CN108220188A - A kind of molten bacillus and its application - Google Patents

A kind of molten bacillus and its application Download PDF

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Publication number
CN108220188A
CN108220188A CN201711452367.7A CN201711452367A CN108220188A CN 108220188 A CN108220188 A CN 108220188A CN 201711452367 A CN201711452367 A CN 201711452367A CN 108220188 A CN108220188 A CN 108220188A
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molten bacillus
fragrance
bacterial strain
molten
bacillus
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Inventor
周国福
段焰青
杨洪明
刘秀明
廖头根
李文均
雷声
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China Tobacco Yunnan Industrial Co Ltd
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China Tobacco Yunnan Industrial Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/24—Treatment of tobacco products or tobacco substitutes by extraction; Tobacco extracts
    • A24B15/26—Use of organic solvents for extraction
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B3/00—Preparing tobacco in the factory
    • A24B3/12—Steaming, curing, or flavouring tobacco
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a kind of molten bacillus and its applications.The molten bacillus of the strain was named (Lysobacter sp) C8 1 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number CGMCC No.1.16271 on the 28th in September in 2017.Show that the bacterial strain is a kind of new bacterium by Microbiological Characteristics analysis and 16S rRNA gene sequencings.The bacterial strain can generate the volatile compounds such as benzaldehyde and native monosodium glutamate, elemene, elemol, a eucalyptols, and tobacco aromaticss can be prepared after fermented.Gained fragrance has very special fragrance, can strengthen cigarette style, enriches cigarette perfume, makes fragrance more mellow and full full, and the activity with antibacterium and fungi, effect stability and it is lasting.In addition, the bacterial strain also has the great potential for therefrom developing novel, efficient, nontoxic natural drug.

