CN116731881A - Mucor HY6-19 and application thereof in preparation of semen Hoveniae extract - Google Patents
Mucor HY6-19 and application thereof in preparation of semen Hoveniae extract Download PDFInfo
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- CN116731881A CN116731881A CN202310725606.0A CN202310725606A CN116731881A CN 116731881 A CN116731881 A CN 116731881A CN 202310725606 A CN202310725606 A CN 202310725606A CN 116731881 A CN116731881 A CN 116731881A
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- semen hoveniae
- extract
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- hovenia dulcis
- water
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Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/72—Rhamnaceae (Buckthorn family), e.g. buckthorn, chewstick or umbrella-tree
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- 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
- C12R2001/785—Mucor
Abstract
The invention discloses a Mucor HY6-19 and application thereof in preparation of semen hoveniae extract, wherein the method aims at purposefully separating and screening the semen hoveniae cell wall capable of being hydrolyzed, obtaining a new microorganism strain Mucor HY6-19 through mutagenesis, moderately growing in semen hoveniae powder added with sucrose to generate cellulase with higher activity and other various hydrolytic enzymes, then adding water into the fermented semen hoveniae powder for heat preservation, and carrying out enzymolysis on cellulose, hemicellulose, pectin and other substances in the cell wall, thereby facilitating dissolution of substances combined with cellulose and intracellular substances in the cell wall, and remarkably improving the yield of the extract and the total flavone content. Before the hovenia dulcis thunb extract is prepared by a hot water ultrasonic method, microbial fermentation treatment is added, and compared with a direct hot water ultrasonic extraction method, the yield of the hovenia dulcis thunb extract can be increased by 34.4%, and the total flavone content is increased by 23.3%.
Description
Field of the art
The invention belongs to the field of application of biotechnology in extraction of plant active ingredients, and particularly relates to mucor HY6-19 and application thereof in preparation of hovenia dulcis thunb extract.
(II) background art
The semen Hoveniae is mature seed of Hoveniae (Hovenia dulcis), hoveniae (Hovenia acerba) and Hoveniae Mao Guozhi (Hovenia trichocarpa). Hovenia dulcis thunb is mainly distributed in North China, east China, south China, southwest, shanxi, gansu and other places; mao Guozhi the main distribution of hovenia dulcis is Zhejiang, jiangxi, hubei, hunan, guangdong and Guizhou. Hovenia dulcis thunb is a medicinal and edible traditional Chinese medicine, has the effects of clearing heat and promoting urination and dispelling alcohol toxicity, and mainly treats diseases such as alcohol toxicity, dysphoria with smothery sensation, thirst, vomiting, inconvenient urination and the like, and has the expression of 'Qianzhu hovenia dulcis thunb' in ancient medical books, namely medical prescription examination, hovenia dulcis (also called chicken feet) is recorded, and alcohol dispelling and flower of kudzuvine are also recorded. It should be used in the future for those who are injured in the middle energizer. The semen Hoveniae contains a large amount of bioactive components including polysaccharide, flavone, triterpene, alkaloids, etc. Modern medical research has also proved that semen Hoveniae extract can accelerate ethanol metabolism of human body, reduce blood alcohol concentration after drinking, enhance liver ethanol dehydrogenase activity, reduce liver lipid peroxidation risk, reduce liver injury caused by ethanol, etc., and has effects of relieving alcoholism, quenching thirst, relieving restlessness, relieving vomiting, and relieving constipation.
At present, there are reports on preparation methods of hovenia dulcis thunb extract, and the methods can be divided into two types, namely a water extraction method and an organic solvent extraction method according to solvents used for extraction. The composition of the substances extracted by different solvents is greatly different, and the water extract mainly contains water-soluble substances such as polysaccharide, oligosaccharide, amino acid and the like; the organic solvent extraction method mainly uses ethanol or ethyl acetate, wherein ethanol is the majority, and the extract mainly contains flavone, triterpene, alkaloid and other substances. On the basis of the two methods, ultrasonic wave, microwave and enzymolysis are assisted to strengthen the extraction, so that the yield of the extract can be improved. The equipment requirement of ultrasonic assisted extraction is low, the extraction cost is not obviously increased, but the effect of improving the extraction yield is not ideal; microwave-assisted extraction is short in time consumption and high in efficiency, but the structure of the extracted active ingredients is often damaged due to high internal temperature; the enzymolysis method adopts cellulase, pectase or protease, etc., and hydrolyzes plant materials under proper conditions to hydrolyze cell walls, thereby facilitating the dissolution of intracellular active ingredients during extraction. The enzymolysis auxiliary method not only can remarkably improve the extraction rate, but also has mild conditions and can not damage the structure of active ingredients in the extract; furthermore, enzymatic hydrolysis can reduce the molecular weight of polysaccharides and convert some glycosides into aglycones, and the biological activity of the extract is better, so that in recent years, enzymatic hydrolysis is increasingly used for extracting plant active ingredients.
Microorganisms can produce a plurality of enzymes for hydrolyzing plant tissues, and plant branches and abortive leaves in nature are decomposed by enzymes produced by the microorganisms. In particular, some fungi in nature are capable of producing a variety of enzymes including cellulases, hemicellulases, ligninases, pectinases, and proteases. Therefore, if the hovenia dulcis thunb is directly fermented and pretreated by using a proper mould, the cell wall of the hovenia dulcis thunb is decomposed by enzyme only by controlling the growth degree, but the active ingredients are not decomposed, so that the dissolution of the active ingredients is promoted, and the extract yield can be improved. Compared with the enzymolysis auxiliary extraction method, the method has better hydrolysis effect of various enzymes produced by microorganisms on plant cell walls, and has the advantages that the polysaccharide is properly degraded, the molecular weight is reduced, the activity is improved, some glycosyl of flavonoid glycoside is hydrolyzed, flavonoid aglycone is released, and the biological activity of the extract is better.
