CN101254027A - Producing method of novel tobacco fermenting enzyme preparations - Google Patents
Producing method of novel tobacco fermenting enzyme preparations Download PDFInfo
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- CN101254027A CN101254027A CNA2007100853381A CN200710085338A CN101254027A CN 101254027 A CN101254027 A CN 101254027A CN A2007100853381 A CNA2007100853381 A CN A2007100853381A CN 200710085338 A CN200710085338 A CN 200710085338A CN 101254027 A CN101254027 A CN 101254027A
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Abstract
The invention relates to a novel production method of a tobacco-leaf fermentation enzymatic preparation, which aims at solving the technical problems how to improve tobacco-leaf fermentation quality and realize the activity protection of the tobacco-leaf fermentation enzymatic preparation in the tobacco-leaf spontaneous fermentation field. The enzymatic preparation is composed of glucose oxidase, chlorophyll oxidase, carotenoid oxidase, protease, and nicotine-degradation enzyme. Fresh tobacco leaves are processed through cell disruption, and processed through secondary fractional precipitation by (NH4)2SO4 to obtain crude enzyme liquid; enzyme molecules adopt Ca<2+> and Mg <2+> to act metal ion exchange to complete molecular modification; macromolecule combining modification is completed by adopting sucrose oligomer of 0.01 percent, so that the half-life of the enzymatic preparation can be prolonged, and the heat-resisting ability thereof can be increased markedly. Experimental results which are obtained by using the enzymatic preparation show that the nicotine is reduced 9.3 percent, total nitrogen is reduced 5.7 percent, protein is reduced 7.1 percent, smoke total particulate matter is reduced 8.4 percent, the quantity of the tar is reduced 5.1 percent, the nicotine content in smoke is reduced 28.0 percent, and carbon monoxide is reduced 1.6 percent. The enzymatic preparation is used in the secondary moistening process before threshing and re-drying of the tobacco leaves.
Description
Technical field
The tobacco natural fermentation.
Background technology
In the tobacco leaf modulated process, it is to improve and improve very important techniques link of quality of tobacco that the tobacco leaf after roasting and redrying is just fermented under suitable temperature and humidity condition fully.Therefore, the research to fermenting mechanism is the focus that relevant both at home and abroad scholar and production of cigarettes merchants pay close attention to for many years always.One of them important research field is exactly the explaination and the application of the enzyme mechanism of tobacco fermentation.(Frankenburg W.G.) just has the scholar to propose tobacco fermentation by enzymatic theoretical frame as far back as nineteen fifty.Along with research deeply and accumulation, generally believe that now the contained redox enzymes of tobacco leaf itself dominates fermentation, and this sweat is the symphyogenetic result of plurality of enzymes, thereby impels the effective conversion of substrate to multiple aroma substance composition.These enzymatic reaction processes mainly comprise the following aspects: 1, terpene compound degradation process.Carotenoid is a most important terpenoid compound in the tobacco leaf, and it is the precursor of more than the 50 kind of peculiar fragrance component of tobacco leaf; 2, Mailard reaction.This course of reaction is that reduced sugar and amino acid generate 56 kinds of heterocyclic compound-melanoids behind molecular rearrangement, and the formation of melanoid makes tobacco leaf show old cigarette fragrance.Although Mailard reaction itself is not by enzymatic reaction, its substrate-reduced sugar and amino acid are the products of enzymatic reaction.Protein is hydrolyzed into amino acid under the effect of protease, the hydrolysis under the effect of hydrolase of starch and sugar generates reduced sugars such as glucose, fructose.Therefore, promote the conversion of polysaccharide and protein will help the carrying out of Mailard reaction; 3, chlorophyllous degradation reaction.Remaining chlorophyll is cigarette products produces blue foreign smell in sucking process root in the tobacco leaf, should be controlled in modulated process.Although the tobacco leaf chlorophyll overwhelming majority is degraded in the tobacco flue-curing process, remaining chlorophyll still can bring harmful effect to cigarette products; 4, intense stimulus and bad pleasant impression are eliminated in free nicotine degraded; 5, acid amides and easily the deamination volatilization of cracked ammonium nitrogen compound.Tobacco leaf ammonia nitriding compound deaminate during the fermentation generates organic acid and ammonia, and the organic acid accumulation can improve ferment effect with the volatilization of ammonia nature.These five aspects are enzymatic reaction processes main in the tobacco fermentation process, and other also comprises the slow physics volatilization process of some small-molecule substances and less important tobacco leaf inclusion conversion reaction.Yet the drying and dehydrating of tobacco leaf in bake process makes tobacco leaf endoenzyme group overwhelming majority inactivation, therefore is unfavorable for the carrying out of above-mentioned enzymatic reaction.Based on these understanding, in the tobacco fermentation process, add selectivity enzyme system or compound enzyme and promote tobacco fermentation, acceleration tobacco leaf inclusion to transform and become an approach that increases tabacco fragrance, accelerated fermentation processes.
