CN102942636A - Method for extracting oligosaccharides from flue-cured tobacco leaf - Google Patents
Method for extracting oligosaccharides from flue-cured tobacco leaf Download PDFInfo
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- CN102942636A CN102942636A CN2012105130727A CN201210513072A CN102942636A CN 102942636 A CN102942636 A CN 102942636A CN 2012105130727 A CN2012105130727 A CN 2012105130727A CN 201210513072 A CN201210513072 A CN 201210513072A CN 102942636 A CN102942636 A CN 102942636A
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Abstract
The invention relates to a method for extracting oligosaccharides from flue-cured tobacco leaf, which comprises the following steps: pulverizing tobacco leaf, pretreating the tobacco leaf with an acetonitrile water solution, carrying out ultrasonic-assisted extraction, centrifuging, filtering, and filtering with a PVDF (polyvinylidene fluoride) membrane to obtain a mixture containing fructooligosaccharide and maltooligosaccharide. The method provided by the invention can effectively extract oligosaccharides from flue-cured tobacco leaf, wherein the fructooligosaccharide product concentration is 21-30 ppm, and the maltooligosaccharide product concentration is 35-45 ppm. The obtained products can be directly used in subsequent industrial applications.
Description
[technical field]
The present invention relates to chemical field, be specifically related to a kind of method of from cured tobacco leaf, extracting the mixture that contains oligose.
[background technology]
Sugar is important component part of tobacco leaf, and carbohydrate and cigarette taste, fragrance have direct relation in the cigarette.Therefore, the first step of carbohydrate research is the relation that exists between the fragrance of carbohydrate and cigarette in the clear and definite tobacco leaf and the taste in the tobacco.There are some researches show that the cured tobacco leaf that cultivates contains abundant fructose, glucose, sucrose under the air of high-quality and sufficient sunlight.In addition, there is research that the carbohydrate in blue or green cured tobacco leaf and the flue-cured tobacco finished product is analyzed.Yet these researchs mainly concentrate on monose, disaccharides and polysaccharide.Information aspect oligose is limited then, the planteose in the tobacco seed, and raffinose and stachyose in the green tobacco leaf, the pyrans Portugal base sucrose in the flue-cured tobacco leaf also was in the news.
Oligose is distributed widely in plant, the algea and bacteria.Wherein.Oligofructose is the fructose that has been condensed in the fructose residues thing, and relevant with the beta-glucan that has single glucose glycan molecule.According to contacting between glucosides and the fructose residues, oligofructose is divided into three main Types: inulin, Polylevulosan and Gramineae Polylevulosan.The fructose of height polymerization is Polylevulosan, is found in plant, algea and bacteria equally.About 15% flowering plant all can accumulate Polylevulosan.As typical chrysanthemum order, the Polylevulosan of inulin type mainly occurs in the dicotyledons, and Polylevulosan type and Gramineae Polylevulosan usually occur in the monocotyledons.Although Polylevulosan is distributed widely in the plant, tobacco still is listed in the plant that does not have the Polylevulosan storage, several research reports, and its reason is that tobacco lacks the effective converting system that produces Polylevulosan.On the other hand, Oligomeric maltose is to be subjected to α 1-4 key to restrict the glucose that is condensed in the glucose residue equally.Maltose has the glucose of two units, is looked simultaneously and does a kind of Oligomeric maltose (polymerization degree is 2), and it is present in the tobacco leaf.Yet seldom having the polymerization degree to surpass 2 Oligomeric maltose is in the news.The hydrolysis of passing through starch of Oligomeric maltose produces in the cured tobacco leaf.