Description

A kind of molten bacillus and its application
Technical field
The invention belongs to microorganisms technical fields, are specifically related to a kind of new molten bacillus.Meanwhile the invention further relates to institutes State the application of molten bacillus.
Background technology
Microbe species enrich, and for a long time, people carry out the microorganism in environment investigation using pure culture technigne and grind Study carefully and develop and use.With the development of science and technology, importance of the microbial resources in Technological research and biological industry is increasingly It highlights.Many countries are all collecting, are preserving various microbial resources in the world, enrich constantly the hair for China's biotechnology Solid material base is established in exhibition.
Flavoring and casing is the key link in cigarette process, is effective promotion cigarette smoking quality, forms the peculiar wind of cigarette One of effective means of lattice.The tobacco aromaticss added in China tobacco at present are allocated again using conventional method extraction mostly Gained although production operation is simple, there is also fragrance dullness, improves the shortcomings of cigarette smoking effect unobvious.Utilize micro- life Object ferments to perfume material, can increase the content of a variety of aroma substances in tradition extraction fragrance, it is rich to promote fragrance.And Existing industry aroma-producing microbe is there are type curing, the problems such as fermentation fragrance type is limited, new there is an urgent need to find and screen Can ferment generate special aroma microbial strains, to expand the rich of tobacco aromaticss, meet production of cigarettes demand.
Domestic and international tobacco worker has done many work, but more in terms of tobacco aromaticss are produced using microbial fermentation It is limited to aroma-producing yeast.A kind of isolated for the first time molten bacillus of the present invention, is identified as microorganism novel species, do not have at present it is any about The molten bacillus and the open report of related application.
Invention content
The purpose of the present invention, which first consists in, provides a kind of new molten bacillus.
The present invention also aims to provide application of the molten bacillus in production of cigarettes.
The purpose of the present invention is achieved by the following technical programs.
A kind of molten bacillus, Classification And Nomenclature are molten bacillus (Lysobacter sp.) C8-1, and the bacterial strain was in September in 2017 28 days It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number CGMCC No.1.16271。
The 16S rRNA gene nucleotide series of the molten bacillus C8-1 are as shown in sequence table.
The molten bacillus C8-1 well-grown, Gram's staining on most of culture mediums are negative, in ISP 2 It is smooth that surface is formed on (International Streptomyces Project) culture medium, neat in edge, intermediate projections Yellow color colonies.Bacterial strain shows growth characteristics in the range of 10-40 DEG C, pH 6.0-9.0, is resistant to 1% NaCl.It is dry Casein, starch, gelatin liquefaction, polysorbas20,40,60 and 80 hydrolysis are positive, cellulose hydrolysis, H2The generation of S is negative.Energy Utilize glucose, galactolipin, fructose, mannose, sodium acetate, acetoacetate, sucrose, N- acetyl-d-glucosamine, Song Ertang, wheat Bud sugar.The polar lipid that cell membrane contains mainly includes cardiolipin, phosphatidyl glycerol, phosphatidyl-ethanolamine and phosphatidyl first Ethylethanolamine.Breathing quinone is CoQ8 (Q-8).Main aliphatic acid is iso-C15:0,iso-C17:0and iso-C17:1ω9c。
The molten bacillus C8-1 isolated, solids from the tobacco rhizosphere soil on Yunnan Province Kunming tobacco planting ground In incubation, finding the microorganism of the overwhelming majority has the ability for generating volatile materials, the gas generated after liquid fermentation It ceases significantly different.In the isolation of pure culture bacterial strain of acquisition, screening obtains to generate on the culture medium containing simple carbon nitrogen source Volatility and for peat-reek breath bacterial strain C8-1.
The molten bacillus C8-1 has the function of generation special aroma, and GC-MS qualification results show that bacterial strain C8-1 can Generate a few class main volatile compounds, benzaldehyde and native monosodium glutamate, elemene, elemol, a- eucalyptols.Therefore can be used for sending out Ferment is prepared in the industrial production of tobacco aromaticss, to expanding the rich of tobacco aromaticss, meets production of cigarettes demand with good Application prospect.
The preferred method of tobacco aromaticss is prepared using the molten bacillus C8-1 fermentations, by molten bacillus C8-1 after expanding and cultivating It is inoculated in fermentation medium, after 30 DEG C of shaking flask culture 6d, carries out refluxing extraction, supernatant is concentrated under reduced pressure filtering and removing slag To required tobacco aromaticss.The fragrance has very special fragrance, can strengthen cigarette style, enriches cigarette perfume, makes fragrance more It is mellow and full full.
The beneficial effects of the invention are as follows：From tobacco rhizosphere soil in isolated pure culture bacterial strain, screening obtains can Volatility is generated on the culture medium containing simple carbon nitrogen source, and is the molten bacillus strain C8-1 of peat-reek breath.By micro- Biological property analysis and 16S rRNA gene sequencings show that the bacterial strain is a kind of new bacterium.The bacterial strain can generate benzene A few class main volatile compounds such as formaldehyde and native monosodium glutamate, elemene, elemol, a- eucalyptols.Wherein, benzaldehyde has fragrance Breath, in microorganism more typically, but yield is not high；Native monosodium glutamate is the common compounds that actinomyces generate, itself has fishy smell.Elemi Alkene has the function of to inhibit cancer cell, is the main component in current a kind of listing anticarcinogen, has higher economic value, especially It is that microorganism is independently metabolized generation.Elemol is commonly used for flavor of raw material.Eucalyptol has certain fragrance in itself, and can make fixastive To use.Using the strain fermentation, the tobacco aromaticss with special aroma can be obtained, gained fragrance, which has, strengthens cigarette wind Lattice enrich cigarette perfume, the effect of mellow and full flue gas, and the activity with antibacterium and fungi, effect stability and it is lasting.Molten Bacillus Bacterial strain has the laudatory title of " bacterium predator ", can generate the compound with multiple biological activities, which also has exploitation The great potential of novel, efficient, nontoxic natural drug.
The explanation of preservation biomaterial
In September in 2017 28 days, it is common to be preserved in China Committee for Culture Collection of Microorganisms for the bacterial strain of the present invention Microorganism center CGMCC, the centre address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research Institute, the strain classification are named as：Molten bacillus (Lysobacter sp.) C8-1, deposit number CGMCC No.1.16271.
