CN115948292B - Lactobacillus reuteri strain A21041 with anti-inflammatory and antioxidant functions and application thereof - Google Patents
Lactobacillus reuteri strain A21041 with anti-inflammatory and antioxidant functions and application thereof Download PDFInfo
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Abstract
The invention provides a lactobacillus reuteri strain with anti-inflammatory and antioxidant functionsLimosilactobacillus reuteri) Strain a21041 and application thereof, belonging to the technical field of functional microorganisms. The lactobacillus reuteri A21041 is separated from a sample of intestinal microbial flora of the century old, and the preservation number is GDMCC No. 62832. The strain A21041 has the characteristics of well tolerating the stimulation of artificial gastrointestinal fluid and intestinal bile salts and inhibiting the growth of pathogenic bacteria, has anti-inflammatory effect, can effectively relieve ulcerative colitis of mice, and also has the effects of scavenging free radicals and enhancing antioxidant enzyme systems. Therefore, the invention provides the lactobacillus reuteri A21041 with anti-inflammatory and antioxidant effects, which can be widely used in foods, health-care products or medicines so as to achieve the purpose of preventing or relieving colonitis.
Description
Technical Field
The invention belongs to the technical field of functional microorganisms, and particularly relates to a lactobacillus reuteri (Limosilactobacillus reuteri) strain A21041 with anti-inflammatory and antioxidant functions and application thereof.
Background
Probiotics are beneficial microorganisms for human bodies, can be used as targets for regulating intestinal microecology balance, and can relieve intestinal stress, inhibit inflammatory reaction and further improve intestinal syndrome by regulating the abundance of intestinal flora and metabolic products thereof. In recent years, the incidence rate of oxidative stress colitis is obviously increased in China, and the colitis has recurrent characteristics and also has the risk of canceration. Clinically, western medicines such as non-steroidal anti-inflammatory drugs, glucocorticoids, immunosuppressants and the like are commonly used for relieving symptoms, but the long-term use of the traditional Chinese medicine composition can cause the problems of reduced curative effect, increased adverse reaction and the like.
At present, more and more researches are conducted on the treatment of stress colitis through the action mechanisms of inhibiting inflammatory response, relieving oxidative stress, regulating intestinal flora imbalance and the like. Many studies have shown that probiotics can be used to treat oxidative stress colitis, and the patent publication No. CN115011518A discloses a lactic acid bacteria mixture comprising bifidobacterium bifidum H3-R2 and lactococcus lactis KLDS4.0325, which can improve the oxidative damage of colon tissue of CAC mice, reduce the level of inflammatory factors IL-6 and TNF-alpha, and simultaneously up-regulate the level of anti-inflammatory cytokine IL-10 in colon and serum, and has the effect of relieving colorectal cancer related to colitis. The patent of publication No. CN112625964B discloses lactobacillus rhamnosus which has good biological characteristics, relieves the weight loss of ulcerative colitis mice, improves the stool characteristics and hematochezia condition, improves the oxidative damage of colon mucous membrane, reduces the content of pro-inflammatory factors TNF-alpha, IL-1 beta, IL-6 and IFN-gamma in colon, improves the richness and diversity of intestinal flora, and plays the roles of anti-inflammatory and antioxidant together to treat oxidative stress colitis. Therefore, the research and development of the probiotics for preventing and treating the stress colitis have clinical application value, and the probiotics can assist in enhancing the drug effect of western medicines for treating intestinal syndromes, reduce the dosage of the western medicines and avoid serious adverse reactions caused by accumulation of the western medicines.
However, there is no report on the effect of lactobacillus reuteri in inhibiting inflammatory response and relieving oxidative stress.
Disclosure of Invention
Therefore, the invention aims to provide the lactobacillus reuteri (Limosilactobacillus reuteri) strain A21041 which has the functions of anti-inflammatory and anti-oxidation and provides a new means for preventing and treating the oxidative stress colonitis.
The invention provides a lactobacillus reuteri (Limosilactobacillus reuteri) strain A21041 with anti-inflammatory and antioxidant functions, wherein the preservation number of the strain A21041 is GDMCC No. 62832.
The invention provides a probiotic agent, which comprises lactobacillus reuteri strain A21041 with anti-inflammatory and antioxidant functions and auxiliary materials.
