CN115029252B - Neurospora crassa WC2022 strain and application thereof - Google Patents

Neurospora crassa WC2022 strain and application thereof Download PDF

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CN115029252B
CN115029252B CN202210676359.5A CN202210676359A CN115029252B CN 115029252 B CN115029252 B CN 115029252B CN 202210676359 A CN202210676359 A CN 202210676359A CN 115029252 B CN115029252 B CN 115029252B
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soybean isoflavone
neurospora crassa
aglycone
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王洁
朱晓璇
陈国浚
林蕾
方祥
廖振林
钟青萍
王丽
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South China Agricultural University
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Abstract

The invention discloses a Neurospora crassa WC2022 strain and application thereof. The invention separates and obtains a strain of mairei (Neurosporasp.) WC2022 which can mediate the conversion of combined soybean isoflavone glycoside to generate free soybean isoflavone aglycone, and the strain is preserved in the microorganism strain preservation center of Guangdong province in the 6 th month of 2022, and the preservation number is GDMCC NO:62523. The strain can completely convert the combined soybean isoflavone glycoside into free soybean isoflavone aglycone within 120 hours, and the contents of the converted soybean aglycone, genistein and glycitein are respectively increased by 23.15 times, 38.09 times and 12.08 times; meanwhile, the oxidation resistance of the converted product is obviously improved. The invention provides excellent strain resources for producing the free soybean isoflavone aglycone.

Description

Neurospora crassa WC2022 strain and application thereof
Technical Field
The invention belongs to the technical field of agricultural microorganisms. More particularly, it relates to a strain of Neurospora crassa WC2022 and its use.
Background
Soy isoflavones are secondary metabolites of flavonoids produced by plants such as soybean or alfalfa, a typical phytoestrogen, with weak estrogenic activity. The soybean isoflavone can play a role in regulating estrogen-like or anti-hormone bi-directionally, has the activities of resisting oxidation, tumor, bacteria and the like, and is closely related to human health when being eaten deeply. However, the active components of soybean isoflavone mainly exist in the form of glycoside in a combined state, and the bioavailability is extremely low, which restricts the full utilization of the active components.
Researches show that intestinal flora can mediate biological transformation of combined soybean isoflavone glycoside to produce free soybean isoflavone aglycone, remarkably improve bioavailability and promote full play of the physiological activity of soybean isoflavone. In addition to bacteria, fungi and their enzymes can also transform the bound soy isoflavone glycoside to produce free soy isoflavone aglycones, as disclosed in the prior art, the amount of aglycones can be increased by fermenting soy foods with rhizopus stolonifer; genistein and daidzein can be produced by fermenting Penicillium citrinum in potato dextrose broth rich in soy isoflavone soy extract; glycosidases can also be produced using Aspergillus niger or Aspergillus oryzae, which constantly convert the bound soy isoflavone glycoside to a biologically active aglycone form. The fungi can convert and produce the soybean isoflavone aglycone, but the strains capable of converting the soybean isoflavone aglycone are fewer at present, so that more new strain resources capable of mediating the biological conversion of the combined soybean isoflavone glycoside to produce the free soybean isoflavone aglycone are needed to be developed, more efficient and rapid conversion sources and ways are provided for converting the free soybean isoflavone aglycone, and the method is fully utilized in the processing of bean products.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the problems and provide a Neurospora crassa WC2022 strain and application thereof.
The first object of the present invention is to provide a Neurospora crassa WC2022 strain.
A second object of the invention is to provide the use of said WC2022 strain.
The third object of the invention is to provide a microbial inoculum for producing free soybean isoflavone aglycone.
The fourth object of the invention is to provide a method for preparing free soybean isoflavone aglycone.
