CN109358170A - A kind of biological cellulose content of detection, folate content and vitamin B12The method of content - Google Patents

A kind of biological cellulose content of detection, folate content and vitamin B12The method of content Download PDF

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CN109358170A
CN109358170A CN201811250181.8A CN201811250181A CN109358170A CN 109358170 A CN109358170 A CN 109358170A CN 201811250181 A CN201811250181 A CN 201811250181A CN 109358170 A CN109358170 A CN 109358170A
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content
vitamin
microwell plate
sample
clear liquid
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刘学文
邓倩南
陈捷
罗志荣
陈海宁
余构彬
高裕锋
王桂华
郭剑雄
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Guangdong Institute of Bioengineering Guangzhou Cane Sugar Industry Research Institute
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Guangdong Institute of Bioengineering Guangzhou Cane Sugar Industry Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/04—Dairy products
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

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Abstract

The present invention relates to technical field of food detection, and in particular to a kind of biological cellulose content of detection, folate content and vitamin B12The method of content, including Step 1: extracting the clear liquid of sample；Step 2: dilution standard product and sample clear liquid；Step 3: standard items and sample clear liquid after culture dilution；Step 4: formulating the standard curve and test sample of standard items；This method can extract biotin, folic acid and vitamin B simultaneously12, to improve biotin, folic acid and vitamin B12Detection efficiency, have the advantages that easily operated and detection sensitivity is high.

Description

A kind of biological cellulose content of detection, folate content and vitamin B12The method of content
Technical field
The present invention relates to technical field of food detection, and in particular to a kind of biological cellulose content of detection, folate content and Wei Sheng Plain B12The method of content.
Background technique
Biotin, folic acid and vitamin B12It is to maintain human body natural's growth, development and normal human's functional health institute necessary Nutrient, therefore, biological cellulose content, folate content and vitamin B in food12Content increasingly by the concern of people, Especially for vitamin B in infant food and dairy products12Measurement be also most important.Since biological cellulose content, folic acid contain Amount and vitamin B12For the substance of different structure, and most of food are by biotin, folic acid and vitamin B now12As battalion Feeding hardening agent is thought to be added in food.Standard GB 5009.259-2016 measurement biotin is respectively adopted in the prior art, adopts Vitamin B is measured with standard GB 5009.211-2014 measurement folic acid, standard GB 5413.14-201012.As it can be seen that above-mentioned survey Calibration standard all independently carries out, that is, when measuring a kind of content of material, needs to extract respectively and surveys biotin, folic acid and vitamin B12, therefore, will lead to the biological cellulose content of measurement, folate content and vitamin B12It needs individually independent mention for 3 times when content Sample is taken, not only influences detection efficiency in this way, also waste testing cost.
Summary of the invention
For the prior art, there are above-mentioned technical problems, and it is an object of that present invention to provide a kind of detection biological cellulose contents, leaf Acid content and vitamin B12The method of content, this method can extract biotin, folic acid and vitamin B simultaneously12, to improve Biotin, folic acid and vitamin B12Detection efficiency, and have the advantages that easily operated and detection sensitivity is high.
