CN102217794B - Tobacco leaf biochemical additive and preparation method and application thereof - Google Patents
Tobacco leaf biochemical additive and preparation method and application thereof Download PDFInfo
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- CN102217794B CN102217794B CN 201110042983 CN201110042983A CN102217794B CN 102217794 B CN102217794 B CN 102217794B CN 201110042983 CN201110042983 CN 201110042983 CN 201110042983 A CN201110042983 A CN 201110042983A CN 102217794 B CN102217794 B CN 102217794B
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Abstract
The invention discloses a tobacco leaf biochemical additive (Van-1) and a preparation method and an application thereof, belonging to the technical field of additives for cigarettes. A strain used in the tobacco leaf biochemical additive (Van-1) is classified and named as (Bacilluspumilus) Van35; and the collection number is CGMCC No. 3411. After the biochemical additive Van-1 is used for processing tobacco leaves for 8 months, the changing of the content of macromolecular compounds in the tobacco leaves can be effectively promoted, for example, the content of total sugar and reducing sugar is increased and the content of total nitrogen, nicotine, protein and cellulose is reduced; simultaneously, the content of aroma components in the tobacco leaves is improved and the quality of the tobacco leaves is close to that of a tobacco leaf sample which is naturally alcoholized for 18-24 months. The biochemical additive Van-1 is conductive to transformation among chemical components in the tobacco leaves and formation and accumulation of the aroma components and can effectively improve the alcoholization effect of the tobacco leaves and shorten the alcoholization time.
Description
Technical field
The invention belongs to the technical field of cigarette additive, more particularly, the present invention relates to a kind of biochemical additive.Simultaneously, the invention still further relates to the preparation method of described additive and the application in tobacco industry.
Background technology
Tobacco is a kind of to suck the special economic crop as purpose, and its use value depends on its quality.Perfume is jealous to be the important indicator of estimating quality of tobacco, and sound tobacco requires cigarette fragrance matter pure, and perfume quantity is sufficient, and jealous pure and mild, pleasant impression is comfortable.Tobacco leaf rolls into the system engineering that cigarette product is a complexity from planting to, during need to experience the series of steps such as beating and double roasting, tobacco mellowing.Wherein, beating and double roasting is to solve the basic skills that has defective on the results quality of tobacco, first roasting the dress cigarette (also claiming former cigarette) that is about to purchase at the cigarette station transported to the redrying workshop and carried out the baking second time, the full wafer cigarette is made into the fragment cigarette of certain specification, desinsection in the beating and double roasting process promotes blue or green cigarette to turn yellow, abirritate and assorted gas, and the control moisture content in leaves prevents tobacco leaf infested going mouldy in the storage process in 11%～13% scope.Fragment leaf in the prior art behind the beating and double roasting need to be deposited in the tobacco mellowing warehouse after packing through machinery, stores under field conditions (factors) 2～3 years, improves the suction quality of tobacco leaf by natural alcoholization.This natural alcoholization method can suppress to be attached to the beneficial microorganism growth of tobacco leaf surface because tobacco leaf is too dry, and required tobacco mellowing overlong time has increased warehouse cost.
Although can because the kind of external source biology enzyme is too single, cause the kind of fragrance material to reduce by adding the external source biology enzyme to shorten the alcoholization time, fragrance be felt naturally, stereovision weakens to some extent.Simultaneously, the mode of production of this interpolation external source biology enzyme natural alcoholization, cost artificial and enzyme preparation is higher, and all so that existing production method excessive cycle need to take substantial contribution and storehouse, production cost is higher for these.How to overcome the problems referred to above, there is no desirable solution in the prior art.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, a kind of new biochemical additive is provided.
Another object of the present invention provides the preparation method of described biochemical additive.
Further aim of the present invention provides the application of described biochemical additive in tobacco mellowing.
Purpose of the present invention is achieved by following technical proposals.
* except as otherwise noted, the percentage that adopts among the present invention is mass percent.
A. the invention provides a kind of biochemical additive, this additive be by to bacillus pumilus (
Bacillus pumilus) tunning of Van35 extracts.
The present invention is separated to a strain bacterium Van35 from the flue-cured tobacco blade face, qualification result shows that this bacterium belongs to bacillus pumilus, this bacterial strain called after (
Bacillus pumilus) Van35.This bacterial strain has been kept at China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCG); This centre address: No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city; Preservation date: on November 5th, 2009; Preserving number is CGMCC No. 3411.
