CN103387947A - JY-Q bacterial strain of Pseudomonassp. and application of bacterial strain in degradation of nicotine in concentrated liquor of reconstituted tobacco - Google Patents

JY-Q bacterial strain of Pseudomonassp. and application of bacterial strain in degradation of nicotine in concentrated liquor of reconstituted tobacco Download PDF

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Publication number
CN103387947A
CN103387947A CN2013103206798A CN201310320679A CN103387947A CN 103387947 A CN103387947 A CN 103387947A CN 2013103206798 A CN2013103206798 A CN 2013103206798A CN 201310320679 A CN201310320679 A CN 201310320679A CN 103387947 A CN103387947 A CN 103387947A
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bacterial strain
nicotine
pseudomonas
application
degradation
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CN103387947B (en
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舒明
高阳
汪华文
周国俊
周强
陶丰
唐恒杰
边红燕
钟卫鸿
焦洋
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China Tobacco Zhejiang Industrial Co Ltd
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Abstract

The invention relates to the field of microorganisms, and in particular relates to a novel effective nicotine degrading bacterium and an application of the bacterium in degradation of nicotine in concentrated liquor of reconstituted tobacco. The JY-Q bacterial strain of Pseudomonassp. is preserved in China Center For Type Culture Collection (CCTCC), wherein the address is Wuhan University, Wuhan, China, the preserving data is 28th, May, 2013, and the preserving number is CCTCCNO:M2013236. The bacterial strain provided by the invention is screened from a tobacco waste mother liquid provided by Hangzhou Liqun environment-friendly paper company, nicotine with the content of 5g/L in a minimal medium can be completely degraded within 24 hours, and the bacterial strain has wide industrialized application prospect in a production process of reconstituted tobacco by a paper-making process.

Description

Pseudomonas JY-Q bacterial strain and be used for the application of degraded reproduced tobacco concentrated liquid Nicotine
Technical field
The present invention relates to microorganism field, relate in particular to the new Nicotine efficient degrading bacteria of a strain and the Nicotine of the reproduced tobacco concentrated liquid that is used for degrading.
Background technology
Nicotine (Nicotine, nicotine), chemical name are 1-methyl-2 (3-pyridyl)-tetramethyleneimine, account for main component in tobacco plant as natural alkaloid, are one of important pollutents of environment.Some microorganisms can utilize Nicotine by different pathways metabolisms (as the pyridine approach, pyrroles's approach, demethylation approach etc.), and the some of them bacterial strain has using value.In recent years, utilize the microbiological deterioration Nicotine to become one of focus of domestic and international research.
Nicotine content in reproduced tobacco concentrated liquid is higher is present thin slice enterprise practical problems anxious to be resolved.In the papermaking-method reconstituted tobaccos production technique, offal and offal again return and are coated onto on reconstituted tobacco sheet base after being become mother liquor, be condensed into concentrated solution by water extraction.And contain the nicotine of the 2-3 % that has an appointment in offal, easily cause the nicotine content of sheet product higher and can not regulate and control.At present main offal concentrated solution by low nicotine offal concentrated solution and high-nicotine content match well realize, but this allotment by physical means (as routine in the ginseng proportioning that increases the offal mother liquor) is larger to the negative impact of thin slice quality.Thereby need research and utilize other technologies (as biochemical technology) directly to process concentrated solution, realization is adjustable controlled to the Nicotine index.
Summary of the invention
In order to solve above-mentioned technical problem, an object of the present invention is to provide a pseudomonas ( PseudomonasSp.) JY-Q bacterial strain, this bacterial strain are that separation screening obtains from the offal mother liquor of papermaking-method reconstituted tobaccos line of production technology, and can be applicable to the biological degradation of Nicotine in the concentrated solution of line of production technology.Two purposes of the present invention are to provide the application of this bacterial strain in the degraded Nicotine.Two purposes of the present invention are to provide the enrichment medium for described pseudomonas JY-Q bacterial strain,
In order to realize first above-mentioned purpose, the present invention has adopted following technical scheme:
Pseudomonas ( PseudomonasSp.) JY-Q bacterial strain, this bacterial strain are preserved in Chinese Typical Representative culture collection center (CCTCC), address: China. Wuhan. and Wuhan University, preservation date: on May 28th, 2013, deposit number: CCTCC NO:M 2013236.
In order to realize second above-mentioned purpose, the present invention has adopted following technical scheme: the application of above-mentioned pseudomonas JY-Q bacterial strain, this bacterial strain is used for the degraded of Nicotine.