In order to improve the yield and the bioactivity of the hovenia dulcis thunb extract, the invention carries out microbial fermentation on the hovenia dulcis thunb, and then uses hot water for ultrasonic extraction, so that the yield of the extract is improved, and the content of total flavonoids can be obviously increased.
(III) summary of the invention
The invention aims to provide a novel microorganism strain Mucor HY6-19 and application thereof in preparation of semen hoveniae extract, wherein a microorganism fermentation technology is applied to preparation of the semen hoveniae extract, the semen hoveniae is subjected to microorganism fermentation and then subjected to hot water ultrasonic extraction, the extract yield is obviously improved, and the total flavone content is increased.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a new microorganism strain, mucor sp.) HY6-19, deposited in the Guangdong province microorganism strain collection center, deposit number GDMCC No. 63391, deposit date 2023, 4 months and 24 days, address: building 5 of No. 59 of Qinghui No. 100 college in Guangzhou City of Guangdong; postal code 510070.
The mucor HY6-19 is an excellent strain for fermenting hovenia dulcis thunb, which is obtained by separating from soil, screening and mutagenizing and breeding. The morphological characteristics of the mucor HY6-19 are as follows: culturing on Potato Dextrose Agar (PDA) plate culture medium at 28deg.C, and turning the initial mycelium into off-white; after 1 day, the grey color is slightly deepened, the hypha is longer and fluffy, and a small amount of grey spores are generated on the surface. The conidiophores are observed to be single-born under an optical microscope, are densely layered, stand upright and all of the aposporangia are grown, and the sporangia are larger, spherical, gray brown and sporangia spherical, and have the diameter of 4-5 mu m. A photograph of a colony of Mucor HY6-19 cultured for 2d at 28℃on PDA plate medium is shown in FIG. 1.
The nucleotide sequence of a transcription spacer (RibosomaL DNA internal transcribed spacer, rDNA-ITS) in the ribosomal DNA of the mucor HY6-19 is shown in SEQ ID NO. 1.
The invention also provides an application of the trichoderma HY6-19 in preparing a hovenia dulcis thunb extract, and the application method comprises the following steps: (1) Inoculating Mucor sp spore solution of HY6-19 into semen Hoveniae powder, stirring, and fermenting at 30-32deg.C for 52-60 hr to obtain semen Hoveniae fermented product; (2) Adding deionized water into semen Hoveniae fermented product, stirring, maintaining at 35-40deg.C for 4-6 hr, ultrasonic extracting with hot water, filtering, and concentrating the filtrate to obtain semen Hoveniae water concentrated solution; (3) Vacuum drying the water extract concentrate of semen Hoveniae, and pulverizing to obtain semen Hoveniae extract.
Further, the Hovenia dulcis thunb in the step (1) is a mature seed of Hovenia dulcis thunb (Hovenia dulcis), hovenia acerba (Hovenia acerba) or Hovenia dulcis thunb (Hovenia trichocarpa) of the Rhamnaceae family; the semen Hoveniae powder is fine powder obtained by oven drying semen Hoveniae at 85deg.C, pulverizing, and sieving with 60 mesh sieve.
Further, the preparation method of the mucor HY6-19 spore liquid in the step (1) comprises the following steps: inoculating the spores of the mucor HY6-19 preserved at low temperature on a Potato Dextrose Agar (PDA) flat plate culture medium, culturing at the constant temperature of 30 ℃ for 48-72 hours, adding a sterile sucrose aqueous solution into the culture, and stirring by an inoculating loop to suspend the spores to obtain spore liquid; preferably, the spore solution is adjusted to 5×10 spore concentration by using sterile sucrose water solution 6 –9×10 6 And each mL. The concentration of sucrose in the sterile sucrose aqueous solution is 10-15g/L, and the sterile sucrose aqueous solution is sterilized by high-pressure steam at 115 ℃ for 15min. The PDA plate culture medium is a finished potato dextrose agar culture medium (Qingdao sea Bo biotechnology Co., ltd.) and is prepared by tap water according to the concentration of 46g/L, the pH is natural, and the high-pressure steam is used for sterilization for 15min at 121 ℃.
Further, the spore liquid volume amount of the mucor HY6-19 in the step (1) is 2-3mL/g based on the mass of the semen hoveniae powder.
Further, the preparation method of the hovenia dulcis thunb water extraction concentrated solution in the step (2) comprises the following steps: adding deionized water into semen Hoveniae fermented product, stirring uniformly, preserving heat for 4-6h at 35-40 ℃, transferring into an ultrasonic cleaner with water temperature of 75-85 ℃, performing ultrasonic extraction at 160-200W for 60-80min, filtering with 200-mesh filter cloth when the ultrasonic extraction is finished, concentrating the filtrate under reduced pressure at 60 ℃ and minus 0.1MPa to 1/10-1/25 of the original volume, and obtaining semen Hoveniae water extract concentrated solution, wherein the adding amount of the deionized water volume is 20-25mL/g based on the mass of semen Hoveniae powder before fermentation.
