CN103773728B - Application of sphingomonas strain xp in degrading polyphenolic compounds in tobacco sheets - Google Patents
Application of sphingomonas strain xp in degrading polyphenolic compounds in tobacco sheets Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms strains, and in particular relates to a sphingomonas strain xp, wherein the sphingomonas strain xp is classified and named as (i) Sphingomonas (/i) sp (i). (/i); the preservation number of the strain xp in China Center for Type Culture Collection is CCTCC No: M2011453. The sphingomonas strain xp is applied to degrading polyphenolic compounds in tobacco stems, and is good in degradation effect.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of Sphingomonas bacterial strain xp and the application in degrading tobacco thin slice in polyphenolic compound thereof.
Background technology
Tobacco sheet is also known as regeneration tobacco leaf or homogeneous tobacco leaf, and it is the product of comprehensive utilization during tobacco industry is produced and retrofit, adds the composition such as sizing agent and other additives primarily of offal, fragment, offal or discarded tobacco leaf.In fact all tobacco materials may be used to the manufacturing of tobacco sheet, by offal discarded in whole production of cigarettes process, stalk label, fragment and part discarded tobacco leaf, short stalk through different working methods, make proterties close to the tobacco sheet being even better than natural tobacco, and the tobacco sheet made has, and cost is low, filling properties well also can reduce the advantages such as the coal-tar middle oil content of flue gas.According to the difference of production and processing technology, mainly contain paper process, thick slurry processes and roll-in method three productions technique.Wherein paper process refers to tobacco material first with water extraction, and enter paper machine after slurry made by insoluble substance or interpolation natural fiber and form thin slice sheet base, water-soluble extract is coated with in the lump and sheet base with additive after concentrated, is finished product after drying.During processing and manufacturing, can extract from water-soluble substances or remove some tobacco component, or add physical chemistry and the combustionproperty that some new compositions improve thin slice.The main component of thin slice or tobacco leaf and offal, if so these starting material are not through to ferment completely and degrade, innerly contain starch tobacco being burnt and sucked to side effect, greatly reduce the quality of tobacco sheet.
Sphingol single-cell in the environment distributed pole is extensive, and has very strong patience (as all once found the existence of Sphingol single-cell in the extreme environment such as polar region, severe radiation) to severe environment.The degraded substrate spectrum of Sphingol single-cell is wide, and the degraded as the aromatic compounds such as Dui dioxin, phenols, azoic dyestuff and xenobiotic polymer etc. has relevant report.Because Sphingol single-cell has metabolic capacity very widely to aromatic compound, and some bacterial classification of this Pseudomonas can synthesize the outer biopolymer of valuable born of the same parents, and therefore, Sphingol single-cell becomes the focus being concerned and studying in recent years.
Tobacco polyphenol is the aromatic cycle compound with one or more hydroxyl at cigarette strain leaf and the synthesis of stem two main portions, and its content in tobacco is higher, is about the 0.5-2.5% of cured leaf weight.The polyphenolic compound found in tobacco comprises tannins (isomer of chlorogenic acid, coffic acid and quinic acid), coumarins (glycosides derivatives of scopoletin and scopoletin), flavonoid (fragrant glycosides, flavones, rhamnosyl, flavonol) and anthocyan (cyanidin(e)-3-rutinoside, pelargonidin-3-violaguercitrin, keampferol) four large class about 135 kinds altogether.Its Content of Chlorogenic Acid and violaguercitrin are topmost polyphenols in tobacco, and chlorogenic acid accounts for 75-95% of polyphenols total amount.Polyphenol is the important latent aromatic substance in tobacco leaf, polyphenol content with baking after tobacco leaf color and cigarette flavor closely related.In smoking process, polyphenol and pyrolysis product thereof are transferred in flue gas, have a direct impact smoke perfume.Under rational modulation, aging condition, plant polyphenol compounds generates many kinds of substance through series reaction degraded, these degraded products can give the fragrance of tobacco product gracefulness, increase the perfume quantity of tobacco product, have extremely important effect to the quality improving tobacco product.
