CN113355256B - Northern industrial and commercial sporosarcina sarcina strain and application thereof - Google Patents

Abstract

The invention discloses a new strain of sarcina beigong commercial spore and application thereof, wherein the strain is sarcina beigong commercial spore (Sporosarcina beigonggshangensis) with the preservation number of CGMCC NO.19211 in the common microorganism center of China Committee for culture Collection of microorganisms. Experiments prove that the new strain provided by the invention is isolated from white spirit cellar mud, can produce substances with strong fragrance and flavor, and can be used for fermentation and brewing.

Description

Northern industrial and commercial sarcina strain and application thereof

Technical Field

The invention relates to the technical field of brewing microorganisms, and in particular relates to a new strain of sarcina beigongensis and application thereof.

Background

The whole inner wall of the pit is covered with pre-cultured pit mud, and 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 in 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 maturation 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, and are deeply loved by the consumers. 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 all known microorganisms, and no new microorganism species is found.

Disclosure of Invention

Therefore, the invention provides a new strain of sarcina hokkaiensis and application thereof.

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

the invention provides a northern industrial and commercial sarcina beiggonggensis (Sporosarcina beiggonggensis) with the preservation number of CGMCC NO.19211 in China general microbiological culture Collection center.

The 16S rDNA sequence of the sarcina produced in North Industrial science of the invention is shown in SEQ ID No. 1.

The invention also provides application of the sarcina beigongensis or the fermentation product or the culture product thereof in producing flavor substances in the brewing process.

The microbial inoculum containing the sarcina beigongensis also belongs to the protection scope of the invention.

The invention also provides a screening and culturing method of the sarcina beigongensis, which comprises the steps of diluting pit mud, coating the diluted pit mud on a TSA culture medium, and screening to obtain the sarcina beigongensis.

In one embodiment of the invention, the TSA medium comprises the following mass numbers of raw materials: 15g of tryptic digest of casein, 5g of papain digest of soybean meal, 5g of sodium chloride and 15g of agar.

In one embodiment of the invention, the time of the screening culture is 20-28 h.

In one embodiment of the invention, the screening culture temperature is 35-40 ℃.

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

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

The invention has the following advantages:

experiments prove that the new strain, namely Sporosarcina beigongensis, is separated from pit mud, can produce substances with strong fragrance and flavor, and can be used for fermenting and brewing wine.

The TSA culture medium is firstly adopted for separating the strains, the common bacteria are relatively slow in reproduction, the bacteria of the sarcina of the newly-cultured spodophyllum difficult to culture grow relatively fast on the same culture dish, and the influence of the conventional microorganisms is small; 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 sarcina (Sporosarcina beigongganggensis) is deposited in the China general microbiological culture Collection center (CGMCC) at 12 and 16 days 2019, and the deposition address is as follows: the preservation number of the microbial research institute of Chinese academy of sciences, No. 3 Xilu No.1 of Beijing, Chaoyang, and the institute of microbiology, is CGMCC NO. 19211.

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 a diagram of 16S rDNA sequence and evolutionary tree of Sporosarcina beigongensis (Sporosarcina beignogonggensis) provided by the present invention;

FIG. 2 is a graph showing the result of detecting respiratory quinone ubiquinone MK-7 in Sporospora beigondii (Sporosarcina beigongshangensis) according to the present invention;

FIG. 3 is a diagram showing the results of detecting polar esters of Sporosarcina beigongshangensis (Sporosarcina beigongshangensis) provided by the present invention.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. 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 Sporosarcina beigongensis

Isolation of novel species of Sporosarcina beigongensis

Step one, preparing a TSA culture medium: pancreatin digest of casein 15g, papain digest of soybean flour 5g, sodium chloride 5g, and agar 15 g.

Step two, taking 10g of pit mud, and diluting the pit mud to 10g by using sterile distilled water ^-5 Coating on a prepared TSA culture medium culture dish for aerobic culture at 28 ℃;

step three, after aerobic culture is carried out for 36 hours at 28 ℃, single colony is picked, and three areas are streaked and purified for 3 times to obtain pure culture bacteria;

and step four, extracting bacterial DNA from the pure culture bacteria, amplifying a 16SrDNA sequence by utilizing a PCR technology, and preliminarily determining a new strain of the northern commercial sarcina beignogongshangensis through sequence comparison.

