CN108300748A - A kind of method that holoenzyme method prepares alternan oligosaccharides - Google Patents
A kind of method that holoenzyme method prepares alternan oligosaccharides Download PDFInfo
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- CN108300748A CN108300748A CN201810148066.3A CN201810148066A CN108300748A CN 108300748 A CN108300748 A CN 108300748A CN 201810148066 A CN201810148066 A CN 201810148066A CN 108300748 A CN108300748 A CN 108300748A
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/22—Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose
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Abstract
The invention discloses a kind of methods that holoenzyme method prepares alternan oligosaccharides, belong to technical field of food biotechnology.The present invention is using potato starch and sucrose as raw material, adjust the ratio of potato starch and sucrose, high-temperature acidic alpha amylase, beta amylase, Pullulanase, maltogenic amylase and alternating sucrase enzyme is added successively to be catalyzed when reacting 72h, final substrate turns glycosides efficiency up to 88.9%, lays a good foundation for the preparation of industrialization of alternan oligosaccharides.
Description
Technical field
The present invention relates to a kind of methods that holoenzyme method prepares alternan oligosaccharides, are exactly to utilize potato starch and sugarcane
Sugar is raw material, and the method that the alternan oligosaccharides of degree of polymerization 3-8 is prepared using holoenzyme method belongs to technical field of food biotechnology.
Background technology
Alternan oligosaccharides is made of glucose unit.Its main chain is that glucose unit passes through α-glucopyra 1,3- glucosides
Key and α -1,6- glucopyranose glycosidic bonds are alternately formed by connecting.
Alternan oligosaccharides all has having been widely used in every field.
In the food industry, alternan oligosaccharides can be used for preparing low glycemic syrup.Such syrup has relatively low
Glycemic index, while product clarity can be further increased, played an important role in food formula.
In pharmaceuticals industry, it is used as carrier and stabilizer, as the excipient of active constituent such as in drug.
In clinical application, alternan oligosaccharides is great effect as the prebiotics of control enteric bacteria pathogen.It is logical
The compositions-treated animal with the one or more alternan oligosaccharides for effectively facilitating beneficial bacteria increment is crossed, can fully be reduced
Or inhibit intestinal pathological bacteria group, especially control salmonella strain, enteropathogenic E.Coli and C.perfringens
Especially effectively.
Currently, reacting production alternan oligosaccharides with receptor oligosaccharides using alternately sucrase enzyme catalysing sucrose both at home and abroad, wherein
Maltose can produce the alternan oligosaccharides of degree of polymerization 3-8 as good receptor.The degree of the alternan oligosaccharides degree of polymerization can be with sugarcane
Sugar and concentration and the relative ratios of receptor maltose and change.Reaction product is usually by the mixing of the oligosaccharides with different polymerization degree
Object forms.In relatively high sucrose and maltose ratio, more glycosyl units are transferred in glucan, and are had
The product of more high polymerization degree.In contrast, in low sucrose and maltose ratio, main reaction product is single glycosyl list
Member is transferred to receptor and the product that generates.
The country is seldom about the research for preparing alternan oligosaccharides and report, only about preparing oligomeric four in alternan oligosaccharides
The part research of sugar, and the external research about alternan oligosaccharides, also only reside within using maltose as receptor, to product
Composition has carried out Components identification, does not do careful research to the production of alternan oligosaccharides.During industrialized production, if only
When only preparing alternan oligosaccharides as raw material using sucrose and maltose, since the high cost of material of maltose replaces to preparation of industrialization
Sugared oligosaccharides brings huge cost pressure.
Invention content
Be with cheap potato starch and sucrose it is original the present invention is intended to provide a kind of method preparing alternan oligosaccharides
Material prepares alternan oligosaccharides with holoenzyme method, lays the foundation for industrialized production alternan oligosaccharides.
The method includes pretreatment of raw material, gelatinization, liquefaction, saccharification successively and generates alternan oligosaccharides.