Further, the preparation method of the hovenia dulcis thunb extract in the step (3) comprises the following steps: drying semen Hoveniae water extract concentrate at 65deg.C under-0.1 MPa under vacuum to constant weight, and pulverizing to obtain semen Hoveniae extract.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the method is aimed at purposefully separating and screening the cell walls of the hovenia dulcis thunb, and obtaining a new microorganism Mucor HY6-19 through mutagenesis, wherein Mucor HY6-19 moderately grows in the hovenia dulcis thunb powder added with sucrose to generate cellulase with higher activity and other various hydrolytic enzymes, and then the fermented hovenia dulcis thunb powder is added with water and is subjected to heat preservation, so that substances such as cellulose, hemicellulose and pectin in the cell walls are subjected to enzymolysis, substances combined with cellulose and intracellular substances in the cell walls are dissolved out, the yield of the extract is obviously improved, and the total flavone content is also obviously improved. Before the hovenia dulcis thunb extract is prepared by a hot water ultrasonic method, microbial fermentation treatment is added, and compared with a direct hot water ultrasonic extraction method, the yield of the hovenia dulcis thunb extract can be increased by 34.4%, and the total flavone content is increased by 23.3%.
(IV) description of the drawings
FIG. 1 is a photograph showing colony morphology of Mucor HY6-19 cultured on PDA at 28℃for 2 d.
FIG. 2 shows a standard curve of spectrophotometry for total flavonoids (rutin is a standard substance).
FIG. 3 shows a standard curve of reducing sugar measurement by the DNS method (glucose is a standard).
(fifth) detailed description of the invention
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
The Hovenia dulcis thunb used in the embodiment of the present invention is a mature seed of Hovenia dulcis thunb (Hovenia acerba) belonging to the family Rhamnaceae; the semen Hoveniae powder is fine powder obtained by oven drying semen Hoveniae at 85deg.C, pulverizing, and sieving with 60 mesh sieve.
The yield of the hovenia dulcis thunb extract in the embodiment of the invention is calculated according to a formula 1:
example 1: microbial strain isolation and screening of fermented hovenia dulcis thunb
The microbial strain of fermented semen Hoveniae is obtained by separating and screening according to the following steps:
(1) Taking 1g of soft and moist soil from woodland with more fallen leaves, diluting 1×10 with sterile physiological saline -4 、1×10 -5 、1×10 -6 、1×10 -7 After doubling, 0.1mL of the dilution was aspirated and spread on Potato Dextrose Agar (PDA) plate medium, and incubated at 30℃for 48 h. Selecting mould colonies with different colors and forms, transferring to fresh PDA plate culture medium, and keeping constant temperature at 30deg.CCulturing for 72h to obtain pure culture mould strain 10, wherein the serial numbers of the strains are shown in table 1;
(2) Adding 10mL sterile sucrose aqueous solution into fresh plate culture of 10 strains, stirring with inoculating loop to suspend spores, transferring spore solution into sterile test tube, adjusting spore concentration with sterile sucrose aqueous solution to make spore solution of different strains at 5×10 6 –9×10 6 In the range of individual/mL, spore liquid of each strain is obtained. The concentration of the sterile sucrose water solution is 15g/L, and the sterile sucrose water solution is sterilized for 15min at the temperature of 115 ℃ by high-pressure steam;
(3) Adding 5g of semen Hoveniae powder into 10 150-mL triangular flasks subjected to 160 ℃ and 2h dry heat sterilization, respectively adding 15mL of each mold spore liquid prepared in the step (2) (the volume dosage is 3mL/g based on the mass of the semen Hoveniae powder), uniformly stirring, tying the triangular flasks with 8 layers of gauze, and culturing for 60h at 30 ℃ to obtain semen Hoveniae fermented products;
(4) And (3) adding 100mL of deionized water (the feed liquid ratio is 1:20 (g: mL)) into all hovenia dulcis thunb fermented products, uniformly stirring, preserving heat for 6h in a water bath at 35 ℃, and then transferring into an ultrasonic cleaner at 75 ℃ for ultrasonic extraction at 160W for 80min. Filtering with 200 mesh filter cloth when the ultrasonic extraction is finished, and collecting filtrate;
(5) Concentrating the filtrate obtained in the step (4) to 10mL (1/10 of the original volume) under reduced pressure at 60 ℃ and minus 0.1MPa to obtain the hovenia dulcis thunb water extraction concentrated solution. Drying the water extract concentrate at 65 deg.C under-0.1 MPa under vacuum to constant weight, and pulverizing to obtain semen Hoveniae extract.
Starting from the step (3), adding 15mL of sterile sucrose aqueous solution (concentration of 15 g/L) into 5g of semen Hoveniae powder, preparing semen Hoveniae extract according to the steps (3) - (5), and making blank fermentation control without inoculating mould; starting from the step (4), adding 100mL of deionized water into 5g of semen Hoveniae, directly extracting according to the steps (4) and (5), and preparing semen Hoveniae extract, and performing unfermented control. The extract yields and total flavone content of hovenia dulcis thunb fermented with different strains and the control are shown in table 1.
TABLE 1 extract yields and Total Flavonoids of Hovenia dulcis Thunb fermented by different strains and controls
As can be seen from the data in Table 1, the addition of the sterile aqueous sucrose solution without inoculating the blank fermentation control of the mold spores, the extract yield was increased by 7.38% compared to the unfermented control, but the total flavone content of the extract was reduced by 6.09% because of the small amount of mixed bacteria growth. The extract yield is not obviously improved after the hovenia dulcis thunb is fermented by most strains, and is respectively improved by 12.3%, 13.1% and 14.8% after the HY4, HY6 and HY9 strains are fermented, but compared with the hovenia dulcis thunb, the total flavone content in the hovenia dulcis thunb extract after the HY6 fermentation is higher, and is improved by 9.57% compared with 11.5% of the hovenia dulcis thunb which is not fermented by a control, the HY6 strain is selected as a fermentation strain of the hovenia dulcis thunb.
