CN102559548B - Amylase high-producing strain and method for improving quality of low-grade tobacco through same - Google Patents
Amylase high-producing strain and method for improving quality of low-grade tobacco through same Download PDFInfo
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- CN102559548B CN102559548B CN 201110435707 CN201110435707A CN102559548B CN 102559548 B CN102559548 B CN 102559548B CN 201110435707 CN201110435707 CN 201110435707 CN 201110435707 A CN201110435707 A CN 201110435707A CN 102559548 B CN102559548 B CN 102559548B
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Abstract
The invention discloses a diastase high-producing strain and a technical method, wherein the diastase high-producing strain is a Bacillus sp.65 strain; and the technical method utilizes fermentation diastase to process flue-cured tobacco leaves so as to improve the quality of low-grade tobacco. The unit enzyme activity of diastase is 806 U/ml, and the enzyme application amount is 2.82 U/g of the tobacco leaves; in addition, the smoking assessment result which is obtained after processing shows that smoke of shredded tobacco is smooth and soft, the concentration of smoke is higher, and the aroma quantity, the miscellaneous gas, the taste and the texture of smoke can be well improved. The technical method achieves a simple technology and a low cost.
Description
Technical field
The present invention relates to a strain Alpha-amylase high yielding strain, the bacterial strain preserving number is: CGMCC No.5312, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: No. 3, BeiChen West Road, Chaoyang District, BeiJing City l institute, preservation date on September 29th, 2011, and the technical field of utilizing bacterial strain fermentation liquor that junior tobacco leaf is processed, understand specifically and utilize the biological starch zymotechnic to improve the technological method of low-quality tobacco quality.
Background technology
Tobacco fermentation is the important procedure that improves quality of tobacco, tobacco leaf without fermentative processing can not be directly used in production of cigarettes, only have by fermentation and process, just can overcome to some extent the bad quality factor of new cigarette and low-quality tobacco, reduce or eliminate new cigarette with cyan, reduce pungency and peculiar smell, make and inhale flavor and suitable, increase elasticity and the incendivity of tobacco leaf.In Tobacco Fermentation Process, microorganism and enzyme play very important effect.
The effect of microorganism in Tobacco Fermentation Process is inseparable with enzymatic reaction because microorganism can be to the different types of enzyme of exocytosis in its growth and development process, these enzymes can the catalysis tobacco leaf in the decomposition of Cucumber or synthetic.In tobacco leaf when the contents of saccharide that exists with the starch form is too much, can affect combustionvelocity and combustion completion when burning and sucking cigarette, produce during burning and stick with paste burnt flavour, tobacco flavor is degenerated, so starch content is determining determining to a certain extent the interior quality of tobacco leaf and the quality of sucking flavor.
China's Starch in Flue-cured will exceed 3% left and right than external sound tobacco according to the study.In tobacco leaf, residual starch is that ripening degree reaches not tobacco leaf palliating degradation degree of starch in the course of processing and causes not owing to gathering.The decomposition of Starch in Tobacco, conversion, consumption, accumulation all will have a huge impact outward appearance and the interior quality of tobacco leaf.
In order to reduce the content of Starch in Tobacco, can utilize and add the diastatic method of external source, to reduce the content of Starch in Tobacco, improve quality of tobacco.Apply a certain amount of amylase under certain condition in tobacco leaf, the tobacco leaf oeverall quality is improved.
Summary of the invention
The present invention aims to provide a strain Alpha-amylase high yielding strain, utilizes the bacterium producing multi enzyme preparation fermented liquid to improve low inferior tabacum sensory, improves the industrial applicability of low-quality tobacco and processes the just method of flue-cured tobacco.
Technical scheme of the present invention is as follows: 1, the bacterial strain of genus bacillus (Bacillus.sp) genus, and the bacterial strain preserving number is: CGMCCNo.5312, should separate from the red large ageing tobacco leaf of bacterium producing multi enzyme preparation obtaining, be a strain Alpha-amylase high yielding strain.
Screening is to utilize to produce amylase plate screening substratum (g/L) preliminary screening product amylase strain;
Then with transfering loop, pure growth is accessed in liquid LB substratum, 37 ℃ of shaking culture 60h, fermented liquid centrifuging and taking supernatant, employing DNS method detects the amylase activity in each fermented liquid, sifts out again and produces diastatic bacterial strain;
Described substratum g/L is: starch 20g, and peptone 10g, yeast powder 5g, NaCL 10g, pH 7.0.