Morphology and the Physiology and biochemistry nature and characteristic of bacillus pumilus Van35 bacterial strain are: thalline is shaft-like, and Gram-positive is given birth in the gemma ellipse or end is given birth to, and does not expand, and gemma is present in the thalline than thalline is little.Bacterium colony is rounded, and is flat without projection, milky, and glossy, the edge is irregular.Conventional Physiology and biochemistry experiment shows: ND12 is aerobic bacteria, the catalase experiment, and the H2S experiment is produced in the VP experiment, and gelatin liquefaction experiment and lecithinase experiment are once positive; The NH3 experiment is produced in the hydrolyzed starch experiment, the methyl red experiment, and the indoles experiment, hydrolyzed fat experiment and nitrate reduction experiment are negative.
The Molecular Identification of bacillus pumilus Van35 is characterized as: utilize universal primer 20F(5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1500R(5 '-GGTTACCTTGTTACGA CT-3 '), obtain PCR product about 1500 bp with the genomic DNA of bacillus pumilus Van35 as template amplification, reclaim part 16S rRNA gene order 1510 bp of the Van35 bacterial strain that obtains after order-checking, the correction, this sequence has been submitted international nucleotide sequence database (GenBank), and sequence index number is: GU290547.
B. the invention provides a kind of preparation method of tobacco leaf biochemical additive, the method adopts following step:
(1) with bacillus pumilus (
B. pumilus) Van35 enlarges fermented and cultured;
50 mL liquid seed culture mediums are sub-packed in the 250 mL triangular flasks, and inoculating strain Van35 cultivates 24 h in 30oC vibration (180 r/min), obtains liquid seeds.The liquid seeds of inoculation 8% is cultivated 24 h in 30oC vibration (180 r/min) in the 1000 mL triangular flasks that 240 mL liquid seed culture mediums are housed, obtain the liquid seeds of cultivating for enlarging.
(2) by filtering or centrifugal collection zymotic fluid;
Then, sterile working is inoculated into 8% liquid seeds in the 100 L fermentation tanks that the liquid culture medium of 60 L is housed, and puts tank in 36 hours in the 30oC static culture.Centrifugal (8000 rpm, 20 min) collect zymotic fluid.
(3) by the protein in the ammonium sulfate precipitation zymotic fluid, centrifugal collecting precipitation;
According to fermentating liquid volume add ammonium sulfate (56%, w/v), stir 2 h, again centrifugal (8000 rpm, 20 min) collecting precipitation
(4) ammonium sulfate is removed in dialysis, namely gets required biochemical additive.
Precipitation is dissolved in the PH7.0 phosphate buffer of 600 ml, ammonium sulfate is removed in dialysis, obtains the Van35 extractive from fermentative, and this extract is biochemical additive Van-1.
C. the invention provides described biochemical additive as the application of tobacco mellowing additive.
Compared with prior art, the present invention has following beneficial effect.
(1) the present invention makes the alcoholization time of tobacco leaf than natural alcoholization significantly shortening arranged, and quality of tobacco is significantly improved.Can effectively promote the change of macromolecular compound content in the tobacco leaf, for example, total reducing sugar and content of reducing sugar raise.And total nitrogen, nicotine, protein and cellulosic content descend to some extent; Simultaneously, the content of the aroma component in the tobacco leaf improves,
(2) shortening of tobacco mellowing time helps tobacco enterprise saving storage room and time, has improved the utilization rate in alcoholization workshop, is conducive to the accelerated development of tobacco enterprise.
(3) biochemical additive provided by the invention is the important breakthrough to conventional art, can effectively improve the alcoholization process of tobacco leaf, and the quality of tobacco leaf is significantly improved, and simultaneously, has increased the utilization rate of tobacco leaf.
(4) simple, the operability of technique of the present invention, standardization and controllability are strong, and raw material is easy to get, and cost is low, is convenient to realize large-scale industrial production, has good development prospect.
Description of drawings
Fig. 1 is the process chart that the present invention prepares bacterial strain Van35 extractive from fermentative.
The specific embodiment
Example only in order to explain the present invention, is not intended to limit the present invention.
