CN110747135B - Trichoderma aureoviride and application thereof - Google Patents

Trichoderma aureoviride and application thereof Download PDF

Info

Publication number
CN110747135B
CN110747135B CN201911270355.1A CN201911270355A CN110747135B CN 110747135 B CN110747135 B CN 110747135B CN 201911270355 A CN201911270355 A CN 201911270355A CN 110747135 B CN110747135 B CN 110747135B
Authority
CN
China
Prior art keywords
trichoderma
fermentation
solid
xylanase
liquid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201911270355.1A
Other languages
Chinese (zh)
Other versions
CN110747135A (en
Inventor
张梅娟
马天意
钱朋智
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qiqihar University
Original Assignee
Qiqihar University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qiqihar University filed Critical Qiqihar University
Priority to CN201911270355.1A priority Critical patent/CN110747135B/en
Publication of CN110747135A publication Critical patent/CN110747135A/en
Application granted granted Critical
Publication of CN110747135B publication Critical patent/CN110747135B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Botany (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Trichoderma aureoviride and application thereof relate to the technical field of microorganisms, and in particular relate to trichoderma aureoviride and application thereof. The Trichoderma pleuroticola ZJ-03 is preserved in Guangdong province microbial strain collection center at the preservation address of No. 59, No. 5, 6-6 days in 2019 and the preservation number of GDMCC NO.60689 of No. 100 Zhonglu-Jie of preforgia, Guangzhou. The trichoderma pleuroticola is used for producing xylanase. The solid fermentation of the trichoderma pleuroticola produces xylanase preparation, and the trichoderma pleuroticola xylanase is used for preparing xylo-oligosaccharide.

