CN101785578B - Enzyme preparation using glucose oxidase as assistant for tobacco processing and preparation and application method - Google Patents
Enzyme preparation using glucose oxidase as assistant for tobacco processing and preparation and application method Download PDFInfo
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- CN101785578B CN101785578B CN201010039167.0A CN201010039167A CN101785578B CN 101785578 B CN101785578 B CN 101785578B CN 201010039167 A CN201010039167 A CN 201010039167A CN 101785578 B CN101785578 B CN 101785578B
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Abstract
An enzyme preparation using glucose oxidase as an assistant for tobacco processing and a preparation and application method belong to the enzyme preparation added before tobacco redrying to improve the tobacco grade. In the method, the known glucose oxidase source is decontaminated and refined, the activity of protease is inhibited by PMSF, the stock solution of 10,000-15,000U/mL is prepared by refining the obtained sediment with 0.0001% cane sugar oligomer liquid, the optimum prepared stock solution of 12,000U/mL becomes the activity additive of the tobacco, and when in use, the additive is diluted with water so as to replace original second conditioning water in strip redrying, the single indicator of the sugar content in tobacco can be well degraded, and the invention has the functions of effectively coordinating and balancing the change of various substances and improving the quality of the tobacco.
Description
Technical field
The invention belongs to the zymin of the raising tobacco grade of adding before tobacco redrying.
Background technology
Glucose oxidase is a kind ofly glucose catalyticing oxidation can be become to gluconic acid and H
2o
2the more general physiological enzyme of (hydrogen peroxide), is prevalent in animals and plants and microbe, can be obtained by microorganism fermentative production.Aspergillus niger (Asperillus niger) and some mould (Penicillium notatum) are research and the conventional bacterial classification of preparing glucose oxidase.Because aspergillus niger strain yield of enzyme is high, suitability for industrialized production adopts aspergillus niger more.As, utilize aspergillus niger, through fermentation, smudge cells, obtain glucose oxidase in born of the same parents, then through ultrafiltration stoste, ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, SephadexG-100 gel-filtration separation and purification [Wang Zhixin, etc.The extraction purifying of Glucose Oxidase from Aspergillus Niger A 9.Chinese food journal, 2007,7 (1): 64～68].This enzyme is comparatively common in Physiologic Studies, is widely used in measuring in clinical medicine the research of glucose.In the enzyme system of glucose analysis, apply more be glucose oxidase and peroxidase (POD) coupling dual-enzyme system [Jiang Xiuming, etc.Glucose oxidase-peroxidase coupling system glucose analysis.Zhengzhou Engineering College's journal, 2003,24 (2): 85～87.Zhang Ning, etc.Glucose oxidase---the evaluation of methodology of oxygen consumption rate method.Luzhou Medical College journal, 2002,25 (4): 327～328].Wang Zhi newly waits the zymologic property of research Glucose Oxidase from Aspergillus Niger A 9, the molecular weight of finding the enzyme of purifying gained is 138200Da, the molecular weight of subunit is 70210Da, is less than the molecular weight 150000Da left and right of the recombinant Aspergillus niger Glucose Oxidase of report, and subunit is respectively 80000.The optimal pH of zymoprotein approaches neutral, is different from the generally value between 5.6～6.0 of report, has wider pH stable range in pH40～8.0.Optimum temperuture is 30 ℃, and under 50 ℃ of conditions, activity remains unchanged substantially, also preserves nearly 50% enzyme activity at 60 ℃.Enzyme is not prevented by EDTA, SDS, and Cu
2+, Fe
2+show restraining effect a little, Ca
2+, Na
+, Mg
2+, Zn
2+deng enzyme is had certain activation [Wang Zhixin, etc.The zymologic property research of Glucose Oxidase from Aspergillus Niger A 9.Jouranl of Agricultural University of Hebei, 2006,29 (4): 69～72,83].Hu Changying etc. also study the main bacterium producing multi enzyme preparation of the Penicillium of malaga carbohydrate oxidase, and some mould HW2203 glucose oxidase enzymatic property and application characteristic, and unit of enzyme activity is defined as under 26 ℃, pH value 5.6 conditions, and per minute catalysis discharges a micromolar H
2o
2required enzyme amount.Enzyme activity determination adopts o-(two) methyl oxyaniline spectrophotometry, that is, under aerobic existence condition, the dehydrogenation of glucose oxidase enzyme catalysis glucose produces H
2o
2under peroxidase (POD) effect, oxygen donor o-(two) methyl oxyanilines (DH2) are oxidized to brown product.In 460 millimicrons of variations of locating to measure absorbancy [Hu Changying, etc.The mutagenic and breeding of some mould high yield glucose oxidase bacterial strain.Hebei Academy of Sciences journal, 2006,23 (3): 11～15; Hu Changying, etc.The research of some mould HW2203 glucose oxidase application characteristic.Biology magazine, 2008,25 (1): 31～34].Zhang Qian etc. study character and the separation and purification of notatin too, and the optimal pH of enzyme catalysis glucose oxidase reaction is 5.6, and optimum temperuture is 40 ℃, enzyme in pH5.5～8.5 interval and temperature stablize at lower than 50 ℃, Al
3+, Zn
2+to enzyme have certain activation [Zhang Qian, Fu Wanhui, Kang Jinghe, Chen Qingxi, etc.The separation and purification of notatin and property research.Xiamen University's journal (natural science edition), 2009,48 (1): 99～102].
