CN108642125A - A kind of inositol quantitative detecting method based on microwell plate - Google Patents
A kind of inositol quantitative detecting method based on microwell plate Download PDFInfo
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- CN108642125A CN108642125A CN201810441370.7A CN201810441370A CN108642125A CN 108642125 A CN108642125 A CN 108642125A CN 201810441370 A CN201810441370 A CN 201810441370A CN 108642125 A CN108642125 A CN 108642125A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
Abstract
The invention discloses a kind of inositol quantitative detecting method based on microwell plate, belongs to microorganism detection field.The present invention includes the following steps：A. bacterium recovery recovery strain will be detected；B. recovery slant strains are washed down using 0.9% sterile saline, be collected by centrifugation cell again with 0.9% Sterile Saline suspension cell, concussion mixing be prepared into bacterium base fluid；C. it chooses suitable bacterium base fluid to be diluted using 0.9% Sterile Saline, controls the light absorption value of bacteria suspension concentration between 0.1~0.3, be as inoculated with bacteria suspension；D. inositol standard solution or sample solution to be detected and sterile water are added in microwell plate according to a certain percentage, are supplemented inositol assay broth；E. it will be inoculated in bacterial suspension inoculation to microwell plate, be placed in high speed concussion shaking table, 14~17h of shake culture；F. bacterium solution light absorption value in microwell plate is measured using microplate reader after cultivating, draw inositol standard curve and calculates the inositol content in sample.The present invention has the characteristics that at low cost, easy to operate, detection time is short and flux is big.
Description
Technical field
The present invention relates to a kind of inositol quantitative detecting method based on microwell plate, it is especially a kind of based on microwell plate culture
The method of high throughput detection food mysoinositol content, belongs to microorganism detection field.
Background technology
Inositol is the cyclitol containing 6 carbon, and medical field is divided in vitamins, referred to as vitamin B8.Inositol and
Its derivative and cyclohexanehexol compound are present in all animal tissues, the content highest in heart and brain.Inositol is extensive at present
Applied to fields such as medical industry, food, feed and daily-use chemical industries.Many experiments show inositol be a kind of required nutrition because
Son, inositol in human and animal's body can itself synthesis, but come from food mostly.To the detection of food mysoinositol content
Through an important indicator as food inspection.
Inositol quantitative analysis method mainly has classical mass method, sodium periodate method, high performance liquid chromatography, gas-chromatography
Method and microbial method.Mass method cannot exclude glucose using harmful substances such as a large amount of chloroforms
It influences；Sodium periodate method is only applicable to inositol sample as main component and measures, and glucose and glycerine are equal to flesh in complex sample
The organic principle that alcohol structure is similar or functional group is similar can influence measurement result very big；High performance liquid chromatography and gas phase color
Spectrometry not only needs expensive determining instrument, specific chromatographic column, will also by technologies such as complicated derivatizations, cost is higher,
It operates cumbersome；Microbial method, it is quantitative to survey using saccharomyces uvarum bacterium (ATCC-9080) to the specificity and sensitivity of inositol
Make the content of food mysoinositol.Compared with above-mentioned other methods, microbial method is with quick, easy, detection limit is low, sensitivity
The advantages that high is that two in national food safety standard (measurement GB5009.270-2016 of food mysoinositol) recommend detection
One of method.
Existing micro- measurement inositol method of thinking of a way mainly includes the following steps that：1, the strain that inoculation inclined-plane preserves is to new wheat
It on bud leaching powder agar slant culture-medium and cultivates, obtains recovery strain；2, by recovery strain transfer to fructus hordei germinatus leaching powder culture medium,
16-24h is cultivated at defined temperature；3, lawn is scraped in the sterile centrifugation tube equipped with physiological saline using oese, into
It goes and centrifuges, cleans, so cleaning 3-4 times, a certain amount of bacterium solution immigration of absorption, which is equipped in 10mL normal saline solutions, to be connect
Kind bacteria suspension makees blank using spectrophotometer with physiological saline, controls the light transmittance at 550 nm of the bacterium solution 60%
~80%；4, finally take inoculation bacterial suspension inoculation to containing inositol assay broth and inositol standard working solution or sample to be tested
In culture tube (culture solution total volume is 10mL), 22-24h is cultivated, culture cell suspending liquid is taken, absorbance is measured after dilution, from
And it obtains inositol standard curve and calculates sample to be tested mysoinositol content.
