CN103642851A - Method for preparing ferulic acid from wheat bran by using two-enzyme method - Google Patents
Method for preparing ferulic acid from wheat bran by using two-enzyme method Download PDFInfo
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- CN103642851A CN103642851A CN201310666358.3A CN201310666358A CN103642851A CN 103642851 A CN103642851 A CN 103642851A CN 201310666358 A CN201310666358 A CN 201310666358A CN 103642851 A CN103642851 A CN 103642851A
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Abstract
The invention provides a method for preparing ferulic acid from wheat bran by using a two-enzyme method. Wheat bran is subjected to enzymolysis by utilizing the synergistic effect of reAorFaeA and reAoXynllA from a laboratory, so as to release ferulic acid and obtain a crude ferulic acid extract. The crude ferulic acid extract is purified by using an HPD-300 weal-polarity macroporous resin, and the fraction with the ferulic acid obtained from purification is dried in vacuum so as to obtain a ferulic acid product. By adopting the method, raw materials for extracting and preparing the ferulic acid are cheap and easily available, in the product purity is high, the preparation process is simple, and the method is simple, convenient and feasible in operation, environment-friendly and applicable to industrial expansion production.
Description
Technical field
The present invention relates to a kind of method of utilizing biological enzyme to prepare forulic acid from Testa Tritici, belong to technical field of bioengineering.
Background technology
China is agriculture production big country, and the agricultural byproducts cell wall substance output such as annual wheat bran, corn cob, rice bran are about 3,000 ten thousand tons, are dropped more or as animal-feed, make a low multiple use, and economic worth are not high.The chemical name of forulic acid (ferulic acid) is Ferulic acid, it is one of derivative of TRANSCINNAMIC ACID, in the cereal such as wheat bran, rice bran, bagasse, content is higher, it is a kind of natural antioxidant, has anticoagulant, promotes thrombocytolysis, antithrombotic formation, anti-oxidant, the physiological action such as antitumor, comes into one's own in industries such as medicine and food.From Testa Tritici, extract preparation forulic acid and both can extend Wheat indurstry chain, improve the added value of wheat, can obtain again the free forulic acid of pharmaceutical use and nourishing function simultaneously, its economic implications, social effect are huge.
The production method of forulic acid mainly comprises chemosynthesis and alkaline hydrolysis at present, but these two kinds of methods all exist certain defect.Chemical synthesis reactions steps is many, the cycle is long; Raw material thiaminogen content in rice bran of alkaline hydrolysis is low, and output is limited; In addition, these two kinds of methods all have contaminative, so Production by Enzymes forulic acid has greater advantage.Feruloyl esterase is as a subclass of carboxylic ester hydrolase, can hydrolyzing plant cell walls in the ester bond that is connected with forulic acid of polysaccharide, and discharge free forulic acid monomer or dimer.Single use feruloyl esterase, enzyme can not fully react with Mierocrystalline cellulose ester bond, causes percent hydrolysis low.If adopt feruloyl esterase and polysaccharide hydrolase acting in conjunction in cell walls, interrupt forulic acid ester bond and glycosidic link simultaneously, will the hydrolysis productive rate of oligose be improved undoubtedly, simultaneously again can production forulic acid.Yet enzymolysis solution is a complicated system, therefrom isolates forulic acid still more difficult.The purifying process of finding suitable forulic acid is also a bottleneck realizing the required breakthrough of forulic acid suitability for industrialized production.
Summary of the invention
The object of the invention is to be to provide a kind of and from Testa Tritici, extract preparation forulic acid, and Environmental Safety, the cycle is short, and technique simple to operate is the Production by Enzymes based theoretical of forulic acid.
Technical scheme of the present invention:
Utilize restructuring feruloyl esterase (reAorFaeA) and recombined xylanase (reAoXynllA) double-enzyme method hydrolyzed wheat wheat bran, obtain forulic acid crude extract, through HPD-300 macroporous resin purification, prepare highly purified forulic acid product.
Involved have restructuring feruloyl esterase (reAorFaeA) and the active engineering strain GS115/AorfaeA of recombined xylanase (reAoXynllA) (number of patent application: 201210562540.X) and GS115/AoxynllA (Ann Microbiol, 2013,63:1109-1120) by this laboratory, build and preserve.