Polyphenolic substance content in tobacco has great effect to cigarette quality.Within the specific limits, polyphenolic substance content and quality of tobacco are proportionate.This is because in modulation, alcoholization and combustion processes, polyphenolic compound is oxidable decomposes the compound generating some and can give the graceful faint scent of tobacco product and increase tobacco product perfume quantity, and therefore they play an important role to the quality improving tobacco product.But, when polyphenolic compound content exceeds certain limit, tobacco leaf in various degree with multiple quality defect, as: have blue foreign smell and pungency, aroma quality be dull, perfume quantity is not enough, can not directly be used for producing cigarette.What is more important, excessive polyphenolic substance is toxic to human body, can affect the health of smoker.Therefore, how reducing the content of polyphenolic compound in tobacco is the important step in tobacco industry.Now there are some researches show that microbial technique plays a significant role in degrading tobacco polyphenolic substance process.Therefore, current pole needs to develop the microorganism strains that better can play polyphenolic compound degradation effect being applicable to tobacco sheet under suitable conditions.
Summary of the invention
The object of the invention is to provide a kind of Sphingol single-cell xp, uses it for the polyphenolic compound in degrading tobacco thin slice, good degrading effect.
For achieving the above object, the present invention adopts following technical scheme:
A kind of Sphingomonas xp, its Classification And Nomenclature is
sphingomonassp
., this bacterial strain xp at the preserving number of China typical culture collection center is: CCTCC No:M 2011453, preservation date: on December 9th, 2011, depositary institution address: No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province (Wuhan University).
The application of described Sphingomonas xp in degrading tobacco thin slice in polyphenolic compound.
The application of described Sphingomonas xp in degrading tobacco thin slice in polyphenolic compound, be specially: Sphingomonas bacterial strain xp LB substratum is recovered (cultivating 24h for 30 DEG C), then be inoculated in the minimal medium containing chlorogenic acid or tobacco vat liquor, collected by centrifugation thalline after 28-32 DEG C of cultivation 2-24 h, after minimal medium cleaning 1-5 time, with the resuspended thalline of minimal medium, be adjusted to OD
600value is 2.0-5.0, and gained suspension is seed culture fluid; By seed culture fluid 28-32 DEG C, be evenly sprayed on tobacco sheet surface under humidity 55-65% condition or put in the water-soluble vat liquor of paper process thin slice.
Concrete, described containing in the minimal medium of chlorogenic acid or tobacco vat liquor, chlorogenic acid concentration is 0.2-1.0g/L, and tobacco vat liquor concentration is 0.05-0.2 ml/L.Chlorogenic acid or the sole carbon source of tobacco vat liquor in order to grow as Sphingomonas xp.Sphingol single-cell xp of the present invention is that when cultivating in the minimal medium of 0.2-1.0g/L, growing state is good at chlorogenic acid addition, and the chlorogenic acid of greater concn can suppress the growth of Sphingol single-cell xp.
Described tobacco vat liquor can obtain through following method: get 1g offal and offal, adds 55-65% ethanol and shake up once in 45-55 DEG C of every 10 min of water-bath extraction 30-60min(after pulverizing), extraction terminates rear filtration, and filtrate is tobacco vat liquor.
Preferably, the formula of described minimal medium is: containing 2.44g Na in every 1000ml deionized water
2hPO
4, 1.52g KH
2pO
4, 0.5g (NH
4)
2sO
4, 0.2g MgSO
47H
2o, 0.05g CaCl
22H
2o and 10ml trace metal salts solution S L-4; Wherein, described trace metal salts solution S L-4 consists of: containing 0.50g EDTA, 0.20g FeSO in 1000ml deionized water
47H
2o and 100ml trace metal salts solution S L-6; Described trace metal salts solution S L-6 consists of: containing 0.10g ZnSO in 1000ml deionized water
47H
2o, 0.03g MnCl
24H
2o, 0.30g H
3bO
3, 0.20g CoCl
26H
2o, 0.01g CuCl
22H
2o, 0.02g NiCl
26H
2o and 0.03g Na
2moO
42H
2o.