Existing microorganism detection method has the following problems：1, the consumption of inositol assay broth is very big, testing cost compared with
It is high.It is 5mL (about 2 yuan of cost) that every culture tube mysoinositol, which measures the additive amount of culture medium, in detection process, standard curve
It measures and needs 10 culture tubes, every needs to consume 5mL inositol assay broths (about 20 yuan of cost)；Each sample to be tested national standard
Middle requirement dilutes 3 concentration, each concentration do 3 it is parallel, single sample detection needs to consume 45mL inositol assay broth (samples
About 18 yuan of product testing cost).2, the detecting step after cultivating is cumbersome, and required time is longer.Existing inositol detection process point
For two stages：Tube propagation stage and spectrophotometer detection-phase.Cultivation stage carries out in culture tube, culture terminate with
After can not directly measure, need to by the culture bacterium solution in test tube dilute after, be placed in cuvette and sample be measured one by one, sample
Detection process is cumbersome, and the time is long.3, used in examining is yeast cells, and yeast cells individual is larger, easily settles, bacterium solution
Absorbance detection Step Time is long, is easy to influence the accuracy and repeatability of inspection result.4, further, since detecting step compared with
It is cumbersome, great work intensity, it is difficult to realize and the high throughput of a large amount of samples is detected.To solve the above problems, needing to develop a kind of behaviour
Make the new method of high-throughput detection simple, quick, at low cost and easy to implement.
Invention content
The object of the present invention is to provide a kind of methods detecting inositol content using microwell plate, with easy to operate, inspection
Survey time short, at low cost, high-throughput feature.
The method of the invention includes the following steps：
Step A：Bacterium is used in the detection of recovery inositol.Inositol detection can use saccharomyces uvarum bacterium ATCC-9080 with bacterium, or
Schizosaccharomyces pombe, heatproof flocculating yeast etc. have the auxotrophic bacterial strain of inositol；It is preferred that saccharomyces uvarum bacterium ATCC-
9080；
Step B：Prepare inoculation bacterium base fluid：After the detection that recovery obtains repeatedly is washed with bacterium with Sterile Saline, it is added to
In Sterile Saline, bacterium base fluid is made；
Step C：It draws suitable bacterium base fluid to be diluted to obtain bacteria suspension using 0.9% Sterile Saline, with sterile salt
Water makees blank, and using the light absorption value of the bacteria suspension under spectrophotometric determination 600nm wavelength, control bacteria suspension light absorption value exists
Between 0.1~0.3, it is as inoculated with bacteria suspension；
Step D：Inositol standard solution or sample solution to be detected are added in microwell plate, inositol is supplemented and measures culture
Base；
Step E：It takes in the microwell plate (96 hole microwell plate) in the inoculation bacterial suspension inoculation to step D in step C, microwell plate
It is placed in the high speed concussion shaking table of 1200~1350r/min, in 30 ± 1 DEG C of 14~17h of shake culture；Shaking table is shaken using high speed
Shake culture is carried out to microwell plate, can prevent yeast cells from settling, yeast cells can be dispersed in culture solution, profit
Cell concentration is detected in later-stage utilization microplate reader, shortens detection incubation time；
Step F：For being added to the microwell plate of inositol standard solution in step D, after the shake culture of step E, utilize
Microplate reader measures light absorption value at 600nm, and using light absorption value as ordinate, and standard curve is drawn by abscissa of inositol content；
For being added to the microwell plate of detected sample solution in step D, after the shake culture of step E, using microplate reader in 600nm
Place measures light absorption value, according to standard curve, calculates the inositol content in sample.
In one embodiment of the invention, the dress sample volume in step E in single micropore is 150 μ of μ L~300 L, excellent
Select 200 μ L.
In one embodiment of the invention, the volume of the bacteria suspension accessed in single micropore in step E is 3 L~10 μ
μ L, preferably 5 μ L.
Specifically, the method that the microbial method of microtitre plate format quantitatively detects inositol, includes the following steps.
