CN106676161A - Micro-porous plate for quantitatively detecting nicotinic acid by microbiological method, kit and preparation method thereof - Google Patents
Micro-porous plate for quantitatively detecting nicotinic acid by microbiological method, kit and preparation method thereof Download PDFInfo
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Abstract
The invention provides a micro-porous plate for quantitatively detecting nicotinic acid by a microbiological method. The micro-porous plate is prepared by the following steps: 1) inoculating activated lactobacillus plantarum strains and removing supernatant; 2) adding 10ml to 12ml of a trehalose/sucrose and calcium chloride mixed solution; after uniformly mixing, centrifuging for 1min to 2min; removing supernatant and repeating the operation for one time; uniformly mixing to prepare bacterium suspension; sucking 1ml to 2ml of the bacterium suspension into 10ml to 12ml of the trehalose/sucrose and calcium chloride mixed solution, and uniformly mixing to prepare a bacterium solution for testing; 3) adding 2mul to 4mul of the bacterium solution for testing into each pore of the micro-porous plate; slightly heating the bacterium solution for testing under a low-vacuum environment, and boiling the bacterium solution for testing under a temperature condition of 33 DEG C to 37 DEG C and generating foam; evaporating and drying under the low-vacuum environment and at room temperature for 36h to 48h; or after freezing, drying for 36h to 48h in a sublimation manner. The invention further provides a kit comprising the micro-porous plate and a preparation method of the kit. According to the micro-porous plate, the kit and the preparation method thereof, the loss of strains in the kit is reduced and the accuracy of a nicotinic acid testing result is improved.
Description
Technical field
The present invention relates to a kind of microwell plate, its test kit for microbial method detection by quantitative nicotinic acid and preparation method thereof,
Belong to microbial method detection field.
Background technology
At present, it is all more complicated with regard to the detection method of nicotinic acid both at home and abroad, typically there are microbial method, high-efficient liquid phase technique and enzyme
Linked immunosorbent assay.Wherein, the lower limit of high-efficient liquid phase technique detection will be higher by much compared with microbial method, and precision is preferable, however it is necessary that expensive
Precision instrument and reagent, sample handling processes are complex, it is difficult to popularization and application are carried out in production；In addition, enzyme linked immunological
Method testing result is quite different with actual numerical value.And microbial method can delicately detect the nicotinic acid with biological activity, this is
Microbial method is different from the maximum feature and advantage of additive method.
Existing microbial process utilizes specificity of the Lactobacillus plantarum (Lactobacillus plantarum) to nicotinic acid
And susceptiveness, quantitative determine out the content of nicotinic acid in sample.The institute supplied in measure culture medium in addition to nicotinic acid is nutritious
Composition, the light transmittance that such growth of microorganism is produced will be with the content of nicotinic acid in standard curve working solution and unknown solution to be measured
It is corresponding；Standard curve is drawn with concentration of the light transmittance of variable concentrations standard solution relative to each concentration level standard substance,
The content of nicotinic acid in sample can be calculated according to standard curve.
But but there is many defects in the test kit for being coated with Lactobacillus plantarum, existing method makes in microwell plate at -80 DEG C
Microbial suspension freezing dormancy, then by the lyophilization under vacuo of the microbic mass in microwell plate.Some microorganisms are
Adaptation adverse environment, morphs or recombinates, therefore adds after water or sample, and some microorganisms are under conditions of without vitamin
Growth is remained to, causes high detection background, and also result in error result on rare occasion.In addition, existing method is adopted
Lyophilization under vacuum, can also result at least about 25% strain loss.
Accordingly, it would be desirable to further improving microbial process utilizes Lactobacillus plantarum (Lactobacillus plantarum)
Method to nicotinic acid detection.
The content of the invention
In order to overcome the shortcomings of above-mentioned existing method, the present invention provides a kind of for the micro- of microbial method detection by quantitative nicotinic acid
Orifice plate, can reduce strain loss, improve the accuracy of the testing result of nicotinic acid.
Another object of the present invention is to provide the preparation method of above-mentioned microwell plate.
The a further object of the present invention is to provide the test kit comprising the microwell plate.