The above object of the present invention is achieved by the following technical scheme:
the invention identifies and obtains a Neurospora crassa (Neurospora sp.) WC2022 strain, and the morphological characteristics of the strain are as follows: the initial mycelium is white mycelium, the mycelium is sparse and extends to the periphery, and then the mycelium is light orange yellow villus; the mycelium is transparent and has branches and partitions; aerial hyphae irregularly grow around to form branch chains at the top; spherical or nearly spherical conidia are produced, which are initially yellowish and later orange-red or red. The nucleotide sequence of the WC2022 strain is shown as SEQ ID NO. 1, and sequence comparison and phylogenetic tree construction are carried out, so that the similarity between the ITS gene sequence of the Neurospora crassa WC2022 strain provided by the invention and Neurospora crassa (GenBank: JN 198494.1) is up to 99.13%. Thus, the WC2022 strain was identified as Neurospora (Neurospora sp.) -Neurospora crassa (Neurospora crassa), which was deposited at the collection of microorganism strains in the cantonese province at month 7 of 2022, accession number: GDMCC NO 62523, deposit address: guangzhou city first middle road No. 100 college No. 59 building 5.
The research of the invention shows that the adoption of WC2022 strain to carry out whole-cell transformation of combined soybean isoflavone glycoside produces free soybean isoflavone aglycone, and the contents of daidzein, genistein and glycitein in the transformation products are respectively increased by 23.15 times, 38.09 times and 12.08 times; the DPPH removing capability of the converted product is stronger, and the oxidation resistance of the soybean isoflavone is obviously improved after the soybean isoflavone is subjected to in-vitro biotransformation by Neurospora crassa.
The invention provides application of a Neurospora crassa WC2022 strain and/or fermentation liquor thereof in converting combined soybean isoflavone glycoside to generate free soybean isoflavone or in preparing a microbial inoculum for converting combined soybean isoflavone glycoside to generate free soybean isoflavone aglycone.
The invention provides a microbial inoculum for producing free soybean isoflavone aglycone, which contains a Neurospora crassa WC2022 strain and/or fermentation liquor thereof.
Preferably, the fermentation broth is a spore suspension.
Preferably, the spore suspension has a concentration of 10 2 ~10 8 Individual spores/mL.
The invention provides a preparation method of free soybean isoflavone aglycone, which comprises the steps of adding a Neurospora crassa WC2022 strain and/or fermentation liquor thereof into a nutrient solution containing soybean isoflavone or purified soybean isoflavone glycoside, fermenting and culturing, collecting fermentation liquor after culturing, and removing hyphae to obtain a conversion product free soybean isoflavone aglycone. Since soybean isoflavone is a flavonoid compound, and a compound group with 3-benzopyrone as a parent nucleus is a mixture containing various substances such as daidzin, genistin, glycitin and the like, free soybean isoflavone aglycone can be produced by directly using soybean isoflavone or purified soybean isoflavone glycoside for fermentation culture.
Preferably, the purified soybean isoflavone glycoside is one or more of daidzin, daidzin or genistin.
Preferably, the formula of the nutrient solution is as follows: adding 20% potato and 2% glucose into distilled water, sterilizing, and adding 0.1-10% soybean isoflavone or purified soybean isoflavone glycoside.
More preferably, the nutrient solution adopts 1000mL of distilled water, 200g of potato, 20g of glucose and sterilization at 121 ℃ for 20min; after cooling, 0.2% soy isoflavones are added.
Preferably, the fermentation culture conditions are: the temperature is 20-40 ℃, the rotating speed is 100-220 rpm, and the fermentation time is 0-144 h.
More preferably, the fermentation time is 120 hours.
The invention has the following beneficial effects:
the Neurospora crassa (Neurospora sp.) WC2022 strain provided by the invention has the capability of converting combined soybean isoflavone glycoside to generate free soybean isoflavone aglycone, and completely converts soybean glycoside, genistin and soybean xanthosine in 120h, wherein the content of the converted soybean aglycone is increased by 23.15 times, the content of genistein is increased by 38.09 times, and the content of glycitein is increased by 12.08 times. Meanwhile, the DPPH free radical clearance of the fermentation broth transformed by the WC2022 strain is higher and is 1.92 times of that of unconverted soybean isoflavone, which shows that the WC2022 strain effectively improves the DPPH free radical clearance of the transformed product after the soybean isoflavone is transformed by whole cells, and has good antioxidation effect. The Neurospora crassa WC2022 strain provided by the invention not only can be used for efficiently converting and producing free soybean isoflavone aglycone, but also has high antioxidant activity and high protease yield, and a novel strain is provided for producing soybean isoflavone.
