CN112300944A

CN112300944A - Hordeum radiobacter strain and application thereof in preparation of fragrant substances

Info

Publication number: CN112300944A
Application number: CN201910680985.XA
Authority: CN
Inventors: 赵瑞琳; 刘安琪; 任晋玮; 曹槟
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2019-07-26
Filing date: 2019-07-26
Publication date: 2021-02-02
Anticipated expiration: 2039-07-26
Also published as: CN112300944B

Abstract

The invention discloses a rhodoporus radiobacter strain and application thereof in preparing a fragrant substance. The invention discloses a fumarole-glomus-shot bacterial strain, the preservation number of which in China general microbiological culture Collection center is CGMCC No. 17071. The fumarole-like rhodobacter sphaeroides strain is cultured to obtain fragrant substances such as benzaldehyde, linalool and benzyl alcohol, and the substances can be used as raw materials for preparing essences and spices. The fomes fumosoroseus is a fragrant fungus with potential commercial application value, and has important significance for developing large fungus fragrance resources and natural essence.

Description

Hordeum radiobacter strain and application thereof in preparation of fragrant substances

Technical Field

The invention relates to a fumaria fumosorosea strain and application thereof in preparing a fragrant substance, belonging to the technical field of biology.

Background

Nowadays, the production of flavors and fragrances has important application in the food, medicine and daily chemical industries. At present, the essence and flavor are divided into two aspects of natural essence and artificial essence. The production of natural essence mainly adopts several ways of extracting from aromatic plant, extracting from animal musk and fermenting from aroma-producing microorganism. Wherein, the microorganism fermentation method is one of the most developing methods for producing natural essence at present.

The aroma-producing microorganism utilizes natural raw materials to synthesize a large amount of natural aroma components, belongs to green chemistry, and has the advantages of short period, high quality, safety, environmental protection and the like. At present, most of the aroma-producing microorganisms are researched by yeasts and molds, but many reports about aroma production of large fungi are not found. And the volatile components of the aroma-producing microorganisms are various in composition and are easily influenced by culture conditions, and the culture conditions need to be optimized to obtain the optimal volatile component combination.

Benzaldehyde is colorless or pale yellow volatile oily liquid with strong refractive index, has bitter almond flavor, is miscible with ethanol, diethyl ether, volatile oil and non-volatile oil, and is slightly soluble in water. It is an important chemical raw material.

Linalool, also known as linalool, is a terpene alcohol, one of the well-known perfume compounds. The colorless oily liquid has sweet, tender and fresh flower fragrance and is like lily of the valley fragrance. Linalool with alcohol content of more than 95% is an important spice and is used in fragrance industry such as floral essence, perfume, perfumed soap and the like. The alcohol content of 92.5 percent is a raw material drug of isophytol which is an important intermediate for preparing vitamin E in the pharmaceutical industry. It can also be used as raw material for preparing esters such as linalyl acetate which is a very valuable perfume.

Benzyl alcohol, also known as benzyl alcohol, is a colorless, transparent, viscous liquid with a faint aromatic odor. It is mainly present in floral essential oils in free form or in the form of esters.

Disclosure of Invention

The invention aims to provide a fumaria fumosorosea strain capable of producing fragrant substances

The preservation number of the fomes fumosoroseus strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 17071.

The invention also provides a microbial inoculum, and the active component of the microbial inoculum is the rhodobacter sphaeroides strain.

In the above microbial inoculum, the microbial inoculum may further comprise a carrier. The carrier may be an edible solid carrier or a liquid carrier. In the microbial inoculum, the active ingredient may be present in the form of cultured live cells or dead cells, a mixture of live cells or dead cells and fermentation broth. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a stabilizer (such as an antioxidant), a pH regulator and the like.

The present invention also provides a process for the preparation of a fragrance material, the process comprising: culturing the said strain to obtain fermentation liquid containing the said fragrant substances.

In the above method, culturing the strain of fomes fumosoroseus may be carried out using a medium for culturing the strain of fomes fumosoroseus.

In the above method, the culture medium for culturing the fomes japonicus strain can be a liquid culture medium, the liquid culture medium is composed of a solute and a solvent, the solvent is water, and the solute and the concentration thereof in the liquid culture medium are respectively 20g/L glucose, 2g/L peptone, 2g/L yeast powder and 3g/L KH₂PO₄，1.5g/L MgSO₄，0.01g/LV_B1。

In the above method, culturing the strain of fomes fumosoroseus may be carried out at 25-27 ℃.

Culturing the strain of the fomes officinalis Linn can be carried out at the rotating speed of 150 r/min. The time for culturing the strain of the Porphyra radiobacter can be enough to smell the fragrance, such as 8-10 days. In one embodiment of the invention, the cultivation time is 9 days.