It is still another object of the present invention to provide a kind of preparation side of the test kit for microbial method detection by quantitative nicotinic acid
Method.
To achieve these goals, a kind of microwell plate for microbial method detection by quantitative nicotinic acid of the present invention, its
It is prepared from by following steps：
1) Lactobacillus plantarum is activated, the lactobacillus plantarum strain after activation is inoculated in lactobacilluss broth bouillon, 32-
36 DEG C of culture 16-20h, 2200 turns/min centrifugation 1-2min, abandoning supernatant；
2) 10-12ml trehaloses/sucrose and calcium chloride mixed liquor are added, 1-2min, abandoning supernatant, weight is centrifuged after mixing
Multiple aforementioned operation once, then adds 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, mixes, and bacteria suspension is obtained；Inhale
Into 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, test bacterium solution is made in mixing to 1-2ml bacteria suspensions；
3) the test bacterium solution 2-4 μ l are added into each hole of microwell plate, respectively 20mTorr, 55mTorr,
The low vacuum environment end of 510mTorr, 5Torr, 45Torr, 210Torr slightly Thermal test bacterium solution, makes test bacterium solution in 33-37
DEG C temperature conditionss under boiling and produce foam, and evaporation drying 36-48h under the low vacuum environment and room temperature, Huo Zheleng
After jelly again by distil in the way of be dried 36-48h.
Wherein, step 1) in activation Lactobacillus plantarum adopt activation method：
11g defatted milk powder is added in 89ml distilled water, is mixed, 115 DEG C sterilize 15 minutes, as fluid medium；
0.3g powder plants lactobacillus strain is added in fluid medium described in 10ml, 37 ± 1 DEG C are cultivated up to curdled milk,
Curdy strain is obtained, 0-4 DEG C of preservation is positioned over；
Cultured curdy strain in step on 0.3ml is added in fluid medium described in 10ml, 37 ± 1 DEG C of cultures
Until curdled milk；
Previous step is repeated several times by, the strain for except for the difference that using every time is the culture that previous step is obtained, and waits to coagulate
Show that strain is activated after the newborn time is stable.
The lactobacillus plantarum strain is Lactobacillus plantarum ATCC8014.
Step 2) in trehalose/sucrose and calcium chloride mixed liquor, respective concentration is respectively 180-190mM, 10-
12mM。
In order to realize another object of the present invention, there is provided a kind of to prepare for the microwell plate of microbial method detection by quantitative nicotinic acid
Method, it is prepared from by following steps：
1) Lactobacillus plantarum is activated, the lactobacillus plantarum strain after activation is inoculated in lactobacilluss broth bouillon, 32-
36 DEG C of culture 16-20h, 2200 turns/min centrifugation 1-2min, abandoning supernatant；
2) 10-12ml trehaloses/sucrose and calcium chloride mixed liquor are added, 1-2min, abandoning supernatant, weight is centrifuged after mixing
Multiple aforementioned operation once, then adds 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, mixes, and bacteria suspension is obtained；Inhale
Into 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, test bacterium solution is made in mixing to 1-2ml bacteria suspensions；
3) the test bacterium solution 2-4 μ l are added into each hole of microwell plate, respectively 20mTorr, 55mTorr,
The low vacuum environment end of 510mTorr, 5Torr, 45Torr, 210Torr slightly Thermal test bacterium solution, makes test bacterium solution in 33-37
DEG C temperature conditionss under boiling and produce foam, and evaporation drying 36-48h under the low vacuum environment and room temperature, Huo Zheleng
In the way of distilling 36-48h is dried again after jelly, so as to detection by quantitative nicotinic acid microwell plate is obtained.
Wherein, step 1) in activation Lactobacillus plantarum adopt activation method：
11g defatted milk powder is added in 89ml distilled water, is mixed, 115 DEG C sterilize 15 minutes, as fluid medium；
0.3g powder plants lactobacillus strain is added in fluid medium described in 10ml, 37 ± 1 DEG C are cultivated up to curdled milk,
Curdy strain is obtained, 0-4 DEG C of preservation is positioned over；
Cultured curdy strain in step on 0.3ml is added in fluid medium described in 10ml, 37 ± 1 DEG C of cultures
Until curdled milk；
Previous step is repeated several times by, the strain for except for the difference that using every time is the culture that previous step is obtained, and waits to coagulate
Show that strain is activated after the newborn time is stable.