Zhang Lichang has been reported a kind of enzyme preparation of adding when being adapted at beating and double roasting, and the quality of natural alcoholization and artificial fermentation's tobacco leaf is improved significantly.This enzyme preparation can obviously increase cigarette flavor, alleviates blue foreign smell and excitant, improves flavor quality of cigarette.Zhao Ming admire to wait formulated tobacco fermentations such as the AMS that utilizes 4 kinds of biologically actives, protease to increase the matter agent to carry out tobacco fermentation, test shows that tabacco fragrance matter is improved, perfume quantity increases, the intrinsic assorted gas of tobacco leaf and excitant alleviate, main chemical compositions such as the sugar of tobacco leaf inside, nitrogen, alkali and ratio is tending towards coordinating, balance.Used enzyme is mainly carbohydrase, cellulase, amylase, protease in these research process.
Ruan Xiang surely waits the Chinese patent CN200510042773.7 of application to disclose a kind of complex enzyme formulation of being made up of multiple external source biology enzyme (cellulase, protease, pectase, amylase, carbohydrase) and dusty yeast, it is characterized in that multiple biology enzyme and dusty yeast directly carry out assembly according to specific ratio.Patent CN03114578.7 discloses the enzyme preparation of being made up of cellulase, protease, invertase, maltose, lysozyme.Patent CN87101362.2 discloses the microbial compound enzyme by the plurality of enzymes system of cellulase, pectase, ligninase and protease.
A common trait of above document and patent is directly to adopt external source (little) biology enzyme directly to carry out manual operations after special ratios mixes to use, and to how protective enzyme is active and how to satisfy the cigarette enterprise batch production requirement and be not described.
Summary of the invention
The present invention proposes a kind of preparation and production technology of tobacco fermenting enzyme preparations.The complex enzyme group of this enzyme preparation comes from new fresh tobacco leaf, obtains the complex enzyme formulation of being made up of glucose oxidase, chlorophyll oxidizing ferment, carotenoid oxidizing ferment, protease and nicotine degradation enzyme five fermentoids after tissue homogenate, enzyme group purifying, the modification of prothetic group displacer molecule and the protection of sucrose oligomer.Concrete production technology following (Fig. 1):
1, gets fresh and alive tobacco leaf or top wooden fork smudge cells, survey its volume, add buffer A (volume ratio V
Juice: V
Buffer solution=1: 9);
2, in 4 ℃, the centrifugal 20min of 2600g gets supernatant and surveys volume, slowly adds (NH
4)
2SO
4(144g/L) to containing 25% (NH
4)
2SO
4(W/V);
3,4 ℃ are slowly mixed 30min, 4 ℃ of centrifugal 40min of 15000g;
4, get supernatant and survey volume, add (NH4)
2SO
4(390g/L) to containing 80% (NH
4)
2SO
4(W/V);
5, slowly mix 2h or spend the night, 4 ℃ of centrifugal 2h of 15000g, collecting precipitation adds buffer A to fully dissolving of precipitation;
6, test solution adds buffer B in 4 ℃ of precipitation 20min by 1: 0.1 volume ratio, and the centrifugal 10min of 8000g obtains supernatant, abandons precipitation;
7, equal-volume adds 0.1%EDTA buffer solution (pH8.0), carries out the 10-20min chelating in 1-5 ℃, filters and abandons precipitation;
8, supernatant successively adds 0.3%Ca (NO by 1: 0.001 volume ratio
3) 24H
2O and MgSO
4Solution, abundant mixing leaves standstill 8-12hr in 1-5 ℃ of cold house or spends the night;
9, add 0.01% sucrose oligomer solution by 1: 0.01 volume ratio, leave standstill 10-15hr in 1-5 ℃;
10, solution example is concentrated into 1/8 o'clock of original volume in-8 ℃ of dryings and stops 4 ℃ of packing of thawing.
Buffer A
0.1M Tris-HCL，pH7.7，0.1M PMSF
Buffer B
0.1M Tris-HCL，pH7.7，0.1％EDTA(W/W)
The finished product enzyme preparation is diluted with water to 0.4% before using, two profits before beating and double roasting the time evenly spray consumption 200ml/50kg tobacco leaf.See Fig. 2.