Description

Trichoderma aureoviride and application thereof
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to trichoderma pleuroticola and application thereof.
Background
Trichoderma aureoviride, a common pollution mold in oyster mushroom cultivation, is a mold in Trichoderma, and currently, researches on Trichoderma aureoviride by scholars at home and abroad mainly focus on the field of biological plant disease control.
The invention discloses a salt-tolerant trichoderma pleuroticola strain and application thereof in an authorized patent (patent number ZL 201310442613.6). The trichoderma pleuroticola strain has the characteristics of high growth speed, large sporulation amount, strong salt tolerance and stress resistance, capability of fast and mass colonization at plant rhizosphere and the like, and has wide application prospects in the aspects of improving the utilization rate of phosphate fertilizers, improving soil structures and increasing the yield and quality of crops.
A patent of Trichoderma aureoviride and application thereof (patent No. ZL201510502268.X) introduces a biological control preparation of Trichoderma aureoviride and a preparation method thereof, the Trichoderma aureoviride has the characteristics of high growth speed, large spore production amount, wide action spectrum, strong stress resistance, rapid and large-scale colonization at plant rhizosphere and the like, and the biological control preparation prepared by the Trichoderma aureoviride can control a plurality of soil-borne plant diseases, including one or more of pepper wilt, chestnut branch blight, pomegranate dry rot and the like, and is a biological control preparation with great application prospect.
A patent of Trichoderma aureobasidum and application thereof in control of anthracnose of winter jujubes (patent application No. 201810343839.3) introduces Trichoderma aureobasidum and application thereof in control of anthracnose of winter jujubes, Trichoderma aureobasidum strain SDLTr16 is obtained by separating from river mud of Xiaoqing river in Dongying City, and the prepared Trichoderma aureobasidum biological control preparation has good effect on anthracnose of winter jujubes, and the control effect is 76.83-92.40%.
In addition to the above studies on the biocontrol effect of the scholars at home and abroad by using trichoderma reesei, some scholars have studied on the production of enzymes by trichoderma reesei.
Rojuncai et al (2011) have conducted sequence analysis and protein structure prediction studies on Trichoderma reesei T2-1 phytase gene, and registered Trichoderma reesei T2-1 phytase gene sequences in Gen Bank (accession No.: GQ 325590). This is the first complete trichoderma phytase coding gene registered in genbank in china at present.
Zhang super (2012) carries out the research of the gene cloning and prokaryotic expression of the Trichoderma aureoviride phytase, determines the optimal reaction temperature of the Trichoderma aureoviride phytase to be 45-55 ℃, and lays a foundation for the further research of the Trichoderma phytase.
However, Trichoderma reesei has not been reported for xylanase production.
Disclosure of Invention
The invention aims to provide trichoderma pleuroticola and application thereof.
The Trichoderma pleuroticola ZJ-03 is preserved in Guangdong province microbial strain collection center, the preservation address is No. 59, No. 5, No.6 of Michelia furiosa No. 100, Guangzhou city, the preservation date is 2019, 6 months and 6 days, and the preservation number is GDMCC NO. 60689.
The trichoderma pleuroticola is used for producing xylanase.
The Trichoderma reesei (Trichoderma pleuroticola) ZJ-03 of the present invention showed rapid colony growth on PDA plate medium. The colony in the early growth stage is cotton-shaped, compact and flat, has an obvious concentric spore production area, the color is finally converted into yellow green from light yellow, and the color is converted into dark green when the colony is aged. The back of the colony starts to be colorless, and some of the colony becomes yellow with the age of the fungus (as shown in FIGS. 1 to 3).
The invention has the beneficial effects that:
the invention relates to a xylanase preparation produced by solid fermentation of trichoderma pleuroticola, which is used for preparing xylo-oligosaccharide.
The invention can obtain trichoderma pleuroticola and application thereof.
Drawings
FIG. 1 is a photograph showing the morphology of colonies of Trichoderma reesei (Trichoderma pleuroticola) ZJ-03 at the initial stage of growth on PDA plate medium;
FIG. 2 is a photograph of the morphology of the positive colony in the late growth phase of Trichoderma reesei (Trichoderma pleuroticola) ZJ-03 on PDA plate medium;
