CN115161237B - Bacillus coagulans capable of degrading lipopolysaccharide and inhibiting alpha-glucosidase and application thereof - Google Patents
Bacillus coagulans capable of degrading lipopolysaccharide and inhibiting alpha-glucosidase and application thereof Download PDFInfo
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- CN115161237B CN115161237B CN202210833845.3A CN202210833845A CN115161237B CN 115161237 B CN115161237 B CN 115161237B CN 202210833845 A CN202210833845 A CN 202210833845A CN 115161237 B CN115161237 B CN 115161237B
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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Abstract
The invention discloses bacillus coagulans SA12 capable of degrading lipopolysaccharide and inhibiting alpha-glucosidase and application thereof. The SA12 strain was stored in the Guangdong province strain collection at 6/2022, with the storage number: GDMCC NO. 62518. The bacillus coagulans SA12 is separated from mulberry enzyme, has strong lipopolysaccharide degrading capability, alpha-glucosidase inhibiting capability and DPPH free radical inhibiting capability, has a lipopolysaccharide removing rate of 80EU/mL for 4 hours as high as 91.55%, and can realize the effect of relieving diabetes by inhibiting alpha-glucosidase which is a key enzyme causing diabetes. The bacillus coagulans SA12 provided by the invention not only can remove lipopolysaccharide so as to achieve anti-inflammatory effect, but also can relieve diabetes, and provides a novel microorganism strain for degrading lipopolysaccharide and inhibiting alpha-glucosidase.
Description
Technical Field
The invention belongs to the technical field of microorganisms. More particularly, it relates to a bacillus coagulans strain capable of degrading lipopolysaccharide and inhibiting alpha-glucosidase and application thereof.
Background
Lipopolysaccharide is the most predominant component on the outer membrane of gram-negative bacteria, also known as endotoxin, and is a potent stimulator of inflammation. It has been found that Toll-like receptor 4 (TLR 4)/nuclear factor κb (NF- κb) signaling pathway is the most important downstream pathway of lipopolysaccharide-mediated signaling pathway, and that TLR4 receptor recognizes and binds lipopolysaccharide, and further activates NF- κb signaling pathway to induce release of inflammatory factors, resulting in inflammation, and tissue damage such as bacterial infection, neurological disease, cardiovascular disease, renal failure, metabolic syndrome, and endotoxin sepsis. Both cofactors Lipopolysaccharide Binding Protein (LBP) and leukocyte differentiation antigen 14 (CD 14) allow lipopolysaccharide to be effective in stimulating inflammatory responses. When lipopolysaccharide binds to TRL4 receptor, NF- κB signal factor can be activated, and the expression of inflammatory factors such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-6 (IL-6) can be started and regulated, and the expression of NLRP3 inflammatory corpuscle can be up regulated, so that lots of endogenous bioactive inflammatory factors can be secreted and released, and systemic inflammatory reaction syndrome can be caused.
Diabetes is a disease of the endocrine system caused by inheritance or the acquired environment, and is also a disease threatening human life and health. In the intestinal flora of type 2 diabetics, the relative abundance of gram-negative bacteria is high, while the main compound of the outer membrane of gram-negative bacteria is lipopolysaccharide, which is continuously produced in the intestinal tract after death of gram-negative bacteria, and can strongly stimulate the release of key induction factors of insulin resistance. Lipopolysaccharide binds to TLR-4 activated signaling cascades that are capable of inhibiting insulin in islets, but have no inhibitory effect on TLR-4 deficient mouse insulin secretion. When lipopolysaccharide binds to CD14, it can be used as a carrier to trigger obesity/insulin resistance caused by high fat ingestion, and CD14 knockout mice are resistant to diet-induced obesity and related diseases such as liver insulin resistance. Whereas α -glucosidase, as a key enzyme on the mucous membrane of the small intestine, regulates postprandial blood glucose by affecting the release and absorption of glucose. Therefore, the inhibition of postprandial blood glucose elevation of diabetics can be achieved by inhibiting the activity of alpha-glucosidase, reducing the polysaccharide content to avoid obesity/insulin resistance caused by high fat ingestion and delaying the absorption of carbohydrates by the intestinal tract.
