CN103926367A - Method for extracting biotin from molasses and thin layer chromatography (TLC) scanning detection method of biotin - Google Patents
Method for extracting biotin from molasses and thin layer chromatography (TLC) scanning detection method of biotin Download PDFInfo
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Abstract
The invention discloses a method for extracting biotin from molasses. The method is characterized by using molasses and diatomite as raw materials, carrying out extraction with methanol, carrying out extraction with N,N-dimethylformamide, carrying out washing and impurity removal with absolute ethyl alcohol, carrying out impurity removal with acetone, carrying out impurity removal with chloroform and carrying out extraction and impurity removal with butyl acetate and cyclohexane, thus finally obtaining a sample solution containing biotin. The invention also provides a thin layer chromatography (TLC) scanning detection method of biotin. The detection method comprises the following steps: (1) dropping the sample solution to be detected on a silica gel plate, adding a developer to a developing cylinder, developing the sample solution to be detected in the developing cylinder, and then taking out the thin layer plate and putting the thin layer plate in a fume hood to be aired; (2) spray-dyeing the developed and aired plate with a dyeing solution to obtain spots, in obvious contrast to the background, of standard substances and samples; (3) carrying out TLC scanning by utilizing a thin layer scanner in the absorption wavelength of 530nm, and computing the mass concentration of biotin in the sample solution. The methods have the advantages of simplicity, quickness, accuracy, reliability, stability and the like.
Description
Technical field
The present invention relates to a kind of method and thin-layer chromatography scanning detection method thereof that extracts biotin from molasses.
Background technology
Biotin is one of vitamin essential in vital movement, is the accessory factor of many enzymes in body, and carboxylation, decarboxylation and the dehydrogenation reaction of participating in body, participate in the metabolism of carbohydrate, fat, protein three major nutrient.Biotin has been widely used in the fields such as food, chemical industry, medicine, herding, biofermentation.In glutamic acid fermentation is produced, the control of biotin consumption directly affects the height of growth, propagation, metabolism and cell membrane, cell permeability of the membrane and the glutamic acid acid production rate of producing bacterium, because most of glutamic acid bacterium are all biotin deficiencies, so the content of biotin plays vital effect to producing, therefore carry out the research that biotin is detected and have great importance.
Molasses are a kind of thickness, pitchy, be the dynamic material of semi-fluid, in sugar industry, to be used for again the waste material of boiling sugar products, but the rich content of biotin in molasses, can be used as raw material and the adjuvant of good glutamic acid fermentation, it is the main source of biotin during current glutamic acid is produced.But because wherein the content of biotin and source, the production batch etc. of molasses are closely related, therefore, the composition that must pay close attention to molasses for steady production, raising acid production rate changes, and Accurate Determining biotin content wherein, to realize the control to biotin consumption in glutamic acid fermentation.
The detection method of the biotin of prior art bibliographical information has microbial method, fluorescence method, spectrophotometric method, euzymelinked immunosorbent assay (ELISA), vapor-phase chromatography, high performance liquid chromatography, thin layer chromatography scanning etc.At present, to the detection of biotin in molasses, microbial method and high performance liquid chromatography are the most conventional, but have following defect: microbial method is because most of bacterial strains are not narrow spectrum, therefore can bring larger detection error, and whole operating process is time-consuming, effort.High performance liquid chromatography investment is large, solvent load is large, cost is higher, again due to molasses thickness, color depth, to contain impurity more, therefore sample preparation is the extraction purge process difficulty of biotin, and high performance liquid chromatography is higher again to the purity requirement of sample,, impurity high to complicated component, pigment content disturbs large molasses sample, should not detect by this method.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of method of extracting biotin from molasses, the present invention also provides a kind of thin-layer chromatography scanning detection method.
