CN104807797A - Method for measuring antioxidant activity of lactic acid bacteria based on cellular level - Google Patents
Method for measuring antioxidant activity of lactic acid bacteria based on cellular level Download PDFInfo
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Abstract
The invention discloses a method for measuring the antioxidant activity of lactic acid bacteria based on a cellular level and belongs to the technical field of microorganisms. According to the method, a full-wave fluorescent scanning type microplate reader with a 96-hole ferment plate based on the cellular level is utilized to replace a conventional spectrophotometer so as to measure the antioxidant capability of a treated sample; a fluorescent probe and a free-radical initiator are utilized to perform batched rapid and accurate measurement on lactic acid bacteria within 1 hour, so that a rapid detection method is provided for researching the antioxidant activity of the lactic acid bacteria at the cellular level; by utilizing the method, the antioxidant activity of the different strains of lactic acid bacteria can be measured within ultrashort time, and the antioxidant level of the lactic acid bacteria can be rapidly and accurately reflected.
Description
Technical field
The present invention relates to a kind of method measuring lactic acid bacteria antioxidation activity based on cellular level, belong to microbial technology field.
Background technology
Oxidative stress is that between being cut down by the generation of free radical and inherent antioxidant system, imbalance causes, and plays an important role, can destroy albumen, cause DNA mutation, the modification of oxidative cell membrane phospholipid and low-density lipoprotein under different physiological and pathological conditions.Excessive active oxygen (Reactive Oxygen Species, ROS) intracellular destruction can be caused, then cause the generation of chronic disease further: as atherosclerotic, arthritis, diabetes, neural change, angiocardiopathy and cancer etc.In order to antioxidation, human body is by self synthesis polyphenoils and absorb polyphenoils from food, jointly sets up biological antioxidant barrier.But this system of resisting still can not protect the damage self avoiding oxidative stress in some cases, therefore, increase oxidation resistance is kept fit with this and is still needed continuous exploration.
Food and Agricultural Organization of the United Nations definition probio is the microorganism that a class is lived, and can pass on health care effect in time taking in sufficient amount.At present, because it can reduce the generation of ROS, health care effect is played to human body, to be widely used in food industry and to be put into Generally Recognized as Safe (GRAS).From this view point, the antioxidation of development effective method reaction lactic acid bacteria, thus effective measure the ability that it removes ROS, can be used in being formed new probiotic products or adjuvant thus plays the effect resisting free radical and relevant disease.
At present spectrophotometric determination lactic acid bacteria DPPH, Scavenging action to hydroxyl free radical are mainly adopted to the detection of lactic acid bacteria antioxidation activity, reducing power, inhibition of peroxidation carries out, the chemical method of these routines has several shortcoming: 1. length consuming time, after sample preparation completes, each sample still needs the reaction time of 1-12h; 2. many by sample amount, the sample size at every turn needed at least 1mL; 3. poor repeatability, due to sample in atmosphere open-assembly time long, changing greatly between sample repeated measures; 4. practicality is weak, complex operation, is merely able to measure minority flora at every turn, can not measure in batches; 5. weak effect, is usually only applicable to the comparison of several single chemical substance antioxidation activity, is difficult to the antioxidant effect evaluating whole material, for inapplicable lactic acid bacteria; 6. accuracy is low, does not consider that antioxidant content is at intracellular bioavailability, absorbs and the situation such as metabolism, therefore can not physiological condition in reacting cells.Although animal model and human trial can react oxidation resistance more accurately, this test cost is higher and very time-consuming, and key is that following use animal used as test will be more difficult.Therefore the method for these conventional determining lactic acid bacteria antioxidation activities can not meet the needs of miscellaneous lactic acid bacteria scientific research far away, although the antioxidation mechanism of lactic acid bacteria is not also illustrated completely, but they be live microorganism cultures be different from pure chemicals, they remove the generation of oxidation material in enteron aisle or tissue oxidizing material, regulating intestinal canal flora, improve epithelial barrier, and the mechanism reducing intestinal inflammatory is set up.Therefore, consider the antioxidation mechanism of lactic acid bacteria complexity, a kind of method of quick and precisely reacting oxidation resistance in lactic acid thalline must be set up.The present invention aims to provide one can Fast Measurement lactic acid bacteria oxidation resistance, reacts the method for its thalline TAC.
