CN105238770A - Method for preparing endoinulinase through in-situ orientation dissolution and process for preparing fructo-oligosaccharide - Google Patents
Method for preparing endoinulinase through in-situ orientation dissolution and process for preparing fructo-oligosaccharide Download PDFInfo
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Abstract
The invention discloses a method for preparing endoinulinase through in-situ orientation dissolution. The method comprises the following steps: mixing a substrate with cellulose with a microorganism generated natural inulase, adding a pH buffer liquid, mixing till the solid-liquid mass ratio is 1g:(7-50)mL, controlling the pH value of the system to be 4-6, adsorbing for 0.5-2 hours at 5-55 DEGC, performing solid-liquid separation on the system, discarding the solid, and retaining the clear liquid, thereby obtaining an endoinulinase liquid. As most endoinulinase in the natural inulase system is removed because of in-situ orientation dissolution, the content of endoinulinase in the natural inulase system is greatly reduced, the yield of fructo-oligosaccharide is increased, the content of monosaccharide free of physiological activity in a product is reduced, and an efficient and low-cost novel method is provided for preparing fructo-oligosaccharide from the microorganism natural inulase.
Description
Technical field
The invention belongs to the microorganism enzymolysis sugar refining field in biochemical industry, be specifically related to inulin raw material after the enzyme biological degradation of microorganism secretion, prepare the technology of functional foodstuff oligofructose.
Background technology
Inulin (inulin), also known as synanthrin, inulin, is natural polymer, is the long-chain polyfructosan be formed by connecting with β-2,1 glycosidic link by D – fructofuranose.Usually its reducing end connects a glucosyl group, and in linear chain structure, the polymerization degree 2 ~ 60, molecular weight is 3000 ~ 5000, and its molecular size is different with floristics, weather condition and harvest season difference.Inulin is slightly soluble in cold water, can dissolve very soon in the hot water, and with iodine not in color reaction, without reductibility, have opticity, be left-handed in water.Inulin is very wide in distributed in nature, is present in plant, marine alga, fungus and bacterium; But be mainly derived from plant, various plants and vegetables are present in form of energy, especially in a large number feverfew is present in, such as, in jerusalem artichoke (JurusalemArtichoke is commonly called as Jerusalem artichoke), witloof (Chicory), Garden Dahlia (dahliapinnata), burdock (arctiumlappa) etc.Industrially much more general from jerusalem artichoke root, extract inulin.
Oligofructose (Fructooligosaccharide is called for short FOS), also known as oligofructose or fructooligosaccharide, is the C of the residue of fructose at sucrose
1the upper oligose be combined into by the fructose of β-1,2-glycosidic link and 1 ~ 3 molecule in position, mainly by kestose (1-kestose, abbreviation GF
2), GF3 (nystose, be called for short GF
3) and GF4 (1-F-β-fructofranosylnystose, abbreviation GF
4) mixture that forms.Oligofructose is extensively present in occurring in nature, as pears, honey, beer, bacterium, yeast, onion, chive, burdock, jerusalem artichoke, witloof, banana, wheat, rye, oat etc. all contain FOS.The content of the oligofructose wherein in inulin is the highest, accounts for about 92% greatly.Oligofructose is the one of functional oligose, and its main physiological function regulates human body intestine microenvironment and activates associated immune system.At present, the method for suitability for industrialized production oligofructose has two kinds.One is that sucrose is prepared through fructose-transferring enzyme catalysis, and product oligomeric fructose is GFn type; Two is utilize inulinase directionally hydrolyzing inulin to prepare, and product oligomeric fructose is the mixed type of GFn and Fm.Prepare in oligofructose at fructose-transferring enzyme catalysing sucrose, byproduct of reaction glucose is the inhibitor of enzyme, low conversion rate, and containing a large amount of dextrose plus saccharose in product, oligofructose only accounts for less than 55 ~ 60%, and complex process cost is high.It is a step list enzyme reaction that inulinase directionally hydrolyzing inulin prepares oligofructose, and technique is simple, and in product, oligofructose content is up to more than 80%.Therefore, be that material, enzyme method one one-step hydrolysis inulin wherein produces oligofructose with jerusalem artichoke, have the advantages that technique is simple, transformation efficiency is high, by product is few.
