CN105543108A - Penicillium purpurescens QL-9204 and application for preparing phloretin during phlorizin conversion - Google Patents

Penicillium purpurescens QL-9204 and application for preparing phloretin during phlorizin conversion Download PDF

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CN105543108A
CN105543108A CN201610051127.5A CN201610051127A CN105543108A CN 105543108 A CN105543108 A CN 105543108A CN 201610051127 A CN201610051127 A CN 201610051127A CN 105543108 A CN105543108 A CN 105543108A
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phloretin
phlorizin
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CN105543108B (en
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梅建凤
李靓
金航
应国清
王鸿
易喻
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses penicillium purpurescens QL-9204 and application for preparing phloretin during phlorizin conversion. The penicillium purpurescens QL-9204 is a strain free of toxicity and harm and safe to use. The penicillium purpurescens QL-9204 grows rapidly, and is high in hybrid bacteria resistance, easy to culture and stable in batch; a fermentation medium is simple in components and low in market price, thereby being low in production cost; a substrate plorizin is directly put into a crude enzyme for conversion, the enzyme separation and purification steps are omitted, the technology is simple, and the penicillium purpurescens QL-9204 is easy to apply industrially; the phloretin conversion yield is high and can reach 92.5% to the maximum, and the penicillium purpurescens QL-9204 has the advantages that few by-products are produced, and the product is easy to extract.

Description

Purple stain mould QL-9204 and the application prepared at conversion phlorizin in Phloretin
(1) technical field
The present invention relates to a kind of preparation method of Phloretin, particularly strain purple stain mould (Penicilliumpurpurogenum) QL-9204, and this bacterial strain prepares the application in Phloretin field at conversion phlorizin.
(2) background technology
Phloretin (phloretin), have another name called three hydroxyl phenol-acetones, CAS 60-82-2, belongs to dihydrochalcone compound.Chemical structure is 3-(4-hydroxy phenyl)-1-(2,4,6-trihydroxy-phenyl)-1-acetone, and molecular formula is C 15h 14o 5, molecular weight is 274.27, and sterling is pink, is soluble in methyl alcohol, ethanol and acetone, water-soluble hardly, dissolves in basic solution.Phloretin has well anti-oxidant, anti-radical action, and energy check melanin cytoactive, has good desalination effect to color spot; Moreover, Phloretin also has good anti-inflammatory and immunosuppressive action; Current research shows that Phloretin also has antitumor and antitumour activity.
Mostly the form that Phloretin exists naturally is to exist with glycoside forms, i.e. phlorizin (phlorizin, CAS 60-81-1).Phlorizin is extensively present in the fruit such as apple, lichee, mainly concentrates in rhizome in these plants or root skin.The method of producing Phloretin can adopt organic chemistry procedures to synthesize, and also can be obtained through acid and alkali hydrolysis or enzymolysis process by natural phlorizin.At present, having patent report to adopt the method for acid and alkali hydrolysis or enzymolysis to transform phlorizin is Phloretin, but the application of these technology has certain restriction, as adopted acid and alkali hydrolysis method, although have the advantage that cost is low, there is the deficiencies such as soda acid is residual, conversion specificity is poor and product need decolour; Adopt enzymolysis rule to need commodity lytic enzyme, and enzyme can not recycle and reuse, and causes process costs too high.
Microorganism has the ability of very powerful enzyme system Sum decomposition transformation substance, the strong microorganism of specificity is transformed if can filter out, the corresponding Glycosylase that fermentation produces, with the crude enzyme liquid of filtering thalline as transformation system, Phloretin is converted under the effect of phlorizin Glycosylase in crude enzyme liquid, the deficiency of acid and alkali hydrolysis and enzymolysis process can be overcome, therefore there is reaction conditions gentleness, specificity is strong, production cost is low and advantages of environment protection.
(3) summary of the invention
The object of the invention is to provide microorganism strains-purple stain mould (Penicilliumpurpurogenum) QL-9204 that a strain produces Glycosylase, and prepare application in Phloretin transforming phlorizin, overcome specificity difference and the low problem of conversion yield in existing acid and alkali hydrolysis method and enzymolysis process preparation preferably, this technique has that cost is low, flow process is simple, conversion yield is high and conversion byproducts such as to lack at the advantage.