Description of the drawings
Fig. 1 is electromicroscopic photographs of the molten bacillus C8-1 of the present invention on 2 culture mediums of ISP.
Fig. 2 is the system hair of the molten bacillus C8-1 and part related strain of the present invention according to 16S rRNA gene orders structure Educate tree.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with drawings and examples pair The present invention is described in further detail.The specific embodiments described herein are merely illustrative of the present invention, is not to this The restriction of inventive technique scheme.
Embodiment 1
(1) --- the separation of molten bacillus C8-1
Tobacco rhizosphere pedotheque is acquired in Yunnan Province Kunming tobacco planting, plastic bag sealing is taken back, and 4 DEG C of preservations are treated With.It is accurate to weigh 5g soil samples, add in 45mL sterile waters, 30 DEG C, 180rpm shaking tables oscillation 1h.10 are diluted to sterile water-1、10-2、10-3、10-4、10-5Concentration, takes 0.2mL to be coated on LB plating mediums respectively, each sets 3 repetitions.One is cultivated at 30 DEG C Zhou Hou from picking difference single bacterium colony on the tablet of every part of soil sample, presses bacterium solution volume 20% after purification on corresponding solid medium Glycerine is added in, -80 DEG C of refrigerators is placed in and saves backup.
Isolated microorganism is inoculated in respectively in 2 fluid nutrient mediums of ISP, in 30 DEG C, the oscillation training of 160rpm shaking tables It supports, Bacteria Culture is overnight OD=2.0 to bacterial concentration, spare as seed liquor.The seed liquor of each strain to be tested is pressed 1% It is seeded in screening and culturing medium, in 30 DEG C, 160rpm shaking tables shaken cultivation 3-5 days, observes zymotic fluid appearance daily and fragrance is special Situation of change is levied, screening obtains to generate the microorganism of peat-reek breath, number C8-1.
(2) --- the identification of molten bacillus C8-1
The C8-1 isolated and purified carries out morphology, Physiology and biochemistry and molecular biology identification.C8-1 Classification And Nomenclatures are molten bar Bacterium (Lysobacter sp.).
Electromicroscopic photographs of the C8-1 on 2 culture mediums of ISP is shown in attached drawing 1.
C8-1 well-grown, Gram's staining on most of culture mediums are negative, in 2 (International of ISP Streptomyces Project) form surface on culture medium smooth, neat in edge, yellow color colonies of intermediate projections.Bacterial strain exists 10-40 DEG C, growth characteristics are shown in the range of pH 6.0-9.0, are resistant to 1% NaCl.Casein, starch, gelatin solution Change, polysorbas20,40,60 and 80 hydrolysis are positive, cellulose hydrolysis, H2The generation of S is negative.Glucose, gala can be utilized Sugar, fructose, mannose, sodium acetate, acetoacetate, sucrose, N- acetyl-d-glucosamine, Song Ertang, maltose.Cell membrane contains Polar lipid mainly include cardiolipin, phosphatidyl glycerol, phosphatidyl-ethanolamine and phosphatidyl methylethanolamine.Breathe quinone For CoQ8 (Q-8).Main aliphatic acid is iso-C15:0,iso-C17:0and iso-C17:1ω9c.(being shown in Table 1 and 2)
The physiological and biochemical property of 1 C8-1 of table
The carbon nitrogen source utilization power of 2 C8-1 of table
The 16S rRNA sequences of C8-1 are as shown in sequence table.
The sequence carries out BLAST comparative analysis with the known array in GenBank databases, and obtains correlation from database The 16S rRNA gene orders of kind, phylogenetic tree construction see Fig. 2.It is found through comparative analysis, molten bacillus of the invention C8-1 and bacterial strain (LysobactercavernaeYIM C01544T) affiliation is nearest, 16S rRNA gene orders with LysobactercavernaeYIM C01544TThere is 98.6 homology, and DNA-DNA hybridization values are less than<70%, system into Change and form independent branch on tree.Therefore, the features such as comprehensive morphological, Physiology and biochemistry, cytochemistry and phylogenetic analysis, molten bar Bacterium C8-1 and molten Bacillus reference strain LysobactercavernaeYIM C01544TThere is notable difference, show the present invention's Molten bacillus C8-1 is a new species, is named as Lysobacter sp.
C8-1 is in the 16S rRNA gene order accession number MF153474 of GenBank databases.Chinese microorganism strain is protected It is CGMCC 1.16271 to hide administration committee's common micro-organisms center culture presevation number.C8-1 and part related strain are according to 16S The phylogenetic tree of rRNA gene orders structure is shown in attached drawing 2.
Embodiment 2
--- applications of the above-mentioned molten bacillus C8-1 filtered out in fermenting and producing tobacco aromaticss is as follows：
(1) inclined-plane culture：Culture medium：Glucose 0.5g；Yeast extract 0.5g；Peptone 0.5g；Acid hydrolyzed casein 0.5g； Soluble starch 0.5g；Sodium Pyruvate 0.3g；Dipotassium hydrogen phosphate 0.3g；Magnesium sulfate 0.05g；Agar 15g；Distilled water 1000ml, pH7.2；Cultivation temperature is 30 DEG C, incubation time 7d.
(2) seed culture：It is accessed in seed liquor from inclined-plane picking part mycelium, shaking flask culture 36 hours.Seed culture Base：Dextrin 120g；Soy meal 40g；Yeast extract 2g；Tryptophan 0.5g；Beta-alanine 5g；Magnesium sulfate 0.5g；Ammonium phosphate 0.2g；It steams Distilled water 1000ml, pH7.2, cultivation temperature is 30 DEG C.
(3) fermented and cultured：It is connected in fermentation medium by 10% inoculum concentration, shaking flask culture 6d.Culture medium：Soy meal 10g；Glucose 10g；Peptone 3g；Sodium chloride 2.5g；Calcium carbonate 2g；Distilled water 1000ml, pH7.2, cultivation temperature is 30 DEG C, Incubation time is 6d.
Zymotic fluid is subjected to refluxing extraction, supernatant is concentrated under reduced pressure to give required tobacco aromaticss by filtering and removing slag.
The fragrance is not clear brown liquid, it is added to by 0.2 ‰ amount in cigarette shreds, and control group adds in equivalent Distilled water.The pipe tobacco of processing group and control group is put into climatic chamber, in humidity 60% ± 2, under the conditions of 22 DEG C ± 2 of temperature After balancing for 24 hours, cigarette is made.Sensory evaluating smoking is carried out by the group of smokeing panel test that the expert that smokes panel test forms.It is very special that the fragrance has Fragrance can strengthen cigarette style, enrich cigarette perfume, make fragrance more mellow and full full, and the activity with antibacterium and fungi, Effect stability and persistently (table 3, table 4).
The physical and chemical index of 3 C8-1 of table fermentation fragrance
Sensory evaluating smoking's table of 4 C8-1 of table fermentation fragrance
Sequence table
<110>Cigarette industry Co., Ltd in Yunnan
<120>A kind of molten bacillus and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>Molten bacillus C8-1 16S rRNA (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
rysbactrta bacsrgnsnc taagat 26

