CN104745558A - Pectinase produced through mixed bacterium fermentation, and application thereof in tobacco sheet processing - Google Patents
Pectinase produced through mixed bacterium fermentation, and application thereof in tobacco sheet processing Download PDFInfo
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Abstract
The invention relates to the technical field of tobacco preparation, and in particular relates to a pectinase produced through mixed bacterium fermentation, and an application thereof in tobacco sheet processing. The pectinase is obtained through the steps of strain activation, seed liquid preparation, fermentation culture, enzyme solution crude extraction, and the like. The crude enzyme solution is diluted by 0-20 times, and the diluted crude enzyme solution is used for treating tobacco sheets by enzymolysis for 2-6h at a temperature of 40-50 DEG C according to a ratio that tobacco sheet to pectinase is 1:(1-5). An overall technical route of the application comprises the following steps: specific strains are mixed and fermented, wherein an optimal fermentation condition is obtained by screening and optimization, such that the crude enzyme solution with highest pectinase enzyme activity is obtained; the crude enzyme solution is used for treating tobacco sheets, such that pectin content in the tobacco sheets can be reduced, and a best degradation effect can be obtained. The maximal enzyme activity of the crude enzyme solution provided by the invention can be higher than 3200U/mL. When the crude enzyme solution is used in tobacco sheet treatment, the pectin content in the tobacco sheets can be reduced by approximately 1/4, and tobacco smoking quality can be well improved.
Description
Technical field
The invention belongs to tobacco preparing technical field, be specifically related to the polygalacturonase of a kind of mixed fungus fermentation production and the application in the tobacco sheet course of processing thereof.
Background technology
Polygalacturonase is a kind of prozyme be made up of various ingredients, and each component can work in coordination with the degraded of pectin, in food-processing, feed manufacturing, weaving, papermaking, environment protection etc., have purposes comparatively widely.The production of polygalacturonase has the history of more than 40 year, mainly contains solid state fermentation and solution fermentation two kinds of modes of production.Many microorganisms all have the ability producing polygalacturonase, and in prior art, polygalacturonase produces bacterium producing multi enzyme preparation mainly fungi, and comparatively extensive with aspergillus bacterium application.Generally speaking, solid state fermentation produces polygalacturonase enzyme and lives higher, but there is equipment amplifies difficulty, the defects such as level of automation is on the low side, and processing parameter is wayward; And although liquid fermenting enzyme is lived and be there is reduction to a certain degree, due to can continuous automatic production, and easily expand the scale of production, and fermenting process is easy to control, because becoming the main mode of production of polygalacturonase production.
The suitability for industrialized production of polygalacturonase starts from nineteen fifties, and there are Germany, France, Italy, Denmark, Holland, Japan etc. in major country of production.It is early stage that China originates in last century the eighties for the research of polygalacturonase, although in recent years achieve significant progress in fermentation strain seed selection, zymotechnique, application art improvement etc., but along with for high yield, high-quality polygalacturonase demand, existing polygalacturonase production technique still needs to optimize further.
In recent years, mixed fermentation with various bacterium technology obtains rapid progress.Generally speaking, multi-cultur es, as a biological mixed system, has the effect that growth metabolism is coordinated between each bacterial classification, and thus multiple bacteria compound fermentation has more advantages than single strain fermentation product enzyme.But due to complicacy, the diversity of microbial metabolites, add that competition, the restraining effect between different microorganisms fermentation is also intricate, thus often need inquire into different optimum fermentation condition combinations for different antimicrobial composition.