The technical solution used in the present invention is:
The invention provides a strain new strains-purple stain mould (Penicilliumpurpurogenum) QL-9204, be preserved in China typical culture collection center, deposit number: CCTCCNo:M2015781, preservation date on December 25th, 2015, address: China, Wuhan, Wuhan University; Postcode: 430072.
Purple stain mould QL-9204 of the present invention is from taking Pericarpium Mali pumilae as the strain excellent that microbe-derived enrichment culture thing, Isolation and screening obtains.The colony characteristics of described purple stain mould QL-9204 is as follows: be seeded on potato culture (PDA) agar plate, cultivate after 2 days for 28 DEG C, planar surface grows the mycelium of white flock, aerial hyphae length is at 1 ~ 2mm, colony edge indentation is irregular, continue cultivation after 3 days, bacterium colony front becomes greyish-green gradually, and the back side gradually becomes red-purple.Basis of microscopic observation thallospore silk is broom shape, 1 ~ 2 layer of stigma, and conidium string is the chain of not branch, oval, faint yellow, surface irregularity.
The partial nucleotide sequence of the 18srDNA of described purple stain mould QL-9204 is as shown in SEQIDNO:1.
SEQIDNO:1：
CACTCTTTTACTGTGAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCCTACTACATGGATACCTGTGGTAATTCTAGAGCTAATACATGCGCAAAACCCCGACTTCGGAAGGGGTGTATTTATTAGATAAAAAACCAATGCCCTTCGGGGCTCCTTGGTGATTCATAATAACTTCACGAATCGCATGGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAGTGGCCTACCATGGTGGCAACGGGTAACGGGGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGATACGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGTAATTGGAATGAGAACAATCTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAACCTTGGGCCCGTCCTGCCGGTCCGCCTCACCGCGAGTACTGGTCCGGATGGGCCTTTCTTTCTGGGGAATCCCATGGCCTTCACTGGCTGTGGCGGGGAACCAGGACTTTTACTGTGAAAAAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGGATACATTAGCATGGAATAATAGAATAGGACGTGCGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGATAGTCGGGGGCGTCAGTATTCAGCTGTCAGAGGTGAAATTCTTGGATTTGCTGAAGACTAACTACTGCGAAAGCATTCGCCAAGGATGTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGGCGGGGTTTCTATGATGACCCGCTCGGCACCTTACGAGAAATCAAAGTTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACAAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAAAATAAGGATTGACAGATTGAGAGCTCTTTCTTGATCTTTTGGATGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCGTTTGCGGGCCGCTGGCTTCTTAGGGGAC。
The present invention also provides a kind of described purple stain mould QL-9204 to prepare application in Phloretin at bio-transformation phlorizin, concrete described application produces the filtrate of the fermentation liquor suction filtration acquisition after enzyme cultivation for biological catalyst with purple stain mould QL-9204, take phlorizin as substrate, be that solubility promoter forms transformation system and (preferentially selects dissolve with methanol phlorizin with methyl alcohol, and then mix with biological catalyst), 28 ~ 30 DEG C, carry out conversion reaction under 200 ~ 250r/min condition, after conversion reaction terminates, by conversion fluid separation and purification, obtain Phloretin.
Further, the final concentration of described substrate phlorizin is 20 ~ 80mg/L transformation system, the volume final concentration of described solubility promoter methyl alcohol is 2 ~ 8ml/L transformation system, and described catalyst levels counts 2.5 ~ 6.0g/L transformation system (preferably 2.5 ~ 5.0g/L) with dry mycelium weight in suction filtration primary fermentation liquid.