The present invention is achieved by the following technical solutions:
A method of extracting biotin from molasses, step is as follows:
(1) make molasses diatomite in powder taking molasses and zeyssatite as raw material: get molasses and zeyssatite, mixing mixes, and 40～60 DEG C of oven dry, obtain molasses diatomite in powder; Wherein, molasses and diatomaceous weight ratio are 3:1～3;
(2) in molasses diatomite in powder, add methyl alcohol, stir and extract 30～60min, filter to obtain filter residue; The addition of described methyl alcohol is: every 1 gram of molasses diatomite in powder adds 3～5ml methyl alcohol;
(3) in above-mentioned filter residue, add DMF, at 70～100 DEG C of water-baths, stir and extract 1～2 hour, filter to obtain supernatant and filter residue; Filter residue absolute ethanol washing 1～3 time, obtains ethanol cleansing solution; The addition of described DMF is: every 1 gram of filter residue adds 4～6mlN, dinethylformamide; The addition of described absolute ethyl alcohol is: the amount of at every turn washing absolute ethyl alcohol used is 0.8～1.2 times of DMF volume;
(4) combining step (3) gained supernatant and ethanol cleansing solution, evaporation and concentration, adds the acetone of 3～5 times of amounts (volume multiple), leave standstill, remove by filter precipitation, again evaporation and concentration, add the acetone of 3～5 times of amounts (volume multiple), leave standstill, remove by filter precipitation, again evaporation and concentration, add the chloroform of 3～5 times of amounts (volume multiple), leave standstill, remove by filter precipitation, obtain extract;
(4) in extract, add butyl acetate and cyclohexane, the addition of butyl acetate, cyclohexane is identical with the volume of extract, stratification; Get supernatant, be biotin solution (solution that contains biotin).
In above method, evaporation and concentration adopts Rotary Evaporators to carry out, and the temperature of rotary evaporation is 40～60 DEG C.
A thin-layer chromatography scanning detection method for biotin, step is as follows:
(1) get detected sample liquid (being the biotin solution that said method prepares) 4 μ l, point silica gel plate (preferably 10cm × 10cm scribble silica G F
254efficient thin layer plate) upper, add developping agent to expansion cylinder, in expansion cylinder (10cm × 12cm × 15cm), launch, the saturation time of developping agent is 60 minutes, development distance is 6cm, and duration of run is 15 minutes, then thin layer plate is taken out to be placed in vent cabinet and dries; Meanwhile, adopt one point external standard method to put the biotin standard solution of 4 μ l variable concentrations, launch simultaneously;
Described developping agent is methylene chloride-chloroform-methanol-glacial acetic acid mixed liquor, and wherein, the volume ratio of methylene chloride, chloroform, methyl alcohol, glacial acetic acid is 1～3:1～3:1～2:0.04～0.06, preferably 3:3:2:0.06;
Described biotin standard solution comprises that concentration is respectively 0.05 μ g/ μ l, 0.2 μ g/ μ l, 0.25 μ g/ μ l, 0.3 μ g/ μ l, the series of standards product solution of 0.4 μ g/ μ l;
The compound method of described biotin standard solution is: precision takes biotin standard items (purchased from Sigma company) 1.29mg, adds DMF 1.29ml, is mixed with the standard solution that concentration is 1mg/ml; Accurate absorption 50,200,250,300, the standard solution of 400 μ l1mg/ml, is mixed with concentration 0.05 μ g/ μ l respectively, 0.2 μ g/ μ l, 0.25 μ g/ μ l, 0.3 μ g/ μ l, the series of standards product solution of 0.4 μ g/ μ l;
(2) by 2,4-dimethylamino cinnamic acid, sulfuric acid and absolute ethyl alcohol mix, be made into dyeing liquor, wherein 2,4-dimethylamino cinnamic acid final concentration is 0.1～0.2% (quality volume fraction, the g/ml of unit), sulfuric acid final concentration is 1～2% (quality volume fraction, the g/ml of unit); The panel of having dried after having opened up, uses dyeing liquor spray dyeing, obtains contrasting the spot of obvious standard items and the spot of sample (orange red) with background;
(3) utilize Camag3 type thin-layer chromatogram scanner to carry out thin-layer chromatography scanning with 530nm absorbing wavelength, sweep velocity is 20mm/s, and resolution is 50 μ m/step, obtains Rf=0.69; Irradiation light is tungsten lamp, detection computations obtains the regression equation of relation of biotin standard solution quality and peak area integrated value, and (by thin layer chromatograph management software, winCATS1.4.1 calculates, for conventional means), then the peak area integrated value of liquid calculates the mass concentration of biotin in sample liquid per sample.
