CN105316390A - High-throughput screening method for efficient urea utilizing strains - Google Patents
High-throughput screening method for efficient urea utilizing strains Download PDFInfo
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- CN105316390A CN105316390A CN201510828914.1A CN201510828914A CN105316390A CN 105316390 A CN105316390 A CN 105316390A CN 201510828914 A CN201510828914 A CN 201510828914A CN 105316390 A CN105316390 A CN 105316390A
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Abstract
The invention discloses a high-throughput screening method for efficient urea utilizing strains, and belongs to the field of metabolic engineering. The method comprises the steps that after flat-plate preliminary screening is performed on strains to be screened, seed liquid is prepared through a deep hole plate and then fermented and cultured to obtain fermented supernatant, 40 muL of the supernatant to be detected, 50 muL of an iron-phosphoric acid solution and 10 muL of diacetyl monoxime thiosemicarbazide liquid are added in a PCR plate to be subjected to a reaction for 10 min in a PCR instrument at 100 DEG C, an OD525 absorption value is determined through a microplate reader, the concentration x of urea in a sample to be detected is calculated according to a formula y=45.45x+0.062, R2=0.999, then the result that the urea concentration is lower is obtained, and the strains with the high urea utilizing capacity are obtained. According to the method, the detection cost of a single sample is decreased by nearly 95p percent, and meanwhile the advantages of being short in detection time, high in efficiency and accuracy, capable of simultaneously detecting multiple samples and the like are achieved.
Description
Technical field
The present invention relates to a kind of method of high flux screening urea efficiency utilization bacterial strain, belong to metabolic engineering field.
Background technology
Urea (urea), also known as carboxamide (carbamide), the diamide of carbonic acid, molecular formula is H
2nCONH
2(CO (NH
2)
2, be the key substance in nitrogen metabolism and ornithine cycle.In addition, in pure mellow wine and yellow rice wine, the urea produced by yeast saccharomyces cerevisiae metabolism is all EC (urethanum) topmost precursor substance.During the fermentation, the urea in yeast cell produces primarily of arginine metabolism.When existing without yeast preference type nitrogenous source (glutamine, l-asparagine etc.) in fermention medium, urea can be degraded to carbonic acid gas and ammonia by ureohydrolase further.But when yeast preference type nitrogenous source exists, the expression of ureohydrolase can be subject to strong suppression.This phenomenon, by nitrogen repression effect (Nitrogencataboliterepression the is called for short NCR) regulation and control of yeast saccharomyces cerevisiae, can ensure that yeast cell preferential utilization can be beneficial to the nitrogenous source of its existence most in the living environment of complexity.The regulation and control of NCR effect can cause the accumulation of high concentration urea in yeast cell.Because urea is to the toxic effect of brewing yeast cell, therefore urea is transported to outside born of the same parents in the mode of active transport by urea permease by cell.In fermented liquid outside born of the same parents, urea spontaneously in the process of fermentation can form EC (urethanum) with ethanol synthesis.
Therefore reducing urea concentration is the inevitable choice reducing carcinogenic substance in yellow rice wine, at present for addressing this problem, constructed a large amount of engineering bacterias, but it exists the restriction of the problems such as food safety.Therefore, screening from occurring in nature the bacterial strain obtaining urea efficiency utilization is problem demanding prompt solution.
Conventional highly effective carbamide utilizes bacterial strain to be method by screening from yellow wine fermentation wine with dregs, and change method complex steps, specific aim is weak, cannot realize high-throughput.At present, carbamide detection method has colorimetry, integrated enzyme reaction method, high performance liquid chromatography, but urea measures test kit (enzyme linked reaction method) relates to two kinds of enzymes (urase, glutamate dehydrogenase), and its cost is high, poor stability; And high performance liquid chromatography--it is high that fluorimetric detector method detects accuracy, scope is comparatively wide, but it is not suitable for the high throughput testing of carrying out urea.And the colorimetric method of routine, comprise Diacetylmonoxime spectrophotometry, paradimethy laminobenzaldehyde method, Diacetylmonoxime sulphur ammonium urea method.But the display of (Wang Liyuan, 2010) bibliographical information, Diacetylmonoxime spectrophotometry many, the shortcoming such as extension rate is high consuming time; Diacetylmonoxime sulphur ammonium urea method needs to carry out under boiling water bath condition in colorimetric cylinder, reaction times, sample volume of a specified duration, required was large, boiling water bath can cause there is reaction system and easily lose, and data fluctuations is comparatively large, the rate of recovery goodness of fit is poor, sensing range is narrower, be not suitable for mass detection etc.
