CN1840673A - Process for preparing feruoylated oligosaccharide by enzymolysis of wheat bran - Google Patents
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- CN1840673A CN1840673A CN 200610037952 CN200610037952A CN1840673A CN 1840673 A CN1840673 A CN 1840673A CN 200610037952 CN200610037952 CN 200610037952 CN 200610037952 A CN200610037952 A CN 200610037952A CN 1840673 A CN1840673 A CN 1840673A
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Abstract
The invention relates to a method for enzymolyzing wheat bran to prepare asafetida oligosaccharide, wherein it uses wheat bran as raw material; uses enzymatical to remove amidon, remove protein to prepare wheat bran insoluble fiber; then uses bacillus subtilis xylanase to hydrolyze wheat bran insoluble fiber to prepare the asafetida oligosaccharide. And the density of asafetida oligosaccharide will reach 1.497mmol/L. the invention can effectively utilize wheat bran, while prepared asafetida oligosaccharide has significant biological activity, to accelerate the generation of bifidobacteria, and restrain the erythrocyte oxidisability damage indused by free group, with better social and economic benefits.
Description
Technical field
A kind of method of preparing feruoylated oligosaccharide by enzymolysis of wheat bran belongs to agricultural byproducts development of resources, functional food additives and dietary supplements technical field.
Background technology
Wheat is one of most important food crop in the world, particularly in developed country, is the important component part of human diet.China's wheat yield is at the forefront in the world.In recent years, the Testa Tritici of the annual processing of China was about about 2,000 ten thousand tons.Testa Tritici is the fine source of human diet fiber, and as the economic worth obviously decline of feed ingredient, existing abroad a lot of bibliographical information Testa Triticis are useful to body health as the human diet fiber.In recent years, the attention of Testa Tritici research is concentrated on the physiologically active substance of wheat bran, on araboxylan, phytic acid and forulic acid, especially araboxylan and forulic acid.Testa Tritici mainly is made up of cell wall polysaccharides, is rich in hemicellulose, and wherein araboxylan accounts for 40%, is replaced by-L-arabinose usually on O-3, the O-2 of xylose residues on the xylan skeletal chain or O-3 and O-2 position.Another notable feature is exactly that forulic acid is connected on the pectinose residue by ester bond.In the phytology field, utilize polysaccharide hydrolase hydrolyzing plant cell wall polysaccharides to prepare feruloylated oligosaccharides, mainly be in order to study the structure of plant cell wall.Up to the present, the Arabic glycosyl xylo-oligosaccharide of asafoetide acyl for preparing from the grass cell walls has structurally demonstrated good consistence, promptly-L-furan type pectinose residue is connected with-1, on the wood sugar O-3 position on the D-xylan skeletal chain that the 4-glycosidic link connects, link to each other on the O-5 position on forulic acid and the pectinose residue.
The anti-oxidant activity of feruloylated oligosaccharides, feruloylated oligosaccharides has remarkable biological activity, can suppress the oxidation of low-density lipoprotein effectively, in that prevent or alleviate may be significant aspect the arteriosclerosis process, in this, free forulic acid can't be compared with it.
Antioxidant is one of the material guarantee of 21 century human health.Because the antioxidant of synthetic causes negative impact as BHA (BHA), fourth hydroxytoluene (BHT), tertiary butylated hydroquinone (TBHQ) meeting under a cloud to human body, abroad, above-mentioned synthetized oxidation preventive agent is used in food and is subjected to strict restriction.Gradually attention is turned in recent years and contain abundant natural antioxidants in the plant, particularly those are present in the antioxidant component in daily bread, vegetables, the fruit, wherein the plant phenols acid compound is exactly the more class of research, is expected to become the important source of novel antioxidant.Forulic acid is the ubiquitous a kind of phenolic acid of vegitabilia, especially in grass.Forulic acid in the cereal links to each other with polysaccharide in the cell walls by ester bond, exists with the form of combined.People such as Rondini report, the forulic acid of the forulic acid specific ionization that exists with combined has better bioavailability.So the research to the forulic acid (comprising feruloylated oligosaccharides) of combined has caused great interest.Up to the present, the research of relevant feruloylated oligosaccharides aspect is domestic does not also report.As everyone knows, xylo-oligosaccharide is a kind of bifidus factor of superior performance.Feruloylated oligosaccharides also contains hydrophilic xylo-oligosaccharide body except that containing the asafoetide acyl group, whether it has the effect that promotes bifidus bacillus propagation, both at home and abroad all less than report.
