CN1900693A - Detecting method for monacolin compound content in red yeast vinegar - Google Patents
Detecting method for monacolin compound content in red yeast vinegar Download PDFInfo
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Abstract
The method includes following steps: (1) extracting and separating Monacolin compound; (2) preparing standard curve of Monacolin K; (3) dual wavelength detection of ultraviolet spectrophotometer; (4) calculating result. Through extraction method, the invention extracts and separates compound of Monacolin class in red rice vinegar by using trichloromethane. The invention does following procedures: volatilizing solvent; redissolution by using 75% ethanol; testing compound of Monacolin class in redissolution of ethanol by dual wavelength detection of ultraviolet spectrophotometer. Ratio between difference of absorbance of Monacolin under 246nm, 254nm wavelengths and concentration is accorded with beers law. Features are: accuracy, and linear relation accorded with requirement of spectrophotometric analysis, obtained better result for detecting red rice vinegar.
Description
Technical field
The present invention relates to a kind of health food functional component detection method, refer to the assay method of Monacolin compounds content in a kind of monascus vinegar especially.
Background technology
Red colouring agent for food, also used as a Chinese medicine (also name red yeast rice) is to be raw material with the rice, and a kind of aubergine rice song that forms through monascus (Monascus) fermentation is so claim red song again.The invention of red colouring agent for food, also used as a Chinese medicine is the history in existing more than 1,000 year so far.Be widely used in wine brewing, aspects such as system vinegar, food color and Chinese medicine.
The Monacolin compounds is the major physiological active substance of monascus, can effectively reduce total plasma cholesterol and LDL-C content.Because Monacolin class material toxic and side effect is little, better tolerance just has been applied to clinical since finding very soon.The Monacolin compound is applied to some as the health food functional component again and has the health food of hypolipemic function and the welcome that is subjected to consumers in general in recent years.
Vinegar also has a long history in China, is not only one of flavouring that China people like, aspect treatment disease, the health care good effect is being arranged also.The king of Qing Dynasty generation hero has effects such as " appetizing, nourishing the liver, strong muscle, warm bones, sober up, help digestion, the therapeutic method to keep the adverse qi flowing downward, exorcise evil spirits, separate fish, all poison of crab aquatic animals " at " arbitrarily occupy diet spectrum " record vinegar.Modern medicine is found, contains multiple organic acid, amino acid, ester class, vitamin and mineral matter in the vinegar, has the metabolism of the human body of improvement, prevents and lessens fatigue, and is hypotensive and prevent effect such as atherosclerotic.Monascus vinegar is the health-care vinegar of producing by red koji fermentation with lipid-reducing function, unique flavor not only, and have the good health care function.In the Jiangsu and Zhejiang Provinces, Fujian, Taiwan one is with very popular.In order to guarantee product quality, just seem very important for the detection of Monacolin class material in production procedure and the product.
The mensuration to Monacolin class material in the red koji fermentation product of report mainly adopts high performance liquid chromatography both at home and abroad at present, be characterized in the accuracy height, but instrument costs an arm and a leg, the detection required time is long, and need the professional and technical personnel to operate, inapplicable for numerous medium-sized and small enterprises of domestic production monascus vinegar goods.Therefore need explore a kind of easy, quick, effective portable measuring method for measuring at actual production.
Because the Monacolin compounds has similar structure and function, all at 230nm, 238nm, there is the feature uv absorption at 246nm wavelength place, therefore makes us record Monacolin total amount of compound in the sample by ultraviolet spectrophotometer is easy.But problem is a vinegar is tunning repeatedly, and contained multiple metabolic product except that the Monacolin compounds can produce serious the interference to the mensuration of Monacolin compounds in the ultraviolet region, be caused difficulty with spectrophotometric determination.This patent discloses the portable measuring method for measuring of Monacolin compounds content in a kind of monascus vinegar, the method can be disturbed in easy, quick eliminating, finishes with the quantitative measurement of simple ultraviolet spectrophotometer to the Monacolin compounds content in the monascus vinegar sample.
