis. After the sample was tested, the library was constructed, and the sequencing test was performed on the machine after the library passed the quality inspection. Using PacBio third-generation sequencing technology, the genome size of Lactobacillus fermentans A10 was 1,914,876 bp, with a total of 2,202 genes, an average gene length of 869 bp, and an average G+C content of 51.13%.

[0076] It was further found that fermentation Lactobacillus mucilaginosus A10 contained genes related to regulation of exopolysaccharide synthesis, as shown in Table 4. The key genes Wzx and Wzy regulating exopolysaccharide synthesis were identified in the whole genome data of fermentation Lactobacillus mucilaginosus A10, and it was further determined that the exopolysaccharide synthesis pathway of fermentation Lactobacillus mucilaginosus A10 was a Wzx / Wzy-dependent pathway, and the synthesized exopolysaccharide was a Wzy-type exopolysaccharide. The exopolysaccharide synthesis of fermentation Lactobacillus mucilaginosus A10 includes five steps ( Figure 5): (1) Phosphorylation and transport of lactose, galactose and glucose. Galactose and lactose enter the cell under the action of galactose permease and lactose permease respectively. Lactose entering the cell is broken down into galactose by β-galactosidase and further phosphorylated by galactosidase. At the same time, lactose and glucose can also be transferred and phosphorylated by pTS-lactose and pTS-glucose. In addition, β-galactosidase catalyzes the conversion of galactose into phosphorylated galactose and phosphorylated glucose. (2) Production of sugar nucleotides. Galactose-1-phosphate is converted into glucose-1-phosphate under the action of galactose-1-phosphate uridylyltransferase. And 6-phosphate lactose is converted into 1-phosphate glucose by glucokinase. In addition, lactose can also be converted into 1-phosphate glucose by β-galactosidase and glucokinase, and finally converted into UDP-glucose by UTP-1-phosphate glucose uridylyltransferase. Among them, glucose-6-phosphate is converted into GDP-mannose by glucose-6-phosphate isomerase, mannose-6-phosphate isomerase and phosphomannose isomerase 2. (3) Synthesis of repeating units. These sugar nucleotide precursors are assembled into galactose-glucose-mannose repeating units in the endoplasm under the catalysis of glycosylase. (4) Transfer of repeating units. The formed galactose-glucose-mannose repeating units are transferred to the periplasmic space or outer membrane under the action of flipping enzymes. (5) Polymerization and release of galactose-glucose-mannose repeating units. The repeating units are assembled into long chains under the action of polymerases, and under the regulation of chain length regulatory proteins, the long chains are released into the extracellular space and finally form exopolysaccharides.

[0077] Table 4 Genes related to exopolysaccharide synthesis in fermentative Lactobacillus mucilaginosus A10

[0078]

[0079] Example 5 Isolation and purification of extracellular polysaccharide from fermented Lactobacillus mucilaginosus A10 and determination of monosaccharide composition

[0080] Take the fermentation mucus Lactobacillus A10 activated to the third generation, inoculate it into sterile fresh MRS liquid medium at a volume ratio of 5%, culture it at 37℃ for 30h to obtain the fermentation broth, then put it into boiling water for 10min to inactivate it, cool it and centrifuge it (4℃, 7500r / min) for 20min to obtain the cell-free fermentation supernatant, add 80% trichloroacetic acid to the supernatant to a final concentration of 4% (w / v), stir it for 3min, let it stand at 4℃ for 10h, and centrifuge to remove the protein. Then purify it to 1 / 3, mix it with 3 times the amount of pre-cooled alcohol, precipitate it at 4℃ overnight, and centrifuge to obtain a precipitate. Then dissolve the precipitate in deionized water, dialyze it at 4℃ for 48h, change the water every 8h, and freeze-dry the dialyzate to obtain exopolysaccharides. The main exopolysaccharide component 1 (EPS-1) was purified by DEAE-52 cellulose column chromatography.

[0081] Determination of Monosaccharide Composition of Exopolysaccharide Fermented by Lactobacillus mucilaginosus A10

[0082] The monosaccharide composition of the main component of the extracellular polysaccharide EPS-1 of fermented Lactobacillus mucilaginosus A10 was determined by high performance liquid chromatography. Figure 6 As shown, EPS-1 is a heteropolysaccharide composed of rhamnose, galactose, glucose, mannose and glucuronic acid; the molar ratio of rhamnose, galactose, glucose, mannose and glucuronic acid is 0.13: 1.97: 1.09: 1.00:0.26.