Enzyme preparation total protein content and specific activity of enzyme are measured
The enzyme preparation product total protein content adopts the film-phenol determination method, is defined as the protein content (mg/100ml) in per 100 milliliters of enzyme preparations; The ratio vigor of five kinds of enzymes adopts AAS to carry out quantitatively.Specific activity of enzyme is defined as the unit quantity (U/ml) of enzyme in every milliliter of enzyme preparation.Measurement result table 1:
Table 1 enzyme preparation protein content and specific activity of enzyme
Molecular modification and oligomer protection are to the influence of enzymatic activity
Compare with existing tobacco fermenting enzyme preparations, have the certain line of anti-redrying hot properties through the novel tobacco fermenting enzyme preparations of molecular modification and oligomer protection, effectively the protective enzyme activity is greatly improved the enzyme preparation effect.
For comparing native enzyme (without Ca (NO
3) 24H
2O and MgSO
4Modify and protection with 0.01% sucrose oligomer) and modification enzyme vigor characteristic, the spy carries out following test:
Enzyme preparation product is accurately measured 1ml with after 9 times of the buffer B dilutions, contain centrifuge tube in 1.5ml, get native enzyme group's solution that 1～6 step obtained in the 1ml summary of the invention, contain centrifuge tube in 1.5ml, two pipes are temperature bath 15min in 75 ℃ of water-baths simultaneously, corresponding control sample is put-20 ℃ of refrigerators, 5 repetitions.Measure more different enzyme enzymes and live, the results are shown in Table 2.
Table 2 enzyme preparation modification enzyme and the contrast of native enzyme heat endurance
Table 2 shows, can significantly improve enzyme group's heat endurance after prothetic group displacement and sucrose oligomer are protected, and enzyme loss alive all is lower than 30%, has the demand that satisfies the tobacco fermentation suitability for industrialized production.
The characteristics of tobacco fermenting enzyme preparations and effect
This technical matters has satisfied the automation requirement of tobacco threshing and redrying line, and promptly the enzyme group is moistened back the maintenance of (75-80 ℃) enzymatic activity under the wet hot conditions two, thereby guarantees enzyme group performance catalytic action conscientiously in the tobacco fermentation process, accelerates substrate conversion.Therefore, this enzyme preparation can improve the availability and the security of tobacco leaf, increases the pure natural cigarette perfume (or spice) of tobacco leaf, shortens fermentation period, improves quality of tobacco.
Compare with existing enzyme preparation, have the characteristics of the line of anti-redrying hot properties, quickening substrate conversion, shortening spontaneous fermentation time, raising quality of tobacco with the novel tobacco fermenting enzyme preparations of present technique explained hereafter.Concrete manifestation is as follows:
1, high temperature resistant.Still can keep enzymatic activity even stand 80 ℃ of blade face temperature 10-15min;
2, use conveniently, satisfy the requirement of cigarette enterprise automation;
3, safe.All enzymes all come from new fresh tobacco leaf, absolute guarantee are arranged sucking in the security;
4, the remaining chlorophyll of degraded tobacco leaf is eliminated blue foreign smell;
5, the inner terpene compound of catalytic decomposition tobacco leaf for the generation of aroma substance provides precursor, reaches to strengthen the natural fragrance of tobacco leaf, the potential quality of performance tobacco leaf;
6, the acidization of accelerating fermentation of leaf tobacco by use.The general unit of pH value that reduces;
7, accelerate the volatilization of ammonia nitriding compound deamination, reduce the tobacco leaf total nitrogen content, reduce excitant, improve jealous;
8, promote free nicotine to decompose, improve the tobacco leaf edible safety, coordinate chemical constituent;
9, shorten spontaneous fermentation time 5-6 doubly.Conventional 2-3 shortens to 100-150 days summer, 150-200 days winter.
10, high repayment rate.Per kilogram tobacco leaf amount of application 4ml (0.4%), cost are roughly equal to 0.40 yuan, generally improve level of tobacco leaf grade, calculate 2.0 yuan of increments by market value, and profit margin is 80%.
The embodiment (see figure 3)
1, chooses the representational cloud 85 top junior tobacco leaf B2F in Hongta area, Yuxi;
2, according to enzyme preparation explanation preparation 0.4% solution, evenly spray B2F cigarette sample under the normal temperature condition, put 28 ℃ of temperature, 65% time alcoholization of relative humidity; (A)
3, simulation redrying process conditions (75 ± 5 ℃), the method by 1 is handled the cigarette sample, deposits in 28 ℃ (B1) and 38 ℃ (B2) respectively, refines under relative humidity 65% condition;
4, after 60 days to (A), (B1) and (B2) the cigarette sample carry out that specialty is smoked panel test, smoking machine analysis and conventional chemical analysis;
5, use the MATLAB statistical analysis software and carry out data analysis.