Summary of the invention
The invention provides the assay method of Monacolin compounds content in a kind of monascus vinegar, it can solve the deficiency of the above method.
In order to realize such scheme, the assay method of Monacolin compounds content in a kind of monascus vinegar of the present invention, it may further comprise the steps:
1) extract and separate of Monacolin compound;
2) preparation of Monacolin K typical curve;
3) the ultraviolet spectrophotometer dual wavelength detects;
4) result's calculating.
The assay method of Monacolin compounds content in the described monascus vinegar, the extract and separate step of Monacolin compound comprises in the above-mentioned steps (1): accurately measure the 10mL monascus vinegar in the 50mL separating funnel, add the 20mL methenyl choloride, vibrated 15 minutes, after leaving standstill 15 minutes, collect the methenyl choloride layer, re-extract is three times altogether; Collect the chloroform extraction liquid that merges three times and be total to 60mL in the 100mL distilling flask, place 28 ℃ of water-baths, use vacuum pump decompression rotary evaporation to flinging to whole solvents; In cucurbit, add a small amount of 75% dissolve with ethanol solution extract, and fully be transferred in the 50mL volumetric flask, add 75% ethanol constant volume; Shake up, lucifuge is placed after 15 minutes and is measured with ultraviolet spectrophotometer.
The assay method of Monacolin compounds content in the described monascus vinegar, the preparation process of Monacolin K typical curve comprises in the above-mentioned steps (2): precision takes by weighing 3.6mg Monacolin K standard items and is dissolved in 50mL 75% ethanol, is made into the solution of 72 μ g/mL; Measure 0.05,0.5,1.0,2.0,3.0,4.0 respectively, 5.0mL, to 10mL, make the Monacolin K standard solution that concentration is respectively 0.36,3.6,7.2,14.4,21.6,28.8,36.0 μ g/mL with 75% ethanol constant volume; Do blank with 75% ethanol, measure this series standard solution absorbency value at 246nm, 254nm wavelength place respectively with ultraviolet spectrophotometer; Poor (rA=A246-A254) with the absorbance of Monacolin compound under 246nm, 254nm two wavelength is ordinate, is horizontal ordinate with the concentration corresponding with it, the drawing standard curve.
The assay method of Monacolin compounds content in the described monascus vinegar, above-mentioned steps (3) medium ultraviolet spectrophotometer dual wavelength detects step and comprises: do blank with 75% ethanol, measure sample absorbance after pre-treatment respectively at 246nm and 254nm wavelength place with ultraviolet spectrophotometer, and calculate the difference (rA=A246-A254) of the absorbance under two wavelength.
Result's calculation procedure comprises in the assay method of Monacolin compounds content in the described monascus vinegar, above-mentioned steps (4): the absorbance difference with sample is being found Monacolin compound concentration in the sample of pre-treatment on the horizontal ordinate of typical curve.Be calculated as follows Monacolin compounds content in the monascus vinegar again:
In the formula: Monacolin kind compound content in the X-monascus vinegar, mg/100ml
C-is at the Monacolin compound concentration of finding on the horizontal ordinate of typical curve in the sample of pre-treatment, mg/ml
Constant volume after the 50-sample pre-treatments, ml
The amount of getting of 10-vinegar, ml.
The assay method of Monacolin compounds content in the described monascus vinegar, when active constituent content in the above-mentioned sample is crossed when low, the amount of taking that can increase vinegar maybe can be dwindled constant volume after the sample pre-treatments, and its result of calculation is changed " 10 " and " 50 " these two numerals in the formula as the case may be.
