CN103773727A - Application of sphingomonas strain xp in degrading polyphenolic compounds in tobacco stems - Google Patents
Application of sphingomonas strain xp in degrading polyphenolic compounds in tobacco stems Download PDFInfo
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- CN103773727A CN103773727A CN201410047482.6A CN201410047482A CN103773727A CN 103773727 A CN103773727 A CN 103773727A CN 201410047482 A CN201410047482 A CN 201410047482A CN 103773727 A CN103773727 A CN 103773727A
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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 Sphingol single-cell strain xp and the application aspect polyphenolic compound in degraded offal.
Background technology
Sphingol single-cell distributed pole in environment is extensive, and severe environment are had to very strong patience (as all once found the existence of Sphingol single-cell in the extreme environment such as polar region, severe radiation).The degraded substrate scope of Sphingol single-cell is wide, as the degraded of 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 that is concerned and studies in recent years.
Offal is the important component part of tobacco leaf, after separating, can make the offal that accounts for tobacco leaf gross weight 25% left and right by beating and double roasting.The character of the chemical composition of offal is basic identical with blade, but variant on content, and the stem that causes making with the offal deficiency of sounding in the process burning and sucking, inhales taste flat, and pungency and strength are less, and the gas of mixing is heavier, the especially wooden property gas weight of mixing.Mainly to be made into the stem that height is filled for the utilization of offal at present, as the fillibility raw material of cigarette, thus the stronger effect of making of performance stem, air flow when increasing the filling value of pipe tobacco and burning and sucking, reaches and reduces consumption, improves the combustible object of cigarette.This mode does not change its chemical composition, therefore reduces wooden assorted gas and irritating degree limited, causes the utilization ratio of offal lower.
Tobacco polyphenol is that its content in tobacco is higher, is about the 0.5-2.5% of cured leaf weight at the synthetic aromatic cycle compound with one or more hydroxyls of cigarette strain leaf and two main positions of stem.The polyphenolic compound of finding in tobacco comprises approximately 135 kinds altogether of tannins (isomer, coffic acid and the quinic acid of chlorogenic 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 classes.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 fragrance precursor in tobacco leaf, and tobacco leaf color and cigarette flavor after polyphenol content and baking are closely related.In smoking process, polyphenol and pyrolysis product thereof are transferred in flue gas, and smoke perfume is had a direct impact.Under rational modulation, ageing condition, plant polyphenol compounds generates many kinds of substance through series reaction degraded, these degraded products can be given the fragrance of tobacco product gracefulness, increase the perfume quantity of tobacco product, have extremely important effect to improving the quality of 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, and the oxidable decomposition of polyphenolic compound generates some compounds that 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 of improving tobacco product.But when polyphenolic compound content exceeds certain limit, tobacco leaf is in various degree with multiple quality defect, as: there are blue foreign smell and pungency, fragrance matter dullness, perfume quantity deficiency etc., can not be directly used for producing cigarette.What is more important, excessive polyphenolic substance is toxic to human body, can affect smoker's health.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, the utmost point need to be developed the microorganism strains that can better bring into play polyphenolic compound degradation effect under low temperature and low humidity condition at present.
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 degraded offal, good degrading effect.
For achieving the above object, the present invention adopts following technical scheme:
A kind of Sphingol single-cell strain xp, its Classification And Nomenclature is
sphingomonassp
., this bacterial strain xp at the preserving number at Chinese Typical Representative 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).
Described Sphingol single-cell strain xp is the application aspect polyphenolic compound in degraded offal.
Described Sphingol single-cell strain xp is the application aspect polyphenolic compound in degraded offal, be specially: by Sphingol single-cell strain xp LB substratum recovery (cultivating 24h for 30 ℃), then be inoculated in the minimal medium that contains chlorogenic acid or offal vat liquor, centrifugal collection thalline after 28-32 ℃ of cultivation 2-24 h, minimal medium cleans after 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; Seed culture fluid being evenly sprayed under 28-32 ℃, humidity 55-65% condition to stalk filaments of tobacco stalks surface or joining bubble at 28-32 ℃ has in the aqueous solution of offal.