Summary of the invention
Object of the present invention is for providing a kind of method of the mensuration lactic acid bacteria oxidation resistance based on cellular level, in conjunction with cell-lactic acid bacteria-fluorescence probe, reflect lactic acid bacteria oxidation resistance by the all-wave ability that fluorescent scanning microplate reader mensuration lactic acid bacteria Fluorophotometry material is formed frequently.
Technical solution of the present invention mainly comprises the following steps:
(1) cultured cell, the esterase energy decomposition non-blooming 2 ' of described cell itself, 7 '-dichlorofluorescein diacetate (DCFH-DA), forms DCFH, DCFH are very easily become hyperfluorescenceZeng Yongminggaoyingguang dichlorofluorescein (DCF) by oxygen radical or active oxygen oxidizes.
(2) typical curve of variable concentrations Quercetin Fluorophotometry material Forming ability is set up: with variable concentrations Quercetin solution pre-treatment step (1) the gained cell containing DCFH-DA, add 2,2-azo diisobutyl amidine dihydrochloride (ABAP) stimulates DCFH to be oxidized to the dichlorofluorescein (DCF) of hyperfluorescenceZeng Yongminggaoyingguang, carries out scanning the fluorescent value obtaining different time in fluorescence microplate reader; Quercetin solution is replaced in contrast with cell culture medium, using do not add ABAP as blank, after deduction blank, Quercetin compares photograph, the ratio that the integral area of fluorescent value to the time reduces is cellular anti-oxidant capacity value (CellularAntioxidantActivity, CAA), with CAA value, quercetin concentration is made to the typical curve of variable concentrations Quercetin Fluorophotometry material Forming ability.
(3) mensuration of lactic acid bacteria oxidation resistance: after lactic acid bacteria activation is prepared into bacteria suspension, replaces the Quercetin solution used in step (2), measure and calculate CAA value; Reference standard curve, is converted into quercetin concentration value by CAA value, is equivalent to micromole's equivalent of Quercetin to represent the oxidation resistance of lactic acid bacteria with the CAA value of every mL bacteria suspension.
Lactic acid bacteria of the present invention comprises: the conventional food lactic acid bacteria kinds such as Lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacilluscasei), lactobacillus fermenti (Lactobacillus fermenti).
Step (1) but described cell human liver cancer cell (Human hepatocellular carcinoma, HepG2), Human colon adenocarcinoma cell line Caco-2, Human umbilical vein endothelial cells EA.hy926 or rat macrophage RAW264.7.By cell culture passages to 2-20 generation.
The described suspension bacterium of step (3) can also replace with the metabolin of bacterium, such as cell-free extract and fermented supernatant fluid etc.
In one embodiment of the invention, the foundation of step (2) described typical curve, adopts following steps:
1. Quercetin [4H-1-Benzopyran-4-one, 2-(3,4-dihydroxyphenyl)-3 is accurately taken, 5,7-trihydroxy-Flavone], with 95% ethanol preparation mother liquor, and with not being diluted to 2 containing the DMEM nutrient culture media of microbiotic and hyclone, 4,8,16,32, the gradient concentration reactant liquor of 64 μMs, obtains Quercetin solution;
2. on 96 orifice plates with 6 × 10
4the density in individual/hole inoculates the DEME nutrient culture media of 100 μ L containing HepG2 cell, removes nutrient solution, clean each inoculation hole with phosphate buffer after cultivating 24h; Then every hole adds the Quercetin solution containing two chlorine fluorescein ethyl acetate of 25 μMs of 100 μ L, at 37 DEG C, 5%CO
2continue under condition to cultivate 1h; Take out 96 orifice plates, after cleaning each hole with 100 μ L phosphate buffers, add that 100 μ L are dissolved in 600 μMs of Hank balanced salt solution 2,2-azo diisobutyl amidine dihydrochloride, 96 orifice plates are put into fluorescence microplate reader scan, keep constant temperature 37 DEG C, excite at wavelength 538nm place, at wavelength 485nm place, release measures every 5min, measures the situation of change of fluorescent value in 1h;
3. with Quercetin pretreatment cell is Quercetin group, replaces Quercetin solution as a control group with DMEM nutrient culture media, using do not add ABAP as blank group, after deduction blank, Quercetin compares photograph, and the integral area of fluorescent value to the time subtracted
Few ratio is cellular anti-oxidant capacity value (Cellular Antioxidant Activity, CAA), CAA computing formula:
CAA(％)＝{1-(∫SA/∫CA)}×100％
Wherein ∫ SA represents the integral area of fluorescent value to time curve of Quercetin group, and ∫ CA represents the integral area of fluorescent value to time curve of control group;
4. with CAA value, quercetin concentration is made to the typical curve of variable concentrations Quercetin Fluorophotometry material Forming ability.