The invention also provides a fermentation method of the fomes fumarosa strain, which comprises the step of culturing the fomes fumarosa strain by using the culture medium for culturing the fomes fumarosa strain to realize the fermentation of the fomes fumarosa strain.

The invention also provides any one of the following applications of the rhodobacter sphaeroides strain or the microbial inoculum:

x1) in the preparation of food products;

x2) in the preparation of essence;

x3) in the preparation of industrial products.

The food, the essence and the industrial goods contain a fragrant substance.

The invention also provides a product for preparing the fragrant substance, and the active ingredient of the product is the strain of the fumaria fumosorosea.

In the present invention, the fragrant substance may be benzaldehyde, linalool, benzyl alcohol, p-tolualdehyde, 2-methylbutanol, n-hexanol, 3-methylbutanol, phenethyl alcohol, 4-methyl-5- (beta-hydroxyethyl) thiazole, furfural, 2-methylbutyric acid, 3-dimethylacrylic acid, hydroxyacetone, 3-hydroxy-2-butanone, furfuryl alcohol, 2-ethylhexanol, 5-methylfurfural and/or hexanoic acid.

The fumarole-like rhodobacter sphaeroides with the preservation number of CGMCC No.17071 is cultured to obtain benzaldehyde, linalool, benzyl alcohol and other fragrant substances which can be used as raw materials for preparing essences and spices. The fomes fumosoroseus is a fragrant fungus with potential commercial application value, and has important significance for developing large fungus fragrance resources and natural essence.

Biological material preservation instructions

Classification nomenclature of biological materials: phlebiporia sp.

Strain number of biological material: GX20172167

Deposit name of biological material: china general microbiological culture Collection center

The preservation unit of the biological material is abbreviated as: CGMCC (China general microbiological culture Collection center)

Deposit unit address of biological material: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101

Preservation date of biological material: year 2019, month 01, day 11

Accession number to the collection of biological materials: CGMCC No.17071

Drawings

FIG. 1 shows the form of the strain of Porphyra radiata with the preservation number of CGMCC No. 17071.

FIG. 2 is a phylogenetic tree based on ITS in combination with LSU sequences. The bold font is the rhodoporus radiatus with the preservation number of CGMCC No. 17071.

FIG. 3 is a GC-MS total ion flow diagram of the fermentation liquid of the fomes fumosoroseus strain with the preservation number of CGMCC No. 17071.

FIG. 4 shows benzaldehyde (A), linalool (B) and benzyl alcohol (C) in the structure of volatile compounds of fomes fumosoroseus.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

Example 1 acquisition of Fomitopsis fumosorosea species

Bacterial strain source and obtaining method

The invention separates and obtains a pulse-shooting fomes officinalis strain (Phleboporia sp.) which is separated from the fresh fruit body of the wild pulse-shooting fomes officinalis (specimen collection number GX20172167) of the Dayaoshan county, Jinxiu county, Guangxi Zhuang nationality. The strain is obtained by separating a sporocarp tissue separation method: collecting fruiting bodies in the field, carrying out experimental operation in a sterile area near the flame of an alcohol burner, cutting a gap on fresh fruiting bodies of wild Horizonta pulse by using a sterilized scalpel, pinching two sides with two hands, pulling outwards to expose internal tissues, placing part of tissues in a sterile culture dish filled with solid PDA, carrying out dark culture at 25 ℃, carrying out periodical reexamination, picking germinated hyphae into other culture media, and purifying to obtain the hypha, thereby obtaining the Horizonta pulse strain, wherein the shape of the strain is shown in figure 1.

The strain is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Siro 1 of Beijing, Chaoyang, and North Cheng) on 11.01.2019, and the preservation number of the strain is CGMCC No. 17071.

Second, molecular phylogenetic analysis of the strains

Extraction of laboratory DNA: culturing the fomes fumarofaciens strain with the preservation number of CGMCC No.17071 obtained in the step one on a PDA plate, extracting DNA by using a rapid plant DNA magnetic bead method extraction kit produced by Zhiang biotechnology company, performing PCR amplification by using general ITS primers ITS1 (5'-TCCGTAGGTGAACCTGCGC-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), sequencing the PCR amplification product, and obtaining a PCR product sequence which is a sequence 1 in a sequence table. And the LSU primer LR5 (5-

TCCTGAGGGAAACTTCG-3 ') and LROR (5'-ACCCGCTGAACTTAAGC-3'), and sequencing the PCR product, wherein the sequence of the obtained PCR product is the sequence 2 in the sequence table.