Sensory evaluating smoking result shows, (A) method is handled quality of tobacco had more obviously and improved, and shows as strength, excitant weakens, and fragrance matter, mouthfeel improve; (B) method mainly shows with (A) similar, note, fragrance matter is improved more obvious, stimulates and weakens to some extent.Three kinds of processing method difference are not obvious, illustrate that this enzyme preparation can keep the enzymatic activity (table 3,4,5) under the hot conditions preferably.
Table 3 processing with enzyme preparation cigarette sample smoking result
| Grade | Numbering | Note | Perfume quantity | Fragrance matter | Concentration | Stimulate | Strength | Assorted gas | Clean degree | Wettability | Aftertaste | Add up to |
| B2F | Contrast CKA1 | 7.67 | 12 | 12.33 | 7.5 | 12.58 | 4.5 | 7.33 | 7.33 | 3.33 | 3.58 | 78.15 |
| B2F | 7-A1 | 7.7 | 12.3 | 12.2 | 7.7 | 12.2 | 4.33 | 7.5 | 7.4 | 3.6 | 3.4 | 78.33 |
| B2F | Contrast CKB1 | 7.4 | 12.3 | 12.1 | 7.5 | 12.6 | 4.5 | 7.3 | 7.6 | 3.6 | 3.3 | 78.2 |
| B2F | 7-B1 | 7.5 | 12.4 | 12.4 | 7.6 | 12.8 | 4.4 | 7.5 | 7.3 | 3.9 | 3.7 | 79.5 |
| B2F | Contrast CKB2 | 7.5 | 12 | 11.8 | 7.7 | 12.5 | 4.7 | 7.5 | 7.7 | 3.8 | 3.7 | 78.9 |
| B2F | 7-B2 | 7.6 | 12 | 12.2 | 7.7 | 12.4 | 4.7 | 7.5 | 7.5 | 3.8 | 3.6 | 79 |
The smoking machine analysis result shows that the flue gas TPM has on average descended 8.4%, and tar content has descended 5.1%, and nicotine content in smoke has descended 28.0%, and carbon monoxide has descended 1.6%, very helps cigarette product reducing tar and reducing harm (table 4).
Table 4 smoking machine flue gas analysis result
| Grade | Numbering | Average weight (g/ props up) | Burn and suck a mouthful number (mouth) | TPM (mg) | Tar content (mg) | Flue gas alkali number (mg) | Carbon monoxide (mg) |
| B2F | Contrast CKA1 | 1.06 | 10.6 | 19.76 | 14.85 | 2.40 | 14.8 |
| B2F | 7-A1 | 1.04 | 10.7 | 18.20 | 14.30 | 1.86 | 14.5 |
| B2F | Contrast CKB1 | 1.04 | 11.2 | 19.96 | 15.0 | 2.32 | 14.9 |
| B2F | 7-B1 | 1.04 | 10.6 | 18.22 | 14.0 | 1.84 | 14.7 |
| B2F | Contrast CKB2 | 1.05 | 11 | 19.66 | 15.0 | 2.32 | 15.6 |
| B2F | 7-B2 | 1.02 | 11 | 18.38 | 14.4 | 1.80 | 15.4 |
Routine chemical components the analysis showed that nicotine descends 9.3%, and total nitrogen descends 5.7%, and protein descends 7.1%, and the chemical composition of tobacco leaf is coordinated (table 5).