The analysis determining method of described forulic acid:
High performance liquid phase (HPLC) is analyzed the burst size of forulic acid.Concrete operations are: accurately take 5mg trans-ferulaic acid standard substance, be settled to 50mL after adding appropriate dissolve with ethanol, obtain the trans-ferulaic acid standardized solution of 0.1mg/mL.With damping fluid, forulic acid standardized solution is diluted to different concns, by high performance liquid chromatography (HPLC), measures peak area.Take ferulaic acid content as X-coordinate, and corresponding peak area is ordinate zou drawing standard curve.After being crossed to film (0.45 μ m), sample carries out HPLC analysis.Chromatographic condition: wear peace UltiMate-3000 high performance liquid chromatograph, ZW & A-C18 reverse-phase chromatographic column, UV-detector, detect wavelength 320nm, column temperature is 30 ℃, and moving phase is methyl alcohol and 1% acetic acid two step gradient elutions (in 0-10min, methanol concentration rises to methanol concentration in 50%, 10-20min by 20% and rises to 80% by 50%), flow velocity is 1mL/min, and sample size is 20 μ L.According to typical curve, calculate ferulaic acid content.
Described double-enzyme method is extracted the forulic acid in Testa Tritici:
(1) take 50g Testa Tritici, add wherein the liquor kalii acetici of appropriate 0.3% (w/v), after 95 ℃ of immersion 30min, with a large amount of distilled water, repeatedly rinse, and dry to constant weight at 105 ℃, pulverize, cross 60 mesh sieves standby.
(2) accurately take 0.5g destarching wheat bran in 100mL Erlenmeyer flask, add 8mL reAorFaeA enzyme liquid (about 16.88U) and 0.5mL reAorXynllA enzyme liquid (about 72.5515U), with the phosphate buffered saline buffer of 0.1mmol/L, pH5.0, be settled to 30mL, in 40 ℃ of enzymolysis 10h, boil the 5min enzyme that goes out and live, centrifugal collection supernatant liquor is forulic acid crude extract.It is dry that crude extract is concentrated to 1mL final vacuum through rotary evaporation, obtains forulic acid and slightly carry product, and HPLC method is analyzed the content of forulic acid.
Described macroporous resin purification forulic acid crude extract:
(1) forulic acid crude extract being concentrated into forulic acid mass concentration is 0.1mg/mL, and concentrated solution is adjusted pH to 4.0, selects HPD-300 type macroporous resin, and under room temperature, resin loadings is 20mL.By the forulic acid crude extract 100mL handling well with 1mL/min upper prop dynamic adsorption after, standing 2h, washing pillar to effluent liquid is colourless, the flow velocity wash-out with 100mL50% ethanol with 1mL/min, quantitative collection.
(2) it is dry that the cut of collecting is concentrated to 1mL final vacuum through rotary evaporation, obtains forulic acid product, and HPLC method is analyzed the content (Fig. 1) of forulic acid.
Beneficial effect of the present invention: the invention provides a kind of method of preparing forulic acid of extracting from agricultural wastes Testa Tritici.Realize double-enzyme method synergy hydrolyzed wheat wheat bran and extracted forulic acid, avoided alkaline process in the past to prepare all drawback of forulic acid, utilize macroporous type low-pole polymeric adsorbent simultaneously and under the lower alcohol condition of suitable concn, can elute the principle of forulic acid, produced forulic acid has been carried out to further fractionation by adsorption, effectively enrichment forulic acid, cost is low, and the cycle is short, pollution-free.For further industrialization green production forulic acid is laid a good foundation.
Accompanying drawing explanation
Fig. 1: the HPLC of forulic acid product detects collection of illustrative plates
Embodiment
Below in conjunction with specific embodiment, further set forth working method of the present invention.But these embodiment only, for describing the present invention in detail, limit the scope of the invention and be not used in.
Embodiment 1 double-enzyme method is extracted the forulic acid in Testa Tritici
Accurately take 0.5g Testa Tritici, be placed in 100mL Erlenmeyer flask.Add 8mL reAorFaeA enzyme liquid (about 16.88U) and 0.5mL reAorXynllA enzyme liquid (about 72.5515U), with the phosphate buffered saline buffer of 0.1mmol/L, pH5.0, be settled to 30mL, in 40 ℃ of enzymolysis 10h, boil the 5min enzyme that goes out and live, centrifugal collection supernatant liquor is forulic acid crude extract, and crude extract is concentrated into 1mL through rotary evaporation, vacuum-drying obtains forulic acid and slightly carries powder, be weighed as 1.75g, through HPLC, detect, ferulaic acid content is 0.13%.