Tobacco sheets by paper making method (Paper-Process Reconstituted Tobacco) is a product growing tobacco resource regeneration.It utilizes some tobacco wastes, scrap stock, if offal, tobacco leaf fragment, offal etc. are raw material, through lixiviate, concentrated, separation, making beating, defibrination, manufacture paper with pulp, dry, perfuming process process, make tobacco sheet, as cigarette weighting material.Tobacco sheets by paper making method, not only there is advantage improving tobacco leaf product structural strength, combustionproperty and reduce in the smoking nicotine of cigarette and tar content etc., can also unit consumption of tobacco leaf be reduced simultaneously, save production cost, can be described as tobacco industry one of most important achievement in recent years.And in recent years, biotechnology and tobacco sheets by paper making method technology connected applications aborning, not only played the process advantage of tobacco sheets by paper making method, and utilize biotechnology to change tobacco components selectively, significantly improve the quality of tobacco sheet.
Research shows, the Mierocrystalline cellulose in tobacco material, xylogen, pectin and protein at high temperature all can produce arene compounds.Wherein pectin is a kind of colloidality material, is deposited on primary cell wall and middle lamella, and is cross-linked with each other with Mierocrystalline cellulose, hemicellulose, xylogen and some protein.Pectin is the objectionable impurities such as main methanol in pyrolytic process.The removal of pectin can not only reduce the generation of this kind of material, but also can change tobacco cell structure, and what allow cell become is looser ventilative, is conducive to improving result of combustion of tobacco ability, reduces the generation of pyrolysis.
In prior art, the degraded for pectic substance in tobacco sheet mainly adopts traditional polygalacturonase to carry out.But due to complicacy and the diversity of pectin structure in tobacco sheet, and polygalacturonase ubiquity of the prior art is strong for pectin degrading specific aim in tobacco sheet, degraded is abundant not, the unmanageable defect of degradation process, be thus badly in need of inquiring into new polygalacturonase and the application in tobacco sheet processing thereof.
Summary of the invention
The object of the invention is to provide a kind of polygalacturonase adopting specific bacterial strain mixed fungus fermentation to produce, and this polygalacturonase may be used for for the pectin in degrading tobacco thin slice in the tobacco sheet course of processing simultaneously, thus improves cigarette quality.
Technical scheme of the present invention is specific as follows.
The polygalacturonase that mixed fungus fermentation is produced, adopts following steps to obtain:
(1) actication of culture, by microbial strains respectively from picking storage medium, is inoculated in activation medium, cultivates;
Described microbial strains comprises: the preserving number of China General Microbiological culture presevation administrative center (CGMCC) preservation is the aspergillus niger (Aspergillus niger) of 3.3287, and the preserving number of Chinese industrial Culture Collection (CICC) preservation is the yeast saccharomyces cerevisiae of 1012;
The activation medium of described aspergillus niger is PDA substratum, adopts method of scoring inoculation, cultivate 3d during actication of culture;
The activation medium of described yeast saccharomyces cerevisiae is PDA substratum, adopts method of scoring inoculation, cultivate 2d during actication of culture;
(2) prepare seed liquor, the bacterial classification after activation in step (1) is inoculated in seed culture medium respectively, preparation seed liquor;
Preparing aspergillus niger seed liquor aspergillus niger seed culture used based formulas is: pectin 5g/L, yeast powder 10g/L, FeSO
40.1g/L, MgSO
40.5g/L, KH
2pO
41g/L, pH 6, liquid amount 100/ 250mL Erlenmeyer flask, 28 DEG C, 160 r/min shaking tables cultivation 48h;
Preparing yeast saccharomyces cerevisiae seed liquor yeast saccharomyces cerevisiae seed culture used based formulas is: glucose 5g/L, peptone 10g/L, yeast powder 10g/L, pH nature, liquid amount 100/ 250mL Erlenmeyer flask, 30 DEG C, 160 r/min shaking tables cultivate and can be used as seed liquor inoculation when 12h adularescents precipitation is formed and use;
(3) fermentation culture, by seed liquor mixing fermentation culture prepared in step (2), concrete steps are: be first inoculated in fermention medium by aspergillus niger seed liquor, and 28 DEG C, 160 r/min shaking tables cultivation 3.95 ~ 14.05h, preferably cultivate 12h; Then inoculation enters yeast saccharomyces cerevisiae seed liquor, 28 DEG C, 160 r/min shaking tables cultivation 36h;
Described fermentative medium formula is: pectin 23.18 ~ 56.82 g/L, yeast powder 0.98 ~ 6.02 g/L, 28 DEG C, pH5; Screening formulation is: pectin 50g/L, yeast powder 2.0g/L, 28 DEG C, pH5;
The inoculum size that the inoculation of aspergillus niger seed liquor enters fermention medium calculates by 4% volume fraction;
The inoculum size that the inoculation of yeast saccharomyces cerevisiae seed liquor enters fermention medium calculates by 3% volume fraction;
(4) slightly carry enzyme liquid, liquid fermentation liquid through suction filtration elimination thalline, then obtains crude enzyme liquid through ultrafiltration and concentration, measures after enzyme is lived and is polygalacturonase finished product provided by the present invention.