Table 5 tobacco leaf conventional analysis partial results
| Grade | Numbering | The total reducing sugar amount | Reduced sugar | The nicotine amount | Nitrogen pool | Nicotine nitrogen | Protein | The nitrogen base ratio |
| B2F | Contrast CKA1 | 29.7 | 24.35 | 4.19 | 2.62 | 0.29 | 6 | 0.94 |
| B2F | 7-A1 | 24.54 | 22.77 | 3.72 | 2.46 | 0.24 | 5.62 | 0.99 |
| B2F | Contrast CKB1 | 29.18 | 26.37 | 4.23 | 2.56 | 0.31 | 5.9 | 0.92 |
| B2F | 7-B1 | 26.41 | 25.60 | 3.88 | 2.37 | 0.22 | 5.55 | 0.96 |
| B2F | Contrast CKB2 | 30.4 | 27.3 | 4.2 | 2.7 | 0.26 | 6.1 | 0.93 |
| B2F | 7-B2 | 28.1 | 24.2 | 3.95 | 2.63 | 0.2 | 5.64 | 0.96 |
Claims (4)
1. complex enzyme preparation special that is used for tobacco fermentation, this enzyme preparation is made up of glucose oxidase, chlorophyll oxidizing ferment, carotenoid oxidizing ferment, protease and nicotine degradation enzyme five fermentoids, the enzyme of its each component is lived than being: glucose oxidase 12354, chlorophyll oxidizing ferment 368, carotenoid oxidizing ferment 231, protease 3 6310, nicotine degradation enzyme 186; Its preparation method is as follows: choose new fresh tobacco leaf, and tissue homogenate, the enzyme group is through twice (NH
4)
2SO
4Obtain crude enzyme liquid behind the fractional precipitation purifying; This crude enzyme liquid carries out the enzyme metal ion-chelant, chelating condition pH8.0,1-5 ℃, chelating time 10-20min with the buffer solution that contains 0.1%EDTA; Use 0.3%Ca (NO then
3)
24H
2O and MgSO
4Carry out ion exchange, permutizer condition is pressed crude enzyme liquid: the reaction of 1: 0.001 volume ratio of substitutional solution, and 1-5 ℃, time swap 8-12hr or spend the night finishes the enzyme molecular modification; Modified enzyme molecule carries out the secondary modification protection with 0.01% sucrose oligomer under 1-5 ℃ of condition, the reaction time is at least more than the 10hr; Through handling like this, can obtain the described high temperature resistant complex enzyme preparation special that is used for tobacco fermentation.
2. the production method of the described complex enzyme formulation of claim 1, it is characterized in that: this enzyme preparation is made up of glucose oxidase, chlorophyll oxidizing ferment, carotenoid oxidizing ferment, protease and nicotine degradation enzyme five fermentoids, the enzyme of its each component is lived than being: glucose oxidase 12354, chlorophyll oxidizing ferment 368, carotenoid oxidizing ferment 231, protease 3 6310, nicotine degradation enzyme 186; Its preparation method is as follows: choose new fresh tobacco leaf, and tissue homogenate, the enzyme group is through twice (NH
4)
2SO
4Obtain crude enzyme liquid behind the fractional precipitation purifying; This crude enzyme liquid is carried out the enzyme metal ion-chelant, chelating condition pH8.0,1-5 ℃, chelating time 10-20min with the buffer solution that contains 0.1%EDTA; Use 0.3%Ca (NO then
3)
24H
2O and MgSO
4Carry out ion exchange, permutizer condition is pressed crude enzyme liquid: the reaction of 1: 0.001 volume ratio of substitutional solution, and 1-5 ℃, time swap 8-12hr or spend the night finishes the enzyme molecular modification; Modified enzyme molecule carries out the secondary modification protection with 0.01% sucrose oligomer under 1-5 ℃ of condition, the reaction time is at least more than the 10hr; Through handling like this, can obtain the described high temperature resistant complex enzyme preparation special that is used for tobacco fermentation.
3. the using method of the described complex enzyme formulation of claim 1 is, the finished product enzyme preparation evenly sprays consumption 200ml/50kg tobacco leaf when carrying out two profits before tobacco threshing and redrying after being 0.4% with distilled water diluting.
4. the purposes of the described complex enzyme formulation of claim 1 is characterized in that, the described complex enzyme formulation of claim 1 is applied to tobacco fermentation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102217794A (en) * | 2011-02-23 | 2011-10-19 | 红云红河烟草(集团)有限责任公司 | Tobacco leaf biochemical additive and preparation method and application thereof |
| CN101787362B (en) * | 2009-12-30 | 2012-05-30 | 云南万芳生物技术有限公司 | Enzyme preparation using protease as assistant for tobacco processing and preparation and application method |
| CN101781638B (en) * | 2010-01-14 | 2012-07-04 | 云南万芳生物技术有限公司 | Enzyme preparation using lipoxygenase as tobacco processing accessories, preparation method and application method |
-
2007
- 2007-03-01 CN CNA2007100853381A patent/CN101254027A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101787362B (en) * | 2009-12-30 | 2012-05-30 | 云南万芳生物技术有限公司 | Enzyme preparation using protease as assistant for tobacco processing and preparation and application method |
| CN101781638B (en) * | 2010-01-14 | 2012-07-04 | 云南万芳生物技术有限公司 | Enzyme preparation using lipoxygenase as tobacco processing accessories, preparation method and application method |
| CN102217794A (en) * | 2011-02-23 | 2011-10-19 | 红云红河烟草(集团)有限责任公司 | Tobacco leaf biochemical additive and preparation method and application thereof |
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