As preferably, this bacterial strain is used for the degraded of reproduced tobacco concentrated liquid Nicotine.As preferred again, the pH of described reproduced tobacco concentrated liquid is 6.5-7.0.
In order to realize the 3rd above-mentioned purpose, the present invention has adopted following technical scheme:
The enrichment medium that is used for described pseudomonas JY-Q bacterial strain, is characterized in that this substratum comprises that following component forms: Na 2HPO 45.57 gL -1, KH 2PO 42.44 gL -1, (NH 4) 2 SO 41 gL -1, MgCl 26H 2O 0.2 gL -1, MnCl 24H 2O 0.0004 gL -1, FeCl 36H 2O 0.001 gL -1, CaCl 20.001 gL -1, Nicotine 2 gL -1, pH=7.0.
Bacterial strain of the present invention is to screen and obtain from the offal mother liquor that Hangzhou profit group rings guarantor paper company provides, and 24h can be that the Nicotine of 5 g/L is degradable with content in minimum medium, and industrial applications prospect is widely arranged in the papermaking-method reconstituted tobaccos production technique.
Description of drawings
Fig. 1. Electronic Speculum picture Electron micrograph (25000 *).
Fig. 2. the genome electrophorogram.
Fig. 3. 16s rDNA PCR electrophorogram.
Fig. 4. based on the phylogenetic tree of bacterial strain 16S rDNA sequence construct.
Fig. 5. different nicotine concentrations are on Pseudomonas sp. JY-Q degraded impact figure.
Fig. 6. differing temps is on Pseudomonas sp.JY-Q degraded impact figure.
Fig. 7. different pH are on Pseudomonas sp.JY-Q degraded impact figure.
Fig. 8. the Pseudomonas sp.JY-Q application drawing of degrading in 5% concentrated solution.
The microbial preservation statement
Pseudomonas ( PseudomonasSp.) JY-Q bacterial strain, this bacterial strain are preserved in Chinese Typical Representative culture collection center (CCTCC), address: China. Wuhan. and Wuhan University, preservation date: on May 28th, 2013, deposit number: CCTCC NO:M 2013236.
Embodiment
Embodiment 1 bacterial strain screening and evaluation
1.1 bacterial strain screening
1.1 .1 sample
Offal mother liquor in the papermaking-method reconstituted tobaccos production technique, protecting paper company by Hangzhou profit group rings provides.
1.1 the .2 bacterial strain screening separates
Substratum
Basic inorganic salt substratum (basic inorganic salt medium, BSM): Na 2HPO 45.57 gL -1, KH 2PO 42.44 gL -1, K 2SO 41 gL -1, MgCl 26H 2O 0.2 gL -1, MnCl 24H 2O 0.0004 gL -1, FeCl 36H 2O 0.001 gL -1, CaCl 20.001 gL -1, Nicotine (content interpolation as required), pH=7.0.
Enrichment medium: Na 2HPO 45.57 gL -1, KH 2PO 42.44 gL -1, (NH 4) 2 SO 41 gL -1, MgCl 26H 2O 0.2 gL -1, MnCl 24H 2O 0.0004 gL -1, FeCl 36H 2O 0.001 gL -1, CaCl 20.001 gL -1, Nicotine 2 gL -1, pH=7.0.
Substratum is using after sterilizing 30 min under 115 ℃.
The screening and separating of nicotine degradation bacterium: add 10 mL offal mother liquors in 100 mL sterilized waters, 180 rmin -1Lower vibration 10 min; Get 10 mL leach liquors and add (nicotine content 2g/L) in 100 mL enrichment mediums to, at 180 rmin -1, enrichment culture 5 days under 30 ℃ of conditions.Enrichment culture liquid is diluted to 10 -1-10 -5Doubly, be coated on (nicotine content 2g/L) on the BSM nutrient agar, cultivate 48 h under 30 ℃.Picking list bacterium colony, line are on fresh BSM nutrient agar, until obtain the bacterium of purifying.The bacterium of the purifying that will obtain again is seeded in liquid B SM, and 30 ℃ of shaking tables are cultivated, and observes nutrient solution and changes.
1.2 identification of strains
1.2.1 nicotine degradation bacterium PseudomonaS sp. JY-Q Morphological Identification
As shown in Figure 1, bacterium colony is light yellow, and metalluster is arranged, circle, and projection, moist, neat in edge, without gemma, the electron microscopic observation thalline is shaft-like, and size is (0.6 ~ 0.8) μ m * (2.0 ~ 1.2) μ m, and end is given birth to flagellum, several 1 ~ 2 of flagellum.