Inulinase is the compound enzyme of a class catalysis hydrolysis of inulin, is made up of endoinulase (Endoinulinase) and exoinulinase (Exoinulinase).Inulin is thoroughly degraded into monose under the synergy of endoinulase and exoinulinase in inulinase system.The mechanism of inulinase hydrolytic inulin is that first Inulin polysaccharide cuts off β-2 at endoinulase from inulin molecules internal random, 1-fructofuranose glycosidic bond, obtain oligofructose, then exoinulinase is hydrolyzed β-D fructose glycosidic bond successively from the non-reducing end of oligofructose molecule, generate the Polylevulosan of a part fructose and a few fructose molecule, final product is fructose and glucose.Can find out from inulinase hydrolytic inulin mechanism, oligofructose is the intermediate product that hydrolysis of inulin becomes fructose and glucose process, the therefore accumulation of intermediate product oligofructose in controlled hydrolysis process, is the key obtaining high yield pulp1 oligofructose.Be not difficult to find out from inulinase Synergistic Mechanisms, if the amount of exoinulinase is lower in inulinase enzyme system, then in hydrolysis of inulin process intermediate product oligofructose to be hydrolyzed into the amount of fructose and glucose further then fewer, hydrolysis of inulin becomes the yield of oligofructose higher.
Microorganism that great majority have industrial application potentiality, that can synthesize inulinase, all contains the exoinulinase of some amount in the inulinase enzyme system of its secretion.And the preparation of oligofructose, be that its exoinulinase content (or vigor) is more low better to the requirement of inulinase, namely require the inulinase of low exoinulinase vigor.In order in the technique of On Fructo-Oligosaccharides Produc Tion By Microbial Enzymatic Reaction, reduce the vigor of exoinulinase as much as possible, mainly take following approach conduct a research and make some progress both at home and abroad:
(1) adopt physics, chemomorphosis or engineered method transformation microorganism, to obtaining new mutant strain or engineering bacteria, they can secrete the inulinase enzyme system of low exoinulinase content;
(2) culture condition of microorganism is changed by the means of regulation and control fermentation, to reduce the secretion of microorganism to exoinulinase;
Summary of the invention
Technical problem to be solved by this invention is the deficiency of inulinase when inulin of degrading prepares oligofructose for general microorganism secretion, finds a kind of novel method being prepared endoinulase by the natural inulinase system of production by biological.
The technical problem that the present invention also will solve aforesaid method is applied to the novel process that enzyme process prepares oligofructose.
Research shows, occurring in nature most of microbe to produce inulinase be the mixed enzyme be made up of endoinulase and exoinulinase, and the high 5 prime excision enzyme activity of most performance.Inulin is thoroughly degraded into monose under the synergy of endoinulase and exoinulinase in inulinase system, the mechanism of inulinase hydrolytic inulin is that first Inulin polysaccharide mainly forms oligofructose under the effect of restriction endonuclease, and oligofructose is thoroughly degraded into fructose and glucose then under the effect of excision enzyme.Therefore, be that target product prepared by raw material is different with inulin, different to the requirement of the enzyme architecture of inulinase, be that in the processing requirement inulinase system of target product, restriction endonuclease and excision enzyme ratio suitably, are conducive to synergy with fructose; And be that in the processing requirement inulinase system of target product, excision enzyme content is as far as possible low with oligofructose, with the hydrolysis yield improving oligofructose and the contents of monosaccharides reduced without physiologically active in hydrolysate.
Therefore, the inulinase screening low excision enzyme vigor is produced bacterial strain or found in hydrolysis of inulin process the endoinulase preparation method suppressing the method for excision enzyme vigor or exploitation to be applicable to industrially scalable will be the key utilizing inulin to produce oligofructose.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of original position orientation splits the method preparing endoinulase, natural to the substrate of cellulose and production by biological inulinase is mixed, adding pH damping fluid, to be mixed to solid-liquid mass ratio be 1g: 7 ~ 50mL, system pH controls 4 ~ 6, under 5 ~ 55 DEG C of conditions, adsorb 0.5 ~ 2h, system, through solid-liquid separation, discards solid substance, retain clear liquid, obtain endoinulase liquid.
Wherein, the substrate of described cellulose is any one or several combinations in the paper pulp of xylooligosaccharides production waste residue, Microcrystalline Cellulose, absorbent cotton and cellulose.The substrate of above-mentioned cellulose can carry out pre-treatment and re-use, and pretreated object is that in the substrate of raising cellulose, Mierocrystalline cellulose, to the accessibility of inulinase, can adopt the pretreatment process of physics, chemistry, biology or more several method combined utilization.
Wherein, the natural inulinase of described production by biological is the inulinase secreted by Aspergillus (Aspergillussp.) or Penicillium (Penicilliumsp.).
Wherein, described pH damping fluid is the acetic acid/sodium acetate buffer of 0.1M.
Wherein, described solid-liquid separation, its means are centrifugal or Plate Filtration.
