CN112410262B

CN112410262B - New strain of sphingosine monad of North industry and commerce and application thereof

Info

Publication number: CN112410262B
Application number: CN202011418829.5A
Authority: CN
Inventors: 任清; 徐嘉良; 孙占斌; 闫怡; 孙乐平; 邢旋
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2022-08-30
Anticipated expiration: 2040-12-07
Also published as: CN112410262A

Abstract

The invention discloses a new strain of Sphingomonas sobria and application thereof, wherein the strain is Sphingomonas sobria benthica (Sphingomonas beignongshangensis) and the preservation number of the strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC NO. 19204. Experiments prove that the new strain Sphingomonas beigongensis (Sphingomonas beigongensis) is provided, is separated from white spirit pit mud, can generate substances with strong aroma and flavor, and can be used for fermenting and brewing wine.

Description

New strain of Beigong commercial sphingosine monad and application thereof

Technical Field

The invention relates to the technical field of brewing microorganisms, and particularly relates to a new strain of sphingomonas sobria and application thereof.

Background

White spirit is one of three distilled spirits in the world, is produced by adopting a unique fermentation technology and has a history of thousands of years. The whole inner wall of the pit is covered with pre-cultured pit mud. The fermented raw materials are cooked, mixed, crushed and distilled. Adding a saccharification leaven into the steamed raw materials, then putting the steamed raw materials into a cellar for fermentation, taking the fermented materials out of the cellar, and distilling the fermented materials to prepare the Chinese liquor. Microorganisms of pit mud produce various flavor components such as butyric acid, caproic acid and ethyl caproate. Ethyl caproate is considered as a key ingredient affecting the flavor and quality of Luzhou-flavor liquor. In the brewing process, the pit mud microorganisms have important aroma-producing and flavor-enhancing effects, and the quality of the Luzhou-flavor liquor is improved along with the increase of the age of the wine pit. The high quality of the Luzhou-flavor liquor is attributed to the ripening process of pit mud, which results in the balance of microbial community structures and the diversity of pit mud, thereby generating unique flavor.

The strong aromatic Chinese spirits have the flavor characteristics of strong cellar aroma, sweet and mellow taste, harmonious aroma and long aftertaste, are deeply favored by consumers and are sold in the annual amount of about 70 percent of the total amount of Chinese spirits. Microorganisms in pit mud can generate various flavor components such as butyric acid, caproic acid and ethyl caproate, and the ethyl caproate is considered as a key component influencing the flavor and the quality of the Luzhou-flavor liquor. The pit mud microbial flora is a main factor influencing the quality of pit mud and white spirit, and is an important microbial source in the brewing process of the white spirit. The microorganisms in the pit mud are various and have very rich species diversity, but most of the microorganisms are still in a difficult culture state. Although the culturability of microorganisms in different environments is different, the culturability is very low, for example, the culturability in seawater is less than 0.1 percent, and the culturability in soil is about 0.3 percent. Only a small proportion of bacteria in a natural habitat can be cultured using conventional bacterial culture methods. The microorganisms cultured by the traditional method are known microorganisms, and no new microorganism species is found.

Disclosure of Invention

Therefore, the invention provides a new strain of sphingomonas sobria and application thereof.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a new strain, which is Sphingomonas beigongensis (Sphingomonas beigongensis) and the preservation number of the strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC NO. 19204.

In one embodiment of the invention, the 16S rDNA sequence of the strain of Sphingomonas beigongensis (Sphingomonas beigongshansensis) is shown in SEQ ID No. 1.

The invention also provides a microbial inoculum, the active ingredient of which is the strain of claim 1.

The invention also provides application of the bacterial strain or the microbial inoculum in the following (A1) or (A2): (A1) generating strong aromatic flavor substances; (A2) preparing the product with the strong aromatic flavor substances.

The invention also provides application of the bacterial strain or the microbial inoculum in the following (B1) or (B2): (B1) brewing wine; (B2) and (5) preparing a wine brewing product.

The invention also provides a screening method of the strain, which comprises the following steps: diluting pit mud, coating the diluted pit mud on an improved Gao's No.1 culture medium, carrying out aerobic culture at 37 ℃ for 24h, carrying out three-region streak purification culture for 3 times to obtain pure culture bacteria, carrying out PCR amplification on the pure culture bacteria to obtain 16SrDNA, and carrying out sequence comparison analysis to obtain the Sphingomonas beigondii.