---the preparation of culture medium
1) the slant preservation culture medium (grams per liter, g/v): tryptone 10, beef extract 3, NaCl 5, agar 15 is used 20% Na
2CO
3Regulate pH 8.5,121oC 20 min that sterilize;
2) liquid seed culture medium (grams per liter, g/v): tryptone 10, beef extract 3, NaCl 5, use 20% Na
2CO
3Regulate pH 8.5,121oC 20 min that sterilize;
3) liquid culture medium (%): wheat bran 1.5, soy peptone 1.5, casein 1.0, CaCl
20. 1%, K
2HPO
40. 1 %, KH
2PO
40.125%, MgSO
47H
2O, 0.05%, FeSO
40.0004%, pH 8.0.121oC 20 min that sterilize.
---the preparation of bacterial strain Van35 extractive from fermentative
As shown in Figure 1, mainly comprise the steps such as seed culture, fermentation, centrifugal, ammonium sulfate precipitation.
Concrete flow process is as follows: first 50 mL liquid seed culture mediums are sub-packed in the 250 mL triangular flasks, inoculating strain Van35 cultivates 24 h in 30oC vibration (180 r/min), obtains liquid seeds.The liquid seeds of inoculation 8% is in the 1000 mL triangular flasks that 240 mL liquid seed culture mediums are housed, cultivate 24 h in 30oC vibration (180 r/min), obtain for the liquid seeds that enlarges cultivation. then, sterile working is inoculated into 8% liquid seeds in the 100 L fermentation tanks that the liquid culture medium of 60 L is housed, and puts tank in 36 hours in the 30oC static culture.Centrifugal (8000 rpm, 20 min) collect zymotic fluid, according to fermentating liquid volume add ammonium sulfate (56%, w/v), stir 2 h, centrifugal (8000 rpm again, 20 min) collecting precipitation is dissolved in precipitation in the PH7.0 phosphate buffer of 600 ml, and ammonium sulfate is removed in dialysis, obtain the Van35 extractive from fermentative, this extract is biochemical additive Van-1.Biochemical additive Van-1 contains multiple hydrolase after testing, comprises the plurality of enzymes such as amylase, carbohydrase, cellulase, hemicellulase, pectase, protease.
Embodiment 3
---product feature and physicochemical property
This product is the transparent supernatant liquid of light brown, and the physicochemical property of product is shown in Table 1.
The physicochemical property of table 1. biochemical additive
Embodiment 4
---the tobacco mellowing experiment
Prepare biochemical additive, the additive of getting suitable volumes (5%～10%) evenly is sprayed at 100g tobacco leaf surface (control moisture content in leaves content is 12-13%), mixes as treatment samples, alcoholization workshop alcoholization 8 months.The deionized water of directly spraying same amount is at 100g tobacco leaf surface (control moisture content in leaves content is 12-13%) sample in contrast.Carrying out 5 times repeats.
Embodiment 5
Biochemical additive Van-1 processed tobacco leaf after 8 months, can effectively promote the change of macromolecular compound content in the tobacco leaf, raise such as total reducing sugar and content of reducing sugar, total nitrogen, nicotine, protein and cellulosic content descend to some extent, simultaneously, the content of the aroma component in the tobacco leaf improves, and the quality of tobacco leaf and natural alcoholization 18-24 month tobacco sample approaches.Illustrate that biochemical additive processes formation and the accumulation help the conversion between the chemical composition and aroma component in the tobacco leaf, can effectively improve the alcoholization effect of tobacco leaf, shorten the alcoholization time.
Embodiment 6
The preparation method of biochemical additive Van-1 is with embodiment 1, the additive of getting suitable volumes (5%-10%) evenly is sprayed at the red large B4F tobacco leaf surface of 100g (control moisture content in leaves content is 12-13%), mix as treatment samples, alcoholization workshop alcoholization 8 months.The deionized water of directly spraying same amount is at 100g tobacco leaf surface (control moisture content in leaves content is 12-13%) sample in contrast.Select simultaneously 24 months red large B4F tobacco leaf of natural alcoholization in contrast, the routine chemical components of test sample and aroma component are further estimated the effect that biochemical additive is processed the cigarette sample by the sensory evaluating smoking simultaneously.
