CN117904004A - Salt-tolerant bacillus and application thereof in degradation of carotenoid - Google Patents

Salt-tolerant bacillus and application thereof in degradation of carotenoid Download PDF

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CN117904004A
CN117904004A CN202410282750.6A CN202410282750A CN117904004A CN 117904004 A CN117904004 A CN 117904004A CN 202410282750 A CN202410282750 A CN 202410282750A CN 117904004 A CN117904004 A CN 117904004A
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salt
tobacco leaves
bacillus
tolerant bacillus
carotenoid
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CN117904004B (en
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沈延源
陈淑君
李志星
邱磊
胡文效
赵先炎
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Qilu University of Technology
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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
    • 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
    • A24B15/20—Biochemical treatment
    • 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
    • C12R2001/07—Bacillus

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Abstract

The invention relates to the technical field of microorganisms and biology, in particular to a salt-tolerant bacillus and application thereof in carotenoid degradation. The salt-tolerant bacillus Bacillus halotolerans is preserved in China general microbiological culture Collection center (China center for type culture Collection), and the preservation number is: CGMCC No.29097; deposit unit address: beichen, qing dynasty district of Beijing, postal code: 100101; classification naming: salt-tolerant bacillus Bacillus halotolerans; meanwhile, the application of the strain in degrading carotenoid in tobacco leaves is provided, the degradation of carotenoid in tobacco leaves can be effectively promoted, and the contents of substances such as farnesyl acetone, methyl heptenone, megastigmatrienone and the like in tobacco leaves are increased.