Drawings
FIG. 1 is a schematic representation of colony morphology, hyphae, and conidia of WC2022 strain on PDA medium;
FIG. 2 is a phylogenetic tree result diagram of the ITS sequence construction of the WC2022 strain;
FIG. 3 is a diagram showing the content change of the glycoside and aglycone substances (daidzin, daidzein, genistin, genistein, daidzein, glycitin) of the converted soybean isoflavone by high performance liquid chromatography;
FIG. 4 is a schematic diagram showing DPPH radical scavenging ability of whole cell transformed soybean isoflavone by WC2022 strain.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The reagents used in the following examples were:
dimethyl sulfoxide DMSO (analytically pure) was purchased from the company of fine chemical engineering, prefecture, the division of the Tianjin city;
soy isoflavones (BR, 40%) were purchased from shanghai source leaf biotechnology limited;
standard substances (more than or equal to 98%) such as daidzin, daidzein, genistin, daidzein, glycitein and genistein are all purchased from Shanghai source leaf biotechnology limited company;
methanol (chromatographic purity), acetonitrile (chromatographic purity) were all purchased from sammer feishier technologies.
The media used in the following examples were:
potato Dextrose Broth (PDB): 200g of potato, 20g of glucose, adding water to a constant volume of 1L, and sterilizing at 121 ℃ for 20min for later use.
PDA medium: after adding 2% agar to PDB, sterilizing at 121deg.C for 20 min.
The formula of the whole cell transformed nutrient solution comprises the following steps: 200g of potato, 20g of glucose, 1000mL of distilled water, natural pH and sterilization at 121 ℃ for 20min; after cooling, 0.2% soy isoflavones are added.
Example 1 isolation and identification of strains
1g of traditional red fungus bean curd in Pingyou county of Guangdong province is weighed, added into 90mL of sterile normal saline, crushed, shaken uniformly, diluted in a gradient manner, coated on a PDA solid medium, cultured at 30 ℃, and separated to obtain 1 strain of fungus named WC2022.
The colony morphology, hypha and conidium of WC2022 strain on PDA culture medium are shown in figure 1, the initial stage of the mycelium in figure 1A is white hypha, the hypha is sparse and extends to the periphery, and the later stage is light orange yellow villus as shown in figure 1B; in FIG. 1C, the hyphae are transparent, branched and segregated; aerial hyphae irregularly grow around to form branch chains at the top; in fig. 1D, spherical or nearly spherical conidia are produced, initially yellowish, followed by reddish orange or red.
And amplifying the strain ITS sequence by adopting a universal primer ITS1/ITS4, and sequencing by adopting a conventional method. The ITS sequence of the strain is shown as SEQ ID NO. 1, the length is about 620bp, the sequence of the WC2022 strain is compared with that of the strain at NCBI, and the phylogenetic tree constructed by the ITS sequence of the WC2022 strain is shown as figure 2, and the phylogenetic tree is found by analysis: WC2022 strain has a similarity of up to 99.13% with Neurospora crassa (GenBank: JN 198494.1). Thus, by combining the morphological characteristics and phylogenetic tree analysis described above, the WC2022 strain was identified as Neurospora (Neurospora sp.) -Neurospora crassa (Neurospora crassa), designated Neurospora crassa (Neurospora sp.) WC2022, which was deposited at the cantonese microorganism strain deposit center at month 7 of 2022, deposit number: GDMCC NO 62523, deposit address: guangzhou city first middle road No. 100 college No. 59 building 5.
EXAMPLE 2 Whole cell transformation of the WC2022 Strain with conjugated Soy isoflavone glycoside to its free aglycone
The Neurospora crassa WC2022 strain isolated in example 1 was used, and the WC2022 strain was cultured in Neurospora crassa WC2022 seed medium (potato 200g, glucose 20g, agar 20g, distilled water 1000mL, pH was natural, and sterilization was performed at 121℃for 20 min) under the following conditions: and (3) carrying out stationary culture on the eggplant in a bottle at a culture temperature of 30 ℃ for 5 days.