The testing result of routine chemical components shows: after red large B4F tobacco leaf was processed 8 months through biochemical additive, larger change has occured in the content of the large biological molecule compound of tobacco leaf, wherein obvious raising has occured in the content of reduced sugar and total reducing sugar, decline has occured in the content of total nitrogen, cellulose and nicotine, volatilization alkali and ligroin extraction total amount descend to some extent, the variation of the content of these compounds is conducive to the sucking quality of tobacco leaf, and the content of the chemical composition of the tobacco leaf of process biochemical additive processing and 24 months tobacco leaves of natural alcoholization approaches simultaneously.In addition, utilize GC-MS that the aroma component of tobacco sample is detected, the result shows, after red large B4F tobacco leaf process natural alcoholization or biochemical additive are processed, the increase that mostly occurred greatly of the aroma component of tobacco leaf, the aroma component total amount of the tobacco sample of simultaneously biochemical additive processing increases manyly than tobacco natural alcoholization, illustrates that the biochemical additive processing helps formation and the accumulation of aroma component in the tobacco leaf.Simultaneously, adding the aroma component of 8 months red large B4F tobacco leaf of biochemical additive alcoholization and the aroma component of 24 months B3F tobacco leaf of natural alcoholization approaches.To sum up, add the chemical composition of 8 months B4F tobacco leaf of biochemical additive alcoholization near 24 months B3F tobacco leaf of natural alcoholization.Sensory evaluating smoking result shows, the tobacco leaf B4F that sensory evaluating smoking's mass ratio of 8 months the red large B4F of tobacco leaf of process biochemical additive processing does not refine has increased significantly, reach the organoleptic quality of 24 months tobacco leaf B4F of natural alcoholization, the difference of smoke panel test score and 24 months the tobacco leaf B3F of natural alcoholization of 8 months tobacco leaf B4F of process biochemical additive processing is little simultaneously, illustrates that their aesthetic quality approaches.Above result shows that the application of biochemical additive Van-1 accelerated the speed of red large B4F tobacco mellowing, has shortened the alcoholization cycle, has improved the quality of tobacco leaf.
Embodiment 7
The preparation method of biochemical additive Van-1 is with embodiment 1, the additive of getting suitable volumes (5%-10%) evenly is sprayed at 100g tobacco leaf MZF surface (control moisture content in leaves content is 12-13%), mix as treatment samples, alcoholization workshop alcoholization 8 months.The deionized water of directly spraying same amount is at 100g tobacco leaf surface (control moisture content in leaves content is 12-13%) sample in contrast.Select simultaneously 18 months tobacco leaf MZF of natural alcoholization in contrast, the routine chemical components of test sample and aroma component are further estimated the effect that biochemical additive is processed the cigarette sample by the sensory evaluating smoking simultaneously.
The testing result of routine chemical components shows: after tobacco leaf MZF processed 8 months through biochemical additive, larger change has occured in the content of the large biological molecule compound of tobacco leaf, wherein obvious raising has occured in the content of reduced sugar and total reducing sugar, decline has occured in the content of total nitrogen, cellulose and nicotine, volatilization alkali and ligroin extraction total amount descend to some extent, the variation of the content of these compounds is conducive to the sucking quality of tobacco leaf, and the content of the chemical composition of the tobacco leaf of process biochemical additive processing and 18 months tobacco leaves of natural alcoholization approaches simultaneously.In addition, utilize GC-MS that the aroma component of tobacco sample is detected, the result shows, after tobacco leaf MZF process natural alcoholization or biochemical additive are processed, the increase that mostly occurred greatly of the aroma component of tobacco leaf, the aroma component total amount of the tobacco sample of simultaneously biochemical additive processing increases manyly than tobacco natural alcoholization, illustrates that the biochemical additive processing helps formation and the accumulation of aroma component in the tobacco leaf.Sensory evaluating smoking result shows that the tobacco leaf MZF that sensory evaluating smoking's mass ratio of 8 months tobacco leaf MZF of process biochemical additive processing does not refine has increased significantly, and reaches the organoleptic quality of 18 months tobacco leaf MZF of natural alcoholization.Above result shows that the application of biochemical additive Van-1 accelerated the speed of tobacco leaf MZF alcoholization, has shortened the alcoholization cycle, has improved the quality of tobacco leaf.