Preferably, the live bacteria concentration of lactobacillus reuteri strain a21041 is 10 8 ～10 10 CFU/g；
The probiotic preparation is in the form of oral preparation.
The invention provides application of the lactobacillus reuteri strain A21041 or the probiotics in preparation of antibacterial products.
Preferably, the species of the antibacterial middle bacteria include pathogenic bacteria;
preferably, the pathogenic bacteria include staphylococcus aureus and/or escherichia coli.
The invention provides application of lactobacillus reuteri strain A21041 or the probiotics in preparation of a medicament for preventing and/or treating colonitis.
Preferably, the lactobacillus reuteri strain A21041 inhibits the expression of COX-2/iNOS, inhibits the expression of downstream pro-inflammatory factors IL-6 and TNF-alpha, increases the expression of anti-inflammatory factors IL-10, reduces the release of NO, and further achieves the effect of preventing and treating colonitis.
The invention provides application of lactobacillus reuteri strain A21041 or the probiotics in preparing anti-inflammatory and antioxidant products.
Preferably, the lactobacillus reuteri strain A21041 has superoxide dismutase and glutathione peroxidase activities and has the capability of scavenging superoxide anions and DPPH free radicals.
Preferably, the product comprises at least one of: pharmaceutical and cosmetic products.
The invention provides lactobacillus reuteri (Limosilactobacillus reuteri) strain A21041 with anti-inflammatory and antioxidant functions, and the preservation number is GDMCC No. 62832. The strain A21041 has good biological characteristics of resisting the stimulation of artificial gastrointestinal fluid and intestinal bile salts and inhibiting the growth of escherichia coli and staphylococcus aureus pathogenic bacteria. Strain A21041 can obviously reduce the expression of inflammatory factors COX-2, iNOS, IL-6 and TNF-alpha produced by RAW264.7 cells induced by LPS, up-regulate the expression of anti-inflammatory factor IL-10 and effectively reduce the release of NO; a21041 can also remarkably prolong the length of colon after DSS induces colonitis of mice, inhibit the expression of IL-6 in colon tissue, increase the expression of IL-10 and effectively relieve ulcerative colitis of the mice; a21041 also can remove DPPH and O 2 - Free radical and the function of enhancing SOD and GSH-Px antioxidant enzyme system. Therefore, the invention provides lactobacillus reuteri A21041 with anti-inflammatory and antioxidant effects, which can be used for or added into foods, health-care foods or medicines and can be used for preventing and treating oxidative stress colitis.
Drawings
FIG. 1 is a photomicrograph of A21041;
FIG. 2 is a graph of A21041 growth curve;
FIG. 3 is a graph showing the results of tolerance of A21041 to artificial gastrointestinal fluids;
FIG. 4 is a graph showing the results of resistance of A21041 to bovine bile salts;
FIG. 5 shows the results of inhibition of E.coli and Staphylococcus aureus by A21041;
FIG. 6 shows the relative expression level of COX-2\iNOS gene in RAW264.7 cell inflammatory state by A21041; and (3) injection: * Represents the comparison of LPS group with Control group, # represents the comparison of LPS+A21041 group with LPS group;
FIG. 7 shows the effect of A21041 on IL-6\TNF- α\IL-10\iNOS protein expression in RAW264.7 cell inflammatory state; and (3) injection: * Represents the comparison of LPS group with Control group, # represents the comparison of LPS+A21041 group with LPS group;
FIG. 8 shows the results of NO release in inflammatory conditions of RAW264.7 cells by A21041; and (3) injection: * Represents the comparison of LPS group with Control group, # represents the comparison of A21041 group with LPS group;
FIG. 9 is a graph showing the effect of A21041 on colon length in colon inflammatory mice; * Represents the comparison of the model group with the normal group, # represents the comparison of the mesalamine group or the A21041-H group with the model group
FIG. 10 is a graph showing the effect of A21041 on IL-6\IL-10 protein expression in colon tissue of a colon inflammatory mouse, and is annotated: * The model group is shown in comparison with the normal group, and # shows that the mesalamine group, the A21041-H group or the A21041-D group is compared with the model group.
Biological material preservation information
Lactobacillus reuteri (Limosilactobacillus reuteri) strain A21041 was deposited with the microorganism strain collection in Guangdong, the unit is GDMCC, the address is building 5 No. 59 of the 100 th university of Mitsui, guangdong province, the deposit number is GDMCCNo 62832, and the deposit date is 2022, 9 months and 23 days.