Bacterium is inoculated in 10ml-100ml LB liquid nutrient medium, is placed on 37 ℃ of constant-temperature tables and cultivates 12h, rotating speed is 150r/min; Seed liquor is inoculated in 500ml LB substratum by 1% amount, 37 ℃ of similarity conditions, and pH 7.0 condition fermentation 60h carry out ultrafiltration and concentration, the centrifugal removal thalline of 5000r/min, supernatant carries out ultrafiltration and concentration with 10,000MWCO, namely gets refining amylase crude enzyme liquid;
Enzyme activity determination adopts the DNS method, gets enzyme liquid 100 μ l, then adds the Zulkovsky starch of 100 μ l 1%, 37 ℃ of constant temperature accurately react 30min, drip 400 μ l DNS, and boiling water boils the 5min colour developing, measure the light absorption value under 540nm, take the reactive system that adds inactivator liquid as blank;
Give a definition at this reaction conditions: every min generates the 1 required enzyme amount of μ g glucose and is defined as the enzyme unit (U) that lives, and in No. 65 bacterial strain crude enzyme liquids, diastatic unit enzyme is lived and is 826U/ml after measured.
with the tobacco leaf conditioning of intending processing, chopping, accurately take the 100g pipe tobacco, be divided into two minutes, portion evenly sprays pipe tobacco with the enzyme liquid for preparing with aseptic miniaturised nebuliser, the amount of spraying is that 0.35% enzyme liquid adds 9.65% sterilized water, the enzyme amount of executing of fermenting enzyme liquid is the 2.82U/g tobacco leaf, another part control group is processed with 10% sterilized water, pack into after mixing and be positioned over room temperature in valve bag and place 12h, then the baking oven of putting into 90 ℃ dries by the fire 20-30min, make enzyme deactivation under hot conditions, then put into (22 ℃ of fixed temperature and humidity incubators, 55%) balance pipe tobacco 24h in, the rear obvious low-quality tobacco quality of improving of smokeing panel test.
Of the present invention have a following advantage:
1. directly utilize the Alpha-amylase high yielding strain that separates on tobacco leaf to ferment, the enzyme that produces due to the bacterial strain from tobacco leaf relatively adapts to the tobacco leaf environment, acts on the substrate on tobacco leaf, can better improve quality of tobacco.
2. fermention medium of the present invention is the LB substratum, fermentation time is 60 hours, simple to operate, be easy to control, it is second-rate cloud 8587 junior tobacco leafs that enzyme liquid is processed tobacco leaf, the tobacco leaf aesthetic quality that bacterial strain 65 is processed improves successful, and fermention medium is on the not impact of sucking quality of tobacco leaf.
Description of drawings
Fig. 1 glucose typical curve of the present invention.
Embodiment
Utilization of the present invention separates from red large ageing tobacco leaf the bacterium producing multi enzyme preparation that obtains, and is numbered 65, and through being accredited as the bacterial strain of bacillus (Bacillus sp.), the bacterial strain preserving number is (CGMCC No.5312).37 ℃, pH 7.0 condition fermentation 60h carry out ultrafiltration and concentration, namely get the amylase crude enzyme liquid, and enzyme activity determination adopts the DNS method.
Amylase enzyme unit alive (U) is defined as every 1min and generates the 1 required enzyme amount of μ g glucose.After measured, in No. 65 bacterial strain crude enzyme liquids, diastatic unit enzyme is lived and is 826U/ml.It is the lower cloud of quality 8587 junior tobacco leafs (B3F) that enzyme liquid is processed the cigarette sample, with the tobacco leaf conditioning of processing, chopping, accurately take a certain amount of pipe tobacco, the applied amount of enzyme is that 0.35% enzyme liquid adds 9.65% sterilized water, contrast is processed with 10% sterilized water, the enzyme liquid that dilution is good evenly sprays pipe tobacco with aseptic miniaturised nebuliser, the valve bag of packing into after the pipe tobacco mixing is built in room temperature places 12h, then put into baking oven and dry by the fire 20-30min, make enzyme deactivation under hot conditions, then put into fixed temperature and humidity incubator balance pipe tobacco.
The tobacco cartridge that balance is good is made cigarette, is completed by Hongta Group's technique center person of smokeing panel test and smokes panel test, and adopts " Yuxi high-quality tobacco single-tobacco-typed cigarette aesthetic quality smoke panel test method " (Yunnan Province's provincial standard, standard number DB53/T 182.3-2006) (table 1).Mainly 10 of note, perfume quantity, fragrance matter etc. are estimated, total points 100 minutes is finally weighed aesthetic quality's level with integrate score.Through the discovery of smokeing panel test, the pipe tobacco that bacterial strain 65 is processed is better harmonious, and flue gas prolongs continuous sense better, and mouthfeel is more comfortable, and assorted gas is lighter.Rhythm, fragrance matter, clean degree, aftertaste etc. all be improved significantly, assorted gas alleviates, the pipe tobacco sensory evaluation is good.