The PDA plate culture medium is a finished potato dextrose agar culture medium (Qingdao sea Bo biotechnology Co., ltd.) prepared by tap water according to the concentration of 46g/L, the pH is natural, the PDA plate culture medium is tied up by 8 layers of gauze in a triangular flask, sterilized for 15min by high-pressure steam at 121 ℃, and poured into sterile culture dishes with the diameter of 9cm before solidification, and 15-20mL of each dish.
The content of total flavone in the hovenia dulcis thunb extract is determined by a spectrophotometry, and the specific method comprises the following steps of: preparing semen Hoveniae extract into aqueous solution with concentration of 0.5mg/mL with deionized water, centrifuging at 5000r/min for 5min, collecting 2.5mL supernatant, and adding sodium nitrite (NaNO) with mass concentration of 5% into 10-mL graduated tube 2 ) 0.4mL of aqueous solution, standing for 6min, adding 10% aluminum nitrate [ Al (NO) 3 ) 3 ]0.6mL of aqueous solution, standing for 6min, adding 2mL of aqueous solution of sodium hydroxide (NaOH) with mass concentration of 20%, fixing volume to 10mL with aqueous solution of ethanol with volume concentration of 60%, shaking, standing for 15min, and measuring absorbance (A) at wavelength of 510nm with spectrophotometer 510 ) And calculating the total flavone concentration in the sample according to a rutin standard curve, and multiplying the concentration by the volume of the measured sample to obtain the total flavone mass.
Drawing a rutin standard curve: and preparing a rutin standard substance solution with the concentration of 0.25g/L by using a 60% ethanol water solution by volume. Respectively taking rutin standard substance solutions 0, 0.5, 1.0, 1.5, 2.0, 2.5mL and 10mLSupplementing 2.5mL of 60% ethanol aqueous solution by volume concentration into a mL-scale test tube, adding 0.4mL of 5% sodium nitrite aqueous solution by mass concentration, standing for 6min, adding 0.6mL of 10% aluminum nitrate aqueous solution by mass concentration, standing for 6min, adding 2mL of 20% sodium hydroxide aqueous solution by mass concentration, and fixing the volume of the aqueous solution by volume concentration of 60% ethanol to 10FeSO 4 mL, shaking, standing for 15min, and measuring absorbance at 510nm (A) 510 ). Rutin concentration is taken as an abscissa, A 510 A standard curve is plotted for the ordinate (fig. 2).
Example 2: microorganism strain mutation breeding of fermented semen Hoveniae
The strain HY6 screened in the embodiment 1 is subjected to mutation breeding, and the strain with excellent fermentation performance is screened by the specific method:
(1) Preparation of spore liquid: after the strain HY6 was subjected to activation culture for 48 hours at 30℃in PDA plate medium, 5mL of sterile physiological saline was added, and spores were suspended by stirring with an inoculating loop. 1mL of the spore liquid was transferred to a triangular flask (containing 20-30 glass beads) containing 50mL of sterile physiological saline, and the mixture was shaken at room temperature for 15min. Filtering spore liquid to remove mycelium (filling small mass of fluffy absorbent cotton at bottom of triangular funnel), counting spores in spore liquid with blood cell counting plate under microscope, diluting with sterile physiological saline, and adjusting spore amount to 1.34×10 7 And each mL.
(2) Mutagenesis: under the illumination of red light, 1.5mL of the spore liquid and a piece of sterile paperclip are respectively taken in 6 culture dishes with the diameter of 6cm, the culture dishes are respectively arranged on a magnetic stirrer, and 1, 2, 3, 4, 5 and 6min are respectively irradiated at the position of 30cm away from a 15W ultraviolet lamp which is preheated for 30 min. 0.5mL of the spore liquid after the irradiation treatment was diluted with a proper multiple of sterile physiological saline, and 0.1mL of the PDA-coated plate medium was removed. In the same manner, a spore liquid dilution plating plate without ultraviolet irradiation was used as a control to calculate the mortality rate. The inoculated PDA plates were wrapped with black cloth, incubated for 48h at 28℃upside down, colonies on the plates were counted and mortality was calculated.
(3) Screening: colonies on PDA plates with the mortality rate of more than 90% are picked up and transferred to fresh PDA plate culture medium, and cultured for 72 hours at 30 ℃ to obtain 50 strains. 10mL of sterile physiological saline is added to the plate culture of each strain, and spores are suspended by stirring with an inoculating loop, so that spore liquid of each strain is obtained. Taking 2.5mL of spore liquid of each strain, inoculating into 50mL of enzyme-producing culture medium, carrying out shaking culture at 30 ℃ and 200r/min for 72h, carrying out suction filtration on the fermentation liquid by using a Buchner funnel, collecting filtrate (namely crude enzyme liquid), and measuring cellulase activity of fermentation filtrate of each strain. Selecting 15 strains with relatively higher enzyme productivity than the original strain HY6, inoculating semen Hoveniae powder with spore liquid of the strains to ferment according to the method of example 1, performing hot water ultrasonic extraction, and fermenting semen Hoveniae with mutant strains to obtain extract with total flavone content shown in Table 2.