Step A：The saccharomyces uvarum bacterium ATCC-9080 that inclined-plane preserves is seeded to new fructus hordei germinatus leaching powder agar slant culture
On base and culture obtains recovery strain；
Step B：Prepare inoculation bacterium base fluid；
The recovery strain of step A is washed down using 0.9% Sterile Saline, is collected by centrifugation cell, then with 0.9% it is sterile
Brine suspension cell again collects thalline again, and repeated washing 2-3 times adds 10mL Sterile Salines, and concussion mixing is prepared into
Bacterium base fluid；
Step C：It chooses suitable bacterium base fluid to be diluted using 0.9% Sterile Saline, with spectrophotometer, with sterile
Brine makees blank, the light absorption value of the bacteria suspension is measured under 600nm wavelength, control bacteria suspension light absorption value is between 0.1~0.3, i.e.,
To be inoculated with bacteria suspension；
Step D：Inositol standard solution or sample solution to be detected are added in microwell plate, supplement inositol measures culture
Base；
Step E：It taking in the inoculation bacterial suspension inoculation to microwell plate in step B, microwell plate is placed in high speed concussion shaking table,
1350r/min, 30 ± 1 DEG C of 14~17h of shake culture；
Step F：The light absorption value of the micropore containing inositol standard solution in microwell plate is measured at 600nm using microplate reader,
And using light absorption value as ordinate, standard curve is drawn by abscissa of inositol content；It is measured with phase co-wavelength and adds test sample to be checked
The light absorption value of product solution micropore calculates the inositol content in sample according to standard curve.
The present invention utilizes Microdilution plate method culture saccharomyces uvarum bacterium ATCC-9080 for detecting food mysoinositol content.With
Inositol content method is measured in national standard using Tube propagation to compare, method mysoinositol provided by the invention measures the use of culture medium
Amount is only the 1/50 of national standard method, can substantially reduce the use for measuring culture medium, can greatly reduce inositol and measure culture
The use cost of base；It is cultivated in high speed concussion shaking table using microwell plate, final result is examined simultaneously using microplate reader
It surveys, avoids test tube and go to the cumbersome operation of cuvette, reduce since yeast settles the influence to measurement result, improve inspection
Survey the accuracy and repeatability of result；And entire detection time is short, only needs shake culture 14-17h in the present invention, and national standard
In need to cultivate 22-24h, incubation time decrease beyond 1/4；Detection needs to carry out strain culturing, labour using test tube in national standard
Intensity is big, cumbersome, it is difficult to realize high-throughput detection, the present invention is cultivated and detected using microwell plate, at low cost, behaviour
Make simply, the quick detection of multiple samples can be realized in single 96 hole microwell plate, it is easy to accomplish high throughput detection.
The present invention has substantive distinguishing features and significant progress following prominent：What assay method through the invention was drawn
Calibration curve coefficient correlation and the existing detection method of national standard are close, and detection process greatly reduces cost and checkout procedure
Fussy degree shortens detection time, increases the accuracy and repeatability of testing result, at the same can be high-throughput to sample
Product are detected.
Description of the drawings
Fig. 1 Microdilution plate method inositols measure flow diagram.
Fig. 2 differences fill sample volume inositol bioassay standard curve graph (a：Fill sample volume 150 μ L, b：Fill 200 μ L, c dresses of sample volume
300 μ L of sample volume).
Fig. 3 different vaccination amount inositol bioassay standard curve graphs (a：Inoculum concentration 3 μ L, b：Inoculum concentration 5 μ L, c：10 μ of inoculum concentration
L)。
Fig. 4 national standard GB5009.270-2016 mysoinositol microbiological method inositol bioassay standard curve graphs.
Fig. 5 sample detection mysoinositol bioassay standard curve graphs.
Specific implementation mode
For following embodiment with the inositol in the microbial method quantitative measurement standard solution of microtitre plate format, bioassay standard is bent
Line, in conjunction with attached drawing, the invention will be further described.
Embodiment 1
Include the following steps using microorganism to the method for standard solution mysoinositol content being detected with reference to figure 1.