Detailed Description
The invention provides a lactobacillus reuteri (Limosilactobacillus reuteri) strain A21041 with anti-inflammatory and antioxidant functions, wherein the preservation number of the strain A21041 is GDMCC No. 62832.
In the invention, lactobacillus reuteri a21041 is isolated from a sample of intestinal microbiota of the century old. The strain is deposited in the Guangdong province microorganism strain collection center, the deposition number is GDMCC No. 62832, and the deposition date is 2022, 9 and 23. The microscopic morphology of the strain A21041 was observed by gram staining, and the growth state of the strain A21041 was examined by ultraviolet spectrophotometry, which revealed that the strain A21041 was a gram-positive bacterium, the cell morphology was a short rod shape, and the cell size (width. Times. Length) was (0.5 to 1.0) μm. Times (1.0 to 2.0) μm. The growth period is 3h before the stationary adaptation period, 3h-12h is logarithmic growth phase and OD 600 The value change is very large, the strain is rapidly increased, the strain is in a plateau period from 12h to 30h, the growth trend of the strain tends to be gentle, and the decay period does not occur; a21041 was subjected to 16S rDNA and whole genome assayAnd, lactobacillus reuteri (Limosilactobacillus reuteri).
In the invention, the lactobacillus reuteri A21041 has the functions of resisting artificial gastrointestinal fluid, intestinal canal bile salt and in-vitro bacteriostasis, and has stronger functions of resisting the artificial intestinal fluid with pH 8 and the bovine bile salt solution than the commercial strain 17938, and the viable count of the treated A21041 reaches 10 after 8 hours 9 CFU, survival rate is more than 80%, and preliminary indicates that A21041 can reach the intestinal tract smoothly through gastrointestinal fluid stimulation, and plays a role in the intestinal tract.
In the present invention, the method for amplifying lactobacillus reuteri a21041 preferably comprises the steps of:
and (3) picking single bacterial colony of the A21041, inoculating the single bacterial colony to an MRS liquid culture medium, and culturing the single bacterial colony at 37 ℃ for 12-14 hours to obtain suspension of the A21041 bacteria in a para-anagen phase.
The invention provides a probiotic agent, which comprises lactobacillus reuteri strain A21041 with anti-inflammatory and antioxidant functions and auxiliary materials.
In the present invention, the viable bacteria concentration of the Lactobacillus reuteri strain A21041 is preferably 10 8 ～10 10 CFU/g；
In view of the good properties of the lactobacillus reuteri strain a21041 against gastrointestinal fluids and bovine bile salt solutions, the probiotic is preferably in the form of an oral dosage form, e.g. powder, oral liquid, tablet, etc.
The invention provides application of the lactobacillus reuteri strain A21041 or the probiotics in preparation of antibacterial products.
In the present invention, the species of the antibacterial bacteria preferably include pathogenic bacteria. The pathogenic bacteria preferably include staphylococcus aureus and/or escherichia coli. The in vitro antibacterial experiment result shows that the strain A21041 obviously inhibits the growth of escherichia coli and staphylococcus aureus, and the inhibition effect is obviously better than that of the strain 17938.
The invention provides application of lactobacillus reuteri strain A21041 or the probiotics in preparation of a medicament for preventing and/or treating colonitis.
In the present invention, the lactobacillus reuteri strain a21041 preferably inhibits expression of downstream pro-inflammatory factors IL-6 and TNF-alpha by inhibiting expression of COX-2\iNOS, increases expression of anti-inflammatory factor IL-10, reduces release of NO, and further achieves the effect of preventing and treating colonitis. In the embodiment of the invention, a DSS induced mouse colonitis model is taken as an experimental object, and the result of the bacterium liquid of the intragastric mouse model A21041 shows that compared with a normal group, the colon of the mouse in the model group is extremely obviously shortened; compared with the model group, group A21041-H (1X 10 10 CFU/ml,0.2 ml/mouse) colon increased significantly and group a21041-H mice responded agilely, psychologically active, smooth hair and increased body weight. This indicates that strain a21041 has good effect of treating colitis and has good biosafety.
The invention provides application of lactobacillus reuteri strain A21041 or the probiotics in preparing anti-inflammatory and antioxidant products.