The application of polygalacturonase in the tobacco sheet course of processing that described mixed fungus fermentation is produced, after gained crude enzyme liquid dilution 0 ~ 20 times, by solid-liquid ratio, tobacco sheet: polygalacturonase=1g:1 ~ 5mL ratio, 40 ~ 50 DEG C, enzymolysis 2 ~ 6h; More excellent treatment condition are after crude enzyme liquid dilutes 10 times, by solid-liquid ratio, and tobacco sheet: polygalacturonase=1:3 ratio, 50 DEG C, enzymolysis 6h process.
General technical route of the present invention is: by specified strain first mixed fermentation, screening and optimizing optimal conditions of fermentation, obtain the crude enzyme liquid that most high fructose enzyme enzyme is lived, then crude enzyme liquid is directly used in the process of process tobacco sheet and reduces its polygalacturonase content, by optimization process technique, obtain best degradation effect, thus improve cigarette quality.
On a series of activities basis, the present invention adopts the enzyme work of specified strain mixed fermentation gained crude enzyme liquid to reach more than 3200U/mL, has good application potential.After being further used for tobacco sheet process, pectin content in tobacco sheet can be reduced nearly 1/4, degradation rate reaches 24.58%.After tobacco sheet after process is used for cigarette, further sense organ suction evaluation shows, be added with the cigarette sample of tobacco sheet after enzymolysis compared to untreated sample, cigarette flavor is finer and smoother, and the flue gas thoroughly property sent out is better, sweet sense strengthens to some extent and pungency alleviates, the astringent degree of mouthfeel reduces, and pleasant impression is pure comfortable, strength foot, and incendivity is better, thus improve the sucking quality of tobacco preferably.
Embodiment
Below in conjunction with embodiment the present invention will be further explained explanation.
embodiment 1
The present embodiment mainly produces the process of polygalacturonase for mixed fungus fermentation and the optimizing process of relevant substratum briefly introduces as follows.
The polygalacturonase that mixed fungus fermentation is produced, adopts following steps to obtain:
(1) actication of culture, by microbial strains respectively from picking storage medium, be inoculated in activation medium, cultivate;
Described microbial strains comprises: the preserving number of China General Microbiological culture presevation administrative center (CGMCC) preservation is the aspergillus niger (Aspergillus niger) of 3.3287, and the preserving number of Chinese industrial Culture Collection (CICC) preservation is the yeast saccharomyces cerevisiae of 1012; These two kinds of bacterial classifications are all bought from corresponding depositary institution and are obtained;
The activation medium of described aspergillus niger is PDA substratum (potato 300g/L, glucose 20g/L, agar 20g/L, pH nature, 121 DEG C of sterilizings), adopts method of scoring inoculation, cultivate 3d during actication of culture;
The activation medium of described yeast saccharomyces cerevisiae is PDA substratum, adopts method of scoring inoculation, cultivate 2d during actication of culture.