Described purple stain mould QL-9204 bacterial strain is before product enzyme is cultivated, usual needs are first through slant medium activation culture, or again through seed culture medium enlarged culturing, then carry out product enzyme with spore or seed liquor access fermention medium to cultivate, described purple stain mould QL-9204 produces enzyme cultural method: (1) activation culture: by purple stain mould QL-9204 bacterial classification spore inoculating in slant medium, in 28 ~ 30 DEG C of constant temperature culture 2 ~ 3 days, obtain slant pore, described slant medium is potato dextrose agar (PDA); PDA substratum consists of: (potato cleans peeling to potato 100 ~ 200g/L, be cut into small pieces, add 5 times of quality water boil 20 ~ 30min, 4 layers of filtered through gauze remove slag and stay juice), sucrose 10 ~ 20g/L, agar 15 ~ 20g/L, solvent is water, pH nature, high pressure steam 121 DEG C of sterilizing 15min; (2) seed enlarged culturing: after picking step (1) activation culture, purple stain mould QL-9204 slant pore is seeded in seed culture medium, in 28 ~ 30 DEG C, cultivate 2 ~ 3 days under 200 ~ 250r/min constant temperature oscillation condition, obtain seed liquor, described seed culture medium is potato dextrose medium (PDB), PDB nutrient solution is not except containing agar, and other compositions and compound method are with PDA substratum; (3) produce enzyme to cultivate: seed liquor is seeded in fermention medium with the inoculum size of volumetric concentration 5 ~ 8%, in 28 DEG C, cultivate 4 ~ 5 days under 200r/min constant temperature oscillation condition, obtain fermented liquid; Described fermention medium consists of: sucrose 2 ~ 5g/L, (NH 4) 2sO 44 ~ 6g/L, NaCl5g/L, KH 2pO 45g/L, MgSO 41g/L, MnSO 41g/L, solvent is water, pH6 ~ 7.
Further, preferred described fermention medium consists of: sucrose 2g/L, (NH 4) 2sO 46g/L, NaCl5g/L, KH 2pO 45g/L, MgSO 41g/L, MnSO 41g/L, solvent is water, and pH is 6.
Further, the condition of preferred described conversion reaction is: 28 ~ 30 DEG C, transform 8 ~ 15h under 200 ~ 250r/min constant temperature oscillation condition.
The method of separation and purification of the present invention is: after bioconversion reaction terminates, the extraction into ethyl acetate of transformation system equating volume 3 times, combining extraction liquid, in round-bottomed flask, at 45 DEG C after evaporated under reduced pressure ethyl acetate, then adds the dissolve with methanol residue of equivalent former transformation system volume.After methanol solution filter paper filtering, proceed in another clean round-bottomed flask, at 45 DEG C after evaporated under reduced pressure methyl alcohol, add a small amount of dissolve with methanol residue, methanol solution proceeds in clean beaker, namely obtains pale yellow powder Phloretin after drying under reduced pressure.
Beneficial effect of the present invention is mainly reflected in: (1) the invention provides a strain new strains-purple stain mould QL-9204, and this bacterium is that a strain is nontoxic, the bacterial classification of use safety; (2) purple stain mould QL-9204 grows fast, anti-miscellaneous bacteria ability and cultivates by force, easily, batch stable; (3) fermentation medium components is simple, and market value is low, thus production cost is lower; (4) substrate phlorizin is directly put in crude enzyme liquid and is transformed by the present invention, eliminates the purification procedures of enzyme, and technique is simple, is easy to industrial applications; (5) conversion yield of Phloretin is high, reaches as high as 92.5%, and it is few to have by product, and product such as easily to extract at the advantage.
(4) accompanying drawing explanation
Fig. 1 phlorizin is converted into the chemical equation of Phloretin;
The typical curve that Fig. 2 phlorizin and Phloretin HPLC mass concentration are analyzed, A is phlorizin typical curve, and B is Phloretin typical curve;
Fig. 3 HPLC is to the analysis collection of illustrative plates transforming sample.Curve A is the HPLC collection of illustrative plates of standard substance phlorizin and Phloretin; Curve B is the control sample HPLC collection of illustrative plates that phlorizin transforms 0h; Curve C is the HPLC collection of illustrative plates of phlorizin after bio-transformation 15h (embodiment 5 sample).
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: transform bacterial strain enrichment be separated
The fresh apple skin of about 50g through pulverizing is added, in 28 DEG C of constant temperature culture 5 days in 250mL triangular flask.The enriched substance sterilized water dilution 1 × 10 of mould will be covered with 6coat on PDA plate culture medium doubly, in 28 DEG C of constant temperature culture 3 days, the mold colony switching PDA slant medium that picking form is different with color, be placed in 28 DEG C of constant temperature culture 3 days, obtain inclined-plane bacterial strain 11 strain that spore is abundant, numbered respectively (QL-9201 ~ QL-9211), be preserved in 4 DEG C of refrigerators for subsequent use.