Domestic and foreign literature has been reported the method for various analysis oligosaccharides.Nowadays, the technology of the most frequently used analysis oligose is high-performance anion-exchange chromatography and impulse current measurement method (HPAEC-PAD).Although the impulse current measurement method has effect to quantizing oligose very much, a lot of relevant its structural informations but can not be provided, and when experiment, sample purity be arrived the degree of certain high-quality.Several methods based on mass spectrometric detection have been reported recently.Compared with the impulse current measurement method, mass spectroscopy has more advantage, and a large amount of information for example peak purity, molecular weight and molecular structure especially can be provided.In addition, substance assistant laser desorpted/ionization technique (MALDI) adds that flight time mass spectrum (TOF-MS) or Fourier transform ion cyclotron resonance mass spectroscopy (FT-ICR-MS) need not also to provide the relevant polymerization degree under the condition of chromatographic separation, the information of molecular structure and concentration.
The present invention is devoted to study the pre-treating process of oligofructose and Oligomeric maltose in a kind of sharp separation cured tobacco leaf, and sets up the with it detection method of supporting quick discriminating material composition.
[summary of the invention]
The purpose of this invention is to provide a kind of method that contains oligose of from cured tobacco leaf, extracting.
The invention provides a kind of method of extracting oligose from cured tobacco leaf, step is as follows:
(1) tobacco leaf is pulverized: get the tobacco leaf powder that cured tobacco leaf is ground into diameter 1-3mm;
(2) pre-treatment: in weight part, weighing 1g tobacco leaf powder, the acetonitrile solution of adding 40ml 50 volume % is at room temperature with 200 rev/mins of pre-treatment 20-40min on shaking table;
(3) ultrasonic-assisted extraction: step (2) is placed on ultrasonic instrument, ultrasonication 20-40min through pretreated mixture;
(4) step (3) is removed through the suspended substance of the top of mixture of ultrasonication, with 3000 rev/mins of centrifugal 5-10 minutes, filter with ultra-fine filter again, obtain filtrate;
(5) get the filtrate 20ml of step (4), remove acetonitrile 75-81 ℃ of lower distillation, add water to residual filtrate to be settled to 10ml, obtain settled solution;
(6) settled solution with step (5) filters with pvdf membrane, obtains containing the mixture of oligofructose and Oligomeric maltose.
According to preferred embodiment a kind of, the ultrasonic power of step (3) is 300-1000W.
In the present invention, ultrasonic equipment for example uses the ultrasonoscope that U.S. Branson Ultrasonic company sells.
Preferably, the ultra-fine filter of step (4) is Amicon Ultra-15 ultra-fine filter (10000 dalton, Millipore company).
According to preferred embodiment a kind of, the pvdf membrane aperture of step (5) is 0.2-1um.
Pvdf membrane is polyvinylidene fluoride film, is filtration, the water treating equipment of commonly using, and for example adopts the pvdf membrane in the 0.45um aperture of the auspicious bio tech ltd sale of Shanghai soldier.
The production concentration that method of the present invention can effectively be extracted the oligofructose in the cured tobacco leaf is 21-30ppm, and the production concentration of Oligomeric maltose is 35-45ppm.Products therefrom can be directly used in the follow-up industrial application.
[embodiment]
Can understand better the present invention by following embodiment.
Embodiment 1
Get the cured tobacco leaf capacity, be crushed to the tobacco leaf powder of diameter 1-2mm.Weighing 1g tobacco leaf powder adds the acetonitrile solution of 40ml 50 volume %, at room temperature with 200 rev/mins of pre-treatment 20min on shaking table, and then is placed on the ultrasonoscope that U.S. Branson Ultrasonic company sells and processes 30min with 1000W.
After processing finishes, remove the suspended substance of top of mixture, with 3000 rev/mins centrifugal 10 minutes, filter with ultra-fine filter and to obtain filtrate.
Get filtrate 20ml, remove acetonitrile 80 ℃ of lower distillations, remaining concentrated filtrate is 5ml approximately, adds water to residual filtrate to be settled to 10ml, obtains settled solution, and the pvdf membrane by 0.2um filters again, obtains containing the mixture of oligofructose and Oligomeric maltose.