FIG. 3 is a photograph of the colony morphology on the back of Trichoderma reesei (Trichoderma pleuroticola) ZJ-03 at the late stage of growth on PDA plate medium;
FIG. 4 is a photograph of the positive morphology of Trichoderma reesei xylanase production on PDA plate medium;
FIG. 5 is a photograph of a morphology of the reverse side of Trichoderma reesei xylanase production on PDA plate medium.
Detailed Description
The first embodiment is as follows: in the embodiment, Trichoderma pleuroticola ZJ-03 is stored in Guangdong province microbial strain collection center at the storage address of No. 59, No. 5 of No. 100 college of Michelia furiosa of Guangzhou city, the storage date is 2019, 6 months and 6 days, and the storage number is GDMCC NO. 60689.
In the present embodiment, Trichoderma pleuroticola ZJ-03 (Trichoderma pleuroticola) had a rapid colony growth on PDA plate medium. The colony in the early growth stage is cotton-shaped, compact and flat, has an obvious concentric spore production area, the color is finally converted into yellow green from light yellow, and the color is converted into dark green when the colony is aged. The back of the colony starts to be colorless, and some of the colony becomes yellow with the age of the fungus (as shown in FIGS. 1 to 3).
The second embodiment is as follows: in this embodiment, trichoderma pleuroticola is used for producing xylanase.
The beneficial effects of the embodiment are as follows:
in the embodiment, the solid fermentation of the trichoderma pleuroticola produces the xylanase preparation, and the trichoderma pleuroticola xylanase is used for preparing xylooligosaccharide.
The following examples were used to demonstrate the beneficial effects of the present invention:
experiment 1, isolation and purification of trichoderma pleuroticola:
the trichoderma pleuroticola is separated and purified by the following steps:
1.1 Collection of auspicious substances
The sample collection time is 4 months to 6 months in 2019, the collection place is an edible fungus cultivation room of the institute of food and bioengineering, university of Qiqi haer, oyster mushroom blocks polluted by mixed fungi are collected in the process of full growth of cultivation bottles, and the oyster mushroom blocks are stored in a refrigerator at 4 ℃ for later use.
1.2 separation and purification
Selecting mould spores from the collected fungus blocks, culturing for 5-7 days at 28 ℃ on a PDA (potato dextrose agar) plate culture medium, immediately purifying and culturing after bacterial colonies grow out, purifying for 2-3 times, and numbering the purified strains in sequence.
Experiment 2, strain identification:
2.1 culture method
And (3) performing inverted culture on the PDA plate culture medium for 5-7 days at 28 ℃ under a dark condition.
2.2 cultural characteristics
Colonies grew rapidly on PDA plate medium at 28 ℃. The colony in the early growth stage is cotton-shaped, compact and flat, has an obvious concentric spore production area, the color is finally converted into yellow green from light yellow, and the color is converted into dark green when the colony is aged. The back of the colony starts to be colorless, and some of the colony becomes yellow with the age of the fungus (as shown in FIGS. 1 to 3).
2.3 molecular biological identification
2.3.1 genomic DNA extraction
According to the instructions of the TIANGEN bacterial genome extraction kit, the centrifugal adsorption column which can be efficiently and specifically combined with DNA and a unique buffer solution system are adopted to extract the genome DNA.
2.3.2PCR amplification
(1) PCR amplification of 18S r DNA sequences
PCR amplification of 18 sr DNA used universal primers: NS1 and NS 8. Total reaction system 50. mu.L, including 10 XPCR buffer 5. mu.L, Mg2+3 μ L (25mmol/L), 1.2 μ L of dNTP mix (10mmol/L), 0.8 μ L each of NS1 and NS8 (both at 25 μmol/L), 0.4 μ L (5U/μ L) of Taq DNA polymerase, 20ng of template, and sterile water to 50 μ L. The PCR reaction program is 94 ℃ for 5min, 94 ℃ for 30s, 53 ℃ for 45s, 72 ℃ for 30s and 30 cycles; extension was then carried out at 72 ℃ for 10 min. After the PCR product electrophoresis is finished, a gel imaging system is used for photographing. Sending the PCR amplification product to Qingdao scientific quality detection limited company for sequencing; wherein, the PCR amplification primer sequence is as follows:
NS1：5′-GTAGTCATATGCTTGTCTC-3′；
NS8：5′-TCCGCAGGTTCACCTACGGA-3′；
(2) PCR amplification of ITS sequences