Prepare a technique for oligofructose, it comprises the steps:
(1) the substrate original position orientation of cellulose splits: mixed by natural to the substrate of cellulose and production by biological inulinase, adding pH damping fluid, to be mixed to solid-liquid mass ratio be 1g: 7 ~ 50mL, system pH controls 4 ~ 6,0.5 ~ 2h is adsorbed under 5 ~ 55 DEG C of conditions, system is through solid-liquid separation, discard solid substance, retain clear liquid, obtain endoinulase liquid;
(2) oligofructose is prepared in hydrolysis: in the endoinulase liquid obtain step (1), add inulin and pH damping fluid, solid-liquid mass ratio is made to be 1g: 7 ~ 50mL, pH value controls 4 ~ 6, the ratio control of the work of endoinulase enzyme and inulin quality is at 1 ~ 50IU/g, at the Water Under solution 24 ~ 72h of 45 ~ 55 DEG C, hydrolysed mix is carried out solid-liquid separation, and the clear liquid obtained is oligofructose solution.
In step (1), the substrate of described cellulose is any one or several combinations in the paper pulp of xylooligosaccharides production waste residue, Microcrystalline Cellulose, absorbent cotton and cellulose.The substrate of above-mentioned cellulose can carry out pre-treatment and re-use, and pretreated object is that in the substrate of raising cellulose, Mierocrystalline cellulose, to the accessibility of inulinase, can adopt the pretreatment process of physics, chemistry, biology or more several method combined utilization.
In step (1), the natural inulinase of described production by biological is the inulinase secreted by Aspergillus (Aspergillussp.) or Penicillium (Penicilliumsp.).Inulinase unit of force (IU/mL) of living is defined as the enzyme amount that per minute under standard reaction condition generates 1 μm of ol reducing sugar.
In step (1), preferably adding pH damping fluid, to be mixed to solid-liquid mass ratio be 1g: 10mL.
In step (1) and (2), described pH damping fluid is the acetic acid/sodium acetate buffer of 0.1M.
In step (2), preferably add inulin and pH damping fluid, make solid-liquid mass ratio be 1g: 50mL.
In step (1) or (2), described solid-liquid separation, its means are centrifugal or Plate Filtration.
Beneficial effect: after the present invention adopts the substrate original position of cellulose orientation to split the exoinulinase in natural inulinase system, because the exoinulinase vigor in hydrolyzation system significantly reduces, improve prepare oligofructose selectivity, thus the content that inulin is degraded into oligofructose in the yield of oligofructose and product is improved further, reduce the content without the monose (fructose) of physiologically active in product, for the natural product inulinase of microorganism prepares the new way that oligofructose technique provides efficient a, low cost.
Accompanying drawing explanation
Fig. 1 is substrate kind to the influence curve of substrate adsorbs endoinulase and exoinulinase, wherein, and 1-paper pulp, 2-xylooligosaccharides production waste residue, 3-Microcrystalline Cellulose, 4-absorbent cotton, 5-inulin; Its experiment condition is: substrate add-on is 10g/100mL, enzyme dosage 0.1mL, pH value 4.6, adsorption time 1.5h, and adsorption temp is 4 DEG C;
Fig. 2 is adsorption time to the influence curve of substrate adsorbs endoinulase and exoinulinase, and its experiment condition is: substrate add-on is 10g/100mL, enzyme dosage 0.1mL, pH value 4.6, and temperature is 4 DEG C;
Fig. 3 is temperature to the influence curve of substrate adsorbs endoinulase and exoinulinase, and its experiment condition is: substrate add-on is 10g/100mL, enzyme dosage 0.1mL, pH value 4.6, adsorption time 1h.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1: obtain natural inulinase with aspergillus niger CBS513.88 preparation.
1) aspergillus niger (A.niger) activation culture based component (g/L): glucose 10.0; Peptone 1.0; Ammonium sulfate 1.4; Urea 0.3; Potassium primary phosphate 2.0; Calcium dichloride dihydrate 0.4; Magnesium sulfate heptahydrate 0.3; Iron vitriol 0.005; Manganese sulfate monohydrate 0.0016; Zinc Sulphate Heptahydrate 0.0014; Cobalt chloride 0.0037; Tween802 drips/50mL.
2) aspergillus niger (A.niger) Inulinase production medium component (g/L): glucose 1.0; Inulin 15.0; SODIUMNITRATE 0.6; Potassium primary phosphate 2.0; Calcium Chloride Powder Anhydrous 0.3; Magnesium sulfate heptahydrate 0.4; Iron vitriol 0.005; Manganese sulfate monohydrate 0.0016; Zinc Sulphate Heptahydrate 0.0014; Initial pH value 4.6.
3) activation culture of aspergillus niger: 50mL Mycelium culture base is placed in 250mL triangular flask, sterilizing 30min at 121 DEG C, be cooled to room temperature, access is preserved in the aspergillus niger spore of test tube slant in right amount, shaking flask be placed in constant-temperature table in 30 ± 3 DEG C, cultivate 24h under 170rpm condition after for producing enzyme.