And (3) constructing a phylogenetic tree by using a maximum likelihood method, wherein a GTRGAMMA model is selected for the raxmlGUI, and the operation is carried out 1000 times, wherein the result is shown in the figure. The P.radiata strain with the preservation number of CGMCC No.17071 is gathered with Phlebiporia and has 95 percent of self-expanding support rate (see figure 2).

Example 2 analysis of volatile Components of Fomitopsis fumago

First, liquid fermentation

Using a liquid culture medium (the culture medium is composed of a solute and a solvent, the solvent is water, the concentration of the solute and the concentration of the solute in the culture medium are respectively 20g/L glucose, 2g/L peptone, 2g/L yeast powder and 3g/L KH₂PO₄，1.5g/L MgSO₄，0.01g/L V_B1) Culturing hyphae by the following steps:

perforating a flat plate of the mycelium of the fomes punctatus with the preservation number of CGMCC No.17071 obtained in the first step of overgrowing with a sterilized perforator in a super clean bench, inoculating the mycelium blocks overgrown with the mycelium into 250ml conical flasks filled with 125ml of liquid culture medium, and inoculating 10 mycelium blocks into each culture flask. Then putting the mixture into a shaking table, and culturing the mixture for 9 days at the temperature of 25 ℃ and the rotating speed of 150r/min to obtain fermentation liquor.

Second, volatile substance separation method and component analysis

Filtering the fermentation liquor obtained in the step one by using filter paper, collecting filtrate, performing solid phase extraction by using macroporous resin (HP20) as an adsorption material, and performing primary extraction and enrichment on aroma components; eluting the macroporous resin with anhydrous ethanol to obtain ethanol eluate (strain extractive solution) containing aroma components.

The volatile aroma components of the strain extracting solution are detected by a gas chromatography-mass spectrometry (GC-MS) method, and the detection conditions are as follows:

the analysis was carried out by gas chromatography-mass spectrometry (GC-MS) combined technique, and the apparatus was GCMS-QP 2010Ultra, Shimadzu corporation. GC conditions were as follows: the chromatographic column is Rtx-Wax capillary column (30m 0.25mm 0.25 μm), the initial column temperature is 35 deg.C, the temperature is programmed to 5 deg.C/min to 230 deg.C, the temperature is maintained for 15min, the column flow is 1ml/min, and the sample injection is not split. MS conditions: EI ion source, ion source temperature 230 ℃, interface temperature 230 ℃, scanning range 29-500amu, solvent delay 4.5min, and quantitative analysis by adopting an area normalization method.

The total ion flow diagram (TIC) is shown in fig. 3. The main chromatogram extraction mass spectrum and similarity search through the NIST 11 database carried by shimadzu GC-MS showed that 118 substances were detected in total, of which 18 compounds had utility for the preparation of fragrances or flavorants, the 18 substances being benzaldehyde, linalool, benzyl alcohol (fig. 4) and p-methylbenzaldehyde, 2-methylbutanol, n-hexanol, 3-methylbutanol, phenethyl alcohol, 4-methyl-5- (beta-hydroxyethyl) thiazole, furfural, 2-methylbutyric acid, 3-dimethylacrylic acid, hydroxyacetone, 3-hydroxy-2-butanone, furfuryl alcohol, 2-ethylhexanol, 5-methylfurfural, hexanoic acid (table 1).

TABLE 1 ingredient table of aroma compounds detected by GC-MS of fermentation broth of fomes officinalis

<110> institute of microbiology of Chinese academy of sciences

<120> an Oreoporus fumosus strain and application thereof in preparation of fragrant substances

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 615

<212> DNA

<213> Phlebiporia sp.

<400> 1

gaacctgcgg aaggatcatt aacgagtttt tgaaaggggt tgtagctggc caaacaaagg 60

catgtgcaca ccctgctcaa tccactctca aacccctgtg cacttattgt aggctatggt 120

gggataattg atttcggtcg atttgaaagc cgtgtctatg tttattacaa acgcttcagt 180

taaagaatgt tatctcgcgt ttaacgcatt atatacaact ttcagcaacg gatctcttgg 240

ctctcgcatc gatgaagaac gcagcgaaat gcgataagta atgtgaattg cagaattcag 300

tgaatcatcg aatctttgaa cgcaccttgc gctccttggt attccgagga gcatgcctgt 360

ttgagtgtca tggaattatc aacttctata gcttttgtta tagaggcttg gatgtggagg 420

ttgtgttggc ttgcaaaggt caactcctct gaaatgcatt agtgtgaacc atacggaacg 480

ccttcagtgt gataattatc tacgctgtgg tgctgaagta tattgtgtca tgcttataac 540

cgtccgcaag gacaattact tgacaatctg acctcaaatc aggtaggact acccgctgaa 600

cttaagcata tcaat 615

<210> 2

<211> 1206

<212> DNA

<213> Phlebiporia sp.