Hebei province institute of microbiology, from the beginning of the eighties in last century, selects aspergillus niger strain research, prepares glucose oxidase essence enzyme, the application of supply Clinical Laboratory glucose content.Afterwards, again successively Application and Development in purposes such as beer, fruit juice fruit wine, flower characters improvement, sea-food and meat preservation, feed add, and set up glucose oxidase professional production workshop [Hu Changying, etc.The mutagenic and breeding of some mould high yield glucose oxidase bacterial strain.Hebei Academy of Sciences journal, 2006,23 (3): 11～15].It is aerobic dehydrogenase that Li Yan etc. are summarized as glucose oxidase, soluble in water, is insoluble to organic solvent.Industrial production is many to be extracted from aspergillus niger, is widely used in, in the industries such as food, medicine, feed, having played the effects such as removal glucose, deoxidation, sterilization.The purifying process that extracts glucose oxidase GOX is: cultivate aspergillus niger cell → filtrations reserved filtrate → be acidified to pH4.0～4.5 → ion exchange column separation → by the saturated throw out → crystallization → lyophilize of buffer solution elution → centrifugal → collect → obtain sterling.The method of measuring enzymic activity mainly contains: spectrophotometry, fluorescent method, electrochemical process etc.Influence factor in mensuration process mainly contains: temperature, pH value, oxygen-supplying amount etc.Highly purified glucose oxidase is light yellow crystal, soluble in water, is insoluble to ether, chloroform, glycerine etc.The maximum light absorption wavelength X max of glucose oxidase is 377nm and 455nm.Under UV-light, without fluorescence, still after heat, acid or alkaline purification, there is special green.The stable pH value scope of glucose oxidase is 3.5～6.5, and operative temperature is generally 30 ℃～60 ℃.Solid enzyme preparation is preserved and is at least stablized 2 years at 0 ℃, can stablize 8 years at-15 ℃.Glucose oxidase enzyme reaction mechanism conventionally with a redox enzyme system of catalase composition.Glucose oxidase can generate D-Glucose acid lactone by oxidizing glucose under molecular oxygen exists, simultaneously oxygen consumed Hydrogen Peroxide.Hydrogen peroxide endonuclease capable decompose to generate water and 1/2 oxygen by hydrogen peroxide, then water be combined with Gluconolactone again produce gluconic acid [Li Yan, etc.Glucose oxidase and application thereof.Food engineering, 2006, (3): 9～11].
The enzyme activity determination method of glucose oxidase has, with excessive NaOH neutralized reaction product gluconic acid, with HCl overtitration NaOH.It is 1 unit (U) [Guo Yong, chief editor that enzyme activity unit is defined as the enzyme amount that per minute catalysis glucose oxidase generates 1 μ mol gluconic acid.Enzyme engineering.Beijing: China Light Industry Press, 1994, p325～326].The principle of glucose oxidase enzymatic determination, according to being optionally by glucose oxidase, quantitatively produces gluconic acid and H
2o
2, utilize the quantitative assay of product further to weigh glucose oxidase enzyme activity, because H
2o
2under the catalysis of POD, quantitatively generate colored compound, the H that colorimetric estimation generates under certain wavelength with dianisidine or 4-AA-phenol or naphthyl alcohol-β-potassium sulfonate etc.
2o
2can be used for determine weighing glucose oxidase [Jiang Xiuming, etc.Glucose oxidase-peroxidase coupling system glucose analysis].Aforesaid assay method no matter, or adopt o-(two) methyl oxyaniline spectrophotometry, and other improve one's methods, and are all the methods of enzyme activity of weighing for the amount of product.