In one embodiment of the invention, the method of the activation of step (3) lactic acid bacteria be with the inoculum concentration of 1% (v/v) by lactobacillus inoculum frozen for glycerine pipe in MRS nutrient culture media, cultivate 20h under 37 DEG C of conditions after again with the switching of same inoculum concentration once.
In one embodiment of the invention, after the activation of step (3) bacterial strain, MRS fluid nutrient medium is inoculated in 1% inoculum concentration, 37 DEG C of quiescent culture 20h, collected by centrifugation thalline, the thalline of collection washs 3 times through phosphate buffer, is resuspended in phosphate buffer, adjustment bacterium is dense, for subsequent use.
The non-blooming indicator mixture 2 ' of cellular esterases decomposition itself, the DCFH that 7 ' dichlorofluorescein diacetate (DCFH-DA) is formed very easily is become fluorescence dichlorofluorescein (DCF) by oxygen radical or active oxygen oxidizes, 2,2 isobutyl amidine dihydrochlorides (ABAP) are dispersed in cell and Auto-decomposition forms peroxylradicals ROO*, and DCFH can be stimulated to produce the DCF with fluorescence signal.Lactic acid bacteria thalline can stop it to enter cell in conjunction with oxygen radical at cell membrane exterior, or be combined with ROS* and ROO* in cell membrane inside and block the process that DCFH is oxidized to DCF, thus reduce the formation of DCF, the fluorescence signal that fluorescence microplate reader is read out reduces, and shows as the fluorescent value area reduction that lactic acid bacteria causes.
Advantage of the present invention and good effect as follows:
1 more close to the physiological situation in body.Because the method is set up on a cellular level, fully take into account antioxidant content in situations such as intracellular bioavailability, absorption and metabolism;
2 take short.Do not need to carry out zoopery and human trial, after sample preparation completes, each sample only needs 1h, and spended time is far below zoopery and human trial spent time;
3 is few by sample amount.The sample size of each needs is only 100 μ L, less than 1/10 of conventional method;
4 is reproducible.Because sample is measured fast, open-assembly time is short in atmosphere, and the change between sample repeated measures is less, reproducible;
5 is practical.Can measure multi-strain bacteria strain in bulk simultaneously, be highly suitable for the comparative studies of miscellaneous lactic acid bacteria different strains oxidation resistance.
The present invention be a kind of can the method for rapid and accurate determination lactic acid bacteria oxidation resistance based on cellular level, can be studied the lactic acid bacteria oxidation resistance of different genera fast and efficiently by this technology, lactic acid bacteria Study of Antioxidation is had great importance.
Accompanying drawing explanation
Fig. 1 is CAA value-quercetin concentration typical curve, (A): with the fluorescent value of the Quercetin group of variable concentrations Quercetin pretreatment cell at different time; (B): CAA value is to the typical curve of quercetin concentration.
Fig. 2 is Lactobacillus rhamnosusCCFM237, Lactobacillus rhamnosusCCFM469, LactobacillusplantarumCCFM238, Lactobacillus plantarumCCFM239, Lactobacillus acidophilusCCFM6, Lactobacillus acidophilusCCFM137, Lactobacillus caseiCCFM9, Lactobacillus caseiCCFM5, Lactobacillus fermentiCCFM381, Lactobacillus fermentiCCFM424 fluorescence intensity change curve in time.
Fig. 3 is every milliliter of Lactobacillus rhamnosusCCFM237, Lactobacillus acidophilusCCFM6, Lactobacillus plantarumCCFM238, Lactobacillus caseiCCFM9, Lactobacillus fermenti CCFM381 bacterium liquid CAA value be equivalent to micromole's equivalent of Quercetin.