Concrete, described in contain chlorogenic acid or offal vat liquor minimal medium in, chlorogenic acid concentration is 0.2-1.0g/L, offal vat liquor concentration is 0.05-0.2 ml/L.Chlorogenic acid or offal vat liquor are in order to the sole carbon source as Sphingomonas xp growth.While cultivation in the minimal medium that Sphingol single-cell xp of the present invention is 0.2-1.0g/L at chlorogenic acid addition, growing state is good, and the chlorogenic acid of greater concn can suppress the growth of Sphingol single-cell xp.
Described offal vat liquor can obtain through following method: get 1g offal, after pulverizing, add 55-65%(volumn concentration) ethanol shakes up once in every 10 min of 45-55 ℃ of water-bath extraction 30-60min(), extraction finishes rear filtration, and filtrate is offal vat liquor.
Preferably, the formula of described minimal medium is: in every 1000ml deionized water, contain 2.44g Na
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: in 1000ml deionized water, contain 0.50g EDTA, 0.20g FeSO
47H
2o and 100ml trace metal salts solution S L-6; Described trace metal salts solution S L-6 consists of: in 1000ml deionized water, contain 0.10g ZnSO
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 Sphingol single-cell strain xp(Sphingomonas sp. provided by the present invention) in minimal medium, utilize chlorogenic acid or offal vat liquor for sole carbon source growth after, the polyphenolic compound that can degrade in offal.Utilizing Sphingol single-cell xp(Sphingomonas sp.) be sprayed on stalk filaments of tobacco stalks surface, the polyphenolic compound content degradation rate after two weeks in offal exceedes 25%; Be applied in the bubble stalk aqueous solution and more than 4 hours the polyphenolic compound content in offal can be degraded more than 30%.In the method for alcoholizing utilizing at present, the required alcoholization time is longer, tends to affect the cycle of product.The present invention, compared with existing method for alcoholizing, has advantages of fast degradation, and this has more advantage for the practical application in cured tobacco production alcoholization process.
Accompanying drawing explanation
Fig. 1 is the phylogenetic tree building based on microorganism strains xp 16S rRNA gene order;
Fig. 2 is that Sphingomonas xp is at the growing state containing 48h in the minimal medium of chlorogenic acid;
Fig. 3 is bacteria concentration while being OD3.0, and Sphingomonas xp is containing the degraded situation to chlorogenic acid in the minimal medium of different chlorogenic acids.
Embodiment
(1) Sphingol single-cell (
sphingomonassp
.) screening of bacterial strain xp:
Because chlorogenic acid is the most representative polyphenols in tobacco, the application, using chlorogenic acid as substrate, is made into agar plate.Under 30 ℃, the condition of 24hr, use the vega soil aqueous solution to carry out coated plate cultivation, plate culture, after the subzero treatment 24-72hr of 4 ℃, is inverted the decomposition effect of microscopic examination bacterium to chlorogenic acid by fluorescence.Because chlorogenic acid is at the lower aobvious blue-fluorescence of UV-light (365nm), the substratum with the periphery of bacterial colonies of chlorogenic acid capacity of decomposition dies down than the fluorescence in background is thin out.In the blue-fluorescence background of chlorogenic acid, can directly separate the bacterial strain not being bacterial contamination.Measure according to the relation of the activity of chlorogenic acid enzyme and transparent circle diameter the chlorogenic acid enzyme activity that different strains produces.The enterprising row filter of agar plate at pH value 4.0-6.0 is cultivated, and can obtain acid resistance bacterial strain xp.
(2) Sphingol single-cell (
sphingomonassp
.) evaluation of bacterial strain xp:
The bacterial strain xp that screening obtains is Gram-negative bacteria, without spore, with one-sided raw polar flagellum motion, is yellow.Entrust Chinese Typical Representative culture collection center to carry out physio-biochemical characteristics evaluation 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.
the comparison result of table 3 based on 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 at Chinese Typical Representative culture collection center is: CCTCC No:M 2011453.
(3) application of Sphingol single-cell strain xp polyphenolic compound in degraded offal
If no special instructions, LB culture medium prescription used is following embodiment: in every 1000 ml deionized waters, containing 2.5g yeast extract paste, 5g peptone, 1g glucose, adjust pH is 7.0.