4) preparation of the natural inulinase of aspergillus niger: 50mL culture medium is in 250mL triangular flask, at 121 DEG C, sterilizing 30min, is cooled to room temperature, by 10% inoculum size access seed, cultivate in the shaking table of 170rpm, after 1d temperature 30 ± 3 DEG C, 1d, temperature controls at 28 ± 3 DEG C.
5) cultivate 3d, the solid matter in nutrient solution is separated removing, i.e. inulinase liquid with whizzer centrifugal 10min under 3000rpm condition.
The mensuration of endoinulase vigor: add enzyme liquid 0.9mL and 5% (w/v) the inulin suspension (acetic acid/sodium acetate buffer preparation with 0.1mol/L, pH4.6) that 0.1mL suitably dilutes in 25mL scale test tube, cover plastic cloth, tightening with bungee is placed in thermostatical water bath, in amplitude 80,30min is incubated at 55 DEG C, take out, add 3mLDNS reagent, 5min is boiled in 100 DEG C of boiling water, after being cooled to room temperature, add water and be settled to 25mL, after fully shaking up, under 520nm wavelength, measure absorbance A value.The quantity of fructose that reaction generates is tried to achieve according to fructose typical curve.Enzyme liquid is replaced to make blank with 0.1mL distilled water.Each sample does 2 ~ 3 Duplicate Samples, averages.Inulinase unit of force (IU/mL) of living is defined as the enzyme amount that per minute under standard reaction condition generates 1 μm of ol reducing sugar.
The mensuration of exoinulinase vigor: add enzyme liquid 0.9mL and 5% (w/v) the sucrose liquid (acetic acid/sodium acetate buffer preparation with 0.1mol/L, pH4.6) that 0.1mL suitably dilutes in 25mL scale test tube, cover plastic cloth, tightening with bungee is placed in thermostatical water bath, in amplitude 80,30min is incubated at 55 DEG C, take out, add 3mLDNS reagent, 5min is boiled in 100 DEG C of boiling water, after being cooled to room temperature, add water and be settled to 25mL, after fully shaking up, under 520nm wavelength, measure absorbance A value.The quantity of fructose that reaction generates is tried to achieve according to fructose typical curve.Enzyme liquid is replaced to make blank with 0.1mL distilled water.Each sample does 2 ~ 3 Duplicate Samples, averages.Inulinase unit of force (IU/mL) of living is defined as the enzyme amount that per minute under standard reaction condition generates 1 μm of ol reducing sugar.
Result shows, aspergillus niger is that carbon source synthesizes inulinase with inulin, and cultivate 3d, endoinulase vigor is 6.94IU/mL, and exoinulinase vigor is 0.78IU/mL.
Embodiment 2: obtain natural inulinase with mould TN-88 preparation.
1) mould (Penicilliumsp.) activation culture based component (g/L): glucose 10.0; Peptone 1.0; Ammonium sulfate 1.4; Urea 0.3; Potassium primary phosphate 2.0; Calcium dichloride dihydrate 0.4; Magnesium sulfate heptahydrate 0.3; Iron vitriol 0.005; Manganese sulfate monohydrate 0.0016; Zinc Sulphate Heptahydrate 0.0014; Cobalt chloride 0.0037; Tween802 drips/50mL.
2) mould (Penicilliumsp.) Inulinase production medium component (g/L): glucose 1.0; Inulin 15.0; Peptone 3; Potassium primary phosphate 2.0; Calcium Chloride Powder Anhydrous 0.3; Magnesium sulfate heptahydrate 0.4; Iron vitriol 0.005; Manganese sulfate monohydrate 0.0016; Zinc Sulphate Heptahydrate 0.0014; Initial pH value 4.6.
3) activation culture of mould: 50mL Mycelium culture base is placed in 250mL triangular flask, sterilizing 30min at 121 DEG C, be cooled to room temperature, access is preserved in the Penicillium notatum of test tube slant in right amount, shaking flask be placed in constant-temperature table in 30 ± 3 DEG C, cultivate 24h under 170rpm condition after for producing enzyme.
4) preparation of the natural inulinase of mould: 50mL culture medium is in 250mL triangular flask, at 121 DEG C, sterilizing 30min, is cooled to room temperature, by 10% inoculum size access seed, cultivate in the shaking table of 170rpm, after 1d temperature 30 ± 3 DEG C, 1d, temperature controls at 28 ± 3 DEG C.
5) cultivate 3d, the solid matter in nutrient solution is separated removing, i.e. inulinase liquid with whizzer centrifugal 10min under 3000rpm condition.
The mensuration of endoinulase vigor and the mensuration of exoinulinase vigor are with embodiment 1.
Result shows, mould is that carbon source synthesizes inulinase with inulin, and cultivate 3d, endoinulase vigor is 5.45IU/mL, and exoinulinase vigor is 1.24IU/mL.
Embodiment 3: the substrate original position orientation of cellulose splits the impact of endoinulase and exoinulinase in the natural inulinase system of Aspergillus Niger.