Step A：Recovery inositol is inoculated with bacterium to inclined-plane, obtains recovery strain
The inositol inoculation bacterium is saccharomyces uvarum bacterium (ATCC-9080), and wheat is inoculated into after saccharomyces uvarum bacterium is activated
Bud soaks on powder agar slant culture-medium, is cultivated at 30 ± 1 DEG C for 24 hours, then obtains recovery strain, fructus hordei germinatus leaching powder fine jade after 2-3 generations of transferring
Fat inclined-plane culture based formulas：Fructus hordei germinatus leaching powder 30g, soy peptone 3g, agar powder 15g add water to 1L, 121 DEG C of sterilizing 20min,
PH is adjusted to 5.6 ± 0.2；
Step B：Prepare inoculation bacterium base fluid：
By on the recovery strain transfer to fructus hordei germinatus leaching powder agar medium of step A, 30 ± 1 DEG C of cultures for 24 hours, utilize 10mL's
0.9% sterile saline washes hypothallus, is transferred in sterile centrifugation tube, and 3000r/min centrifuges 5min, removes supernatant,
0.9% sterile saline for adding 10mL, is cleaned.3000r/min centrifuges 5min again, removes supernatant, weight
It cleans 2-3 times again, adds the Sterile Saline of 10mL, concussion mixing is bacterium base fluid；
Step C：It chooses suitable bacterium base fluid to be diluted using 0.9% Sterile Saline, with spectrophotometer, with sterile
Brine makees blank, and the light absorption value of the bacteria suspension is measured under 600nm wavelength, and control bacteria suspension light absorption value obtains between 0.1~0.3
To inoculation bacteria suspension.The ingredient and preparation method of inositol assay broth are referring to national food safety standard GB5009.270-
2016《The measurement of food mysoinositol》.
Step D：Using inoculation bacteria suspension, standard curve is made
Step D1：Preparing new microwell plate, setting general assembly sample volume is 150 μ L, 200 μ L, 300 check experiments of μ L tri-,
With a row of microwell plate for 1 group, every group has 8 concentration gradients, and 3 control groups are arranged in each sample volume that fills.Respectively according to table 1,
The sterile water, inositol standard working solution and inositol assay broth of different volumes are added into each standard microplate for table 2, table 3.
Table 1 fills sterile water, inositol standard working solution and the inositol assay broth that 150 μ L microwell plates of sample volume are added
Table 2 fills sterile water, inositol standard working solution and the inositol assay broth that 200 μ L microwell plates of sample volume are added
Table 3 fills sterile water, inositol standard working solution and the inositol assay broth that 300 μ L microwell plates of sample volume are added
Step D2：10 μ L inoculation bacterial suspension inoculations are added into each standard culture micropore, it is enterprising in high speed concussion shaking table
Row shake culture, 1350r/min, 30 ± 1 DEG C of shake culture 14h.
Step D3：Utilize the absorbance of bacterium solution in micropore in microplate reader bioassay standard group, the measurement knot of difference dress sample volume
Fruit is shown in Table 4 (average values of 3 control groups), and using the inositol content of inositol standard working solution as abscissa, absorbance is ordinate
Standard curve is made, standard curve is shown in that Fig. 2, the related coefficient of standard curve are shown in Table 4.
The micropore light absorption value and calibration curve coefficient correlation of the different dress sample volumes of table 4
The present embodiment is to study in the present invention, and microwell plate difference fills reliability of the sample volume for testing result of the present invention
Influence, reflected by the related coefficient of standard curve, dress sample volume be 200 μ L when, the linearisation of standard curve is best.
Embodiment 2
The detection method of the present embodiment and embodiment 1 difference lies in：
Step D1, be added into each standard microplate according to table 2 sterile waters of different volumes, inositol standard working solution and
Inositol assay broth, total volume are 200 μ L.
Step D2, inoculation bacterial suspension inoculation, 3 μ L of setting inoculation volume, 5 μ L, 10 μ are added into each standard culture micropore
Tri- different vaccination amount inoculations of L.
Step D3, the measurement result of different vaccination amount is shown in Table 5, and the standard curve of making is shown in Fig. 3.
The micropore light absorption value and calibration curve coefficient correlation of 5 different vaccination amount of table
The present embodiment is to study in the present invention, and microwell plate is determining identical dress sample volume, different inoculum concentrations for
The influence of the reliability of testing result of the present invention.Reflected by the related coefficient of standard curve, 200 μ L are determined in dress sample volume
When, the linearisation of standard curve is best when inoculum concentration is 5 μ L.