To sum up, current Natural Selection difficulty is large, screening efficiency is low, and existing carbamide detection method exists the shortcomings such as accuracy, stability and poor accuracy, cannot realize the high flux screening to urea efficiency utilization bacterial strain.
Summary of the invention
In order to overcome the problems referred to above, sensing range controls at 0.5 ~ 20mg/L by optimizing reaction by this research, and achieves urea high throughput testing.The method of a kind of high flux screening urea efficiency utilization bacterial strain of the present invention, chemical levels is few, single pattern detection cost can be made to reduce nearly 95%, and detection time is short, efficiency is high, can detects multiple sample simultaneously.
First object of the present invention is to provide a kind of method that high flux screening urea utilizes bacterial strain, it is characterized in that, described method is first is that treating the solid medium of only nitrogen source is sieved in bacterial strain from being grown on urea, the relatively large bacterial strain of choosing colony is as primary dcreening operation bacterial strain, then shaking culture in primary dcreening operation inoculation to deep-well plates is prepared seed liquor, again seed liquor is seeded in another deep-well plates with identical inoculative proportion and carries out fermentation culture, after fermentation culture terminates, deep-well plates is centrifugal, the supernatant liquor got after supernatant liquor or dilution is sample to be tested; By the Diacetylmonoxime thiamines urea liquid of the iron-phosphoric acid solution of the sample to be tested of 40 μ L, 50 μ L and 10 μ L, add in PCR plate, in PCR instrument, 100 DEG C of reaction 10min, add reacted liquid in 96 orifice plates, measure OD by microplate reader
525absorption value y, according to formula y=45.45x+0.062 (R
2=0.999) urea concentration x in sample to be tested is calculated, and then directly obtain or to convert the urea concentration obtained in supernatant liquor according to Dilution ratio, the bacterial strain corresponding to the hole that the urea concentration in supernatant liquor is relatively little is the urea that multiple sieve obtains and utilizes bacterial strain; Wherein, x span is 0.5-20, unit mg/L.
In one embodiment of the invention, treat described in that sieve bacterial strain is yeast saccharomyces cerevisiae.
In one embodiment of the invention, treat described in that sieve bacterial strain is rice wine production bacterial strain.
In one embodiment of the invention, described seed liquor is in 48 or 96 deep-well plates, in 900r/min, 30 DEG C, cultivate 48h and obtain.
In one embodiment of the invention, described inoculation is that the seed liquor of 100 μ L is seeded in 48 or 96 deep-well plates containing 900 μ L fermention mediums.
In one embodiment of the invention, in the fermention medium that described fermentation culture uses, by g/L, containing glucose 20, peptone 20, yeast powder 10.
In one embodiment of the invention, described fermentation culture is at 30 DEG C of fermentation culture 72h.
In one embodiment of the invention, described fermentation culture is carried out in 48 or 96 deep-well plates.
In one embodiment of the invention, described Diacetylmonoxime thiamines urea liquid is dissolved Diacetylmonoxime 600mg, thiamines urea 30mg adding distil water, and add water and be settled to that 100mL obtains.
In one embodiment of the invention, ferric ammonium sulfate 600mg is dissolved in 100mL strong phosphoric acid and obtains by described iron-phosphoric acid solution.
Usefulness of the present invention:
(1) establish a kind of method of high flux screening urea efficiency utilization bacterial strain, greatly simplifie whole screening process.The inventive method is by optimizing reagent adding proportion etc., single pattern detection cost can be made to reduce nearly 95% (reagent dosage is reduced to 100 μ L from 5mL), the screening cycle is short, 96 samples can be detected simultaneously, efficiency is high, easy (can the volley of rifle fire be used, the sample in 12 holes can be added simultaneously).
(2) accuracy of the present invention is high, stability is high, typical curve y=45.45x+0.062 (R
2=0.999), in yellow rice wine, mark-on reclaims and measures, and the rate of recovery is: 98.2% ~ 101.18%, and average recovery rate is 99.47%.Detected result of the present invention and high performance liquid chromatography detected result basically identical.