After adopting said method, the principle of this assay method is:
The Monacolin compounds is soluble in methenyl choloride, available its solvent as extraction Monacolin compound from monascus vinegar.Monascus vinegar has been removed most of impurity after extract and separate, volatilize solvent, again the crystal of separating out with 75% dissolve with ethanol.Sample impurity such as remaining small amount of coloring matter still after extract and separate, although they also have uv absorption, but in the 240-260nm wavelength coverage, absorbance is in minimum, and very mild, do not influence of the variation of the Monacolin compound absorption spectrum of stack at 246nm, 254nm wavelength place absorbance difference.The Monacolin compound is at 246nm, poor (rA=A246-A254) of the absorbance under 254nm two wavelength meets Beer law with the ratio of its concentration, therefore this method elder generation difference working sample is in the absorbance at 246nm and 254nm wavelength place, calculate the content of Monacolin compound in the solution again with the difference of these two absorbances, compare with high performance liquid chromatography, it is cheap that this method has a required instrument and equipment, easy and simple to handle, characteristics fast, the accuracy of this method, precision and linear relationship meet the spectrophotometric analysis requirement, monascus vinegar is detected can obtain better result.
Description of drawings
Fig. 1 is a standard Monacolin K ultra-violet absorption spectrum;
Fig. 2 is the ultra-violet absorption spectrum that does not contain the monascus vinegar of Monacolin compounds;
Fig. 3 is the ultra-violet absorption spectrum that contains the monascus vinegar of Monacolin compounds;
Fig. 4 is a Monacolin K typical curve.
Embodiment
The detection of Monacolin kind compound content in the functional red yeast rice vinegar
Material:
Reagent: Monacolin K (Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences);
Absolute ethyl alcohol (Beijing Chemical Plant) is analyzed pure;
Methenyl choloride (Beijing Chemical Plant) is analyzed pure;
Instrument and equipment: ultraviolet-visible pectrophotometer (SHIMADZU, UV-2450);
Rigorous analysis balance (Mettler AE-100);
Rotary evaporator (BUCHI)
Separating funnel (volume is 50mL)
Sample: monascus vinegar, functional red yeast rice vinegar (the red small stream in Yiwu Wine Co., Ltd)
Experimental technique and result
Sample is handled and is measured: accurately measure the 10mL monascus vinegar in the 50mL separating funnel, add the abundant mixing of 20mL methenyl choloride, vibrated 15 minutes, after leaving standstill 15 minutes, collect the methenyl choloride layer, re-extract is three times altogether, collect the chloroform extraction liquid that merges three times and be total to 60mL in the 100mL distilling flask, place 28 ℃ of left and right sides water-baths, use vacuum pump decompression rotary evaporation to flinging to whole solvents, in cucurbit, add a small amount of 75% dissolve with ethanol solution extract, and fully be transferred in the 50mL volumetric flask constant volume.Shake up, lucifuge is placed after 15 minutes with ultraviolet spectrophotometer respectively in wavelength 246nm and 254nm place mensuration solution absorbency value, and calculates poor (rA=A246-A254) of the absorbance under two wavelength.
Detect wavelength determination: take by weighing Monacolin K standard items 40.6mg, to 25mL, making concentration is the Monacolin K standard solution of 1.624mg/mL with 75% ethanol constant volume.Get 0.5mLMonacolin K standard solution, to 50mL, obtain Monacolin K standard application liquid with 75% ethanol constant volume, concentration is 16.24 μ g/mL.In the 190nm-300nm wavelength coverage, carry out length scanning with ultraviolet spectrophotometer, obtain standard Monacolin K ultra-violet absorption spectrum (as shown in Figure 1), from Fig. 1, can be clear that three characteristic absorption peaks that the Monacolin compounds is common.
Measure 2 parts of monascus vinegars respectively, each 10mL, a copy of it is removed the Monacolin compounds with charcoal absorption.Two duplicate samples are measured Monacolin content (shown in Fig. 2,3) with ultraviolet spectrophotometer respectively after steps such as above-mentioned extraction, rotary evaporation, redissolution.Fig. 2 removes the monascus vinegar of Monacolin compounds through the ultra-violet absorption spectrum under the 190-300nm wavelength after the pre-treatment with charcoal absorption, as we can see from the figure, in the dissolve with ethanol liquid after extraction, still have a small amount of remaining impurity such as pigment in the 190-300nm wavelength coverage, to have light absorption, but in the 240-260nm wavelength coverage, absorbance is in minimum, and very mild, and this becomes possibility with regard to the interference of using the double wave regular way to get rid of these impurity.