Bacterial strain Sphingomonas bacterial strain xp(Sphingomonas sp. provided by the present invention) in minimal medium, utilize chlorogenic acid or tobacco vat liquor for after sole carbon source growth, can polyphenolic compound in degrading tobacco thin slice.Utilizing Sphingol single-cell xp(Sphingomonas sp.) be sprayed on tobacco sheet surface, the polyphenolic compound content degradation rate after a week in Flue-cured is more than 25%; And bacterial strain xp is put in the water-soluble vat liquor of paper process thin slice, the polyphenolic substance content degraded after 4 hours in vat liquor is more than 30%.In the method for alcoholizing utilized at present, the required alcoholization time is longer, often affects the cycle of product.The present invention, compared with existing method for alcoholizing, has the advantage of fast degradation, and this has more advantage for the practical application in tobacco sheet production alcoholization process.
Accompanying drawing explanation
Fig. 1 is the phylogenetic tree built based on microorganism strains xp 16S rRNA gene order;
Fig. 2 is the growing state of Sphingomonas xp 48h in the minimal medium containing chlorogenic acid;
Fig. 3 is bacteria concentration when being OD3.0, the degraded situation of Sphingomonas xp to chlorogenic acid in containing the minimal medium of different chlorogenic acid.
Embodiment
(1) Sphingol single-cell (
sphingomonassp
.) screening of bacterial strain xp:
Because chlorogenic acid is the representational polyphenols of most in tobacco, the application, using chlorogenic acid as substrate, is made into agar plate.30 DEG C, use the vega soil aqueous solution to carry out coated plate cultivation under the condition of 24hr, plate culture, after the subzero treatment 24-72hr of 4 DEG C, is inverted microscopic examination bacterium to the discomposing effect of chlorogenic acid by fluorescence.Due to chlorogenic acid aobvious blue-fluorescence under UV-light (365nm), have that the substratum of the periphery of bacterial colonies of chlorogenic acid capacity of decomposition is more thin out than the fluorescence in background to die down.In the blue-fluorescence background of chlorogenic acid, can directly be separated the bacterial strain be not bacterial contamination.The chlorogenic acid enzyme activity of different strains generation is measured according to the activity of chlorogenic acid enzyme and the relation of transparent circle diameter.Cultivate at the enterprising row filter of the agar plate of pH value 4.0-6.0, acid resistance bacterial strain xp can be obtained.
(2) Sphingol single-cell (
sphingomonassp
.) qualification of bacterial strain xp:
Screening the bacterial strain xp obtained is Gram-negative bacteria, without spore, with one-sided raw polar flagellum motion, in yellow.Entrust China typical culture collection center to carry out physio-biochemical characteristics qualification to it, the results are shown in Table 1 and table 2, its 16S rRNA gene order comparison result is in table 3, and the phylogenetic tree data of structure are shown in accompanying drawing 1.
+: positive reaction;-: negative reaction
table 3 is based on the comparison result of microorganism strains xp 16S rRNA gene order
according to the phylogenetic tree of above-mentioned physio-biochemical characteristics, 16S rRNA gene order comparison result and structure, identify that this bacterial strain xp belongs to the food new sphingomonas bacteria of aromatic hydrocarbons (Novosphingobium aromaticivorans), bacterial strain xp at the preserving number of China typical culture collection center is: CCTCC No:M 2011453.
(3) application of Sphingomonas bacterial strain xp polyphenolic compound in degrading tobacco thin slice
If no special instructions, LB culture medium prescription used is following embodiment: containing 2.5g yeast extract paste, 5g peptone, 1g glucose in every 1000 ml deionized waters, adjust pH is 7.0.
Minimal medium formula used is: containing 2.44g Na in every 1000ml deionized water
2hPO
4, 1.52g KH
2pO
4, 0.5g (NH
4)
2sO
4, 0.2g MgSO
47H
2o, 0.05g CaCl
22H
2o and 10ml trace metal salts solution S L-4; Wherein, described trace metal salts solution S L-4 consists of: containing 0.50g EDTA, 0.20g FeSO in 1000ml deionized water
47H
2o and 100ml trace metal salts solution S L-6; Described trace metal salts solution S L-6 consists of: containing 0.10g ZnSO in 1000ml deionized water
47H
2o, 0.03g MnCl
24H
2o, 0.30g H
3bO
3, 0.20g CoCl
26H
2o, 0.01g CuCl
22H
2o, 0.02g NiCl
26H
2o and 0.03g Na
2moO
42H
2o.