To achieve the above object, the present invention the following technical schemes are provided:
A kind of biological cellulose content of detection, folate content and vitamin B are provided12The method of content, comprising the following steps:
Step 1: extracting the clear liquid of sample
(1) sample is weighed into centrifuge tube, and the buffer solution of potassium phosphate of preheating is first added to centrifuge tube, keeps sample sufficiently molten Solution, adds buffer solution of potassium phosphate, so that the weight ratio of sample and buffer solution of potassium phosphate is 1:35~45；
(2) the centrifuge tube boiling water bath of above-mentioned (1) is handled, when boiling water bath shakes the centrifuge tube, and the boiling water bath time is 25 ~40min obtains sample and buffer solution of potassium phosphate mixed liquor；
(3) the centrifuge tube ice-water bath of above-mentioned (2) is handled, sample and buffer solution of potassium phosphate mixed liquor is made to be cooled to 3~5 ℃；
(4) it uses centrifuge and is cooled to 3~5 DEG C, then described in centrifuge tube after cooling in above-mentioned (3) merging Centrifuge carries out centrifugal treating, obtains upper-layer fat, middle layer clear liquid and lower sediment；
Step 2: dilution
(3) dilute sample clear liquid: measuring three portions of clear liquids, which is the clear liquid for measuring biotin, measurement leaf respectively The clear liquid and measurement vitamin B of acid12Clear liquid, then dilute three portions of clear liquids respectively, obtain three parts of sample diluting liquids, this three parts The content of sample diluting liquid it is corresponding in biotin standard curve range, in folic acid standard curve range and vitamin B12Standard In curve ranges, and the clear liquid volume after dilution is no less than 500 μ L；
(4) biotin standard items, folic acid standard items and vitamin B dilution standard product: is respectively adopted12Standard items and by its point Different content is not diluted to it
Step 3: culture
(3) clear liquid after resulting three dilutions of step 2 and the standard items after three dilutions are separately added into and are embedded with spy Surely it tests in the microwell plate of strain, and is separately added into corresponding culture medium, wherein after the culture medium and dilution in each microwell plate The weight ratio of liquid be 0.8~1.5:1；
(4) film is affixed on microwell plate, then microwell plate is placed in aluminium box with cover, the bottom bedding of the aluminium box With the sterile gauze of sterile water-soaked, the microwell plate is erected on the sterile gauze by bracket；
(3) the above-mentioned aluminium box equipped with microwell plate is placed on 46~50h of culture in 30~40 DEG C；
Step 4: measurement
(1) standard curve
A, the film on the microwell plate after step 3 culture is compacted, then turns upside down and shake microwell plate, makes microwell plate The bacterium of bottom is uniformly distributed in the medium, forms bacteria suspension；
B, biotin standard items, folic acid standard items and the vitamin of the different content after dilution are measured respectively using microplate reader B12The OD value of standard items, respectively using the different content of standard items as abscissa, OD value corresponding to the different content with standard items For ordinate, 4 order polynomial regression curves are made, biotin standard curve, folic acid standard curve and vitamin B12 are respectively obtained Standard curve；
(2) sample
A, the film on the microwell plate after step 4 culture is compacted, then turns upside down and shake microwell plate, makes microwell plate The bacterium of bottom is uniformly distributed in the medium, forms bacteria suspension；
B, it is measured respectively using microplate reader for biotin, folic acid and vitamin B12Bacterium corresponding to detection sample micropore is outstanding Measured OD value is substituted into 4 order polynomials of corresponding standard curve, and calculated multiplied by corresponding dilution by the OD value of liquid Biotin, folic acid and vitamin B out12The content of three.
Wherein, in the step 1, the temperature of the buffer solution of potassium phosphate of preheating is 45~55 DEG C.
Wherein, in the step 1, in first 5 minutes in boiling water bath, the primary centrifugation is shaken every rotation in one minute Pipe, subsequent every 5min, which turns upside down, shakes the centrifuge tube 3 times.
Wherein, in the step 1, the revolving speed of the centrifuge is 10000r/min, and centrifugation time is 10~20min.
Wherein, in the step 1, removal upper-layer fat is toppled under clean environment, keeps centrifuge tube transverse presentation, and clear liquid is held It is continuous slowly to pour out, the clear liquid of 100~200 μ L is drawn as spare in deep-well plates.
Wherein, in the step 2, clear liquid is diluted using sterilized level-one water.
Wherein, in the step 4, the volume of culture medium and the volume of sample diluting liquid are 150uL in microwell plate.
Wherein, in the step 4, the microwell plate is erected on the sterile gauze by cross.
Wherein, in the step 5, the wavelength of microplate reader is 630nm.
Wherein, the pH value of the buffer solution of potassium phosphate is 7~7.5.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1.