The PCR amplification of ITS sequences employs universal primers: ITS1 and ITS 4. Total reaction system 50. mu.L, including 10 XPCR buffer 5. mu.L, Mg2+3 μ L (25mmol/L), 1.3 μ L of dNTP mix (10mmol/L), 0.8 μ L each of ITS1 and ITS4 (both at 25 μmol/L), 0.3 μ L (5U/μ L) of Taq DNA polymerase, 20ng of template, and sterile water to 50 μ L. The PCR reaction program is 94 ℃ for 5min, 94 ℃ for 30s, 55 ℃ for 45s, 72 ℃ for 40s, and 30 cycles; extension was then carried out at 72 ℃ for 10 min. After the PCR product electrophoresis is finished, a gel imaging system is used for photographing. Sending the PCR amplification product to Qingdao scientific quality detection limited company for sequencing; wherein, the PCR amplification primer sequence is as follows:
ITS1：5′-TCCGTAGGTGAACCTGCGG-3′；
ITS4：5′-TCCTCCGCTTATTGATATGC-3′；
(3) similarity analysis
The length of 18S r DNA of Trichoderma pleuroticola ZJ-03 is 1716bp (shown as a sequence table Seq ID No: 1), the 18S r DNA sequence is analyzed by Blast in NCBI, the similarity of the strain and sequences with accession numbers of JN941680, JN941677.1, JN941676.1 and the like is higher, and the similarity is 99%, which indicates that the strain belongs to Trichoderma (Trichoderma);
the ITS DNA length of Trichoderma pleuroticola ZJ-03 is 614bp (shown as sequence table Seq ID No: 2), and the ITS sequence is analyzed by Blast in NCBI, so that the strain has high similarity and 99% similarity with the sequences with accession numbers JQ040377.1, MF687194.1, MH863369.1 and the like, which indicates that the strain belongs to Trichoderma pleuroticola (Trichoderma pleuroticola), and the strain is preserved in Guangdong province microbial culture collection 6 months and 6 months in 2019 and has the preservation number GDMCC NO. 60689.
The PCR primers were synthesized by Shanghai Bioengineering Co., Ltd.
Experiment 3, application of trichoderma pleuroticola:
3.1 characterization of xylanase produced by Trichoderma reesei
3.1.1 qualitative method
The transparent circle method for qualitative determination of xylanase produced by trichoderma pleuroticola is visual, simple, convenient and quick.
3.1.2 detailed procedures
Xylanase production qualitative plate culture medium: 0.5g of beef extract, 1g of peptone, 0.5g of NaCl, 2g of agar, 0.5g of xylan and 100mL of distilled water.
And (3) punching the purified trichoderma aureobasicola plate culture medium with a puncher with the diameter of 0.5mm, inoculating a strain piece to the center of the plate culture medium, and culturing for 5-7 days at 28 ℃ in an incubator. The growth of colonies and the formation of hydrolysis rings were observed daily.
3.1.3 results
The qualitative results of the plate culture medium are shown in FIGS. 4 to 5, and the plate culture medium has obvious hydrolysis cycle on day 3 and the strain grows well. And determining the cellulase produced by the trichoderma pleuroticola.
3.2 production of xylanase preparation by solid fermentation of Trichoderma aureoides
3.2.1 preparation of spore suspensions
Preparing spore suspension from the grown Trichoderma reesei slant with sterile water, wherein the final spore suspension has a spore number of 2 × 106one/mL for inoculation into a solid fermentation medium;
3.2.2 solid State fermentation
(1) Solid fermentation medium
Nutrient solution: 1g of glucose mother liquor, 1g of threonine mother liquor and 100mL of distilled water.
Solid medium: the solid-liquid ratio of the aronia melanocarpa residues to the oyster mushroom waste mushroom residues (1:1) to the nutrient solution is 1: 1.8;
(2) fermentation process
The solid culture medium is fully and uniformly stirred and then is filled into a polypropylene cuboid box (28cm multiplied by 18cm multiplied by 10cm), the height of a material layer in the polypropylene cuboid box is 3cm, and sterilization is carried out for 2 hours at 121 ℃. The suspension of the spores of the fungus Pleurotus ostreatus was inoculated under aseptic conditions, the inoculum size being 5%. After inoculation, the mixture is placed on a culture rack of a fermentation chamber (the fermentation chamber is disinfected in advance) with the relative humidity of 80 percent for culture, the fermentation temperature is 28 ℃, and the fermentation time is 120 hours. The fermentation chamber is provided with a cooling and heating air conditioner, a humidifier and a full-automatic hygrothermograph, so that the environmental conditions can be adjusted in time according to the culture condition;
3.2.3 preparation of enzyme preparations
After fermentation is finished, adding 25 times of water by volume into trichoderma aureobasicola solid fermentation starter, leaching for 6h at 30 ℃, centrifuging for 10min under the condition of 4000r/min, taking supernatant as crude enzyme liquid, wherein the enzyme activity is 120IU/mL, and is 3000IU/g larger than that of the culture medium in terms of dry weight.
3.3 preparation of xylooligosaccharide from Trichoderma aureoides xylanase
