CN107142220B - Trichosporon for producing gamma-decalactone and application thereof - Google Patents

Abstract

The invention discloses a strain of Piptoporus sp. A-9 that produces γ-decalactone, and the strain has been deposited in the "China Microorganism Culture Collection" on May 25, 2017 General Microbiology Center of the Management Committee", accession number CGMCC No. 13895. The present invention also discloses the application of the Tritonobacterium in the preparation of γ-decalactone. Experiments have confirmed that the yield of γ-decalactone produced by the fermentation of the Tritonobacterium of the present invention can be up to 6.5 g/L or more, which explores a new direction and approach for liquid fermentation to produce γ-decalactone, and has a relatively high yield. It has great application value and has great industrial application prospects.

Description

A γ-decalactone-producing Trichopycium and its application

technical field

The invention relates to a gamma-decalactone-producing Trichopycium bacterium and its application in the preparation of gamma-decalactone, belonging to the field of biotechnology.

Background technique

Lactone flavors occupy an important position in the flavor industry with their unique aroma quality, and can be used in the preparation of food and daily necessities flavors. Among them, the most widely used one is γ-decalactone. γ-decalactone (gamma-decalactone, GDL), also known as gamma-decalactone, its molecular formula is C ₁₀ H ₁₈ O ₂ , the aroma threshold of γ-decalactone is 11 μg/kg, and the aroma threshold is very low. The aroma is strong, with sweet fruity, peach, coconut, vanilla and creamy notes. Gamma-decalactone is listed as a food additive generally recognized as safe by the U.S. Food and Drug Administration. It is also listed as a permitted food spice in China's Hygienic Standard for the Use of Food Additives. Gamma-decalactone is a common raw material for formulating food flavors such as milk, cream, butter, peaches, apricots, berries, mangoes, apples, strawberries, coconuts, citrus, and chocolate.

Natural gamma-decalactone is present in many fruits, such as peaches, strawberries, coconuts, mangoes, apricots, etc. Extraction from these natural raw materials is the main method for preparing natural γ-decalactone. However, because the production of plant raw materials is limited by regions and seasons, it is difficult to achieve continuous and large-scale supply. At present, the main production method of γ-decalactone is chemical synthesis. However, the residual chemical substances in the production process of synthetic fragrances may be detrimental to human health, and the γ-decalactone synthesized by chemical synthesis is an optically non-active racemic mixture, while the use of microorganisms can produce optically active and relatively Pure products are a big trend in the production of natural flavors.

At present, it has been found that the microorganisms that can synthesize or transform γ-decalactone are mainly yeast and some filamentous fungi. Among them, the most studied is Yarrowia lipolytic. -The technology of decanolide has been protected by foreign patents.

γ-Decalactone is an intermediate product of metabolism of various microorganisms, and γ-decalactone can be produced by microbial fermentation with castor oil as a precursor. However, according to the current literature on the production of γ-decalactone by microorganisms, most of the reports are related to the theoretical research on the metabolic pathway of γ-decalactone, and a few reports on the production of γ-decalactone are also genetically engineered strains. Only gram-level production capacity can be achieved by manipulating mutant strains.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the problem to be solved by the present invention is to provide a γ-decalactone-producing Trichoderma and its application in the preparation of γ-decalactone. The strain of the present invention can effectively transform castor oil to generate γ-decalactone, and realize the desire to obtain γ-decalactone as the main target product through a microbial fermentation method.

The γ-decalactone-producing Piptoporus sp. A-9 of the present invention belongs to the genus Piptoporus sp. of the family Polyporaceae, and is characterized in that: the strain is named Piptoporus sp. A-9, and the strain has been released in 2017 It was deposited in the "General Microbiology Center of China Microorganism Culture Collection and Management Committee" on May 25, 2009, and the preservation number is CGMCC No.13895.

The above-mentioned Tritoporus A-9, which produces γ-decalactone and triterpenoids, is a Tritoporus isolated and purified by the inventor's self-picking in the field. , the size is about 20 × 15cm, the thickness of a single cover is about 0.5-1.5cm, and the cover is arranged in imbricates. The front of the fruiting body is pale yellow to orange, the edge is darker, wavy or petal-shaped, and more round, the back of the fruiting body is white, and the edge is yellow, full of fungal pores. Mycelial system disomic, with reproductive and skeletal hyphae. Reproductive hyphae have the distinctive feature of basidiomycete hyphae, namely lock-like association. At the same time, the reproductive hyphae of the bacteria also have simple diaphragms, but the number of simple diaphragms is small, and they often appear at the front end of the hyphae. Reproductive hyphae vary in diameter from 2-3 μm to 5-7 μm.