Identification of Sporosarcina beigongensis (Sporosarcina beigongensis)

The new bacteria separated and screened from the pit mud are gram-negative bacteria, namely, the genus Sporosarcina, named as: sporospora coerulea (Sporosarcina beigongshengensis)

1. The strain of Sporosarcina beigongensis (Sporosarcina beigongensis) is subjected to morphological, physiological, biochemical, cytochemical and gene level research. Physiological and biochemical characteristics of sarcina beigongensis REN13(Sporosarcina beigongensis)

Colony morphology of northern industrial and commercial sarcina (Sporosarcinabeignonshangensis) is round, and the surface is smooth, yellowish and free of spores; gram-positive bacteria, the optimum temperature is 28 ℃, the optimum pH value is 7, the optimum sodium chloride concentration is 1%, the starch can be hydrolyzed, the catalase is positive, the alkaline phosphatase, the esterase (C4), the leucine arylamine, the valine arylamine, the cystine arylamine, the chymotrypsin, the acid phosphatase, the naphthol-AS-BI-phosphohydrolase, the beta-glucuronidase and the beta-glucosidase are in positive reaction, and the nitrate is reduced to hydrolyze the glucosidase-esculin.

2. Cytochemical characteristic detection of sarcina (Sporosarcina beigongshengnsis)

The cytochemical components of the species fatty acids, quinones, polar lipids and the like of Sarcina beijerinckii (Sporogenic microorganisms) were detected by GC, HPLC and TLC thin-layer chromatography (Sasser M. identification of bacterial by gas chromatography, DE: MIDIinc; 1990.Minnikin DE, O' Donnel AG, GoodFellfem, Alderson G, Atharye M et al. Integrated procedure for the extraction of bacterial of microbial lipids, JMicrobiol 1984; 233: method 241).

Sarcina beigongensis (Sporosarcina beigongganggensis) cell fatty acid component: saturated fatty acids: c _16:0 ，Antesio-C _15:0 ，Antesio-C _17:0 ，Iso-C _15:0 ，Iso-C _16:0 ，Iso-C _17:0 (ii) a Unsaturated fatty acid: c _16:0 N alcohol. The cellular fatty acid composition is shown in Table 1

TABLE 1

As shown in figure 2, in the detection of the cell respiration quinone component of the strain Sporosarcina beigongshanensis (Sporosarcina beignonggensis), the main advantage of the strain is quinone ubiquinone MK-7, and quinone compounds are a class of oxidation active substances which are widely existed in natural products, anti-tumor drugs, in-vivo biochemical metabolites, environmental pollutants or polycyclic aromatic hydrocarbon metabolism. Each bacterium has a main quinone component, and the difference in the kind and amount 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 sarcina beijeringshangensis (Sporosarcina beigongensis) comprises the following specific steps: polar lipid extraction: 100mg of lyophilized mycelia of sarcina beicommercially available were suspended in 9.5mL of chloroform: methanol: 0.3% NaCl (2.5:5: 2). Placing the thallus solution in water bath at 80 deg.C 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 by 10cm Silica gel plate (25 TLC alumina sheets 20cm by 20cm Silica gel 60F254 from Merck) was activated in an oven at 110 ℃ for 1 hour, and then removed and cooled. Pipette 2 μ L of total lipid sample onto TLC plate, and spot 3 times.

And (3) placing the TLC thin plate into a first chromatographic cylinder for layer development, wherein the first spreading agent is chloroform, methanol and water (65:25:4, v/v), taking out the thin plate after the solvent is spread to the top, drying the thin plate by blowing, placing the thin plate into a second chromatographic cylinder, and 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, sarcina are commercially available from north, and contain 4 polar esters, DPG: diphosphatidylglycerol, diphosphatidylglycerol; PG: phosphatidylglycerol, phosphonoglycerolpethophipopid; PE phosphatidylethanolamine, phosphatidyl ethanolamines; PL: phospholipids, Phospholipids. And the protein, the sugar and the like form cell membranes, and play an important role in substance transportation, metabolism and normal osmotic pressure maintenance. The phospholipidic components of different genera of bacteria are different, and are one of the important characteristics for identifying the genus, and are indispensable classification indicators in chemical classification projects.