The lactobacillus plantarum strain is Lactobacillus plantarum ATCC8014.
Step 2) in trehalose/sucrose and calcium chloride mixed liquor, respective concentration is respectively 180-190mM, 10-
12mM。
The present invention also provides a kind of test kit comprising above-mentioned microwell plate.
The test kit, also comprising nicotinic acid standard substance, nicotinic acid test media and sterilized water.
Specifically, test kit of the present invention, comprising the above-mentioned micropore for being coated with Lactobacillus plantarum ATCC8014 bacterial strains
Plate, 3 × 3.2 μ g nicotinic acid standard substance, 3 × (0.75-0.76) g nicotinic acid test medias (12.0g/L casamino acids,
40.0g/L glucoses, 20.0g/L sodium acetates, 0.4g/L L-Cystine, 0.2g/L DL-Trps, 20.0mg/L sulphuric acid glands are fast
Purine, 20.0mg/L guanine hydrochlorides, 20.0mg/L uracil, 200.0 μ g/L thiamine hydrochlorides, 200.0 μ g/L calcium pantothenates,
400.0 μ g/L pyridoxine hydrochlorides, 400.0 μ g/L riboflavin, 200.0 μ g/L p- amino benzoic Acid, 0.8 μ g/L biotin,
1.0g/L dipotassium hydrogen phosphates, 1.0g/L sodium dihydrogen phosphate, 0.4g/L magnesium sulfate, 20.0mg/L Sodium Chloride, 20.0mg/L sulphuric acid are sub-
Ferrum, 20.0mg/L manganese sulfates) and 3 × 30ml sterilized water.
The preparation method of test kit of the present invention, it comprises the steps：
A. the microwell plate for being coated with lactobacillus plantarum strain is first prepared：
1) Lactobacillus plantarum is activated, the lactobacillus plantarum strain after activation is inoculated in lactobacilluss broth bouillon, 32-
36 DEG C of culture 16-20h, 2200 turns/min centrifugation 1-2min, abandoning supernatant；
2) 10-12ml trehaloses/sucrose and calcium chloride mixed liquor are added, 1-2min, abandoning supernatant, weight is centrifuged after mixing
Multiple aforementioned operation once, then adds 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, mixes, and bacteria suspension is obtained；Inhale
Into 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, test bacterium solution is made in mixing to 1-2ml bacteria suspensions；
3) the test bacterium solution 2-4 μ l are added into each hole of microwell plate, respectively 20mTorr, 55mTorr,
The low vacuum environment end of 510mTorr, 5Torr, 45Torr, 210Torr slightly Thermal test bacterium solution, makes test bacterium solution in 33-37
DEG C temperature conditionss under boiling and produce foam, and evaporation drying 36-48h under the low vacuum environment and room temperature, Huo Zheleng
In the way of distilling 36-48h is dried again after jelly, so as to detection by quantitative nicotinic acid microwell plate is obtained；
B. then prepare comprising nicotinic acid standard substance and nicotinic acid test media (12.0g/L casamino acids, 40.0g/L
Glucose, 20.0g/L sodium acetates, 0.4g/L L-Cystine, 0.2g/L DL-Trps, 20.0mg/L adenine sulfates,
20.0g/L guanine hydrochlorides, 20.0mg/L uracil, 200.0 μ g/L thiamine hydrochlorides, 200.0 μ g/L calcium pantothenates, 400.0 μ g/
L pyridoxine hydrochlorides, 400.0 μ g/L riboflavin, 200.0 μ g/L p- amino benzoic Acid, 0.8 μ g/L biotin, 1.0g/L phosphoric acid hydrogen
Dipotassium, 1.0g/L sodium dihydrogen phosphate, 0.4g/L magnesium sulfate, 20.0mg/L Sodium Chloride, 20.0mg/L ferrous sulfate, 20.0mg/L sulfur
Sour manganese).
