CN104342482B - Myrosin activity test method in probiotic bacteria - Google Patents
Myrosin activity test method in probiotic bacteria Download PDFInfo
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- CN104342482B CN104342482B CN201410530104.3A CN201410530104A CN104342482B CN 104342482 B CN104342482 B CN 104342482B CN 201410530104 A CN201410530104 A CN 201410530104A CN 104342482 B CN104342482 B CN 104342482B
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- sinigrin
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Abstract
The invention discloses myrosin activity test method in a kind of probiotic bacteria. It is probably the situation of inducible enzyme for myrosin, uses the reaction substrate sinigrin of myrosin that thalline is induced, then measure the vigor of myrosin again. Replace glucose to carry out primary dcreening operation first by sinigrin, tentatively judge that bacterial strain is with or without the ability decomposing sinigrin. Re-use sinigrin induction thalline and produce myrosin, the metabolic pathway that thalline produces myrosin is made to activate, then complete thalline is joined in finite concentration sinigrin solution and carry out metabolism, centrifugal segregation thalline after certain time, measure the supernatant absorbance at 340nm place, it has been found that absorbance now is changed significantly compared with the absorbance of the sinigrin solution of the same concentrations being added without thalline. Therefore, in induction thalline method mensuration probiotic bacteria, the method for myrosin activity is feasible. The method is simple to operate, and the detection cycle is short.
Description
Technical field
The present invention relates to a kind of method detecting probiotic bacteria myrosin activity.
Background technology
Myrosin formal name used at school is glucosinase, the β that is otherwise known as-thioglycoside hydrolytic enzyme, is mainly found in crucifer, is unique β-thioglucoside hydrolytic enzyme found in the Nature up to now. Myrosin has multiple isozyme, is respectively present in different crucifer tissue. The myrosin purified from Brassica campestris L and Caulis et Folium Sinapis albee is a protein dimer, and its molecular mass is about 135kD-150kD. Myrosin seldom exists with free state, but assists the coenzyme of albumen one class to form protein complex with such as myrosin associated proteins or myrosin. Generally; myrosin in crucifer can form relatively independent sulfur glycoside glucoside-myrosin system with sulfur glycoside glucoside; just contacting with each other both only when plant tissue is damaged reacts generates poisonous rhodanate, isothiocyanate and nitrile; wherein isothiocyanic acid salt pair insecticide has higher toxicity, and plant can play certain protective effect. Myrosin activity is subject to the impact of external environment, as temperature and pH can large effect reaction time enzymatic activity.Additionally, ascorbic acid, Fe2+And Cu2+Also certain impact is produced. Wherein, ascorbic acid shows as low concentration kinase activity, high concentration inhibitory enzyme activity. It is generally acknowledged Fe2+For zymoexciter, Cu2+For enzyme inhibitor.
The activity of myrosin is determined typically via the change of the growing amount or substrate sulfur glycoside glucoside concentration that measure product glucose. Growing amount for glucose, it is possible to use glucose kit measures, commonly used method when this is also myrosin determination of activity. Spectrophotography is also feasible, carrying out series reaction with sinigrin for enzymolysis substrate such as what SandroPalmieri introduced, when generating by detecting NADPH at 340nm place, the change of absorbance measures myrosin activity in brassicaceous vegetable. Additionally gas chromatogram fixative, pH value method and electrode method can measure myrosin vigor in vegetable. But these methods are subject in sample extracts impure interference containing impurity glucose and myrosin, or there is the problems such as requirement of experiment condition is higher, experimental implementation is not strong. The change of sulfur glycoside glucoside concentration can also adopt spectrophotometry, Zhang Lei etc. carry out enzymolysis with sinigrin for substrate, and the size reduced by detecting the absorbance caused because sinigrin concentration reduces measures myrosin activity in arabidopsis. The method sampling amount is little, is suitable for batch and measures, but there is also enzyme activity relatively low time be difficult to detect or the problem such as result is inconspicuous. Additionally, high performance liquid chromatography (HPLC) method and HPLC-MS (HPLC-MS) method are also conventional measure the method for myrosin activity in brassicaceous vegetable.