The extracting method of biotin of the present invention, simple to operate, reliable, the detection method of biotin of the present invention, has the advantages such as simple, quick, accurate, reliable, stable.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1 carries out the extraction of biotin taking molasses as raw material
15g molasses and 5g zeyssatite are stirred, obtain dry molasses diatomite in powder 40 DEG C of oven dry, add 60ml methyl alcohol, stir at normal temperatures 1 time, mixing time 30 minutes, filters afterwards.In processed molasses diatomite in powder, add 80mlN, dinethylformamide stirs extraction in water-bath, and temperature is at 75 DEG C, time is 1 hour, filters and obtains supernatant, and use absolute ethanol washing filter residue, each 80ml, washs 3 times, merges supernatant and cleansing solution.After mixed liquor filters, steam instrument evaporation and concentration to 50ml with revolving, add 150ml acetone, stir and evenly mix rear leaving standstill, be settled out partial impurities, after filtration, again steam instrument evaporation and concentration to 40ml with revolving, then add wherein 120ml acetone, stir and evenly mix rear leaving standstill, be settled out partial impurities, concentrated with revolving steaming instrument after filtering, be concentrated to 30ml, add the chloroform of 90ml, stir and evenly mix rear leaving standstill, after filtering, concentrated by rotary evaporation is to 3ml; Mix with 3ml butyl acetate and 3ml cyclohexane, stratification, gets supernatant concentration to 2ml, is biotin solution (as sample liquid).Described rotary evaporation temperature is 55 DEG C.
Embodiment 2 carries out the extraction of biotin taking molasses as raw material
15g molasses and 10g zeyssatite are stirred, obtain dry molasses diatomite in powder 50 DEG C of oven dry, add 100ml methyl alcohol to stir at normal temperatures 2 times, mixing time 45 minutes, filters afterwards.Processed molasses diatomite in powder is added to 125mlN, and dinethylformamide stirs extraction in water-bath, temperature is at 85 DEG C, and the time is 1.5 hours, filters and obtains supernatant, and use absolute ethanol washing filter residue, each 125ml washing 4 times, merges supernatant and cleansing solution.After filtering, mixed liquor steams instrument evaporation and concentration to 80ml with revolving, add the stirring of 320ml acetone and be settled out partial impurities, after filtration, again steam instrument evaporation and concentration to 50ml with revolving, add wherein again 200ml acetone precipitation to go out partial impurities, concentrated with revolving steaming instrument after filtering, be concentrated to the chloroform precipitated impurities that 20ml adds 80ml, after filtering, concentrated by rotary evaporation is to 3ml.Mix with 3ml butyl acetate and 3ml cyclohexane, stratification, gets supernatant concentration to 2ml, is biotin solution (as sample liquid).Rotary evaporation temperature is 60 DEG C.
Embodiment 3 carries out the extraction of biotin taking honey as raw material
15g molasses and 15g zeyssatite are stirred, obtain dry molasses diatomite in powder 60 DEG C of oven dry, add 150ml methyl alcohol to stir at normal temperatures 3 times, mixing time 60 minutes, filters afterwards.Processed molasses diatomite in powder is added to 180mlN, and dinethylformamide stirs extraction in water-bath, temperature is at 95 DEG C, and the time is 2 hours, filters and obtains supernatant, and use absolute ethanol washing filter residue, each 180ml washing 5 times, merges supernatant and cleansing solution.After filtering, mixed liquor steams instrument evaporation and concentration to 60ml with revolving, add the stirring of 300ml acetone and be settled out partial impurities, after filtration, again steam instrument evaporation and concentration to 40ml with revolving, add wherein again 200ml acetone precipitation to go out partial impurities, concentrated with revolving steaming instrument after filtering, be concentrated to the chloroform precipitated impurities that 20ml adds 100ml, after filtering, concentrated by rotary evaporation is to 3ml; Mix with 3ml butyl acetate and 3ml cyclohexane, stratification, gets supernatant concentration to 2ml, is biotin solution (as sample liquid).Rotary evaporation temperature is 65 DEG C.