(3) according to method of the present invention, can screen the bacterial strain of the urea efficiency utilization in yellow rice wine, obtain a strain efficiency utilization bacterium, may be used for yellow rice wine or pure mellow wine is brewageed, reduce the urea in product and ethyl carbamate content.
Embodiment
The mensuration of urea: high performance liquid chromatography--fluorimetric detector (HPLC)
Instrument: Agilent1260 high performance liquid chromatograph (joining UV-vis detector, fluorimetric detector and workstation), chromatographic condition:
Chromatographic column: ZORBAXEclipseXDBC18 (250 × 4.6mm, 5 μm)
Moving phase: 2mMCH
3cOONa
Flow velocity: 1mL/min
Column temperature: 35 DEG C
Sample size: 20 μ L
UV-detector wavelength: 210nm (detecting α-ketoglutaric acid and pyruvic acid)
Fluorimetric detector wavelength: excitation wavelength: 213nm emission wavelength: 308nm
Sample preparation: 1mL fermented liquid is centrifugal 5min under 12,000rpm, gets the hydrochloric acid 100 μ L that 400 μ L supernatant liquors add 20M9-xanthenes alcohol 600 μ L and 1.5M, dark reaction 30min.Then through 0.22 μ L membrane filtration, filtrate is for efficient liquid phase chromatographic analysis.
Substratum:
Seed culture medium (g/L): glucose 20g, peptone 20g, yeast powder 10g.115 DEG C of sterilizing 15min.
Primary dcreening operation substratum (g/L): glucose 20g, YNB1,74g, urea 5g, agar 20g.115 DEG C of sterilizing 15min.
Fermention medium (g/L): glucose 20g, peptone 20g, yeast powder 10g.115 DEG C of sterilizing 15min.
Embodiment 1: Specification Curve of Increasing
That standardized solution is diluted to 0.1mg/L, 0.3mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 3.0mg/L, 4.5mg/L, 5.0mg/L, 6.0mg/L, 8.0mg/L, 10.0mg/L, 20mg/L, 30mg/L by standard urea solution gradient dilution, then sample (standard urea solution), Diacetylmonoxime thiamines urea and iron-phosphoric acid solution are mixed with volume ratio 4:1:5, in PCR instrument, 100 DEG C of reaction 10min, can cooling room temperature detector survey.
Adopting microplate reader full wavelength scanner determination maximum absorption band to be 525nm, is X-coordinate with urea concentration, with OD
525absorption value is ordinate zou, Criterion curve y=45.45x+0.062 (R
2=0.999), in yellow rice wine, mark-on reclaims and measures, and the rate of recovery is: 98.2% ~ 101.18%, and average recovery rate is 99.47%.
Meanwhile, present method and high performance liquid chromatography are detected urea result basically identical.
Embodiment 2: the screening of superior strain
(1) sieve waiting the primary dcreening operation substratum that inoculation is only nitrogen source in urea, bacterial strain 400 strain that choosing colony is relatively large, as primary dcreening operation bacterial strain;
(2) by the seed culture medium in primary dcreening operation inoculation to 96 deep-well plates, 48h is cultivated in 900r/min30 DEG C of high-throughput deep-well plates constant temperature oscillator, obtain seed liquor, again by seed liquor with 10% inoculum size be seeded in 48 deep-well plates, 900r/min, 30 DEG C of fermentation culture 72h, after fermentation culture terminates by centrifugal for deep-well plates in the centrifugal 10min of 3500r/min.
(3) supernatant liquor got after supernatant liquor or dilution is sample to be tested; By the Diacetylmonoxime thiamines urea liquid of the iron-phosphoric acid solution of the sample to be tested of 40 μ L, 50 μ L and 10 μ L, add in PCR plate (96 orifice plate), in PCR instrument, 100 DEG C of reaction 10min, add reacted liquid in 96 orifice plates, measure OD by microplate reader
525absorption value y, according to formula y=45.45x+0.062 (R
2=0.999) (x span is 0.5-20 to calculate urea concentration x in sample to be tested, unit mg/L, not in this scope, by Sample Dilution or concentrated after detect again), and then directly obtain or to convert the urea concentration obtained in supernatant liquor according to Dilution ratio;
(4) choose the bacterial strain corresponding to hole that urea concentration in supernatant liquor is relatively little, be the urea that multiple sieve obtains and utilize bacterial strain.