Summary of the invention
The method that the purpose of this invention is to provide a kind of preparing feruoylated oligosaccharide by enzymolysis of wheat bran, realize the effective increment and the utilization of Testa Tritici, prepared feruloylated oligosaccharides has remarkable biological activity, can promote the erythrocyte oxidisability injury of bifidobacterium growth and inhibition free yl induction, HUMAN HEALTH is highly profitable, has very big economic benefit and social benefit.
Technical scheme of the present invention: the present invention is to be raw material with the Testa Tritici, handle successively through alpha-amylase, proteolytic enzyme, saccharifying enzyme, mixture carries out centrifugal must the precipitation, after washing with hot distilled water, volume fraction 95% ethanol and acetone, the gained precipitation obtains the Testa Tritici insoluble dietary fibre, carry out enzyme digestion reaction with zytase again, the enzymolysis supernatant liquor prepares the feruloylated oligosaccharides crude product through concentrated, spraying drying.The used zytase of enzyme digestion reaction is subtilis (Bacillus subtilis) zytase.The enzymolysis substrate is the Testa Tritici insoluble dietary fibre, and the substrate mass concentration is 90～120g/L, and the enzyme mass concentration is 2～5g/L in the system, and hydrolysis temperature is 30～50 ℃, and enzymolysis time is 20～40h, and enzymatic hydrolysis system pH is 4.5～5.5.
It is 120g/L for the substrate mass concentration that the optimal conditions of Testa Tritici insoluble dietary fibre enzyme digestion reaction draws the enzyme digestion reaction optimal conditions through center combination Rotation Design scheme optimization model, the enzyme mass concentration is 4.8g/L in the system, hydrolysis temperature is 42 ℃, enzymolysis time is 35h, and enzymatic hydrolysis system pH is 5.2.
Testa Tritici mainly is made up of cell wall polysaccharides, wherein araboxylan accounts for the overwhelming majority, on O-3, the O-2 of xylose residues on the xylan skeletal chain or O-3 and O-2 position, replaced by-L-arabinose usually, in addition, also be connected with forulic acid on the pectinose residue on some O-2 position.Prepare feruloylated oligosaccharides, in the preparation process of Testa Tritici insoluble dietary fibre, must be able to not destroy the ester bond between forulic acid and cell wall polysaccharides, make the forulic acid of esterification on polysaccharide all remain, to help the catalytic hydrolysis of endo-xylanase.In research process, we adopt usual method to utilize high temperature resistant α-Dian Fenmei, alkaline endo proteolytic enzyme and refining saccharifying enzyme that raw material is carried out destarch, isolating protein on the one hand; On the other hand, the sample of specially enzyme being handled utilizes ethanol and acetone and other organic solvent to wash again, removing partial pigment, free phenolic acid compound etc., thereby has obtained the higher Testa Tritici insoluble dietary fibre of esterification ferulaic acid content.
The basic chemical composition analysis result (%) of Testa Tritici and Testa Tritici insoluble dietary fibre is as shown in table 1.
Table 1
| Ash content | Crude fat | Crude protein | Thick starch | Piperylene | Forulic acid | Other a | |
| Testa Tritici Testa Tritici insoluble dietary fibre | 5.10 2.40 | 4.30 2.80 | 15.70 5.10 | 14.60 0.10 | 21.40 54.70 | 0.48 0.90 | 38.42 34.00 |
aUndetermined.
Testa Tritici is handled 45min with high pressure steam at 121 ℃, so that its endogenous enzyme inactivation.The Testa Tritici of handling is suspended in the water of certain volume, 60 ℃ of following continuously stirring 16h make its abundant swelling, then add the heatproof α-Dian Fenmei of 75mL/kg wheat bran after, mixture stirs 40min in boiling water bath.After suspension is cooled to 60 ℃, transfer pH to 7.5, the hydrolysising protease Alcalase that adds the 30mL/kg wheat bran again, 60 ℃ of following continuously stirring 30min, transfer pH to 4.5, the refining saccharifying enzyme that adds the 35mL/kg wheat bran again, 60 ℃ of following continuously stirring 30min, mixture is centrifugal, abandoning supernatant, precipitation hot distilled water repetitive scrubbing, suspension does not have muddiness when washing with cold distilled water, reusable heat distilled water, volume fraction 95% ethanol and acetone is washed twice repeatedly successively, and centrifugal gained throw out obtains the Testa Tritici insoluble dietary fibre at 40 ℃ of following vacuum-drying 24h.Heatproof α-Dian Fenmei, hydrolysising protease and refining saccharifying enzyme provide by Novozymes company.