Embodiment 4 carries out the extraction of biotin taking molasses as raw material
15g molasses and 10g zeyssatite are stirred, obtain dry molasses diatomite in powder 55 DEG C of oven dry, add 100ml methyl alcohol to stir at normal temperatures 3 times, mixing time 50 minutes, filters afterwards.Processed molasses diatomite in powder is added to 100mlN, and dinethylformamide stirs extraction in water-bath, temperature is at 85 DEG C, and the time is 75min, filters and obtains supernatant, and use absolute ethanol washing filter residue, each 100ml washing 4 times, merges supernatant and cleansing solution.After filtering, mixed liquor steams instrument evaporation and concentration to 60ml with revolving, add the stirring of 240ml acetone and be settled out partial impurities, after filtration, again steam instrument evaporation and concentration to 40ml with revolving, add wherein again 200ml acetone precipitation to go out partial impurities, concentrated with revolving steaming instrument after filtering, be concentrated to the chloroform precipitated impurities that 20ml adds 100ml, after filtering, concentrated by rotary evaporation is to 3ml; Mix with 3ml butyl acetate and 3ml cyclohexane, stratification, gets supernatant concentration to 2ml, is biotin solution (as sample liquid).Rotary evaporation temperature is 65 DEG C.
Embodiment 5 biotin thin-layer chromatography scanning detection methods
Precision takes biotin standard items (Sigma company) 1.29mg, adds DMF 1.29ml, is mixed with the standard solution that concentration is 1mg/ml.Accurate absorption 50,200, the standard solution of 250,300,400 μ l1mg/ml, be mixed with concentration 0.05 μ g/ μ l respectively, 0.2 μ g/ μ l, 0.25 μ g/ μ l, 0.3 μ g/ μ l, the series of standards product solution of 0.4 μ g/ μ l, sample liquid 4 μ l and the titer 4 μ l points of getting respectively embodiment 1 gained are scribbling silica G F
254on efficient thin layer plate (10cm × 10cm), with methylene chloride (v): chloroform (v): methyl alcohol (v): the ratio preparation 8.06ml developping agent of glacial acetic acid (v)=3:3:2:0.06, chromatography in expansion cylinder (10cm × 12cm × 15cm), the saturation time of developping agent is 60 minutes, development distance is 6cm, duration of run is 15 minutes, then thin layer plate is taken out to be placed in vent cabinet and dries.2,4-dimethylamino cinnamic acid, sulfuric acid and absolute ethyl alcohol are mixed, be made into dyeing liquor, wherein 2,4-dimethylamino cinnamic acid concentration is 0.1%, and sulfuric acid concentration is 1%, the dyeing liquor spray dyeing of thin layer plate after drying, obtains and the background obvious orange-red standard items spot of contrast and sample point.
Utilize Camag3 type thin-layer chromatogram scanner to carry out thin-layer chromatography scanning with 530nm absorbing wavelength, sweep velocity is 20mm/s, and data resolution is 50 μ m/step, obtains Rf=0.69.Irradiation light is that tungsten lamp carries out detection computations, taking the quality (ng) of biotin standard items as horizontal ordinate (X), peak area integrated value is ordinate (Y), directly obtained the regression equation Y=67.1435+2.8351X of standard items and peak area integrated value relation by thin layer chromatograph management software winCATS1.4.1, r=0.99933, RSD=2.58%.Result shows, sample liquid is linear good at 0.2～1.6 μ g/ spot, and in molasses, the total content of biotin is 26.525 μ g/g as calculated.