(2) seed liquor is prepared, the bacterial classification after activation in step (1) is inoculated in seed culture medium respectively, preparation seed liquor;
Preparing aspergillus niger seed liquor aspergillus niger seed culture used based formulas is: pectin 5g/L, yeast powder 10g/L, FeSO
40.1g/L, MgSO
40.5g/L, KH
2pO
41g/L, pH 6, liquid amount 100/ 250mL Erlenmeyer flask, 28 DEG C, 160 r/min shaking tables cultivation 48h;
Concrete steps are: get two pieces of 0.5cm with the position of punch tool near strain growth edge on activation medium flat board
2, the inoculation block that covers with newborn mycelia, be inoculated in aspergillus niger seed culture medium; Shaker fermentation adds the magnetic bead of sterilizing and appropriate granulated glass sphere after cultivating and terminating, and uses magnetic stirrer 2h, to make culture smash, is convenient to use for further fermentation culture amplification as seed liquor inoculation;
Preparing yeast saccharomyces cerevisiae seed liquor yeast saccharomyces cerevisiae seed culture used based formulas is: glucose 5g/L, peptone 10g/L, yeast powder 10g/L, pH nature, liquid amount 100/ 250mL Erlenmeyer flask, 30 DEG C, 160 r/min shaking tables cultivate and can be used as seed liquor inoculation when 12h adularescents precipitation is formed and use; Concrete operations, with reference to aspergillus niger seed liquor preparation process, also conveniently can be inoculated and operate, namely scrape a ring fresh yeast with transfering loop from activation medium flat board and be inoculated in yeast saccharomyces cerevisiae liquid seed culture medium.
(3) fermentation culture, by seed liquor mixing fermentation culture prepared in step (2), concrete steps are: be first inoculated in fermention medium by aspergillus niger seed liquor, 28 DEG C, 160 r/min shaking tables cultivation 12h; Then inoculation enters yeast saccharomyces cerevisiae seed liquor, 28 DEG C, 160 r/min shaking tables cultivation 36h;
Described fermentative medium formula is: pectin 50g/L, yeast powder 2.0g/L, pH5;
The inoculum size that the inoculation of aspergillus niger seed liquor enters fermention medium calculates by 4% volume fraction;
The inoculum size that the inoculation of yeast saccharomyces cerevisiae seed liquor enters fermention medium calculates by 3% volume fraction.
(4) enzyme liquid is slightly carried, liquid fermentation liquid through suction filtration elimination thalline, then obtains crude enzyme liquid through ultrafiltration and concentration, measures after enzyme is lived and is polygalacturonase finished product provided by the present invention.
fermentation culture conditions optimization is introduced
Because the formula of substratum in bacterial classification mixed fermentation process and different strain all have considerable influence for ferment effect turn-on time, for bacterial classification turn-on time, if namely access distillery yeast in the starting stage of fermentation, now black-koji mould bulk concentration is still lower, add the competition consumption of nutritive substance, aspergillus niger with the competition of distillery yeast in do not preponderate, thus the growth of aspergillus niger is suppressed, be unfavorable for the generation of polygalacturonase, the turn-on time thus for bacterial classification needs to optimize in detail.Under regard to part optimization factor in fermentation culture process procurement process briefly introduce as follows.
The optimization of fermentation culture conditions adopts center combination design (Central Composite Design) method, with major influence factors: (inoculum size is: the inoculum size that the inoculation of aspergillus niger seed liquor enters fermention medium calculates by 4% volume fraction for pectin content (A), yeast powder content (B), access yeast time (C); The inoculum size that the inoculation of yeast saccharomyces cerevisiae seed liquor enters fermention medium calculates by 3% volume fraction) be independent variable(s), pectinase activity (Y) designs for response value, adopt Desigan Expert 8.05b (Stat-Ease Inc, Minneapolis, USA) software completes relevant test design, data analysis and model foundation, wherein, d(axial point) be 1.682.
Center combination test variable and be horizontally disposed with as shown in the table.
Test-results is as shown in the table.
Carry out variance analysis by the variance analysis function in Design Expert 8.0.5b software to above-mentioned testing data, result is as shown in the table.