The gained mixture is detected
(1) oligose detects: Agilent 1200 HPLC and 6410 triple level Four bar MSD couplings (U.S. Agilent company), liquid-phase chromatographic column (Develosil RP Aqueous post 150 * 1.5mm, Nomura Chemical Co., Ltd., Japan), mobile phase A (5mM ammonium acetate), Mobile phase B (pure acetonitrile); Flow velocity: 0.1mL/min; A:0-15 minute, 100%; A:15-30 minute, the 100%-85%(linear gradient); A:30-40 minute, 85%; Rear working time, A:20 minute, 100%; Column temperature: 30 ℃; Sample size: 5uL;
MSD condition: negatively charged ion pattern; Capillary column voltage 4kV; Dry gas temperature: 350 ℃; Sheath airshed 11L/min, atomizer pressure: 35psi; Acquisition time: 0.2s; Collision gas: argon gas; Collision atmospheric pressure: 0.2Pa; Collision energy: 5-35V;
(2) oligose is mainly in the final cured tobacco leaf of differentiating: oligofructose, Oligomeric maltose concentration are respectively: 22.7ppm, 32.1ppm.
Embodiment 2
Get the cured tobacco leaf capacity, be crushed to the tobacco leaf powder of diameter 1-3mm.Weighing 1g tobacco leaf powder, the acetonitrile solution of adding 40ml 50 volume %, at room temperature with 200 rev/mins of pre-treatment 20min on shaking table, then 300w processes 40min on ultrasonoscope.
After processing finishes, remove the suspended substance of top of mixture, with 3000 rev/mins centrifugal 5 minutes, filter with ultra-fine filter and to obtain filtrate.
Get filtrate 20ml, remove acetonitrile 75 ℃ of lower distillations, remaining concentrated filtrate is 5ml approximately, adds water to residual filtrate to be settled to 10ml, obtains settled solution, and the pvdf membrane by 0.45um filters again, obtains containing the mixture of oligofructose and Oligomeric maltose.
With the detection method identical with embodiment 1 the gained mixture is detected, consequently oligofructose and Oligomeric maltose concentration are respectively: 22.4ppm, 31.8ppm.
Embodiment 3
Get the cured tobacco leaf capacity, be crushed to the tobacco leaf powder of diameter 1-3mm.Weighing 1g tobacco leaf powder, the acetonitrile solution of adding 40ml 50 volume % at room temperature with 200 rev/mins of pre-treatment 30min on shaking table, is then processed 30min at ultrasonoscope.
After processing finishes, remove the suspended substance of top of mixture, with 3000 rev/mins centrifugal 8 minutes, filter with ultra-fine filter and to obtain filtrate.
Get filtrate 20ml, remove acetonitrile 81 ℃ of lower distillations, remaining concentrated filtrate is 5ml approximately, adds water to residual filtrate to be settled to 10ml, obtains settled solution, and the pvdf membrane by 1um filters again, obtains containing the mixture of oligofructose and Oligomeric maltose.
With the detection method identical with embodiment 1 the gained mixture is detected, consequently oligofructose and Oligomeric maltose concentration are respectively: 21.9ppm, 33.1ppm.Products therefrom can be directly used in the follow-up industrial application.
Claims (5)
1. method of extracting oligose from cured tobacco leaf is characterized in that step is as follows:
(1) tobacco leaf is pulverized: get the tobacco leaf powder that cured tobacco leaf is ground into diameter 1-3mm;
(2) pre-treatment: weighing 1g tobacco leaf powder, the acetonitrile solution of adding 40ml 50 volume % is at room temperature with 200 rev/mins of pre-treatment 20-40min on shaking table;
(3) ultrasonic-assisted extraction: step (2) is placed on ultrasonic instrument, ultrasonication 20-40min through pretreated mixture;
(4) step (3) is removed through the suspended substance of the top of mixture of ultrasonication, with 3000 rev/mins of centrifugal 5-10 minutes, filter with ultra-fine filter again, obtain filtrate;
(5) get the filtrate 20ml of step (4), remove acetonitrile 75-81 ℃ of lower distillation, add water to residual filtrate to be settled to 10ml, obtain settled solution;
(6) settled solution with step (5) filters with pvdf membrane, obtains containing the mixture of oligofructose and Oligomeric maltose.