Description

Salt-tolerant bacillus and application thereof in degradation of carotenoid
Technical Field
The invention relates to the technical field of microorganisms and biology, in particular to a salt-tolerant bacillus and application thereof in carotenoid degradation.
Background
Carotenoids are lipophilic isoprenoid pigments found in plants and microorganisms, 700 or more are known, mainly including beta-carotene, alpha-carotene, lutein, lycopene, etc., with the beta-carotene being most widely distributed. Beta-carotene has high unsaturation because of containing 3-15 conjugated double bonds, and can be degraded by enzyme or photo-oxidation to generate derivatives of C-13, C-11, C-10, C-9 and the like, wherein the derivatives comprise important perfume substances such as beta-ionone, megastigmatrienone, geranylacetone and the like, so the beta-carotene and degradation products thereof have wide application in industries such as food, cosmetics, alcoholic flavor fermentation, tobacco processing and the like. Beta-carotene cleavage products, beta-ionone and large Ma Shitong, are the main flavor components of tomatoes; the isoprenoid C-10 compound component formed by beta-carotene degradation has great influence on the fragrance of the citrus juice; the isoprene derivatives (such as beta-damascenone, beta-ionone, etc.) of the main aroma component of the wine are also beta-carotene degradation products. Besides the above, the carotenoid in the fresh tobacco leaves also has new lutein, purple flavin, phytoene and the like, the total content of the carotenoid is not high, generally 0.1% -1.1%, and the flavor quality of cigar tobacco leaves can be effectively improved by adding the beta-carotene into tobacco shreds.
Cigar is a tobacco product with unique flavor, and the cigar sample prepared by the processes of airing, agricultural fermentation, stacking fermentation, curing alcoholization and the like has complex flavors such as flower flavor, baking flavor, nut flavor and the like. In recent years, cigar products are increasingly sold and demanded in China, but domestic cigars are inferior to imported cigars in tobacco aroma, quality and the like, and the development of domestic cigar industry is limited, industrial availability is not high and other factors are unfavorable for the development of domestic cigars and the competitiveness of domestic cigars in the international market.
In order to better improve the aroma and quality of tobacco leaves, a great deal of researches have been made on improving the aroma and quality of tobacco leaves of low-grade tobacco leaves through screening and application of functional strains, such as fermenting tobacco leaves by using geotrichum candidum (G.candidum), and generating volatile aroma substances such as esters, alcohols, ketones and the like; the Bacillus pumilus 0855-9 is utilized for fermentation to prepare fermentation protease liquid, and the crude protease liquid is uniformly sprayed to the tobacco shreds of low-grade tobacco leaves, so that the quality and aroma quality of the tobacco leaves are obviously improved, the miscellaneous gas is reduced, and the suction taste is good; long Zhangde et al (Long Zhangde, wang Min, xue Yun, etc.) screening and identifying beta-carotene high-efficiency degradation strains in tobacco leaves, optimizing fermentation conditions [ J ]. Light industry report, 2022, 37 (4): 86-93), found that a Western Schwann yeast (Schwanniomyces occidentalis) can degrade beta-carotene, and increase the content of substances such as tobacco leaf dihydroactinolide, nerylacetone, etc. The oxidative degradation of the carotenoid has positive influence on the aroma quality, aroma quantity and tobacco quality of tobacco, and the carotenoid can be broken into various aroma substances at different positions of a carbon chain under the action of oxidase such as microorganism peroxidase, carotenoid degradation oxygenase, lipoxygenase and the like, so that the screening and application research on the carotenoid degradation strain of the tobacco is less at present.
A method for improving tobacco leaf quality disclosed in patent document CN111657536a, comprising: screening strains with carotenoid degradation capacity; preparing the screened strain into a biological preparation; the biological agent is applied to tobacco mellowing to obtain tobacco with carotenoid degradation products; the strain is bacillus highland separated from flue-cured tobacco. A method for improving tobacco quality by synergistic treatment with a bacterial enzyme disclosed in the publication No. CN114376257A uses Bacillus pumilus and a carotenoid dioxygenase to synergistically mix and treat tobacco.
Disclosure of Invention
The application provides a salt-tolerant bacillus strain, and simultaneously provides application of the strain in degrading carotenoid in tobacco leaves, which can effectively promote degradation of carotenoid in the tobacco leaves and increase contents of substances such as farnesyl acetone, methyl heptenone, megastigmatrienone and the like in the tobacco leaves. The strain also has the functions of improving Maillard reaction products, aromatic amino acid degradation products, chlorophyll degradation products and cembrane degradation products in tobacco leaves, and simultaneously has the function of reducing protein in the tobacco leaves.
The technical scheme of the application is as follows:
The salt-tolerant bacillus A4 is salt-tolerant bacillus Bacillus halotolerans, and is preserved in China general microbiological culture Collection center (China center for type culture collection) at the month 11 and 22 of 2023, with the preservation number: CGMCC No. 29097; deposit unit address: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, beichen West way, beijing, chao's area.
The application of the salt-tolerant bacillus A4 in degradation of carotenoid.
Preferably for degrading carotenoids in tobacco leaves.