In the invention, the lactobacillus reuteri strain A21041 preferably has superoxide dismutase and glutathione peroxidase activities and has the capability of scavenging superoxide anions and DPPH free radicals.
In the present invention, the product preferably comprises at least one of the following: pharmaceutical and cosmetic products.
The lactobacillus reuteri strain a21041 having anti-inflammatory and antioxidant functions and its application according to the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Separation and purification and identification method of lactobacillus reuteri strain A21041
Intestinal microbial flora samples of 102-year old people in Guangxi Hepu county are diluted according to a double ratio, spread on an MRS plate, and cultured for about 48 hours at 37 ℃ in a biochemical incubator to obtain single colonies. Single colonies were picked, inoculated in MRS liquid medium, and shake cultured at 37℃for 24 hours. And then picking the bacterial liquid, streaking on an MRS plate, and continuously culturing single bacterial colonies. And then picking single bacterial colony, re-streaking, continuously culturing the single bacterial colony, and obtaining pure lactobacillus after 3 times of streaking. A single colony of the isolated Lactobacillus was picked, gram stained, and 16S rDNA (SEQ ID NO: 1) was identified as Lactobacillus reuteri A21041. A21041 single colony is cultured in MRS liquid culture medium, a microplate reader detects the growth state, and graphpad prism 5 software draws a growth curve.
Fig. 1 shows the growth cycle of a 21041: the first 3h is a stationary adaptation period, 3h-12h is a logarithmic growth period, the OD600 value is greatly changed, the strain is rapidly increased, 12h-30h is a platform period, the growth trend of the strain is gradually gentle, and no decay period occurs. FIG. 2 shows the microscopic morphology of A21041, which is a gram-positive bacterium, the cell morphology is short rod-like, and the cell size (width. Times. Length) is (0.5 to 1.0) μm X (1.0 to 2.0) μm.
Example 2
Lactobacillus reuteri strain A21041 is resistant to artificial gastric juice and intestinal juice
The purified Lactobacillus reuteri strain 17938 and Lactobacillus reuteri strain A21041 were picked up as single colonies, inoculated in MRS liquid medium, and shake cultured overnight at 37 ℃. Inoculating bacterial liquid into artificial gastric juice with pH=2.5 according to 10% of inoculum size, setting 3 time points (0 h, 1h and 3 h), respectively taking 100 μl of bacterial liquid to be coated on an MRS plate, culturing for 48h at 37 ℃ in a biochemical incubator, and counting the number of single colonies; bacterial liquid after 3h of artificial gastric juice treatment is inoculated into the artificial gastric juice with pH=8 according to 10 percent of inoculum size, 4 time points (0 h, 2h, 4h and 8 h) are set, 100 μl of each bacterial liquid is coated on an MRS plate, after the bacterial liquid is cultured for 48h at 37 ℃ in a biochemical incubator, the number of single bacterial colonies is counted, and the survival rate is calculated according to a formula I. Survival (%) = (number of single colonies at different time points/number of single colonies at 0 h) ×100% formula I
As shown in fig. 3, both 17938 and a21041 can tolerate ph=2.5 artificial gastric juice for 3 hours, and the survival rates are about 90%; the A21041 has stronger resistance to the artificial intestinal juice with pH=8 than 17938, and the viable count of the A21041 is up to 10 after 8 hours 9 CFU, survival rate is above 80%, preliminary showing that a21041 can successfully reach the intestinal tract to play a role through gastrointestinal fluid stimulation.
Example 3
Lactobacillus reuteri strain A21041 tolerates bovine bile salt solution conditions
The purified Lactobacillus reuteri strain 17938 and Lactobacillus reuteri strain A21041 were picked up as single colonies, inoculated in MRS liquid medium, and shake cultured overnight at 37 ℃. Inoculating bacterial liquid into 0.5g/L concentration ox gall salt solution according to 10% of inoculation amount, culturing for 2 hours at 37 ℃ in a biochemical incubator, diluting by 100 mu L times, and coating on an MRS plate; culturing for 24h, diluting with 100 μl times, and coating on MRS plate; after culturing the MRS plates for 48 hours in a biochemical incubator at 37 ℃, the number of single colonies was counted, and the survival rate was calculated.
As shown in FIG. 4, A21041 was able to tolerate 0.5g/L of ox gall salt solution and the viable count was as high as 10 9 CFU, survival rate is more than 80%, and A21041 tolerance ox gall salt is better than 17938, preliminary demonstration A21041 can stay smoothly in intestinal canal and play a role.