External current research shows, sulfur glycoside glucoside can by the microbial hydrolysis in intestinal. As observed in mouse test, glucoraphenin is broken down into isothiocyanate. Researcher makes glucoraphenin and Sulforaphane break-even entrance mice caecum so that it is is hydrolyzed, detected isothiocyanate in intestinal mucosa serum. Bacteroides thetaiotaomicron in human body intestinal canal is transplanted to mouse intestinal by LilaElfoul etc., it has been found that it can decompose sinigrin and form isothiocyanate. And in testing in vitro, mouse intestinal flora also can decompose glucoraphenin and produce the isothiocyanate of low concentration. D.-L.Cheng etc. are in vitro it was found that, three kinds of bacillus bifiduss: false chain bacillus bifidus, bifidobacterium adolescentis and bifidobacterium longum be hydrolyzeds sulfur glycoside glucoside generation isothiocyanate when pH=7, and have only generated nitrile when pH drops to 5.2. The studies above shows, the bacillus class probiotic bacteria such as bacteroides thetaiotaomicron and bacillus bifidus has myrosin activity more. But more than research is merely by the generation measuring catabolite isothiocyanate to prove that enteric microorganism has myrosin activity, do not carry out myrosin further purifying analyzing or its enzyme activity being measured, and above research adopts mass spectrography or chromatography more, experimental facilities and experimental cost are required higher.
Summary of the invention
It is an object of the invention to provide myrosin activity test method in a kind of probiotic bacteria, the method adopts the method that enzymolysis and ultrasonic bacterial cell disruption combine that the enzymatic activity in bacterium solution is carried out Sensitive Detection.
It is an object of the invention to be achieved through the following technical solutions:
One, being inoculated in MRS liquid culture medium with 2% inoculum concentration by probiotic bacteria to be measured under aseptic condition, in 37 DEG C of constant temperature culture 24h, activation can make for 2-3 time bacterial strain rejuvenate so repeatedly.
In this step, described probiotic bacteria is bacillus class probiotic bacteria (lactobacillus and bacillus bifidus).
In this step, the compound method of described MRS liquid culture medium is as follows: peptone 10g, beef leaching thing 10g, yeast extract 5.0g, sodium acetate 5.0g, sodium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, Magnesium sulfate heptahydrate 0.2g, seven water manganese sulfate 0.05g, it is dissolved in water, then add 1ml Tween 80, be adjusted to 1000ml with water.
Two, primary dcreening operation: be 5mmol/L containing sinigrin concentration, modified MRS culture fluid 5mL without glucose cultivates and treats sieve bacterial strain, incubation time 48h, cultivation temperature 30 DEG C, observes strain growth situation, it is possible to the bacterial strain of normal growth is for next step experiment.
In this step, described containing sinigrin concentration be 5mmol/L, the compound method of modified MRS culture fluid without glucose as follows: aseptic 100mmol/L sinigrin mixes with the volume ratio of 1:20 with MRS liquid culture medium.
Three, multiple sieve: configuration glucose and sinigrin concentration are the modified MRS culture fluid of 10mmol/L, bacterial strain to be screened inoculum concentration routinely is inoculated into 30 DEG C of incubated overnight 12-18h of this modified MRS culture fluid, it is then centrifuged for taking thalline, add 10mLMES wash buffer thalline, thalline is resuspended in 3mLMES buffer after recentrifuge, bacteria suspension is mixed according to the volume ratio of 1-1.5:1 with the MES buffer of 10mmol/L sinigrin, 24-48h is cultivated at 30 DEG C, last centrifuging and taking supernatant measures absorbance at 340nm place, filter out absorbance difference bacterial strain more than 0.2.
In this step, described 10mmol/L glucose, 10mmol/L sinigrin modified MRS compound method as follows: the 1. preparation of 10mmol/L glucose modified MRS: peptone 10g, beef leaching thing 10g, yeast extract 5.0g, sodium acetate 5.0g, sodium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, Magnesium sulfate heptahydrate 0.2g, seven water manganese sulfate 0.05g, 1.8g glucose is dissolved in water, then add 1ml Tween 80, be adjusted to 1000ml with water. 2. 10mmol/L glucose modified MRS mixes with the volume ratio of 20:1 with aseptic 100mmol/L sinigrin.