And then, washing the solid flat-plate culture medium fully grown with the neurospora crassa spores after the culture by using sterile water containing 0.05% of Tween-80, and lightly scraping the surface of the culture medium by using a pipette tip to obtain the neurospora crassa conidium suspension. Adding the conidium suspension into whole cell transformation nutrient solution, wherein the final concentration of the conidium is as follows: 10 6 Individual spores/mL. Culturing at 30 deg.c and shaking table rotation speed of 150-200 rpm/min for 144 hr.
In whole cell transformation nutrient solution culture, soybean isoflavone transformation products were taken out at regular intervals of 24 hours, and Neurospora crassa hyphae were removed, and filtration was performed using a 0.22 μm filter membrane. After the filtrate is reserved, the content change of the glycoside and aglycone substance (daidzin, daidzein, genistin, genistein, daidzein, glycitin) of the converted soybean isoflavone is detected by high performance liquid chromatography.
Setting parameters of the high performance liquid chromatography detection method: the mobile phase is A: primary aqueous solution, B:100% pure methanol solution, C:100% pure acetonitrile solution; column temperature: 36 ℃; sample injection amount: 10. Mu.L; detection wavelength: 260nm; flow rate: 0.8mL/min. The gradient elution conditions for soy isoflavones are shown in table 1 below.
TABLE 1 gradient elution conditions of Soy isoflavone
As shown in FIG. 3, WC2022 strain can convert the conjugated soybean isoflavone glycoside into free soybean isoflavone aglycone, wherein daidzin in the soybean isoflavone glycoside is converted into daidzein, genistin is converted into genistein, and daidzein is converted into glycitein; in FIG. 3A, daidzin was completely converted at 120h, daidzein content increased gradually with the conversion of daidzin, with a peak at 120h and a 23.15-fold increase in content. In FIG. 3B, genistin was completely converted at 120h, genistein content was gradually increased with the conversion of genistin, the peak reached at 120h, and the content was increased 38.09 times. In FIG. 3C, huang Douhuang glycoside was completely converted at 120h, glycitein content was gradually increased with the conversion of daidzein, the peak was reached at 120h, and the content was increased by 12.08 times.
In conclusion, the MAICHO WC2022 can convert soybean isoflavone glycoside into corresponding aglycone through liquid fermentation, and can be used as a novel microbial resource for producing free soybean isoflavone aglycone through soybean isoflavone conversion.
EXAMPLE 3 DPPH radical scavenging ability test of soybean isoflavone conversion products
The whole cell transformed nutrient solution prepared in example 2 was cultured with sterile water and the fermentation broth was diluted for use. Preparing DPPH-ethanol solution: weighing 20mgDPPH reagent in beaker, dissolving with absolute ethanol, fixing volume in 250mL volumetric flask, and preserving in dark place.
Respectively sucking 2mL of diluted fermentation liquor, adding the diluted fermentation liquor into a test tube, mixing the fermentation liquor and the 2mL of diluted fermentation liquor, standing the mixture at room temperature in the dark for reaction for 30min, measuring the absorbance at 517nm wavelength, and marking the absorbance as Ai. Absolute ethyl alcohol is used to replace the sample to be tested and DPPH-ethanol solution in the above steps respectively, and the absorbance value is measured under the same condition and recorded as A 0 And Ar. Experiments were repeated 3 times. DPPH radical scavenging (K) was calculated according to the following formula.