Example 4
A21041 inhibits colipathogenic bacteria and staphylococcus aureus in vitro
Culturing pathogenic O157H 7 escherichia coli (ATCC 35150) and staphylococcus aureus (ATCC 43300) as indicator bacteria, respectively taking 100ul of indicator bacteria liquid to be coated on an LB plate after 12 hours, placing oxford cups after the plates are dried, adding 200 ul of 17938 and A21041 bacteria liquid into each oxford cup, and taking MRS culture liquid as a control. The LB plate was placed at 4℃for 6 hours and then placed in a 37℃incubator for 12 hours. Observing whether the inhibition zone exists or not and measuring the diameter of the inhibition zone.
The results are shown in Table 1.
Table 1A21041 and 17938 in vitro antibacterial conditions
The results are shown in FIG. 5, wherein A21041 significantly inhibited the growth of E.coli and Staphylococcus aureus, and the inhibition was significantly better than 17938.
Example 5
Effect of A21041 on expression of inflammatory factors and NO Release in inflammatory State of LPS-induced RAW264.7 cells
(1) Mouse mononuclear macrophage cell line RAW264.7 cells were cultured in DMEM complete medium (10% Gibco foetal calf serum). Selecting log-of-lifeLong-term RAW264.7 cells according to 5X 10 5 cells/well cells were seeded in 12-well plates, 4 groups were set, control group, LPS group, a21041 group, lps+a21041 group, 3 wells/group, respectively. After cell attachment (about 2 h), 2ml of DMEM complete medium was added to the Control group, 2ml of DMEM complete medium containing 100ng/ml LPS was added to the LPS group, and 2ml of DMEM complete medium containing 10 was added to the A21041 group 6 Complete DMEM medium for CFU cells, LPS+A21041 group was added with 2ml containing 100ng/ml LPS and 10 6 DMEM complete medium of CFU thallus at 37 ℃ and 5% CO 2 Culturing for 24 hours under the condition.
(2) Collecting cells, usingSuper total RNA extraction kit (Shanghai Propofol Bio-supplied) extracts total RNA; synthesizing cDNA according to a FastKing cDNA first strand synthesis kit (provided by Tiangen organism) by taking RNA as a template; use->Tip Green qPCR SuperMix kit (TRAN provided) was PCR amplified to detect COX-2, iNOSmRNA expression.
The qPCR amplification system (total volume 20.0 uL) is shown in Table 2.
TABLE 2qPCR amplification System
TABLE 3 mouse COX-2, iNOS gene primers
qPCR reaction procedure was as follows:
as a result, as shown in FIG. 6, the LPS group significantly increased the expression of COX-2 and iNOS genes as compared with the Control group; whereas compared with LPS group, A21041 significantly reduced the expression of COX-2 and iNOS genes, and was statistically significant (p <0.01 or p < 0.05).
(3) Cell supernatants were collected and assayed for IL-6, IL-10, iNOS, TNF- α protein expression using ELISA kits (provided by Jiang Lai organisms).
As shown in FIG. 7, the LPS group significantly increased the expression of IL-6, TNF-. Alpha.and iNOS proteins, while the expression of anti-inflammatory factor IL-10 protein was not affected, compared to the Control group; compared with LPS group, A21041 can obviously reduce the expression of IL-6, TNF-alpha and iNOS proteins, and can obviously raise the expression of IL-10 protein, so that it has statistical significance (p <0.01 or p < 0.05).
(4) And collecting cells and cell supernatant, detecting nitrite content by using a total NO detection kit, and indirectly calculating total NO release.
As a result, as shown in fig. 8, the LPS group significantly increased NO release compared to the Control group; whereas a21041 significantly reduced the release of NO compared to the LPS group, it was statistically significant (p <0.01 or p < 0.05).