Preferably, the content of geranylacetone, farnesylacetone, methylheptenone, megastigmatrienone and beta-ionone in the degradation products of tobacco leaves is improved; more preferably, the content of megastigmatrienone, geranylacetone and farnesylacetone in the tobacco degradation products is increased.
Another object of the present invention is to provide a method for fermenting tobacco leaves, which comprises fermenting tobacco leaves with a preparation comprising the above-mentioned salt-tolerant bacillus A4.
Preferably, the concentration of the salt-tolerant bacillus A4 in the preparation is more than or equal to 1 multiplied by 10 8 CFU/mL; and/or
The fermentation conditions are as follows: fermenting at 35-39deg.C and 68-72% humidity for 5-10d; and/or the dosage ratio of the preparation to the tobacco leaves is (40-50) mL:200g.
Preferably, the formulation is prepared by the steps of: inoculating the salt-tolerant bacillus A4 into a culture medium, and culturing for 24-48 hours at the temperature of 35-40 ℃ and the rpm of 200-240 rpm.
Preferably, the salt-tolerant bacillus A4 is inoculated into the LB liquid medium with an inoculum size of more than or equal to 1 percent.
The application screens out a strain of degradable carotenoid from tobacco leaves, uses bacterial universal primers 27F and 1492R to carry out colony PCR on the screened carotenoid degradation strain, sends the obtained PCR product to Qingdao Lai company for sequencing, uses BLAST to carry out 16S rRNA sequence alignment on NCBI, has homology of 81.4 percent, and is identified as salt-tolerant bacillus (Bacillus halotolerans). It grows in LB agar medium, its colony features are: the colony has larger diameter, is milky white and has irregular shape.
Information on preservation of strains
Preservation time: 2023, 11, 22 days,
Preservation unit: the China general microbiological culture Collection center,
Preservation number: CGMCC No. 29097,
Deposit unit address: beichen, qing dynasty district of Beijing, postal code: 100101;
Classification naming: salt-tolerant bacillus Bacillus halotolerans.
The beneficial effects of the invention are that
The invention provides a salt-tolerant bacillus A4, which is applied to cigar tobacco leaves, can effectively reduce carotenoid content in the cigar tobacco leaf fermentation process, increase the content of substances such as farnesyl acetone, megastigmatrienone, geranylacetone and the like, strengthen bean aroma, costustoot and nut aroma of the tobacco leaves, improve aroma quality and aroma quantity of the tobacco leaves, reduce sensory indexes such as miscellaneous gas, irritation and the like, and provide a research thought for developing novel characteristic aroma cigar products and cigar industry microbial preparations.
Drawings
FIG. 1 is a photograph of a colony of salt-tolerant Bacillus;
Fig. 2 is a sensory evaluation analysis result.
Detailed Description
The following is a specific embodiment of the present invention, and the technical solution of the present invention is further described with reference to the accompanying drawings, but the present invention is not limited to these examples.
Example 1 isolation, screening and identification of salt tolerant bacillus strains.
1. Isolation of strains
The method comprises the following specific steps:
tobacco leaf sample 10g provided by Shandong Zhongyan industry Limited company is taken, liquid nitrogen is used for grinding to promote the release of endophytes, ground powder is added into 90 mL LB culture medium according to 20g/L, and the culture medium is obtained by shaking culture under the conditions of 37 ℃ and 220 rpm for 48 h.
2. Screening of strains
The method comprises the following specific steps:
(1) The obtained culture solution is diluted to 10 -7 in a gradient way, the culture solution is coated on a screening culture medium for culturing 24 h, and the strain with the transparent ring is selected for re-screening. Weighing 10 mg tobacco leaves in a 250 mL triangular flask, adding 100mL of sterile water for soaking 24 h, carrying out suction filtration, taking filtrate 2mL, adding into an enrichment medium, and culturing at 37 ℃ at a rotating speed of 150 r/min for 2 d to obtain a bacterial source. And (3) carrying out gradient dilution on a proper amount of bacterial sources, selecting 10 -3,10-4,10-5 concentration gradients, carrying out plate coating on a separation culture medium, coating 3 plates on each concentration gradient in parallel, culturing the coated plates in an incubator at 37 ℃ for 2-3 d, selecting single bacterial colony with good morphology and having transparent rings, and repeatedly carrying out streak separation and purification.
(2) The single colony with obvious transparent ring is selected and inoculated in 100mL fermentation medium for culturing 2d at 28 deg.c and 150 r/min, and the degradation product is detected through observing the color of shake flask and GC-MS analysis, and the strain with carotenoid degrading capacity is further screened and marked as A4.
3. Identification of strains
The method comprises the following specific steps:
(1) The obtained strain A4 was amplified with the bacterial primer 27F having nucleotide sequences shown in SEQ ID No.1 (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R and the bacterial primer having nucleotide sequence shown in SEQ ID No.2 (5'-CTACGGCTACCTTGTTACGA-3') to the strain 16S R RNA gene.
(2) PCR was performed using 30. Mu.L of the reaction mixture, and Taq DNA Polymerase (Takara) enzyme was selected, and the PCR conditions were pre-denatured at 95℃and 5, 5 min; 35 cycles of amplification stage, according to 95 ℃,30 s;58 ℃,30 s; and (3) carrying out at 72 ℃ for 1min for 30 s to obtain a PCR product.
(3) The PCR product obtained in the step (2) was analyzed by 1% agarose gel electrophoresis and sent to Qingdao Laiyitose company for sequencing.
