CN107629979B

CN107629979B - California burclover and application thereof

Info

Publication number: CN107629979B
Application number: CN201710830950.0A
Authority: CN
Inventors: 段焰青; 陈兴; 李源栋; 陶鹰; 汪显国; 李文均; 曾熠程; 张健
Original assignee: China Tobacco Yunnan Industrial Co Ltd
Current assignee: China Tobacco Yunnan Industrial Co Ltd
Priority date: 2017-09-15
Filing date: 2017-09-15
Publication date: 2020-04-03
Anticipated expiration: 2037-09-15
Also published as: CN107629979A

Abstract

The invention discloses a sporulation bacterium and application thereof. The strain is named as a cavernous bacillus sp (Blastococcus calvernae sp.) SYSUZL-2, and is preserved in China center for type culture Collection CCTCC at 2016, 12 and 9 days, with the preservation number M2016739. The microbiological characteristic analysis and 16S rRNA gene sequence analysis show that the strain is a new bacterium. The strain can produce citronellol, geraniol, camphol, terpineol, linalool and other volatile compounds, and can be used for preparing the tobacco flavor after fermentation. The obtained spice has special fragrance, can strengthen cigarette style, enrich cigarette fragrance, make fragrance rich and full, reduce irritation to oral cavity and nasal cavity, and has antibacterial and antifungal activity.

Description

California burclover and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a cavernous bacillus. Meanwhile, the invention also relates to application of the cavernous bacillus.

Background

The microorganisms are abundant in variety, and people use pure culture technology to investigate, develop and utilize the microorganisms in the environment for a long time. With the development of science and technology, the importance of microbial resources in scientific research and biological industry is increasingly prominent. Various microbial resources are collected and stored in many countries in the world, and the continuous enrichment of the microbial resources lays a solid material foundation for the development of biotechnology in China.

The flavoring and casing are key links in the cigarette process, and are one of effective means for effectively improving the smoking quality of the cigarettes and forming special cigarettes with qualified quality. At present, most of tobacco flavors added in tobacco production in China are extracted by a traditional method and then blended, and although the production operation is simple, the defects of monotonous aroma, unobvious cigarette smoking effect improvement and the like exist. The fermentation of the perfume raw materials by using microorganisms can increase the content of various aroma substances in the traditional extracted perfume and improve the richness of the perfume. However, the existing industrial aroma-producing microorganisms have the problems of species solidification, limited type of fermented flavors and the like, so that a new microbial strain capable of producing special aroma by fermentation needs to be searched and screened urgently to expand the richness of tobacco flavors and meet the production requirements of cigarettes.

Tobacco workers at home and abroad have done a lot of work on producing tobacco flavors by microbial fermentation, but the production is mostly limited to aroma-producing yeast. The invention separates a cavernous coccus for the first time, and the cavernous coccus is identified as a new microorganism species, and no public report about the cavernous coccus and related applications exists at present.

Disclosure of Invention

The invention aims to provide a novel cavernous bacillus.

The invention also aims to provide application of the cavernous bacillus in cigarette production.

The invention achieves the above object through the following technical scheme.

The bacillus cave is classified and named as bacillus cave SYSUZL-2(blast coccus vertenasus. SYSUZL-2), and is deposited in China Center for Type Culture Collection (CCTCCNO) at 2016, 12, 9 days: m2016739.

The nucleotide sequence of the 16S rRNA gene of the bacillus cave SYSUZL-2 is shown in the sequence table.

The spodiococcus cave SYSUZL-2 grows well on most culture media, and hyphae in the media are grey to yellow brown. The gelatin liquefaction, the starch hydrolysis and the nitric acid reduction are positive, the milk coagulation and the peptone conversion are negative, and hydrogen sulfide and melanin are not generated. Glucose, mannose, xylose, ribose, inositol, mannitol, glycine, histidine, methionine can be utilized. The cell wall contains meso-DAP and the whole cell hydrolysate contains galactose and arabinose.

The bacillus cave SYSUZL-2 is obtained by separating karst cave soil adjacent to an image tobacco field in Shilin county of Yunnan province, and during solid culture, most microorganisms are found to have the capacity of generating volatile substances, and the gas information generated after liquid fermentation is obviously different. Among the obtained pure culture isolated strains, a strain SYSUZL-2 which can generate volatility on a culture medium containing a simple carbon and nitrogen source and is special flavor is obtained by screening.

The bacillus cave SYSUZL-2 has the function of generating special fragrance, and GC-MS identification results show that the strain SYSUZL-2 generates several types of main volatile compounds, namely citronellol, geraniol, camphol, terpineol and linalool. Camphol has clinical anti-inflammatory and analgesic effects, linalool has fresh and sweet smell and can be used for perfuming and impurity removing of cigarettes, and citronellol has antifungal and antioxidant activities and is intensively applied to the aspect of medicines. Terpineol can be used as food essence and cosmetic substrate, and has wide application.

