Method for measuring antioxidant activity of lactic acid bacteria based on cellular level

A cell-level, lactic acid bacteria technology, applied in the field of microbiology, can solve problems such as animal difficulties, cumbersome operations, and inability to respond to physiological conditions, and achieve the effects of short exposure time, good repeatability, and high-efficiency research

Active Publication Date: 2015-07-29
YANGZHOU YANGDA KANGYUAN DAIRY +1
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Problems solved by technology

[0004] At present, the detection of the antioxidant activity of lactic acid bacteria mainly uses a spectrophotometer to measure lactic acid bacteria DPPH, hydroxyl radical scavenging rate, reducing ability, and ability to inhibit lipid peroxidation. These conventional chemical methods have several disadvantages: 1. Time-consuming, After the sample processing is completed, each sample still needs a reaction time of 1-12h; 2. The amount of sample used is large, and the sample volume required each time is at least 1mL; 3. The repeatability is poor. Due to the long exposure time of the sample in the air, the sample The variation between repeated measurement values ​​is large; 4. The practicability is weak and the operation is cumbersome, and only a small number of bacterial groups can be measured each time, and batch determination is not possible; 5. The effect is poor, usually only suitable for the antioxidant activity of several single chemical substances In comparison, it is difficult to evaluate the antioxidant effect of the whole substance, which is not suitable for lactic acid bacteria; 6. The accuracy is low, and the bioavailability, absorption and metabolism of antioxidant components in the cell are not considered, so it cannot reflect the intracellular Physiological conditions
Although animal models and human experiments can more accurately reflect the antioxidant capacity, such experiments are expensive and time-consuming. The key is that it will be more difficult to use experimental animals in the future
Therefore, these conventional methods for measuring the antioxidant activity of lactic acid bacteria are far from meeting the needs of scientific research on a wide variety of lactic acid bacteria. Although the antioxidant mechanism of lactic acid bacteria has not been fully elucidated, they are living microbial tissues different from pure chemical substances. They clear Mechanisms for the production of oxidative substances in the gut or tissue oxidative substances, regulation of intestinal flora, improvement of the epithelial barrier, and reduction of intestinal inflammation have been established

Method used

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  • Method for measuring antioxidant activity of lactic acid bacteria based on cellular level
  • Method for measuring antioxidant activity of lactic acid bacteria based on cellular level
  • Method for measuring antioxidant activity of lactic acid bacteria based on cellular level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Determination of antioxidant capacity of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus fermentum

[0035] 1 cell culture

[0036] Human hepatocellular carcinoma (HepG2) was grown in Dulbecco's Modified Eagle's Medium-high glucose (DMEM) medium (containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin) environment, and at 37 °C, 5% CO 2 cultured under conditions. The number of generations of cells used in the present embodiment is 12-20 generations;

[0037] 2 Quercetin standard curve establishment

[0038] ① Accurately weigh quercetin [4H-1-Benzopyran-4-one,2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-Flavone], prepare mother solution (100μM) with 95% ethanol, and use Dilute the DMEM medium without antibiotics and fetal bovine serum into a reaction solution with gradient concentrations (2, 4, 8, 16, 32, 64 μM) to form a quercetin solution;

[0039] ② On a 96-well plate wit...

Embodiment 2

[0054] Embodiment 2 adopts the method for measuring the antioxidant capacity of different measures to measure the antioxidant capacity of lactic acid bacteria

[0055] 1 Determination of lactic acid bacteria DPPH scavenging free radical ability

[0056] Take 1 mL of the cell-free extract of lactic acid bacteria and add it to a test tube, then add 1 mL of 0.2 mmol / L DPPH-absolute ethanol solution, mix well, and react in the dark for 30 minutes, then take the supernatant and measure the absorbance at 517 nm to determine An equal volume of PBS was used as a control group instead of the sample solution.

[0057] Calculate according to the following formula: DPPH free radical scavenging rate=(1-A 517样品 / A 517对照 )×100%, where: A 517样品 and A 517对照 are the absorbance values ​​measured at 517nm of the sample group and the control group, respectively

[0058] We are used for the analysis of the antioxidant activity of 5 kinds of 10 strains of lactic acid bacteria shown in table 1 w...

Embodiment 3

[0069] The coefficient of variation of embodiment 3 CAA method measurement result

[0070] For determining the reproducibility of the CAA method of the present invention, we compared the coefficient of variation of the antioxidant capacity of quercetin and 10 strains of lactic acid bacteria determined by the CAA method, the results are as shown in table 4, and the coefficient of variation of visible quercetin and 10 strains of lactic acid bacteria The range is 1.23-3.25%, which is lower than 10%, indicating that the method of the present invention has good repeatability.

[0071] Table 4 Coefficient of variation of different sample determination results

[0072]

[0073] After the quercetin standard curve of the present invention is established, it can be applied to the determination of each sample in the future, and can measure the antioxidant capacity of different species of lactic acid bacteria in batches in a very short time, and the determination speed is about 10 time...

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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 [0001] The invention relates to a method for measuring the antioxidant activity of lactic acid bacteria based on the cell level, and belongs to the technical field of microorganisms. Background technique [0002] Oxidative stress is caused by an imbalance between the production of free radicals and the depletion of the intrinsic antioxidant system, which plays an important role in different physiological and pathological conditions, can damage proteins, cause DNA mutations, oxidize cell membrane phospholipids and low-density lipoproteins modification. Excessive reactive oxygen species (Reactive Oxygen Species, ROS) can lead to intracellular damage, and then further lead to the occurrence of chronic diseases: such as atherosclerosis, arthritis, diabetes, neurodegeneration, cardiovascular disease and cancer. In order to resist oxidation, the human body synthesizes antioxidant substances by itself and ingests antioxidant substances from food to jointly establis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 陈卫王刚邢家溧印伯星房东升张秋香田丰伟刘小鸣范大明张白曦赵国忠郭敏赵建新张灏
Owner YANGZHOU YANGDA KANGYUAN DAIRY
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