Minimal medium formula used is: in every 1000ml deionized water, contain 2.44g Na
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: in 1000ml deionized water, contain 0.50g EDTA, 0.20g FeSO
47H
2o and 100ml trace metal salts solution S L-6; Described trace metal salts solution S L-6 consists of: in 1000ml deionized water, contain 0.10g ZnSO
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 ℃ of sterilizing 15min.
embodiment 1:
sphingol single-cell xp utilizes the ability that chlorogenic acid is grown to measure
1) prepare seed culture fluid: by the recovery of bacterial strain xp LB substratum, then be inoculated in the minimal medium containing 0.5g/L chlorogenic acid, 30 ℃ of shaking tables are cultivated 24 h(rotating speed 200rpm) rear centrifugal collection thalline, minimal medium cleans 3 times, use again the resuspended thalline of minimal medium, be adjusted to OD
600value is 3.0, and gained suspension is seed culture fluid.
2) energy for growth of Sphingol single-cell: seed culture fluid is joined in minimal medium, obtain initial OD
600value is 0.15 bacterium liquid, 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 be by the chlorogenic acid degraded 80% above (see figure 3) of concentration 0.5 g/L in 48 h.
embodiment 2: Sphingol single-cell xp utilizes the ability that offal vat liquor is grown to measure
1) prepare offal vat liquor: take 1g offal, add 60% ethanol 20ml in 50 ℃ of water-bath extraction 50min after pulverizing, every 10 min shake up once, and extraction finishes rear filtration, and filtrate is offal vat liquor.
2) prepare seed culture fluid: by the recovery of bacterial strain xp LB substratum, then be inoculated into containing in the minimal medium of 0.1 ml/L offal vat liquor, 30 ℃ of shaking tables are cultivated 24 h(rotating speed 200rpm) rear centrifugal collection thalline, minimal medium cleans 3 times, use again the resuspended thalline of minimal medium, be adjusted to OD
600value is 3.0, and gained suspension is seed culture fluid.
3) energy for growth of Sphingol single-cell: seed culture fluid is joined in minimal medium, obtain initial OD
600value is 0.15 bacterium liquid, then adds 0.1 ml/L offal vat liquor, in different time sampling, detects the turbidity of bacterium liquid and the content of polyphenolic compound.
Result: Sphingol single-cell xp can be by the chlorogenic acid degraded 60% in concentration 0.1 ml/L offal vat liquor in 48 h.
embodiment 3: after Sphingomonas xp cultivates take chlorogenic acid as sole carbon source, the mensuration on polyphenolic compound degradation capability in offal and offal is sucked to the impact of mouthfeel
1) prepare seed culture fluid: preparation method is with embodiment 1.
2) degradation capability of Sphingol single-cell xp: under 30 ℃, humidity 60% condition, 50ml seed culture fluid is evenly sprayed on to 500g stalk filaments of tobacco stalks surface, in different time sampling, detects the content of polyphenol in offal.Control group uses 50ml distilled water to be evenly sprayed on 500g stalk filaments of tobacco stalks surface.Test offal used and be selected from Henan offal in 2009.
The mensuration of polyphenol content is selected Forint phenol method: get 1g offal and pack centrifuge tube into, add 60% ethanol 20ml to shake up once in 50 ℃ of every 10 min of water-bath lixiviate 50min(), filter, collect offal vat liquor.Get 50 μ g offal vat liquors in 25ml volumetric flask, add 3 ml forint phenol reagents, fully shake up, reaction 5 min, add 6 ml 20% sodium carbonate solutions, shake up the constant volume that adds water, 30 ℃ of lucifuge placing response 30 min colour developings.Measure 710 nm place light absorption values.
Test-results confirms: Sphingol single-cell xp can be by the polyphenolic compound degraded 7% in stalk filaments of tobacco stalks in 3 weeks.Wherein, spray after the offal 20% of cultivating after 14 days is added into standard cigarettes and suck mouthfeel data in table 4.As can be seen from Table 4, control group: total score 40.96; Test group: total score 42.33.The conclusion of smokeing panel test: control group: assorted gas is larger, fragrance matter, 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 fragrance matter, perfume quantity promote to some extent.
Table 4, utilize Sphingol single-cell xp to spray to cultivate the offal of offal after 14 days and suck mouthfeel
| ? | Fragrance matter | 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 | 6.07 | 6.20 | 5.80 | 6.25 | 5.70 | 6.04 | 6.27 | 42.33 |
embodiment 4: the mensuration on polyphenolic compound degradation capability in offal after Sphingol single-cell xp cultivates take offal vat liquor as sole carbon source and offal is sucked to the impact of mouthfeel
1) prepare seed culture fluid: preparation method is with embodiment 2.