In one embodiment of the invention, the pretreatment, which refers to, suspends 5~30% (g/100mL) potato starch
In 20~200mM phosphate buffers (pH 3~7).
In one embodiment of the invention, the gelatinization refers to continuously stirring 1~10min in boiling water bath to make starch
Gelatinization.
In one embodiment of the invention, the gelatinization is that 5~10% (m/v) potato starch are suspended in 50mM
In phosphate buffer (pH 4.5), it is stirred to react in boiling water bath.
In one embodiment of the invention, the liquefaction refers to addition 5~100U/g high temperature acid alpha-amylases,
After continuously stirring 1~10min in boiling water bath, 0~10M hydrochloric acid is added, pH is adjusted to 2.0~6.0 hereinafter, to terminate liquefaction.
In one embodiment of the invention, the liquefaction is that 5~10U/g high temperature acid alphas-are added into gelatinization product
Amylase after continuously stirring 4~10min in boiling water bath, is added 1~10M hydrochloric acid and pH is adjusted to 2.0~4.0, with terminate liquid
Change.
In one embodiment of the invention, the saccharification refers to that 5~100U/g beta amylases, 1~100U/ is first added
G Pullulanases react 5~240h in 30~80 DEG C of shaking baths；5~100U/g maltogenic amylases are added, 30~
80 DEG C of 5~100h of reaction.
In one embodiment of the invention, described be saccharified includes：Primary saccharification：Liquefied starch is cooled to room
Temperature adjusts pH to 5.2, and 5~15U/g beta amylases and 1~5U/g Pullulanases is added, is reacted for 24 hours in 60 DEG C of shaking baths；
Dextrine conversion：Reaction solution pH is adjusted to 5.5, is separately added into 5~10U/g maltogenic amylases, 10h is reacted at 60 DEG C.
In one embodiment of the invention, the generation alternan oligosaccharides, is the enzyme deactivation after dextrine conversion, according to
1~30% sucrose of secondary addition and 0.1~100U/g replace sucrase enzyme, and 5~240h is reacted in 30~80 DEG C of shaking baths.
In one embodiment of the invention, the generation alternan oligosaccharides, is the enzyme deactivation after dextrine conversion, according to
20~30% sucrose of secondary addition and 0.1~5U/g replace sucrase enzyme, and 34h is reacted at 40 DEG C.
In one embodiment of the invention, the maltogenic amylase is Bacillusstearothermophilus
Source.
In one embodiment of the invention, the alternately sucrase enzyme is the sources Leuconostoc citreum.
Advantageous effect：A kind of holoenzyme method of providing of the present invention prepares alternan oligosaccharides, is with potato starch and sucrose
Raw material adjusts the ratio of potato starch and sucrose, addition high temperature acid alpha-amylase, beta amylase, Pullulanase, maltose
When amylase and alternately sucrase enzyme are successively catalyzed reaction 72h, final substrate turns glycosides efficiency up to 88.9%, is alternan oligosaccharides
Preparation of industrialization is laid a good foundation.
Description of the drawings
The HPLC collection of illustrative plates of Fig. 1 products.From left to right appearance be successively solvent peak, monosaccharide, disaccharides, trisaccharide, tetrose, pentasaccharides,
Six sugar, seven sugar.(product is the mixture of the oligosaccharides of different polymerization degree)
The HPLC collection of illustrative plates of Fig. 2 products.From left to right appearance be successively solvent peak, monosaccharide, disaccharides, trisaccharide, tetrose, pentasaccharides,
Six sugar.(product is the mixture of the oligosaccharides of different polymerization degree)
The HPLC collection of illustrative plates of Fig. 3 products.Appearance is solvent peak, monosaccharide, disaccharides, trisaccharide, tetrose, pentasaccharides successively from left to right
(product is the mixture of the oligosaccharides of different polymerization degree)
Specific implementation mode
Maltogenic amylase enzyme activity determination method：The soluble starch for weighing final concentration of 1% (m/v), is dissolved in 50mM
In the phosphate buffer of pH5.5, and 10min is preheated in 60 DEG C of water-baths.The enzyme solution of 100ul is added, is added after reacting 30min
3mLDNS boils 7min and cools down rapidly, adds distilled water to be settled to 15ml, absorbance is surveyed under 540nm.