TABLE 2 extract yield and Total Flavonoids of Hovenia dulcis Thunb fermented with mutant strain and control
As can be seen from the data in Table 2, the activity of the cellulase produced by fermentation of the strain No. HY6-19 among the 15 strains selected was 62.7U/mL, which is 22.3% higher than that of the strain HY6 of the wild strain. After the strain is used for fermenting hovenia dulcis thunb, the extract yield is 15.3%, and is improved by 12.5% compared with 13.6% of a wild strain HY6, and is improved by 25.4% compared with 12.2% of a non-fermented control; the total flavone content in the extract is 13.5%, which is 7.14% higher than the 12.7% of the wild strain HY6 and 17.4% higher than the 11.5% of the unfermented control, so the HY6-19 strain is selected as the microorganism strain of the fermented semen Hoveniae.
The enzyme-producing culture medium comprises the following components: wheat bran 50g/L, (NH) 4 ) 2 SO 4 6g/L peptone 4g/L KH 2 PO 4 2g/L，MgSO 4 ·7H 2 O 1g/L，CaCl 2 0.5g/L, tap water as solvent, pH 6.0. 250-mL triangular flask is filled with 50mL of enzyme-producing culture medium, 8 layers of gauze are tied up, and high-pressure steam is used for sterilization at 121 ℃ for 20min.
And the cellulase activity is determined: 1.5mL of 10g/L sodium carboxymethyl cellulose solution is respectively added into 10-mL graduated test tubesThe solution (prepared by pH 6.0 and 0.2mol/L phosphate buffer solution) and 0.5mL of crude enzyme solution are subjected to water bath heat preservation for 30min at 50 ℃, then 3mL of DNS reagent is added, boiling is carried out for 5min, deionized water is added to a constant volume of 10mL after running water is cooled, and stirring is uniform; the same treatment was carried out with the inactivated crude enzyme solution after boiling at 100deg.C for 10min as a reference, and absorbance at 540nm was measured by a spectrophotometer (A 540 ) The glucose concentration in the sample was calculated from the glucose standard curve (FIG. 3), and cellulase activity (U/mL) was calculated. Definition of cellulase activity: the amount of enzyme required to hydrolyze sodium carboxymethylcellulose to 1. Mu.g glucose per minute at pH 6.0 and 50℃was 1 enzyme activity unit (U).
Cellulase activity was calculated according to equation 2.
In formula 2, C: glucose concentration (μg/mL) calculated from the standard curve; v (V) 1 : the volume of the enzyme reaction system is 2mL; t: the reaction time is 30min; v (V) 2 : the crude enzyme solution volume, i.e., 0.5mL.
Drawing a glucose standard curve: adding standard glucose aqueous solution 0,0.2, 0.4, 0.6, 0.8, 1.0 and 1.2mL with concentration of 1mg/mL into 7-branch 10-mL graduated test tubes, respectively, adding phosphate buffer solution 2.0, 1.8, 1.6, 1.4, 1.2, 1.0 and 0.8mL with pH of 6.0,0.2mol/L, 1.8, 1.6, 1.2, 1.0 and 0.8mL with pH of 0, respectively, adding DNS solution 3.0mL, boiling the mixed solution in boiling water bath for 5min, cooling with running water, fixing volume to 10mL with deionized water, stirring, and measuring A with spectrophotometer 540 On the abscissa, the glucose concentration, A 540 A standard curve is plotted for the ordinate (fig. 3).
Preparing a DNS reagent: 6.3g of 3, 5-dinitrosalicylic acid and 262mL of 2mol/L NaOH aqueous solution are added into 500mL of hot aqueous solution containing 182g of sodium tartrate, 5g of distilled phenol and 5g of sodium sulfite are added, stirred and dissolved, cooled, added with deionized water to a volume of 1L, stored in a brown bottle and used after 7 d.
Example 3: classification and identification of strain HY6-19
The strain HY6-19 is streaked and inoculated on a PDA plate culture medium for culturing at 28 ℃, and the initial mycelium is grey white; after 1 day, the grey color is slightly deepened, the hypha is longer and fluffy, and a small amount of grey spores are generated on the surface. The conidiophores are observed to be single-born under an optical microscope, are densely layered, stand upright and all of the aposporangia are grown, and the sporangia are larger, spherical, gray brown and sporangia spherical, and have the diameter of 4-5 mu m. A photograph of a colony of Mucor HY6-19 cultured for 2d at 28℃on PDA plate medium is shown in FIG. 1.
The rDNA-ITS nucleotide sequence of strain HY6-19 was determined to be SEQ ID NO.1, which shows that there were only 2 strains with homology of more than 95%, i.e. Mucor lusitanicus CBS 108.17 (96.56% and Mucor phayaoensis MFLUCC-0044 (95.50%), BLAST alignment at NCBI (National Center for Biotechnology Information, https:// www.ncbi.nlm.nih.gov), so that depending on the ITS sequence alignment, it could be determined that the HY6-19 strain is a Mucor strain, and that HY6-19 could not be identified as a species, and therefore, classification of HY6-19 belongs to the fungus kingdom (Fungi) of the genus (NCBI, http:// www.ncbi.nlm.nih.gov), the phylum (Mucor mycetas), the subgenera (Pezizomycetin), mao Meigang (Mucor mycetes), the order (Mucorales), the family of Mucoraceae (Mucor), and one species of the genus (Mucor).