Embodiment 6 biotin thin-layer chromatography scanning detection methods
According to the chromatography method of embodiment 5, difference is to adopt the biotin sample liquid of embodiment 2 gained, with methylene chloride (v): chloroform (v): methyl alcohol (v): the ratio preparation 7.04ml developping agent of glacial acetic acid (v)=3:2:1:0.04, dyeing liquor wherein 2,4-dimethylamino cinnamic acid concentration is 0.2%, sulfuric acid concentration is 2%, and the content that calculates biotin in molasses according to equation of linear regression is 26.515 μ g/g.
Embodiment 7 biotin thin-layer chromatography scanning detection methods
According to the chromatography method of embodiment 5, difference is to adopt the biotin sample liquid of embodiment 3 gained, with methylene chloride (v): chloroform (v): methyl alcohol (v): the ratio preparation 7.05ml developping agent of glacial acetic acid (v)=2:3:2:0.05, dyeing liquor wherein 2,4-dimethylamino cinnamic acid concentration is 0.15%, sulfuric acid concentration is 1.5%, and the content that calculates biotin in molasses according to equation of linear regression is 26.545 μ g/g.
Embodiment 8 biotin thin-layer chromatography scanning detection methods
According to the chromatography method of embodiment 5, difference is to adopt the biotin sample liquid of embodiment 4 gained, with methylene chloride (v): chloroform (v): methyl alcohol (v): the ratio preparation 7.05ml developping agent of glacial acetic acid (v)=2:2:3:0.05, dyeing liquor wherein 2,4-dimethylamino cinnamic acid concentration is 0.15%, sulfuric acid concentration is 2%, and the content that calculates biotin in molasses according to equation of linear regression is 26.535 μ g/g.
Claims (9)
1. a method of extracting biotin from molasses, is characterized in that: step is as follows:
(1) make molasses diatomite in powder taking molasses and zeyssatite as raw material: get molasses and zeyssatite, mixing mixes, dry, obtain molasses diatomite in powder; Wherein, molasses and diatomaceous weight ratio are 3:1～3;
(2) in molasses diatomite in powder, add methyl alcohol, stir and extract 30～60min, filter to obtain filter residue; The addition of described methyl alcohol is: every 1 gram of molasses diatomite in powder adds 3～5ml methyl alcohol;
(3) in above-mentioned filter residue, add DMF, at 70～100 DEG C of water-baths, stir and extract 1～2 hour, filter to obtain supernatant and filter residue; Filter residue absolute ethanol washing 1～3 time, obtains ethanol cleansing solution; The addition of described DMF is: every 1 gram of filter residue adds 4～6mlN, dinethylformamide; The addition of described absolute ethyl alcohol is: the amount of at every turn washing absolute ethyl alcohol used is 0.8～1.2 times of DMF volume;
(4) combining step (3) gained supernatant and ethanol cleansing solution, evaporation and concentration, adds the acetone of 3～5 times of amounts, leave standstill, remove by filter precipitation, again evaporation and concentration, add the acetone of 3～5 times of amounts, leave standstill, remove by filter precipitation, again evaporation and concentration, add the chloroform of 3～5 times of amounts, leave standstill, remove by filter precipitation, obtain extract;
(4) in extract, add butyl acetate and cyclohexane, the addition of butyl acetate, cyclohexane is identical with the volume of extract, stratification; Get supernatant, be biotin solution.
2. the method for extracting biotin from molasses according to claim 1, is characterized in that: described evaporation and concentration adopts Rotary Evaporators to carry out, and the temperature of rotary evaporation is 40～60 DEG C.