Described plate culture medium and slant medium are potato dextrose agar (PDA), by following composition and method preparation: potato is cleaned peeling and is cut into small pieces, take 200g, add tap water 1000mL, boil 30min, 4 layers of filtered through gauze remove slag, filtrate supplies 1000mL, add sucrose 20g, agar 18g again, pH nature (actual measurement 6.5), be heated to agar dissolve rear packing test tube or in triangular flask, shelve inclined-plane or pour sterile petri dish into after high pressure steam 121 DEG C of sterilizing 15min.
Embodiment 2: the screening and the taxonomic identification that transform bacterial strain
Spore 2 ring of each bacterial strain slant strains obtained by transfering loop difference picking embodiment 1, in access 100mL fermention medium (250mL triangle is bottled), in 28 DEG C, produce under 200r/min constant temperature oscillation condition after enzyme cultivates 5 days, use Büchner funnel suction filtration, the filtrate of collection is crude enzyme liquid.Get 50mL crude enzyme liquid and be placed in 250mL triangular flask, 1mg phlorizin 0.1mL dissolve with methanol, then join in crude enzyme liquid and form reaction system (cumulative volume is in 50mL), in 28 DEG C, transform 8h under 200r/min constant temperature oscillation condition after; Get the extraction into ethyl acetate 2 times of conversion fluid 10mL equal volume, combined ethyl acetate is in round-bottomed flask, with 1mL dissolve with methanol residue after ethyl acetate evaporated under reduced pressure, use 0.45 μm of filtering with microporous membrane again, by the concentration of phlorizin and Phloretin in high performance liquid chromatography (HPLC) analytic sample.
HPLC method analyzes the concentration that the standby crude enzyme liquid of different strains fermentation transforms phlorizin and Phloretin in sample, and comparing different strains product enzymatic conversion phlorizin is thus the capacity of water of Phloretin.Through comparing, the crude enzyme liquid prepared by the strain fermentation of numbering QL-9204 transforms phlorizin, and mole conversion yield generating Phloretin is the highest, and reach 34.7%, in conversion fluid, concentration reaches 4.11mg/L.
Be seeded in by bacterial strain QL-9204 on PDA plate culture medium, cultivate after 2 days for 28 DEG C, planar surface grows the mycelium of white flock, aerial hyphae length is at 1 ~ 2mm, and colony edge indentation is irregular, continues cultivation after 3 days, bacterium colony front becomes greyish-green gradually, and the back side gradually becomes red-purple.Basis of microscopic observation thallospore silk is broom shape, 1 ~ 2 layer of stigma, and conidium string is the chain of not branch, oval, faint yellow, surface irregularity.
This bacterial strain transfers to Sangon Biotech (Shanghai) Co., Ltd. to carry out 18SrDNA order-checking, and recording sequence size is 1318bp (shown in SEQIDNO:1).This sequence is carried out BLAST comparison on GenBank, has the homology of more than 99% with the 18SrDNA sequence of purple stain penicillium bacterial strains more than 30 strains.The comprehensive morphological specificity of QL-9204 bacterial strain and the sequential analysis of 18SrDNA, can determine that QL-9204 bacterial strain is a strain purple stain mould (Penicilliumpurpurogenum), name as purple stain mould QL-9204, be preserved in China typical culture collection center, deposit number: CCTCCNo:M2015781, preservation date on December 25th, 2015.
Described PDA plate culture medium composition and preparation method are with embodiment 1.
Described fermention medium is by following composition and method preparation: sucrose 5g/L, peptone 5g/L, NaCl5g/L, KH 2pO 45g/L, MgSO 41g/L, MnSO 41g/L, solvent is water, the bottled 100mL fermention medium of triangle of pH7,250mL, 8 layers of gauze tying, high pressure steam 121 DEG C of sterilizing 15min.
Described HPLC analytical procedure is: LC-20AD high performance liquid chromatograph (Japanese Shimadzu Instrument Ltd.), and chromatographic column is PhenomenexLunaC18 bonded silica gel post (5 μm, 250mm × 4.6mm), column temperature 25 DEG C; Moving phase is methyl alcohol and the water mixture of volume ratio 7:3; Flow velocity 0.8mL/min, determined wavelength 280nm, sample size 10 μ L.By the standard substance phlorizin mass concentration under same analysis condition-peak area typical curve (in Fig. 2 A), calculate the mass concentration of the phlorizin in sample, by standard substance Phloretin mass concentration-peak area typical curve (in Fig. 2 B), calculate the mass concentration of the Phloretin in sample.