Maltogenic amylase enzyme activity defines：Hydrolysis soluble starch per minute is generated into enzyme needed for the glucose sugar of 1 μm of ol
Amount is defined as the enzyme activity (U) of a unit of maltogenic amylase.
Alternately sucrase enzyme enzyme activity determination method：Enzyme solution to be measured is kept the temperature into 30min at 45 DEG C, 0.4mL is taken to be added to
In 3.6mL sucrose NaAc-HAc buffer solutions (50mmol/L, pH5.4), it is 10% to make sucrose ultimate density, is preheated at 40 DEG C
The enzyme solution of 100ul is added in 10min, and 3mLDNS is added after reacting 30min, boils 7min and cools down rapidly, distilled water is added to be settled to
Absorbance is surveyed under 15ml, 540nm.
Alternately sucrase enzyme enzyme activity definition：Sucrose hydrolysis per minute is generated into enzyme amount needed for the fructose of 1 μm of ol and is defined as sugarcane
The enzyme activity (U) of one unit of saccharophosphorylase.
The HPLC of product is detected：The amount of sucrose, glucose, fructose and monosaccharide in end reaction system uses HPLC
To determine.Chromatographic condition is：Agilent 1200HPLC chromatographs, Agilent autosamplers, chromatographic column NH2-504E
(4.6mm × 250mm), Composition distribution are Agilent G1362A；Mobile phase is molten using the mixing of 75% (v/v) acetonitrile and water
Liquid, flow velocity 0.8mL/min, column temperature are set as 35 DEG C.Using external standard method, determine that respective quadrature is replaced according to retention time and peak area
The concentration of sugared oligosaccharides.
The calculation formula for turning glycosides efficiency D of alternan oligosaccharides is：
D (%)=M1/M2* 100%
Wherein D：Sucrose inversion is that alternan oligosaccharides mole turns glycosides efficiency (%)；
M1：The molal quantity (mol) of the fructose generated in enzymatic conversion reaction；
M2：Put into the molal quantity (mol) of the sucrose in reaction solution.
Holoenzyme method prepares alternan oligosaccharides under 1 optimum reaction condition of embodiment
The enzymatic conversion system of 10mL is carried out in the closed container of 50mL.Successively utilize high temperature acid alpha-amylase, β-
Amylase, Pullulanase, maltogenic amylase and alternating sucrase enzyme prepare alternan oligosaccharides, and steps are as follows：
Gelatinization：10% (m/v) potato starch is suspended in 50mM phosphate buffers (pH 4.5), it is continuous in boiling water bath
Stirring 1.5min makes starch gelatinization.
Liquefaction：10U/g high temperature acid alpha-amylases are added, after continuously stirring 4min in boiling water bath, 1M hydrochloric acid is added will
PH is adjusted to 4.0 hereinafter, to terminate liquefaction.
Primary saccharification：Liquefied starch is cooled to room temperature, pH to 5.2 is adjusted, 15U/g beta amylases are added and 1U/g is general
Shandong orchid enzyme, reacts (rotating speed 150rpm) for 24 hours in 60 DEG C of shaking baths.
Dextrine conversion：Reaction solution pH is adjusted to 5.5, is separately added into 9.3U/g maltogenic amylases, is shaken in 60 DEG C of water-baths
Reaction 10h (rotating speed 150rpm) in bed.