Embodiment
The mensuration of embodiment 1 Lactobacillus rhamnosus, lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, lactobacillus fermenti oxidation resistance
1 cell chulture
Human liver cancer cell (Human hepatocellular carcinoma, HepG2) (10% hyclone is comprised at Dulbecco ' s Modified Eagle ' sMedium-high glucose (DMEM) nutrient culture media, 100U/mL penicillin, 100 μ g/mL streptomysins) grow under environment, and at 37 DEG C, 5%CO
2cultivate under condition.The cell algebraically that the present embodiment uses is in 12-20 generations;
2 Quercetin typical curves are set up
1. Quercetin [4H-1-Benzopyran-4-one, 2-(3,4-dihydroxyphenyl)-3 is accurately taken, 5,7-trihydroxy-Flavone], with 95% ethanol preparation mother liquor (100 μMs), and with not being diluted to gradient concentration (2 containing the DMEM nutrient culture media of microbiotic and hyclone, 4,8,16,32,64 μMs) reactant liquor, build up Quercetin solution;
2. on 96 orifice plates with 6 × 10
4the density in individual/hole inoculates 100 μ LHepG2 cells, removes nutrient solution, clean each inoculation hole with phosphate buffer after cultivating 24h.Then every hole adds the Quercetin solution containing two chlorine fluorescein ethyl acetate of 25 μMs of 100 μ L, at 37 DEG C, 5%CO
2continue under condition to cultivate 1h.Take out 96 orifice plates, phosphate buffer is removed after cleaning each hole with 100 μ L phosphate buffers, add 100 μ L2,2-azo diisobutyl amidine dihydrochloride (ABAP) (600 μMs, be dissolved in Hank balanced salt solution), 96 orifice plates are put into fluorescence microplate reader and scans, keep constant temperature 37 DEG C, excite at wavelength 538nm place, at wavelength 485nm place, release measures 1h to every 5min;
3. Quercetin solution is replaced in contrast with DMEM, with do not add ABAP for blank, after deduction blank, Quercetin compares photograph, the ratio that the integral area of fluorescent value to the time reduces is cellular anti-oxidant capacity value (Cellular AntioxidantActivity, CAA), computing formula:
CAA(％)＝{1-(∫SA/∫CA)}×100％
Integral area when wherein ∫ SA represents Quercetin under m-fluorescent value curve, ∫ CA represents the contrast time
-integral area under fluorescent value curve;
4. the typical curve of quercetin concentration-CAA value is calculated
3 lactic acid bacteria activation culture
The Lactobacillus of Lactobacillus rhamnosus shown in table 1 rhamnosus, lactobacillus acidophilus Lactobacillus acidophilus, Lactobacillus plantarum Lactobacillus plantarum, Lactobacillus casei Lactobacillus casei, lactobacillus fermenti Lactobacillusfermenti are carried out activation culture respectively.Bacterial strain activation method be with the inoculum concentration of 1% (v/v) by frozen lactobacillus inoculum in MRS nutrient culture media, cultivate 20h under 37 DEG C of conditions after again with same inoculum concentration switching once.
The bacterial strain that table 1. is used
4 lactic acid bacteria bacteria suspensions prepare
After bacterial strain activation, be inoculated in MRS fluid nutrient medium with 1% inoculum concentration, the centrifugal 10min of 37 DEG C of quiescent culture 20h, 3000rpm collects thalline, and the thalline of collection washs 3 times through phosphate buffer, is resuspended in phosphate buffer, adjustment bacterium number to 10
9cfu/mL, for subsequent use.
5 lactic acid bacteria determination oxidatives
1. replace the Quercetin solution in step 2 with testing sample bacteria suspension, all the other operate with step 2, the then CAA value of calculation sample bacteria suspension;
2. CAA value is converted into the quercetin concentration value with equivalent antioxidant effect, the micromole's equivalent being equivalent to Quercetin with every mL bacteria suspension represents.
3. compare 10 kinds of lactic acid bacteria oxidation resistances and see Fig. 3.
Embodiment 2 adopts the different method measuring oxidation resistance to measure the inoxidizability of lactic acid bacteria
The mensuration of 1 lactic acid bacteria DPPH Scavenging ability
Extracting lactic acid bacterium cell-free extract 1mL adds in test tube, then adds the DPPH-ethanol solution of 1mL 0.2mmol/L, fully after mixing, lucifuge reaction 30min, then get supernatant, measure light absorption value in 517nm place, replace sample solution as a control group with isopyknic PBS.
According to following formulae discovery: DPPH free radical scavenging activity=(1-A
517 samples/ A
517 contrasts) × 100%, in formula: A
517 samplesand A
517 contrastsbe respectively the light absorption value that sample sets and control group record at 517nm place
The DPPH Scavenging action to hydroxyl free radical method of the method for embodiment 1 and the present embodiment in-vitro evaluation lactic acid bacteria antioxidation activity is used for the analysis of the antioxidation activity of shown 5 kind of 10 strains of lactic acid bacteria of table 1 by us, and compare the correlativity of these 2 kinds of methods, result shows that 10 strains of lactic acid bacteria measured are 3 concentration (10
7-10
9cfu/mL) correlativity is not had under, specifically in table 2.