1) xylooligosaccharides production waste residue, Microcrystalline Cellulose, the paper pulp of cellulose, absorbent cotton and inulin 2.0g is taken respectively in 50mL triangular flask, add inulinase liquid 0.1mL, add pH damping fluid, controlling absorption system water volume is about 20mL, pH4.6, is placed in constant temperature oscillation water-bath and adsorbs 0.5 ~ 2h under pH value about 4.6, temperature 10 ~ 50 DEG C of conditions.
2) after absorption terminates, centrifugal 10min under 3000rpm, obtain clear liquid, measure endoinulase vigor and exoinulinase vigor in liquid phase, the substrate calculating different cellulose to the adsorption rate of endoinulase and exoinulinase vigor, and is that the result that adsorbate contrasts contrasts with inulin.Adsorbate substrate kind, adsorption time and temperature affect result as accompanying drawing 1 ~ 3 to adsorption rate.
The adsorption rate method of calculation of substrate to endoinulase and exoinulinase of cellulose are:
Adsorption rate (%)=[(in initial enzyme activity-absorption system liquid phase enzyme activity)/initial enzyme activity] × 100
Wherein in initial enzyme activity, absorption system liquid phase, enzyme activity represents with the total activity contained, unit IU.
Accompanying drawing 1 ~ 3 shows, the adsorption of substrate on the endoinulase in inulinase system and exoinulinase of cellulose is less by adsorption conditions (temperature, time) impact.With maximum to exoinulinase adsorption rate and minimum for inspection target to endoinulase adsorption rate, optimum adsorption conditions: the substrate of cellulose is paper pulp, temperature 50 C, the adsorption time 1h of the pretreated cellulose of sulphite.With this understanding, the adsorption rate of paper pulp to endoinulase and exoinulinase is respectively 2.57% and 97.10%.Solid-liquid separation after absorption, a large amount of exoinulinase can be directed removal with the separation of solid substance.
Table 1 paper pulp is that the original position orientation of substrate to endoinulase and exoinulinase in natural black aspergillus inulinase system splits
| Endoinulase vigor | Exoinulinase vigor | |
| Before absorption | 0 | 0 |
| After absorption | 2.57％ | 97.10％ |
Paper pulp is that original position orientation split result (with substrate adsorption rate to enzyme represent) of substrate to endoinulase and exoinulinase in natural inulinase system is as table 1.
Embodiment 4: the substrate original position orientation of cellulose splits the impact that mould produces endoinulase and exoinulinase in natural inulinase system.
1) xylooligosaccharides production waste residue, Microcrystalline Cellulose, the paper pulp of cellulose, absorbent cotton and inulin 2.0g is taken respectively in 50mL triangular flask, add inulinase liquid 0.1mL, add damping fluid, controlling absorption system water volume is about 20mL, pH4.6, is placed in constant temperature oscillation water-bath and adsorbs 0.5 ~ 2h under pH value about 4.6, temperature 10 ~ 50 DEG C of conditions.
2) after absorption terminates, centrifugal 10min under 3000rpm, obtain clear liquid, measure endoinulase vigor and exoinulinase vigor in liquid phase, the substrate calculating different cellulose to the adsorption rate of endoinulase and exoinulinase vigor, and is that the result that adsorbate contrasts contrasts with inulin.
The adsorption rate method of calculation of substrate to endoinulase and exoinulinase of cellulose are:
Adsorption rate (%)=[(in initial enzyme activity-absorption system liquid phase enzyme activity)/initial enzyme activity] × 100
Wherein in initial enzyme activity, absorption system liquid phase, enzyme activity represents with the total activity contained, unit IU.
Equally, the adsorption of substrate on the endoinulase in inulinase system and exoinulinase of cellulose is less by adsorption conditions (temperature, time) impact.With maximum to exoinulinase adsorption rate and minimum for inspection target to endoinulase adsorption rate, optimum adsorption conditions: the substrate of cellulose is paper pulp, temperature 50 C, adsorption time 1h.With this understanding, the adsorption rate of the paper pulp that sulphite is pretreated to endoinulase and exoinulinase is respectively 3.08% and 95.39%.Solid-liquid separation after absorption, a large amount of exoinulinase can be directed removal with the separation of solid substance.
Paper pulp is that original position orientation split result (with substrate adsorption rate to enzyme represent) of substrate to endoinulase and exoinulinase in natural black aspergillus inulinase system is as table 2.
The pretreated paper pulp of table 2 sulphite is that the original position orientation of substrate to endoinulase and exoinulinase in natural inulinase system splits
| Endoinulase vigor | Exoinulinase vigor | |
| Before absorption | 0 | 0 |
| After absorption | 3.08％ | 95.39％ |
Embodiment 5: utilize original position orientation to split the aspergillus niger endoinulase hydrolytic inulin obtained and prepare oligofructose.