To examine detection time for the influence of the testing result of the present invention, in detection inoculum concentration for present invention detection knot
The influence of fruit is carried out at the same time following operation.
It selects to detect bacterium solution in micropore using microplate reader when sample earthquake culture 12h, 14h, 18h of 5 μ L of inoculum concentration
Light absorption value, produces the standard curve of different incubation times, and related coefficient is shown in Table 6.It is observed it can be found that microwell plate in 12h
The growing state of middle yeast is poor, therefore without detection, the light absorption value after 18h is detected, light absorption value is all higher than 1, exceeds
Microplate reader detection range is detected, and is detected after 24h, light absorption value no longer increases, and reaches the growth upper limit.
The related coefficient for the standard curve that 6 inoculum concentration of table, 5 μ L difference incubation times make
| Detection time (h) | The related coefficient of standard curve |
| 12 | 0.9127 |
| 14 | 0.9765 |
| 18 | 0.9096 |
Comparative example
The detection method of this comparative example refers to national food safety standard GB5009.270-2016《The survey of food mysoinositol
It is fixed》The standard curve of middle microbial method, making is shown in Fig. 4.
The related coefficient of standard curve in Fig. 4 detects the related coefficient of ultimate criterion curve less than microwell plate, with micropore
The related coefficient for the standard curve that plate detection is not optimized is close, it was demonstrated that the reliability of detection method of the invention.The present invention
Inositol assay broth used in detection is only 1/50 in national standard method, and detection time is 1/4 in national standard, the present invention
It is middle that result is detected using microplate reader, the fussy degree of detection process is reduced, while eliminating because yeast cells exists
Testing result poor repeatability is caused because of sedimentation in detection process, the low influence of accuracy, to greatly improve testing result
Reliability.
Embodiment 3
It is the detection reliability of the invention to sample detection result, the present embodiment examines skimmed milk power mysoinositol content
It surveys.The present embodiment and embodiment 1 difference lies in：
Step D1, be added into each standard microplate according to table 2 sterile waters of different volumes, inositol standard working solution and
Inositol assay broth (total volume is 200 μ L).
Step D2,5 μ L inoculation bacterial suspension inoculations are added into each standard culture micropore, are carried out on high speed concussion shaking table
Culture, 30 DEG C of culture 14h.
Step D3, the standard curve of making is shown in Fig. 5.
Step E, by sample treatment at prepare liquid, and the absorbance of prepare liquid is measured
Weigh 1 portion of skimmed milk power, quality 2.1254g, by sample through dissolving with hydrochloric acid, digestion, pH value adjusting, constant volume, mistake
Prepare liquid is made after filter.Dilution is adjusted, makes the concentration of prepare liquid inositol within the scope of 0.1 μ of μ g/mL~1 g/mL.
Distilled water, prepare liquid and the inositol assay broth being added in the different test group micropores of table 7
| Sample number into spectrum | Sample A | Sample B | Sample C | Sample D | Sample E | Sample F | Sample G | Sample H |
| Prepare liquid (μ L) | 100 | 80 | 60 | 50 | 40 | 30 | 20 | 10 |
| Sterile water (μ L) | 0 | 20 | 40 | 50 | 60 | 70 | 80 | 90 |
| Measure culture medium (μ L) | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
Two parallel control groups are arranged in sample, be added into each micropore by table 7 sterile waters of different volumes, prepare liquid and
Inositol assay broth is respectively labeled as sample A, sample B, sample C, sample D, sample E, sample F, sample G, sample H, each
5 μ L inoculation bacterial suspension inoculations are added in the micropore of test group, after culture, the absorbance of prepare liquid are measured by microplate reader, as a result
It is shown in Table 8.
Sample：M=2.1254g
The absorbance and sample inositol content of 8 prepare liquid of table
Step F, the content of sample mysoinositol is calculated
According to the absorbance of prepare liquid in table 8, the quality of prepare liquid mysoinositol is checked in from standard curve shown in fig. 5,
The content that sample mysoinositol in each test microvia is calculated according to dilution gfactor and sample weighting amount, the results are shown in Table 8, by average
The inositol content that value calculates sample is 47.09mg/100g, and computational methods refer to national food safety standard GB5009.270-
2016《The measurement of food mysoinositol》.