Carry out regression analysis to upper table data, the Quadratic response regression equation that each factor obtains after regression fit is: Y=2157.90+357.54*A-149.71*B+508.72*C+367.75*A*B+180.18*A * C-242.55*B*C.
Above-mentioned equation and related experiment result are analyzed, during mixed culture, enzyme is lived the most Gao Shikeda 3179.72U/mL of theoretical value, now corresponding culture medium prescription is: pectin 50g/L, yeast powder 3.0g/L, inoculation time is 12h, and the enzyme namely inoculating yeast saccharomyces cerevisiae after aspergillus niger cultivation 12h is lived the highest.
Again verify with this optimal conditions, namely culture medium prescription is: pectin 50g/L, yeast powder 3.0g/L, and inoculation time is 12h, and enzyme trial value of living is 3228.86U/mL, is similar to, shows that enzymic activity provided by the present invention can comparatively be stablized, reliably with theoretical value.
enzyme activity determination method
In the present invention, enzyme activity determination is with reference to Miller (1959) method.Be specially:
A. get 0.4mL l% pectin solution in test tube, add the 0.04mol/L Na of 1.0mL pH5
2hPO
4-0.02mol/L citrate buffer solution, 45 DEG C of water-bath balance 5min;
B. add the enzyme liquid that 0.1mL suitably dilutes, 45 DEG C of reaction 30min(blank groups replace with the enzyme liquid boiling inactivation);
C. 3.0mLDNS solution termination reaction is added, boiling water bath 5min;
D. cool, be settled to 15mL, under spectrophotometer 540 nm wavelength, survey absorbancy, obtain the amount of galacturonic acid in reaction system.
Enzyme activity unit: 1mL enzyme liquid 45 DEG C, under the condition of pH5.0, the enzyme amount of every min bottom exploded produce raw l μ g galacturonic acid is defined as 1 unit polygalacturonase (PG) enzyme and lives, and represents with U/mL.
Enzyme activity calculation formula: U=(C
experiment-C
contrast) * N*1000* (V
always/ V
enzyme)/t;
Wherein, N-enzyme liquid extension rate, V
always-reaction cumulative volume, V
enzyme-enzyme liquid amasss, t-reaction times (min).
The drafting of DNS method typical curve: the galacturonic acid standardized solution of configuration 1mg/mL.Get 1 mg/mL galacturonic acid solution 0,0.2,0.4,0.6,0.8,1.0 mL in test tube, moisturizing to 1 mL, adds DNS reagent 3 mL, boils 7 min after mixing in boiling water, be settled to 15mL after cooling, under spectrophotometer 540 nm wavelength, survey absorbancy.Take absorbancy as ordinate zou, galacturonic acid amount is X-coordinate, drawing standard curve.
embodiment 2
Crude enzyme liquid prepared in embodiment 1 is used for tobacco sheet degraded by the present embodiment, thus reduces the pectin content in tobacco sheet, and carries out sensory evaluation analysis further, briefly introduces as follows.
The application of polygalacturonase in the tobacco sheet course of processing that in embodiment 1, mixed fungus fermentation is produced, by (theoretical after gained crude enzyme liquid dilution 0 ~ 20 times, any enzyme live crude enzyme liquid all can be used for depolymerized pectin enzyme, the present embodiment is only for enzyme the highest crude enzyme liquid alive, i.e. enzyme 3228.86U/mL alive, inquire into the degradation effect of polygalacturonase and the improvement situation to cigarette quality), carry out biological degradation process by different feed liquid proportioning.Detailed process is:
Crude enzyme liquid is evenly sprayed on 10 g tobacco sheets with the consumption miniaturised nebuliser of different feed liquid ratio (ratio of tobacco sheet dry weight (g) and crude enzyme liquid volume (mL)), under 40 DEG C ~ 50 DEG C conditions, enzymolysis 2 ~ 6h; After enzymolysis terminates, process rapidly the baking oven that tobacco sheet afterwards puts into 70 DEG C to dry, made polygalacturonase inactivation, more repeatedly clean to remove soluble sugar with 75 DEG C of distilled water and (it should be noted that, actually do not need cleaning when preparing cigarette sample), measure the pectin content in processing sample; Under same operation conditions, to spray fermentoid liquid for control group, final calculating pectin degrading rate.