Most of the prior researches are to relieve diabetes by inhibiting alpha-glucosidase, and only Bifidobacterium (Bifidobacterium) F-35, lactobacillus rhamnosus (Lactobacillus rhamnosus) GG, lactobacillus plantarum (Lactobacillus plantarum), lactobacillus acidophilus NM (Lactobacillus acidophilus) and the like can inhibit the alpha-glucosidase. However, the microorganism disclosed in the prior art can only inhibit alpha-glucosidase and does not have the function of degrading lipopolysaccharide, for example, the prior art discloses that the inhibition rate of bacillus coagulans JA845 on the alpha-glucosidase is 39.51%, and the inhibition rate is still to be improved. It can be seen that there are too few microbial strains capable of inhibiting α -glucosidase and degrading lipopolysaccharide, and there is a lack of microbial strains capable of inhibiting α -glucosidase while also degrading lipopolysaccharide. Therefore, in order to obtain more microorganism strains with the functions of inhibiting alpha-glucosidase and degrading lipopolysaccharide, more new multifunctional microorganism strains with better efficiency need to be screened, and a microorganism strain resource library is enriched.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the problems and provide bacillus coagulans capable of degrading lipopolysaccharide and relieving diabetes and application thereof.
The invention aims to provide a bacillus coagulans (Weizmannia coagulans) SA12 strain.
Another object of the invention is to provide the use of said Bacillus coagulans SA12 strain.
It is a further object of the present invention to provide a formulation with lipopolysaccharide degrading and/or alpha-glucosidase inhibiting properties.
It is a further object of the present invention to provide a method for degrading lipopolysaccharide and/or inhibiting alpha-glucosidase.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a bacillus coagulans (Weizmannia coagulans) SA12 strain which is separated from naturally fermented mulberry residues and is preserved in the microorganism strain collection (GDMCC) of Guangdong province in 2022, 6 months and 6 days, wherein the preservation number is as follows: GDMCC NO. 62518. The research of the invention shows that the SA12 strain has stronger DPPH free radical removal capability, and the DPPH free radical removal rate reaches 37.95 percent; has the capability of efficiently degrading lipopolysaccharide, and the clearance rate of the lipopolysaccharide reaches 91.55 percent; meanwhile, the SA12 strain can inhibit alpha-glucosidase, the inhibition effect on the alpha-glucosidase reaches 42.59 percent compared with an enzyme inhibitor acarbose, the inflammatory reaction can be effectively reduced, and the diabetes mellitus can be relieved.
The invention provides an application of bacillus coagulans SA12 strain and/or fermentation liquor thereof in degrading lipopolysaccharide and/or preparing lipopolysaccharide degrading bacterial agents, inhibiting alpha-glucosidase and/or preparing alpha-glucosidase inhibitors, removing DPPH free radicals or preparing preparations for removing DPPH free radicals, and preparing medicines for relieving diabetes.
The invention provides a preparation for degrading lipopolysaccharide and/or inhibiting alpha-glucosidase, which contains bacillus coagulans SA12 strain and/or fermentation liquor thereof.
Preferably, the SA12 strain is added in an amount of 0.1 to 10%.
More preferably, the SA12 strain is added in an amount of 0.2%
Preferably, the fermentation broth is a supernatant from which the cells are removed.
Preferably, the fermentation conditions are: 30-40 ℃, 10-15 h and 150-200 r/min.
More preferably, the fermentation conditions are: 37 ℃ and 12h,180r/min.
The invention provides a method for degrading lipopolysaccharide and/or inhibiting alpha-glucosidase, which adopts bacillus coagulans SA12 strain and/or fermentation liquor thereof to treat the lipopolysaccharide and/or the alpha-glucosidase.
The invention has the following beneficial effects:
the invention provides a bacillus coagulans (Weizmannia coagulans) SA12 strain, which is obtained by separating from mulberry ferment in natural fermentation, wherein the SA12 strain has stronger acid production capacity and DPPH free radical removal capacity, and the clearance rate of the SA12 strain to the DPPH free radical reaches 37.95%. The research shows that the SA12 strain has the capacity of efficiently degrading lipopolysaccharide, the content of the lipopolysaccharide in the added Bacillus coagulans SA12 is reduced from the original concentration of 80U/mL to 6.76U/mL, and the clearance rate of the lipopolysaccharide is 91.55%. The bacillus coagulans SA12 has stronger lipopolysaccharide removing capability and can effectively reduce inflammatory response induced by lipopolysaccharide. Meanwhile, the bacillus coagulans SA12 also has the capacity of inhibiting alpha-glucosidase, and the common alpha-glucosidase inhibitor, acarbose, is used as a positive control (the inhibition rate of the positive control to the alpha-glucosidase is close to 100%), the inhibition rate of the SA12 strain to the alpha-glucosidase is 42.59%, and the diabetes can be relieved by inhibiting the alpha-glucosidase which is a key enzyme causing diabetes. The bacillus coagulans SA12 provided by the invention not only can remove lipopolysaccharide so as to achieve anti-inflammatory effect, but also can relieve diabetes, and provides a novel microorganism strain for degrading lipopolysaccharide and inhibiting alpha-glucosidase.