The results of the determination of the gene sequences of 18S rRNA and ITS for the γ-decalactone-producing Trichomonas A-9 CGMCC No. 13895 of the present invention show that the length of its 18S rDNA sequence is 1679bp, and its nucleotide sequence is as shown in SEQ ID NO. .1; its ITS sequence fragment size is 727bp, and its nucleotide sequence is shown in SEQ ID NO.2. It was preliminarily identified as Piptoporus sp. by morphological observation and alignment of 18SrRNA and ITS gene sequences.

The basic method for the selection and breeding of the γ-decanolactone-producing Trichoderma A-9 strain of the present invention is:

The strains were collected from the fruiting bodies of Basidiomycetes in the wild. The fresh fruiting bodies were cut with a sterile scalpel, and a little bacterial flesh was torn with ophthalmic tweezers and inoculated on a PDA plate, and cultured at 30°C. When the diameter of the mycelium pad reaches 2-3 cm, pick a little mycelium from the edge of the mycelium and inoculate it on a new PDA plate, and transfer it repeatedly until the pure strain is isolated. The strain was named Piptoporus sp. A-9, and the strain was deposited in the "General Microbiology Center of China Microorganism Culture Collection and Management Committee" on May 25, 2017, and the deposit number is CGMCC No.13895.

The application of the γ-decalactone-producing Piptoporus sp. A-9 in the preparation of γ-decalactone.

Wherein, the sequence of steps involved in the application method is as follows:

(1) strain selection: select Piptoporus sp. A-9 CGMCC No.13895;

(2) slant culture activation: inoculate the strain on the slant medium, under the condition of 27～33 ℃, static culture for 96～102 hours, standby;

(3) Seed cultivation: connect the strains cultivated in step (2) to 90-100 mL liquid seed medium with inoculation loops for 1-2 loops under aseptic conditions, set the rotation speed to 160-180 rpm, rotate On a shaker with a radius of 40mm, culture at 27-33°C for 72-96 hours to obtain seed liquid;

(4) Fermentation culture with γ-decalactone as the target product

Shake flask fermentation: with an inoculum volume of 8 to 10% by volume, inoculate the seed liquid in a shake flask containing 80 to 100 mL (500 mL of triangular flask) fermentation medium, at 27 to 33°C, and at a rotational speed of 150 to 180 rpm /min, the pH is 5.0±0.5, and the shake flask is fermented for 144-168 hours to obtain a fermentation product containing γ-decalactone;

Or, 5L fermentation tank fermentation: with an inoculation amount of 8 to 10% by volume, inoculate the seed liquid in a 5L fermentation tank with 3.3 to 3.5 L fermentation medium, and carry out aeration and stirring culture at 27 to 33 ° C, wherein The stirring speed is 200～300r/min, the ventilation rate is 8L/min, the pressure in the tank is 1.0±0.02MPa, the fermentation time is 144～168 hours, and the initial pH value of the fermentation broth is 5.0～6.0. The dry weight of the bacterial cells and the concentration of γ-decalactone, when the concentration of γ-decalactone in the fermentation broth no longer rises, the fermentation is terminated, and the fermentation product containing γ-decalactone is obtained;

Wherein: the above-mentioned slant culture medium consists of: 180-220g of potato, 20g of glucose, 2g of peptone, 2g of yeast extract, 1L of deionized water, peeled and cut into small pieces, boiled in water for 30min, filtered with 8 layers of gauze, Add glucose to the filtrate, make up water to 1L, the pH is natural; add 1.5% agar powder to the solid medium, namely PDA;

The above-mentioned liquid seed medium is composed of: 180-220 g of potato, 20 g of glucose, 2 g of peptone, 2 g of yeast extract, 1 L of deionized water, peeled and cut into small pieces, boiled in water for 30 minutes, filtered with 8 layers of gauze, and added to the filtrate. Add glucose, make up water to 1L, pH is natural;

The composition of the above-mentioned fermentation medium with γ-decalactone as the object product is: soluble starch 10～40g/L, soybean meal powder 10～40g/L, castor oil 100g, Tween-20 volume ratio 0.05%, magnesium sulfate heptahydrate 0.5g/L, potassium dihydrogen phosphate 0.46g/L, disodium hydrogen phosphate dodecahydrate 1.2g/L; deionized water 1L, pH5±0.5, sterilized at 117℃, 30min.