Embodiment 1: screening and the evaluation of producing amylase strain
Bacterium producing multi enzyme preparation from the large gold dollar leaf of high-quality Yuxi sound tobacco safflower is numbered 65.Utilization product amylase plate screening substratum (g/L) (starch 20g, peptone 10g, yeast powder 5g, NaCL 10g, pH 7.0) preliminary screening product amylase strain.Then with transfering loop, pure growth is accessed in liquid LB substratum, 37 ℃ of shaking culture 60h, fermented liquid centrifuging and taking supernatant, employing DNS method detects the amylase activity in each fermented liquid, sifts out again and produces diastatic bacterial strain.The DNS ratio juris: starch is hydrolyzed to glucose under diastatic effect, glucose is reducing sugar, heats under certain condition by 3,5-dinitrosalicylic acid to be oxidized to saccharic acid and other product, 3,5-dinitrosalicylic acid is reduced to henna 3-amino-5-NITROSALICYLIC ACID.Within the specific limits, the depth relation in direct ratio of the amount of reducing sugar and brown materials color is measured light absorption value under the 540nm wavelength, checks typical curve (Fig. 1) and calculates, and just can obtain the content of glucose in sample.
Utilize a pair of universal primer of 16S rRNA gene to carry out pcr amplification, connection carrier transforms and checks order, result is submitted NCBI to, carry out the sequence homology retrieval analysis by BLAST, then carry out Multiple Sequence Alignment with CLUSTAL X software and calculate strains tested and the reference bacterial strain between sequence similarity, adopt ortho position phase connection (Neighbor-Joining), use MEGA4 software building systematic evolution tree.This bacterial strain is carried out sequencing analysis, it is initially identified as the bacterial strain of bacillus (Bacillus sp.65), the bacterial strain preserving number is (CGMCC No.5312).
Embodiment 2: the preparation of amylase liquid
Utilize bacterial strain to ferment, make crude enzyme liquid.Bacterium is inoculated in a small amount of LB liquid nutrient medium, is placed on 37 ℃ of constant-temperature tables and cultivates 12h, rotating speed is 150r/min.Seed liquor is inoculated in 500ml LB substratum by 1% amount, similarity condition fermentation 60h, and the centrifugal removal thalline of 5000r/min, supernatant carries out ultrafiltration and concentration with 10,000MWCO, namely gets refining crude enzyme liquid.
Adopt the DNS method, get enzyme liquid 100 μ l, then add the Zulkovsky starch of 100 μ l 1%, 37 ℃ of constant temperature accurately react 30min, drip 400 μ l DNS, and boiling water boils the 5min colour developing.Measure the light absorption value under 540nm.Take the reactive system that adds inactivator liquid as blank.Give a definition at this reaction conditions: every min generates the 1 required enzyme amount of μ g glucose and is defined as the enzyme unit (U) that lives.After measured, in No. 65 bacterial strain crude enzyme liquids, diastatic unit enzyme is lived and is 826U/ml.
Embodiment 3: amylase is to the processing of pipe tobacco and the sensory quality assessment of pipe tobacco
Processing the cigarette sample is cloud 8587 junior tobacco leafs (B3F), with tobacco leaf conditioning, chopping, accurately take the 100g pipe tobacco, the enzyme liquid for preparing is evenly sprayed pipe tobacco with aseptic miniaturised nebuliser, the amount of spraying is that 0.35% enzyme liquid adds 9.65% sterilized water, the enzyme amount of executing of 65 fermenting enzyme liquid is the 2.82U/g tobacco leaf, contrast is processed with 10% sterilized water, the valve bag of packing into after mixing is built in room temperature and places 12h, and the baking oven of then putting into 90 ℃ dries by the fire 20-30min, makes enzyme deactivation under hot conditions, then put into balance pipe tobacco 24h in fixed temperature and humidity incubator (22 ℃, 55%).
The tobacco cartridge that balance is good is made cigarette, is completed by Hongta Group's technique center person of smokeing panel test and smokes panel test, and adopts " Yuxi high-quality tobacco single-tobacco-typed cigarette aesthetic quality smoke panel test method " (Yunnan Province's provincial standard, standard number DB53/T 182.3-2006) (table 1).Mainly 10 of note, perfume quantity, fragrance matter etc. are estimated, total points 100 minutes is finally weighed aesthetic quality's level with integrate score.