The present invention is directed to myrosin and be probably the situation of inducible enzyme, use the reaction substrate sinigrin of myrosin that thalline is induced, then measure the vigor of myrosin again. Replace glucose to carry out primary dcreening operation first by sinigrin, tentatively judge that bacterial strain is with or without the ability decomposing sinigrin. Re-use sinigrin induction thalline and produce myrosin, the metabolic pathway that thalline produces myrosin is made to activate, then complete thalline is joined in finite concentration sinigrin solution and carry out metabolism, centrifugal segregation thalline after certain time, measure the supernatant absorbance at 340nm place, it has been found that absorbance now is changed significantly compared with the absorbance of the sinigrin solution of the same concentrations being added without thalline. Therefore, in induction thalline method mensuration probiotic bacteria, the method for myrosin activity is feasible.
The present invention adopts sinigrin as specific substrate to bring out strain secretes high-activity black myrosase, thus substantially increasing the sensitivity of myrosin detection; Additionally, the method is simple to operate, the detection cycle is short.
Detailed description of the invention
Below technical scheme is further described, but is not limited thereto, every technical solution of the present invention modified or equivalent replaces, without deviating from the spirit and scope of technical solution of the present invention, all should be encompassed in protection scope of the present invention.
The invention provides a kind of method utilizing sinigrin induction thalline method to measure myrosin activity, it specifically comprises the following steps that
1, solution preparation
MES buffer: 30mmol/LMES(30mol/L2-(N-morpholine) ethyl sulfonic acid solution), 6mmol/LMgCl2, 2mmol/LVc, NaOH regulates pH=6.5,121 DEG C of high pressure steam sterilization 15min.
2, experimental procedure
(1) strain recovery: this experiment test strains used is lactobacillus and bacillus bifidus, including: bacillus acidophilus, Lactobacillus bulgaricus, lactobacillus casei, Deshi Lactobacillus, bacillus acidophilus, Lactobacillus plantarum, Lactobacillus coryniformis, Lactobacillus paracasei, bacillus bifidus. Test strains is inoculated in MRS liquid culture medium with 2% inoculum concentration under aseptic condition, in 37 DEG C of constant temperature culture 24h. So activation can make for 2-3 time bacterial strain rejuvenate repeatedly.
(2) primary dcreening operation: be 5mmol/L containing sinigrin concentration, modified MRS culture fluid 5mL without glucose cultivates and treats sieve bacterial strain, incubation time 48h, cultivation temperature 30 DEG C. Observe strain growth situation (with a blank cuvette controlled observation), as its can normal growth, then illustrate that this bacterial strain is likely to utilize sinigrin to produce the energy, namely this bacterial strain is likely to be of myrosin activity. Experimental result is: Lactobacillus plantarum and bacillus bifidus are 5mmol/L in sinigrin concentration, equal energy normal growth in the modified MRS culture fluid without glucose, therefore can carry out next step and sieve experiment again.
(3) multiple sieve: when there being glucose, treats that sieve bacterial strain can using glucose as energy substance, and after glucose exhausts, sinigrin can be induced and be treated that it is carried out decomposition and utilizes to obtain energy by sieve bacterial strain.
Configuration glucose and sinigrin concentration are the modified MRS culture fluid of 10mmol/L, are distributed into 10mL every test tube. Bacterial strain Lactobacillus plantarum to be screened and bacillus bifidus are inoculated into 30 DEG C of incubated overnight (12-18h) of this modified MRS culture fluid, it is then centrifuged for (4 DEG C, 10min, 7000g) take thalline, add 10mLMES wash buffer thalline, recentrifuge (4 DEG C, 10min, 7000g) after thalline is resuspended in 3mLMES buffer, take 1mL bacteria suspension and mix with the 1mL MES buffer containing 10mmol/L sinigrin, take two by all means altogether, cultivate 24h and 48h respectively at 30 DEG C. Finally be centrifuged (4 DEG C, 10min, 10000g) take supernatant at 340nm place mensuration absorbance (returning to zero with the MES buffer containing 5mmol/L sinigrin). Absorbance when assaying reaction just starts simultaneously, and calculate difference between the two, the two difference is more big, then show that sinigrin is decomposed, and illustrates to treat that the activity of sieve bacterial strain myrosin is more big. Treat the absorbance change of sieve bacterial strain decomposition sinigrin as shown in table 2.