In the above application, the culture temperature in steps (2), (3) and (4) is preferably 30°C.

In the above application, the incubation time in step (2) is preferably 100 hours.

In the above application, the incubation time in step (3) is preferably 80 hours.

In the above application, the incubation time in step (4) is preferably 150-160 hours.

In the above application, the initial soluble starch concentration of the fermentation medium in step (4) is preferably 20 g/L to 30 g/L.

In the above application, the pH of the fermentation medium in step (4) is preferably 5.0.

Above-mentioned γ-decalactone is measured by the following method:

Gas chromatography detection: standard sample preparation: take 6.33 μl of γ-decalactone standard sample and add it to 3 ml of chromatographically pure butyl acetate to obtain a γ-decalactone standard sample with a concentration of 2 g/l. Standard samples of 1g/l, 0.5g/l, 0.25g/l and 0.1g/l.

GC analysis conditions:

Inlet SPL1: temperature 250℃, pressure: 23Mpa, control mode linear velocity, linear velocity 10.3cm/min, total flow 12.5ml/min, column flow 0.5ml/min, purge flow 2ml/min, split ratio 20: 1. The injection volume is 1 μl.

Column temperature: the initial temperature was 100 °C, increased to 250 °C at 8 °C/min, and held at 250 °C for 20 min.

Detector: hydrogen flame ion detector FID, detector temperature 275°C, hydrogen flow rate 40ml/min, air flow rate 400ml/min, makeup flow rate 3ml/min.

Carrier gas: high purity nitrogen

When sampling, take 1ml of culture into a 5ml centrifuge tube, add 1ml of analytically pure butyl acetate, fully shake and mix, centrifuge at 12000rpm for 10min, take 0.8ml of the upper organic phase into a 1.5ml centrifuge tube, centrifuge at 12000rpm for 10min, centrifugation is complete Take an appropriate amount with a sterile syringe, filter it with a 0.22 μm organic filter, and perform gas chromatography detection.

The invention discloses a strain of Piptoporus sp. A-9 that produces γ-decalactone. As a new strain that has been independently picked, isolated and identified, the inventor has not found any information on using Piptoporus sp. A-9 to produce γ-decanolactone. -Decalactone as the target product was reported in the literature, and no literature report on the production of γ-decalactone by other strains of the genus Trichotonia or with the high yield of CGMCC No.13895 was found. Experiments have confirmed that castor oil can be fermented and transformed by the CGMCC No. 13895 of the present invention to directly generate and obtain the target product γ-decalactone, the conversion rate of castor oil to generate γ-decalactone is high, and the fermentation performance of the strain is stable. , is a γ-decalactone-producing strain with great research and development value. It explores a new direction and approach for liquid fermentation production of γ-decalactone, and lays a foundation for future fermentation production.

The present invention adopts the method of biological fermentation, and uses soluble starch, sucrose, soybean meal powder and the like as raw materials to produce γ-decanolide, which has the characteristics of simple production method, mild conditions, abundant raw material sources, low production cost and the like. The highest ester yield can reach more than 6.5g/L, which has high application value and has great industrial application prospects.

Detailed ways

Example 1 Isolation and purification of Trichomonas strains producing γ-decalactone

The strains were collected from the fruiting bodies of Basidiomycetes in the wild. The fresh fruiting bodies were cut with a sterile scalpel, and a little bacterial flesh was torn with ophthalmic tweezers and inoculated on a PDA plate, and cultured at 30°C. When the diameter of the mycelium pad reaches 2-3 cm, pick a little mycelium from the edge of the mycelium and inoculate it on a new PDA plate, and transfer it repeatedly until the pure strain is isolated. The objective strain was re-screened by shake flask fermentation and further isolated and screened to obtain a γ-decalactone-producing strain with stable performance, which was named Piptoporus sp. A-9.

The above strain A-9 has been deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee on May 25, 2017. The deposit address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences , zip code: 100101, and its deposit number is CGMCC N0.13895.

Composition of the above-mentioned liquid seed medium (g/L): 180-220 g of potato, 20 g of glucose, 2 g of peptone, 2 g of yeast extract, 1 L of deionized water, peel the potatoes and cut them into small pieces, boil them in water for 30 min, and use 8 Layer gauze filtration, add glucose to the filtrate, make up water to 1L, and pH is natural.