A kind of detection biology cellulose content, folate content and vitamin B of the present embodiment12The method of content, including following step It is rapid:
Step 1: extracting the clear liquid of sample
(1) spiral shell with a scale of drying of 1g (the being accurate to 0.0001g) sample to 50mL without ingredient to be measured is accurately weighed In mouth centrifuge tube, addition is preheating to 50 DEG C of kaliumphosphate buffer about 30mL, abundant sample dissolution, and is determined with kaliumphosphate buffer Hold to 40mL, wherein the pH of kaliumphosphate buffer is 7；
(2) boiling water bath centrifuge tube 30min in first 5 minutes, shakes the primary centrifuge tube every rotation in one minute, then Every 5min, which turns upside down, shakes the centrifuge tube 3 times；
(3) centrifuge tube described in ice-water bath makes centrifuge tube be cooled to 4 DEG C；
(4) centrifuge is cooled in advance 4 DEG C, centrifuge is centrifuged the centrifuge tube, centrifugation time with the revolving speed of 10000r/min For 15min, upper-layer fat, middle layer clear liquid and lower sediment are obtained；
(5) under clean bench or other clean environments, topple over removal upper-layer fat, keep centrifuge tube transverse presentation, will under Supernatant liquor in layer liquid continues slowly to pour out state, draws 200 μ L clear liquids as spare in deep-well plates.Wherein, keep from Heart pipe transverse presentation is very crucial step, if established after toppling over fat deposit or other accidentally operation cause liquid in centrifuge tube to hit back bottom Portion can be such that bottom precipitation is resuspended, and clear liquid is muddy, and background turbidity increases, and bring error；
Step 2: dilution clear liquid
1) three portions of clear liquids are measured, which is the clear liquid for measuring biotin, the clear liquid for measuring folic acid and measurement respectively Then the clear liquid of vitamin B12 dilutes three portions of clear liquids respectively, obtains three parts of sample diluting liquids, three parts of sample diluting liquids Content it is corresponding within the scope of biotin standard working solution, within the scope of folic acid standard working solution and vitamin standard working solution range It is interior, and the clear liquid after dilution is no less than 500 μ L；
Step 3: preparing standard curve and preparation training using commercial kit and according to commercial kit specification Support base；
Step 4: culture
1) corresponding 150 μ L of culture medium and sample diluting liquid are separately added into the microwell plate for being embedded with fc-specific test FC strain 150μL；
2) film is affixed on microwell plate, prevents moisture from evaporating；
3) microwell plate is placed in aluminium box with cover, aluminium cassette bottom layer covers the sterile gauze with sterile water-soaked, gauze One frame word of upper placement, microwell plate is placed on gauze, to be not directly contacted with gauze.
4) the aluminium box equipped with microwell plate is placed in 40 DEG C and cultivates 50h.
Step 5: measurement
(1) standard curve is formulated
A, biotin standard items, folic acid standard items and vitamin B is respectively adopted12It is simultaneously diluted to difference by standard items respectively Content；
B, biotin standard items, the leaf of the different content using microplate reader after measuring dilution under wavelength is 630nm respectively The OD value of sour standard items and vitamin B12 standard items, respectively using the different content of standard items as abscissa, with the difference of standard items OD value corresponding to content is ordinate, makes 4 order polynomial regression curves, respectively obtains biotin standard curve, folic acid mark Directrix curve and vitamin B12Standard curve；
(2) it detects
A, the film on the microwell plate after step 4 culture is compacted, then turns upside down and shake microwell plate, makes microwell plate The bacterium of bottom is uniformly distributed in the medium, forms bacteria suspension；
B, microplate reader is used to be measured corresponding to biotin, folate content and vitamin B12 respectively in the case where wavelength is 630nm Measured OD value is substituted into 4 order polynomials of corresponding standard curve, measures biotin, folic acid by the OD value of bacteria suspension The content of content and vitamin B12 three.
Embodiment 2.