[0083] Example 6 Analysis of structural characteristics of extracellular polysaccharides from fermented Lactobacillus mucilaginosus A10

[0084] Preparation and purification of exopolysaccharide from fermented Lactobacillus mucilaginosus A10

[0085] Microstructure of the main component of extracellular polysaccharide EPS-1 of fermented Lactobacillus mucilaginosus A10 at 200× (A), 500× (B), 1,000 (C), and 5,000× (D). Figure 7 Scanning electron microscopy showed that the surface of EPS-1 was rough, the structure was loose and porous, and there were some flakes and many small particles mixed in.

[0086] FTIR spectroscopy analysis

[0087] The functional group structure of the main component of extracellular polysaccharide EPS-1 of fermented Lactobacillus mucilaginosus A10 was analyzed by FTIR. Figure 8 The results showed that EPS-1 has a characteristic absorption peak of polysaccharides, 3,411 cm -1 (3,200-3,600 cm -1 The typical absorption peak at 2,930 cm is caused by the stretching vibration of the OH bond of the extracellular polysaccharide, indicating that there are a large number of hydroxyl groups in the extracellular polysaccharide molecules; -1 The absorption peak at 1648 cm is the CH stretching vibration peak of the extracellular polysaccharide molecule; -1 and 1378 cm -1 The absorption peak at 1059.25 cm corresponds to the C=O or CO stretching vibration peak in the carboxyl group, indicating the presence of a carboxyl group; -1 The absorption peak at 580.08 cm is caused by the stretching vibration of the COC or COH bond, indicating the presence of pyranose. -1 The absorption peak at also proves the existence of pyranose ring.

[0088] Zeta potential and particle size analysis

[0089] Zeta potential and particle size of the main component of extracellular polysaccharide EPS-1 of fermented Lactobacillus mucilaginosus A10 Fig. 9 The zeta potential of EPS-1 is -18 mV ( Fig. 9 A), which is higher than the 1.3 mV of Bacillus SCU-E108 exopolysaccharide and -14.7 mV of Lactobacillus fermentum A51 in the existing literature. It is reported that negatively charged exopolysaccharides have stronger biological activity than neutral ones. The particle size of exopolysaccharide is 146nm ( Fig. 9 B), which is smaller than the 446.8 nm of the exopolysaccharide of Pediococcus pentosaceus M41 and the 525.5 nm of Lactobacillus plantarum C70 reported in the existing literature.

[0090] Thermal stability analysis

[0091] The TGA curve, DTG curve and DSC curve of the main component of extracellular polysaccharide EPS-1 of fermented Lactobacillus mucilaginosus A10 in the temperature range of 25-600℃ were analyzed by TGA-DSC method. Fig.10 As shown. Fig.10 As shown in Figure A, EPS-1 exhibited three stages of thermal mass loss within the temperature range of 25-600 °C. After treatment at 600 °C, the mass loss rate of exopolysaccharide was 65.8%. Exopolysaccharide showed a broad exothermic reaction peak at 97.30 °C, indicating that its melting point (Tm) and enthalpy change (ΔH) were 1131.31 mJ and 164.65 J / g, respectively. In addition, EPS-1 showed two small absorption reaction peaks at 257.80 °C and 305.14 °C. In addition, according to the DTG and DSC curves, the thermal degradation and dissolution temperatures of EPS-1 were 252.4 °C and 236.5 °C, respectively.

[0092] XRD spectrum analysis

[0093] XRD spectral analysis showed that there were no sharp peaks in the XRD spectrum of EPS-1, the main component of extracellular polysaccharide of fermented Lactobacillus mucilaginosus A10, indicating that it has typical amorphous properties. In addition, a strong diffraction peak was observed in the range of 16°-30°, indicating that EPS-1 has a crystalline structure ( Fig.11 ). Therefore, XRD spectrum analysis shows that EPS-1 belongs to an amorphous crystal structure, which directly affects the physical properties of extracellular polysaccharides (including solubility, tensile strength, swelling ability or viscosity).

[0094] Example 7 Analysis of the Antioxidant Activity of Fermented Exopolysaccharide from Lactobacillus mucilaginosus A10

[0095] In vitro antioxidant activity

[0096] DPPH free radical scavenging experiment: Take 200μL (10mg / mL) of fermented Lactobacillus mucilaginosus A10 extracellular polysaccharide solution, add 2mL of 1.8mM DPPH ethanol solution, shake thoroughly, react in the dark at room temperature for 20min, and measure the absorbance at 517nm. Calculate the DPPH free radical scavenging rate according to the following formula:

[0097]

[0098] Where: A is the absorbance of the solution when the sample is added; A0 is the absorbance of the solution when distilled water is added; A1 is the absorbance of the DPPH free radical working solution.