Wherein, the activation Lactobacillus plantarum adopts activation method：
11g defatted milk powder is added in 89ml distilled water, is mixed, 115 DEG C sterilize 15 minutes, as fluid medium；
0.3g powder plants lactobacillus strain is added in fluid medium described in 10ml, 37 ± 1 DEG C are cultivated up to curdled milk,
Curdy strain is obtained, 0-4 DEG C of preservation is positioned over；
Cultured curdy strain in step on 0.3ml is added in fluid medium described in 10ml, 37 ± 1 DEG C of cultures
Until curdled milk；
Previous step is repeated several times by, the strain for except for the difference that using every time is the culture that previous step is obtained, and waits to coagulate
Show that strain is activated after the newborn time is stable.
The lactobacillus plantarum strain is Lactobacillus plantarum ATCC8014.
In the trehalose/sucrose and calcium chloride mixed liquor, respective concentration is respectively 180-190mM, 10-12mM.
Microwell plate and its test kit for microbial method detection by quantitative nicotinic acid of the present invention, by adding in preparation
Enter the trehalose/sucrose and calcium chloride mixed liquor protective agent of certain concentration and consumption, and improve bacterium solution drying meanss it is (specific
The low vacuum environment and drying mode of gradient), it is possible to decrease the strain loss in test kit, improve the standard of nicotinic acid testing result
Really property, Jing count plates, the active bacteria number preserved in the Lactobacillus plantarum dried foam in the microwell plate of preparation, exceed
95% (general freezer seasoning at least loses about 25% strain) of strain sum in added bacterium solution.
Description of the drawings
Fig. 1 is the examination criteria curve for microbial method detection by quantitative nicotinic acid of the present invention.
Specific embodiment
Below with reference to the accompanying drawings illustrating embodiments of the invention.Retouch in an a kind of accompanying drawing or embodiment of the present invention
In combination with element that the element stated and feature can be illustrated in one or more other accompanying drawings or embodiment and feature.Should
Work as attention, for purposes of clarity, eliminate known to unrelated to the invention, those of ordinary skill in the art in accompanying drawing and explanation
Part or process expression and description.
The present invention is described further below in conjunction with the accompanying drawings.
Embodiment 1
1. prepared by microwell plate
(1) Lactobacillus plantarum ATCC 8014 is activated
By 11g defatted milk powder add 89ml distilled water in, mix, with 10ml as unit subpackage to test tube in, 115 DEG C sterilizing
15 minutes, this solution was fluid medium.
The bacterial strains of 0.3g powder plant lactobacilluss ATCC 8014 are added in 10ml culture medium, 37 ± 1 DEG C of cultures are until solidifying
Breast, is positioned at 4 DEG C of refrigerator and preserves.
Cultured strain in step on 0.3ml is added in 10ml culture medium, 37 ± 1 DEG C of cultures are until curdled milk.
Previous step is repeated several times by, shows that strain is activated after the curdled milk time is stable.
(2) test bacterium solution to prepare
By in the inoculations of Lactobacillus plantarum ATCC 8014 after activation to lactobacilluss broth bouillon, 36 DEG C ± 1 DEG C is trained
Foster 24h, 2200 turns/min are centrifuged 2min so as to stop culture, abandoning supernatant；
Add 190mM trehaloses and 10mM CaCl2Mixed solution 10ml, mixes, 2200 turns/min centrifugation 2min, discards
Supernatant, then add 10ml protective agents, mix.Such as front centrifugally operated, abandoning supernatant.Again plus 12ml protective agents, mix.Inhale 1ml
Into 10ml protective agents, test bacterium solution is made in mixing to the bacteria suspension.
(3) microwell plate coating
By 2 μ l test bacterium solution etc. point additions into each hole of microwell plate, successively 20mTorr, 55mTorr, 510mTorr,
The slightly hot bacterium solution of the low vacuum environment end of 5Torr, 45Torr, 210Torr (can now be such that microorganism is preferably coated on micro-
On orifice plate, and the activity of microorganism is kept to greatest extent), make bacterium solution seethe with excitement under 35 DEG C of room temperature condition so as to produce bubble
Foam, and under the low vacuum environment and room temperature evaporation drying about 48 hours so as to the nicotinic acid detection microwell plate is obtained.