The above-mentioned slant culture medium is composed of (g/L): 180-220 g of potato, 20 g of glucose, 2 g of peptone, 2 g of yeast extract, 1 L of deionized water, peeled and cut into small pieces, boiled in water for 30 minutes, and then used for 8 layers. Filter with gauze, add glucose to the filtrate, make up water to 1L, and pH is natural. Solid medium plus 1.5% agar powder, namely PDA.

Composition (g/L) of above-mentioned fermentation medium: soluble starch 10～40g/L, soybean meal powder 10～40g/L, castor oil 100g, Tween-20 0.05% (volume ratio), magnesium sulfate (heptahydrate) 0.5 g/L, potassium dihydrogen phosphate 0.46g/L, disodium hydrogen phosphate (dodecahydrate) 1.2g/L. Deionized water 1L, pH5, sterilized at 117℃, 30min.

Example 2 Sequencing of 18S rDNA and ITS sequence of strain A-9 CGMCC No.13895

The γ-decalactone-producing strain obtained by separation and purification in Example 1, that is, the CGMCC NO.1869 strain, was entrusted to Boshang Biological Company for 18S rDNA sequencing.

The experimental method is: pick the slant culture, extract the genome as a template, amplify the 18srRNA gene with the universal primers NS1 and NS8 respectively, the fragment size is about 1.8kb, and use the universal primers ITS1, ITS4 to amplify the ITS sequence, and the fragment size is about 750bp. .

Take 5 μl for agarose gel electrophoresis, use TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) to cut the gel to recover the target fragment, and perform DNA sequencing on the recovered product.

Sequencing result:

The length of the 18S rDNA sequence of CGMCC No.13895 strain is 1679bp, and its nucleotide sequence is shown in SEQ ID NO.1; the size of the ITS sequence fragment of CGMCC No.13895 strain is 727bp, and its nucleotide sequence is shown in SEQ ID NO.2 Show. The primers used are shown in Table 1.

Table 1 List of primers used

Example 3 Application of Trichomonas A-9 CGMCC No.13895 in the preparation of γ-decalactone by shaking flask culture

The sequence of steps involved in applying the method is as follows:

(1) strain selection: select Piptoporus sp. A-9 CGMCC No.13895;

(2) slant culture activation: inoculate the bacterial strain on the slant medium, under the condition of 30 ℃, statically cultivate for 100 hours, for use;

(3) Seed cultivation: connect the strains cultivated in step (2) with 1 to 2 loops of inoculation in 90 to 100 mL (500 mL conical flasks) liquid seed medium under sterile conditions, and set the rotational speed to 160 rpm/ Incubate for 80 hours at 30°C on a shaker with a rotation radius of 40mm and obtain seed liquid;

(4) Shake flask fermentation: with an inoculation volume of 8 to 10% by volume, inoculate the seed liquid in a shake flask containing 90 to 100 mL (500 mL of conical flask) fermentation medium, at 30 to 35° C., with a rotating speed of 150 ~180 r/min, shake flask for 150 hours to obtain a fermentation product containing γ-decalactone;

The above-mentioned slant culture medium is composed of: 180-220 g of potato, 20 g of glucose, 2 g of peptone, 2 g of yeast extract, 1 L of deionized water, peeled and cut into small pieces, boiled in water for 30 min, filtered with 8 layers of gauze, and added to the filtrate. Add glucose and make up water to 1L, pH is natural. Solid medium plus 1.5% agar powder, namely PDA;

The above-mentioned liquid seed medium is composed of: 180-220 g of potato, 20 g of glucose, 2 g of peptone, 2 g of yeast extract, 1 L of deionized water, peeled and cut into small pieces, boiled in water for 30 minutes, filtered with 8 layers of gauze, and added to the filtrate. Add glucose, make up water to 1L, pH is natural;

The composition of above-mentioned fermentation medium is: soluble starch 20g/L, soybean meal powder 30g/L, castor oil 100g, Tween-20 volume ratio 0.05%, magnesium sulfate (heptahydrate) 0.5g/L, potassium dihydrogen phosphate 0.46g /L, disodium hydrogen phosphate (dodecahydrate) 1.2g/L, deionized water 1L; pH5, sterilization at 117°C, 30min.

