CN107037159B - A kind of Medicated Leaven fermentation process online quality control method - Google Patents
A kind of Medicated Leaven fermentation process online quality control method Download PDFInfo
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Abstract
The present invention provides a kind of Medicated Leaven fermentation process online quality control systems, it includes the following steps: a, samples in Medicated Leaven fermentation process different time points；B, the Liquid Chromatography data of each sample is obtained using high performance liquid chromatography；Measure respectively qinghaosu in each sample, Quercetin, rutin and amarogentin content；C, proteinase activity power is predicted: y=0.0021x1+3.1067x2‑1.4643x3+0.0116x4+0.2095x5+27.6106；The enzyme activity of amylase is predicted: y=0.3054x1+89.2468x2‑16.0689x3‑0.0748x4+0.2888x5+352.1262；Wherein, x1For qinghaosu, x2For Quercetin, x3For rutin, x4For amarogentin, x5For fermentation time, y is amylase and/or prolease activity.The on-line detecting system of Medicated Leaven fermentation process quality index of the present invention, be conducive to the dynamic change for understanding fermentation process intermediate products quality in real time, the developing direction for being conducive to the control optimization intermediate products quality by technological parameter, to improve the quality pass rate of finished product.
Description
Technical field
The present invention relates to a kind of Medicated Leaven fermentation process high performance liquid chromatography online quality control methods.
Background technique
Medicated Leaven also known as six bent, Divine Comedy, are recorded earliest in " haigoushen ", are most representative traditional Chinese medicine fermentations simply
Processed product, its composition of ministry standard are polygonum flaccidum 500g, Siberian cocklebur grass 500g, sweet wormwood 500g, semen armeniacae amarae 100g, rde bean
100g, wheat bran 5000g, flour 2500g have effects that strengthening the spleen and stomach, in the tune that helps digestion.
Application of fermentation technique during Chinese medicine preparation has a long history, it refers in certain humidity and temperature
Under the conditions of, medicinal material or medicinal material after processing or net system, which are mixed, adds auxiliary material etc., using the decomposition catalytic action of enzyme and microorganism, makes
Drug foaming, the concocting method for giving birth to clothing.
Currently, relying on subjective experience for the control of Medicated Leaven traditional zymotic concocting process, control technology lag lacks more
Objective index and effective method, the quality stability of Medicated Leaven fermented product are bad, the quality between different batches of product
It differs greatly.Medicated Leaven using the production of modern zymotechnique is also only to technological parameters such as temperature, the humidity of fermentation process
It is controlled, only has prescription, preparation method, character and usage and dosage etc. in ministry standard and provincial standard and be briefly described, and to production
Main body --- the fermentation materials in dynamic change and the detection without correlated quality index, lack effective component identify and
The routine inspections project such as content, moisture, ash content, it is difficult to control quality of medicinal material.
Therefore, in order to understand the dynamic change of fermentation process intermediate products quality in real time, be conducive to through control technique ginseng
The developing direction of number optimization intermediate products quality, to improve the quality pass rate of finished product, need it is a kind of can more comprehensively, it is quasi-
Really, reliably reflect the online test method of Medicated Leaven fermentation process quality index.
Summary of the invention
The present invention relates to a kind of Medicated Leaven fermentation process high performance liquid chromatography online quality control methods.
The present invention provides a kind of Medicated Leaven fermentation process online quality control methods, it includes the following steps:
A, it is sampled in Medicated Leaven fermentation process different time points；
B, the Liquid Chromatography data of each sample is obtained using high performance liquid chromatography；Sweet wormwood in each sample is measured respectively
Element, Quercetin, rutin and amarogentin content；
C, proteinase activity power is predicted:
Y=0.0021x1+3.1067x2-1.4643x3+0.0116x4+0.2095x5+27.6106；
The enzyme activity of amylase is predicted:
Y=0.3054x1+89.2468x2-16.0689x3-0.0748x4+0.2888x5+352.1262；
Wherein, x1For qinghaosu, x2For Quercetin, x3For rutin, x4For amarogentin, x5For fermentation time, y is amylase
And/or prolease activity.
D, according to proteinase activity power, the Fermentation progress of amylase enzyme activity monitoring Medicated Leaven.
Wherein, the fermentation of Medicated Leaven described in a step is spontaneous fermentation；Fermentation condition are as follows: 30-35 DEG C of temperature, humidity 70-
80%.
Wherein, high performance liquid chromatography condition described in b step are as follows:
Chromatographic column: C18(250mm×4.6mm,5μm)；Mobile phase is acetonitrile (A) and water (B), linear gradient elution, elution
Program is 0~15min, 5% → 10% (A)；15~50min, 10% → 30% (A)；50~80min, 30% → 60% (A)；
80~100min, 60% → 95% (A)；100~110min, 95% (A)；110~120min, 95% → 5% (A)；120~
125min, 5% (A)；Detection wavelength 210nm；Flow velocity 1mL/min；Column temperature: 30 DEG C.