Minimal medium and LB substratum are before use in 121 DEG C of sterilizing 15min.
embodiment 1:
the ability that Sphingol single-cell xp utilizes chlorogenic acid to carry out growing measures
1) seed culture fluid is prepared: recovered by bacterial strain xp LB substratum, then be inoculated in the minimal medium containing 0.5g/L chlorogenic acid, 24 h(rotating speed 200rpm cultivated by 30 DEG C of shaking tables) collected by centrifugation thalline afterwards, minimal medium cleans 3 times, use the resuspended thalline of minimal medium again, be adjusted to OD
600value is 3.0, and gained suspension is seed culture fluid.
2) energy for growth of Sphingol single-cell: joined by seed culture fluid in minimal medium, obtains initial OD
600value is the bacterium liquid of 0.15, then adds 0.5g/L chlorogenic acid, in 48h sampling, detects the turbidity of bacterium liquid and the content (determination of chlorogenic acid employing high performance liquid chromatography) of chlorogenic acid, the results are shown in Figure 2.
Conclusion: Sphingol single-cell xp can by the chlorogenic acid of concentration 0.5 g/L degraded more than 80% (see figure 3) in 48 h.
embodiment 2: the ability that Sphingol single-cell xp utilizes tobacco vat liquor to carry out growing measures
1) prepare tobacco vat liquor: take 1g offal and offal (1:1), add 60% ethanol 20ml after pulverizing in 50 DEG C of water-bath extraction 50min, every 10 min shake up once, and extraction terminates rear filtration, and filtrate is tobacco vat liquor.
2) seed culture fluid is prepared: recovered by bacterial strain xp LB substratum, then be inoculated in the minimal medium containing 0.1 ml/L tobacco vat liquor, 24 h(rotating speed 200rpm cultivated by 30 DEG C of shaking tables) collected by centrifugation thalline afterwards, minimal medium cleans 3 times, use the resuspended thalline of minimal medium again, be adjusted to OD
600value is 3.0, and gained suspension is seed culture fluid.
3) energy for growth of Sphingol single-cell: joined by seed culture fluid in minimal medium, obtains initial OD
600value is the bacterium liquid of 0.15, then adds 0.1 ml/L tobacco vat liquor, in different time sampling, detects the turbidity of bacterium liquid and the content of polyphenolic compound.
Result: Sphingol single-cell xp can by the chlorogenic acid degraded 60% in concentration 0.1 ml/L tobacco vat liquor in 48 h.
embodiment 3: Sphingomonas xp is after sole carbon source is cultivated with chlorogenic acid, on the mensuration of polyphenolic compound degradation capability in tobacco sheet and the impact on smoking property of cigarette mouthfeel
1) seed culture fluid is prepared: preparation method is with embodiment 1.
2) degradation capability of Sphingol single-cell xp: in 30 DEG C, 50ml seed culture fluid is evenly sprayed under humidity 60% condition 500g tobacco sheet surface, in different time sampling, detect the content of polyphenol in tobacco sheet.Control group uses 50ml distilled water to be evenly sprayed on 500g tobacco sheet surface.Test thin slice used and be selected from the thin board thin slice of Xuchang, Henan in 2012 gold.
The mensuration of polyphenol content selects Forint phenol method: get 1g thin slice and load centrifuge tube, add 60% ethanol 20ml and shake up once in 50 DEG C of every 10 min of water-bath lixiviate 50min(), filter, collect tobacco vat liquor.Get 50 μ g tobacco vat liquors in 25ml volumetric flask, add 3 ml forint phenol reagents, fully shake up, react 5 min, add 6 ml 20% sodium carbonate solutions, shake up, add water constant volume, and 30 DEG C of lucifuge placing response 30 min develop the color.Measure 710 nm place light absorption values.