In one embodiment of the invention, the modified Gauss No.1 culture medium comprises the following raw materials in mass number: 1.0g of potassium nitrate, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 0.01g of ferrous sulfate, 0.5g of sodium chloride, 20.0g of soluble starch, 15.0g of agar, 7.2-7.4 of pH value and 25 ℃.

The invention has the following advantages:

experiments prove that the new strain, Sphingomonas beigongensis, is separated from cellar, can produce aroma flavor substances, and can be used for fermenting and brewing wine.

According to the invention, the improved Gao's 1 culture medium is adopted firstly, common bacteria are slow to reproduce, and bacteria of the difficult-to-culture new sphingomonas genus grow fast on the same culture dish and are little influenced by conventional microorganisms; meanwhile, after the culture time is prolonged for 36 hours, a single colony is picked, and new microorganisms difficult to culture grow fully for a long time until the colony is visible.

Bacterial preservation description: the Sphingomonas beigongensis (Sphingomonas beigonggangshensis) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 2019, 12 months and 16 days, and the preservation addresses are as follows: the collection number of the microorganism research institute of Chinese academy of sciences, No. 3 Xilu No.1 of Beijing, Chaoyang, and the institute of microbiology, is CGMCC NO. 19204.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is the 16S rDNA sequence and the evolutionary tree diagram of Sphingomonas beigongensis provided by the present invention;

FIG. 2 is a diagram showing the result of detection of coenzyme Q-10 of Sphingomonas beigongensis (Sphingomonas beigongshansensis) according to the present invention;

FIG. 3 is a diagram showing the results of detecting polar esters of Sphingomonas beigongensis (Sphingomonas beigongshangensis) according to the present invention.

Detailed Description

The present invention is described in terms of specific embodiments, and other advantages and benefits of the present invention will become apparent to those skilled in the art from the following disclosure. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 isolation and characterization of Sphingomonas beigongensis

Isolation of novel Sphingomonas beigongensis

Step one, preparing an improved Gao's No.1 culture medium: 1.0g of potassium nitrate, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 0.01g of ferrous sulfate, 0.5g of sodium chloride, 20.0g of soluble starch, 15.0g of agar, 7.2-7.4 of pH value and 25 ℃.

Step two, taking 10g of pit mud, and diluting the pit mud to 10g by using sterile distilled water ^-5 Coating on a prepared improved Gao's No.1 culture medium culture dish for aerobic culture at 28 ℃;

step three, carrying out aerobic culture at 28 ℃ for 36 hours, then selecting a single colony, and carrying out streak purification on three regions for 3 times to obtain pure culture bacteria;

and step four, extracting bacterial DNA, amplifying a 16SrDNA sequence by utilizing a PCR technology, and preliminarily determining a new strain of Sphingomonas beigongensis (Sphingomonas beigongensensis) through sequence comparison.

Identification of Sphingomonas beigongensis

The new bacteria separated and screened from pit mud are gram-negative bacteria, sphingomonas, and are named as: sphingomonas beigongensis (Sphingomonas beigongshengsis)

1. The morphology, physiology, biochemistry, cytochemistry and gene level research of the strain is carried out by the Beigong commercial Sphingomonas (Sphingomonas beigongshansensis). Physiological and biochemical characteristics of Sphingomonas beigongensis REN5(Sphingomonas beigongensis)

The colony morphology of the Sphingomonas beigongensis (Sphingomonas beigongensis) is round, the surface is smooth and orange; gram reaction is negative, gram negative bacteria are obtained, the optimal temperature is 30 ℃, the optimal pH value is 5-6, the optimal sodium chloride concentration is 0.5%, and the hydrolysis product can hydrolyze starch, is catalase negative, is beta-galactosidase positive and hydrolyzes glucosidase-esculin.

2. Cytochemical characteristic detection of Sphingomonas beigongensis

The cytochemical components of Sphingomonas sobria (Sphingomonas beignogngshanensis) such as fatty acids, quinone types, polar lipids, etc. were detected by GC gas chromatography, HPLC liquid chromatography and TLC thin layer chromatography (Sasser M. identification of bacterial by gas chromatography of cellular lipids, MIDI Technical Note 101.Newark, DE: MIDIinc; 1990.Minnikin DE, O' Donnell AG, Goodfelow M, Alderson G, Atharye M et al. Integrated procedure for the extraction of bacterial isophenone reagents. J. Microbiol. Methods 1984; 2: 233-.