[0099] ABTS free radical scavenging experiment: Take 500μL (10mg / mL) of fermented Lactobacillus mucilaginosus A10 extracellular polysaccharide solution, add 3.8mL of ABTS free radical dilution working solution with a concentration of 5mM, pH=7.4, and an absorbance of 0.7-0.8 at 734nm, react at room temperature for 6 minutes, and measure the absorbance value at 734nm. Distilled water is used as a blank control. Calculate the ABTS free radical scavenging rate according to the following formula:

[0100]

[0101] Where: A is the absorbance when adding sample; A0 is the absorbance of the solution when adding distilled water; A1 is the absorbance of ABTS free radical working solution.

[0102] Hydroxyl free radical scavenging experiment: aspirate 400 μL of fermented Lactobacillus mucilaginosus A10 extracellular polysaccharide solution and add it into a 5 mL centrifuge tube, add 400 μL of salicylic acid-ethanol solution (9 mmol / L), 400 μL of FeSO4 solution (9 mmol / L) and 400 μL of H2O2 solution (8.8 mmol / L), stir evenly, and measure the absorbance at 510 nm.

[0103]

[0104] In the formula: A0 is 400μL water + 400μL salicylic acid-ethanol solution + 400μL FeSO4 solution + 400μL H2O2 solution; A1 is 400μL sample + 400μL salicylic acid-ethanol solution + 400μL FeSO4 solution + 400μL H2O2 solution; A2 is 400μL sample + 400μL salicylic acid-ethanol solution + 400μL FeSO4 solution + 400μL distilled water.

[0105] The in vitro antioxidant activity of exopolysaccharide from fermented Lactobacillus mucilaginosus A10 was evaluated by scavenging experiments on DPPH free radical, ABTS free radical and hydroxyl free radical. Fig.12As shown. The DPPH radical, ABTS radical and hydroxyl radical of the exopolysaccharide of Lactobacillus mucilaginosus A10 fermented were dose-dependent. The DPPH radical scavenging activity of 10 mg / mL fermented exopolysaccharide of Lactobacillus mucilaginosus A10 was 81.8%, which was higher than the 41.79%-78.66% of the exopolysaccharide of thermophilic Streptococcus CGMCC 7.179 and the 22.61%-56.03% of halophilic Tetra Streptococcus (10 mg / mL) in the existing literature. The ABTS radical scavenging activity of 10 mg / mL fermented exopolysaccharide of Lactobacillus mucilaginosus A10 was 71.7%, which was higher than the 22%-42% of halophilic Tetra Streptococcus (10 mg / mL) in the existing literature. In addition, the scavenging rate of 10 mg / mL fermented Lactobacillus mucilaginosus A10 exopolysaccharide on hydroxyl radicals was 63.8%, which was higher than the 20%-40% of exopolysaccharide of Streptococcus thermophilus CGMCC 7.179 in the existing literature. The above results show that the exopolysaccharide of fermented Lactobacillus mucilaginosus A10 has good free radical scavenging activity and can act as an electron or hydrogen donor to scavenge free radicals.

[0106] Cellular antioxidant activity

[0107] The protective effect of exopolysaccharide of fermented Lactobacillus mucilaginosus A10 on HepG2 cells damaged by H2O2 was determined by MTT method. Firstly, the toxic effect of exopolysaccharide on HepG2 cells was evaluated. HepG2 cells in logarithmic growth phase were cultured at 1.0×10 5 The cells were inoculated into 96-well plates at a density of 100 μg / mL and cultured at 37°C and 5% CO2 for 24 h. After the culture, 50 μL of different concentrations of extracellular polysaccharides (0.125, 0.25, 0.5, 1.0 and 2.0 mg / mL) were added and cultured for another 24 h. Then, the degree of oxidative damage to HepG2 cells by different concentrations of H2O2 (200, 400, 600, 800, 1,000, 1,200 and 1,400 μmol / L) was determined in the same way to determine the optimal H2O2 concentration for inducing oxidative damage. The protective effect of extracellular polysaccharides on HepG2 at the optimal H2O2-induced damage concentration (1,000 μmol / L) was determined. In the experimental group, cells were treated with different concentrations of extracellular polysaccharides (0.125-2.0 mg / mL) for 24 h, and then treated with 1,000 μmol / L H2O2 for 4 h. Untreated cells served as the control group. Then add MTT solution and continue to culture for 4 hours, then replace the culture medium with 150 μL dimethyl sulfoxide and shake the plate. Finally, measure the absorbance value at 570 nm with an ELISA reader. The calculation method of cell viability is as follows:

[0108]

[0109] Where: A T is the absorbance of the protection group, AC is the absorbance of the control group.