Example 6
Effects of A21041 on changes in colon Length and expression of inflammatory factors in DSS-induced mouse colitis
(1) 24 female C57BL/6 mice, SPF grade 6 weeks old, with an initial body weight of 15-17g, purchased from Daikovia Biotechnology Co., ltd, laboratory animal license number (Scxk (Hunan) 2019-0013). After 1 week of adaptive feeding, mice were randomly divided into 3 normal groups, 3 model groups, 6 mesalamine groups, a21041 low concentration group (concentration 1×10) 9 CFU/ml, abbreviated as A21041-D group) 6, A21041 high concentration group (concentration 1X 10 10 CFU/ml, abbreviated as A21041-H) 6. Wherein, normal group and model group mice are irrigated with 0.2 ml/mouse physiological saline every day; group A21041-D mice had a daily lavage concentration of 1X 10 9 CFU/ml bacterial liquid 0.2 ml/ml; daily gastric lavage of group A21041-H miceDegree of 1×10 10 CFU/ml bacterial liquid 0.2 ml/ml; mesalamine group mice were perfused with 300mg/kg mesalamine solution per day for 3 weeks at 0.2 ml/mouse. Wherein, after week 2, the mice of the other groups except the normal group were changed to drink water freely with 2.5% dss in sterile water for 1 week.
(2) Mice were observed for motor status, mental status, coat gloss and weight change: model group mice were slow to move, mental retardation, rough hair loss, bow back and weight loss; the mesalamine group and the A21041-H group mice respond agilely, psychologically active, smooth hair and gain weight.
(3) Colon length measurement in mice: after the mice were sacrificed and dissected on day 22, the entire colon from the anus to the distal cecum was taken, the blood trace was washed and naturally developed, and the length was measured, and the results are shown in fig. 9: the colon of the mice in the model group was extremely significantly shortened (p < 0.01) compared to the normal group; the colon of mice in group a21041-H increased significantly (p < 0.05) compared to the model group and was statistically significant.
(4) Determination of the colon inflammatory factors IL-6 and IL-10 in mice: taking 1cm of colon tissue of a mouse, placing the colon tissue into 1.5mL centrifuge tubes, placing 3 steel balls into each centrifuge tube, adding pre-cooled sterile PBS (phosphate buffer solution) with the volume of 9 times, placing the mixture into a high-speed tissue grinder for grinding, centrifuging the mixture at 1000 Xg for 15min at 4 ℃, and collecting supernatant for detecting inflammatory factors. The content of inflammatory factors IL-6 and IL-10 in colon tissues of mice is detected according to an enzyme-linked immunosorbent assay kit (Jiang Lai provided by organisms).
The results of the measurement are shown in FIG. 10, which shows that the expression level of IL-6 in colon tissue of mice in the model group is extremely remarkably increased and IL-10 is extremely remarkably decreased as compared with that in the normal group (p<0.01). Compared with the model group, high concentration of a21041 significantly reduced the expression of IL-6 in colonic tissue of colitis mice (p<0.05 Low concentration a21041 inhibited IL-6 but not significantly (p>0.05). A21041 (concentration 1X 10) 10 CFU/mL or 1X 10 9 CFU/mL) significantly reduced IL-10 in mice induced by DSS, with statistical significance (p)<0.05)。
Conclusion: a21041 may reduce colonic inflammation by decreasing the expression of pro-inflammatory factor IL-6 and increasing the expression of anti-inflammatory factor IL-10.
Example 7
A21041 has the ability of eliminating DPPH/O2-free radical and the activity of SOD and GSH-Px contained therein
Single colonies of 17938 and A21041 after purification were picked and inoculated in MRS liquid medium, respectively, and shake cultured overnight at 37 ℃.
(1) Detecting the concentration of the bacterial liquid with the capability of A21041 for removing 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals to be adjusted to 10 8 Centrifuging CFU/mL, collecting thallus, adding DPPH absolute ethanol 1mL with concentration of 0.2mmol/L, mixing, standing at room temperature, performing light-proof reaction for 30min, centrifuging at 600 r/min for 10min, collecting supernatant, and measuring absorbance value A at 517nm i . The samples were averaged 3 times in parallel. The blank group uses equal volume absolute ethyl alcohol to replace DPPH solution, and the absorbance value A is measured j The method comprises the steps of carrying out a first treatment on the surface of the The control group uses distilled water with equal volume to replace the sample solution to measure the absorbance value A 0 The method comprises the steps of carrying out a first treatment on the surface of the And the blank zeroing is carried out by using the mixed solution of distilled water and absolute ethyl alcohol.
DPPH clearance W1 calculation is completed according to formula II.