When the ultraviolet spectrum of compound shows that when overlapping, in fact the intensity of viewed absorption band and maximum absorption wavelength are not the performances of single transition, but different transition bands of a spectrum add and overlapping absorption band.Fig. 3 is the uv absorption spectra of monascus vinegar after the pre-treatment extract and separate.Distinctive three characteristic absorption peaks of Monacolin compound as we can see from the figure.Just owing to still contain some impurity in the sample, three characteristic absorption peaks of Monacolin compound have been added on the absorption spectrum of these impurity.Obviously, in this case, if only with the pairing wavelength of one of them absorption peak as the wavelength that detects the Monacolin compound, the absorbance that determines is actually impurity and the common generation of Monacolin compound.Remove to calculate the Monacolin compounds content with this absorbance and certainly lead to very big positive error, make testing result higher.Because light absorption very mild (see figure 2) in the 240-260nm wavelength coverage of impurity, after the stack of Monacolin compound absorption spectrum, the degree that Monacolin compound absorption spectrum is elevated in the 240-260nm wavelength coverage is consistent.Be that this raising do not influence the variation of Monacolin compound at 246nm, 254nm wavelength place absorbance difference.Therefore this method selects 246nm and 254nm wavelength for detecting wavelength.Working sample calculates the content of Monacolin compound in the solution again in the absorbance at 246nm and 254nm wavelength place with the difference of these two absorbances respectively earlier.So just can get rid of the spectrum that remains in the impurity in the sample solution disturbs.
According to Lambert-Beer's law as can be known:
-log(I1/I0)＝Aλ1＝ελ1lc+As1
-log(I2/I0)＝Aλ2＝ελ2lc+As2
Wherein: I0 represents incident intensity;
ε is a molar absorptivity;
A is the absorbance log of solution to light;
As1 and As2 are respectively the absorption of impurity at λ 1 and λ 2.
As1=As2 here, λ 1=254nm, λ 2=246nm.
RA=A246-A254=-log (I246/I254)=(ε λ 2-ε λ 1) lc then
Hence one can see that, and sample is directly proportional with Monacolin compounds content in the sample in the difference of the absorbance at 246nm and 254nm wavelength place, promptly can carry out quantitatively the Monacolin compound in the sample by this method.
The drafting of Monacolin K typical curve: precision takes by weighing 3.6mg Monacolin K standard items and is dissolved in 50mL 75% ethanol, is made into the solution of 72 μ g/mL.Measure 0.05,0.5,1.0,2.0,3.0,4.0 respectively, the 5.0mL constant volume is to 10mL, makes its concentration be respectively 0.36,3.6,7.2,14.4,21.6,28.8,36.0 μ g/mL.(246nm, 254nm) measures its content with the double wave regular way, and with drawing standard curve as a result as shown in Figure 4.Its equation of linear regression is y=0.0397x+0.0092, and the γ value is 0.9999.Show that Monacolin K is good linear relationship with rA246-254 in the concentration range of 0.36-36.0 μ g/mL, meet Beer law.
The mensuration of the method recovery: take by weighing 40.6mg Monacolin K standard items with 75% ethanol constant volume to 25mL, be that concentration is 1.624mg/mL, shake up the back precision and measure 2mL and put into the 50mL separating funnel, measure the 10mL monascus vinegar again and add wherein, as the mark-on sample.Measure the 10mL monascus vinegar and join in another separating funnel, add 2mL 75% ethanol, as mark-on sample not.Two duplicate samples said method operation repetitive is measured the Monacolin compounds content respectively, reaches the not recovery of mark-on test result of samples computing method according to mark-on, repeats 5 times.
Table 1 adds standard Monacolin K sample recovery rate measurement result
| Sequence number | Addition (mg) | Detected level (mg) | The recovery (%) | X±SD |
| 1 2 3 4 5 | 3.25 3.25 3.25 3.25 3.25 | 3.06 3.02 3.12 2.99 3.15 | 94 93 96 92 97 | 94±2 |
The recovery of this method is more than 90% as can be seen from Table 1.