For obtaining best degradation rate, contriver adopts the method for orthogonal experimental design, selects L
9(3
4) orthogonal table is optimized test to pectin degrading condition (enzyme liquid extension rate, solid-liquid ratio, hydrolysis temperature, enzymolysis time), each influence factor design is as shown in the table.
Test-results under various combination condition and significance of difference analytical results as shown in the table.
Carry out analysis to upper table known, the impact of each factor on degradation rate is descending to be followed successively by: extension rate > solid-liquid ratio > reaction times > temperature of reaction.And the optimum combination of 4 kinds of factors to pectin degrading rate is A2B2C3D3, namely crude enzyme liquid dilute 10 times, solid-liquid ratio 1:3, temperature of reaction 50 DEG C, treatment time 6h.Test further with this understanding, result shows, in tobacco sheet, pectin degrading rate reaches maximum, and be 24.58%, pectin degrading effect is better.
In order specifically to evaluate after biological degradation process, tobacco sheet is for the improvement effect of smoking property of cigarette quality, and contriver, for the tobacco sheet of optimum enzymolysis condition process, has carried out tobacco sucking evaluation further.Briefly introduce as follows.
Tobacco sheet after optimum enzymolysis condition process is suitably dried under 80 DEG C of conditions (to humidity 65%) chopping afterwards, be placed in climatic chamber inner equilibrium (relative humidity (60 ± 5) %, temperature (22 ± 2) DEG C) 48 h.Namely take 0.12 g sample by pipe tobacco weight 15%() with balance after 85% the Homogeneous phase mixing that cuts filler after manual cigarette, this is test group.The cigarette prepared by tobacco sheet of inactivator process is adopted to be control group under the same terms, with the cigarette prepared by the tobacco sheet of distilled water process for blank group.
(often prop up a cigarette gross weight 0.80 ± 0.01 g) by by each group of cigarette sample prepared by Standard, quality is carried out to cigarette to smoke panel test qualification and evaluating according to the national existing tobacco product standard GB5606.4-2005 that smokes panel test balance 24 h in the climatic chamber of temperature (22 ± 2) DEG C, relative humidity (60 ± 5) % after.
Concrete evaluation result is as shown in the table.
As can be seen from above-mentioned evaluation result, tobacco sheet after enzymolysis processing add with cigarette after, the fragrance of cigarette can be made finer and smoother, the flue gas thoroughly property sent out is better, can increase sweet sense and abirritate simultaneously, and the astringent degree of mouthfeel reduces, pleasant impression is pure comfortable, strength foot, and incendivity is better, the sucking quality of cigarette obtains better improvement.