Subtilis (Bacillus subtilis) zytase (abbreviation zytase) is raised biological company limited by Wuhan Xinhua and is provided.
Temperature is very complicated to the situation that influences of microbial enzyme, it not only influences the native conformation of zymoprotein, the dissociated state of participation enzymatic reaction functional groups, but also have influence on the avidity of enzyme-to-substrate, the decomposition of enzyme-substrate complex compound, even also influence avidity of enzyme and activator, inhibitor etc.PH also is an important parameter of decision enzymatic activity, except that the dissociated state of the conformation that influences enzyme, zymophore catalytic group and conjugated group, also influences the electriferous state of substrate.Therefore, temperature and pH are very obvious to the catalyzed reaction speed influence of enzyme.
In lower temperature range (30 ℃～50 ℃), xylanase activity power raises with temperature and increases; In the comparatively high temps scope (60 ℃～80 ℃), xylanase activity power raises with temperature and reduces rapidly, because temperature raises, the thermally denature deactivation rate of zymoprotein is accelerated; The optimum temperuture of this enzyme reaction is 50 ℃, and this enzyme has 97% vigor in the time of 60 ℃, and the vigor of enzyme only has 6% in the time of 80 ℃.Suitably the enzyme liquid of dilution is incubated 1h under differing temps, and cooling immediately after the taking-up is measured relative enzyme according to a conventional method and lived.The result shows this enzyme the most stable below 50 ℃, can keep 38% enzyme work in the time of 60 ℃, the basic complete deactivation of enzyme in the time of 80 ℃.
Zytase is when pH3.0, and vigor is 37% relatively; When pH6.0, vigor is 91% relatively; When pH9.0, vigor only is 26% relatively; Optimum pH is 5.0 during this enzyme reaction.Enzyme is suitably diluted with different pH damping fluids, at room temperature be incubated 1h, measure relative enzyme according to a conventional method and live, the pH stable range of this enzyme is 4.0～6.0.
Subtilis (Bacillus subtilis) zytase can hydrolyzed wheat wheat bran insoluble dietary fibre, arbitrarily disconnects the glycosidic link on the araboxylan main chain in the food fibre, discharges feruloylated oligosaccharides.Along with the increase of enzyme concn, the concentration of feruloylated oligosaccharides also constantly increases.When the enzyme amount reached 4.0g/L, the concentration increase of feruloylated oligosaccharides was milder.
When low concentration of substrate 10g/L, the maximum value of feruloylated oligosaccharides concentration is less relatively, and 0.090mmol/L is only arranged.Yet when concentration of substrate surpassed 50g/L, the peak concentration increase of feruloylated oligosaccharides was very fast relatively, and when concentration of substrate 70g/L, the maximum value of feruloylated oligosaccharides concentration reaches 1.217mmol/L.
B.subtilis zytase optimal reactive temperature is at 50 ℃; Optimal reaction pH value is 5.0; The insulation enzyme is alive basicly stable down at 50 ℃ for this enzyme, and its stable pH range is pH4.0～pH6.0.
45 ℃ of selective reaction temperature, pH5.5, reaction times 36h, enzyme amount 3.0g/L, concentration of substrate 50g/L is as the center condition of center combination Rotation Design.
Center combination Rotation Design scheme and the experimental result such as the table 2 of each factor.