Summary of the invention
The present invention seeks to not change original technique of sheet cigarette modulation operations,, after fresh tobacco roasting, in the course of processing before redrying, utilize glucose oxidase zymin, reduce sugar content in tobacco and reach the effect that improves cigarette quality in suitability for industrialized production.
Another object of the present invention is to propose a kind of method of measuring enzyme activity in conjunction with glucose oxidase enzyme preparation technique, to meet stability and the reliability of suitability for industrialized production product.
Object of the present invention realizes in the following manner:
Using glucose oxidase as the auxiliary zymin of adding of tobacco processing, and said preparation glucose oxidase has the effect that oxidation-reduction catalysis reaction reduces sugar content in tobacco, and its preparation enzyme activity is 10000～15000U/mL.
The best enzyme activity of described preparation glucose oxidase is 12000U/mL.
The method of preparing zymin of the present invention:
Said preparation has the glucose oxidase of redox catalysis reaction, and its enzyme activity is 10000～15000U/mL.
The enzyme activity of described preparation glucose oxidase is 12000U/mL.
In described preparation, there is PMSF arrestin enzymic activity, avoid it to destroy glucose oxidase.
Prepare glucose oxidase as a method for the auxiliary enzymes preparation of tobacco processing, comprise the following steps:
(1) get glucose oxidase crude product solid or liquid and add that to contain concentration be that the damping fluid of 0.1mol/L proteinase inhibitor PMSF dissolves or dilution, glucose oxidase crude product is 1g: 20mL with the proportioning that contains PMSF damping fluid or is 1mL: 10mL, the centrifugal 20min of 3500r/min, get supernatant liquor, add (NH
4)
2sO
4, making ammonium sulfate final concentration is 55%, standing 24h, and then 4 ℃ of centrifugal 30min of 15000r/min, get supernatant liquor, add (the NH that concentration is 10% (W/V)
4)
2sO
4spend the night, then 4 ℃ of centrifugal 30min of 15000r/min, collecting precipitation, merging;
Above-mentioned glucose oxidase crude product is microbe-derived unpurified glucose oxidase:
(2) detect the enzyme activity of glucose oxidase in precipitation, add the sucrose oligopolymer liquid of concentration 0.0001% to be modulated to proenzyme liquid, making its enzyme activity is 10000～15000U/mL, and the best is 12000U/mL.
In the present invention, microbe-derived glucose oxidase raw product can be bought commercial goods enzyme product or obtain by corresponding malaga carbohydrate oxidase microorganism strains fermentative production.
More than for refining stoste, make the process of zymin of the present invention, Britton-Robinsion damping fluid used refers to [Yin Yongjia.University chemistry handbook.Jinan: Shandong Science Press, 1985] compound method.The method of improving and be modulated into pH5.6 with reference to [Liu Shiqing, etc.In agro-ecology environment, basal enzyme technical study method is built discussion.Agricultural research, 2008, (10): 162～166,175] use.
The present invention also provide a kind of substrate with catalyzed oxidizing reaction weigh glucose oxidase enzyme activity method, specifically:
With 3,5-dinitrosalicylic acid (DNS) colour developing, OD
540nmglucose quantitation mensuration is carried out in analysis;
The required enzyme amount of the oxidized minimizing 1 μ g glucose of per minute in 1.0000mg/mL concentration glucose standard substrate solution of take under 30 ℃ of temperature and pH5.6 condition is 1U, and enzyme sample is expressed as: U/mL or U/g.
Described measuring method is further: by needing sample product to be diluted to the extension rate of suitable enzymatic determination, add 0.3g Ca (NO in the every 100mL of damping fluid used
3)
2with 0.3g MgSO
4as basic enzyme, stable and activator strengthens enzymic activity, and calculates detected result in detection.
Glucose quantitation mensuration process refer to [Li Hesheng, etc.Plant physiology and biochemistry experimental principle and technology.Beijing: Higher Education Publishing House, 2000, p197～199; Liu Shiqing, etc.In agro-ecology environment, basal enzyme technical study method is built discussion.Agricultural research, 2008, (10): 162～166,175].
Apply above-mentioned glucose oxidase and as tobacco, process the method for auxiliary enzymes preparation:
The former liquid formulation of glucose oxidase is diluted to 250 times of waters of leaves moisting for the second time before the redrying of sheet cigarette, and its enzyme activity scope is 40～60U/mL.
Described application method is further that its best enzyme activity is 48U/mL using the water of leaves moisting for the second time before the redrying of sheet cigarette after concentrated glucose oxidase preparation dilution.