The correlativity of table 2.2 kind of method
*p<0.05
2 build H
2o
2cause cell oxidative damage model
We analyze CAA method of the present invention further respectively and screen the ability that alleviation hydrogen peroxide that the high anti-oxidation bacterial strain CCFM9 that obtains and DPPH method screen the high anti-oxidation bacterial strain CCFM237 obtained causes cell oxidative damage.
HepG2 cell is divided into 5 processed group G1, G2, G3, G4 and G5, G1 group is the control group do not processed, and G2 group is exposed to 500 μMs of H for HepG2 cell
2o
2continue 6h; G3 group is first exposed to 500 μMs of H with 100 μMs of Quercetin pre-service 12h again for HepG2 cell
2o
2continue 6h; G4 group screens the Quercetin that the high anti-oxidation bacterial strain that obtains replaces in G3 group for CAA method, and G5 group screens the Quercetin in the high anti-oxidation bacterial strain replacement G3 group obtained for DPPH method, and other processing modes are identical with G3.Cultivate and terminate, measure the activities of antioxidant enzymes of each group respectively: (1) total antioxidant activity T-AOC, (2) superoxide dismutase SOD, (3) glutathione peroxidase GSH-PX, (4) cat catalase, (5) peroxidase POD.
Result is as shown in table 3, visible CAA method is screened the high anti-oxidation bacterial strain obtained and is screened than DPPH hydroxy radical the bacterial strain obtained and have the ability that stronger alleviation hydrogen peroxide causes cell oxidative damage, and this illustrates that CAA method is to the actual oxidation resistance of the evaluation of bacterial strain inoxidizability ability closer to bacterial strain.
Table 3: activities of antioxidant enzymes
Note: different letter representation significant correlation p<0.05.
The coefficient of variation of embodiment 3 CAA method measurement result
For determining the repeatability of CAA method of the present invention, we compare the coefficient of variation of the oxidation resistance of Quercetin that CAA method determines and 10 strains of lactic acid bacteria, result is as shown in table 4, the coefficient of variation scope of visible Quercetin and 10 strains of lactic acid bacteria is 1.23 – 3.25%, lower than 10%, illustrate that the inventive method is reproducible.
The coefficient of variation of the different sample determination result of table 4
Quercetin typical curve of the present invention can be applicable to later each sample determination after setting up, and within the extremely short time, can measure the oxidation resistance of different genera lactic acid bacteria by batch, finding speed improves about 10 times than conventional method.The present invention obviously has fast, efficient, accurately, the advantages such as physiologically active can be reacted, thus provide one based on cell for lactic acid bacteria Study of Antioxidation, closer to the assay method in body, the research of lactic acid bacteria oxidation resistance is of great significance and value.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (7)
1., based on a method for the mensuration lactic acid bacteria oxidation resistance of cellular level, it is characterized in that, mainly comprise the following steps:
(1) cultured cell, the esterase of described cell can decompose 2 ', 7 '-dichlorofluorescein diacetate, forms DCFH, DCFH are very easily become hyperfluorescenceZeng Yongminggaoyingguang dichlorofluorescein by oxygen radical or active oxygen oxidizes;
(2) typical curve of variable concentrations Quercetin Fluorophotometry material Forming ability is set up: with variable concentrations Quercetin solution pre-treatment step (1) the gained cell containing DCFH-DA, then 2 are added, 2-azo diisobutyl amidine dihydrochloride stimulates DCFH to be oxidized to the dichlorofluorescein of hyperfluorescenceZeng Yongminggaoyingguang, carries out scanning the fluorescent value obtaining different time in fluorescence microplate reader; Quercetin solution is replaced in contrast with cell culture medium, using do not add ABAP as blank, after deduction blank, Quercetin compares photograph, the ratio that the integral area of fluorescent value to the time reduces is cellular anti-oxidant capacity value CAA, with CAA value, quercetin concentration is made to the typical curve of variable concentrations Quercetin Fluorophotometry material Forming ability;
(3) mensuration of lactic acid bacteria oxidation resistance: after lactic acid bacteria activation is prepared into bacteria suspension, replaces the Quercetin solution used in step (2), measure and calculate CAA value; Reference standard curve, is converted into quercetin concentration value by CAA value, is equivalent to micromole's equivalent of Quercetin to represent the oxidation resistance of lactic acid bacteria with every mL bacteria suspension.