A kind of detection biology cellulose content, folate content and vitamin B of the present embodiment12The method of content, including following step It is rapid:
Step 1: extracting the clear liquid of sample
(1) spiral shell with a scale of drying of 1g (the being accurate to 0.0001g) sample to 50mL without ingredient to be measured is accurately weighed In mouth centrifuge tube, addition is preheating to 45 DEG C of kaliumphosphate buffer about 30mL, abundant sample dissolution, and is determined with kaliumphosphate buffer Hold to 40mL, wherein the pH of kaliumphosphate buffer is 7.5；
(2) 25~40min of boiling water bath centrifuge tube in first 5 minutes, shakes the primary centrifuge tube every rotation in one minute, Subsequent every 5min, which turns upside down, shakes the centrifuge tube 3 times；
(3) centrifuge tube described in ice-water bath makes centrifuge tube be cooled to 5 DEG C；
(4) centrifuge is cooled in advance 5 DEG C, centrifuge is centrifuged the centrifuge tube, centrifugation time with the revolving speed of 10000r/min For 10min, upper-layer fat and lower liquid are obtained；
(5) under clean bench or other clean environments, topple over removal upper-layer fat, keep centrifuge tube transverse presentation, will under Supernatant liquor in layer liquid continues slowly to pour out state, draws 100 μ L clear liquids as spare in deep-well plates.
Step 2: dilution clear liquid
1) three portions of clear liquids are measured, which is the clear liquid for measuring biotin, the clear liquid for measuring folic acid and measurement respectively Then the clear liquid of vitamin B12 dilutes three portions of clear liquids respectively, obtains three parts of sample diluting liquids, three parts of sample diluting liquids Content it is corresponding within the scope of biotin standard working solution, within the scope of folic acid standard working solution and vitamin standard working solution range It is interior, and the clear liquid after dilution is no less than 500 μ L；
Step 3: preparing standard curve and preparation training using commercial kit and according to commercial kit specification Support base；
Step 4: culture
(1) corresponding 150 μ L of culture medium and sample dilution are separately added into the microwell plate for being embedded with fc-specific test FC strain 150 μ L of liquid；
(2) film is affixed on microwell plate, prevents moisture from evaporating；
(3) microwell plate is placed in aluminium box with cover, aluminium cassette bottom layer covers the sterile gauze with sterile water-soaked, gauze One frame word of upper placement, microwell plate is placed on gauze, to be not directly contacted with gauze.
(4) the aluminium box equipped with microwell plate is placed in 30 DEG C and cultivates 46h.
Step 5: measurement
(1) standard curve is formulated
A, biotin standard items, folic acid standard items and vitamin B is respectively adopted12It is simultaneously diluted to difference by standard items respectively Content；
B, biotin standard items, the leaf of the different content using microplate reader after measuring dilution under wavelength is 630nm respectively The OD value of sour standard items and vitamin standard items is contained respectively using the different content of standard items as abscissa with the difference of standard items The corresponding OD value of amount is ordinate, makes 4 order polynomial regression curves, respectively obtains biotin standard curve, folic acid standard Curve and vitamin B12Standard curve；
(2) it detects
A, the film on the microwell plate after step 4 culture is compacted, then turns upside down and shake microwell plate, makes microwell plate The bacterium of bottom is uniformly distributed in the medium, forms bacteria suspension；
B, microplate reader is used to measure biotin, folate content and vitamin B respectively in the case where wavelength is 630nm12Corresponding Measured OD value is substituted into 4 order polynomials of corresponding standard curve, measures biotin, folic acid by the OD value of bacteria suspension Content and vitamin B12The content of three.
Embodiment 3.
Step 1: extracting the clear liquid of sample
(1) spiral shell with a scale of drying of 1g (the being accurate to 0.0001g) sample to 50mL without ingredient to be measured is accurately weighed In mouth centrifuge tube, addition is preheating to 55 DEG C of kaliumphosphate buffer about 30mL, abundant sample dissolution, and is determined with kaliumphosphate buffer Hold to 40mL, wherein the pH of kaliumphosphate buffer is 7.2；
(2) boiling water bath centrifuge tube 40min in first 5 minutes, shakes the primary centrifuge tube every rotation in one minute, then Every 5min, which turns upside down, shakes the centrifuge tube 3 times；
(3) centrifuge tube described in ice-water bath makes centrifuge tube be cooled to 5 DEG C；
(4) centrifuge is cooled in advance 5 DEG C, centrifuge is centrifuged the centrifuge tube, centrifugation time with the revolving speed of 10000r/min For 20min, upper-layer fat and lower liquid are obtained；
(5) under clean bench or other clean environments, topple over removal upper-layer fat, keep centrifuge tube transverse presentation, will under Supernatant liquor in layer liquid continues slowly to pour out state, draws 150 μ L clear liquids as spare in deep-well plates.