Method collimation precision is measured: same even sample is divided into 5 parts, begins to experimentize with said method is parallel from pre-treatment step, 5 duplicate samples measurement results see Table 2.
Table 2 collimation precision measurement result
| Sequence number | Monacolins measurement result (mg/100mL) | Mean value | RSD |
| 1 2 3 4 5 | 21.2 21.0 20.7 20.4 20.7 | 20.8 | 1.48％ |
The result shows that this method has good collimation precision.
Method repeatability precision is measured: same even sample begins to measure once with said method every day from sample pre-treatments under same experiment condition, continuous coverage 5 days, and it the results are shown in Table 3.
The repeated precision measurement result of table 3
| Sequence number | Monacolins measurement result (mg/100mL) | Mean value | RSD |
| 1 2 3 | 21.0 19.3 19.8 | 20.2 | 3.30％ |
| 4 5 | 20.3 20.6 |
The result shows that this method repeatability precision is good.
The mensuration of Monacolin compounds content in the monascus vinegar sample: 2 kinds of monascus vinegar samples, measure wherein Monacolin compounds content with above-mentioned detection method, the results are shown in Table 4.
Monacolin compounds content measurement result in table 4 monascus vinegar
| Sequence number | The sample title | The replication number of times | Monacolins measurement result (μ g/mL) |
| 1 2 | Monascus vinegar functional red yeast rice vinegar | 5 5 | 20.8±0.3 116.7±1.6 |
The actual measurement process of 2 samples is all very smooth, the coefficient of variation that can calculate each sample replication from 2 sample results measured all is no more than 5%, illustrate that the result that detects all within the allowed band of spectrophotometric quantitative test, also illustrates that this method has good practicality.
Monacolin compound in the monascus vinegar: the Monacolin compound is a series of structures that produce in the red koji fermentation process, the material of similar performance, and they have same chromophore and ultraviolet characteristic absorption spectrum thereof.Main chromophore is the different ring conjugated diene structure that replaces in the Monacolin compound, absorbs rule by hexatomic ring diene and triolefin, and can add and calculate at the 238nm place has absorption maximum.Actual ultraviolet absorption peak observed reading is 230,238, and 246nm has promptly formed the distinctive three peak ultraviolet characteristic absorption spectrums of Monacolin compounds.In the monascus vinegar extraction process, these Monacolin compounds are extracted in the chloroform soln, therefore, the absorbance that goes out with spectrophotometric determination, be the coefficient result of chromophore of these Monacolin compounds, promptly the result of Ce Dinging has represented Monacolin total amount of compound in the monascus vinegar.
The lowest detection lower limit of method: after the pre-treatment of above-mentioned experiment condition, adopt the double wave regular way to measure under the lowest detection of Monacolin K standard items and be limited to 0.36 μ g/mL, the monascus vinegar sample is after pre-treatment, the value of absorbance poor (A246-A254) is 0.013, and measuring concentration is 5.05 μ g/mL.Experiment finds that instrument can not detect the characteristic peak of Monacolin compound if contained Monacolin compound is lower than this concentration in the sample, therefore under this experiment condition, is limited to 5.05 μ g/mL under the detection of Monacolin total amount of compound.
Claims (6)
1, the assay method of Monacolin compounds content in a kind of monascus vinegar, it may further comprise the steps:
1) extract and separate of Monacolin compound;
2) preparation of Monacolin K typical curve;
3) the ultraviolet spectrophotometer dual wavelength detects;
4) result's calculating.
2, the assay method of Monacolin compounds content in the monascus vinegar according to claim 1, it is characterized in that: the extract and separate step of Monacolin compound comprises in the above-mentioned steps (1): accurately measure the 10mL monascus vinegar in the 50mL separating funnel, add the 20mL methenyl choloride, vibrated 15 minutes, after leaving standstill 15 minutes, collect the methenyl choloride layer, re-extract is three times altogether; Collect the chloroform extraction liquid that merges three times and be total to 60mL in the 100mL distilling flask, place 28 ℃ of water-baths, use vacuum pump decompression rotary evaporation to flinging to whole solvents; In cucurbit, add a small amount of 75% dissolve with ethanol solution extract, and fully be transferred in the 50mL volumetric flask, add 75% ethanol constant volume; Shake up, lucifuge is placed after 15 minutes and is measured with ultraviolet spectrophotometer.