Embodiment 3: the checking of method reliability and superior strain
(1) method reliability demonstration: the sample to be tested concentration obtained according to embodiment 2, the dense element of random selecting sample to be tested is at 20, sample, 20, the sample of 1.0-3.0mg/L, 20, the sample of 8.0mg/L-10.0mg/L of 0.3-0.5mg/L, then urea concentration in the fermented supernatant fluid of HPLC and enzyme linked reaction method difference tester 60 strain bacterium is used, found that, the detected result of the inventive method and HPLC or enzyme linked reaction method basically identical, the bacterial strain that the urea concentration that present method detects is relatively high, it is also relatively high for using enzyme linked reaction method to detect.Illustrate, the inventive method reliability is strong.
(2) bacterial strain 5 strain corresponding to hole that the urea concentration that obtains of Example 2 is relatively minimum, shake flask fermentation verifies its urea utility.After being activated by bacterial strain, the inoculum size access with 10% is added L-glutamic acid, glutamine, ammonium sulfate and urea and is (500mL shaking flask dress 50mL (after inoculation volume)) in the fermention medium of the YNB of 20mM, 200r/min, 30 DEG C of cultivations.Found that the bacterial strain that urea concentration that embodiment 2 obtains is minimum, after shake flask fermentation, be also that urea concentration is minimum, illustrate, detection method accuracy of the present invention is high.
Embodiment 4: the application of yeast
Yellow rice wine simulation leavening property detects
1. seed liquor preparation: the 10-D bacterial classification list bacterium colony that picking N85, XZ-11 and the method by embodiment 1-3 obtain in 5mLYPD liquid nutrient medium or rice juice substratum, the activated spawn step by step of the inoculum size according to 10%, and be cultured to desired number.Culture condition is
200rmin
-1, 12h.
2. zymotechnique:
Carry out laboratory scale yellow wine fermentation, each bacterium is cooked parallel.Yellow wine fermentation technical process: rice dipping → steamed rice → cooling → mixed song → inoculation → front ferment → rear ferment → filtration clarification.
Rice dipping: get 100g rice, rice layer about 10cm is crossed in water logging,
dipping 2 ~ 3d.
Boiling: atmospheric cooking, maintains about 25min after having a large amount of steam, to uniform particles, interior without the white heart, ripe and not sticky, soft and not rotten.
Add wheat koji and mix meal: ventilating and cooling, spreading for cooling to room temperature (
left and right) after, add wheat koji (17% of rice material) 17g (raw wheat koji 13g, ripe wheat koji 4g), water 170g (material-water ratio: 1:1.7).
Front ferment: by the inoculum size access yeast starter liquid of 5% (V/M) in fermentation liquid, shake up, add fermentation bung, be positioned over
bottom fermentation.After 12h, shaking flask once, and every day, shaking flask was weighed afterwards, works as CO
2weightlessness terminates main ferment when being less than 2.0g.
Rear ferment: triangular flask transfer is placed in
under, fermentation about 15d.
Decoct wine: filter paper filtering.
Get the yeast saccharomyces cerevisiae N85 (saccharomycescerevisiaeN85) that provided by Guyuelongshan Shaoxing Wine Co Ltd, Zhejiang and yeast saccharomyces cerevisiae xz-11 (to thank and extensively send out etal., 2015), apply under above-mentioned yellow rice wine simulation fermentation condition, simulation fermentation period detects the urea concentration of fermented liquid for 20 days afterwards.Result shows, compares with yeast saccharomyces cerevisiae xz-11 with yeast saccharomyces cerevisiae N85, and the urea concentration that the present invention screens the bacterial strain fermentation liquor obtained significantly reduces.