1) the substrate original position orientation of cellulose splits: take paper pulp 2g in 50mL triangular flask, add natural black aspergillus inulinase enzyme liquid 0.1mL, add 0.1M acetic acid/sodium acetate buffer 19.9mL, make absorption system pH value be 4.6.The thermostat water bath being placed in 50 DEG C adsorbs 1h.
2) solid-liquid separation: after above-mentioned system has been adsorbed, under 3000rpm, centrifugal 10min, obtains clear liquid, discards solid substance.
3) oligofructose is prepared in hydrolysis: get the supernatant liquor 0.1mL after above-mentioned centrifugation, add 0.1M acetic acid/sodium acetate buffer 19.9mL, and add inulin 20g/L, the enzyme concentration of endoinulase is 1.5IU/g inulin, abundant mixing, makes pH value of reaction system be 4.6.Be placed in 55 DEG C, the shaking table of 170rpm is hydrolyzed 24h, hydrolysis terminates rear hydrolyzate centrifugal 10min under 3000rpm condition, and the clear liquid obtained is oligofructose hydrolysis sugar liquid.Detect by analysis, oligofructose hydrolysis yield is 96.13%, and oligofructose selective hydrolysis is 92.19%.
Oligofructose hydrolysis yield method of calculation are:
Oligofructose hydrolysis yield (%)=(C
1/ C) × 100
Oligofructose selective hydrolysis method of calculation are:
Oligofructose selective hydrolysis (%)=[C
1/ C
1+ (C
2+ C
3) × 0.9+C
4) × 100
Wherein: the initial inulin content of C--, g/L
C
1--oligofructose content in enzymolysis solution after enzymolysis, g/L
C
2--fructose content in enzymolysis solution after enzymolysis, g/L
C
3--glucose content in enzymolysis solution after enzymolysis, g/L
C
4--sucrose content in enzymolysis solution after enzymolysis, g/L
The gain factor of 0.9--glycan and monose.
Comparative example 1: the aspergillus niger inulinase hydrolytic inulin that the substrate original position orientation without cellulose splits exoinulinase prepares oligofructose
Oligofructose is prepared with the inulinase hydrolytic inulin that aspergillus niger preparation obtains in direct use embodiment 1.Experimental technique is as follows:
Take inulin 0.4g in 50mL triangular flask, add natural black aspergillus inulinase enzyme liquid 0.1mL, add 0.1mol/L citrate buffer solution 19.9mL, the enzyme concentration of endoinulase is 1.5IU/g inulin, fully mixes, and makes pH value of reaction system be 4.6.Be placed in 55 DEG C, the shaking table of 170rpm is hydrolyzed 24h, hydrolysis terminates rear reactant centrifugal 10min under 3000rpm condition, and the clear liquid obtained is oligofructose hydrolysis sugar liquid.Detect by analysis, oligofructose hydrolysis yield is 30.60%, and oligofructose selective hydrolysis is 32.67%.
Oligofructose hydrolysis yield method of calculation are:
Oligofructose hydrolysis yield (%)=(C
1/ C) × 100
Oligofructose selective hydrolysis method of calculation are:
Oligofructose selective hydrolysis (%)=[C
1/ C
1+ (C
2+ C
3) × 0.9+C
4) × 100
Wherein: the initial inulin content of C--, g/L
C
1--oligofructose content in enzymolysis solution after enzymolysis, g/L
C
2--fructose content in enzymolysis solution after enzymolysis, g/L
C
3--glucose content in enzymolysis solution after enzymolysis, g/L
C
4--sucrose content in enzymolysis solution after enzymolysis, g/L
The gain factor of 0.9--glycan and monose.
Result shows: the low circumscribed aspergillus niger inulinase vigor inulinase obtained after the substrate original position orientation of cellulose splits exoinulinase, significantly improves the selectivity that oligofructose is prepared in hydrolysis.With the former inulinase enzyme liquid phase ratio of non-deconsolidation process, in hydrolysate, the selectivity of oligofructose brings up to 92.19% from 32.67% before fractionation.
Embodiment 6: utilize original position orientation to split the mould endoinulase hydrolytic inulin obtained and prepare oligofructose.
1) the substrate original position orientation of cellulose splits: take paper pulp 2g in 50mL triangular flask, add natural mould inulinase enzyme liquid 0.1mL, add 0.1M ethyl sodium damping fluid 19.9mL, make absorption system pH value be 4.6.The thermostat water bath being placed in 50 DEG C adsorbs 1h.
2) solid-liquid separation: after above-mentioned system has been adsorbed, under 3000rpm, centrifugal 10min, obtains clear liquid, discards solid substance.