2. method according to claim 1, it is characterized in that, described lactic acid bacteria comprises Lactobacillus rhamnosus (Lactobacillusrhamnosus), lactobacillus acidophilus (Lactobacillusacidophilus), Lactobacillus plantarum (Lactobacillusplantarum), Lactobacillus casei (Lactobacilluscasei), lactobacillus fermenti (Lactobacillusfermenti).
3. method according to claim 1, is characterized in that, step (1) described cell is human liver cancer cell HepG2, Human colon adenocarcinoma cell line Caco-2, Human umbilical vein endothelial cells EA.hy926 or rat macrophage RAW264.7.
4. method according to claim 1, is characterized in that, the foundation of step (2) typical curve, adopts following steps:
1. accurately take Quercetin, with 95% ethanol preparation mother liquor, and be diluted to the gradient concentration reactant liquor of 2,4,8,16,32,64 μMs with the DMEM nutrient culture media not containing microbiotic and hyclone, obtain Quercetin solution;
2. on 96 orifice plates with 6 × 10
4the density in individual/hole inoculates the DEME nutrient culture media of 100 μ L containing HepG2 cell, removes nutrient solution, clean each inoculation hole with phosphate buffer after cultivating 24h; Then every hole adds the Quercetin solution containing two chlorine fluorescein ethyl acetate of 25 μMs of 100 μ L, at 37 DEG C, 5%CO
2continue under condition to cultivate 1h; Take out 96 orifice plates, after cleaning each hole with 100 μ L phosphate buffers, add that 100 μ L are dissolved in 600 μMs of Hank balanced salt solution 2,2-azo diisobutyl amidine dihydrochloride, 96 orifice plates are put into fluorescence microplate reader scan, keep constant temperature 37 DEG C, excite at wavelength 538nm place, at wavelength 485nm place, release measures every 5min, measures the situation of change of fluorescent value in 1h;
3. with Quercetin pretreatment cell is Quercetin group, Quercetin solution is replaced as a control group with DMEM nutrient culture media, using do not add ABAP as blank group, after deduction blank, Quercetin compares photograph, the ratio that the integral area of fluorescent value to the time reduces is cellular anti-oxidant capacity value CAA, CAA computing formula: CAA (%)={ 1-(∫ SA/ ∫ CA) } × 100%
Wherein ∫ SA represents the integral area of fluorescent value to time curve of Quercetin group, and ∫ CA represents the integral area of fluorescent value to time curve of control group;
4. with CAA value, quercetin concentration is made to the typical curve of variable concentrations Quercetin Fluorophotometry material Forming ability.
5. method according to claim 1, is characterized in that, the described suspension bacterium of step (3) replaces with the metabolin of bacterium.
6. method according to claim 1, it is characterized in that, the method of the activation of step (3) lactic acid bacteria be with the inoculum concentration of 1% by lactobacillus inoculum frozen for glycerine pipe in MRS nutrient culture media, cultivate 20h under 37 DEG C of conditions after again with the switching of same inoculum concentration once.
7. method according to claim 1, it is characterized in that, after the activation of step (3) bacterial strain, MRS fluid nutrient medium is inoculated in, 37 DEG C of quiescent culture 20h, collected by centrifugation thalline with 1% inoculum concentration, the thalline collected washs 3 times through phosphate buffer, be resuspended in phosphate buffer, adjustment bacterium is dense, for subsequent use.
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| CN106950203B (en) * | 2015-10-15 | 2021-04-06 | 浜松光子学株式会社 | Phagocytosis ability evaluation method and fluorescence measurement method |
| CN109364103A (en) * | 2018-10-09 | 2019-02-22 | 昆明理工大学 | The purposes of Chinese sumac fruit extract |
| CN109374542A (en) * | 2018-10-13 | 2019-02-22 | 安发（福建）生物科技有限公司 | A kind of evaluation method of fruit drink antioxidant activity |
| CN110361378A (en) * | 2019-07-25 | 2019-10-22 | 浙江大学 | ORAC antioxidant activity evaluation method and fluorescence indicator based on fluorescein ethyl ester |
| CN110361378B (en) * | 2019-07-25 | 2021-04-02 | 浙江大学 | ORAC antioxidant activity evaluation method based on fluorescein ethyl ester and fluorescent indicator |
| CN113218928A (en) * | 2021-05-21 | 2021-08-06 | 宁德师范学院 | Colorimetric method for rapidly determining antibacterial activity based on fluorescent probe |
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