CN1840674A - Process for preparing xylo-oligosaccharide by enzymolysis of wheat bran - Google Patents
Process for preparing xylo-oligosaccharide by enzymolysis of wheat bran Download PDFInfo
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- CN1840674A CN1840674A CN 200610037953 CN200610037953A CN1840674A CN 1840674 A CN1840674 A CN 1840674A CN 200610037953 CN200610037953 CN 200610037953 CN 200610037953 A CN200610037953 A CN 200610037953A CN 1840674 A CN1840674 A CN 1840674A
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Abstract
The preparation method for oligoxylose comprises: with wheat bran as material, processing at high temperature with amylase, proteinase and saccharifying enzyme in turn to obtain the insoluble fiber; taking enzymolysis reaction with bacillus subtilis xylanase; taking enzymolysed supernatant to concentrate, spray dry and obtain the crude product with 12.5% oligoxylose yield and purity more than 60% while xylobiose and xylotriose more than 30%. The product is very benefit to people health. This invention increases value of wheat bran.
Description
Technical field
A kind of method of preparing xylo-oligosaccharide by enzymolysis of wheat bran belongs to agricultural byproducts development of resources and functional food additives technical field.
Background technology
Plant hemicellulose is the abundantest polysaccharide of occurring in nature content except that Mierocrystalline cellulose.Xylan is the main component of plant hemicellulose, and its main chain is linked to each other by β-D-(1,4)-glycosidic link by xylopyranosyl and constitutes, and L-arabinose, D-glucose, D-semi-lactosi and methyl, ethanoyl etc. constitute side chain.The content of xylan and the branched type of institute are different with substitution value in the different material.As, hemicellulose level accounts for the 25%-40% of dry weight in the cereal cortex, and main component is an araboxylan, and pectinose replaces in O-2, O-3 position or O-2 and the O-3 position of xylose residues.
Xylo-oligosaccharide is the product of xylan degrading.At present, the suitability for industrialized production of xylo-oligosaccharide mainly adopts enzyme process, promptly utilizes zytase directional catalyzing xylan hydrolysis to obtain.The a lot of microorganisms of occurring in nature can both produce zytase, as: subtilis, streptomycete, aspergillus, mould, wood is mould and chactomium globosum etc., mainly is bacterium and fungi.
Because the main effective constituent of xylo-oligosaccharide is disaccharides and trisaccharide, the preparation xylo-oligosaccharide should be selected the higher relatively zytase of endo-xylanase vigor.Simultaneously, because xylan is a kind of baroque non-homogeneous glycan, contain many different substituting groups, for obtaining higher xylo-oligosaccharide yield, the zytase of selecting for use should have enzyme characteristics such as (as arabinofuranosidase/xylosidases) of cutting that contain side chain in the hydrolysis specific substrates in the strong or enzyme system of the ability of special-shaped xylan in the hydrolysis specific substrates, and raw material is different to require to use different microbial xylanase.Therefore the key of preparing xylo-oligosaccharide by enzymolysis of wheat bran is that zytase to being present in the adaptability of the wheat bran xylan in the Testa Tritici insoluble dietary fibre in a large number, promptly selects suitable zytase.
Summary of the invention
The method that the purpose of this invention is to provide a kind of preparing xylo-oligosaccharide by enzymolysis of wheat bran, realize the effective increment and the utilization of Testa Tritici, prepared xylo-oligosaccharide is a kind of functional oligose of brilliance, and HUMAN HEALTH is highly profitable, and has very big economic benefit and social benefit.
Technical scheme of the present invention: with the Testa Tritici is raw material, adopt the common Testa Tritici insoluble dietary fibre that after alpha-amylase, proteolytic enzyme, saccharifying enzyme are handled successively, obtains, the present invention carries out enzyme digestion reaction with zytase again with the Testa Tritici insoluble dietary fibre, and the enzymolysis supernatant liquor prepares the xylo-oligosaccharide 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 4%-7%, and the enzyme mass concentration is 0.4%-0.7% in the system, and hydrolysis temperature is 40-50 ℃, and enzymolysis time is 24-30h, and enzymatic hydrolysis system pH is 5.0.