The preferable method for preparing the tobacco flavor by fermenting the sporulation bacillus SYSUZL-2 comprises the steps of carrying out amplification culture on the sporulation bacillus SYSUZL-2, inoculating the cultured sporulation bacillus SYSUZL-2 into a fermentation culture medium, carrying out shake flask culture at 28 ℃ for 7d, extracting fermentation liquor for 3 times and 1L/time by using ethyl acetate, combining extraction liquor, and concentrating under reduced pressure (50 ℃ and 0.06MPa) to obtain the required tobacco flavor. The spice has very special fragrance, and can strengthen cigarette style, enrich cigarette fragrance, and make fragrance more mellow.

The invention has the beneficial effects that: screening pure culture strains separated from karst cave soil adjacent to the tobacco field to obtain a strain SYSUZL-2 which can generate volatility on a culture medium containing a simple carbon and nitrogen source and has special aroma. The microbiological characteristic analysis and 16S rRNA gene sequence analysis show that the strain is a new bacterium. The strain can produce several main volatile compounds such as citronellol, geraniol, camphanol, terpineol, linalool and the like. Wherein, the camphol has the clinical anti-inflammatory and analgesic efficacy, the linalool has sweet smell and can be used for perfuming and impurity removal of cigarettes, and the citronellol has antifungal and antioxidant activities and is intensively applied to the medicine aspect. Terpineol can be used as food essence and cosmetic substrate, and has wide application. The tobacco flavor with special fragrance can be obtained by utilizing the strain for fermentation, the obtained flavor is rich and full in fragrance, can enhance the cigarette style, can reduce the stimulation of oral cavity and nasal cavity, and has antibacterial and antifungal activities.

Description of preservation of biological Material

The strain of the invention has been preserved in China Center for Type Culture Collection (CCTCC) in 2016, 12 months and 9 days, and the address of the center is as follows: the strain is classified and named as Spodococcus caveolus SYSUZL-2(Blastococcus callunae sp. SYSUZL-2) in Wuhan university, and the preservation number is CCTCC NO: m2016739.

Drawings

FIG. 1 is a micrograph and an electron micrograph of a bacterium cavernosum in R2A medium.

FIG. 2 shows a phylogenetic tree constructed by the spodiococcus cavernosum and some related strains according to the 16S rRNA gene sequence.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and examples. The specific embodiments described herein are merely illustrative of the present invention and do not limit the scope of the invention.

Example 1

Isolation of Bacillus cave SYSUZL-2

Taking a soil sample in a karst cave adjacent to an impression tobacco field in Shilin county of Yunnan province, sealing and bringing back the soil sample by a plastic bag, and storing the soil sample at 4 ℃ for later use. Accurately weighing 10g of soil sample, adding 90mL of sterile water, and shaking by a shaking table at 180rpm and 30 ℃ for 30 min. Diluting with sterile water to 10^-1、10^-2、10^-3、10^-4、10^-5The concentration of the active ingredients in the mixture is,0.2mL of each was plated on LB plate medium in 3 replicates. Culturing at 30 deg.C for 48 hr, selecting different single colonies from each soil sample plate, purifying on corresponding solid culture medium, adding glycerol at an amount of 20% of the liquid, and storing in a refrigerator at-80 deg.C.

The separated microorganisms are respectively inoculated in LB liquid culture medium, shaking culture is carried out on a shaking table at 160rpm and at 30 ℃, and the bacteria are cultured overnight until the concentration of the bacteria liquid OD is 2.0, and the bacteria liquid is used as a seed liquid for standby. Inoculating 1% of seed solution of each strain to be tested in a screening culture medium, shaking and culturing at 30 deg.C and 160rpm for 3-5 days, observing appearance and fragrance characteristic change of fermentation liquor every day, and screening to obtain microorganism capable of producing special fragrance, with number SYSUZL-2.

Example 2

Identification of Bacillus cave SYSUZL-2

And carrying out morphological, physiological, biochemical and molecular biological identification on the separated and purified SYSUZL-2. The SYSUZL-2 classification was named as Blastococcus cavernosum (Blastococcus calcoaceae sp.).

The microscopic and electron micrograph of SYSUZL-2 on R2A medium is shown in figure 1.

SYSUZL-2 grew well on most media with hyphae in the medium gray to tan. Gelatin liquefaction, starch hydrolysis and nitric acid reduction are positive, milk coagulation and peptonization are negative, and hydrogen sulfide and melanin are not generated. Can utilize glucose, mannose, xylose, ribose, inositol, mannitol, glycine, histidine, methionine. The cell wall contains meso-DAP and the whole cell hydrolysate contains galactose and arabinose. (tables 1 to 3)

TABLE 1 culture characteristics of SYSUZL-2

TABLE 2 physio-biochemical characteristics of SYSUZL-2

TABLE 3 utilization of carbon and nitrogen sources of SYSUZL-2

Carbon source utilization	Results	Carbon or nitrogen source utilization	Results
				Glucose	++	Inositol	+++
Lactose	-	Mannitol	+++
				Mannose	+++	Sorbitol	-
Arabinose	-
				Xylose	+	Glycine	++
Soft candy	-	Histidine	+++
				Ribose	++	Methionine	+

The 16S rRNA sequence of SYSUZL-2 is shown in a sequence table.