Embodiment 3: purple stain mould QL-9204 bacterial strain transforms the application 1 that phlorizin generates Phloretin
Screening the purple stain mould QL-9204 obtained with embodiment 2 is conversion bacterial classification, through seed enlarged culturing, the crude enzyme liquid conversion processing phlorizin that fermentation is standby, mole conversion yield of Phloretin comparatively embodiment 2 slightly improves, repeat the result there was no significant difference of experiment 3 batches, show that this bacterial classification transforms the stable performance that phlorizin produces Phloretin, concrete technology step is as follows:
(1) the purple stain mould QL-9204 slant strains of preservation in 4 DEG C of refrigerators is inoculated in fresh PDA slant medium, inclined-plane was in 28 DEG C of constant temperature culture 3 days.Described PDA slant medium composition and preparation method are with embodiment 1;
(2) with purple stain mould QL-9204 spore 2 ring after transfering loop picking step (1) activation culture in seed culture medium, in 28 DEG C, cultivate 3 days under 200r/min constant temperature oscillation condition, obtain the seed liquor that dry mycelium concentration is 5.12g/L.Described seed culture medium is PDB substratum, and except not containing agar, other compositions and compound method are with PDA slant medium;
(3) step (2) seed liquor is seeded in 100mL fermention medium with the inoculum size of volumetric concentration 5% (i.e. 5mL), in 28 DEG C, cultivate 5 days under 200r/min constant temperature oscillation condition, obtain the fermented liquid that dry mycelium concentration is 4.87g/L.Described fermention medium is composed as follows: sucrose 5g/L, peptone 5g/L, NaCl5g/L, KH 2pO 45g/L, MgSO 41g/L, MnSO 41g/L, solvent is water, the bottled 100mL fermention medium of triangle of pH7,250mL, 8 layers of gauze tying, high pressure steam 121 DEG C of sterilizing 15min.
(4) after product enzyme is cultivated and is terminated, step (3) is obtained fermented liquid 100mL Büchner funnel suction filtration, the filtrate 90mL collected is crude enzyme liquid, the 50mL crude enzyme liquid got wherein is placed in 250mL triangular flask, 1mg phlorizin 0.1mL dissolve with methanol, join again in crude enzyme liquid and form reaction system (cumulative volume is in 50mL), in 30 DEG C, after transforming 8h under 200r/min constant temperature oscillation condition, get the extraction into ethyl acetate 2 times of conversion fluid 10mL equal volume, combined ethyl acetate is in round-bottomed flask, after ethyl acetate evaporated under reduced pressure, add 1mL dissolve with methanol residue, use 0.45 μm of filtering with microporous membrane again, then analyze by the HPLC condition in case study on implementation 2.
HPLC analyzes and shows, by the present embodiment method, crude enzyme liquid conversion processing phlorizin prepared by the bacterial strain of purple stain mould QL-9204, transforms Phloretin concentration in sample and reaches 5.46mg/L, mole conversion yield reaches 46.1%, and comparatively in embodiment 2, conversion yield improves 11.4%.Embodiment 3 method for transformation repeats experiment 3 batches, 3 batches of experimental result there was no significant differences, and showing that crude enzyme liquid prepared by purple stain mould QL-9204 bacterial strain transforms phlorizin is that the conversion performance of Phloretin is stablized.
Embodiment 4: purple stain mould QL-9204 bacterial strain transforms the application 2 that phlorizin generates Phloretin
With purple stain mould QL-9204 for zymogenic bacteria kind, after producing enzymic fermentation substratum composition and initial pH, concentration of substrate and transformation time optimization, the crude enzyme liquid conversion processing phlorizin that fermentation is standby, mole conversion yield of Phloretin comparatively embodiment 3 is significantly increased, and concrete technology step is as follows:
(1) the purple stain mould QL-9204 slant strains of preservation in 4 DEG C of refrigerators is inoculated in fresh slant medium, inclined-plane was in 30 DEG C of constant temperature culture 3 days.Described slant medium composition and preparation method are with embodiment 1.
(2) with purple stain mould QL-9204 spore 2 ring after transfering loop picking step (1) activation culture in seed culture medium, in 30 DEG C, cultivate 2 days under 250r/min constant temperature oscillation condition, obtain the seed liquor that dry mycelium concentration is 5.74g/L.Described seed culture medium is PDB substratum, and its composition and preparation method are with embodiment 3.