After dextrine conversion, boiling water bath heats 5min enzyme deactivations, sequentially adds 20% sucrose and 5U/g alternating sucrase enzymes,
34h (rotating speed 150rpm) is reacted in 40 DEG C of shaking baths.Subsequent boiling water bath heating 5min terminates reaction, and reaction solution centrifugation is led to
Cross HPLC analysis products.When turning glycosides efficiency after reaction up to 88.9%, product is the mixture of the oligosaccharides of different polymerization degree,
Chromatographic peak is shown in Fig. 1：Appearance is solvent peak, monosaccharide, disaccharides, trisaccharide, tetrose, pentasaccharides, six sugar, seven sugar successively from left to right.
1 sucrose of comparative example and recipient ratio are to reaction product and turn the influence of glycosides efficiency
The degree of the alternan oligosaccharides degree of polymerization can change with concentration and the relative ratios of sucrose and receptor maltose.Reaction
Product is usually made of the mixture of the oligosaccharides with different polymerization degree.In relatively high sucrose and maltose ratio, more
Glycosyl units be transferred in glucan, and obtain with more high polymerization degree product.In contrast, in low sucrose and wheat
When bud sugar ratio, main reaction product is that single glycosyl units are transferred to receptor and the product that generates.Therefore, by changing sugarcane
Sugar and recipient ratio, can optimized product alternan oligosaccharides the product composition for turning glycosides efficiency and product alternan oligosaccharides.
Ensure reaction conditions and the embodiments 1 one such as enzyme concentration, reaction temperature, reaction time and the reaction pH of enzymic catalytic reaction
It causes, the ratio for adjusting raw material potato starch and sucrose is respectively 1:1、1:2 and 2:1, carry out enzymic catalytic reaction.After reaction
Product is analyzed by HPLC.Measure that turn glycosides efficiency be respectively 71.5%, 88.9% and 55.0%, reaction product be respectively disaccharides~
Six is sugared, disaccharides~seven are sugared, disaccharides~pentasaccharides, and chromatographic peak is shown in Fig. 2, Fig. 1 and Fig. 3.
2 reaction temperature of comparative example is to reaction product and turns the influence of glycosides efficiency
In alternately sucrase enzyme catalyzes and synthesizes the reaction process of alternan oligosaccharides, the temperature of reaction system can influence to replace
The activity of sucrase enzyme, and then influence the yield of reaction product alternan oligosaccharides.Meanwhile according to the literature, it is known that work as reaction
There are when sucrose and another monosaccharide or disaccharide that can be connect with enzyme in substrate, two kinds of sugar of enzymatic generate the oligomeric of small molecule
Sugar.Since acceptor molecule is different from sucrose molecule, thus dextran chain is terminated after acceptor molecule attack glucityl or glucan base
Formation.And due to the presence of space steric effect, the bigger oligosaccharides of the degree of polymerization is more difficult to be formed.Therefore, if the temperature of reaction system
Degree can influence the activity of alternately sucrase enzyme, can will further influence the formation of the larger oligosaccharides of the degree of polymerization so that reaction production
Containing less big degree of polymerization alternan oligosaccharides in object, or even almost without.
Ensure reaction conditions and the embodiments 1 one such as concentration of substrate, enzyme concentration, reaction time and the reaction pH of enzymic catalytic reaction
It causes, carries out enzymic catalytic reaction under 25 DEG C, 40 DEG C, 55 DEG C of reaction temperature respectively, product is analyzed by HPLC after reaction,
Calculate reaction turns glycosides efficiency.After the degree of polymerization of oligosaccharides first increases with the raising of reaction temperature in reaction product alternan oligosaccharides
It reduces, turns glycosides efficiency and reduced afterwards as the raising of reaction temperature first increases.That is, at 40 DEG C, the product degree of polymerization and turn glycosides efficiency
Reach maximum value, reaction product is that disaccharides~seven are sugared, and it is 88.9% to turn glycosides efficiency, and chromatographic peak is shown in Fig. 1.