The optimal conditions of Testa Tritici insoluble dietary fibre enzyme digestion reaction is 5%-6% for the substrate mass concentration, and the enzyme mass concentration is 0.45%-0.55% in the system, and hydrolysis temperature is 45 ℃, and enzymolysis time is 24h, and enzymatic hydrolysis system pH is 5.0.
Owing to contain relatively large starch, protein in the Testa Tritici, in order to improve raw material availability, alleviate the burden of downstream separation purifying, at first handle successively with amylase, proteolytic enzyme, saccharifying enzyme carry out destarch, isolating protein has obtained the Testa Tritici insoluble dietary fibre.
The essentially consist of Testa Tritici is as shown in table 1 by analysis.The essentially consist of gained Testa Tritici insoluble dietary fibre is as shown in table 2 behind destarch, isolating protein.
Table 1 Testa Tritici essentially consist
| Composition | Moisture | Ash content | Starch | Protein | Piperylene | Other |
| Content (%) | 10.98 | 6.98 | 14.21 | 13.97 | 21.40 | Do not survey |
Table 2 Testa Tritici insoluble dietary fibre essentially consist
| Composition | Ash content | Starch | Protein | Piperylene | Other |
| Content (%) | 1.46 | 2.20 | 3.97 | 54.70 | Do not survey |
Usually Testa Tritici is suspended in the hot water, the heatproof α-Dian Fenmei that adds the 75mL/kg wheat bran, 95 ℃ of stir process 1h are cooled to 60 ℃ and transfer pH to 7.5, the hydrolysising protease Alcalase that adds the 30mL/kg wheat bran again, 60 ℃ of following stir process 30min transfer pH to 4.5, add the saccharifying enzyme of 35mL/kg wheat bran again, 60 ℃ of following stir process 30min, the centrifuging and taking precipitation, precipitation is used the hot distilled water repetitive scrubbing, obtains the Testa Tritici insoluble dietary fibre.Heatproof α-Dian Fenmei, hydrolysising protease and refining saccharifying enzyme provide by Novozymes company.
Determine to adopt subtilis (Bacillus subtilis) zytase that the Testa Tritici insoluble dietary fibre is carried out enzyme digestion reaction through screening, because of it has higher enzymolysis efficiency to the Testa Tritici insoluble dietary fibre, this enzyme is raised biological company limited by Wuhan Xinhua and is provided.
Concentration of substrate has very big influence to enzymatic reaction, and when concentration of substrate was low, the concentration that improves substrate can improve speed of response, but when concentration of substrate was higher, the further raising of concentration of substrate, the increase of speed of response became slowly even no longer increase.And the raising of concentration of substrate tends to cause the decline of product yield.Increase enzyme concn and can improve speed of response, but cause that simultaneously cost increases, and should select suitable enzyme concn that enzyme reaction is finished in acceptable time.
In the preliminary enzymolysis process of determining, five factors (concentration of substrate, enzyme concn, temperature, time, pH) that influence enzyme digestion reaction are carried out orthogonal design, its each level is shown in Table 3.
Table 3 empirical factor and water-glass
| Level | Concentration of substrate [S] | Enzyme concn [E] | Temperature T (℃) | Time t (h) | pH |
| 1 2 3 | 4％ 6％ 8％ | 0.3％ 0.5％ 0.7％ | 60 40 50 | 24 30 18 | 4 5 6 |
According to the characteristics of factor and level, we adopt orthogonal table L
18(3
7) test.
Experimental result sees Table 4.