The solid-liquid ratio of the oyster mushroom waste mushroom dregs to water is 1: 8, cooking at 150 ℃ for 2 hours, after high-temperature cooking is finished, cooling a residue-liquid mixture to 45 ℃, adjusting the pH value to 5.0, adding xylanase crude enzyme liquid according to the enzyme activity of 15I U/g oyster mushroom waste fungus residues, cooking 5 kilograms of oyster mushroom waste fungus residues, adding 625mL of enzyme liquid, stirring uniformly, reacting at constant temperature of 45 ℃ for 6 hours, inactivating at 90 ℃ for 15 minutes after enzymolysis is finished, centrifuging by using a three-legged centrifuge to obtain sugar liquid, adding 1% of active carbon powder in total volume into the sugar liquid, decolorizing at 75 ℃ for 30 minutes, performing suction filtration by using a Buchner funnel, performing first concentration on the decolorized liquid through a rotary evaporator until the refraction concentration is 15%, performing second decolorization on the first concentrated liquid with the refraction of 15%, wherein the decolorizing conditions are the same as those of the first decolorization. And (3) feeding the secondary decolorized solution to an ion exchange resin column, carrying out primary ion exchange decolorization and impurity removal according to a positive and negative mode, carrying out secondary concentration on the primary ion exchange decolorized solution, carrying out tertiary decolorization on the secondary concentrated solution, feeding the tertiary decolorized solution to the ion exchange resin column, carrying out secondary ion exchange decolorization and impurity removal according to a positive and negative mode, and carrying out vacuum concentration on the secondary ion exchange solution to obtain light yellow viscous xylo-oligosaccharide syrup with the refraction of 75%, wherein the xylo-oligosaccharide accounts for 72.0% of the total sugar content, and the yield of the xylo-oligosaccharide syrup to the raw material oyster mushroom waste residues is 9.09%.
Sequence listing
<110> university of ziqi hall
<120> Trichoderma aureobasicola and application thereof
<160> 2
<210> 1
<211> 1716
<212> DNA
<213> Trichoderma (Trichoderma).
aggttactaa tttttcttct ctaatgaccg agtttggaga gctttccggc cctgagtggt 60
agttgcccac ctctctgggc cagtccggac gcctcactga gccattcaat cggtagtagc 120
gacgggcggt gtgtacaaag ggcagggacg taatcaacgc aagctgatga cttgcgctta 180
ctagggattc ctcgttgaag agcaataatt gcaatgctct atccccagca cgacggagtt 240
taacaagatt acccaggcct tccggccaag ggagtactcg ctggctccgt cagtgtagcg 300
cgcgtgcggc ccagaacatc taagggcatc acagacctgt tattgcctca aacttccatc 360
ggcttgagcc gatagtccct ctaagaagcc ggcgtactgc caaagcaata cgggctattt 420
agcaggttaa ggtctcgttc gttatcgcaa ttaagcagac aaatcactcc accaactaag 480
aacggccatg caccaccacc cacaaaatca agaaagagct ctcaatctgt caatcctcat 540
tgtgtctgga cctggtgagt ttccccgtgt tgagtcaaat taagccgcag gctccacccc 600
tggtggtgcc cttccgtcaa tttctttaag tttcagcctt gcgaccatac tccccctgga 660
gcccaagcac tttgatttct cgtaaggtgc cgaacgcgtc aaaaatgtaa catcgtccga 720
tccctagtcg gcatagttta tggttaagac tacgacggta tctgatcgtc ttcgatcccc 780
taactttcgt tcctgattaa tgaaaacatc cttggcaaat gctttcgcag tagttagtct 840
tcaataaatc caagaatttc acctctgaca attgaatact gatgcccccc gactgtccct 900
attaatcatt acggcggtcc tagaaaccaa caaaatagaa ccacacgtcc tattctatta 960
ttccatgcta atgtattcga gcataggcct gccttgagca ctctaatttt ttcaaagtaa 1020
aagtcctgtt tccccgccac acccagtgaa gggcatgggg ttccgcagag ggaaaggccc 1080
ggccggacca gtgcacgcgg tgaggcggac cggccagcca ggcccaaggt tcaactacga 1140
gctttttaac cacaacaact ttaatatacg ctattggagc tggaattacc gcggctgctg 1200
gcaccagact tgccctccaa ttgttcctcg ttaagggatt taaattgtac tcattccgat 1260
tacaagaccc aaaagagccc tgtatcagta tttattgtca ctacctcccc gtgtcgggat 1320
tgggtaattt gcgcgcctgc tgccttcctt ggatgtagta gccgtttctc aggctccttc 1380
tccggggtcg agccctaacc ctccgttacc cgttgccacc atgtttggcc aatacccaaa 1440
catcgaaagt tgatagggaa gaaatttgaa tgagccatcg ccggcacaag gcctgtcgat 1500
tcgactagtt atcatgattc accagagagc cccgaggggc attggttttt aatctaataa 1560
atacatccct tccgaagtcg ggattttcag catgtattag ctctagaatt accacggtta 1620
tccaagtagt aaagtattat caaataaacg ataacttata taatgagcca ttcgcagttt 1680
cgcggtataa ttgctatact agacaggtcc gggccc 1716
<210> 2
<211> 614
<212> DNA
<213> Trichoderma pleuroticola (richoderma pleuroticola).
acctgcggag ggatcattac cgagtttaca actcccaaac ccaatgtgaa cgttaccaaa 60
ctgttgcctc ggcgggatct ctgccccggg tgcgtcgcag ccccggagca aggcgcccgc 120
cggaggacca accaaaactc ttactgtata ccccctcgcg ggttttttta taatctgagc 180
cttctcggcg cccctcgtgg gcgttccgaa aatgaatcaa aactttcaac aacggatctc 240
ttggttctgg catcgatgaa gaacgcagcg aaatgcgata agtaatgtga attgcagaat 300
tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccgc cagtattctg gcgggcatgc 360
ctgtccgagc gtcatttcaa ccctcgaacc cctccggggg gtcggcgttg gggatcggcc 420
ctccctctgc ggggggccgt ctccgaaatg cagtggcggt cccgccgcag cctctcctgc 480
gcagtagttt gcacactcgc accgggagcg cggcgcgtcc acagccgtta aacacccaac 540
tttctgaaat gttgacctcg gatcaggtag gaatacccgc tgaacttaag catatcaata 600
ggcgcaaagg aatg 614