Test-results confirms: Sphingol single-cell xp can by the polyphenolic compound degraded 7% in tobacco sheet in 3 weeks.Wherein, spray the tobacco sheet 20% of cultivation after 14 days make an addition to standard cigarettes after suck mouthfeel data in table 4.As can be seen from Table 4, control group: total score 40.96, test group: total score 42.75.To smoke panel test conclusion: control group: assorted gas is comparatively large, aroma quality, amount are general; Test group: assorted gas is less, sugariness increases, and concentration strength increases to some extent, and flue gas is soft and fine, and aroma quality, perfume quantity promote to some extent.
Table 4, utilize Sphingol single-cell xp to spray to cultivate the smoking property of cigarette mouthfeel of tobacco sheet after 14 days
| Aroma quality | Perfume quantity | Concentration | Soft and fine degree | Pleasant impression | Assorted gas | Stimulation degree | Total score | |
| Control group | 5.71 | 5.88 | 5.79 | 6.21 | 5.67 | 5.46 | 6.25 | 40.96 |
| Test group | 6.17 | 6.21 | 5.92 | 6.25 | 6.00 | 6.04 | 6.17 | 42.75 |
embodiment 4: Sphingol single-cell xp with tobacco vat liquor for sole carbon source cultivate after on the mensuration of polyphenolic compound degradation capability in tobacco sheet and the impact on smoking property of cigarette mouthfeel
1) seed culture fluid is prepared: preparation method is with embodiment 2.
2) degradation capability of Sphingol single-cell xp: in 30 DEG C, 50ml seed culture fluid is evenly sprayed under humidity 60% condition 500g tobacco sheet surface, in different time sampling, detect the content of polyphenol in tobacco sheet.Control group uses 50ml distilled water to be evenly sprayed on 500g tobacco sheet surface.Test thin slice used and take from the thin board thin slice of Xuchang gold in 2012.
Test-results confirms: with tobacco vat liquor for the Sphingol single-cell xp that sole carbon source is cultivated can by the polyphenolic compound degraded 7.2% in tobacco sheet in 3 weeks.Wherein, spray the tobacco sheet 20% of cultivation after 14 days and make an addition to the smoking property of cigarette mouthfeel data of standard cigarettes in table 5.As can be seen from Table 5, control group: total score 40.96, test group: total score 42.68.To smoke panel test conclusion: assorted gas is comparatively large, aroma quality, amount are general; Test group: assorted gas is less, sugariness increases, and concentration strength increases to some extent, and flue gas is soft and fine, and aroma quality, perfume quantity promote to some extent.
Table 5, utilize Sphingol single-cell xp to spray to cultivate the smoking property of cigarette mouthfeel of tobacco sheet after 14 days
| Aroma quality | Perfume quantity | Concentration | Soft and fine degree | Pleasant impression | Assorted gas | Stimulation degree | Total score | |
| Control group | 5.71 | 5.88 | 5.79 | 6.21 | 5.67 | 5.46 | 6.25 | 40.96 |
| Test group | 6.10 | 6.25 | 5.85 | 6.27 | 6.01 | 6.00 | 6.20 | 42.68 |
embodiment 5: Sphingol single-cell xp is applied in the water-soluble vat liquor of tobacco sheet the mensuration of polyphenolic compound degradation capability and the impact on smoking property of cigarette mouthfeel
1) seed culture fluid is prepared: preparation method is with embodiment 2.
2) degradation capability of Sphingol single-cell xp: put on by 100ml seed culture fluid under 30 DEG C of conditions in the water-soluble vat liquor (pH value nature, about 6.0) of 10L paper process thin slice, different time samples, and detects the content of polyphenol in tobacco sheet.Control group does not deal with.The vat liquor of test group and control group carries out respectively concentrating and coats the sheet base of tobacco sheet, is made into standard 500g tobacco sheet.
Test-results confirms: with tobacco vat liquor for the Sphingol single-cell xp that sole carbon source is cultivated can by the polyphenolic compound degraded 7.2% in tobacco sheet in 3 weeks.Wherein, seed culture fluid carries out concentrating to vat liquor and coats the sheet base of tobacco sheet, being made into standard tobacco thin slice after applying 4h.Tobacco sheet 20% is made an addition to and in standard cigarettes, sucks mouthfeel data in table 6.As can be seen from Table 6, control group: total score 40.96; Test group: total score 42.54.To smoke panel test conclusion: control group: assorted gas is comparatively large, aroma quality, amount are general; Test group: assorted gas is less, sugariness increases, and concentration strength increases to some extent, and flue gas is soft and fine, and aroma quality, perfume quantity promote to some extent.