3. a thin-layer chromatography scanning detection method for biotin, is characterized in that: step is as follows:
(1) get detected sample liquid, point, on silica gel plate, adds developping agent to expansion cylinder, in expansion cylinder, launches, and then thin layer plate is taken out be placed in vent cabinet and is dried; Meanwhile, adopt one point external standard method to put the biotin standard solution of 4 μ l variable concentrations, launch simultaneously;
Described developping agent is methylene chloride-chloroform-methanol-glacial acetic acid mixed liquor, and wherein, the volume ratio of methylene chloride, chloroform, methyl alcohol, glacial acetic acid is 1～3:1～3:1～2:0.04～0.06;
(2) 2,4-dimethylamino cinnamic acid, sulfuric acid and absolute ethyl alcohol are mixed, be made into dyeing liquor, wherein 2,4-dimethylamino cinnamic acid final concentration is 0.1～0.2%, sulfuric acid final concentration is 1～2%; The panel of having dried after having opened up, uses dyeing liquor spray dyeing, obtains contrasting the spot of obvious standard items and the spot of sample with background;
(3) utilize thin-layer chromatogram scanner to carry out thin-layer chromatography scanning with 530nm absorbing wavelength, irradiation light is tungsten lamp, detection computations obtains the regression equation of the relation of biotin standard solution quality and peak area integrated value, and then the peak area integrated value of liquid calculates the mass concentration of biotin in sample liquid per sample.
4. the thin-layer chromatography scanning detection method of biotin according to claim 3, is characterized in that: described silica gel plate is for scribbling silica G F
254efficient thin layer plate.
5. the thin-layer chromatography scanning detection method of biotin according to claim 3, is characterized in that: the volume ratio of described methylene chloride, chloroform, methyl alcohol, glacial acetic acid is 3:3:2:0.06.
6. the thin-layer chromatography scanning detection method of biotin according to claim 3, it is characterized in that: described biotin standard solution comprises that concentration is respectively 0.05 μ g/ μ l, 0.2 μ g/ μ l, 0.25 μ g/ μ l, 0.3 μ g/ μ l, the series of standards product solution of 0.4 μ g/ μ l.
7. the thin-layer chromatography scanning detection method of biotin according to claim 3, is characterized in that: described regression equation Y=67.1435+2.8351X, r=0.99933, RSD=2.58%.
8. the thin-layer chromatography scanning detection method of biotin according to claim 3, is characterized in that: described sample liquid prepares by the following method:
(1) make molasses diatomite in powder taking molasses and zeyssatite as raw material: get molasses and zeyssatite, mixing mixes, dry, obtain molasses diatomite in powder; Wherein, molasses and diatomaceous weight ratio are 3:1～3;
(2) in molasses diatomite in powder, add methyl alcohol, stir and extract 30～60min, filter to obtain filter residue; The addition of described methyl alcohol is: every 1 gram of molasses diatomite in powder adds 3～5ml methyl alcohol;
(3) in above-mentioned filter residue, add DMF, at 70～100 DEG C of water-baths, stir and extract 1～2 hour, filter to obtain supernatant and filter residue; Filter residue absolute ethanol washing 1～3 time, obtains ethanol cleansing solution; The addition of described DMF is: every 1 gram of filter residue adds 4～6mlN, dinethylformamide; The addition of described absolute ethyl alcohol is: the amount of at every turn washing absolute ethyl alcohol used is 0.8～1.2 times of DMF volume;
(4) combining step (3) gained supernatant and ethanol cleansing solution, evaporation and concentration, adds the acetone of 3～5 times of amounts, leave standstill, remove by filter precipitation, again evaporation and concentration, add the acetone of 3～5 times of amounts, leave standstill, remove by filter precipitation, again evaporation and concentration, add the chloroform of 3～5 times of amounts, leave standstill, remove by filter precipitation, obtain extract;
(4) in extract, add butyl acetate and cyclohexane, the addition of butyl acetate, cyclohexane is identical with the volume of extract, stratification; Get supernatant, be sample liquid.
9. the thin-layer chromatography scanning detection method of biotin according to claim 8, is characterized in that: described evaporation and concentration adopts Rotary Evaporators to carry out, and the temperature of rotary evaporation is 40～60 DEG C.
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| CN102539616A (en) * | 2012-02-14 | 2012-07-04 | 山东师范大学 | Method for extracting and detecting biotin of bird nest |
| CN102735512A (en) * | 2012-07-18 | 2012-10-17 | 山东师范大学 | Method for extracting biotin from Antarctic krill and high-efficiency thin layer scanning analysis method |
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| CN102539616A (en) * | 2012-02-14 | 2012-07-04 | 山东师范大学 | Method for extracting and detecting biotin of bird nest |
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