(5) Product detection: when sampling, take 1ml of culture into a 5ml centrifuge tube, add 1ml of analytically pure butyl acetate, fully shake and mix, centrifuge at 12000rpm for 10min, take 0.8ml of the upper organic phase into a 1.5ml centrifuge tube, centrifuge 12000rpm, 10min, after centrifugation, take an appropriate amount with a sterile syringe and filter with a 0.22μm organic filter for gas chromatography detection;

The content of γ-decalactone in the fermentation broth was detected to be 3.6 g/L.

Example 4 Application of Trichomonas A-9 CGMCC No.13895 in the preparation of γ-decalactone by fermenter culture

The sequence of steps involved in applying the method is as follows:

(1) Bacterial selection: choose Trichomonas A-9 (Piptoporus sp.) CGMCC No.13895;

(2) slant culture activation: inoculate the bacterial strain on the slant medium, under the condition of 35 ℃, statically cultivate for 102 hours, for use;

(3) Seed cultivation: connect the strains cultivated in step (2) with 1 to 2 loops of inoculation in 90 to 100 mL (500 mL conical flask) liquid seed medium under sterile conditions, and set the rotational speed to 180 rpm/ Incubate for 80 hours at 30-35°C on a shaker with a rotation radius of 40mm and obtain seed solution;

(4) Fermentation culture:

Fermentation in a 5L fermenter: with an inoculation volume of 8 to 10% by volume, inoculate the seed liquid in a 5L fermenter with 3.5L of fermentation medium, and perform ventilation and stirring culture at 30°C, wherein the stirring speed is 250r/min , the ventilation rate is 8L/min of the fermentation liquid, the pressure in the tank is 0.08MPa; the initial pH value of the fermentation liquid is 5.0-5.5, and the concentration of γ-decalactone in the fermentation liquid is determined by sampling regularly during the fermentation process, and the fermentation time is 144 hours. Fermentation, namely obtaining the fermentation product containing γ-decalactone;

The above-mentioned slant culture medium consists of: 220 g of potatoes, 20 g of glucose, 2 g of peptone, 2 g of yeast extract, 1 L of deionized water, peeled and cut into small pieces, boiled in water for 30 min, filtered with 8 layers of gauze, and added to the filtrate. Glucose, supplemented with water to 1L, pH is natural, solid medium plus 1.5% agar powder, namely PDA;

The above-mentioned liquid seed medium is composed of: 220 g of potato, 20 g of glucose, 2 g of peptone, 2 g of yeast extract, 1 L of deionized water, peeled and cut into small pieces, boiled in water for 30 min, filtered with 8 layers of gauze, and added to the filtrate Glucose, make up water to 1L, pH is natural;

The composition of above-mentioned fermentation medium is: soluble starch 40g/L, soybean meal powder 40g/L, castor oil 100g, Tween-20 volume ratio 0.05%, magnesium sulfate (heptahydrate) 0.5g/L, potassium dihydrogen phosphate 0.46g /L, disodium hydrogen phosphate (dodecahydrate) 1.2g/L. Deionized water 1L, pH5, sterilized at 117℃, 30min.

(5) Product detection: when sampling, take 1ml of culture into a 5ml centrifuge tube, add 1ml of analytically pure butyl acetate, fully shake and mix, centrifuge at 12000rpm for 10min, take 0.8ml of the upper organic phase into a 1.5ml centrifuge tube, centrifuge 12000rpm, 10min, after centrifugation, take an appropriate amount with a sterile syringe and filter with a 0.22μm organic filter for gas chromatography detection;

The content of γ-decalactone in the fermentation broth was detected to be 6.8 g/L.

The γ-decalactone described in the above embodiment is measured by the following method:

Gas chromatography detection: standard sample preparation: take 6.33 μl of γ-decalactone standard sample and add it to 3 ml of chromatographically pure butyl acetate to obtain a γ-decalactone standard sample with a concentration of 2 g/l. Standard samples of 1g/l, 0.5g/l, 0.25g/l and 0.1g/l.

GC analysis conditions:

Inlet SPL1: temperature 250℃, pressure: 23Mpa, control mode linear velocity, linear velocity 10.3cm/min, total flow 12.5ml/min, column flow 0.5ml/min, purge flow 2ml/min, split ratio 20: 1. The injection volume is 1 μl.

Column temperature: the initial temperature was 100 °C, increased to 250 °C at 8 °C/min, and held at 250 °C for 20 min.