Jing count plates, the active bacteria preserved in the Lactobacillus plantarum dried foam in microwell plate prepared by said method
Number, has exceeded 95% (general freezer seasoning at least loses about 25% strain) for adding strain sum in bacterium solution.
2. prepared by standard substance
The quantitative 3.2 μ g nicotinic acid standard substance that obtain include 3 bottles of standard substance into brown reagent bottle with cover in each test kit.
With the 1 bottle of standard substance of aseptic water dissolution in 2ml test kits when using, the standard substance storing solution of 0.16mg/100ml is obtained, then
Desired concn is diluted to according to operating instruction, 0 standard substance are sterilized water.
3. prepared by culture medium
Weigh 12.0g casamino acids, 40.0g glucoses, 20.0g sodium acetates, 0.4g L-Cystine, 0.2gDL- colors
Propylhomoserin, 20.0mg adenine sulfates, 20.0g guanine hydrochlorides, 20.0mg uracil, 200.0 μ g thiamine hydrochlorides, 200.0 μ g
Calcium pantothenate, 400.0 μ g pyridoxine hydrochlorides, 400.0 μ g riboflavin, 200.0 μ g p- amino benzoic Acid, 0.8 μ g biotin, 1.0g
Dipotassium hydrogen phosphate, 1.0g sodium dihydrogen phosphate, 0.4g magnesium sulfate, 20.0mg Sodium Chloride, 20.0mg ferrous sulfate, 20.0mg sulphuric acid
Manganese, mixes.Weigh 0.75 mixed culture based powders to add in 1 medium bottle, each test kit includes 3 bottles.
4. prepared by sterilized water
By distilled water in 121 DEG C of autoclavings 15 minutes, every bottle of 30ml includes 3 bottles of sterilized water in each test kit.
5. other
2 adhesive foils are also included in test kit.
Embodiment 2
1. prepared by microwell plate
Coating lactobacillus plantarum strain adopts Lactobacillus plantarum ATCC 8014, and step is as follows：By the bacterial strain (activation after activation
Processing mode is with embodiment 1) be inoculated into lactobacillus broth culture medium, 38 DEG C of ± 1 DEG C of culture 18h, with 2200 revs/min from
Heart 3min so as to stop culture, abandoning supernatant.Add 180mM sucrose and 12mM CaCl2Protective agent mixed liquor 12ml, mixes
It is even, then 2min is centrifuged, abandoning supernatant, then add 10ml protective agents, mix.Such as front centrifugally operated, abandoning supernatant.Again plus 12ml
Protective agent, mixes.The 2ml bacteria suspensions are inhaled into 12ml protective agents, test bacterium solution is made in mixing.
By bacterium solution etc. described in 1 μ l point addition into each hole of microwell plate, respectively 20mTorr, 55mTorr, 510mTorr,
The slightly hot bacterium solution of low vacuum environment end of 5Torr, 45Torr, 210Torr, make bacterium solution seethe with excitement under 37 DEG C of room temperature condition from
And foam is produced, then foam freezing is subsequently dried 36 hours in the way of distilling, so as to nicotinic acid detection is obtained with micro-
Orifice plate.
Jing count plates, the active bacteria preserved in the Lactobacillus plantarum dried foam in microwell plate prepared by said method
Number, has exceeded 95% (general freezer seasoning at least loses about 25% strain) for adding strain sum in bacterium solution.
2. prepared by standard substance
The quantitative 3.2 μ g nicotinic acid standard substance that obtain include 3 bottles of standard substance into brown reagent bottle with cover in each test kit.
With the 1 bottle of standard substance of aseptic water dissolution in 2ml test kits when using, the standard substance storing solution of 0.16mg/100ml is obtained, then
Desired concn is diluted to according to operating instruction, 0 standard substance are sterilized water.