The rDNA-ITS nucleotide sequence of the strain HY6-19 is as follows:
TTATCTATTTACTGTGAAACGTATTATTACTTGACGCCTGAGGGATGTTCCATTGCTATAAGGATAGGCAGCGGAAATGTTAACCGAGTCATAATCAAGCTTAGGCTTGGTATCCTATTATTATTTACCAAAAGAATTCAGAATTAATATTGTAACATAGACGTAAAAAATCTATAAAACAACTTTTAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGTAGCAAAGTGCGATAACTAGTGTGAATTGCATATTCAGTGAATCATCGAGTCTTTGAACGCAACTTGCGCTCATTGGTATTCCAATGAGCACGCCTGTTTCAGTATCAAAACAAACCCTCTATCCAACTTTTGTTGAATAGGATGACTGAGAGTCTCTTGATCGTCAGATCTCGAACCTCTTGAAATGTACAAAGGCCTGATCTTGTTTGATGGCTGAACTTTTTTTTAATATTAAAAAAAACTCCTGGGGGAAACTGGGGTGGGGCCCCCCCAATAACCCTTTTTTAAATTTGAACTGAAATCCAGGGGGAATACCCGGTGAACTT。
in summary, the microorganism strain HY6 is isolated from soil, and after ultraviolet mutagenesis, the strain HY6-19, namely Mucor sp.) HY6-19, for fermenting semen Hoveniae is obtained by screening, and is preserved in the Guangdong province microorganism strain collection, with the preservation number GDMCC No. 63391, the preservation date 2023, 4 months and 24 days, address: building 5 of No. 59 of Qinghui No. 100 college in Guangzhou City of Guangdong; postal code 510070.
Example 4: application of Mucor HY6-19 in preparation of semen Hoveniae extract 1
The application of the Mucor HY6-19 in the preparation of the semen Hoveniae extract can be operated according to the following steps:
(1) The Mucor HY6-19 spore powder preserved by freeze drying is inoculated on a fresh PDA flat-plate culture medium and cultured for 72 hours at a constant temperature of 30 ℃. Adding 10mL of sterile sucrose aqueous solution into a culture dish, suspending the spores with inoculating loop, transferring the spore solution into sterile test tube, and adjusting spore concentration to 8.15X10 with sterile sucrose aqueous solution 6 Obtaining mucor HY6-19 spore liquid per mL. The components and the preparation method of the PDA plate culture medium are the same as those of the example 1; the concentration of the sterile sucrose water solution is 15g/L, and the sterile sucrose water solution is sterilized for 15min at the temperature of 115 ℃ by high-pressure steam;
(2) 10g of semen hoveniae powder is put into a 250-mL triangular flask which is sterilized by dry heat at 160 ℃ for 2 hours, 30mL of mucor HY6-19 spore liquid prepared in the step (1) is added (the volume dosage is 3mL/g based on the mass of the semen hoveniae powder), and the mixture is stirred uniformly. Tying the triangular flask with 8 layers of gauze, and culturing at 30deg.C for 60 hr to obtain semen Hoveniae fermented product;
(3) Transferring all the raisin tree seed fermented products in the step (2) into a 500-mL beaker, adding 200mL of deionized water (the feed liquid ratio is 1:20 (g: mL)), stirring uniformly, and then preserving the heat for 6h in a water bath at 35 ℃. Then, transferring the beaker into an ultrasonic cleaner with water temperature of 75 ℃, performing 160W ultrasonic extraction for 80min, filtering with 200-mesh filter cloth while the beaker is hot, and collecting all filtrate;
(4) Concentrating the filtrate prepared in the step (3) to 10mL (1/20 of the original filtrate volume) under reduced pressure at 60 ℃ and minus 0.1MPa to obtain the semen Hoveniae water extraction concentrated solution. All the water extract concentrate is dried to constant weight in a clean culture dish at 65 ℃ and minus 0.1MPa, and crushed to obtain the hovenia dulcis thunb extract.
According to the above steps, 1.51g of extract is extracted from 10g of semen Hoveniae, the yield is 15.1%, and the total flavone content is 13.6%.
Comparative example 1: preparation of semen Hoveniae extract by conventional hot water ultrasonic method (compare with example 4)
(1) 10g of semen hoveniae powder is placed in a 500-mL beaker, 200mL of deionized water (the ratio of the solution to the solution is 1:20 (g: mL)) is added, and after being stirred uniformly, the mixture is kept in a water bath at 35 ℃ for 6h. Then, transferring the beaker into an ultrasonic cleaner with water temperature of 75 ℃, performing 160W ultrasonic extraction for 80min, filtering with 200-mesh filter cloth while the beaker is hot, and collecting filtrate;
(2) Concentrating the filtrate prepared in the step (1) to 10mL (1/20 of the original filtrate volume) under reduced pressure at 60 ℃ and minus 0.1MPa to obtain the semen Hoveniae water extraction concentrated solution. All the water extract concentrate is dried to constant weight in a clean culture dish at 65 ℃ and minus 0.1MPa, and crushed to obtain the hovenia dulcis thunb extract.
According to the above steps, 1.16g extract is obtained from 10g of semen Hoveniae, the yield is 11.6%, and the total flavone content is 11.2%.
The results of comparative example 4 and comparative example 1 can be seen: before the hot water ultrasonic extraction of the semen hoveniae, the fermentation treatment of Mucor HY6-19 is added, the preparation yield of the extract is improved from 11.6% of unfermented extract to 15.1%, the total flavone content in the extract is improved from 11.2% to 13.6%, and the preparation yield is improved by 21.4%.