Sphingomonas beigongensis (Sphingomonas beigonggangshangensis) cell fatty acid component: saturated fatty acids: c14:0, C16:0, Antesio-C15:0, Antesio-C17:0, Iso-C14:0, Iso-C15:0, Iso-C16:0, Iso-C17:0, unsaturated fatty acids: the fatty acid composition of C16:0N alcohol, C16: 1. omega.7C alcohol cells is shown in Table 1

TABLE 1

As shown in figure 2, in the detection of quinone component in cell respiration of Sphingomonas beigongensis (Sphingomonas beigongshangensis), the main advantage of the strain is quinone coenzyme Q-10, and quinone compounds are oxidation active substances which are widely existed in natural products, antitumor drugs, in-vivo biochemical metabolites, environmental pollutants or polycyclic aromatic hydrocarbon metabolism. Each bacterium has a main quinone component, and the difference in the kinds and amounts of quinones in a microbial population reflects the diversity of the population composition, and the quinone spectrum has been widely used in environmental microorganisms as an index of the diversity of the bacterial population composition.

The detection of polar ester of Sphingomonas beigongensis comprises the following steps: polar lipid extraction: 100mg of lyophilized mycelia of Sphingomonas beijerinckii were suspended in 9.5mL of chloroform: methanol: 0.3% NaCl (2.5:5: 2). The thallus solution is placed in a water bath at 80 ℃ for 15 min. After cooling, the filter paper was filtered into a 50mL centrifuge tube, 2.5mL chloroform and 2.5mL 0.3% NaCl were added, and centrifuged at 4000rpm for 5 min. The lower chloroform phase was carefully separated into a clean rotary evaporator-dedicated flask and the chloroform removed by rotary evaporation under reduced pressure on the rotary evaporator, the temperature of the water bath not exceeding 40 ℃. 250 mu.L of chlorine anti-methanol (2:1, v/v) is added and transferred to a brown screw sample bottle, and the bottle is placed in a refrigerator at 4 ℃ for storage and testing.

TLC analysis of polar lipids: a10 cm × 10cm Silica gel plate (Merck 25TLC aluminum sheets 20cm × 20cm Silica gel 60F254) was activated in an oven at 110 deg.C for 1 hour, and then cooled. Pipette 2 μ L of total lipid sample onto TLC plate, and spot 3 times.

And (3) placing the TLC thin plate in a first chromatographic cylinder for spreading, wherein the first spreading agent is chloroform: and (3) spreading methanol, water (65:25:4, v/v) and a solvent to the top, taking out the thin plate, drying the thin plate by blowing, putting the thin plate into a second chromatographic cylinder, wherein the second spreading agent is chloroform: methanol: acetic acid: water (80: 12: 15: 4, V/V), ascending in a direction perpendicular to the first direction, spreading the solvent to the top, taking out the thin plate for drying, spraying phosphomolybdic acid color developing agent to the TLC plate until the TLC plate is completely wet, heating at 100 ℃ for 5-8min, displaying clear spots, immediately scanning the TLC plate on a scanning instrument, and recording the result. As shown in fig. 3, sphingomonas sobria, 4 polar esters, DPG: diphosphatidylglycerol, diphosphatidylglycerol; PG: phosphatidylglycerol, phosphatidylglycerol; PE: phosphatidylethanolamine, phosphatidyl ethanolamines; SGL: sphingomythipids; PC: phosphatidylcholine; UL: unknown polar lipids, unidentified lipids; SGL: glycosphingolipid, sphingoglycolipid; and (4) UPL: the unidentified phospholipid, unidentified phospholipd. Phospholipids (phospholipids) belong to polar lipids (polar lipids), which together with proteins, sugars, etc. form cell membranes and play an important role in substance transport, metabolism and maintenance of normal osmotic pressure. The phospholipide components of different genera of bacteria are different, and the phospholipide components are one of the important characteristics for identifying the genera and are indispensable classification indications in chemical classification projects.

3. 16S rDNA sequence and evolutionary tree of Sphingomonas beigongensis

The strain of Sphingomonas beigongensis (Sphingomonas beigongshanhensis) is subjected to whole genome sequencing by Meiji biological medicine science and technology Limited, Shanghai to obtain a whole genome sequence. The phylogenetic tree is shown in figure 1.