Further to examine reliability of the present invention to food mysoinositol content detection, in the same of sample survey inositol content
Shi Jinhang or less operating procedures pass through the accuracy of recovery of standard addition validation criteria curve.
The specific steps that recovery of standard addition is examined：Sample (skimmed milk power) is weighed, the quality of mark-on sample is 2.1222g,
0.4mg inositols are added, is adjusted through dissolving with hydrochloric acid, digestion, pH value, mark-on prepare liquid is made after constant volume filtering.Dilution is adjusted, is made
The concentration of prepare liquid inositol is within the scope of 0.1 μ of μ g/mL~1 g/mL.
Distilled water, prepare liquid and the inositol assay broth being added in the different mark-on group micropores of table 9
Two parallel control groups are arranged in sample, be added into each micropore by table 9 sterile waters of different volumes, prepare liquid and
Inositol assay broth is respectively labeled as mark-on sample A, mark-on sample B, mark-on sample C, mark-on sample D, mark-on sample E, adds
Sample F, mark-on sample G, mark-on sample H are marked, 5 μ L inoculation bacterial suspension inoculations are added in the micropore of each test group, after culture, lead to
The absorbance that microplate reader measures prepare liquid is crossed, the results are shown in Table 10.
Mark-on sample m=2.1222g
The absorbance and mark-on sample inositol content of 10 mark-on prepare liquid of table
According to the absorbance of mark-on prepare liquid in table 10, from checking in flesh in mark-on prepare liquid in standard curve shown in fig. 5
The quality of alcohol calculates the inositol content in each test group culture tube after sample mark-on according to dilution gfactor and sample weighting amount, knot
Fruit is shown in Table 10, then the inositol content gone out by mean value calculation after sample mark-on is, computational methods are marked with reference to food security country
Quasi- GB5009.270-2016《The measurement of food mysoinositol》.
The overall merit of 11 microwell plate sample detection data reliability of table
The inositol amount that is added in the inositol content and sample before and after mark-on according to sample calculates recovery of standard addition, calculates
Method is as follows：
Sample mark-on amount=mark-on amount/sample quality
Recovery of standard addition=(mark-on sample size-sample size)/(sample mark-on amount × 100) × 100%
Wherein：
The inositol amount that mark-on amount --- recovery of standard addition is added in examining, 0.4mg.
Sample quality --- recovery of standard addition weighs the quality of sample in examining.
The inositol content 63.92mg/100g of mark-on sample size --- mark-on sample.
Sample size --- the inositol content 47.09mg/100g in sample.
It is 90.2% that recovery of standard addition, which is calculated,.
The present embodiment repeats sexual deviation and recovery of standard addition and carries out comprehensive comment to the reliability of detection data by relative deviation
Valence is shown in Table 11.
By table 11 as it can be seen that being detected to the inositol content of sample using Microdilution plate method, the recovery of standard addition of detection meets
The repetition sexual deviation that standard requirement, sample and mark-on sample measure is low, meets precision<10% requirement, no sample and mark-on
The inositol content data of sample>15%, data reliability is high, and measurement result is accurate.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
Claims (9)
1. a kind of method of detection inositol content, which is characterized in that include the following steps：
Step A：Bacterium is used in the detection of recovery inositol；
Step B：Prepare inoculation bacterium base fluid：After the detection that recovery obtains repeatedly is washed with bacterium with Sterile Saline, it is added to sterile
In brine, bacterium base fluid is made；
Step C：It draws suitable bacterium base fluid to be diluted to obtain bacteria suspension using 0.9% Sterile Saline, controls bacteria suspension
Light absorption value is as inoculated with bacteria suspension between 0.1~0.3 at 600nm；
Step D：Inositol standard solution or sample solution to be detected are added in microwell plate, inositol assay broth is supplemented；
Step E：It takes in the microwell plate in the inoculation bacterial suspension inoculation to step D in step C, microwell plate is placed in 1200~1350r/
In the high speed concussion shaking table of min, in 30 ± 1 DEG C of 14~17h of shake culture；
Step F：For being added to the microwell plate of inositol standard solution in step D, after the shake culture of step E, enzyme mark is utilized
Instrument measures light absorption value at 600nm, and using light absorption value as ordinate, and standard curve is drawn by abscissa of inositol content；For
It is added to the microwell plate of detected sample solution in step D, after the shake culture of step E, is surveyed at 600nm using microplate reader
Determine light absorption value, according to standard curve, calculates the inositol content in sample.