Table 4 quadrature is table as a result
| Test number | [S] | [E] | T | t | pH | Reducing sugar yield (%) |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 | 1(4％) 1 1 2(6％) 2 2 3(8％) 3 3 1 1 1 2 2 2 3 3 3 | 1(0.3％) 2(0.5％) 3(0.7％) 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 | 1(60) 2(40) 3(50) 1 2 3 2 3 1 3 1 2 2 3 1 3 1 2 | 1(24) 2(30) 3(18) 2 3 1 1 2 3 3 1 2 3 1 2 2 3 1 | 1(4) 2(5) 3(6) 2 3 1 3 1 2 2 3 1 1 2 3 3 1 2 | 17.08 26.04 16.48 21.18 19.67 23.85 17.80 22.74 21.92 23.13 19.85 22.26 17.97 23.88 18.76 17.07 14.89 23.28 |
From the reducing sugar yield of 18 experiments find out experiment 2 yield higher be 26.04%, corresponding experiment condition is a concentration of substrate 4%, enzyme concn 0.5%, 40 ℃ of temperature, time 30h, pH5.0.
Statistical results show pH value is an important factor, and reaction times, temperature and enzyme concn also are important factors, and concentration of substrate is the less factor of influence.Reaction system is pH5.0,50 ℃ of temperature, enzyme concn 0.5%, concentration of substrate 6% is an optimum level, and the reaction times rate of looking more is high more, and the reaction times can not be too short, the yield increase slows down after the 24h, considers that from the angle of producing it is proper that the reaction times is defined as 24h.
Except that temperature with concentration of substrate is different, the combination of the optimum level of statistical study is identical with enzyme concn and pH that the optimum level that orthogonal table intuitively obtains makes up No. 2 test conditionss.In order further to contrast two kinds of conditions, we carry out enzymolysis Testa Tritici insoluble dietary fibre 24h respectively under these two kinds of conditions.The best level combined result is better than experimental result No. 2, and the reducing sugar yield is respectively 25.80% and 24.71% to the Testa Tritici insoluble dietary fibre.
In sum, we have determined that the subtilis zytase acts on the optimization processing condition of Testa Tritici insoluble dietary fibre: pH 5, reaction times 24h, 45 ℃ of temperature, enzyme concn 0.45%-0.55%, concentration of substrate 5%-6%.To the lyophilize of gained enzymolysis solution weigh under this condition, the xylo-oligosaccharide yield reaches 12.50% to Testa Tritici.
Beneficial effect of the present invention: domestic existing enzymolysis Testa Tritici is produced the research of oligose method; Testa Tritici after the compound enzymic preparation of existing employing phytase, cellulase, proteolytic enzyme and oligosaccharidase soaks crushed after being dried, deionized water carries out enzymolysis, adjusts pH value preparation xylo-oligosaccharide Study on Conditions (seeing Chinese patent CN1438319A); Existing α-Dian Fenmei, the Sumizyme MP of adopting handled Testa Tritici, adopts decolouring again, removes insoluble substance from refining means such as friendships, filtrations and prepare oligose, and content reaches 80% open source literature and reports (seeing Chinese patent CN1438319A).About utilizing the effect characteristics existing open source literature report of Testa Tritici, adopt a kind of (1 → 4)-β-endo-xylanase, two kinds of (1 → 4)-β-endo-xylanases (zytases of 11 families and 10 families), two kinds of pure enzymes (heat-staple zytase XP1 and XP2) enzymolysis Testa Tritici to reach enzymatic hydrolysis condition is studied for the poly-pool of the wood enzyme of substrate research different sources.
Newly do not see and utilize the subtilis zytase hydrolysis prepares xylo-oligosaccharide to the Testa Tritici insoluble dietary fibre through looking into, do not see yield, purity and the xylo-bioses of xylo-oligosaccharide and the open source literature report of xylotriose content; Abroad do not see and adopt α-Dian Fenmei, alkaline endo proteolytic enzyme and refining saccharifying enzyme to handle Testa Tritici, utilize the hydrolysis of subtilis zytase catalysis Testa Tritici insoluble dietary fibre to prepare xylo-oligosaccharide, do not see yield, purity and the xylo-bioses of xylo-oligosaccharide and the open source literature report of xylotriose content.