(4) Sequences were aligned in NCBI database with previously published bacterial 16S rRNA sequences, strain A4 homology was 99%. The bacterial strain A4 is in LB solid medium, and the colony characteristics are as follows: the colony has larger diameter, is milky white and has an irregular shape, as shown in fig. 1.
(5) The strain A4 is Bacillus halotolerans, belongs to bacillus and is preserved in China general microbiological culture collection center (CGMCC), and the number is: 29097;
Example 2 use of salt tolerant bacillus in cigar tobacco fermentation.
The method comprises the following specific steps:
(1) The strain A4 is inoculated into LB liquid medium with 1 percent of inoculation amount, and is cultured for 48 hours at 37 ℃ and 220 rpm to obtain fermentation liquor, the fermentation liquor is added into sterile water according to 10 mL to lead the final volume to be 50mL, and the preparation is obtained after uniform mixing, and the concentration of salt-tolerant bacillus in the preparation is at least 1 multiplied by 10 8 CFU/mL. The preparation was sprayed uniformly onto the surface of 250g of Hainan cigar tobacco leaves using a spray pot.
(2) And (3) placing the tobacco leaves sprayed with the fermentation liquid preparation into a self-sealing bag, and conditioning for 24 hours at room temperature in a constant temperature and humidity incubator.
(3) The temperature of the constant temperature and humidity incubator is set to be 37 ℃, the humidity is 70%, the fermentation is carried out for 5 days, and tobacco leaves are turned once a day.
(4) After fermentation, the mixture is preserved at-20 ℃.
Comparative example 1 aseptic fermentation
The method comprises the following specific steps:
(1) 46mL of sterile water was sprayed evenly onto the surface of Hainan cigar tobacco leaves using a spray can, labeled CK.
(2) And (3) placing the tobacco leaves sprayed with sterile water into a self-sealing bag, and conditioning for 24 hours at room temperature in a constant temperature and humidity incubator.
(3) The temperature of the constant temperature and humidity incubator is set to be 37 ℃, the humidity is 70%, the fermentation is carried out for 5 days, and tobacco leaves are turned once a day.
(4) After fermentation, the mixture is preserved at-20 ℃.
Component analysis
The fermented cigar tobacco leaves in example 2 (A4) and comparative example 1 (CK) were subjected to component analysis, specifically as follows:
(1) Determining the content of carotenoid degradation products in fermented tobacco leaves by adopting a gas chromatography-mass spectrometry (GC-MS), drying the fermented tobacco leaves at a low temperature of 40 ℃ and grinding the dried tobacco leaves into powder, taking 1.5 g tobacco leaves powder into a headspace bottle, and adding phenethyl acetate as an internal standard, wherein the content is 100 mug. Helium flow rate was 1 ml.min -1, oven temperature was fixed at 40 ℃ holding 2 min, heating to 250 ℃ at 15 ℃/min and holding 5 min, ion source temperature was 210 ℃, transmission line temperature was 280 ℃.
(2) The computer spectrum library WILEY 8.0 and NIST14 library are used for comparing and analyzing the analysis compounds, and carotenoid degradation products, maillard reaction products, aromatic amino acid degradation products, chlorophyll degradation products and cembrane degradation products with the similarity of more than 800 are selected for analysis, and the content is calculated by an absolute quantitative method. The results of the carotenoid degradation product content changes are shown in table 1 below, and the other reactions or degradation products are shown in table 2 below.
TABLE 1 variation in carotenoid degradation product content in example 2 (A4) and comparative example 1 (CK)
As can be seen from Table 1, the content of carotenoid degradation products in the tobacco leaves fermented by the strain A4 is increased by 1.08 times compared with that in the comparative example 1, which shows that the strain A4 can promote the carotenoid degradation of the tobacco leaves in the cigar tobacco leaves. Wherein, compared with comparative example 1, the content of megastigmatrienone 2 in the tobacco leaf fermented by A4 is increased by 1.40 times, the content of megastigmatrienone 1 is increased by 1.15 times, the content of geranyl acetone is increased by 1.01 times, and the increase of the content of carotenoid degradation products plays an important role in increasing the aroma of the tobacco leaf, improving the aroma quality of the tobacco leaf and reducing miscellaneous gases.
TABLE 2 content of related products in example 2 (A4) and comparative example 1 (CK)
As can be seen from Table 2, the Maillard reaction products, aromatic amino acid degradation products, chlorophyll degradation products and cembrane degradation products of cigar tobacco leaf fermented by the strain A4 are all increased compared with the control CK group, and the Maillard reaction products, the aromatic amino acid degradation products, the chlorophyll degradation products and the cembrane degradation products are respectively improved by 51.63%, 93.95%, 74.56% and 36.60% compared with the control CK group.
Sensory evaluation analysis
Sensory evaluation of tobacco samples was performed by Shandong Zhongyan industry Limited liability company, scoring the samples for flavor, qualitative index and quantitative index according to 1-5 points, and marking 0 points was absent. The quantitative index is divided into: green, bitter, irritating, burning, and other rhyme are shown in fig. 2.
The results show that the quality scores of the tobacco leaves fermented by the strain A4 are higher than those of the tobacco leaves fermented by the CK group, and the quality improvement of the tobacco leaves fermented by the strain A4 is most obvious. The total sensory quality of A4 is highest, and the green, bitter and irritation of the tobacco leaves fermented by A4 are reduced, wherein the reduction of the irritation is most obvious; and other rhyme feelings are obviously improved.