The above is preferred embodiment of the present invention only, is not limited to the present invention.All any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. a tobacco leaf biochemical additive is characterized in that, this alcoholization additive is to extract by the tunning to bacillus pumilus (Bacillus pumilus) Van35 to obtain; Described bacillus pumilus is kept at China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCCNo.3411; The liquid culture medium of described tunning by mass percent is: wheat bran 1.5, soy peptone 1.5, casein 1.0, CaCl
20.1, K
2HPO
40.1, KH
2PO
40.125, MgSO
47H
2O, 0.05, FeSO
40.0004 pH 8.0; 121 ℃ of sterilization 20min.
2. the preparation method of a tobacco leaf biochemical additive claimed in claim 1 is characterized in that adopting following steps:
(1) the 50mL liquid seed culture medium is sub-packed in the 250mL triangular flask, inoculating strain Van35 in 30 ℃ of shaken cultivation 24h, obtains liquid seeds; The liquid seeds of inoculation 8% in 30 ℃ of shaken cultivation 24h, obtains the liquid seeds of cultivating for enlarging in the 1000mL triangular flask that the 240mL liquid seed culture medium is housed;
(2) sterile working is inoculated into 8% liquid seeds in the 100L fermentation tank that the liquid culture medium of 60L is housed, and puts tank in 36 hours in 30 ℃ of static culture; Centrifugal 8000rpm, 20min collects zymotic fluid, according to envelope-bulk to weight ratio, adds 56% ammonium sulfate in the zymotic fluid, stirs 2h, centrifugal 8000rpm again, 20min collecting precipitation;
(3) precipitation is dissolved in the pH7.0 phosphoric acid liquid of 600ml, analyses and remove ammonium sulfate, obtain the Van35 extractive from fermentative, this extract is required tobacco leaf biochemical additive Van-1.
3. the application of biochemical additive claimed in claim 1 in tobacco mellowing, its concrete practice is as follows: the biochemical additive of getting 5%～10% volume evenly is sprayed at the 100g tobacco leaf surface, the control tobacco leaf surface, control moisture content in leaves content is 12%～13%.
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| CN102816710B (en) * | 2011-12-22 | 2014-05-07 | 红塔烟草(集团)有限责任公司 | Strain capable of generating protease, method for preparing protease liquid and method for improving quality of low-grade tobaccos by using protease liquid |
| CN102559548B (en) * | 2011-12-22 | 2013-06-05 | 红塔烟草(集团)有限责任公司 | Amylase high-producing strain and method for improving quality of low-grade tobacco through same |
| CN105768199B (en) * | 2016-03-25 | 2017-08-08 | 河南中烟工业有限责任公司 | A kind of compound formulation for being used to accelerate tobacco mellowing |
| CN107034162A (en) * | 2016-08-25 | 2017-08-11 | 江西中烟工业有限责任公司 | A kind of bacillus BA 01 and its application in terms of tobacco |
| CN107354109B (en) * | 2017-08-11 | 2020-11-06 | 陕西中烟工业有限责任公司 | Aroma-imparting compound microbial preparation for rapid fermentation of tobacco leaves and application thereof |
| CN109554306B (en) * | 2018-11-14 | 2022-04-19 | 河南中烟工业有限责任公司 | Compound preparation of protease and protein degrading bacteria and application of compound preparation in cigars |
| CN114376257B (en) * | 2022-01-30 | 2023-05-02 | 广西中烟工业有限责任公司 | Method for improving tobacco leaf quality through synergistic treatment of bacterial enzymes |
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| CN1230527C (en) * | 2001-07-13 | 2005-12-07 | 武汉东湖生物技术发展有限公司 | Hemicellulase and its application in preparing chitoligose |
| CN1439713A (en) * | 2002-02-23 | 2003-09-03 | 王革 | Microbe (No.3) and production thereof |
| CN1319478C (en) * | 2005-07-13 | 2007-06-06 | 云南瑞升科技有限公司 | Additive for flue-cured tobacco, prepn. method and application thereof |
| CN101254027A (en) * | 2007-03-01 | 2008-09-03 | 韩伟 | Producing method of novel tobacco fermenting enzyme preparations |
| CN101288505A (en) * | 2007-12-06 | 2008-10-22 | 云南万芳生物技术有限公司 | Method for improving quality of the tobacco leaf expanded cut stem by microorganism enzyme |
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