The prepared xylo-oligosaccharide yield of the present invention is 12.5% (calculating with Testa Tritici), and xylo-oligosaccharide purity surpasses 60%, and wherein the content of xylo-bioses and xylotriose is more than 30%.The present invention realizes the effective increment and the utilization of Testa Tritici, and prepared xylo-oligosaccharide is a kind of functional oligose of brilliance, and HUMAN HEALTH is highly profitable, and has very big economic benefit and social benefit.
Embodiment
Testa Tritici is suspended in the hot water, the heatproof α-Dian Fenmei that adds the 75mL/kg wheat bran, 95 ℃ of stir process 1h, be cooled to 60 ℃ and transfer pH to 7.5, the hydrolysising protease Alcalase that adds the 30mL/kg wheat bran again, 60 ℃ of following stir process 30min, transfer pH to 4.5, the saccharifying enzyme that adds the 35m/Lkg wheat bran again, 60 ℃ of following stir process 30min, centrifuging and taking precipitation, precipitation hot distilled water repetitive scrubbing, obtain the Testa Tritici insoluble dietary fibre, yield is 35%, and promptly the per kilogram Testa Tritici gets 350g Testa Tritici insoluble dietary fibre, and the control concentration of substrate is 4%-7%, press the per kilogram wheat bran then and add 35g subtilis zytase (zytase is prepared with 6.6L pH5.0 sodium-acetate buffer), at 50 ℃ of enzymolysis 24h.Reaction mixture carries out centrifugal, gets supernatant liquor, through concentrated, spraying drying, obtains the xylo-oligosaccharide crude product, is 12.5% in the Testa Tritici yield.
Claims (2)
1. the method for a preparing xylo-oligosaccharide by enzymolysis of wheat bran, comprise that Testa Tritici obtains the Testa Tritici insoluble dietary fibre after alpha-amylase, proteolytic enzyme, saccharifying enzyme are handled successively, it is characterized in that the Testa Tritici insoluble dietary fibre carries out enzyme digestion reaction with zytase again, the enzymolysis supernatant liquor prepares the xylo-oligosaccharide 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 4%-7%, and the enzyme mass concentration is 0.4%-0.7% in the system, and hydrolysis temperature is 40-50 ℃, and enzymolysis time is 24-30h, and enzymatic hydrolysis system pH is 5.0.
2. method according to claim 1, the optimal conditions that it is characterized in that Testa Tritici insoluble dietary fibre enzyme digestion reaction is 5%-6% for the substrate mass concentration, and the enzyme mass concentration is 0.45%-0.55% in the system, and hydrolysis temperature is 45 ℃, enzymolysis time is 24h, and enzymatic hydrolysis system pH is 5.0.