Table 25 levels, the center combination Rotation Design scheme and the experimental result of 5 factors
a
| Sequence number | X 1Temperature (℃) | X 2 pH | X 3Reaction times (h) | X 4Enzyme amount (g/L) | X 5Concentration of substrate (g/L) | FOs **(mmol/L) | |
| Experimental value | Predictor | ||||||
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 | 40(-1) 50(+1) 40(-1) 50(+1) 40(-1) 50(+1) 40(-1) 50(+1) 40(-1) 50(+1) 40(-1) 50(+1) 40(-1) 50(+1) 40(-1) 50(+1) 35(-2) 55(+2) 45(0) 45(0) 45(0) 45(0) 45(0) 45(0) 45(0) 45(0) 45(0) 45(0) 45(0) 45(0) 45(0) 45(0) | 5.0(-1) 5.0(-1) 6.0(+1) 6.0(+1) 5.0(-1) 5.0(-1) 6.0(+1) 6.0(+1) 5.0(-1) 5.0(-1) 6.0(+1) 6.0(+1) 5.0(-1) 5.0(-1) 6.0(+1) 6.0(+1) 5.5(0) 5.5(0) 4.5(-2) 6.5(+2) 5.5(0) 5.5(0) 5.5(0) 5.5(0) 5.5(0) 5.5(0) 5.5(0) 5.5(0) 5.5(0) 5.5(0) 5.5(0) 5.5(0) | 24(-1) 24(-1) 24(-1) 24(-1) 48(+1) 48(+1) 48(+1) 48(+1) 24(-1) 24(-1) 24(-1) 24(-1) 48(+1) 48(+1) 48(+1) 48(+1) 36(0) 36(0) 36(0) 36(0) 12(-2) 60(+2) 36(0) 36(0) 36(0) 36(0) 36(0) 36(0) 36(0) 36(0) 36(0) 36(0) | 2.0(-1) 2.0(-1) 2.0(-1) 2.0(-1) 2.0(-1) 2.0(-1) 2.0(-1) 2.0(-1) 4.0(+1) 4.0(+1) 4.0(+1) 4.0(+1) 4.0(+1) 4.0(+1) 4.0(+1) 4.0(+1) 3.0(0) 3.0(0) 3.0(0) 3.0(0) 3.0(0) 3.0(0) 1.0(-2) 5.0(+2) 3.0(0) 3.0(0) 3.0(0) 3.0(0) 3.0(0) 3.0(0) 3.0(0) 3.0(0) | 70(+1) 30(-1) 30(-1) 70(+1) 30(-1) 70(+1) 70(+1) 30(-1) 30(-1) 70(+1) 70(+1) 30(-1) 70(+1) 30(-1) 30(-1) 70(+1) 50(0) 50(0) 50(0) 50(0) 50(0) 50(0) 50(0) 50(0) 10(-2) 90(+2) 50(0) 50(0) 50(0) 50(0) 50(0) 50(0) | 1.078 0.384 0.332 1.013 0.438 1.046 0.999 0.391 0.514 1.159 1.112 0.440 1.217 0.559 0.497 1.108 0.712 0.921 0.702 0.767 0.588 0.824 0.704 0.896 0.090 1.280 0.927 0.884 0.965 0.911 0.89 0.861 | 1.011 0.381 0.381 1.011 0.439 1.069 1.069 0.439 0.490 1.120 1.120 0.490 1.178 0.548 0.548 1.178 0.896 0.896 0.767 0.767 0.680 0.796 0.787 1.005 0.087 1.347 0.896 0.896 0.896 0.896 0.896 0.896 |
aThe average absolute relative deviation is 5.75%,
*FOs represents feruloylated oligosaccharides.
To sum up analyze, the condition of xylanase hydrolysis Testa Tritici insoluble dietary fibre when using the optimization model can draw feruloylated oligosaccharides concentration maximum, 42 ℃ of enzyme digestion reaction temperature, pH5.2, enzyme digestion reaction time 35h, enzyme amount 4.8g/L, concentration of substrate 120g/L, under optimal conditions, the peak concentration of model prediction feruloylated oligosaccharides is 1.497mmol/L.The feruloylated oligosaccharides that obtains under optimal conditions is made up of forulic acid, pectinose and the wood sugar of esterification.
Beneficial effect of the present invention: with the Testa Tritici is raw material, utilize enzyme process destarch, isolating protein to prepare the Testa Tritici insoluble dietary fibre, utilize subtilis xylanase hydrolysis Testa Tritici insoluble dietary fibre to prepare feruloylated oligosaccharides again, 42 ℃ of temperature of reaction, pH5.2, reaction times 35h, enzyme amount 4.8g/L, under the condition of concentration of substrate 120g/L, feruloylated oligosaccharides concentration reaches 1.497mmol/L.
Feruloylated oligosaccharides not only can external promotion bifidobacterium growth; but also has unique anti-oxidant activity; can protect HRBC to avoid the oxidative stress injury that AAPH causes effectively, with the inductive injury of the collaborative opposing of red corpuscle endogenous antioxidant AAPH institute.
The present invention has realized the effective increment and the utilization of Testa Tritici, prepared feruloylated oligosaccharides has remarkable biological activity, can promote the erythrocyte oxidisability injury of bifidobacterium growth and inhibition free yl induction, HUMAN HEALTH is highly profitable, has very big economic benefit and social benefit.
Newly do not see relevant open source literature report through looking into.