3. 16S rDNA sequence and evolutionary tree of Sporosarcina beigonggangshensis

The strain of Sporospora sarcina beigongshangensis (Sporosarcina beigongshengensis) was subjected to whole genome sequencing by Meiji biological medicine science and technology Co., Ltd, Shanghai, to obtain a whole genome sequence. The phylogenetic tree is shown in figure 1.

Measured REN13 ^T The 16S rRNA sequences were aligned with the model strains in NCBI (https:// www.ncbi.nlm.nih.gov /), and 15 model strains 16S rRNA sequences were selected according to the similarity for phylogenetic tree construction, as shown in FIG. 1, REN13 ^T The strain is close to the related distance of the sarcina protosporum and is an independent branch in a phylogenetic tree, the sarcina protosporum is determined to be the sarcina protosporum, and the similar strain is Sporosarcina psychrophila DSM 6497 according to the similarity (98.98 percent). Then REN13 will be ^T The strain is sent to Shanghai Meiji biological medicine science and technology limited company for whole genome sequencing to obtain a whole genome sequence. At the same time, the whole genome of the similar strain is downloaded from NCBI, and REN13 is added ^T Whole genome and similar strain Sporosarcinaphyrophia DSM 6497 genome wide alignment at DSMZ (http:// ggdc. DSMZ. de/home. php) genomic DNA-DNA homology hybridization (DDH): DNA Homology (DDH) 20.90% [ 18.6-23.3% ]]DDH is less than 70%, and thus it is preliminarily determined as a new species.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing university of Industrial and commercial

<120> new strain of northern industrial and commercial sarcina and application thereof

<130> GG19719814A

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aactggcggc tgctaataca tgcaagtcga gcggattgat gggagcttgc tccctgatat 60

tagcggcgga cgggtgagta acacgtgggc aacctgccct acagatgggg ataactccgg 120

gaaaccgggg ctaataccga ataatcagtt ggttcgcatg aaccaactct gaaagacggc 180

ttcggctgtc actgtaggat gggcccgcgg cgcattagct agttggtggg gtaatggcct 240

accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300

cggcccagac tcctacggga ggcagcagta gggaatcttc cacaatggac gaaagtctga 360

tggagcaatg ccgcgtgagc gaagaaggtt ttcggatcgt aaagctctgt tgtaagggaa 420

gaacacgtac gggagtaact gcccgtacct tgacggtacc ttattagaaa gccacggcta 480

actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540

gtaaagcgcg cgcaggcggt cctttaagtc tgatgtgaaa gcccacggct caaccgtgga 600

gggtcattgg aaactggggg acttgagtac agaagaggaa agcggaattc cacgtgtagc 660

ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcggctttc tggtctgtaa 720

ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840

attaagcact ccgcctgggg agtacggccg caaggctgaa actcaaagga attgacgggg 900

acccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960

tcttgacatc ccactgcccg gtgtagagat acgcctttcc cttcggggac agtggtgaca 1020

ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080

cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140

caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200

cacgtgctac aatggatgat acagagggtt gccaacccgc gagggggagc caatcccata 1260

aaatcattcc cagttcggat tggaggctgc aactcgcctc catgaagccg gaatcgctag 1320

taatcgtgga tcagcatgcc acggtgaata cgttcccggg tcttgtacac accgcccgtc 1380

acaccacgag agtttgtaac acccgaagtc ggtggggtaa cccttacggg agccagccgc 1440

cgaagcgaca aggagt 1456

Claims (5)

1. The culture number of the northern industrial and commercial sarcina beignogongshangensis (Sporosarcina beignogonggensis) in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC NO. 19211.

2. The sarcina beijerinckii of claim 1, which has the 16S rDNA sequence shown in SEQ ID No. 1.

3. A microbial agent comprising sarcina beigongensis according to claim 1.

4. Use of sarcina beijerinckii according to claim 1 or the microbial inoculum according to claim 3 in (a1) or (a2) as follows:

(A1) generating aromatic flavor substances;

(A2) preparing the product with the aroma flavor substances.

5. Use of sarcina beijerinckii according to claim 1 or the microbial inoculum according to claim 3 in (B1) or (B2) as follows:

(B1) brewing wine;

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

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