Drawings
FIG. 1 is a diagram of bacterial colonies;
FIG. 2 is a strain evolutionary tree;
FIG. 3 shows the DPPH clearance of the strain;
FIG. 4 shows the lipopolysaccharide clearance of the strain;
FIG. 5 shows the inhibition ratio of strain alpha-glucosidase.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
MRS liquid medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of beef extract, 20g/L of anhydrous dextrose, 2g/L of dipotassium hydrogen phosphate, 5g/L of anhydrous sodium acetate, 2g/L of tri-ammonium citrate, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate and 1g/L of tween 80 are prepared by distilled water.
MRS solid medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of beef extract, 20g/L of anhydrous dextrose, 2g/L of dipotassium hydrogen phosphate, 5g/L of anhydrous sodium acetate, 2g/L of triammonium citrate, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 1g/L of tween 80, 15g/L of agar and distilled water.
TSB medium: 17g/L tryptone, 3g/100L soyase peptone, 5g/L sodium chloride, 2.5g/L K2HPO4 and 2.5g/L glucose, and distilled water.
Example 1 screening and identification of strains
(1) Isolation of strains
Fresh mulberries are selected from the market, washed clean, put into a fermentation tank, added with sugar water with different concentrations and subjected to light-proof natural fermentation. Weighing appropriate amount of the above fermentation residues, grinding, adding MRS liquid culture medium in conical flask, and culturing in shaking table at 37deg.C for 48 hr. After culturing, 1mL of the mixed bacterial solution was extracted from the culture medium, subjected to gradient dilution with sterile water, and coated on an MRS solid plate with 10 -2 ~10 -6 Five dilutions were performed and incubated at 37℃for 48h. After the cultivation, different single colonies were randomly picked from the plate and streaked on MRS solid medium for purification, and cultivated at 37℃for 48h. The obtained pure culture strain is washed by 20% glycerol, sucked into a glycerol pipe and stored in a refrigerator at the temperature of minus 20 ℃ for standby.
(2) Morphological identification
The strain was inoculated on a medium plate and cultured upside down at 37℃for 24 hours, and the colony morphology was observed. The bacterial colony morphology of the strain cultured in the culture medium for 24 hours is shown in figure 1, the bacterial colony presents opaque milky white, the surface is smooth and moist, the strain is positive through gram staining, and the proper growth temperature range of the strain is 37-50 ℃ and the proper growth pH range is 6-7.
(3) Molecular biological identification of bacteria
The purified bacteria were subjected to DNA extraction by SDS method, and the conserved sequences of the bacteria were amplified using the universal primers 27f and 1492 r. And then sending the amplified product to a sequencing company for sequencing, splicing the obtained sequences, comparing the spliced sequences in an NCBI database to obtain a plurality of different bacteria, and according to repeated comparison results, wherein 22 strains are the same genus, the 16S rRNA gene sequence of the bacteria has the highest homology with Bacillus coagulans (Weizmannia coagulans) (note: bacillus coagulans is updated with Latin names now and is updated with Weizmannia coagulans from original Bacillus coagulans, and the invention adopts the latest Latin names for naming and preserving), wherein the phylogenetic tree of five strains is shown in figure 2. Thus, the strain of the same genus was identified as Bacillus coagulans (Weizmannia coagulans) and designated SA1 to SA22.
And (3) measuring the acid production capacity and DPPH free radical removal capacity of the identified bacillus coagulans, and screening out bacillus coagulans with better DPPH free radical removal capacity by measuring, wherein the DPPH removal rate is shown in a graph 3, and the clearance rate of the SA12 strain to the DPPH free radicals reaches 37.95 percent and is obviously superior to other bacillus coagulans. Thus, the Bacillus coagulans SA12 with excellent performance was determined, the subsequent experiments were carried out by using SA12 strain, and Bacillus coagulans (Weizmannia coagulans) SA12 was deposited with the microorganism strain deposit center (GDMCC) of Guangdong province at 6/2022, with the accession number of GDMCC NO:62518, and the deposit address of: no. 100 is a first-break middle road in the Guangzhou city, vaxiu district.
EXAMPLE 2 lipopolysaccharide degradation experiment with Strain SA12
Since MRS medium interferes with the method of measuring lipopolysaccharide content by limulus reagent dynamic chromogenic method, TSB medium was selected for experiment. Bacillus coagulans SA12 isolated in example 1 was used in an inoculum size of 0.2% in sterilized TSB medium and incubated at 37℃in a 180r/min shaker for 12 h. 5mL of bacterial liquid is sucked from the cultured bacillus coagulans SA12 bacterial liquid and placed in a centrifugal tube without a heat source, the centrifugal tube is centrifuged for 4000r/min and 10min, the supernatant after the suction and centrifugation is placed in a test tube without the heat source, bacterial mud is resuspended for 3 times with sterile water without lipopolysaccharide to wash off residual culture medium, and the residual culture medium is complemented to 5mL with the sterile water without lipopolysaccharide.