Claims (6)

1. a kind of molten bacillus, it is characterised in that：The Classification And Nomenclature of the bacterial strain be molten bacillus (Lysobactersp.) C8-1, preservation Number CGMCC No.1.16271.
2. molten bacillus according to claim 1, it is characterised in that：The 16SrRNA gene nucleotides of the molten bacillus C8-1 Sequence is as shown in sequence table.
3. applications of the molten bacillus C8-1 described in claim 1 in production of cigarettes.
4. applications of the molten bacillus C8-1 described in claim 1 in tobacco aromaticss are prepared.
5. applications of the molten bacillus C8-1 according to claim 4 in tobacco aromaticss are prepared, it is characterised in that：By molten bacillus C8-1 is inoculated in after expanding and cultivating in fermentation medium, after 30 DEG C of shaking flask culture 6d, carries out refluxing extraction, filtering and removing slag will Supernatant is concentrated under reduced pressure to give tobacco aromaticss.
6. applications of the molten bacillus C8-1 described in claim 1 in antibacterials are prepared.
CN201711452367.7A 2017-12-28 2017-12-28 A kind of molten bacillus and its application Pending CN108220188A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109722393A (en) * 2019-01-04 2019-05-07 云南中烟工业有限责任公司 A kind of tobacco yellow soil source bacterium and its separation method and application
CN113215022A (en) * 2021-03-01 2021-08-06 云南中烟工业有限责任公司 Rhizosphere Qidun bacillus and separation method and application thereof

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CN107164258A (en) * 2017-05-10 2017-09-15 云南中烟工业有限责任公司 Move coccus and its application in a kind of cave

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109722393A (en) * 2019-01-04 2019-05-07 云南中烟工业有限责任公司 A kind of tobacco yellow soil source bacterium and its separation method and application
CN113215022A (en) * 2021-03-01 2021-08-06 云南中烟工业有限责任公司 Rhizosphere Qidun bacillus and separation method and application thereof

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Application publication date: 20180629