W1(％)＝{1-(A i -A j )/A 0 } ×100% formula II
(2) Detection of A21041 scavenging superoxide anion (O) 2 - ) Free radical capability
4.5mL of 0.05mol/LTris-HCl buffer was placed in a water bath at 25℃and preheated for 20min. Respectively adding 2mL of bacterial liquid and 2.4mL of sterile physiological saline solution and 0.1mL of 25mmol/L of pyrogallol solution (prepared by 10mmol/L of HCl), uniformly mixing, placing in a water bath at 25 ℃ for reaction for 5min, adding 1mL of 8mol/L of HCl for stopping reaction, and measuring absorbance value A at 325nm x The blank group uses 4.4mL of sterile physiological saline to replace the sample liquid, and the absorbance value A is measured 0 The zeroing tube was replaced with 10mmol/L HCl in the case of pyrogallol HCl solution. O (O) 2 - The clearance W2 calculation is completed according to formula III.
W2(％)＝(1-A x /A 0 ) X 100% formula III
(3) Detection of A21041 superoxide dismutase (SOD) activity
The concentration of the bacterial liquid is adjusted to 5 multiplied by 10 6 CFU/mL, centrifuging to discard supernatant, and detecting according to SOD activityCartridge (Jiang Lai organism supplied) procedure, assayed for SOD-containing activity of a 21041. Results are defined as every 10 4 When the SOD inhibition rate of the CFU single colony in the reaction system reaches 50%, the corresponding SOD amount is one SOD activity unit U.
(4) Detection of A21041 glutathione-containing peroxidase (GSH-Px) Activity
The concentration of the bacterial liquid is adjusted to 5 multiplied by 10 6 CFU/mL, centrifuging to discard supernatant, and determining GSH-Px content in A21041 according to the operation flow of GSH-Px detection kit (provided by Grignard organism). The results are defined as every 10 under reaction conditions of 25 DEG C 4 Each cell oxidizes 1nmol of GSH per minute to 1 enzyme activity unit.
(5) To sum up and clean DPPH/O 2 - The free radical capacity and the activity of SOD and GSH-Px contained in the extract were evaluated for the antioxidant effect of A21041, and the results are shown in Table 2: a21041 clears DPPH/O 2 - The free radical capability is better than 17938, and the activity of the antioxidant enzyme system containing SOD and GSH-Px in A21041 is stronger than 17938. Thus, a21041 has potential antioxidant capacity.
Table 4A21041 DPPH/O 2 - Statistical result of free radical clearance rate and SOD, GSH-Px activity
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. Lactobacillus reuteri with anti-inflammatory and antioxidant functionsLimosilactobacillus reuteri) Strain a21041, wherein said strain a21041 has accession No. GDMCC No. 62832.
2. A probiotic preparation, which is characterized by comprising lactobacillus reuteri strain a21041 with anti-inflammatory and antioxidant functions as claimed in claim 1 and auxiliary materials.
3. The probiotic according to claim 2, characterized in that the live bacteria concentration of lactobacillus reuteri strain a21041 is 10 8 ~10 10 CFU/g；
The probiotic preparation is in the form of oral preparation.
4. Use of lactobacillus reuteri strain a21041 according to claim 1 or a probiotic according to claim 2 or 3 for the preparation of an antibacterial product, the species of bacteria in the antibacterial comprising pathogenic bacteria;
the pathogenic bacteria are staphylococcus aureus and/or escherichia coli.
5. Use of lactobacillus reuteri strain a21041 according to claim 1 or a probiotic according to claim 2 or 3 for the manufacture of a medicament for the prevention and/or treatment of colitis.
6. The use according to claim 5, wherein lactobacillus reuteri strain a21041 inhibits expression of COX-2\inos, but inhibits expression of downstream pro-inflammatory factors IL-6 and TNF- α, increases expression of anti-inflammatory factor IL-10, reduces release of NO, and thereby achieves the effect of preventing and treating colitis.
7. Use of lactobacillus reuteri strain a21041 according to claim 1 or a probiotic according to claim 2 or 3 for the preparation of an anti-inflammatory and anti-oxidative medicament.
8. The use according to claim 7, wherein lactobacillus reuteri strain a21041 has superoxide dismutase and glutathione peroxidase activities and has the ability to scavenge superoxide anions and DPPH radicals.
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| CN117025456B (en) * | 2023-07-31 | 2024-02-06 | 广西爱生生命科技有限公司 | Lactobacillus paracasei A21151 with blood uric acid reducing and anti-inflammatory effects and application thereof |
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