2. method according to claim 1, the ultrasonic power that it is characterized in that step (3) is 300-1000W/h.
3. method according to claim 1, the ultra-fine filter that it is characterized in that step (4) is Amicon Ultra-15 ultra-fine filter.
4. method according to claim 1, the pvdf membrane aperture that it is characterized in that step (5) is 0.2-1um.
5. method according to claim 1 is characterized in that the mixing species that contain oligofructose and Oligomeric maltose at gained, and the concentration of oligofructose is 21-30ppm, and the concentration of Oligomeric maltose is 35-45ppm.
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| CN2012105130727A CN102942636A (en) | 2012-12-04 | 2012-12-04 | Method for extracting oligosaccharides from flue-cured tobacco leaf |
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| CN2012105130727A CN102942636A (en) | 2012-12-04 | 2012-12-04 | Method for extracting oligosaccharides from flue-cured tobacco leaf |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103804507A (en) * | 2014-02-28 | 2014-05-21 | 河南中烟工业有限责任公司 | Maryland tobacco polysaccharide, extracting and purifying method and application thereof as antioxidant |
| CN103910808A (en) * | 2014-03-31 | 2014-07-09 | 河南中烟工业有限责任公司 | Spice tobacco bud polysaccharide, as well as preparation method and application thereof as antioxidant |
| CN104725523B (en) * | 2015-03-31 | 2016-11-09 | 川渝中烟工业有限责任公司 | Utilize the method that homogenate extraction technology discards extraction tobacco polysaccharide fresh tobacco leaf from land for growing field crops |
| CN106404990A (en) * | 2016-08-15 | 2017-02-15 | 华宝香精股份有限公司 | A method of measuring a peroxide value of a tobacco product |
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| US4986286A (en) * | 1989-05-02 | 1991-01-22 | R. J. Reynolds Tobacco Company | Tobacco treatment process |
| CN102230923A (en) * | 2011-04-01 | 2011-11-02 | 红塔烟草(集团)有限责任公司 | Method for determining content of water-soluble sugar in tobaccos and tobacco products |
| CN102507829A (en) * | 2011-11-17 | 2012-06-20 | 云南省烟草烟叶公司 | Method for establishing tobacco HPLC (High Performance Liquid Chromatography) fingerprint database and application thereof |
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2012
- 2012-12-04 CN CN2012105130727A patent/CN102942636A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US4986286A (en) * | 1989-05-02 | 1991-01-22 | R. J. Reynolds Tobacco Company | Tobacco treatment process |
| CN102230923A (en) * | 2011-04-01 | 2011-11-02 | 红塔烟草(集团)有限责任公司 | Method for determining content of water-soluble sugar in tobaccos and tobacco products |
| CN102507829A (en) * | 2011-11-17 | 2012-06-20 | 云南省烟草烟叶公司 | Method for establishing tobacco HPLC (High Performance Liquid Chromatography) fingerprint database and application thereof |
Non-Patent Citations (2)
| Title |
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| ATSUSHI NAGAI等: "Identification of Fructo-and Malto-oligosaccharides in Cured Tobacco Leaves (Nicotiana tabacum)", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103804507A (en) * | 2014-02-28 | 2014-05-21 | 河南中烟工业有限责任公司 | Maryland tobacco polysaccharide, extracting and purifying method and application thereof as antioxidant |
| CN103910808A (en) * | 2014-03-31 | 2014-07-09 | 河南中烟工业有限责任公司 | Spice tobacco bud polysaccharide, as well as preparation method and application thereof as antioxidant |
| CN104725523B (en) * | 2015-03-31 | 2016-11-09 | 川渝中烟工业有限责任公司 | Utilize the method that homogenate extraction technology discards extraction tobacco polysaccharide fresh tobacco leaf from land for growing field crops |
| CN106404990A (en) * | 2016-08-15 | 2017-02-15 | 华宝香精股份有限公司 | A method of measuring a peroxide value of a tobacco product |
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