Embodiment
Testa Tritici is handled 45min with high pressure steam at 121 ℃, so that its endogenous enzyme inactivation.The Testa Tritici of handling is suspended in the water of certain volume, 60 ℃ of following continuously stirring 16h make its abundant swelling, then add the heatproof α-Dian Fenmei of 75mL/kg wheat bran after, mixture stirs 40min in boiling water bath.After suspension is cooled to 60 ℃, transfer pH to 7.5, the hydrolysising protease Alcalase that adds the 30mL/kg wheat bran again, 60 ℃ of following continuously stirring 30min, transfer pH to 4.5, the refining saccharifying enzyme that adds the 35mL/kg wheat bran again, 60 ℃ of following continuously stirring 30min, mixture is centrifugal, abandoning supernatant, must precipitate, be common destarch, isolating protein prepares the common method of Testa Tritici insoluble dietary fibre, and the present invention takes specially with precipitation hot distilled water repetitive scrubbing, suspension does not have muddiness when washing with cold distilled water, reusable heat distilled water, volume fraction 95% ethanol and acetone successively twice of repeated washing to remove partial pigment, free phenolic acid compounds etc., centrifugal gained throw out obtains the Testa Tritici insoluble dietary fibre at 40 ℃ of following vacuum-drying 24h.The Testa Tritici insoluble dietary fibre carries out enzymolysis with the subtilis zytase again, when enzymatic hydrolysis condition was 42 ℃ of temperature of reaction, pH5.2, reaction times 35h, enzyme amount 4.8g/L, concentration of substrate 120g/L, the concentration of feruloylated oligosaccharides was 1.546 ± 0.037mmol/L (experiment repeats 3 times).
Claims (2)
1. the method for a preparing feruoylated oligosaccharide by enzymolysis of wheat bran, comprise that Testa Tritici handles successively through alpha-amylase, proteolytic enzyme, saccharifying enzyme, mixture carries out centrifugal must the precipitation, it is characterized in that obtaining the Testa Tritici insoluble dietary fibre after the gained precipitation is washed with hot distilled water, volume fraction 95% ethanol and acetone, carry out enzyme digestion reaction with zytase again, the enzymolysis supernatant liquor prepares the feruloylated oligosaccharides crude product through concentrated, lyophilize; The used zytase of enzyme digestion reaction is subtilis (Bacillus subtilis) zytase, the enzymolysis substrate is the Testa Tritici insoluble dietary fibre, the substrate mass concentration is 90～120g/L, the enzyme mass concentration is 2～5g/L in the system, hydrolysis temperature is 30～50 ℃, enzymolysis time is 20～40h, and enzymatic hydrolysis system pH is 4.5～5.5.
2. method according to claim 1, the optimal conditions that it is characterized in that Testa Tritici insoluble dietary fibre enzyme digestion reaction is 120g/L for the substrate mass concentration, and the enzyme mass concentration is 4.8g/L in the system, and hydrolysis temperature is 42 ℃, enzymolysis time is 35h, and enzymatic hydrolysis system pH is 5.2.
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| CN102174616A (en) * | 2011-03-14 | 2011-09-07 | 盐城工学院 | Method for preparing feruloyl oligosaccharides by wheat bran fermentation |
| CN101240293B (en) * | 2007-02-08 | 2011-10-19 | 姜贞姬 | Bran extract and cleansing composition comprising bran extract and preparation method thereof |
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| CN101191137B (en) * | 2007-12-26 | 2010-11-03 | 江南大学 | Method for synthesizing feruloylated oligosaccharides by biological catalysis |
| CN101629191B (en) * | 2009-08-18 | 2012-07-04 | 河南工业大学 | Ultrasound-assisted enzymolysis wheat bran method for preparing ferulic acid |
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| CN102174616A (en) * | 2011-03-14 | 2011-09-07 | 盐城工学院 | Method for preparing feruloyl oligosaccharides by wheat bran fermentation |
| CN102334628A (en) * | 2011-06-29 | 2012-02-01 | 江南大学 | Preparation method for prebiotic preparation |
| CN102334628B (en) * | 2011-06-29 | 2012-11-07 | 江南大学 | Preparation method for prebiotic preparation |
| CN102876757B (en) * | 2012-10-15 | 2014-12-10 | 南京农业大学 | Technique for preparing (FOs) feruloyl oligosaccharides by adopting two-period-type combined regulation fermentation technology |
| CN102876757A (en) * | 2012-10-15 | 2013-01-16 | 南京农业大学 | Technique for preparing (FOs) feruloyl oligosaccharides by adopting two-period-type combined regulation fermentation technology |
| CN104212859A (en) * | 2014-10-11 | 2014-12-17 | 郑州新威营养技术有限公司 | Production method for preparing hydrolyzed wheat proteins by fermentation method |
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| CN104830927B (en) * | 2015-04-14 | 2018-03-23 | 无锡群硕谷唐生物科技有限公司 | A kind of method that forulic acid oligosaccharide syrup is prepared using wheat bran |
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