Claims (1)

1. Trichoderma aureoviride is Trichoderma aureoviride ZJ-03 which is preserved in Guangdong province microbial strain collection center at No. 59 building 5 of Mirabilitum 100 Mcjutsu of Guangzhou city with the preservation date of 2019, 6 and 6 days and the preservation number of GDMCC NO. 60689;
the trichoderma pleuroticola is used for producing xylanase, and the xylanase is used for preparing xylo-oligosaccharide syrup;
the specific steps of the trichoderma pleuroticola for producing xylanase are as follows:
firstly, preparation of spore suspension:
preparing spore suspension from the grown Trichoderma reesei slant with sterile water, wherein the final spore suspension has a spore number of 2 × 106one/mL for inoculation into a solid fermentation medium;
secondly, solid state fermentation:
(1) solid fermentation medium
Nutrient solution: 1g of glucose mother liquor, 1g of threonine mother liquor and 100mL of distilled water;
solid medium: the solid-liquid ratio of the aronia melanocarpa residues to the oyster mushroom waste fungus residues and the nutrient solution is 1:1.8, and the mass ratio of the aronia melanocarpa residues to the oyster mushroom waste fungus residues is 1: 1;
(2) fermentation process
Fully and uniformly stirring the solid culture medium, filling the solid culture medium into a polypropylene cuboid box with the thickness of 28cm multiplied by 18cm multiplied by 10cm, wherein the height of a material layer in the polypropylene cuboid box is 3cm, and sterilizing the solid culture medium for 2 hours at the temperature of 121 ℃; inoculating the pleurotus ostreatus spore suspension under aseptic condition, wherein the inoculation amount is 5%; after inoculation, placing the inoculated seeds on a pre-sterilized culture frame of a fermentation chamber with the relative humidity of 80 percent for culture, wherein the fermentation temperature is 28 ℃, and the fermentation time is 120 hours; the fermentation chamber is provided with a cooling and heating air conditioner, a humidifier and a full-automatic hygrothermograph, so that the environmental conditions can be adjusted in time according to the culture condition;
thirdly, preparing an enzyme preparation:
after fermentation, adding 25 times of water by volume into trichoderma aureobasicola solid fermentation starter, leaching for 6h at 30 ℃, centrifuging for 10min under the condition of 4000r/min, taking supernatant as crude enzyme liquid, wherein the enzyme activity is 120IU/mL, and is 3000IU/g larger than that of the culture medium in terms of dry weight;
the specific steps of the xylanase for preparing the xylo-oligosaccharide syrup are as follows:
firstly, preparing xylo-oligosaccharide syrup by using trichoderma pleuroticola xylanase:
the solid-liquid ratio of the oyster mushroom waste mushroom dregs to water is 1: 8, cooking at 150 ℃ for 2 hours, after high-temperature cooking is finished, cooling a residue-liquid mixture to 45 ℃, adjusting the pH value to 5.0, adding xylanase crude enzyme liquid according to the enzyme activity of 15I U/g oyster mushroom waste fungus residues, cooking 5 kilograms of oyster mushroom waste fungus residues, adding 625mL of enzyme liquid, uniformly stirring, reacting at constant temperature of 45 ℃ for 6 hours, inactivating at 90 ℃ for 15 minutes after enzymolysis is finished, centrifuging by using a three-legged centrifuge to obtain sugar liquid, adding 1% of active carbon powder in total volume into the sugar liquid, decolorizing at 75 ℃ for 30 minutes, performing suction filtration by using a Buchner funnel, performing first concentration on the decolorized liquid by using a rotary evaporator until the refractive concentration is 15%, performing second decolorization on the first concentrated liquid with the refractive concentration of 15%, wherein the decolorizing condition is the same as that of the first decolorization; and (3) feeding the secondary decolorized solution to an ion exchange resin column, carrying out primary ion exchange decolorization and impurity removal according to a positive and negative mode, carrying out secondary concentration on the primary ion exchange decolorized solution, carrying out tertiary decolorization on the secondary concentrated solution, feeding the tertiary decolorized solution to the ion exchange resin column, carrying out secondary ion exchange decolorization and impurity removal according to a positive and negative mode, and carrying out vacuum concentration on the secondary ion exchange solution to obtain light yellow viscous xylo-oligosaccharide syrup with the refraction of 75%, wherein the xylo-oligosaccharide accounts for 72.0% of the total sugar content, and the yield of the xylo-oligosaccharide syrup to the raw material oyster mushroom waste residues is 9.09%.
CN201911270355.1A 2019-12-11 2019-12-11 Trichoderma aureoviride and application thereof Active CN110747135B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911270355.1A CN110747135B (en) 2019-12-11 2019-12-11 Trichoderma aureoviride and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911270355.1A CN110747135B (en) 2019-12-11 2019-12-11 Trichoderma aureoviride and application thereof