Detector: hydrogen flame ion detector FID, detector temperature 275°C, hydrogen flow rate 40ml/min, air flow rate 400ml/min, makeup flow rate 3ml/min.

Carrier gas: high purity nitrogen

When sampling, take 1ml of culture into a 5ml centrifuge tube, add 1ml of analytically pure butyl acetate, fully shake and mix, centrifuge at 12000rpm for 10min, take 0.8ml of the upper organic phase into a 1.5ml centrifuge tube, centrifuge at 12000rpm for 10min, centrifugation is complete Take an appropriate amount with a sterile syringe, filter it with a 0.22 μm organic filter, and perform gas chromatography detection.

sequence listing

<110> Shandong University

<120> A γ-decalactone-producing Trichoderma and its application

<141>2017-06-14

<160> 2

<210> 1

<211>1679

<212>rDNA

<213> Piptoporus sp. A-9

<221> 18S rDNA sequence of Trichomonas strain CGMCC No. 13895

<222>(1)…(1679)

<400>1

cagtttgtac tgtgaaactg cgaatggctc attaaatcag ttatagttta tttgatggta 60

ccttgctaca tggataactg tggtaattct agagctaata catgcaatca aagccccgac 120

ttctggaagg ggtgtattta ttagataaaa aaccaacgcg gttcgccgct ccattggtga 180

ttcataataa cttctcgaat cgcatggcct tgtgccggcg atgcttcatt caaatatctg 240

ccctatcaac tttcgatggt aggatagagg cctaccatgg tttcgacggg taacggggaa 300

taagggttcg attccggaga gggagcctga gaaacggcta ccacatccaa ggaaggcagc 360

aggcgcgcaa attacccaat cccgacacgg ggaggtagtg acaataaata acaatatagg 420

gctcttttgg gtcttataat tggaatgagt acaatttaaa tcccttaaca aggaacaatt 480

ggagggcaag tctggtgcca gcagccgcgg taattccagc tccaatagcg tatattaaag 540

ttgttgcagt taaaacgctc gtagttgaac ttcagacctg gccgggtggt ctgcctaacg 600

gtatgtactg tctggctggg tcttacctct tggtgagccg gcatgccctt tactgggtgt 660

gtcggggaac caggactttt accttgagaa aattagagtg ttcaaagcag gcctatgccc 720

gaatacatta gcatggaata ataaaatagg acgtgcggtt ctattttgtt ggtttctaga 780

gtcgccgtaa tgattaatag ggatagttgg gggcattagt attcagttgc tagaggtgaa 840

attcttggat ttactgaaga ctaactactg cgaaagcatt tgccaaggat gttttcatta 900

atcaagaacg aaggttaggg gatcgaaaac gatcagatac cgttgtagtc ttaacagtaa 960

actatgccga ctagggttcg ggcgatctca aatttatgtg tcgctcggca ccttacgaga 1020

aatcaaagtc tttgggttct ggggggagta tggtcgcaag gctgaaactt aaaggaattg 1080

acggaagggc accaccaggt gtggagcctg cggcttaatt tgactcaaca cggggaaact 1140

caccaggtcc agacataact aggattgaca gattgatagc tctttcataa ttttatgggt 1200

ggtggtgcat ggccgttctt agttggtgga gtgatttgtc tggttaattc cgataacgaa 1260

cgagacctta acctgcttaa tagccaggcc ggcttttgct ggtctccggc ttcttagagg 1320

gactgtctgt gtctaacaga tggaagtttg aggcaataac aggtctgtga tgcccttaga 1380

tgttctgggc cgcacgcgcg ctacactgac agaaccagcg agtttttttc tcccttggcc 1440

ggaaggtctg ggtaatcttg tgaaactctg tcgtgctggg gatagagcat tgcaattatt 1500

gctcttcaac gaggaatacc tagtaagcgc gagtcatcag ctcgcgttga ttacgtccct 1560

gccctttgta cacaccgccc gtcgctacta ccgattgaat ggtttagtga ggtcttggga 1620

ttggcttcgg ggagccggca acggcacctt gctgctgaga acttgatcaa acttggtca 1679

<210> 2

<211>727

<212> DNA

<213> Piptoporus sp. A-9

<221> ITS sequence of Trichomonas strain CGMCC No. 13895

<222> (1)…(727)