3) oligofructose is prepared in hydrolysis: get the supernatant liquor 0.1mL after above-mentioned centrifugation, add 0.1M acetic acid/sodium acetate buffer 19.9mL, and add inulin 20g/L, the enzyme concentration of endoinulase is 1.5IU/g inulin, abundant mixing, makes pH value of reaction system be 4.6.Be placed in 55 DEG C, the shaking table of 170rpm is hydrolyzed 24h, hydrolysis terminates rear hydrolyzate centrifugal 10min under 3000rpm condition, and the clear liquid obtained is oligofructose hydrolysis sugar liquid.Detect by analysis, oligofructose hydrolysis yield is 95.25%, and oligofructose selective hydrolysis is 90.23%.
Oligofructose hydrolysis yield method of calculation are:
Oligofructose hydrolysis yield (%)=(C
1/ C) × 100
Oligofructose selective hydrolysis method of calculation are:
Oligofructose selective hydrolysis (%)=[C
1/ C
1+ (C
2+ C
3) × 0.9+C
4) × 100
Wherein: the initial inulin content of C--, g/L
C
1--oligofructose content in enzymolysis solution after enzymolysis, g/L
C
2--fructose content in enzymolysis solution after enzymolysis, g/L
C
3--glucose content in enzymolysis solution after enzymolysis, g/L.
Comparative example 2: the mould inulinase hydrolytic inulin that the substrate original position orientation without cellulose splits exoinulinase prepares oligofructose.
Oligofructose is prepared with the inulinase hydrolytic inulin that mould preparation obtains in direct use embodiment 2.Experimental technique is as follows:
Take inulin 0.4g in 50mL triangular flask, add mould and produce natural inulinase enzyme liquid 0.1mL, add 0.1mol/L citrate buffer solution 19.9mL, fully mix, make pH value of reaction system be 4.6.Be placed in 55 DEG C, the shaking table of 170rpm is hydrolyzed 24h, hydrolysis terminates rear reactant centrifugal 10min under 3000rpm condition, and the clear liquid obtained is oligofructose hydrolysis sugar liquid.Detect by analysis, oligofructose hydrolysis yield is 28.74%, and oligofructose selective hydrolysis is 30.29%.
Oligofructose hydrolysis yield method of calculation are:
Oligofructose hydrolysis yield (%)=(C
1/ C) × 100
Oligofructose selective hydrolysis method of calculation are:
Oligofructose selective hydrolysis (%)=[C
1/ C
1+ (C
2+ C
3) × 0.9+C
4) × 100
Wherein: the initial inulin content of C--, g/L
C
1--oligofructose content in enzymolysis solution after enzymolysis, g/L
C
2--fructose content in enzymolysis solution after enzymolysis, g/L
C
3--glucose content in enzymolysis solution after enzymolysis, g/L
Result shows: the low circumscribed mould inulinase vigor inulinase obtained after the substrate original position orientation of cellulose splits exoinulinase, significantly improves the selectivity that oligofructose is prepared in hydrolysis.With the former inulinase enzyme liquid phase ratio of non-deconsolidation process, in hydrolysate, the selectivity of oligofructose brings up to 90.23% from 30.29% before fractionation.
Claims (10)
1. an original position orientation splits the method preparing endoinulase, it is characterized in that, natural to the substrate of cellulose and production by biological inulinase is mixed, adding pH damping fluid, to be mixed to solid-liquid mass ratio be 1g: 7 ~ 50mL, and system pH controls 4 ~ 6, under 5 ~ 55 DEG C of conditions, adsorb 0.5 ~ 2h, system is through solid-liquid separation, discard solid substance, retain clear liquid, obtain endoinulase liquid.
2. original position orientation according to claim 1 splits the method preparing endoinulase, it is characterized in that, the substrate of described cellulose is any one or several combinations in the paper pulp of xylooligosaccharides production waste residue, Microcrystalline Cellulose, absorbent cotton and cellulose.
3. original position orientation according to claim 1 splits the method preparing endoinulase, it is characterized in that, the natural inulinase of described production by biological is the inulinase secreted by Aspergillus (Aspergillussp.) or Penicillium (Penicilliumsp.).
4. original position orientation according to claim 1 splits the method preparing endoinulase, and it is characterized in that, described pH damping fluid is the acetic acid/sodium acetate buffer of 0.1M.
5. original position orientation according to claim 1 splits the method preparing endoinulase, and it is characterized in that, described solid-liquid separation, its means are centrifugal or Plate Filtration.