Embodiment 2
Alternan oligosaccharides is prepared using 1 identical strategy of embodiment, difference lies in be added 100U/g and replace sucrase enzyme system
Standby alternan oligosaccharides analyzes product by HPLC, and conversion ratio 88.7% converts compared with 5U/g alternating sucrase enzymes are added
Rate is essentially identical.
Embodiment 3
Alternan oligosaccharides is prepared using 1 identical strategy of embodiment, difference lies in after dextrine conversion, in 40 DEG C of water-baths
The reaction response time is for 24 hours, to analyze product, conversion ratio 70.2% by HPLC, hence it is evident that less than reaction time 34h's in shaking table
Conversion ratio.
Embodiment 4
Alternan oligosaccharides is prepared using 1 identical strategy of embodiment, difference lies in pH6.0 is adjusted when, dextrine conversion, is passed through
HPLC analyzes product, conversion ratio 74.3%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
Claims (10)
1. a kind of method preparing alternan oligosaccharides, which is characterized in that using potato starch and sucrose as raw material, add step by step
High temperature acid alpha-amylase, beta amylase, Pullulanase, maltogenic amylase and alternately sucrase enzyme, by gelatinization, liquefaction,
Saccharification and generate alternan oligosaccharides and etc. prepare alternan oligosaccharides.
2. a kind of method preparing alternan oligosaccharides according to claim 1, which is characterized in that the pretreatment refers to horse
Bell sweet potato starch is suspended in the phosphate buffer of 20~200mM, pH 3~7, obtains 5~30% potato starch solution.
3. a kind of method preparing alternan oligosaccharides according to claim 1 or 2, which is characterized in that the gelatinization refers to
1~10min is continuously stirred in boiling water bath makes starch gelatinization.
4. a kind of method preparing alternan oligosaccharides according to claim 1 or 2 or 3, which is characterized in that the liquefaction is
Refer to and 5~100U/g high temperature acid alpha-amylases are added, after continuously stirring 1~10min in boiling water bath, 0~10M hydrochloric acid is added will
PH is adjusted to 2.0~6.0 hereinafter, to terminate liquefaction.
5. a kind of method preparing alternan oligosaccharides according to any one of claims 1 to 4, which is characterized in that the saccharification
Refer to that 5~100U/g beta amylases, 1~100U/g Pullulanases is first added, 5~240h is reacted at 30~80 DEG C；Add 5~
100U/g maltogenic amylases, in 30~80 DEG C of 5~100h of dextrine conversion.
6. according to a kind of any method preparing alternan oligosaccharides of Claims 1 to 5, which is characterized in that the generation
Alternan oligosaccharides is the enzyme deactivation after dextrine conversion, sequentially added into the reaction solution after dextrine conversion 1~30% sucrose and
0.1~100U/g replaces sucrase enzyme, and 5~240h is reacted at 30~80 DEG C.
7. according to a kind of any method preparing alternan oligosaccharides of claim 1~6, which is characterized in that the malt
Saccharogenic amylase is Bacillus stearothermophilus, Bacillus licheniformis, Thermus
Vulgaris, Bacillus cereus or the sources Bacillus subtilis.
8. according to a kind of any method preparing alternan oligosaccharides of claim 1~7, which is characterized in that the alternating
Sucrase enzyme be Leuconostoc citreum, Leuconostoc mesenteroides, Leuconostoc fallax,
The sources Leuconostoc gelidum or Oenococcus oeni.
9. the alternan oligosaccharides being prepared according to any the method for claim 1~8.
10. alternan oligosaccharides described in claim 9 is preparing low glycemic syrup, pharmacy or the application in preparing prebiotics.
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Cited By (1)
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| CN113493812A (en) * | 2020-04-08 | 2021-10-12 | 无锡秋可生物科技有限公司 | Preparation process of oligomeric maltose syrup with high maltotetraose content |
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| CN113493812B (en) * | 2020-04-08 | 2024-01-09 | 无锡秋可生物科技有限公司 | Preparation process of low-polymer maltose syrup with high maltotetraose content |
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