Step 2: dilution clear liquid
1) three portions of clear liquids are measured, which is the clear liquid for measuring biotin, the clear liquid for measuring folic acid and measurement respectively Then the clear liquid of vitamin B12 dilutes three portions of clear liquids respectively, obtains three parts of sample diluting liquids, three parts of sample diluting liquids Content it is corresponding within the scope of biotin standard working solution, within the scope of folic acid standard working solution and vitamin standard working solution range It is interior, and the clear liquid after dilution is no less than 500 μ L；
Step 3: preparing standard curve and preparation training using commercial kit and according to commercial kit specification Support base；
Step 4: culture
(1) corresponding 150 μ L of culture medium and sample dilution are separately added into the microwell plate for being embedded with fc-specific test FC strain 150 μ L of liquid；
(2) film is affixed on microwell plate, prevents moisture from evaporating；
(3) microwell plate is placed in aluminium box with cover, aluminium cassette bottom layer covers the sterile gauze with sterile water-soaked, gauze One frame word of upper placement, microwell plate is placed on gauze, to be not directly contacted with gauze.
(4) the aluminium box equipped with microwell plate is placed in 40 DEG C and cultivates 50h.
Step 5: measurement
(1) standard curve is formulated
A, biotin standard items, folic acid standard items and vitamin B is respectively adopted12It is simultaneously diluted to difference by standard items respectively Content；
B, biotin standard items, the leaf of the different content using microplate reader after measuring dilution under wavelength is 630nm respectively Sour standard items and vitamin B12The OD value of standard items, respectively using the different content of standard items as abscissa, with the difference of standard items OD value corresponding to content is ordinate, makes 4 order polynomial regression curves, respectively obtains biotin standard curve, folic acid mark Directrix curve and vitamin B12Standard curve；
(2) it detects
A, the film on the microwell plate after step 4 culture is compacted, then turns upside down and shake microwell plate, makes microwell plate The bacterium of bottom is uniformly distributed in the medium, forms bacteria suspension；
B, microplate reader is used to measure biotin, folate content and vitamin B respectively in the case where wavelength is 630nm12Corresponding Measured OD value is substituted into 4 order polynomials of corresponding standard curve, measures biotin, folic acid by the OD value of bacteria suspension Content and vitamin B12The content of three.
Detection test
One, test of the kaliumphosphate buffer to the buffering effect of milk powder pH
Test procedure:
(1) it prepares kaliumphosphate buffer: preparing 0.2mol/L K2HPO4·3H2O and 0.2mol/L KH2PO4Both, pass through Ratio adjustment with 0.2mol/L pH=7.2 kaliumphosphate buffer, it is ready-to-use.
(2) 30 formula milk samples of test are chosen, each sample claims 1g milk powder in 50ml extraction tube, and is added and goes Ionized water is settled to 40ml, mixes, is measured with pH meter, obtain the background pH value of Milk Powder Formula For Infants, be shown in Table 1；In addition, weighing again 30 formula milk samples of above-mentioned test, each sample weigh 1g powdered milk sample into 50mL centrifuge tube, slow with potassium phosphate Fliud flushing is settled to 40mL, measures pH value of solution respectively, obtains the pH value for adding the milk powder after kaliumphosphate buffer, is shown in Table 2.
The background pH of 1 Milk Powder Formula For Infants of table
The pH value of milk powder after table 2 plus kaliumphosphate buffer
By above-mentioned Tables 1 and 2 as it can be seen that kaliumphosphate buffer can steadily adjust the pH value of milk powder, and then meets experiment and want It asks.
Two, the present invention detects the effect test of biological cellulose content
Test procedure: choosing 26 formula milk samples of test, then measures milk using the detection method of embodiment 1 The biological cellulose content of powder, wherein pH is adjusted to 7.2；In addition, choose 26 formula milk samples of above-mentioned test, then " GB The measurement of biotin in 5009.259-2016 national food safety standard food " measuring method measure the biotin of milk powder, The measurement result of above-mentioned two method is shown in Table 3.