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| CN101608000B (en) * | 2008-06-18 | 2011-07-20 | 山东省生物药物研究院 | Method and equipment for extracting xylan by continuous cooking and use of xylan |
| CN102334628A (en) * | 2011-06-29 | 2012-02-01 | 江南大学 | Preparation method for prebiotic preparation |
| CN102356910A (en) * | 2011-09-22 | 2012-02-22 | 江南大学 | Preparation method of wheat bran beverage rich in xylo oligosaccharide |
| CN102863548A (en) * | 2012-09-28 | 2013-01-09 | 青岛蔚蓝生物集团有限公司 | Method for extracting xylan from wheat bran |
| CN103073913A (en) * | 2013-01-22 | 2013-05-01 | 山东农业大学 | Method for comprehensively extracting various functional components in color wheat bran |
| US8524471B2 (en) | 2007-03-19 | 2013-09-03 | Sud-Chemie Ip Gmbh & Co. Kg | Generation of chemical building blocks from plant biomass by selective depolymerization |
| CN104544137A (en) * | 2014-12-12 | 2015-04-29 | 湖北省农业科学院农产品加工与核农技术研究所 | Novel method for preparing wheat bran dietary fibers by taking wheat bran as raw material |
| US9314496B2 (en) | 2007-12-19 | 2016-04-19 | Kirin Holdings Kabushiki Kaisha | Insoluble dietary fiber-containing product derived from grain seeds |
| WO2017107527A1 (en) * | 2015-12-25 | 2017-06-29 | 山东龙力生物科技股份有限公司 | Soluble dietary fiber and preparation method thereof |
| CN107254457A (en) * | 2017-08-15 | 2017-10-17 | 青岛澳蓝明东生物科技有限公司 | A kind of complex enzyme preparation special for improving wooden pentasaccharides conversion ratio and its application |
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| CN110973421A (en) * | 2019-11-29 | 2020-04-10 | 中国农业科学院农产品加工研究所 | High-content xylo-oligosaccharide corn bamboo shoot beverage and preparation method thereof |
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| US9314496B2 (en) | 2007-12-19 | 2016-04-19 | Kirin Holdings Kabushiki Kaisha | Insoluble dietary fiber-containing product derived from grain seeds |
| CN101608000B (en) * | 2008-06-18 | 2011-07-20 | 山东省生物药物研究院 | Method and equipment for extracting xylan by continuous cooking and use of xylan |
| 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 |
| CN102356910A (en) * | 2011-09-22 | 2012-02-22 | 江南大学 | Preparation method of wheat bran beverage rich in xylo oligosaccharide |
| CN102356910B (en) * | 2011-09-22 | 2012-09-05 | 江南大学 | Preparation method of wheat bran beverage rich in xylo oligosaccharide |
| CN102863548A (en) * | 2012-09-28 | 2013-01-09 | 青岛蔚蓝生物集团有限公司 | Method for extracting xylan from wheat bran |
| CN103073913A (en) * | 2013-01-22 | 2013-05-01 | 山东农业大学 | Method for comprehensively extracting various functional components in color wheat bran |
| CN104544137B (en) * | 2014-12-12 | 2016-06-29 | 湖北省农业科学院农产品加工与核农技术研究所 | A kind of method preparing wheat-bran dietary fiber for raw material with Testa Tritici |
| CN104544137A (en) * | 2014-12-12 | 2015-04-29 | 湖北省农业科学院农产品加工与核农技术研究所 | Novel method for preparing wheat bran dietary fibers by taking wheat bran as raw material |
| WO2017107527A1 (en) * | 2015-12-25 | 2017-06-29 | 山东龙力生物科技股份有限公司 | Soluble dietary fiber and preparation method thereof |
| US10632136B2 (en) | 2015-12-25 | 2020-04-28 | Shandong Longlive Bio-Technology Co., Ltd | Soluble dietary fiber and preparation method thereof |
| CN107254457A (en) * | 2017-08-15 | 2017-10-17 | 青岛澳蓝明东生物科技有限公司 | A kind of complex enzyme preparation special for improving wooden pentasaccharides conversion ratio and its application |
| CN107254457B (en) * | 2017-08-15 | 2020-06-23 | 青岛澳蓝明东生物科技有限公司 | Special complex enzyme preparation for improving conversion rate of xylopentaose and application thereof |
| CN110742282A (en) * | 2019-10-29 | 2020-02-04 | 武汉新华扬生物股份有限公司 | Method for preparing dietary fiber by using wheat bran and rice bran and product |
| CN110973421A (en) * | 2019-11-29 | 2020-04-10 | 中国农业科学院农产品加工研究所 | High-content xylo-oligosaccharide corn bamboo shoot beverage and preparation method thereof |
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| CN115747262A (en) * | 2021-09-03 | 2023-03-07 | 国投生物科技投资有限公司 | Method for producing ethanol by using wheat |
| CN116195747A (en) * | 2023-03-14 | 2023-06-02 | 山东省农业科学院 | Composite dietary fiber nutrition powder for regulating blood glucose metabolism and preparation method and application thereof |
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