The measured REN5T 16S rRNA sequences are compared with model strains in NCBI (https:// www.ncbi.nlm.nih.gov /), 24 model strains 16S rRNA sequences are selected according to similarity to construct a phylogenetic tree, as shown in FIG. 1, REN5T is close to the genetic distance of Sphingomonas and is an independent branch in the phylogenetic tree, and is determined to be Sphingomonas aquatilis, and the similar strain is known to be Sphingomonas aquatilis according to the similarity (98.66%). Then, REN5T strain is sent to Shanghai Meiji biological medicine science and technology limited to perform whole genome sequencing, so as to obtain a whole genome sequence. At the same time, the whole genome of the similar strain was downloaded from NCBI, and the whole genome of REN5T was compared with that of the similar strain Sphingomonas aquatilis at DSMZ (http:// ggdc. DSMZ. de/home. php) for genomic DNA-DNA homology hybridization (DDH): DNA Homology (DDH) is 23.00% [ 20.8-25.5% ], and DDH is less than 70%, and thus it was preliminarily determined as a new species.

Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Sequence listing

<110> Beijing university of Industrial and commercial

<120> new strain of Beigong sphingosine monad and application thereof

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aacgctggcg gcatgcctaa cacatgcaag tcgaacgaag ccttcgggct tagtggcgca 60

cgggtgcgta acgcgtggga atctgccctt tggttcggaa taacagttgg aaacgactgc 120

taataccgga tgatgacgaa agtccaaaga tttatcgcca gaggatgagc ccgcgtagga 180

ttagctagtt ggtgtggtaa aggcgcacca aggcgacgat ccttagctgg tctgagagga 240

tgatcagcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 300

atattggaca atgggcgaaa gcctgatcca gcaatgccgc gtgagtgatg aaggccctag 360

ggttgtaaag ctcttttacc cgggatgata atgacagtac cgggagaata agccccggct 420

aactccgtgc cagcagccgc ggtaatacgg agggggctag cgttgttcgg aattactggg 480

cgtaaagcgc acgtaggcgg ctttgtaagt tagaggtgaa agcctggagc ttaactccag 540

aactgccttt aagactgcat cgcttgaatc cgggagaggt gagtggaatt ccgagtgtag 600

aggtgaaatt cgtagatatt cggaagaaca ccagtggcga aggcggctca ctggaccggt 660

attgacgctg aggtgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac 720

gccgtaaacg atgataacta gctgtccggg gacttggtct ttgggtggcg cagctaacgc 780

attaagttat ccgcctgggg agtacggccg caaggttaaa actcaaatga attgacgggg 840

gcctgcacaa gcggtggagc atgtggttta attcgaagca acgcgcagaa ccttaccagc 900

gtttgacatg tccggacgac tggcagagat gcctttcttc ccttcgggga ctggaacaca 960

ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1020

cgcaaccctc gcctttagtt accatcattt agttgggtac tctaaaggaa ccgccggtga 1080

taagccggag gaaggtgggg atgacgtcaa gtcctcatgg cccttacgcg ctgggctaca 1140

cacgtgctac aatggcggtg acagtgggca gcaatcccgc gagggtgagc taatctccaa 1200

aagccgtctc agttcggatt gttctctgca actcgagagc atgaaggcgg aatcgctagt 1260

aatcgcggat cagcatgccg cggtgaatac gttcccaggc cttgtacaca ccgcccgtca 1320

caccatggga gttggattca cccgaaggcg ttgcgccaac ccgcaagggg agcaggcgac 1380

cacggtgggt tcagcgactg gggtgaagtc gt 1412

Claims

1. The strain is characterized in that the strain is Sphingomonas beigongensis (Sphingomonas beigongensis), and the preservation number of the strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC NO. 19204;

the application of the strain in the following (A1) or (A2): (A1) generating strong aromatic flavor substances; (A2) preparing the product with the strong aromatic flavor substances.

2. The strain of claim 1,

the 16SrDNA sequence of the strain in the Sphingomonas beigongensis (Sphingomonas beignongshangensis) is shown as SEQ ID No. 1.

3. A bacterial agent characterized in that the active ingredient thereof is the strain according to claim 1.

4. Use of the strain of claim 1 or the microbial agent of claim 3 in (B1) or (B2) as follows:

(B1) brewing wine;

(B2) and (5) preparing a wine brewing product.