BLAST comparative analysis was performed on this sequence with known sequences in the GenBank database, and the 16S rRNA gene sequences of the related species were obtained from the database to construct a phylogenetic tree, as shown in FIG. 2. The comparative analysis shows that the cavernous bacillus and the strain (Blastococus jejuensis KST 3-10) of the invention^T) The genetic relationship between the 16SrRNA gene sequence of the spodiococcus cavernosa and the Blastococcus jejuensis KST3-10 is recent^THas 98.65 homology and DNA-DNA hybridization value lower than<70%, forming independent branches on the phylogenetic tree. Therefore, the reference strain of the Blastococcus jejuensis KST3-10 is obtained by combining the characteristics of morphology, physiological and biochemical analysis, cytochemical analysis, phylogenetic analysis and the like^TThe obvious difference shows that the cavernous bacillus of the invention is a new species named as Blastococcus calvernaesep.

SYSUZL-2 has 16S rRNA gene sequence accession number KY593172 in GenBank database. The strain preservation number of the China Wuhan university patent preservation center is M2016739. SYSUZL-2 and a phylogenetic tree of a part of related strains constructed according to 16S rRNA gene sequences are shown in figure 2.

Example 3

Application of bacillus cave SYSUZL-2 in preparation of tobacco flavor through fermentation

The screened hole buddleja SYSUZL-2 is applied to fermentation production of tobacco flavor, and comprises the following specific steps:

(1) slant culture: culture medium: 0.5g of glucose; 0.5g of yeast extract; peptone 0.5 g; 0.5g of acid hydrolyzed casein; 0.5g of soluble starch; 0.3g of sodium pyruvate; dipotassium phosphate 0.3 g; magnesium sulfate 0.05 g; 15g of agar; 1000ml of distilled water, pH 7.2; the culture temperature is 28 ℃, and the culture time is 48 h.

(2) The seed culture comprises the steps of picking part of mycelia from an inclined plane, inoculating the mycelia into seed liquid, and carrying out shake culture on the mycelia, wherein the seed culture medium comprises 120g of dextrin, 40g of soybean meal, 2g of yeast extract, 0.5g of tryptophan, 5g of β -alanine, 0.5g of magnesium sulfate, 0.2g of ammonium phosphate and 1000ml of distilled water, the pH value is 7.2, the culture temperature is 28 ℃, and the culture time is 36 hours.

(3) Fermentation culture: inoculating the strain into a fermentation medium according to the inoculation amount of 10%, and performing shake flask culture. Culture medium: 10g of soybean meal; 10g of glucose; 3g of peptone; 2.5g of sodium chloride; 2g of calcium carbonate; 1000ml of distilled water, pH7.2, and fermenting for 7d in a full temperature shaker at 28 deg.C with shaking (121 r/min).

Extracting the fermentation liquid with ethyl acetate for 3 times, 1L/time, mixing extractive solutions, and concentrating under reduced pressure (50 deg.C, 0.06MPa) to obtain tobacco flavor. The perfume is clear reddish brown liquid, and is added into tobacco shred at 0.2 ‰, and distilled water of the same amount is added into control group. And (3) putting the cut tobaccos of the treatment group and the control group into a constant-temperature constant-humidity box, and balancing for 24 hours under the conditions that the humidity is 60 +/-2 and the temperature is 22 +/-2 to prepare the cigarettes. The sensory evaluation was performed by an evaluation panel consisting of evaluation experts. The perfume has rich and full fragrance, can strengthen cigarette style, reduce irritation to oral cavity and nasal cavity, and has antibacterial and antifungal activity (Table 4, Table 5).

TABLE 4 physicochemical indices of SYSUZL-2 fermented flavors

TABLE 5 sensory evaluation of SYSUZL-2 fermented flavors

Claims

1. A spodiococcus cave, which is characterized in that: the classification of the strain is named as a cavernous bacillus (Blastococcus calverae sp.) SYSUZL-2 with a preservation number of CCTCC NO: m2016739.

2. Use of a bacillus cave SYSUZL-2 as claimed in claim 1 for the preparation of tobacco flavor by fermentation.

3. The application of the bacillus cave bacterium SYSUZL-2 in preparing the tobacco flavor through fermentation according to claim 2, wherein the application comprises the following steps: carrying out amplification culture on the blastomyces SYSUZL-2, inoculating the blastomyces SYSUZL-2 into a fermentation medium, carrying out shake culture at 28 ℃ for 7d, extracting the fermentation liquor with ethyl acetate for 3 times and 1L/time, combining the extracts, and carrying out reduced pressure concentration at 50 ℃ and 0.06MPa to obtain the tobacco flavor.