3. prepared by culture medium
Weigh 12.0g casamino acids, 40.0g glucoses, 20.0g sodium acetates, 0.4g L-Cystine, 0.2gDL- colors
Propylhomoserin, 20.0mg adenine sulfates, 20.0g guanine hydrochlorides, 20.0mg uracil, 200.0 μ g thiamine hydrochlorides, 200.0 μ g
Calcium pantothenate, 400.0 μ g pyridoxine hydrochlorides, 400.0 μ g riboflavin, 200.0 μ g p- amino benzoic Acid, 0.8 μ g biotin, 1.0g
Dipotassium hydrogen phosphate, 1.0g sodium dihydrogen phosphate, 0.4g magnesium sulfate, 20.0mg Sodium Chloride, 20.0mg ferrous sulfate, 20.0mg sulphuric acid
Manganese, mixes.Weigh 0.76g mixed culture based powders to add in 1 medium bottle, each test kit includes 3 bottles.
4. prepared by sterilized water
By distilled water in 121 DEG C of autoclavings 15 minutes, every bottle of 30ml includes 3 bottles of sterilized water in each test kit.
5. other
2 adhesive foils are also included in test kit.
Nicotinic acid is detected using mentioned reagent box, shows excellent on preci-sion and accuracy.
Experimental example 1
This experimental example is the precision for studying test kit of the present invention.
Using 3 batch test kits (test kit prepared by embodiment 1) detection 1849F American National Standards and technical research institute
Milk powder reference material 1849F (Nicotinic is 10.9mg/100g), does parallel laboratory test, the results are shown in Table 1.Each batch detection milk
1 kind of dilution (ultimate density is in standard curve range) of powder reference material 1849F.
Table 1
The coefficient of variation very little (1.62%) of milk powder reference material concentration is determined, shows that precision is high.Mark between 3 batches
The variation of quasi- product baseline results is less than 10%.
Using 3 batch test kits (test kit prepared by embodiment 2) detection milk powder reference material 1849F【American National mark
Accurate and technical research institute's milk powder reference material, Nicotinic desired value is 10.9mg/100g】, parallel laboratory test is done, the results are shown in Table 2.
1 kind of dilution (ultimate density is in standard curve range) of each batch detection milk powder reference material 1849F.
Table 2
The coefficient of variation very little (1.62%) of milk powder reference material concentration is determined, shows that precision is high.Mark between 3 batches
The variation of quasi- product baseline results is less than 10%.
Experimental example 2
This experimental example is the accuracy for studying test kit of the present invention.
The test kit of the present embodiment 1 is respectively adopted and detects milk powder reference material 1849F with contrast agent box, grasp by respective product
Explaining book carries out sample extraction, and does 4 dilutions (ultimate density is in standard curve range) respectively, and each dilution does 3
It is individual parallel, the results are shown in Table 3.
The preparation method of available reagent box microwell plate：
The culture of Lactobacillus plantarum ATCC 8014 is excessively inoculated in 10ml lactobacilluss culture medium, is cultivated 36 hours.
Culture is stopped by centrifugation (2500G × 5 minute) in exponential phase, cell mass is washed 3 times with 0.85%NaCl solution, hanged
In floating on 10ml analysis culture medium, then diluted with analysis culture medium 1-10 times.The micro- lifes of 3 μ l are added in each micropore of microwell plate
Thing suspension, the microbial suspension freezing dormancy in making micropore at -80 DEG C, then by the microbic mass in micropore under vacuo
Lyophilization.
Accuracy=testing result/actual concentrations × 100%
Table 3
Calibration curve
The scope of the examination criteria curve is 0.016-0.16mg/100g.The content of nicotinic acid in order to obtain sample, it is necessary to
The sample result read from standard curve is multiplied by into extension rate.Data processing software includes the coefficient of dilution in final calculating,
And output result is mg nicotinic acid/100 gram sample.Typical curve is as shown in Figure 1.
Although the present invention and its advantage has been described in detail it should be appreciated that without departing from by appended claim
Various changes can be carried out in the case of the spirit and scope of the present invention for being limited, is substituted and is converted.And, the model of the application
Enclose process, equipment, means, the specific embodiment of method and steps for being not limited only to described by description.In the art is common
Technical staff will readily appreciate that from the disclosure, can be used according to the present invention and be performed and corresponding reality described herein
Apply the essentially identical function of example or obtain process essentially identical with it result, existing and that future is to be developed, equipment,
Means, method or step.Therefore, appended claim is directed at including such process, equipment, handss in the range of them
Section, method or step.