(3) step (2) seed liquor is seeded to 100mL fermention medium with volumetric concentration 8% (i.e. 8mL) inoculum size, in 30 DEG C, cultivate 5 days under 200r/min constant temperature oscillation condition, obtain the fermented liquid that dry mycelium concentration is 2.53g/L.Described fermention medium is composed as follows: sucrose 2g/L, (NH 4) 2sO 46g/L, NaCl5g/L, KH 2pO 45g/L, MgSO 41g/L, MnSO 41g/L, solvent is water, and pH is the bottled 100mL fermention medium of triangle of 6,250mL, 8 layers of gauze tying, high pressure steam 121 DEG C of sterilizing 15min.
(4) after product enzyme is cultivated and is terminated, the fermented liquid 100mL Büchner funnel suction filtration that step (3) is obtained, the filtrate 90mL collected is crude enzyme liquid, the 50mL crude enzyme liquid got wherein is placed in 250mL triangular flask, 4mg phlorizin 0.4mL dissolve with methanol, join again in crude enzyme liquid and form reaction system (cumulative volume is in 50mL), in 30 DEG C, transform 15h under 200r/min constant temperature oscillation condition after; Get the extraction into ethyl acetate 2 times of conversion fluid 10mL equal volume, combined ethyl acetate, in round-bottomed flask, after ethyl acetate evaporated under reduced pressure, adds 1mL dissolve with methanol residue, use 0.45 μm of filtering with microporous membrane again, then analyze by the HPLC condition in case study on implementation 2.
HPLC analyzes and shows, by the present embodiment method, crude enzyme liquid conversion processing phlorizin prepared by the strain fermentation of purple stain mould QL-9204, in conversion fluid, Phloretin concentration is improved largely, and reaches 43.3mg/L, and a mole conversion yield reaches 91.4%.
Embodiment 5: purple stain mould QL-9204 bacterial strain transforms the application 3 that phlorizin generates Phloretin
On the basis of embodiment 3, cultivated by product enzyme and be amplified to 400mL scale, transformation system is amplified to 300mL scale, and concrete technology step is as follows:
(1) the purple stain mould QL-9204 slant strains of preservation in 4 DEG C of refrigerators is inoculated in fresh slant medium, inclined-plane was in 30 DEG C of constant temperature culture 3 days.Described slant medium composition and preparation method are with embodiment 1.
(2) with purple stain mould QL-9204 spore 2 ring after transfering loop picking step (1) activation culture in seed culture medium, in 30 DEG C, cultivate 2 days under 250r/min constant temperature oscillation condition, obtain the seed liquor that dry mycelium concentration is 5.92g/L.Described seed culture medium is PDB substratum, and its composition and preparation method are with embodiment 3.
(3) step (2) seed liquor is seeded to 400mL fermention medium with volumetric concentration 8% (i.e. 32mL) inoculum size, in 30 DEG C, cultivate 4 days under 250r/min constant temperature oscillation condition, obtain the fermented liquid that dry mycelium concentration is 2.68g/L.Described fermention medium is composed as follows: sucrose 2g/L, (NH 4) 2sO 46g/L, NaCl5g/L, KH 2pO 45g/L, MgSO 41g/L, MnSO 41g/L, solvent is water, and pH is the bottled 400mL fermention medium of triangle of 6,1000mL, 8 layers of gauze tying, high pressure steam 121 DEG C of sterilizing 20min.
(4) after product enzyme is cultivated and is terminated, the fermented liquid 400mL Büchner funnel suction filtration that step (3) is obtained, the filtrate 350mL collected is crude enzyme liquid, the 300mL crude enzyme liquid got wherein is placed in 250mL triangular flask, 24mg phlorizin 2.4mL dissolve with methanol, join again in crude enzyme liquid and form reaction system (cumulative volume is in 300mL), in 30 DEG C, transform 15h under 250r/min constant temperature oscillation condition after; Get the extraction into ethyl acetate 2 times of conversion fluid 10mL equal volume, combined ethyl acetate, in round-bottomed flask, after ethyl acetate evaporated under reduced pressure, adds 1mL dissolve with methanol residue, use 0.45 μm of filtering with microporous membrane again, then analyze by the HPLC condition in case study on implementation 2.