The change (λ=340nm) of table 2 test strains culture fluid supernatant absorbance
| Treat sieve bacterial strain | 24h | 48h |
| Lactobacillus plantarum | 0.495±0.348 | 0.426±0.322 |
| Bacillus bifidus | 0.245±0.130 | 0.232±0.117 |
Analyzed by table 2: compared with just having started with reaction, test strains culture fluid supernatant has obvious change at the absorbance of 24h and 48h, and particularly Lactobacillus plantarum absorbance has significant diversity. After this explanation adds the probiotics bacterial suspension after being induced, sinigrin is decomposed, and namely probiotic bacteria thalline has secreted myrosin. It is therefore contemplated that the method that sinigrin induction thalline method measures myrosin activity is feasible.
Claims (1)
1. myrosin activity test method in a probiotic bacteria, it is characterised in that described method step is as follows:
One, probiotic bacteria to be measured is inoculated in MRS liquid culture medium with 2% inoculum concentration under aseptic condition, in 37 DEG C of constant temperature culture 24h, activation 2-3 time so repeatedly, bacterial strain is made to rejuvenate, described probiotic bacteria is bacillus class probiotic bacteria, the compound method of MRS liquid culture medium is as follows: peptone 10g, beef leaching thing 10g, yeast extract 5.0g, sodium acetate 5.0g, sodium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, Magnesium sulfate heptahydrate 0.2g, seven water manganese sulfate 0.05g, it is dissolved in water, then add 1ml Tween 80, be adjusted to 1000ml with water;
Two, primary dcreening operation: treat sieve bacterial strain, incubation time 48h, cultivation temperature 30 DEG C with cultivating containing the modified MRS culture fluid 5mL that sinigrin concentration is 5mmol/L, observe strain growth situation, it is possible to the bacterial strain of normal growth is for next step experiment;
Three, multiple sieve: preparation glucose and sinigrin concentration are the modified MRS culture fluid of 10mmol/L, bacterial strain to be screened inoculum concentration routinely is inoculated into 30 DEG C of incubated overnight 12-18h of this modified MRS culture fluid, it is then centrifuged for taking thalline, add 10mLMES wash buffer thalline, thalline is resuspended in 3mLMES buffer after recentrifuge, bacteria suspension is mixed according to the volume ratio of 1-1.5:1 with the MES buffer of 10mmol/L sinigrin, 24-48h is cultivated at 30 DEG C, last centrifuging and taking supernatant measures absorbance at 340nm place, compared with the absorbance of the MES buffer of the 10mmol/L sinigrin being added without thalline, filter out absorbance difference bacterial strain more than 0.2.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5770789A (en) * | 1995-06-28 | 1998-06-23 | University Of Montana | Heritable reduction in insect feeding on brassicaceae plants |
| CN102754682A (en) * | 2011-09-23 | 2012-10-31 | 中国林业科学研究院资源昆虫研究所 | Method for deactivating Maca myrosinase |
| CN103533842A (en) * | 2011-05-03 | 2014-01-22 | 哥本哈根大学 | A process for the manufacture of products from cruciferous crops |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5770789A (en) * | 1995-06-28 | 1998-06-23 | University Of Montana | Heritable reduction in insect feeding on brassicaceae plants |
| CN103533842A (en) * | 2011-05-03 | 2014-01-22 | 哥本哈根大学 | A process for the manufacture of products from cruciferous crops |
| CN102754682A (en) * | 2011-09-23 | 2012-10-31 | 中国林业科学研究院资源昆虫研究所 | Method for deactivating Maca myrosinase |
Non-Patent Citations (4)
| Title |
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| Total Myrosinase Activity Estimates in Brassica Vegetable Produce;Edward B. Dosz等;《J. Agric. Food Chem.》;20140804;第62卷(第32期);8094-8100 * |
| 盐芥黑芥子酶活性测定方法;金绍静等;《安徽农业科学》;20111231;第39卷(第7期);3788-3789,3795 * |
| 黑芥子酶提取及活性测定方法的改进;张蕾等;《东北师大学报(自然科学版)》;20110331;第43卷(第1期);118-121 * |
| 黑芥子酶研究进展;刘月萍等;《生物技术通讯》;20060930;第17卷(第5期);837-839 * |
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