In above content, for commercially available commercial enzyme preparation, should select food grade enzyme source as far as possible, carry out refining and edulcoration, and needn't buy medical grade enzyme source.
The present invention has following positively effect:
When glucose oxidase preservation condition is suitable, itself just has good stability.In the quality control of product detects, the present invention measures with the DNS method of substrate glucose assays the glucose oxidase enzyme activity that method of cracking is weighed preparation.
Select its object of glucose oxidase of food grade to be to retain in the microbial enzyme system of source to the effective enzyme of sugar.
The present invention also can be used in conjunction with other tobacco processing active zymin, and conduct glucose oxidase preparation part wherein.Meanwhile, the present invention also can be used as one of enzyme component of applicant " a kind of cigarette quality improves and the tobacco processing active complex enzyme preparation improving ".
Accompanying drawing explanation
Fig. 1 is glucose oxidase preparation preparation technology schematic flow sheet of the present invention.
Below in conjunction with accompanying drawing and by embodiment, the present invention will be further described, but embodiment is not as the restriction to other embodiment of the present invention.
Embodiment
(1) preparation of preparation of the present invention and measuring method
Glucose oxidase can directly be bought the product of microorganism fermentation.Or utilize voluntarily corresponding malaga carbohydrate oxidase strain fermentation to produce, by breaking cell wall, discharge and collects the preliminary extraction of fermentation etc. and obtain raw product.Then, with the inventive method, refining, extract main is the enzyme that can reach food grade, and the destruction of protease inhibition to glucose oxidase.
Glucose oxidase is refined as crude product: be by glucose oxidase raw product solid with W/V=1g: 20mL or liquid W/V=1mL: 10mL ratio, add and contain damping fluid dissolving or the dilution that concentration is 0.1mol/L proteinase inhibitor PMSF, the centrifugal 20min of 3500r/min, get supernatant liquor, add (NH
4)
2sO
4, making ammonium sulfate final concentration is 55%, standing 24h, and then 4 ℃ of centrifugal 30min of 15000r/min, get supernatant liquor, add (the NH that concentration is 10% (W/V)
4)
2sO
4spend the night, then 4 ℃ of centrifugal 30min of 15000r/min, collecting precipitation, merging; Detect the enzyme activity of glucose oxidase, then with appropriate, be added with the proenzyme liquid that 0.0001% sucrose oligopolymer liquid is modulated to 10000～15000U/mL, the best is modulated to the proenzyme liquid of 12000U/mL.Finished product is generally the glucose oxidase preparation of enzyme activity 10000～15000U/mL.
Can further the proenzyme liquid of gained be obtained to concentrated compound enzymic preparation by concentrated 8～10 times of-8 ℃ of lyophilizes.
Above finished product glucose oxidase vigour-testing method: the glucose oxidase enzyme testing method that adopts the present invention to propose in summary of the invention is: with 3,5-dinitrosalicylic acid (DNS), develop the color, OD
540nmglucose quantitation mensuration is carried out in analysis; The required enzyme amount of the oxidized minimizing 1 μ g glucose of per minute in 1.0000mg/mL concentration glucose standard substrate solution of take under 30 ℃ of temperature and pH5.6 condition is 1U, and enzyme sample is expressed as: U/mL or U/g.
In mensuration process, needing sample product to be diluted to after the extension rate of suitable enzymatic determination, will in the every 100mL of damping fluid used, add 0.3g Ca (NO
3)
2with 0.3g MgSO
4stable and the activator as basic enzyme.
During use, glucose oxidase preparation is diluted to 250 times of waters of leaves moisting for the second time before the redrying of sheet cigarette, its glucose oxidase enzyme activity scope is 40～60U/mL.
The best enzyme activity of glucose oxidase is 48U/mL, therefore, usings 250 times of the concentrated glucose oxidase preparation dilutions of 12000U/mL as the water of leaves moisting for the second time before the redrying of sheet cigarette,
(2) the present invention simulates the effect of Tobacco Leaves cigarette redrying alcoholization
In laboratory condition Imitating Tobacco Leaves cigarette redrying alcoholization process, investigate the concentrated enzyme finished product of active additive to alcoholization (spontaneous fermentation) impact in early stage.The neutral glucose oxidase preparation of finished product preparation replaces leaves moisting water for the second time as auxiliary activity additive, to contrast with the tap water of same amount as leaves moisting water with tap water.Beat after leaf, redrying, each packing 500g tobacco leaf, each is processed and parallelly repeats to be no less than 3 times, and under the room temperature of relative humidity 65%, natural alcoholization is 60 days, makes respectively pipe tobacco and mixes, sampling analysis and be rolled into cigarette and smoke panel test.