1.2 .2 nicotine degradation bacterium PseudomonaS sp. JY-Q Physiology and biochemistry is identified
Bacterial strain can be take Nicotine as the sole carbon nitrogenous source and energy growth, and aerobic, gramstaining is negative, and 4 ℃ of growths or not for 42 ℃, can utilize glucose, fructose, and catalase test is positive, and methyl red test is positive.
1.2 .3 nicotine degradation bacterium PseudomonaS sp. JY-Q molecular biology identification
16s rDNA order-checking:
E.Z.N.A.TM Bacterial DNA Kit test kit extracts genome, take the genome that extracts as template, (its forward primer sequence: 5 ' AGAGTTTGATCCTGGCTCAG-3 ', the reverse primer sequence is 16s rDNA universal primer: 5 ' CGGCTACCTTGTTACGACTTC-3 ') carry out pcr amplification.
PCR 50 μ L systems:
Takara Taq 0.6μL
10× PCR Buffer （Mg 2+） 5μL
dNTP 4μL
Template 2 μ L
Primer 11 μ L
Primer 21 μ L
ddH 2O 36.4μL。
The PCR program:
1. 94℃ 4min
2. 94℃ 50s
3. 52℃ 1min
4. 72℃ 2min
5. circulation 2---4 step 29 time
6. 72℃ 10min
7. 16 ℃ of constant temperature.
Fig. 2 is the genome electrophorogram, Line 1 DNA marker; Line 2 genome; Fig. 3 is 16s rDNA PCR electrophorogram, Line 1 DNA marker; Line 2 16s rDNA PCR fragment.
16s rDNA sequencing result:
TGGTACCGTCCTCCCGAAGGTTAGACTAGCTACTTCTGGTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTGTGAGATTAGCTCCACCTCGCGGCTTGGCAACCCTCTGTACCGACCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGCATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAAATCTCAAGGATTCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTACCGTACTCTAGCTTGCCAGTTTTGGATGCAGTTCCCAGGTTGAGCCCGGGGCTTTCACATCCAACTTAACAAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAACAGCAAGGTATTAACTTACTGCCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTGGTGAGCCATTACCCCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCGTTCCTTTCGAAACGTTGTCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACCCGTCCGCCGCTGAATCAAGGAGCAAGCTCCCGTCATCCGCTCGACTTGCATGTGT。
Sequencing result is committed to genebank, obtains accession number KC963965.
And bacterial strain is saved to Chinese Typical Representative culture collection center, preserving number is: CCTCC M 2013236.
Through form, Physiology and biochemistry, 16S rDNA homology analysis, identify that this bacterial strain belongs to Pseudomonas., called after Pseudomonas sp. JY-Q.
Embodiment 2 bacterial classifications PseudomonasSp. the degradation characteristic of JY-Q
2.1 different nicotine concentrations pair PseudomonasSp. JY-Q degraded impact
In the 250mL triangular flask, 1mL bacterium liquid is inoculated in the minimum medium that 50mL contains different nicotine contents (1g/L, 2g/L, 3g/L, 4g/L, 5g/L).180r/min, 37 ℃ of shaking tables are cultivated.The degraded of METHOD FOR CONTINUOUS DETERMINATION Nicotine and the upgrowth situation of thalline, as shown in Figure 5. PseudomonasSp. JY-Q has higher Nicotine tolerance and degradation efficiency.12h can be that 2-3 g/L Nicotine is degradable with content in minimum medium, and 24 h can be that 5 g/L Nicotines are degradable with content in minimum medium.
2.2 differing temps pair PseudomonasThe sp.JY-Q impact of degrading
In the 250mL triangular flask, 1mL bacterium liquid is inoculated in the minimum medium that the 50mL nicotine content is 2g/L.180r/min, and differing temps (25 ℃, 30 ℃, 37 ℃) the shaking table cultivation.The degraded of METHOD FOR CONTINUOUS DETERMINATION Nicotine and the upgrowth situation of thalline, as shown in Figure 6. PseudomonasSp. JY-Q is at 25 ℃, 30 ℃, in 37 ℃ of three temperature ranges, under 37 ℃ degraded fastest, 12h can be that the minimum medium of 2g/L is degradable with nicotine content.But differing temps is little on the impact of strains for degrading Nicotine.