Claims (10)
1. a kind of microwell plate for microbial method detection by quantitative nicotinic acid, it is characterised in that it is prepared from by following steps：
1) Lactobacillus plantarum is activated, the lactobacillus plantarum strain after activation is inoculated in lactobacilluss broth bouillon, 32-36 DEG C
Culture 16-20h, 2200 turns/min centrifugation 1-2min, abandoning supernatant；
2) 10-12ml trehaloses/sucrose and calcium chloride mixed liquor are added, 1-2min, abandoning supernatant, before repeating is centrifuged after mixing
State operation once, then add 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, mix, bacteria suspension is obtained；Inhale 1-2ml
Into 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, test bacterium solution is made in mixing to bacteria suspension；
3) the test bacterium solution 2-4 μ l are added into each hole of microwell plate, respectively 20mTorr, 55mTorr, 510mTorr,
Add Thermal test bacterium solution under the low vacuum environment of 5Torr, 45Torr, 210Torr, make temperature conditionss of the test bacterium solution at 33-37 DEG C
Lower boiling and produce foam, again distilling after and evaporation drying 36-48h under the low vacuum environment and room temperature, or freezing
Mode be dried 36-48h.
2. microwell plate according to claim 1, it is characterised in that step 1) in activation Lactobacillus plantarum adopt activation side
Formula is：
11g defatted milk powder is added in 89ml distilled water, is mixed, 115 DEG C sterilize 15 minutes, as fluid medium；
0.3g powder plants lactobacillus strain is added in fluid medium described in 10ml, 37 ± 1 DEG C of cultures are until curdled milk, obtains solidifying
Emulsus strain, is positioned over 0-4 DEG C of preservation；
Cultured curdy strain in step on 0.3ml is added in fluid medium described in 10ml, 37 ± 1 DEG C of cultures until
Curdled milk；
Previous step is repeated several times by, the strain for except for the difference that using every time is the culture that previous step is obtained, when curdled milk
Between it is stable after show that strain is activated.
3. microwell plate according to claim 1, it is characterised in that the lactobacillus plantarum strain is Lactobacillus plantarum
ATCC8014。
4. microwell plate according to claim 1, it is characterised in that step 2) in trehalose/sucrose and calcium chloride mixed liquor
In, respective concentration is respectively 180-190mM, 10-12mM.
5. the method for preparing microwell plate described in claim 1-4 any one, it is prepared from by following steps：
1) Lactobacillus plantarum is activated, the lactobacillus plantarum strain after activation is inoculated in lactobacilluss broth bouillon, 32-36 DEG C
Culture 16-20h, 2200 turns/min centrifugation 1-2min, abandoning supernatant；
2) 10-12ml trehaloses/sucrose and calcium chloride mixed liquor are added, 1-2min, abandoning supernatant, before repeating is centrifuged after mixing
State operation once, then add 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, mix, bacteria suspension is obtained；Inhale 1-2ml
Into 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, test bacterium solution is made in mixing to bacteria suspension；
3) the test bacterium solution 2-4 μ l are added into each hole of microwell plate, respectively 20mTorr, 55mTorr, 510mTorr,
Add Thermal test bacterium solution under the low vacuum environment of 5Torr, 45Torr, 210Torr, make temperature conditionss of the test bacterium solution at 33-37 DEG C
Lower boiling and produce foam, again distilling after and evaporation drying 36-48h under the low vacuum environment and room temperature, or freezing
Mode be dried 36-48h, so as to detection by quantitative nicotinic acid microwell plate is obtained.
6. the test kit of microwell plate described in claim 1-4 any one is included.
7. test kit according to claim 6, it is characterised in that also comprising nicotinic acid standard substance, nicotinic acid test media with
And sterilized water.