2. according to the method described in claim 1, it is characterized in that, it is saccharomyces uvarum bacterium ATCC- that inositol, which is detected with bacterium,
9080, schizosaccharomyces pombe, heatproof flocculating yeast etc. have one kind in the auxotrophic bacterial strain of inositol.
3. method according to claim 1 or 2, which is characterized in that inositol detection is saccharomyces uvarum bacterium ATCC- with bacterium
9080。
4. according to any method of claims 1 to 3, which is characterized in that in step C, draw suitable bacterium base fluid and utilize
0.9% Sterile Saline is diluted to obtain bacteria suspension, makees blank with Sterile Saline, uses spectrophotometric determination 600nm waves
The light absorption value of the bacteria suspension under length, control bacteria suspension light absorption value are as inoculated with bacteria suspension between 0.1~0.3.
5. method according to any one of claims 1 to 4, which is characterized in that the dress sample volume in step E in single micropore is
150 μ of μ L~300 L.
6. according to the method described in claim 5, it is characterized in that, the dress sample volume in step E in single micropore is 200 μ L.
7. method according to any one of claims 1 to 4, which is characterized in that the bacteria suspension accessed in single micropore in step E
Volume be 3 μ of μ L~10 L.
8. the method according to the description of claim 7 is characterized in that the volume of the bacteria suspension accessed in single micropore in step E
It is 5 μ L.
9. according to any method of claim 1~8, which is characterized in that
Step A：The saccharomyces uvarum bacterium ATCC-9080 that inclined-plane preserves is seeded on new fructus hordei germinatus leaching powder agar slant culture-medium
And it cultivates and obtains recovery strain；
Step B：Prepare inoculation bacterium base fluid；
The recovery strain of step A is washed down using 0.9% Sterile Saline, is collected by centrifugation cell, then with 0.9% Sterile Saline
Again suspension cell collects thalline again, and repeated washing 2-3 times adds 10mL Sterile Salines, and concussion mixing is prepared into bacterium base
Liquid；
Step C：It draws suitable bacterium base fluid to be diluted to obtain bacteria suspension using 0.9% Sterile Saline, be made with Sterile Saline
Blank, using the light absorption value of the bacteria suspension under spectrophotometric determination 600nm wavelength, control bacteria suspension light absorption value 0.1~
Between 0.3, it is as inoculated with bacteria suspension；
Step D：Inositol standard solution or sample solution to be detected are added in microwell plate, inositol assay broth is supplemented；
Step E：It takes in the inoculation bacterial suspension inoculation to microwell plate in step B, the high speed concussion that microwell plate is placed in 1350r/min is shaken
Bed, 30 ± 1 DEG C of 14~17h of shake culture；
Step F：Measure the light absorption value of the micropore containing inositol standard solution in microwell plate at the 600nm using microplate reader, and with
Light absorption value is ordinate, and standard curve is drawn by abscissa of inositol content；It is molten that addition detected sample is measured with phase co-wavelength
The light absorption value of liquid micropore calculates the inositol content in sample according to standard curve.
Priority Applications (1)
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| CN111562210A (en) * | 2020-06-16 | 2020-08-21 | 北京挑战农业科技有限公司 | Method for detecting viable count in pre-coated forage microecological preparation product |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109680035A (en) * | 2019-02-27 | 2019-04-26 | 上海市食品药品检验所 | A kind of screening technique of inositol mutant bacterial strain and its application |
| CN109680035B (en) * | 2019-02-27 | 2022-11-04 | 上海市食品药品检验研究院 | Screening method and application of inositol-deficient strain |
| CN111562210A (en) * | 2020-06-16 | 2020-08-21 | 北京挑战农业科技有限公司 | Method for detecting viable count in pre-coated forage microecological preparation product |
| CN111562210B (en) * | 2020-06-16 | 2023-01-03 | 北京挑战农业科技有限公司 | Method for detecting number of viable bacteria in pre-coated feed microecological preparation product |
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