Medicated Leaven is traditional zymotic preparation, and sweet wormwood, polygonum flaccidum, Siberian cocklebur grass, semen armeniacae amarae, rde bean are primary raw material, and composition is multiple
It is miscellaneous.The raw medicinal material quality of Medicated Leaven has large effect to the quality of Medicated Leaven, it is necessary to the matter of these raw medicinal materials
Amount is monitored.But ratio of the above-mentioned raw materials medicinal material in Medicated Leaven is smaller, while raw medicinal material medicinal material during the fermentation
Certain variation occurs at branch, many ingredients are difficult to detect, and find a suitable Testing index to monitor raw medicinal material
Quality is difficult.
The online test method of Medicated Leaven fermentation process quality index of the present invention is conducive to understand among fermentation process in real time
The dynamic change of product quality is conducive to the developing direction of the control optimization intermediate products quality by technological parameter, to mention
The quality pass rate of high finished product.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 Medicated Leaven chromatograms of the present invention
Correlativity between the measured value and predicted value of Fig. 2 proteinase activity power
Correlativity between the measured value and predicted value of Fig. 3 amylase enzyme activity
Fig. 4 prolease activity change curve
Fig. 5 amylase activity change curve
Specific embodiment
Material and reagent: qinghaosu standard reference material, Quercetin standard reference material, rutin standard reference material, amarogentin
Standard reference material；Methanol, acetonitrile, forint phenol, iodine, sodium carbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium thiosulfate, sulfuric acid,
Sodium hydroxide, tyrosine are that analysis is pure；Water is ultrapure water.
Laboratory apparatus:
Sample is all from Sichuan Fu Zheng medicine company Co., Ltd
The foundation of the Medicated Leaven fermentation process high performance liquid chromatography online quality control method of the present invention of embodiment 1
1 materials and methods
The preparation and sampling of 1.1 Medicated Leaven samples
Medicated Leaven raw material is put into climatic chamber and is fermented, 30 DEG C~35 DEG C of temperature of holding, humidity 70%~80%, and
Start timing, since fermentation start carry out primary sample at regular intervals, up to Medicated Leaven ferment to sample surfaces cover with it is white
Mould, the sample of a hour about more than 130, taking-up are saved at -20 DEG C, spare.
The acquisition of 1.2 high-efficient liquid phase color modal datas
1.2.1 the preparation of test solution
The sample 5.00g of crushing is weighed in conical flask, 10mL methanol, weighing is added, ultrasonic extraction 30min is mended later
The methanol weight lost enough, filter membrane are fitted into sample injection bottle, spare.
1.2.2 high-efficient liquid phase color modal data (see Fig. 1)
High performance liquid chromatography condition are as follows: chromatographic column: C18 (250mm × 4.6mm, 5 μm)；Mobile phase is acetonitrile (A) and water
(B), linear gradient elution, elution program are 0~15min, 5% → 10% (A)；15~50min, 10% → 30% (A)；50~
80min, 30% → 60% (A)；80~100min, 60% → 95% (A)；100~110min, 95% (A)；110~120min,
95% → 5% (A)；120~125min, 5% (A)；Detection wavelength 210nm；Flow velocity 1mL/min；Column temperature: 30 DEG C.
With the content of each component in external standard method sample.
Qinghaosu standard curve is y=0.00002330X+0.07005874, R2=0.9999；
Quercetin standard curve is y=0.00000026x+0.00038846, R2=0.9992；
Rutin standard curve is y=0.00000038x+0.00180675, R2=0.9999；
Amarogentin standard curve is y=0.00000285x+0.01223938, R2=0.9998.
1.3 protease and amylase activity measure
1.3.1 prepared by enzyme solution
4.00g Medicated Leaven sample is accurately weighed in iodine flask, 40.00mL distilled water is added, simultaneously in 40 DEG C of waters bath with thermostatic control
Occasional agitation 1h filters to get enzyme solution, is placed in 4 DEG C of preservations, spare.
1.3.2 prolease activity measures
It is measured by forint phenol colorimetric method.Standard curve regression equation is y=0.0797x+0.0264, R2=
0.9982。
1.3.3 amylase activity measures
It is measured by iodimetric titration.
The foundation of 1.4 models
Using MATLAB R2014b software, brings the experimental data measured into equation and carry out nonlinear fitting, obtain albumen
Enzyme and amylase enzyme activity and qinghaosu, Quercetin, rutin, amarogentin and fermentation time relationship prediction model.
2 results
The enzyme activity determination result of protease and amylase in 2.1 Medicated Leavens
The present invention determines the prolease activity and diastatic activity of Medicated Leaven sample using Forint phenol method and iodimetric titration respectively
Power.Measurement result see the table below 1 and table 2.Having a very wide distribution for protease and amylase activity, substantially conforms to modeling demand.
Prolease activity measurement result in 1 Medicated Leaven of table
2 Medicated Leaven amylase activity measurement result of table
The liquid chromatogram map analysis of 2.2 Medicated Leavens
Qinghaosu, Quercetin, rutin and amarogentin are measured in Medicated Leaven sample in the different fermentations time by external standard method
Content.