[0110] The results of the cytotoxicity experiment showed that the extracellular polysaccharide of fermented Lactobacillus mucilaginosus A10 had no cytotoxic effect between 0.125-2.0 mg / mL (13A). Based on the construction of the H2O2-induced HepG2 cell oxidative damage model (13B), 0.5-2.0 mg / mL of extracellular polysaccharide of fermented Lactobacillus mucilaginosus A10 was used to evaluate its protective effect on H2O2-induced HepG2 cell oxidative damage. The results are as follows Fig.13 C. Compared with the control group, the survival rate of HepG2 cells decreased to 52.81% after 4h treatment with 1000μmol / L H2O2, while the cell survival rate was significantly increased after 24h treatment with exopolysaccharides (P < 0.05), indicating that exopolysaccharides can effectively inhibit H2O2-induced oxidative damage. Among them, the cell survival rate of the group treated with exopolysaccharides at a concentration of 2.0mg / mL was as high as 71.40%, which was about 1.50 times higher than that of the group treated with H2O2-induced oxidative damage, indicating that the exopolysaccharide of Lactobacillus mucilaginosus A10 plays an important role in reducing cellular oxidative stress and oxidative damage.

[0111] Example 8: Fermented Lactobacillus mucilaginosus A10 exopolysaccharide exerts antioxidant activity by activating the Keap1-Nrf2 pathway

[0112] Molecular docking analysis of the monosaccharides rhamnose, galactose, glucose, mannose and glucuronic acid in the exopolysaccharide composition of Lactobacillus mucilaginosus A10 with Keap1 protein (PDB ID: 2FLU) was performed using AutoDock Vina software. The three-dimensional structures of rhamnose, galactose, glucose, mannose and glucuronic acid (CID numbers 25310, 6036, 5793, 18950 and 94715, respectively) were downloaded from the PubChem database (http: / / PubChem.ncbi.nlm.nih.gov). The highest-scoring conformations were selected and visually analyzed using PyMOL 2.4.1 software.

[0113] The results of cell experiments showed that the extracellular polysaccharide of fermented Lactobacillus mucilaginosus A10 had good cellular antioxidant activity, and the extracellular polysaccharide may exert antioxidant activity by activating the Keap1-Nrf2 / ARE signaling pathway. Therefore, molecular docking technology was used to study the molecular mechanism by which each monosaccharide component interacts with the Keap1 protein to activate the Keap1-Nrf2 / ARE signaling pathway and exert antioxidant activity. The results are as follows Fig.14As shown. The binding energies of rhamnose, galactose, glucose, mannose and glucuronic acid with Keap1 protein are -6.4, -6.7, -6.6, -6.7 and -7.2 kcal / moL, respectively, indicating that each monosaccharide is tightly bound to Keap1 protein. Hydrogen bonding is the main interaction force between each monosaccharide component and Keap1 protein. Rhamnose, galactose, glucose, mannose and glucuronic acid form 8, 13, 7, 12 and 10 hydrogen bonds with Keap1 protein, respectively. Among them, the hydrogen bonds formed between galactose, glucose, mannose, glucuronic acid and Keap1 protein are mainly NH and OH, while the carboxyl group (-COOH-) in glucuronic acid can bind to the amino group (-NH 3+ ) to form an amide bond, forming an exopolysaccharide-Keap1 electrostatic complex. These results indicate that the exopolysaccharide of fermented Lactobacillus mucilaginosus A10 can exert antioxidant activity by activating the Keap1-Nrf2 / ARE signaling pathway, and is expected to be developed into a natural antioxidant that interacts with Keap1-Nrf2 in a targeted manner.

[0114] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, but rather, the present invention extends to all other methods and applications with the same functions.

Claims

1. A fermented mucus lactobacillus, characterized in that The fermented mucus lactobacillus (Limosilactobacillusfermentum) is named: fermented mucus lactobacillus A10, and was deposited in the China Center for Type Culture Collection on September 25, 2024, with a preservation number of CCTCC NO: M20242082, and the preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A microbial agent containing the fermented mucus lactobacillus according to claim 1.

3. The use of the fermented mucus lactobacillus according to claim 1 or the microbial agent according to claim 2 in antibacterial treatment, characterized in that: The antibacterial application is for non-therapeutic purposes; the antibacterial effect refers to the antibacterial effect on Escherichia coli or Staphylococcus aureus.

4. Use of the fermented mucus lactobacillus according to claim 1 or the microbial agent according to claim 2 in producing exopolysaccharides.

5. An extracellular polysaccharide, characterized in that The exopolysaccharide is obtained by fermentation of the fermented mucus lactobacillus according to claim 1 or the microbial agent according to claim 2.

6. The exopolysaccharide according to claim 5, characterized in that The extracellular polysaccharide is a heteropolysaccharide composed of rhamnose, galactose, glucose, mannose and glucuronic acid; the molar ratio of rhamnose, galactose, glucose, mannose and glucuronic acid is 0.13-0.16:1.80-1.97:1.09-1.11:0.88-1.00:0.26-0.

27.

7. Use of the exopolysaccharide according to claim 5 or 6 as a natural antioxidant.

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