Smoking result (table 2) is described below: the contrast flue gas concentration is higher, and rich good, assorted gas shows slightly, and aftertaste is slightly poor, and is sour, puckery, slightly jagged sense, and matter is coarse, and the pipe tobacco flue gas is fine and smooth, mellow, and flue gas concentration is higher, and texture is improved.From marking situation (table 2), the pipe tobacco sensory quality assessment total points that bacterial strain 65 fermenting enzyme liquid are processed exceeds 1.5 minutes than contrast, from smoking result, the perfume quantity of flue gas, assorted gas, mouthfeel, texture is had preferably improve.Fermention medium LB, can continue to use substantially without impact on note.
Table 1: Hongta Group's monomer tobacco leaf organoleptic quality evaluation method and index
Table 2:65 bacterial strain is processed the pipe tobacco smoking result
Claims (3)
1. the bacterial strain that belongs to of genus bacillus (Bacillus. sp), the bacterial strain preserving number is: CGMCC No. 5312, this bacterial strain separate from red large ageing tobacco leaf and obtain, and are strain Alpha-amylase high yielding strains.
2. right to use requires 1 described genus bacillus (Bacillus. sp) bacterial strain to prepare the method for amylase liquid, it is characterized in that bacterium is inoculated in 10 ml-100ml LB liquid nutrient medium, be placed on 37 ℃ of constant-temperature tables and cultivate 12h, rotating speed is 150r/min; Seed liquor is inoculated in 500ml LB substratum by 1% amount, 37 ℃ of similarity conditions, and pH 7.0 condition fermentation 60h carry out ultrafiltration and concentration, the centrifugal removal thalline of 5000r/min, supernatant carries out ultrafiltration and concentration with 10,000MWCO, namely gets refining amylase crude enzyme liquid;
Enzyme activity determination adopts the DNS method, gets enzyme liquid 100 μ l, then adds the Zulkovsky starch of 100 μ l 1%, 37 ℃ of constant temperature accurately react 30min, drip 400 μ l DNS, and boiling water boils the 5min colour developing, measure the light absorption value under 540nm, take the reactive system that adds inactivator liquid as blank;
Give a definition at this reaction conditions: every min generates the 1 required enzyme amount of μ g glucose and is defined as the enzyme unit (U) that lives, and the unit enzyme work in the above-mentioned amylase that obtains after measured is 826 U/ml.
3. the method for utilizing the thick liquid of amylase that the method for preparing amylase liquid claimed in claim 2 prepares that junior tobacco leaf is processed, it is characterized in that and to intend the tobacco leaf conditioning of processing, chopping, accurately take 100 g pipe tobaccos, be divided into two parts, the a thick liquid of amylase that claim 2 is prepared evenly sprays pipe tobacco with aseptic miniaturised nebuliser, the amount of spraying is that 0.35% enzyme liquid adds 9.65% sterilized water, the enzyme amount of executing of the thick liquid of amylase is 2.82 U/g tobacco leaves, another part control group is processed with 10% sterilized water, pack into after mixing and be positioned over room temperature in valve bag and place 12h, then put into the baking oven baking 20-30 min of 90 ℃, make enzyme deactivation under hot conditions, then put into 22 ℃ of fixed temperature and humidity incubators, relative humidity is balance pipe tobacco 24h in 55% fixed temperature and humidity incubator, the rear obvious low-quality tobacco quality of improving of smokeing panel test.
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| CN103120362B (en) * | 2013-02-28 | 2015-02-04 | 湖北中烟工业有限责任公司 | Method for improving combustibility of cigar tobacco leaves |
| CN104886763A (en) * | 2015-06-25 | 2015-09-09 | 湖北中烟工业有限责任公司 | Application of amylase in cigar fermentation and quality improvement |
| CN106191209B (en) * | 2016-06-30 | 2019-10-18 | 昆明学院 | A method of detection Starch in Tobacco degrading microorganism |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86102596A (en) * | 1986-04-10 | 1986-10-08 | 李祥麟 | The method of strengthen fermentation of tobacco leaf |
| CN1439716A (en) * | 2002-02-23 | 2003-09-03 | 王革 | Microbe(No.J1) and production thereof |
| CN1439714A (en) * | 2002-02-23 | 2003-09-03 | 王革 | Microbe(No.4) and production thereof |
| CN1439715A (en) * | 2002-02-23 | 2003-09-03 | 王革 | Microbe(No.1) and production thereof |
| 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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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86102596A (en) * | 1986-04-10 | 1986-10-08 | 李祥麟 | The method of strengthen fermentation of tobacco leaf |
| CN1439716A (en) * | 2002-02-23 | 2003-09-03 | 王革 | Microbe(No.J1) and production thereof |
| CN1439714A (en) * | 2002-02-23 | 2003-09-03 | 王革 | Microbe(No.4) and production thereof |
| CN1439715A (en) * | 2002-02-23 | 2003-09-03 | 王革 | Microbe(No.1) and production thereof |
| 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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