8. the test kit according to claim 6 or 7, it is characterised in that the nicotinic acid test media is 12.0g/L cheese eggs
Casamino acid, 40.0g/L glucoses, 20.0g/L sodium acetates, 0.4g/L L-Cystine, 0.2g/L DL-Trps, 20.0mg/
L adenine sulfates, 20.0g/L guanine hydrochlorides, 20.0mg/L uracil, 200.0 μ g/L thiamine hydrochlorides, 200.0 μ g/L are general
Sour calcium, 400.0 μ g/L pyridoxine hydrochlorides, 400.0 μ g/L riboflavin, 200.0 μ g/L p- amino benzoic Acid, 0.8 μ g/L are biological
Element, 1.0g/L dipotassium hydrogen phosphates, 1.0g/L sodium dihydrogen phosphate, 0.4g/L magnesium sulfate, 20.0mg/L Sodium Chloride, 20.0mg/L sulphuric acid
Ferrous, 20.0mg/L manganese sulfates.
9. the method for preparing test kit described in claim 6-8 any one, it is characterised in that it comprises the steps：
First prepare the microwell plate for being coated with lactobacillus plantarum strain：
1) Lactobacillus plantarum is activated, the lactobacillus plantarum strain after activation is inoculated in lactobacilluss broth bouillon, 32-36 DEG C
Culture 16-20h, 2200 turns/min centrifugation 1-2min, abandoning supernatant；
2) 10-12ml trehaloses/sucrose and calcium chloride mixed liquor are added, 1-2min, abandoning supernatant, before repeating is centrifuged after mixing
State operation once, then add 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, mix, bacteria suspension is obtained；Inhale 1-2ml
Into 10-12ml trehaloses/sucrose and calcium chloride mixed liquor, test bacterium solution is made in mixing to bacteria suspension；
3) the test bacterium solution 2-4 μ l are added into each hole of microwell plate, respectively 20mTorr, 55mTorr, 510mTorr,
Add Thermal test bacterium solution under the low vacuum environment of 5Torr, 45Torr, 210Torr, make temperature conditionss of the test bacterium solution at 33-37 DEG C
Lower boiling and produce foam, again distilling after and evaporation drying 36-48h under the low vacuum environment and room temperature, or freezing
Mode be dried 36-48h, so as to detection by quantitative nicotinic acid microwell plate is obtained；
Then prepare comprising nicotinic acid standard substance and nicotinic acid test media.
10. the preparation method of test kit according to claim 9, it is characterised in that the trehalose/sucrose and calcium chloride
In mixed liquor, respective concentration is respectively 180-190mM, 10-12mM.
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Cited By (3)
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| CN109957598A (en) * | 2019-03-13 | 2019-07-02 | 深圳容金科技有限公司 | Quickly detect the detection kit and preparation method thereof of vitamin B7 in dairy products |
| CN111635925A (en) * | 2020-06-15 | 2020-09-08 | 深圳市瑞赛生物技术有限公司 | Ready-to-use biotin detection carrier and preparation method thereof |
| CN111705101A (en) * | 2020-06-15 | 2020-09-25 | 深圳市瑞赛生物技术有限公司 | Ready-to-use trace component detection kit and preparation method thereof |
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| CN1193908A (en) * | 1995-06-07 | 1998-09-23 | 廓德伦特控股剑桥有限公司 | Method for stably incorporating substances within dry, foamed glass matrices and compositions obtained thereby |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957598A (en) * | 2019-03-13 | 2019-07-02 | 深圳容金科技有限公司 | Quickly detect the detection kit and preparation method thereof of vitamin B7 in dairy products |
| CN111635925A (en) * | 2020-06-15 | 2020-09-08 | 深圳市瑞赛生物技术有限公司 | Ready-to-use biotin detection carrier and preparation method thereof |
| CN111705101A (en) * | 2020-06-15 | 2020-09-25 | 深圳市瑞赛生物技术有限公司 | Ready-to-use trace component detection kit and preparation method thereof |
| CN111705101B (en) * | 2020-06-15 | 2021-08-24 | 深圳市瑞赛生物技术有限公司 | Ready-to-use trace component detection kit and preparation method thereof |
| CN111635925B (en) * | 2020-06-15 | 2023-08-25 | 深圳市瑞赛生物技术有限公司 | Ready-to-use biotin detection carrier and preparation method thereof |
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| CN106676161B (en) | 2020-01-21 |
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