3 two methods of table measure the Comparative result of biological cellulose content
According to the measurement result of table 3, measurement result is compared, comparison result is shown in Table 4.
4 National Standard Method of table is compared with detection method is to biotin assay result
As shown in Table 4, the designated value of biotin quality-control sample (QC-IP-017) is 22.550 μ g/100g, satisfied Value range is 16.432~28.758 μ g/100g, and two methods testing result all in satisfactory value range, shows two methods data As a result accurate and reliable, and the present invention detects the average content of the method for biological cellulose content closer to designated value, RSD value is also lower, It can be seen that detection method of the invention has feasibility and reliability.
Three, the effect test of present invention detection folate content
Test procedure: taking test formula milk sample, then using the folic acid of the measuring method measurement milk powder of embodiment 1 Content；In addition above-mentioned test formula milk sample is chosen, then " GB 5009.211-2014 national food safety standard food The measurement of middle folic acid " measuring method measure the folic acid of milk powder, the measurement result of above-mentioned two method is shown in Table 5
5 National Standard Method of table is compared with detection method is to folate content measurement result
By table 5 as it can be seen that the designated value of folic acid quality-control sample (QC-IP-016) is 126.0 μ g/100g, satisfactory value range is 75.2~176.8 μ g/100g, two methods testing result show that two methods data result accurately may be used all in satisfactory value range It leans on, and new method average content, closer to designated value, RSD value is also lower.
Four, present invention detection vitamin B12The effect test of content
Test procedure: taking test formula milk sample, and then the dimension using the measuring method measurement milk powder of embodiment 1 is raw Plain B12Content；In addition above-mentioned test formula milk sample is chosen, then " GB 5413.14-2010 national food safety standard Vitamin B in infant food and dairy products12Measurement " measuring method measure the vitamin B of milk powder12, above-mentioned two method Measurement result be shown in Table 6.
6 National Standard Method of table is compared with detection method is to vitamin B12 assay result
By table 6 as it can be seen that vitamin B12The designated value of quality-control sample (QC-IP-009) is 3.550 μ g/100g, satisfactory value model It encloses for 2.290~4.810 μ g/100g, two methods testing result shows that two methods data result is quasi- all in satisfactory value range It is really reliable, and new method average content, closer to designated value, RSD value is also lower.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than the present invention is protected The limitation of range is protected, although explaining in detail referring to preferred embodiment to the present invention, those skilled in the art are answered Work as understanding, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the reality of technical solution of the present invention Matter and range.

Claims (10)

1. a kind of biological cellulose content of detection, folate content and vitamin B12The method of content, it is characterized in that: the following steps are included:
Step 1: extracting the clear liquid of sample
(1) sample is weighed into centrifuge tube, and the buffer solution of potassium phosphate of preheating is first added to centrifuge tube, dissolves sample sufficiently, Buffer solution of potassium phosphate is added, so that the weight ratio of sample and buffer solution of potassium phosphate is 1:35~45；
(2) the centrifuge tube boiling water bath of above-mentioned (1) being handled, when boiling water bath, shakes the centrifuge tube, and the boiling water bath time is 25~ 40min obtains sample and buffer solution of potassium phosphate mixed liquor；
(3) the centrifuge tube ice-water bath of above-mentioned (2) is handled, sample and buffer solution of potassium phosphate mixed liquor is made to be cooled to 3~5 DEG C；
(4) it uses centrifuge and is cooled to 3~5 DEG C, centrifuge tube after cooling in above-mentioned (3) is then placed in the centrifugation Machine carries out centrifugal treating, obtains upper-layer fat, middle layer clear liquid and lower sediment；
Step 2: dilution
(1) dilute sample clear liquid: measuring three portions of clear liquids, which is the clear liquid for measuring biotin, measurement folic acid respectively Clear liquid and measurement vitamin B12Clear liquid, then dilute three portions of clear liquids respectively, obtain three parts of sample diluting liquids, three parts of samples The content of dilution it is corresponding in biotin standard curve range, in folic acid standard curve range and vitamin B12Standard curve In range, and the clear liquid volume after dilution is no less than 500 μ L；