2) degradation capability of Sphingol single-cell xp: under 30 ℃, humidity 60% condition, 50ml seed culture fluid is evenly sprayed on to 500g stalk filaments of tobacco stalks surface, in different time sampling, detects the content of polyphenol in offal.Control group uses 50ml distilled water to be evenly sprayed on 500g stalk filaments of tobacco stalks surface.Test offal used and be selected from Henan offal in 2009.
Test-results confirms: the Sphingol single-cell xp cultivating take offal vat liquor as sole carbon source can be by the polyphenolic compound degraded 7.2% in offal in 3 weeks.Wherein, spray after the offal 20% of cultivating after 14 days is added into standard cigarettes and suck mouthfeel data in table 5.As can be seen from Table 5, control group: total score 40.96; Test group: total score 42.57.The conclusion of smokeing panel test: control group: fragrance matter, 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 fragrance matter, perfume quantity promote to some extent.
Table 5, utilize Sphingol single-cell xp to spray to cultivate the offal of offal after 14 days and suck mouthfeel
| ? | Fragrance matter | 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 | 6.13 | 6.15 | 5.83 | 6.26 | 5.88 | 6.03 | 6.29 | 42.57 |
Claims (5)
1. Sphingol single-cell strain xp application aspect polyphenolic compound in degraded offal, is characterized in that, described Sphingol single-cell strain xp Classification And Nomenclature is
sphingomonassp., this bacterial strain xp at the preserving number at Chinese Typical Representative culture collection center is: CCTCC No:M 2011453.
2. Sphingol single-cell strain xp application aspect polyphenolic compound in degraded offal as claimed in claim 1, it is characterized in that, by LB substratum recovery for Sphingol single-cell strain xp, then be inoculated in the minimal medium that contains chlorogenic acid or offal vat liquor, centrifugal collection thalline after 28-32 ℃ of cultivation 2-24 h, minimal medium cleans after 1-5 time, with the resuspended thalline of minimal medium, is adjusted to OD
600value is 2.0-5.0, and gained suspension is seed culture fluid; Seed culture fluid being evenly sprayed under 28-32 ℃, humidity 55-65% condition to stalk filaments of tobacco stalks surface or joining bubble at 28-32 ℃ has in the aqueous solution of offal.
3. Sphingol single-cell strain xp application aspect polyphenolic compound in degraded offal as claimed in claim 2, it is characterized in that, in the described minimal medium that contains chlorogenic acid or offal vat liquor, chlorogenic acid concentration is 0.2-1.0g/L, and offal vat liquor concentration is 0.05-0.2 ml/L.
4. Sphingol single-cell strain xp application aspect polyphenolic compound in degraded offal as described in claim 2 or 3, it is characterized in that, described offal vat liquor obtains through following method: get 1g offal, after pulverizing, add 60-70% ethanol 10-30ml in 45-55 ℃ of water-bath extraction 30-60min, extraction finishes rear filtration, and filtrate is offal vat liquor.
5. Sphingol single-cell strain xp application aspect polyphenolic compound in degraded offal as described in claim 2 or 3, is characterized in that, the formula of described minimal medium is: in every 1000ml deionized water, contain 2.44g Na
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: in 1000ml deionized water, contain 0.50g EDTA, 0.20g FeSO
47H
2o and 100ml trace metal salts solution S L-6; Described trace metal salts solution S L-6 consists of: in 1000ml deionized water, contain 0.10g ZnSO
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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Cited By (1)
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| CN109234180A (en) * | 2017-07-10 | 2019-01-18 | 伽蓝（集团）股份有限公司 | Sphingol single-cell, its extracellular products and its preparation method and application |
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| CN103060420A (en) * | 2012-12-25 | 2013-04-24 | 天津大学 | Interaction of polyphenol pollutants in the process of biodegradation and a research method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109234180A (en) * | 2017-07-10 | 2019-01-18 | 伽蓝（集团）股份有限公司 | Sphingol single-cell, its extracellular products and its preparation method and application |
| CN109234180B (en) * | 2017-07-10 | 2022-10-14 | 伽蓝（集团）股份有限公司 | Sphingomonas, extracellular products thereof, and preparation method and application thereof |
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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. |