The calculation formula is as follows: k (%) = [1- (Ai-Ar)/A 0 ]×100
As shown in the experimental result in figure 4, the clearance of DPPH free radicals of the fermentation liquor obtained after the soybean isoflavone is converted by the Neurospora crassa WC2022 is the highest (p is less than 0.05) in 120 hours, and is 1.92 times of that of the unconverted soybean isoflavone, which shows that the DPPH free radical clearance is effectively improved after the soybean isoflavone is converted by the whole cell of the Neurospora crassa WC2022, and the fermentation liquor has good antioxidation effect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> agricultural university of south China
<120> a Neurospora crassa WC2022 strain and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 620
<212> DNA
<213> Mai cell-roughening WC2022 Strain ITS sequence (SIPOSEQUENCELISTERING 1.0)
<400> 1
ccgagcactt atttcctccg gccttatgat atgcttaagt tcagcgggta ttcctacctg 60
atccgaggtc aaccttagaa atggggggtt ttacggcaag aacccgccgc acgaccatag 120
cgatgtagag ttactacgct cggtgtgact agcgagcccg ccactgattt tgagggaccg 180
cggacagccg cggatcccca acgcaagcag agcttgatgg ttgaaatgac gctcgaacag 240
gcatgctcgc cagaatactg gcgagcgcaa tgtgcgttca aagattcgat gattcactga 300
attctgcaat tcacattact tatcgcattt cgctgcgttc ttcatcgatg ccagaaccaa 360
gagatccgtt gttgaaagtt ttgacttatt taaaagttta ctcagagaga cataaaatat 420
caagagttta gtttcggcac tccggcgggc agcctcccgc gagcgggaga cccgaggatc 480
cgggagggcc cgagggcctt tccggaccgc cagcgccgag gcaaccgtac gggtaagatt 540
cgcgatggtt tgtgggagtt ttgcaactct gtaatgatcc ctccgcaggt cccccttacg 600
gagggatcat tacagagttg 620

Claims (10)

1. Neurospora crassa (L.) KuntzeNeurosporasp.) WC2022 strain, characterized in that it was deposited with the Guangdong province microbiological culture Collection center (GDMCC) on day 7 of 6.2022, with the accession numberGDMCC NO:62523。
2. Use of the strain neurospora crassa WC2022 and/or its fermentation broth according to claim 1 for converting a bound soy isoflavone glycoside to produce free soy isoflavone aglycone.
3. Use of the neurospora crassa WC2022 strain and/or its fermentation broth according to claim 1 for the preparation of a bacterial agent for converting a bound soy isoflavone glycoside to produce free soy isoflavone aglycone.
4. A microbial preparation for producing free soybean isoflavone aglycone, which comprises the Neurospora crassa WC2022 strain and/or a fermentation liquid thereof according to claim 1.
5. The microbial inoculum of claim 4, wherein the fermentation broth is a spore suspension.
6. The microbial preparation of claim 5, wherein said spore suspension has a concentration of 10 2 ~10 8 Individual spores/mL.
7. A process for preparing free soybean isoflavone aglycone, which is characterized in that the strain WC2022 of Neurospora crassa and/or the fermentation liquor thereof is adopted, added into the nutrient solution containing soybean isoflavone or purified soybean isoflavone glycoside, fermented and cultured, and after the culture is finished, the fermentation liquor is collected, hyphae are removed, and the converted product free soybean isoflavone aglycone can be obtained.
8. The method of claim 7, wherein the purified soy isoflavone glycoside is one or more of daidzein, glycitin, or genistin.
9. The method of claim 7, wherein the nutrient solution is formulated as follows: adding 20% potato and 2% glucose into distilled water, sterilizing, and adding 0.1-10% soybean isoflavone or purified soybean isoflavone glycoside.
10. The method of claim 7, wherein the fermentation culture conditions are: the temperature is 20-40 ℃, the rotating speed is 100-220 rpm, and the fermentation time is 48-144 h.
CN202210676359.5A 2022-06-15 2022-06-15 Neurospora crassa WC2022 strain and application thereof Active CN115029252B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101479380A (en) * 2006-06-26 2009-07-08 帝斯曼知识产权资产管理有限公司 Peptidylarginine deiminase and uses thereof in the production of citrullinated proteins and peptides
CN105886561A (en) * 2014-09-10 2016-08-24 瑞安市普罗生物科技有限公司 Neurospora crassa-based microbial conversion method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101479380A (en) * 2006-06-26 2009-07-08 帝斯曼知识产权资产管理有限公司 Peptidylarginine deiminase and uses thereof in the production of citrullinated proteins and peptides
CN105886561A (en) * 2014-09-10 2016-08-24 瑞安市普罗生物科技有限公司 Neurospora crassa-based microbial conversion method

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