Publications (2)

Publication Number Publication Date
CN110747135A CN110747135A (en) 2020-02-04
CN110747135B true CN110747135B (en) 2022-03-04

Family

ID=69285861

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911270355.1A Active CN110747135B (en) 2019-12-11 2019-12-11 Trichoderma aureoviride and application thereof

Country Status (1)

Country Link
CN (1) CN110747135B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111621425B (en) * 2020-05-19 2021-09-03 齐齐哈尔大学 Application of trichoderma pleuroticola ZJ-03 in corn deep processing
CN111549083B (en) * 2020-05-19 2021-09-03 齐齐哈尔大学 Application of trichoderma pleuroticola ZJ-03 in deep processing of industrial hemp

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468591A (en) * 2013-09-26 2013-12-25 山东省林业科学研究院 Salt-tolerant trichoderma pleuroticola strain and application thereof
CN105018354A (en) * 2015-08-14 2015-11-04 青岛中达生物技术有限公司 Trichoderma pleuroticola and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468591A (en) * 2013-09-26 2013-12-25 山东省林业科学研究院 Salt-tolerant trichoderma pleuroticola strain and application thereof
CN105018354A (en) * 2015-08-14 2015-11-04 青岛中达生物技术有限公司 Trichoderma pleuroticola and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Melih N. Korkmaz 等.Xylanase production from marine derived Trichoderma pleuroticola 08ÇK001 strain isolated from Mediterranean coastal sediments.《J Basic Microbiol》.2017,第57卷第839-851页. *

Also Published As

Publication number Publication date
CN110747135A (en) 2020-02-04

Similar Documents

Publication Publication Date Title
CN110373359B (en) Streptomyces albus X-18 and method for producing epsilon-polylysine by using same
KR101778436B1 (en) Acetobacter sp. SLV-7 as a novel strain with high acetic acid producing ability and use thereof
CN108220169B (en) Separation screening and identification method of strain for degrading polystyrene
CN110747135B (en) Trichoderma aureoviride and application thereof
CN110093285B (en) Acid-resistant lactobacillus fermentum and application thereof
CN108676734B (en) Entomopathogenic fungi and application thereof
CN110791462B (en) Bacillus subtilis and application thereof in fermentation production of adenosine
CN110564580B (en) Method for producing vinegar containing pyrroloquinoline quinone through microbial co-culture fermentation
CN111518710B (en) Enterobacter strain and application thereof in preparation of microbial polysaccharide
CN108841889B (en) Method for producing griseofulvin serving as major component of tranexamycin by microbial fermentation
CN108823110B (en) Strain for producing griseofulvin and application thereof
CN108841743B (en) Cold region straw rotten bacterial strain and preparation method and application thereof
CN114410523B (en) Strain combination for preparing black tea fungus and application thereof
CN115838632B (en) Trichoderma in Phlebopus portentosus and application thereof
CN1737110A (en) Method for producing C. ophioglossouides using liquid submerged culture
CN115927234A (en) application of amt gene and mutant M107 in expression of azotobacter siderophin
CN106085880B (en) A kind of separation method and used medium of smut
CN112410231B (en) Trichoderma viride new strain and application thereof
CN110923153B (en) Straw saprophytic bacteria and application thereof
CN109182154B (en) Pabuvina rhodotorula strain capable of producing protease at high yield
CN110591971A (en) New strain of xanthomonas Tenaci, culture method and application thereof
CN111088292A (en) Sectional type culture method capable of improving pigment yield and growth speed of cacao conidiophores
CN116515647B (en) Aspergillus flavus and application thereof in preparing tannase and/or degrading tannin
CN111172225B (en) Method for producing and purifying production of bacitracin S
CN116515795B (en) Application of Aspergillus tubingensis in preparing phytase and/or degrading phytic acid

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information
CB03 Change of inventor or designer information

Inventor after: Zhang Meijuan

Inventor after: Ma Tianyi

Inventor after: Qian Pengzhi

Inventor before: Zhang Meijuan

Inventor before: Qian Pengzhi

GR01 Patent grant
GR01 Patent grant