Claims (8)

1. The salt-tolerant bacillus A4 is characterized in that the salt-tolerant bacillus A4 is salt-tolerant bacillus Bacillus halotolerans and is preserved in China general microbiological culture Collection center (China center for type culture Collection, with the preservation number of: CGMCC No. 29097; deposit unit address: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, beichen West way, beijing, chao's area.
2. Use of the salt tolerant bacillus A4 of claim 1 for degrading carotenoids.
3. The use according to claim 2, for degrading carotenoids in tobacco leaves.
4. Use according to claim 3, characterized in that the content of geranylacetone, farnesylacetone, methylheptenone, megastigmatrienone, β -ionone in the degradation products of tobacco leaves is increased.
5. A method for fermenting tobacco leaves, characterized in that the tobacco leaves are fermented with a preparation comprising the salt-tolerant bacillus A4 according to claim 1.
6. The method for fermenting tobacco leaves according to claim 5, wherein the concentration of the salt-tolerant bacillus A4 in the preparation is not less than 1X 10 8 CFU/mL; and/or
The fermentation conditions are as follows: fermenting at 35-39deg.C and 68-72% humidity for 5-10d; and/or the dosage ratio of the preparation to the tobacco leaves is (40-50) mL:200g.
7. A method of fermenting tobacco leaf according to claim 5 or 6, wherein the formulation is prepared by: inoculating the salt-tolerant bacillus A4 into a culture medium, and culturing for 24-48 hours at the temperature of 35-40 ℃ and the rpm of 200-240 rpm.
8. The method for fermenting tobacco leaves according to claim 7, wherein the salt-tolerant bacillus A4 is inoculated in the LB liquid medium in an inoculum size of not less than 1%.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110291059A (en) * 2017-01-12 2019-09-27 喜施倍全球股份有限公司 Microbe soil reinforcing agent
WO2021004830A1 (en) * 2019-07-05 2021-01-14 Basf Se Industrial fermentation process for microbial cells using a fed-batch pre-culture
CN114591878A (en) * 2022-05-10 2022-06-07 齐鲁工业大学 Process for preparing mariculture tail water treatment microbial inoculum and tail water treatment method
CN116004468A (en) * 2023-01-09 2023-04-25 山东农业大学 Salt-tolerant bacillus B13 and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110291059A (en) * 2017-01-12 2019-09-27 喜施倍全球股份有限公司 Microbe soil reinforcing agent
WO2021004830A1 (en) * 2019-07-05 2021-01-14 Basf Se Industrial fermentation process for microbial cells using a fed-batch pre-culture
CN114591878A (en) * 2022-05-10 2022-06-07 齐鲁工业大学 Process for preparing mariculture tail water treatment microbial inoculum and tail water treatment method
CN116004468A (en) * 2023-01-09 2023-04-25 山东农业大学 Salt-tolerant bacillus B13 and application thereof

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