<400>2

tccgtaggtg aacctgcgga aggatcatta tcgaattttg aattgggttg tagctggcct 60

ccgggcatgt gcacatcctt ttctttaaca cacacacaca cacacacctt gtgcactcac 120

tgtaggctgg ggttgaaaag ggttgttggg ttgccttcgg gttctaactc ctaaccccag 180

tttatgtttt tatatctacc cactccaagt catagaatgt cattgatgcg tctaacgcaa 240

tgttgggaat aatataactt tcagcaacgg atctcttggc tctcgcatcg atgaagaacg 300

cagcgaaatg cgataagtaa tgtgaattgc agaattcagt gaatcatcga atctttgaac 360

gcaccttgcg ctccttggta ttccgaggag catacctgtt tgagtatcat ggaactctca 420

actcattcat tcttgttata tagtgtgagt gggcttggac ttggaggctt tgctggtgca 480

tgaaatggca tcggctcctc ttgaatgcat tagcttgaac ctatgtgggg tggatcggct 540

atcggtgtga taatatttgt ctatgccggg ttgtgaaaga gccctgggtt acccttctgt 600

ttgggggggg atttcaagct tctaatctct gagttgggac cattttcttg ggaccatttc 660

ttcttacctc tgatctcaaa tcaggtagga ctacccgctg aacttaagca tatcaataag 720

cggagga 727

Claims (3)

1. a strain producing gamma-decalactone Pycnoporus, it is characterized in that: this bacterial strain is named Piptoporus sp. Species Deposit Management Committee General Microbiology Center", the deposit number is CGMCC No.13895. 2. the application of the Tritoid bacteria of producing γ-decalactone described in claim 1 in the preparation of γ-decalactone, the sequence of steps is as follows: (1) strain selection: select Piptoporus sp. A-9CGMCC No.13895; (2) slant culture activation: inoculate the bacterial strain on the slant medium, under the condition of 27～33 ℃, static culture 96～102 hours, standby; Peptone 2g, yeast extract 2g, deionized water 1L; pH natural; solid medium plus 15g/L agar powder, namely PDA; (3) Seed cultivation: connect the strains cultivated in step (2) to 90-100 mL liquid seed medium with inoculation loops for 1-2 loops under aseptic conditions, set the rotation speed to 160-180 rpm, rotate On a shaking table with a radius of 40mm, culture at 27-33°C for 72-96 hours to obtain seed liquid; wherein, the liquid seed medium is composed of: 180-220g of potato, 20g of glucose, 2g of peptone, 2g of yeast extract, deionized Water 1L; pH natural; (4) take γ-decalactone as the target product fermentation culture; It is characterized by: Described in step (4), taking γ-decalactone as the purpose product fermentation culture is shake flask fermentation or fermentation tank fermentation, wherein: Shake flask fermentation: with an inoculation volume of 8 to 10% by volume, inoculate the seed liquid in a shake flask containing 80 to 100 mL of fermentation medium, at 27 to 33°C, with a rotational speed of 150 to 180 rpm, and a pH of 5.0±0.5, shake flask for 144-168 hours to obtain a fermentation product containing γ-decalactone; 5L fermentation tank fermentation: with the inoculation amount of 8～10% volume ratio, inoculate the seed liquid in the 5L fermenter tank with 3.3～3.5L fermentation medium, carry out ventilation stirring culture at 27～33 ℃, wherein stirring speed It is 200～300r/min, the ventilation rate is 8L/min, the pressure in the tank is 1.0±0.02MPa, the fermentation time is 144～168 hours, the initial pH value of the fermentation broth is 5.0～6.0, and the bacteria in the fermentation broth are regularly sampled during the fermentation process to determine the bacteria in the fermentation broth. Dry body weight and the concentration of γ-decalactone, when the concentration of γ-decalactone in the fermentation broth no longer rises, the fermentation is terminated, that is, a fermentation product containing γ-decalactone is obtained; The composition of the above-mentioned fermentation medium with γ-decalactone as the object product is: soluble starch 10～40g/L, soybean meal powder 10～40g/L, castor oil 100g, Tween-20 volume ratio 0.05%, magnesium sulfate heptahydrate 0.5g/L, potassium dihydrogen phosphate 0.46g/L, disodium hydrogen phosphate dodecahydrate 1.2g/L; deionized water 1L; pH5±0.5. 3 . The application according to claim 2 , wherein the initial soluble starch concentration of the fermentation medium in step (4) is 20 g/L to 30 g/L. 4 .