The experimental treatment group is to remove the supernatant of bacillus coagulans SA12 thalli; the control group was blank TSB medium without access to Bacillus coagulans SA12 broth.
Respectively collecting bacterial liquid supernatant, and culturing with TSBIn the medium, TSB blank medium was prepared, and 1mL of lipopolysaccharide of 60EU was added to each medium, and reacted at 37℃for 0h and 4h, respectively. Respectively taking a 0h reacted solution and a 4h reacted solution, and respectively diluting the solution by 200 times, 400 times and 800 times. Then, respectively taking 0.1mL of diluted solution, adding the diluted solution into a pyrogen-removing micro-pore plate, adding 3 holes in each concentration, respectively adding 0.1mL of limulus reagent, uniformly mixing by medium-speed shaking for 10s, putting the micro-pore plate into a preheated endotoxin automatic analyzer ELx808 for detection, simultaneously establishing a standard curve by adopting standard endotoxin with concentrations of 0.005EU/mL, 0.05EU/mL, 0.5EU/mL and 5EU/mL, setting at least 3 parallel holes in each concentration of endotoxin solution, carrying out negative control on the parallel holes, and taking the average value of the results of three parallel treatments. Then according to the standard curve y= -0.3039x+2.8422, R 2 =0.998, y is time(s), x is mass concentration in EU/mL, endotoxin concentration is calculated.
The results are shown in FIG. 4, where TSB medium (without lipopolysaccharide removal) and 60EU lipopolysaccharide at 0h contained a total of 80U/mL lipopolysaccharide. During the period of 0-4 hours, the lipopolysaccharide content in the TSB of the control group was reduced from the original concentration of 80U/mL to 63.24U/mL by 16.76U/mL, while the lipopolysaccharide content in the supernatant group of Bacillus coagulans SA12 of the experimental group was changed from the original concentration of 80U/mL to 6.76U/mL in the control group, and the clearance was calculated as a change amount compared with the original concentration, and the lipopolysaccharide clearance of Bacillus coagulans SA12 was found to reach 91.55%. The results indicate that fermentation supernatant of bacillus coagulans SA12 can significantly degrade lipopolysaccharide.
EXAMPLE 3 experiments on inhibition of alpha-glucosidase Activity by Strain SA12
Taking an equal volume of alpha-glucosidase solution without Bacillus coagulans as a control group; taking an alpha-glucosidase solution without a substrate as a blank group; the commonly used alpha-glucosidase inhibitor acarbose is used as a positive control group (control). Three replicates of each of the above groups were run and inhibition was calculated by measuring absorbance of each group.
The inhibition rate was calculated as follows:
wherein: a is that n : absorbance at 400nm for SA12 strain samples;
A B : absorbance values for the blank group;
A C : is the absorbance of the control group.
As shown in FIG. 5, the inhibition rate of alpha-glucosidase in the acarbose positive control group was 100%, while the inhibition rate of alpha-glucosidase using SA12 strain reached 42.59%, indicating that Bacillus coagulans SA12 strain can inhibit alpha-glucosidase, thereby alleviating diabetes and being a high-efficiency strain for glycemic control.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. Bacillus coagulans strainWeizmannia coagulans) SA12 strain, characterized in that it was deposited with the cantonese province microorganism strain collection (GDMCC) at 6/2022, accession number: GDMCC NO. 62518.
2. The use of bacillus coagulans SA12 strain and/or a fermentation broth thereof according to claim 1 for the preparation of lipopolysaccharide degrading bacterial agents.
3. Use of a bacillus coagulans SA12 strain and/or a fermentation broth thereof according to claim 1 for the preparation of an alpha-glucosidase inhibitor.
4. Use of the bacillus coagulans SA12 strain and/or the fermentation broth thereof according to claim 1 for the preparation of a DPPH radical scavenging formulation.
5. Use of the bacillus coagulans SA12 strain and/or the fermentation broth thereof according to claim 1 for the preparation of a medicament for alleviating diabetes.
6. A preparation for degrading lipopolysaccharide and/or inhibiting alpha-glucosidase, comprising the Bacillus coagulans SA12 strain and/or a fermentation broth thereof according to claim 1.
7. The preparation according to claim 6, wherein the SA12 strain is added in an amount of 0.1 to 10%.
8. The formulation of claim 6, wherein the fermentation conditions are: 30-40 ℃, 10-15 h and 150-200 r/min.
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