(2) biotin standard items, folic acid standard items and vitamin B dilution standard product: is respectively adopted12Standard items are simultaneously distinguished dilute It is interpreted as different content
Step 3: culture
(1) clear liquid after resulting three dilutions of step 2 and the standard items after three dilutions are separately added into and are embedded with specific survey In the microwell plate for trying strain, and it is separately added into corresponding culture medium, wherein the liquid after culture medium and dilution in each microwell plate The weight ratio of body is 0.8~1.5:1；
(2) film is affixed on microwell plate, then microwell plate being placed in aluminium box with cover, the bottom bedding of the aluminium box is used The sterile gauze of sterile water-soaked, the microwell plate are erected on the sterile gauze by bracket；
(3) the above-mentioned aluminium box equipped with microwell plate is placed on 46~50h of culture in 30~40 DEG C；
Step 4: measurement
(1) standard curve
A, the film on the microwell plate after step 3 culture is compacted, then turns upside down and shake microwell plate, makes microwell plate bottom Bacterium it is uniformly distributed in the medium, form bacteria suspension；
B, biotin standard items, folic acid standard items and the vitamin B of the different content after dilution are measured respectively using microplate reader12Mark The OD value of quasi- product, respectively using the different content of standard items as abscissa, OD value corresponding to the different content with standard items is vertical Coordinate makes 4 order polynomial regression curves, respectively obtains biotin standard curve, folic acid standard curve and vitamin B12 standard Curve；
(2) sample
A, the film on the microwell plate after step 4 culture is compacted, then turns upside down and shake microwell plate, makes microwell plate bottom Bacterium it is uniformly distributed in the medium, form bacteria suspension；
B, it is measured respectively using microplate reader for biotin, folic acid and vitamin B12Bacteria suspension corresponding to detection sample micropore Measured OD value is substituted into 4 order polynomials of corresponding standard curve, and calculates and be born multiplied by corresponding dilution by OD value Object element, folic acid and vitamin B12The content of three.
2. a kind of detection biology cellulose content, folate content and vitamin B according to claim 112The method of content, it is special Sign is: in the step 1, the temperature of the buffer solution of potassium phosphate of preheating is 45~55 DEG C.
3. a kind of detection biology cellulose content, folate content and vitamin B according to claim 112The method of content, it is special Sign is: in the step 1, in first 5 minutes in boiling water bath, the primary centrifuge tube is shaken every rotation in one minute, it is then every 5min, which turns upside down, shakes the centrifuge tube 3 times.
4. a kind of detection biology cellulose content, folate content and vitamin B according to claim 112The method of content, it is special Sign is: in the step 1, the revolving speed of the centrifuge is 10000r/min, and centrifugation time is 10~20min.
5. a kind of detection biology cellulose content, folate content and vitamin B according to claim 112The method of content, it is special Sign is: in the step 1, removal upper-layer fat is toppled under clean environment, keeps centrifuge tube transverse presentation, clear liquid continues slowly to incline Out, the clear liquid of 100~200 μ L is drawn as spare in deep-well plates.
6. a kind of detection biology cellulose content, folate content and vitamin B according to claim 112The method of content, it is special Sign is: in the step 2, being diluted using sterilized level-one water to clear liquid.
7. a kind of detection biology cellulose content, folate content and vitamin B according to claim 112The method of content, it is special Sign is: in the step 4, the volume of culture medium and the volume of sample diluting liquid are 150uL in microwell plate.
8. a kind of detection biology cellulose content, folate content and vitamin B according to claim 112The method of content, it is special Sign is: in the step 4, the microwell plate is erected on the sterile gauze by cross.
9. a kind of detection biology cellulose content, folate content and vitamin B according to claim 112The method of content, it is special Sign is: in the step 5, the wavelength of microplate reader is 630nm.
10. a kind of detection biology cellulose content, folate content and vitamin B according to claim 112The method of content, Be characterized in: the pH value of the buffer solution of potassium phosphate is 7~7.5.
CN201811250181.8A 2018-10-25 2018-10-25 A kind of biological cellulose content of detection, folate content and vitamin B12The method of content Pending CN109358170A (en)

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