HPLC analyzes and shows, by the present embodiment method, crude enzyme liquid conversion processing phlorizin prepared by the strain fermentation of purple stain mould QL-9204, in conversion fluid, Phloretin concentration is 43.8mg/L, and mole conversion yield is 92.5%.

Claims (10)

1. purple stain mould (Penicilliumpurpurogenum) QL-9204, is preserved in China typical culture collection center, deposit number: CCTCCNo:M2015781, preservation date on December 25th, 2015, address: China, Wuhan, Wuhan University; Postcode: 430072.
2. described in a claim 1, purple stain mould QL-9204 prepares the application in Phloretin at bio-transformation phlorizin.
3. apply as claimed in claim 2, it is characterized in that described application be with purple stain mould QL-9204 multiparity enzyme cultivate after fermented liquid suction filtration obtain filtrate for catalyzer, take phlorizin as substrate, be that solubility promoter forms transformation system with methyl alcohol, 28 ~ 30 DEG C, carry out conversion reaction under 200 ~ 250r/min condition, after conversion reaction terminates, by conversion fluid separation and purification, obtain Phloretin.
4. apply as claimed in claim 3, it is characterized in that the final concentration of described substrate phlorizin is 20 ~ 80mg/L transformation system.
5. apply as claimed in claim 3, it is characterized in that the volume final concentration of described solubility promoter methyl alcohol is 2 ~ 8ml/L transformation system.
6. apply as claimed in claim 3, it is characterized in that described catalyst levels counts 2.5 ~ 6.0g/L transformation system with dry mycelium weight in suction filtration primary fermentation liquid.
7. apply as claimed in claim 3, it is characterized in that described purple stain mould QL-9204 produces enzyme cultural method and is: (1) activation culture: by purple stain mould QL-9204 bacterial classification spore inoculating in slant medium, in 28 ~ 30 DEG C of constant temperature culture 2 ~ 3 days, obtain slant pore, described slant medium is potato dextrose agar; (2) seed enlarged culturing: after picking step (1) activation culture, purple stain mould QL-9204 slant pore is seeded in seed culture medium, in 28 ~ 30 DEG C, cultivate 2 ~ 3 days under 200 ~ 250r/min oscillating condition, obtain seed liquor, described seed culture medium is potato dextrose medium; (3) produce enzyme to cultivate: seed liquor is seeded in fermention medium with the inoculum size of volumetric concentration 5 ~ 8%, in 28 DEG C, cultivate 5 days under 200r/min constant temperature oscillation condition, obtain fermented liquid; Described fermention medium consists of: sucrose 2 ~ 5g/L, (NH 4) 2sO 44 ~ 6g/L, NaCl5g/L, KH 2pO 45g/L, MgSO 41g/L, MnSO 41g/L, solvent is water, pH6 ~ 7.
8. apply as claimed in claim 7, it is characterized in that described fermention medium consists of: sucrose 2g/L, (NH 4) 2sO 46g/L, NaCl5g/L, KH 2pO 45g/L, MgSO 41g/L, MnSO 41g/L, solvent is water, and pH is 6.
9. apply as claimed in claim 3, it is characterized in that the method for described separation and purification is: after bioconversion reaction terminates, transformation system isopyknic extraction into ethyl acetate 3 times, combining extraction liquid is in round-bottomed flask, at 45 DEG C after evaporated under reduced pressure ethyl acetate, then add the dissolve with methanol residue of equivalent former transformation system volume; After methanol solution filter paper filtering, proceed in another clean round-bottomed flask, at 45 DEG C after evaporated under reduced pressure methyl alcohol, add a small amount of dissolve with methanol residue, methanol solution proceeds in clean beaker, namely obtains Phloretin after drying under reduced pressure.
10. apply as claimed in claim 3, it is characterized in that the condition of described conversion reaction is: 28 ~ 30 DEG C, transform 8 ~ 15h under 200 ~ 250r/min constant temperature oscillation condition.
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CN108384814B (en) * 2018-03-02 2022-05-17 重庆大学 Preparation method of phloretin
CN109136320A (en) * 2018-09-19 2019-01-04 湖南中茂生物科技有限公司 The method that a kind of enzyme bacteria complex system conversion phloridzin prepares phloretin
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