Example 5: application of Mucor HY6-19 in preparation of semen Hoveniae extract 2
The application of the Mucor HY6-19 in the preparation of the semen Hoveniae extract can be operated according to the following steps:
(1) PDA plate spores of Mucor HY6-19 stored at 4deg.C are inoculated on fresh PDA plate culture medium, incubated at 30deg.C for 64h, 10mL of sterile sucrose water solution is added into a culture dish, the spores are suspended by stirring with an inoculating loop, the spore solution is transferred into a sterile test tube, and the spore concentration is regulated to 7.75X10 by using the sterile sucrose water solution 6 Obtaining mucor HY6-19 spore liquid per mL. The components and the preparation method of the PDA plate culture medium are the same as those of the example 1; the concentration of the sterile sucrose water solution is 12.5g/L, and the sterile sucrose water solution is sterilized for 15min at the temperature of 115 ℃ by high-pressure steam;
(2) 10g of semen hoveniae powder is put into a 250-mL triangular flask which is sterilized by dry heat at 160 ℃ for 2 hours, 25mL of mucor HY6-19 spore liquid prepared in the step (1) is added (the volume dosage is 2.5mL/g based on the mass of the semen hoveniae powder), and the mixture is stirred uniformly. Tying the triangular flask with 8 layers of gauze, and culturing at 32deg.C for 56 hr to obtain semen Hoveniae fermented product;
(3) Transferring all the raisin tree seed fermented products in the step (2) into a 500-mL beaker, adding 225mL of deionized water (the ratio of the feed to the liquid is 1:22.5 (g: mL)), uniformly stirring, and then preserving the heat for 5h in a water bath at 37.5 ℃. Then, transferring the beaker into an ultrasonic cleaner with water temperature of 80 ℃, performing ultrasonic extraction for 70min at 180W, filtering with 200-mesh filter cloth while the beaker is hot, and collecting all filtrate;
(4) Concentrating the filtrate prepared in the step (3) to 10mL (1/22.5 of the original filtrate volume) under reduced pressure at 60 ℃ and minus 0.1MPa to obtain the semen Hoveniae water extraction concentrated solution. All the water extract concentrate is dried to constant weight in a clean culture dish at 65 ℃ and minus 0.1MPa, and crushed to obtain the hovenia dulcis thunb extract.
According to the above steps, 1.64g of extract is extracted from 10g of semen Hoveniae, the yield is 16.4%, and the total flavone content is 14.3%.
Comparative example 2: preparation of semen Hoveniae extract by conventional hot water ultrasonic method (compare with example 5)
(1) 10g of hovenia dulcis thunb powder is placed in a 500-mL beaker, 225mL of deionized water (feed liquid ratio: 1:22.5 (g: mL)) is added, and after uniform stirring, the mixture is incubated in a water bath at 37 ℃ for 5h. Then, transferring the beaker into an ultrasonic cleaner with water temperature of 80 ℃, performing ultrasonic extraction for 70min at 180W, filtering with 200-mesh filter cloth while the beaker is hot, and collecting all filtrate;
(2) Concentrating the filtrate prepared in the step (1) to 10mL (1/22.5 of the original filtrate volume) under reduced pressure at 60 ℃ and minus 0.1MPa to obtain the semen Hoveniae water extraction concentrated solution. All the water extract concentrate is dried to constant weight in a clean culture dish at 65 ℃ and minus 0.1MPa, and crushed to obtain the hovenia dulcis thunb extract.
According to the above steps, 1.22g extract is obtained from 10g of semen Hoveniae, the yield is 12.2%, and the total flavone content is 11.6%.
The results of comparative example 5 and comparative example 2 can be seen: before the hot water ultrasonic extraction of the semen hoveniae, the fermentation treatment of Mucor HY6-19 is added, the preparation yield of the extract is improved from 12.2% of unfermented extract to 16.4%, the total flavone content in the extract is improved from 11.6% to 14.3%, and the preparation yield is improved by 23.3%.
Example 6: application of Mucor HY6-19 in preparation of semen Hoveniae extract 3
The application of the Mucor HY6-19 in the preparation of the semen Hoveniae extract can be operated according to the following steps:
(1) PDA plate spores of Mucor HY6-19 stored at 4deg.C are inoculated on fresh PDA plate culture medium, incubated at 30deg.C for 48h, 10mL of sterile sucrose water solution is added into a culture dish, the spores are suspended by stirring with an inoculating loop, the spore solution is transferred into a sterile test tube, and the spore concentration is adjusted to 5.80×10 by using the sterile sucrose water solution 6 Obtaining mucor HY6-19 spore liquid per mL. The components and the preparation method of the PDA plate culture medium are the same as those of the example 1; the concentration of the sterile sucrose water solution is 10g/L, and the sterile sucrose water solution is sterilized for 15min at the temperature of 115 ℃ by high-pressure steam;
(2) 10g of semen hoveniae powder is put into a 250-mL triangular flask which is sterilized by dry heat at 160 ℃ for 2 hours, 20mL of mucor HY6-19 spore liquid prepared in the step (1) is added (the volume dosage is 2mL/g based on the mass of the semen hoveniae powder), and the mixture is stirred uniformly. Tying the triangular flask with 8 layers of gauze, and culturing at 32deg.C for 52 hr to obtain semen Hoveniae fermented product;
(3) Transferring all the raisin tree seed fermented products in the step (2) into a 500-mL beaker, adding 250mL of deionized water (the feed liquid ratio is 1:25 (g: mL)), uniformly stirring, and then preserving the heat for 4h in a water bath at 40 ℃. Then, transferring the beaker into an ultrasonic cleaner with water temperature of 85 ℃, performing ultrasonic extraction for 60 minutes at 200W, filtering with 200-mesh filter cloth while the beaker is hot, and collecting all filtrate;
(4) Concentrating the filtrate prepared in the step (3) to 10mL (1/25 of the original filtrate volume) under reduced pressure at 60 ℃ and minus 0.1MPa to obtain the semen Hoveniae water extraction concentrated solution. All the water extract concentrate is dried to constant weight in a clean culture dish at 65 ℃ and minus 0.1MPa, and crushed to obtain the hovenia dulcis thunb extract.