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 (9)
1. a high flux screening urea utilizes the method for bacterial strain, it is characterized in that, described method is first is that treating the solid medium of only nitrogen source is sieved in bacterial strain from being grown on urea, the relatively large bacterial strain of choosing colony is as primary dcreening operation bacterial strain, then shaking culture in primary dcreening operation inoculation to deep-well plates is prepared seed liquor, again seed liquor is seeded in another deep-well plates with identical inoculative proportion and carries out fermentation culture, after fermentation culture terminates, deep-well plates is centrifugal, the supernatant liquor got after supernatant liquor or dilution is sample to be tested; By the Diacetylmonoxime thiamines urea liquid of the iron-phosphoric acid solution of the sample to be tested of 40 μ L, 50 μ L and 10 μ L, add in PCR plate, in PCR instrument, 100 DEG C of reaction 10min, add reacted liquid in 96 orifice plates, measure OD by microplate reader
525absorption value y, urea concentration x in sample to be tested is calculated according to formula y=45.45x+0.062, and then directly obtain or to convert the urea concentration obtained in supernatant liquor according to Dilution ratio, the bacterial strain corresponding to the hole that the urea concentration in supernatant liquor is relatively little is the urea that multiple sieve obtains and utilizes bacterial strain; Wherein, x span is 0.5-20, unit mg/L.
2. method according to claim 1, is characterized in that, described in treat sieve bacterial strain be yeast saccharomyces cerevisiae.
3. method according to claim 1, is characterized in that, described Diacetylmonoxime thiamines urea liquid, is Diacetylmonoxime 600mg, thiamines urea 30mg are dissolved in water, and adds water and be settled to that 100mL obtains.
4. method according to claim 1, is characterized in that, ferric ammonium sulfate 600mg is dissolved in 100mL strong phosphoric acid and obtains by described iron-phosphoric acid solution.
5. method according to claim 1, is characterized in that, in the fermention medium that described fermentation culture uses, by g/L, containing glucose 20, and peptone 20, yeast powder 10.
6. method according to claim 1, is characterized in that, described inoculation is in 48 or 96 deep-well plates that are seeded to containing 900 μ L fermention mediums of the seed liquor of 100 μ L.
7. method according to claim 1, is characterized in that, described fermentation culture is at 30 DEG C of fermentation culture 72h.
8. method according to claim 1, is characterized in that, described seed liquor is in 48 or 96 deep-well plates, in 900r/min, 30 DEG C, cultivate 48h and obtain.
9. the urea obtained by the arbitrary described method of claim 1-8 utilizes bacterial strain.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113495064A (en) * | 2020-03-19 | 2021-10-12 | 财团法人工业技术研究院 | Detection reagent, detection device and method for detecting primary amide compound |
| CN113913314A (en) * | 2021-11-29 | 2022-01-11 | 江南大学 | Strain for reducing accumulation of urea and ethyl carbamate in yellow wine and application thereof |
| CN114045219A (en) * | 2021-11-17 | 2022-02-15 | 中国药科大学 | Method for rapidly screening high-yield caffeic acid strains in high flux |
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| CN103710278A (en) * | 2013-12-25 | 2014-04-09 | 江南大学 | Industrial rice wine yeast metabolic engineering bacteria with low-yield urea and building method thereof |
| CN104232734A (en) * | 2014-09-18 | 2014-12-24 | 中国科学院天津工业生物技术研究所 | High-throughput screening method of high-yield microbial strains for adenosine |
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| CN103710278A (en) * | 2013-12-25 | 2014-04-09 | 江南大学 | Industrial rice wine yeast metabolic engineering bacteria with low-yield urea and building method thereof |
| CN104232734A (en) * | 2014-09-18 | 2014-12-24 | 中国科学院天津工业生物技术研究所 | High-throughput screening method of high-yield microbial strains for adenosine |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113495064A (en) * | 2020-03-19 | 2021-10-12 | 财团法人工业技术研究院 | Detection reagent, detection device and method for detecting primary amide compound |
| CN113495064B (en) * | 2020-03-19 | 2024-05-03 | 财团法人工业技术研究院 | Detection reagent, detection device, and method for detecting primary amide compound |
| CN114045219A (en) * | 2021-11-17 | 2022-02-15 | 中国药科大学 | Method for rapidly screening high-yield caffeic acid strains in high flux |
| CN113913314A (en) * | 2021-11-29 | 2022-01-11 | 江南大学 | Strain for reducing accumulation of urea and ethyl carbamate in yellow wine and application thereof |
| CN113913314B (en) * | 2021-11-29 | 2023-02-24 | 江南大学 | Strain for reducing accumulation of urea and ethyl carbamate in yellow wine and application thereof |
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