6. prepare a technique for oligofructose, it is characterized in that, it comprises the steps:
(1) the substrate original position orientation of cellulose splits: mixed by natural to the substrate of cellulose and production by biological inulinase, adding pH damping fluid, to be mixed to solid-liquid mass ratio be 1g: 7 ~ 50mL, system pH controls 4 ~ 6,0.5 ~ 2h is adsorbed under 5 ~ 55 DEG C of conditions, system is through solid-liquid separation, discard solid substance, retain clear liquid, obtain endoinulase liquid;
(2) oligofructose is prepared in hydrolysis: in the endoinulase liquid obtain step (1), add inulin and pH damping fluid, solid-liquid mass ratio is made to be 1g: 7 ~ 50mL, pH value controls 4 ~ 6, the ratio control of the work of endoinulase enzyme and inulin quality is at 1 ~ 50IU/g, at the Water Under solution 24 ~ 72h of 45 ~ 55 DEG C, hydrolysed mix is carried out solid-liquid separation, and the clear liquid obtained is oligofructose solution.
7. the technique preparing oligofructose according to claim 6, it is characterized in that, in step (1), the substrate of described cellulose is any one or several combinations in the paper pulp of xylooligosaccharides production waste residue, Microcrystalline Cellulose, absorbent cotton and cellulose.
8. the technique preparing oligofructose according to claim 7, it is characterized in that, in step (1), the natural inulinase of described production by biological is the inulinase secreted by Aspergillus (Aspergillussp.) or Penicillium (Penicilliumsp.).
9. the technique preparing oligofructose according to claim 7, is characterized in that, in step (1) and (2), described pH damping fluid is the acetic acid/sodium acetate buffer of 0.1M.
10. the technique preparing oligofructose according to claim 7, is characterized in that, in step (1) or (2), described solid-liquid separation, its means are centrifugal or Plate Filtration.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591264A (en) * | 2017-03-02 | 2017-04-26 | 福建省农业科学院农业工程技术研究所 | Endoglucanase promotion factor |
| CN106591260A (en) * | 2017-03-09 | 2017-04-26 | 南京林业大学 | Novel method for directional preparation of endoinulinase |
| CN116326684A (en) * | 2021-12-24 | 2023-06-27 | 中国科学院青岛生物能源与过程研究所 | Oligosaccharide composition and application thereof in regulating animal intestinal metabolites |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341709A (en) * | 2001-09-11 | 2002-03-27 | 湖北大学 | Yeast gene engineering bacterium and endoinulase preparation and its application method |
| CN101693910A (en) * | 2009-10-15 | 2010-04-14 | 南京林业大学 | New process for producing cellooligosaccharides by microbe enzyme method |
| CN101914596A (en) * | 2010-02-10 | 2010-12-15 | 南京林业大学 | Method for improving yield of fiber oligosaccharide prepared by enzyme method |
| CN104928330A (en) * | 2015-07-06 | 2015-09-23 | 青海威德生物技术有限公司 | Fructo-oligosaccharide producing method with fossilized endoinulinase in bacterial cellulose |
-
2015
- 2015-11-13 CN CN201510780545.3A patent/CN105238770B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341709A (en) * | 2001-09-11 | 2002-03-27 | 湖北大学 | Yeast gene engineering bacterium and endoinulase preparation and its application method |
| CN101693910A (en) * | 2009-10-15 | 2010-04-14 | 南京林业大学 | New process for producing cellooligosaccharides by microbe enzyme method |
| CN101914596A (en) * | 2010-02-10 | 2010-12-15 | 南京林业大学 | Method for improving yield of fiber oligosaccharide prepared by enzyme method |
| CN104928330A (en) * | 2015-07-06 | 2015-09-23 | 青海威德生物技术有限公司 | Fructo-oligosaccharide producing method with fossilized endoinulinase in bacterial cellulose |
Non-Patent Citations (6)
| Title |
|---|
| TAKAO UCHIYAMA等: "PURIFICATION AND PROPERTIES OF ARTHROBACTER UREAFACIENS INULASE II", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| 刘超纲等: "选择性吸附纤维素酶分离纯化木聚糖酶", 《中南林学院学报》 * |
| 庄蕾: "曲霉L22纤维素酶的分离纯化及性质研究", 《山东大学硕士学位论文》 * |
| 张国青等: "毛壳霉内切菊粉酶的纯化与性质", 《微生物学报》 * |
| 杜明等: "《极端环境中的酶科学与技术》", 31 March 2014, 哈尔滨工业大学出版社 * |
| 郑重: "内切菊粉酶的制备、纯化及应用研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591264A (en) * | 2017-03-02 | 2017-04-26 | 福建省农业科学院农业工程技术研究所 | Endoglucanase promotion factor |
| CN106591260A (en) * | 2017-03-09 | 2017-04-26 | 南京林业大学 | Novel method for directional preparation of endoinulinase |
| CN106591260B (en) * | 2017-03-09 | 2019-08-06 | 南京林业大学 | A kind of novel beam system for endoinulase method |
| CN116326684A (en) * | 2021-12-24 | 2023-06-27 | 中国科学院青岛生物能源与过程研究所 | Oligosaccharide composition and application thereof in regulating animal intestinal metabolites |
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