According to the steps, 1.69g of extract is extracted from 10g of hovenia dulcis thunb, the yield is 16.9%, and the total flavone content is 14.5%.
Comparative example 3: preparation of semen Hoveniae extract by conventional hot water ultrasonic method (compare with example 6)
(1) 10g of semen hoveniae powder is placed in a 500-mL beaker, 250mL of deionized water (the ratio of the solution to the solution is 1:25 (g: mL)) is added, and after being stirred uniformly, the mixture is kept in a water bath at 40 ℃ for 4h. Then, transferring the beaker into an ultrasonic cleaner with water temperature of 85 ℃, performing ultrasonic extraction for 60 minutes at 200W, filtering with 200-mesh filter cloth while the beaker is hot, and collecting all filtrate;
(2) Concentrating the filtrate prepared in the step (1) to 10mL (1/25 of the original filtrate volume) under reduced pressure at 60 ℃ and minus 0.1MPa to obtain the semen Hoveniae water extraction concentrated solution. All the water extract concentrate is dried to constant weight in a clean culture dish at 65 ℃ and minus 0.1MPa, and crushed to obtain the hovenia dulcis thunb extract.
According to the above steps, 1.27g extract is obtained from 10g of semen Hoveniae, the yield is 12.7%, and the total flavone content is 12.1%.
The results of comparative example 6 and comparative example 3 can be seen: before the hot water ultrasonic extraction of the semen hoveniae, the fermentation treatment of Mucor HY6-19 is added, the preparation yield of the extract is improved from 12.7% of unfermented extract to 16.9%, the total flavone content in the extract is improved from 12.1% to 14.5%, and the preparation yield is improved by 19.8%.
Claims (10)
1. Mucor sp.) HY6-19 deposited at the collection of microorganism strains, guangdong province under accession number GDMCCNo 63391, accession date 2023, 4 months 24, address: building 5 of No. 59 of Qinghui No. 100 college in Guangzhou City of Guangdong; postal code 510070.
2. Use of a mucor HY6-19 according to claim 1 for the preparation of a hovenia dulcis thunb extract.
3. The application according to claim 2, wherein the method of application is: (1) Inoculating spore solution of Mucor HY6-19 into semen Hoveniae powder, stirring, and fermenting at 30-32deg.C for 52-60 hr to obtain semen Hoveniae fermented product; (2) Adding deionized water into semen Hoveniae fermented product, stirring, maintaining at 35-40deg.C for 4-6 hr, ultrasonic extracting with hot water, filtering, and concentrating the filtrate to obtain semen Hoveniae water concentrated solution; (3) Vacuum drying the water extract concentrate of semen Hoveniae, and pulverizing to obtain semen Hoveniae extract.
4. The use according to claim 3, wherein the hovenia dulcis thunb powder in the step (1) is fine powder obtained by pulverizing hovenia dulcis thunb after drying at 85 ℃ and sieving with a 60-mesh sieve.
5. The use according to claim 3, wherein the preparation method of the mucor HY6-19 spore liquid in the step (1) comprises the following steps: inoculating the spores of the mucor HY6-19 preserved at low temperature on a PDA flat-plate culture medium, culturing at constant temperature of 30 ℃ for 48-72 hours, adding sterile sucrose aqueous solution into the culture, and stirring by an inoculating loop to suspend the spores to obtain spore liquid.
6. The use according to claim 5, wherein the concentration of sucrose in said sterile aqueous sucrose solution is 10-15g/L and the concentration of spore solution is 5X 10 6 –9×10 6 And each mL.
7. The use according to claim 3, wherein the spore liquid volume amount of mucor HY6-19 in the step (1) is 2-3mL/g based on the mass of hovenia dulcis thunb powder.
8. The use according to claim 3, wherein the preparation method of the hovenia dulcis thunb aqueous extract concentrate of step (2) comprises the steps of: adding deionized water into semen Hoveniae fermented product, stirring, maintaining at 35-40deg.C for 4-6 hr, transferring into ultrasonic cleaner with water temperature of 75-85deg.C, ultrasonic extracting at 160-200W for 60-80min, filtering with 200 mesh filter cloth, and concentrating the filtrate under reduced pressure at 60 deg.C and-0.1 MPa to 1/10-1/25 of original volume to obtain semen Hoveniae water extract concentrate.
9. The use according to claim 3 or 8, characterized in that the deionized water is added in a volume amount of 20-25mL/g by mass of hovenia dulcis thunb powder before fermentation.
10. The use according to claim 3, wherein the preparation method of the hovenia dulcis thunb extract in the step (3) is as follows: drying semen Hoveniae water extract concentrate at 65deg.C under-0.1 MPa under vacuum to constant weight, and pulverizing to obtain semen Hoveniae extract.
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