2.3 different pH couple PseudomonasThe sp.JY-Q impact of degrading
In the 250mL triangular flask, 1mL bacterium liquid is inoculated in the minimum medium that the 50mL nicotine content is 2g/L.180r/min, 37 ℃, different pH(5,6,6.5,7,7.5,8,9) the shaking table cultivation.Detect the degraded of Nicotine and the upgrowth situation of thalline while cultivating 12h, as shown in Figure 7. PseudomonasSp. JY-Q degraded Nicotine optimal pH is 6.5-7, as pH less than 6 or greater than 7.5, all can cause larger restraining effect to the strains for degrading Nicotine.
2.4 PseudomonasThe degraded application of sp.JY-Q in concentrated solution
In the 250mL triangular flask, appropriate bacterium liquid is inoculated in 5% concentrated solution that the 50mL nicotine content is 1.139g/L.180r/min, 37 ℃ of shaking tables are cultivated.The degraded situation of METHOD FOR CONTINUOUS DETERMINATION Nicotine, as shown in Figure 8. PseudomonasSp.JY-Q has Degradation to Nicotine in 5% concentrated solution, and 12h can be with its whole degradeds.
Sequence table
＜110〉China Tobacco Industry Zhejiang Co., Ltd.
＜120〉pseudomonas JY-Q bacterial strain and be used for the application of degraded reproduced tobacco concentrated liquid Nicotine
<160>1
<210> 1
<211> 1412
<212> 16s rDNA
＜213〉pseudomonas ( Pseudomonas sp.) the JY-Q bacterial strain
<400> 1
1 TGGTACCGTC CTCCCGAAGG TTAGACTAGC TACTTCTGGT GCAACCCACT CCCATGGTGT
61 GACGGGCGGT GTGTACAAGG CCCGGGAACG TATTCACCGC GACATTCTGA TTCGCGATTA
121 CTAGCGATTC CGACTTCACG CAGTCGAGTT GCAGACTGCG ATCCGGACTA CGATCGGTTT
181 TGTGAGATTA GCTCCACCTC GCGGCTTGGC AACCCTCTGT ACCGACCATT GTAGCACGTG
241 TGTAGCCCAG GCCGTAAGGG CCATGATGAC TTGACGTCAT CCCCACCTTC CTCCGGTTTG
301 TCACCGGCAG TCTCCTTAGA GTGCCCACCA TAACGTGCTG GTAACTAAGG ACAAGGGTTG
361 CGCTCGTTAC GGGACTTAAC CCAACATCTC ACGACACGAG CTGACGACAG CCATGCAGCA
421 CCTGTGTCAG AGTTCCCGAA GGCACCAATC CATCTCTGGA AAGTTCTCTG CATGTCAAGG
481 CCTGGTAAGG TTCTTCGCGT TGCTTCGAAT TAAACCACAT GCTCCACCGC TTGTGCGGGC
541 CCCCGTCAAT TCATTTGAGT TTTAACCTTG CGGCCGTACT CCCCAGGCGG TCAACTTAAT
601 GCGTTAGCTG CGCCACTAAA ATCTCAAGGA TTCCAACGGC TAGTTGACAT CGTTTACGGC
661 GTGGACTACC AGGGTATCTA ATCCTGTTTG CTCCCCACGC TTTCGCACCT CAGTGTCAGT
721 ATCAGTCCAG GTGGTCGCCT TCGCCACTGG TGTTCCTTCC TATATCTACG CATTTCACCG
781 CTACACAGGA AATTCCACCA CCCTCTACCG TACTCTAGCT TGCCAGTTTT GGATGCAGTT
841 CCCAGGTTGA GCCCGGGGCT TTCACATCCA ACTTAACAAA CCACCTACGC GCGCTTTACG
901 CCCAGTAATT CCGATTAACG CTTGCACCCT CTGTATTACC GCGGCTGCTG GCACAGAGTT
961 AGCCGGTGCT TATTCTGTCG GTAACGTCAA AACAGCAAGG TATTAACTTA CTGCCCTTCC
1021 TCCCAACTTA AAGTGCTTTA CAATCCGAAG ACCTTCTTCA CACACGCGGC ATGGCTGGAT
1081 CAGGCTTTCG CCCATTGTCC AATATTCCCC ACTGCTGCCT CCCGTAGGAG TCTGGACCGT
1141 GTCTCAGTTC CAGTGTGACT GATCATCCTC TCAGACCAGT TACGGATCGT CGCCTTGGTG
1201 AGCCATTACC CCACCAACTA GCTAATCCGA CCTAGGCTCA TCTGATAGCG CAAGGCCCGA
1261 AGGTCCCCTG CTTTCTCCCG TAGGACGTAT GCGGTATTAG CGTTCCTTTC GAAACGTTGT
1321 CCCCCACTAC CAGGCAGATT CCTAGGCATT ACTCACCCGT CCGCCGCTGA ATCAAGGAGC
1381 AAGCTCCCGT CATCCGCTCG ACTTGCATGT GT

Claims (5)

  1. Pseudomonas ( PseudomonasSp.) JY-Q bacterial strain, this bacterial strain are preserved in Chinese Typical Representative culture collection center (CCTCC), address: China. Wuhan. and Wuhan University, preservation date: on May 28th, 2013, deposit number: CCTCC NO:M 2013236.