<400> 2

accacccgga tgttgtggaa acccgagcgt aataaaaaga aagtggatcc ctttgggagc 60

cacgacgccc cataccagcc cttctggacg atatgcggtg agcagtatgt ggacccgaaa 120

gatgggaact tatcctgaat aggggaagcc agagaaactc tgtggaagct cgtagcgatt 180

ctgacgtgca aatcgatctt caaatttggg tatagggcga aagactaatc gaaccatcta 240

gtagcgggtt ggggtgggct ttcctgagcg aaacttcggc aggaaccagc tactagatgg 300

ttcgattagt ctttcgcccc tatacccaaa tttgacgatc gatttgcacg tcagaatcgc 360

tacgagcttc caccagagtt tcctctggct tcaccctatt caggcatagt tcaccatctt 420

tcgggtccca acatacatgc tcaaccgcat atccgtcaca gaaggtctgg tatgggcgtc 480

ggtgctcccc acgacaggga tcccaacttt cactttcatt acgcgctcgg gtttaccacc 540

caaacactcg caggcatgtt agactccttg gtccgtgttt caagacgggt cgcttaaagc 600

cattacgcca acatcctaag cacgtacgtg ggcgaacccc ggccataagg cgtgctgcgg 660

tcctcagtcc caaccaccgt atacaaccaa aggctataac acacccgtag gtgccacatt 720

ccaaaggctt ttatccggca gtcaaaactg atgttggccc gtcaactaga aagtgcacca 780

agcaaaagca aggttgagtt ctagacgacg cgactgactt caagcggttc cctttcagca 840

atttcacgta ctgtttaact ctctttccaa agtgcttttc atctttccct cacggtactt 900

gttcgctatc ggtctctcgc caatatttag ctttagatgg aatttaccac ccattttgag 960

ctgcattccc aaacaactcg actctttgag agcgcatcac aaagcactgg tagtccgtgt 1020

catggacggg attctcaccc tctatgacgc tctgttccaa gagacttgta cacggtccag 1080

cgcggaagac acttctccag actacaactc gggcagccaa agactaccag attttaaatt 1140

tgagcttttc ccgcttcact cgcagttact aggggaatcc ttgttagttt cttttcctcc 1200

gcttat 1206

Claims

1. The preservation number of the fumarole-roselle bacterial strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 17071.

2. A microbial agent comprising as an active ingredient the strain of Clarithromyces rikos according to claim 1.

3. A method of preparing a fragrance material comprising: culturing the strain of fomes officinalis of claim 1 to obtain a fermentation broth containing said flavor substance.

4. The method of claim 3, wherein: the fragrant substance is benzaldehyde, linalool, benzyl alcohol, p-tolualdehyde, 2-methylbutanol, n-hexanol, 3-methylbutanol, phenethyl alcohol, 4-methyl-5- (beta-hydroxyethyl) thiazole, furfural, 2-methylbutyric acid, 3-dimethylacrylic acid, hydroxyacetone, 3-hydroxy-2-butanone, furfuryl alcohol, 2-ethylhexanol, 5-methylfuran aldehyde and/or hexanoic acid.

5. The method according to claim 3 or 4, characterized in that: culturing the fomes fumosoroseus strain is carried out by using a culture medium for culturing the fomes fumosoroseus strain.

6. The method of claim 5, wherein: the culture medium for culturing the Horseradish fumonis strain is a liquid culture medium, the liquid culture medium consists of a solute and a solvent, the solvent is water, the solute and the concentration of the solute in the liquid culture medium are respectively 20g/L glucose, 2g/L peptone, 2g/L yeast powder and 3g/L KH₂PO₄，1.5g/L MgSO₄，0.01g/L V_B1。

7. The method according to any one of claims 3-6, wherein: culturing the strain of fomes officinalis at 25-27 ℃.

8. The method for fermenting a strain of fomes fumosus of claim 1, comprising culturing the strain of fomes fumerica with a medium for culturing the strain of fomes fumerica to effect fermentation of the strain of fomes fumerica.

9. Use of the strain of fomes fumosoroseus of claim 1 or the bacterial agent of claim 2 in any one of the following applications:

x1) in the preparation of food products;

x2) in the preparation of essence;

x3) in the preparation of industrial products.

10. A product for the preparation of a fragrance substance, the active ingredient of which is the strain of rhodoporus fumosus as claimed in claim 1.