The foundation of 3 models
The enzyme activity prediction model of protease are as follows:
Y=0.0021x1+3.1067x2-1.4643x3+0.0116x4+0.2095x5+27.6106；
The enzyme activity prediction model of amylase are as follows:
Y=0.3054x1+89.2468x2-16.0689x3-0.0748x4+0.2888x5+352.1262。
Correlativity between the protease of foundation and the measured value and predicted value of amylase enzyme activity is shown in Fig. 2-3.
The above results show that built enzyme activity prediction model has preferable predictive ability.
The Medicated Leaven fermentation process high performance liquid chromatography online quality control method validation of the present invention of embodiment 2
Medicated Leaven raw material is put into climatic chamber and is fermented, 30 DEG C~35 DEG C of temperature of holding, humidity 70%~80%, and
Start timing, since fermentation start carry out primary sample at regular intervals, up to Medicated Leaven ferment to sample surfaces cover with it is white
Mould, the sample of a hour about more than 130, taking-up are saved at -20 DEG C, spare.
High performance liquid chromatography condition are as follows: chromatographic column: C18 (250mm × 4.6mm, 5 μm)；Mobile phase is acetonitrile (A) and water
(B), linear gradient elution, elution program are 0~15min, 5%~10% (A)；15~50min, 10%~30% (A)；50~
80min, 30%~60% (A)；80~100min, 60%~95% (A)；100~110min, 95% (A)；110~120min,
95%~5% (A)；120~125min, 5% (A)；Detection wavelength 210nm；Flow velocity 1mL/min；Column temperature: 30 DEG C.
Proteinase activity power, amylase enzyme activity are calculated, with actually detected proteinase activity power, amylase enzyme activity pair
Than (see Fig. 4, Fig. 5), the Fermentation progress of Medicated Leaven is judged.
Test proof, the online test method of Medicated Leaven fermentation process quality index of the present invention, the proteinase activity of measurement
Power, amylase enzyme activity are accurate, are conducive to the dynamic change for understanding fermentation process intermediate products quality in real time, are more advantageous to and pass through
The developing direction of the control optimization intermediate products quality of technological parameter, to improve the quality pass rate of finished product.
Claims (2)
1. a kind of Medicated Leaven fermentation process online quality control method, it is characterised in that: it includes the following steps:
A, it is sampled in Medicated Leaven fermentation process different time points；The fermentation of the Medicated Leaven is spontaneous fermentation；Fermentation condition are as follows:
30-35 DEG C of temperature, humidity 70-80%；
B, the Liquid Chromatography data of each sample is obtained using high performance liquid chromatography；Measure respectively qinghaosu in each sample,
Quercetin, rutin and amarogentin content；High performance liquid chromatography condition are as follows:
Chromatographic column: C18, specification 250mm × 4.6mm, 5 μm；Mobile phase: using acetonitrile as mobile phase A, using water as Mobile phase B, linearly
Gradient elution, elution program are 0~15min, mobile phase A 5% → 10%；15~50min, mobile phase A 10% → 30%；50
~80min, mobile phase A 30% → 60%；80~100min, mobile phase A 60% → 95%；100~110min, mobile phase A
95%；110~120min, mobile phase A 95% → 5%；120~125min, mobile phase A 5%；
C, proteinase activity power is predicted:
Y=0.0021x1+3.1067x2-1.4643x3+0.0116x4+0.2095x5+27.6106；
The enzyme activity of amylase is predicted:
Y=0.3054x1+89.2468x2-16.0689x3-0.0748x4+0.2888x5+352.1262；
Wherein, x1For qinghaosu, x2For Quercetin, x3For rutin, x4For amarogentin, x5For fermentation time, y be amylase and/
Or prolease activity；
D, according to proteinase activity power, the Fermentation progress of amylase enzyme activity monitoring Medicated Leaven.
2. online quality control method according to claim 1, it is characterised in that: in chromatography conditions described in b step
Detection wavelength 210nm；Flow velocity 1.0mL/min；Column temperature: 30 DEG C.
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| CN109856274A (en) * | 2019-01-30 | 2019-06-07 | 广西壮族自治区食品药品检验所 | Open the discrimination method of Medicated Leaven ingredient in spleen ball and Qipi oral liquid |
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Non-Patent Citations (4)
| Title |
|---|
| HPLC测定六神曲中青蒿素、芦丁和槲皮素含量;谢彦博 等;《中国酿造》;20141231(第10期);摘要 * |
| 六神曲质量特征及发酵变化研究;练晶军 等;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20110915(第9期);第40页，第44页，第52-53页，第54-55页 * |
| 正交试验优化六神曲中淀粉酶和蛋白酶的提取工艺;孔杰娜 等;《山西医科大学学报》;20120531;第43卷(第5期);全文 * |
| 论六神曲中微生物、消化酶动态检测及整体性研究;王丽芳 等;《时珍国医国药》;20161231;第27卷(第8期);全文 * |
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