the method of calculation of pectin content and degradation rate
Adopt colorimetry of carbazole to measure for the pectin content in tobacco sheet in the present invention, determination step is as follows:
A. sample pretreatment, gets testing sample (after enzymolysis processing or untreated tobacco sheet), grinds, cross 250 mesh sieves, take 10g(and be accurate to 0.001g) sample in 250mL Erlenmeyer flask, add the HCl solution of 0.05mol/L that 150mL is heated to seethe with excitement, load onto condenser, reflux 1h in 90 DEG C of water-baths, after cooling, filtrate is moved in 250mL volumetric flask, the NaOH adding 2mol/L is neutralized to pH=4, shakes up, collect filtrate and namely obtain total pectin extracting soln, for subsequent use;
B. be standard substance with galacturonic acid, colorimetry of carbazole measures pectin content, gets the total pectin extracting soln 1mL of gained in steps A in the test tube containing the 6mL vitriol oil, shakes up; 85 DEG C of heating in water bath 15min; Cooling, adds 0.15% carbazole ethanol solution 0.3mL, shakes up, secretly put 30min; Absorbancy is surveyed under 530nm;
C. colorimetry of carbazole typical curve and formula, tries to achieve liquid pectin content to be measured;
Described formula is: pectin (%)=C × V × (W × 10, K × 100/
6);
Wherein, C: the pectin content (μ g/mL) of the pectin extraction diluent that reference standard curve is tried to achieve, V: pectin extracting soln stoste volume (mL), K: pectin extracting soln extension rate, W: sample quality (g), 10
6: mass unit reduction factor;
The method for drafting of described colorimetry of carbazole typical curve is: first accurately take 0.1000g galacturonic acid standard substance in beaker, and being transferred in 100mL volumetric flask after dissolving with a small amount of distilled water, is the hydrochloric acid constant volume of 3 with pH; Then get 0 respectively, 1,2,3,4,5,6,7mL in 8 100 volumetric flasks, be the HCl constant volume of 3 with pH, obtain the standardized solution that concentration is 0,10,20,30,40,50,60,70 μ g/mL; Finally measure with aforesaid method, then draw the typical curve of colorimetry of carbazole.
In the present invention, pectin degrading rate calculates as follows:
E
i=（C
0－C
i）/C
0×100%，
Wherein, E
i: the degradation rate of pectin, C
0: the amount of the galacturonic acid that hydrolyzed pectin generates in blank sample; C
i: the amount of the galacturonic acid that hydrolyzed pectin generates in the tobacco sheet of ferment treatment; In the tobacco sheet of blank sample and ferment treatment, pectin content measures all to measure by above-mentioned colorimetry of carbazole and carries out.
Claims (7)
1. a polygalacturonase for mixed fungus fermentation production, is characterized in that, this polygalacturonase adopts following steps to obtain:
(1) actication of culture, by microbial strains respectively from picking storage medium, is inoculated in activation medium, cultivates;
Described microbial strains comprises: the preserving number of China General Microbiological culture presevation administrative center preservation is the aspergillus niger of 3.3287, and the preserving number of Chinese industrial Culture Collection preservation is the yeast saccharomyces cerevisiae of 1012;
(2) prepare seed liquor, the bacterial classification after activation in step (1) is inoculated in seed culture medium respectively, preparation seed liquor;
(3) fermentation culture, by seed liquor mixing fermentation culture prepared in step (2), concrete steps are: be first inoculated in fermention medium by aspergillus niger seed liquor, 28 DEG C, 160 r/min shaking tables cultivation 3.95 ~ 14.05h; Then inoculation enters yeast saccharomyces cerevisiae seed liquor, 28 DEG C, 160 r/min shaking tables cultivation 36h;
(4) slightly carry enzyme liquid, liquid fermentation liquid through suction filtration elimination thalline, then obtains crude enzyme liquid through ultrafiltration and concentration, measures after enzyme is lived and is polygalacturonase finished product provided by the present invention.
2. the polygalacturonase of mixed fungus fermentation production as claimed in claim 1, it is characterized in that, described in step (1), activation medium is PDA substratum, adopts method of scoring inoculation, cultivate 3d during aspergillus niger strain activation; Adopt method of scoring inoculation during the activation of yeast saccharomyces cerevisiae bacterial classification, cultivate 2d.
3. the polygalacturonase of mixed fungus fermentation production as claimed in claim 1, it is characterized in that, preparing aspergillus niger seed liquor aspergillus niger seed culture used based formulas in step (2) is: pectin 5g/L, yeast powder 10g/L, FeSO
40.1g/L, MgSO
40.5g/L, KH
2pO
41g/L, pH 6, liquid amount 100/ 250mL Erlenmeyer flask, 28 DEG C, 160 r/min shaking tables cultivation 48h;
Preparing yeast saccharomyces cerevisiae seed liquor yeast saccharomyces cerevisiae seed culture used based formulas is: glucose 5g/L, peptone 10g/L, yeast powder 10g/L, pH nature, liquid amount 100/ 250mL Erlenmeyer flask, 30 DEG C, 160 r/min shaking tables cultivate and can be used as seed liquor inoculation when 12h adularescents precipitation is formed and use.