Routine analysis result: to going 85 upper tobacco leaf total reducing sugars to reduce by 5% left and right, reducing sugar reduces by 3% left and right, to the total reducing sugar of middle part tobacco leaf and reducing sugar 2% left and right that all can decline, to the total reducing sugar of lower tobacco leaf and reducing sugar, decline suitable with reducing sugar rate of descent with the total reducing sugar of middle part tobacco leaf.Because also having caused, sugared variation the variation of other index components protein, without impact, has been caused to the larger variation of schmuck value especially.
Single use for glucose oxidase, from smoking result, by sugar is declined, not that expendable is degraded, but the conversion of oxidation-reduction reaction, although therefore few to the improvement scoring item of quality, but contrast generally quality of tobacco comparatively round who, exquisiteness, reflect and have certain proportionality action to coordinating material variation.
Claims (2)
1. prepare and using glucose oxidase as the method for the auxiliary zymin of tobacco processing, the enzyme activity of said preparation glucose oxidase is 12000U/mL, it is characterized in that the preparation method of this zymin comprises the following steps:
(1) get glucose oxidase crude product solid or liquid and add that to contain concentration be that the damping fluid of 0.1mol/L proteinase inhibitor PMSF dissolves or dilution, glucose oxidase crude product is 1g ︰ 20mL with the damping fluid proportioning that contains PMSF or is 1mL ︰ 10mL, the centrifugal 20min of 3500r/min, get supernatant liquor, add (NH
4)
2sO
4, making ammonium sulfate final concentration is 55%, after standing 24h, 4 ℃ of centrifugal 30min of 15000r/min, get supernatant liquor, add (the NH that concentration is 10% (W/V)
4)
2sO
4spend the night, then 4 ℃ of centrifugal 30min of 15000r/min, collecting precipitation, merging;
Above-mentioned glucose oxidase crude product is microbe-derived unpurified glucose oxidase:
(2) detect the enzyme activity of glucose oxidase in precipitation, add the sucrose oligopolymer liquid of concentration 0.0001% to be modulated to proenzyme liquid, making its enzyme activity is 12000U/mL;
The proenzyme liquid of described step (2) gained, obtains concentrated compound enzymic preparation through concentrated 8～10 times of-8 ℃ of lyophilizes.
2. right to use requires the glucose oxidase described in 1 as tobacco, to process the method for auxiliary enzymes preparation, it is characterized in that glucose oxidase stoste to dilute 250 times of waters of leaves moisting for the second time before the redrying of sheet cigarette, and its enzyme activity is 48U/mL.
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| CN101785578B true CN101785578B (en) | 2014-07-30 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102613695B (en) * | 2012-03-28 | 2014-02-26 | 华宝食用香精香料（上海）有限公司 | Method utilizing functional liquid to reduce content of carbohydrate materials in tobaccos |
| CN105647889A (en) * | 2016-02-25 | 2016-06-08 | 云南万芳生物技术有限公司 | Auxiliary enzyme preparation prepared from commercially available acidic xylanase and used for tobacco processing and application |
| CN106577763A (en) * | 2016-12-22 | 2017-04-26 | 四川省烟草公司达州市公司 | Conditioner capable of reducing proportion of conversion plants of burley tobacco group and method for treating tobacco plant by conditioner |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1066469A (en) * | 1991-05-09 | 1992-11-25 | 广西热带作物研究所 | A kind of basic protease of plant |
| CN101144073A (en) * | 2006-09-13 | 2008-03-19 | 谢勇 | Method for producing tobacco leaf fermenting enzyme preparation |
| CN101260387A (en) * | 2008-03-14 | 2008-09-10 | 云南万芳生物技术有限公司 | Tobacco leaf on-line alcoholized enzyme preparation and preparation method thereof |
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2010
- 2010-01-14 CN CN201010039167.0A patent/CN101785578B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1066469A (en) * | 1991-05-09 | 1992-11-25 | 广西热带作物研究所 | A kind of basic protease of plant |
| CN101144073A (en) * | 2006-09-13 | 2008-03-19 | 谢勇 | Method for producing tobacco leaf fermenting enzyme preparation |
| CN101260387A (en) * | 2008-03-14 | 2008-09-10 | 云南万芳生物技术有限公司 | Tobacco leaf on-line alcoholized enzyme preparation and preparation method thereof |
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