3, the assay method of Monacolin compounds content in the monascus vinegar according to claim 1, it is characterized in that: the preparation process of Monacolin K typical curve comprises in the above-mentioned steps (2): precision takes by weighing 3.6mg Monacolin K standard items and is dissolved in 50mL 75% ethanol, is made into the solution of 72 μ g/mL; Measure 0.05,0.5,1.0,2.0,3.0,4.0 respectively, 5.0mL, to 10mL, make the Monacolin K standard solution that concentration is respectively 0.36,3.6,7.2,14.4,21.6,28.8,36.0 μ g/mL with 75% ethanol constant volume; Do blank with 75% ethanol, measure this series standard solution absorbency value at 246nm, 254nm wavelength place respectively with ultraviolet spectrophotometer; Poor (rA=A246-A254) with the absorbance of Monacolin compound under 246nm, 254nm two wavelength is ordinate, is horizontal ordinate with the concentration corresponding with it, the drawing standard curve.
4, the assay method of Monacolin compounds content in the monascus vinegar according to claim 1, it is characterized in that: above-mentioned steps (3) medium ultraviolet spectrophotometer dual wavelength detects step and comprises: do blank with 75% ethanol, measure sample absorbance after pre-treatment respectively at 246nm and 254nm wavelength place with ultraviolet spectrophotometer, and calculate the difference (rA=A246-A254) of the absorbance under two wavelength.
5, the assay method of Monacolin compounds content in the monascus vinegar according to claim 1 is characterized in that: result's calculation procedure comprises in the above-mentioned steps (4): the absorbance difference with sample is being found Monacolin compound concentration in the sample of pre-treatment on the horizontal ordinate of typical curve.Be calculated as follows Monacolin compounds content in the monascus vinegar again:
In the formula: Monacolin kind compound content in the X-monascus vinegar, mg/100ml
C-find on the horizontal ordinate of typical curve in the sample of pre-treatment
The Monacolin compound concentration, mg/ml
Constant volume after the 50-sample pre-treatments, ml
The amount of getting of 10-vinegar, ml.
6, the assay method of Monacolin compounds content in the monascus vinegar according to claim 5, it is characterized in that: when active constituent content in the above-mentioned sample is crossed when low, the amount of taking that can increase vinegar maybe can be dwindled constant volume after the sample pre-treatments, and its result of calculation is changed " 10 " and " 50 " these two numerals in the formula as the case may be.
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| CN101493420B (en) * | 2009-02-24 | 2011-03-30 | 华南理工大学 | Method for rapidly measuring pentoses and hexose content in hemicellulose extract |
| CN101520411B (en) * | 2009-04-08 | 2011-06-22 | 中国科学院青海盐湖研究所 | Method for measuring azalea oil content of aqueous emulsion by using double-wave length spectrophotometry |
| CN105021555A (en) * | 2015-07-15 | 2015-11-04 | 沈阳大学 | Method for detecting zinc content in oyster product by dual-wavelength spectrophotometric method |
| CN107101956A (en) * | 2015-10-02 | 2017-08-29 | 哈希公司 | Water base sample fluid measurement and analysis |
| WO2019164999A3 (en) * | 2018-02-20 | 2019-12-26 | Kerry Luxembourg S.à.r.l. | Buffered vinegar products with reduced color, odor, and flavor and methods of producing the same |
| CN112423595A (en) * | 2018-02-20 | 2021-02-26 | 凯瑞卢森堡有限责任公司 | Buffered vinegar products with reduced color, odor and flavor and methods of producing same |
| CN109100315A (en) * | 2018-08-21 | 2018-12-28 | 暨南大学 | A kind of Wavelength selecting method based on jamtosignal |
| CN109100315B (en) * | 2018-08-21 | 2020-11-13 | 暨南大学 | Wavelength selection method based on noise-signal ratio |
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