Table 6, utilize Sphingol single-cell xp to apply paper process thin slice vat liquor 4h after smoking property of cigarette mouthfeel
| Aroma quality | Perfume quantity | Concentration | Soft and fine degree | Pleasant impression | Assorted gas | Stimulation degree | Total score | |
| Control group | 5.71 | 5.88 | 5.79 | 6.21 | 5.67 | 5.46 | 6.25 | 40.96 |
| Test group | 6.05 | 6.27 | 5.80 | 6.30 | 6.03 | 5.89 | 6.20 | 42.54 |
Claims (5)
1. the application of Sphingomonas bacterial strain xp in degrading tobacco thin slice in polyphenolic compound, is characterized in that, described Sphingomonas bacterial strain xp Classification And Nomenclature is
sphingomonassp., this bacterial strain xp at the preserving number of China typical culture collection center is: CCTCC No:M 2011453.
2. the application of Sphingomonas bacterial strain xp in degrading tobacco thin slice in polyphenolic compound as claimed in claim 1, it is characterized in that, Sphingomonas bacterial strain xp LB substratum is recovered, then be inoculated in the minimal medium containing chlorogenic acid or tobacco vat liquor, collected by centrifugation thalline after 28-32 DEG C of cultivation 2-24 h, after minimal medium cleaning 1-5 time, with the resuspended thalline of minimal medium, be adjusted to OD
600value is 2.0-5.0, and gained suspension is seed culture fluid; By seed culture fluid 28-32 DEG C, be evenly sprayed on tobacco sheet surface under humidity 55-65% condition or put in the water-soluble vat liquor of paper process thin slice.
3. the application of Sphingomonas bacterial strain xp in degrading tobacco thin slice in polyphenolic compound as claimed in claim 2, it is characterized in that, described containing in the minimal medium of chlorogenic acid or tobacco vat liquor, chlorogenic acid concentration is 0.2-1.0g/L, and tobacco vat liquor concentration is 0.05-0.2 ml/L.
4. the application of Sphingomonas bacterial strain xp in degrading tobacco thin slice in polyphenolic compound as described in Claims 2 or 3, it is characterized in that, described tobacco vat liquor obtains through following method: get 1g offal and offal, 55-65% ethanol 10-30ml is added in 45-55 DEG C of water-bath extraction 30-60min after pulverizing, extraction terminates rear filtration, and filtrate is tobacco vat liquor.
5. the application of Sphingomonas bacterial strain xp in degrading tobacco thin slice in polyphenolic compound as described in Claims 2 or 3, it is characterized in that, the formula of described minimal medium is: containing 2.44g Na in every 1000ml deionized water
2hPO
4, 1.52g KH
2pO
4, 0.5g (NH
4)
2sO
4, 0.2g MgSO
47H
2o, 0.05g CaCl
22H
2o and 10ml trace metal salts solution S L-4; Wherein, described trace metal salts solution S L-4 consists of: containing 0.50g EDTA, 0.20g FeSO in 1000ml deionized water
47H
2o and 100ml trace metal salts solution S L-6; Described trace metal salts solution S L-6 consists of: containing 0.10g ZnSO in 1000ml deionized water
47H
2o, 0.03g MnCl
24H
2o, 0.30g H
3bO
3, 0.20g CoCl
26H
2o, 0.01g CuCl
22H
2o, 0.02g NiCl
26H
2o and 0.03g Na
2moO
42H
2o.
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Address after: 450000 Yulin South Road, Henan, Zheng Dong, No. 16 South Road, Zhengzhou Patentee after: China Tobacco Henan Industrial Co., Ltd. Address before: 450000 Zhengzhou agricultural road, Henan, No. 29 Patentee before: China Tobacco Henan Industrial Co., Ltd. |