4. the polygalacturonase of mixed fungus fermentation production as claimed in claim 1, it is characterized in that, described fermentative medium formula is: pectin 23.18 ~ 56.82 g/L, yeast powder 0.98 ~ 6.02 g/L, pH5.
5. the polygalacturonase of mixed fungus fermentation production as claimed in claim 4, it is characterized in that, described fermentative medium formula is: pectin 50g/L, yeast powder 2.0g/L; Inoculate after fermentation of Aspergillus niger 12h and enter yeast saccharomyces cerevisiae; The inoculum size that the inoculation of aspergillus niger seed liquor enters fermention medium calculates by 4% volume fraction; The inoculum size that the inoculation of yeast saccharomyces cerevisiae seed liquor enters fermention medium calculates by 3% volume fraction.
6. the application of polygalacturonase in the tobacco sheet course of processing of mixed fungus fermentation production described in claim 1, is characterized in that, after gained crude enzyme liquid dilution 0 ~ 20 times, by solid-liquid ratio, i.e. and tobacco sheet: the ratio of polygalacturonase=1:1 ~ 5,40 ~ 50 DEG C, enzymolysis 2 ~ 6h.
7. the application of polygalacturonase in the tobacco sheet course of processing as claimed in claim 6, is characterized in that, after crude enzyme liquid dilutes 10 times, by solid-liquid ratio, i.e. and tobacco sheet: the ratio of polygalacturonase=1:3,50 DEG C, enzymolysis 6h process.
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| CN107129975A (en) * | 2017-05-26 | 2017-09-05 | 河南中烟工业有限责任公司 | A kind of microcapsules pectase and its application in reconstituted tobacoo process |
| CN107177571A (en) * | 2017-05-26 | 2017-09-19 | 河南中烟工业有限责任公司 | Degradation treatment method while pectin in a kind of reconstituted tobacoo process |
| CN107177570A (en) * | 2017-05-26 | 2017-09-19 | 河南中烟工业有限责任公司 | A kind of two step edman degradation Edmans for being directed to pectin in reconstituted tobacoo |
| CN107224003A (en) * | 2017-05-26 | 2017-10-03 | 河南中烟工业有限责任公司 | A kind of enzymolysis alcoholization method for improving reconstituted tobacoo quality |
| CN107338232A (en) * | 2017-05-26 | 2017-11-10 | 河南中烟工业有限责任公司 | A kind of method of three-step approach degrading tobacco thin slice pectin content |
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| CN109984371A (en) * | 2019-05-10 | 2019-07-09 | 南宁雄晋生物科技有限公司 | A kind of biological method for alcoholizing of tobacco leaf |
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| CN107224003A (en) * | 2017-05-26 | 2017-10-03 | 河南中烟工业有限责任公司 | A kind of enzymolysis alcoholization method for improving reconstituted tobacoo quality |
| CN107338232A (en) * | 2017-05-26 | 2017-11-10 | 河南中烟工业有限责任公司 | A kind of method of three-step approach degrading tobacco thin slice pectin content |
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| CN110122920A (en) * | 2019-03-22 | 2019-08-16 | 河南中烟工业有限责任公司 | A method of utilizing microbial flora fermentation process cigar tobacco |
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Address after: 450000 Yulin South Road, Henan, Zheng Dong, No. 16 South Road, Zhengzhou Applicant after: China Tobacco Henan Industrial Co., Ltd. Applicant after: Zhengzhou University of Light Industry Address before: 450000 Zhengzhou agricultural road, Henan, No. 29 Applicant before: China Tobacco Henan Industrial Co., Ltd. Applicant before: Zhengzhou University of Light Industry |
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Application publication date: 20150701 |