Embodiment 2 macroporous resin purification forulic acid crude extracts
It is 0.1mg/mL that forulic acid crude extract is concentrated into forulic acid mass concentration, concentrated solution is adjusted pH to 4.0, select HPD-300 type macroporous resin, resin wet method dress post 20mL, by the forulic acid crude extract 100mL handling well with 1mL/min upper prop dynamic adsorption after, standing 2h, washing pillar to effluent liquid is colourless, flow velocity wash-out with 100mL50% ethanol with 1mL/min, elutriant is concentrated into 1mL through rotary evaporation, vacuum-drying powdered, be weighed as 431mg, through HPLC, detect, ferulaic acid content is 22.5%, and the rate of recovery is 97%.
Claims (2)
1. from Testa Tritici, prepare a method for forulic acid, it is characterized in that take that Testa Tritici extracts forulic acid with feruloyl esterase and zytase double-enzyme method synergy as raw material:
(1) pre-treatment: take 50g Testa Tritici, add wherein the liquor kalii acetici of appropriate 0.3% (w/v), after 95 ℃ of immersion 30min, repeatedly rinse with a large amount of distilled water, and dry to constant weight at 105 ℃, pulverize, cross 60 mesh sieves standby;
(2) enzyme is processed: accurately take 0.5g destarching wheat bran in 100mL Erlenmeyer flask, add 8mL reAorFaeA enzyme liquid (about 16.9U) and 0.5mL reAorXynllA enzyme liquid (about 72.6U), with the phosphate buffered saline buffer of 0.1mmol/L, pH5.0, be settled to 30mL, in 40 ℃ of enzymolysis 10h, boil the 5min enzyme that goes out and live, centrifugal collection supernatant liquor is forulic acid crude extract.
2. utilize macroporous type low-pole polymeric adsorbent and under the lower alcohol condition of suitable concn, can elute the principle of forulic acid, prepare the method for high purity forulic acid:
(1) purifying: it is 0.1mg/mL that forulic acid crude extract is concentrated into forulic acid mass concentration, concentrated solution is adjusted pH to 4.0, select HPD-300 type macroporous resin, under room temperature, resin loadings is 20mL, by the forulic acid crude extract 100mL handling well with 1mL/min upper prop dynamic adsorption after, standing 2h, washing pillar to effluent liquid is colourless, the flow velocity wash-out with 100mL50% ethanol with 1mL/min, quantitative collection;
(2) dry: it is dry that the cut of collecting is concentrated in vacuo to 1mL final vacuum, obtains forulic acid product, is weighed as 431mg, through HPLC, detect, ferulaic acid content is 22.5%, the rate of recovery is 97%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106591382A (en) * | 2016-08-04 | 2017-04-26 | 北京联合大学 | Method for efficiently extracting ferulic acid from wheat bran |
| CN109868292A (en) * | 2019-01-09 | 2019-06-11 | 中国农业科学院农产品加工研究所 | The method that single enzymatic prepares ferulic acid and xylose |
| CN110475483A (en) * | 2017-04-10 | 2019-11-19 | 雀巢产品有限公司 | The method of composition of the preparation comprising ferulic acid |
| CN113667622A (en) * | 2021-09-16 | 2021-11-19 | 陕西海斯夫生物工程有限公司 | Burkholderia gladioli A1-07 and application thereof |
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| CN1425773A (en) * | 2002-12-27 | 2003-06-25 | 暨南大学 | Process for preparing oligosaccharide and trans-ferulaic acid |
| WO2004009804A2 (en) * | 2002-07-18 | 2004-01-29 | Biocatalysts Limited | Feruloyl esterase and uses thereof |
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| WO2004009804A2 (en) * | 2002-07-18 | 2004-01-29 | Biocatalysts Limited | Feruloyl esterase and uses thereof |
| CN1425773A (en) * | 2002-12-27 | 2003-06-25 | 暨南大学 | Process for preparing oligosaccharide and trans-ferulaic acid |
| CN102876753A (en) * | 2012-09-07 | 2013-01-16 | 华侨大学 | Separation and purification method for ferulic acid and xylo-oligosaccharide in spent grains |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591382A (en) * | 2016-08-04 | 2017-04-26 | 北京联合大学 | Method for efficiently extracting ferulic acid from wheat bran |
| CN110475483A (en) * | 2017-04-10 | 2019-11-19 | 雀巢产品有限公司 | The method of composition of the preparation comprising ferulic acid |
| CN109868292A (en) * | 2019-01-09 | 2019-06-11 | 中国农业科学院农产品加工研究所 | The method that single enzymatic prepares ferulic acid and xylose |
| CN113667622A (en) * | 2021-09-16 | 2021-11-19 | 陕西海斯夫生物工程有限公司 | Burkholderia gladioli A1-07 and application thereof |
| CN113667622B (en) * | 2021-09-16 | 2022-02-01 | 陕西海斯夫生物工程有限公司 | Burkholderia gladioli A1-07 and application thereof |
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