  2. 2. the application of pseudomonas JY-Q bacterial strain claimed in claim 1, this bacterial strain is used for the degraded of Nicotine.
  3. 3. the application of pseudomonas JY-Q bacterial strain according to claim 2 is characterized in that: this bacterial strain is used for the degraded of reproduced tobacco concentrated liquid Nicotine.
  4. 4. the application of pseudomonas JY-Q bacterial strain according to claim 3 is characterized in that: the pH of reproduced tobacco concentrated liquid is 6.5-7.0.
  5. 5. be used for the enrichment medium of pseudomonas JY-Q bacterial strain claimed in claim 1, it is characterized in that this substratum comprises that following component forms: Na 2HPO 45.57 gL -1, KH 2PO 42.44 gL -1, (NH 4) 2SO 41 gL -1, MgCl 26H 2O 0.2 gL -1, MnCl 24H 2O 0.0004 gL -1, FeCl 36H 2O 0.001 gL -1, CaCl 20.001 gL -1, Nicotine 2 gL -1, pH=7.0.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108531501A (en) * 2018-04-20 2018-09-14 浙江工业大学 The construction method of nicotine degradation selectivity bacterium and its application in waste tobacco Aqueous extracts
CN109929868A (en) * 2019-03-19 2019-06-25 浙江工业大学 The construction method of pseudomonad JY-Q fructose metabolism gene-deleted strain and application
CN115191636A (en) * 2022-06-03 2022-10-18 云南瑞升烟草技术（集团）有限公司 Reconstituted tobacco with high sugar-alkali ratio and preparation method thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101016528A (en) * 2007-01-22 2007-08-15 山东大学 Pseudomonas putida capable of metabolizing nicotine and application thereof
CN101139563A (en) * 2007-08-16 2008-03-12 浙江工业大学 Novel bacterial strain for nicotine degradation-Pseudomonas ZUTSKD and uses thereof
CN101525579A (en) * 2009-04-09 2009-09-09 浙江中烟工业有限责任公司 Pseudomonad ZCJ bacterial strain applied to nicotine degradation of tobacco and screening method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101016528A (en) * 2007-01-22 2007-08-15 山东大学 Pseudomonas putida capable of metabolizing nicotine and application thereof
CN101139563A (en) * 2007-08-16 2008-03-12 浙江工业大学 Novel bacterial strain for nicotine degradation-Pseudomonas ZUTSKD and uses thereof
CN101525579A (en) * 2009-04-09 2009-09-09 浙江中烟工业有限责任公司 Pseudomonad ZCJ bacterial strain applied to nicotine degradation of tobacco and screening method and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108531501A (en) * 2018-04-20 2018-09-14 浙江工业大学 The construction method of nicotine degradation selectivity bacterium and its application in waste tobacco Aqueous extracts
CN108531501B (en) * 2018-04-20 2021-04-30 浙江工业大学 Construction method of nicotine selective degrading bacteria and application of nicotine selective degrading bacteria in waste tobacco leaf water extract
CN109929868A (en) * 2019-03-19 2019-06-25 浙江工业大学 The construction method of pseudomonad JY-Q fructose metabolism gene-deleted strain and application
CN109929868B (en) * 2019-03-19 2021-03-30 浙江工业大学 Construction method and application of pseudomonas JY-Q fructose metabolic gene deletion strain
CN115191636A (en) * 2022-06-03 2022-10-18 云南瑞升烟草技术（集团）有限公司 Reconstituted tobacco with high sugar-alkali ratio and preparation method thereof

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