CN100510094C - Production method of konjak mannose using cellulase - Google Patents
Production method of konjak mannose using cellulase Download PDFInfo
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- CN100510094C CN100510094C CNB2006100191729A CN200610019172A CN100510094C CN 100510094 C CN100510094 C CN 100510094C CN B2006100191729 A CNB2006100191729 A CN B2006100191729A CN 200610019172 A CN200610019172 A CN 200610019172A CN 100510094 C CN100510094 C CN 100510094C
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- cellulase
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- mannooligo saccharide
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The present invention relates to a production method for preparing konjak mannoligose by using cellulase. Its technological process includes the following several procedures: enzymolysis, filtraction, decoloring, concentration and drying. It is characterized by that said invention adopts non-specific commercial enzyme (including cellulose and hemicellulase), and utilizes centrifugal filtering and membrane filtering combined process, so that said invention can obtain high-purity konjak mannoligose. Besides, said invention also provides the concrete requirements and steps of the above-mentioned every procedure.
Description
Technical field
The present invention relates to microorganism enzymolysis engineering, belong to functional oligosaccharide enzymolysis process production technology.
Technical background
The general name of oligosaccharides (oligose) the little polymkeric substance that to be 2-10 monose be formed by connecting by the glycosidic link of certain position.Mannooligo saccharide (or claiming oligo-glucomannan) is the zymolyte of mannosans (konjac glucomanna), extensively is present in konjaku, guar gum, sesbania gum and the multiple microorganism wall.Mannooligo saccharide has the absorption enteric pathogenic bacteria as a kind of novel little ecologic effect additive and brings into play the dual-use function of immunoregulation effect, is one of most promising microbiotic substitute.Simultaneously, this product all has purposes quite widely in industries such as food medicine.
1, be used for fodder additives, requirement was more than 200,000 tons in domestic year.
Studies show that in a large number mannooligo saccharide can be widely used in various livestock and poultry, aquaculture.Can activate and improve the immunizing power of animal, the quantity discharged of nitrogen prevents to pollute in the incidence of reduction gastrointestinal tract disease and animal dead rate raising day weight gain and feed conversion rate, the reduction ight soil, improves the growing environment of animal.According to relevant departments' statistics, 2004, average addition 0.1% reckoning in the perfect compound feed was pressed in 9,500 ten thousand tons of China's perfect compound feed ultimate productions, 1,500 ten thousand tons of concentrated fodders, 5,000,000 tons of Preblendes, and the demand of mannooligo saccharide is more than 200,000 tons.
2, huge at (health care) food, pharmaceutically application potential, have vast market prospect.
Mannooligo saccharide can significantly improve the human body micro-ecological environment, strengthens the human immunological competence, promotes people's physical and mental health.According to related data, prove that through clinical trial that mannooligo saccharide has is significantly hypotensive, hypoglycemic, cholesterol regulating, prevent cardiovascular and cerebrovascular diseases, and effects such as anti-curing oncoma, relief of constipation, simultaneously, also can be used for fat-reducing.Has huge development potentiality at medicine and protective foods application facet.
At present, domestic production and development kanjak mannan oligosaccharides are mainly realized by polysaccharide hydrolysis chemical method or biologic enzymolysis method.Hydrolysis chemistry law part requires harsh; equipment corrosion is serious; environmental pollution is outstanding; and the biological enzymolysis rule can overcome these shortcomings well; be ideal approach comparatively, but be that the technical study of feedstock production mannooligo saccharide is domestic at the early-stage with the konjaku that the major technique of employing all is with degrade mannosans in the konjaku of mannase; its cost height, efficient are low, can not reach the economy and the technical requirements of scale preparation kanjak mannan oligosaccharides.
Pertinent literature:
Relate to a kind of neutral beta-mannase enzyme liberating konjaku powder production Portugal sweet dew melon and fruit sugar technology, by inoculation Bacillus sp.M10 seed liquor in culture medium selected, produce enzyme incident bottom fermentation in optimization and obtain crude enzyme liquid, again this crude enzyme liquid is used for the enzymolysis konjaku powder, obtains the oligo-glucomannan syrupy product.
Document 2, " preparation, the chemical structure of konjaku Portugal village dew oligosaccharides reach the influence to pancreas islet NO free radical burst size " quick Lv Xiu chrysanthemum of Chen Xiu etc., biochemical engineering National Key Laboratory of Chinese Academy Of Sciences Process Engineering Research Institute, Chinese biological chemistry and molecular biosciences journal .2002,18 (6)
Konjaku powder is after the 'beta '-mannase enzymolysis becomes oligosaccharides, carry out separation and purification with activated carbon column, with different concns (5,10,20) ethanol elution, study different elution fractions and streptozotocin (STZ) is induced the influence of the pancreas islet NO free radical burst size of diabetes model, find ling/ml] can make NO free radical burst size in the pancreas islet nutrient solution 25.4 (P＜=0.05) that on average descend with the oligosaccharides of 5 ethanol elutions, 0.1mg/ml makes NO free radical level 20 (P＜=0.05) that descend with the oligosaccharides of 5 ethanol elutions.
And people such as Zhang Yingqing have proposed to utilize cellulase to prepare the technical scheme of Konjac Glucomannan.
Document 3, " cellulase prepares Konjac Glucomannan "/Zhang Yingqing do letter Xie Bijun, food science and technology institute of Hua Zhong Agriculture University natural product chemistry research department, Hubei Polytichnic College's bioengineering dept, University Of Jishou's journal: natural science edition .2003,24 (3).
Utilize cellulase (viride, Ec3.2.1.4) enzymolysis Rhizoma amorphophalli glucomannan, 10g konjaku powder, the 500U cellulase, at 40 ℃, the pH value is that 5.0 o'clock reaction 2h are optimum condition, gained oligose number average relative molecular weight is 5100 under this condition, and the weight average relative molecular weight is 7160.
Its weak point is: the oligosaccharides molecule number that makes with these processing condition is greatly about 28, and molecular chain is excessive, is unfavorable for activating to greatest extent and improves the immunizing power of animal and improve the human body micro-ecological environment, strengthens the human immunological competence.
Summary of the invention
The object of the present invention is to provide a kind of production method of improved konjak mannose using cellulase, improve enzymolysis efficiency, shorten the mannooligo saccharide molecular chain, reduce its molecule number, improve the immunizing power that mannooligo saccharide activated and improved animal, with the ability of improving human body micro-ecological environment, enhancing body immunity, be that technology is simple, cost is low, the new technology of production high purity mannooligo saccharide.
Technical scheme of the present invention is: a kind of production method of konjak mannose using cellulase, its technical process is: enzymolysis, filtration, decolouring, concentrated, dry, it is characterized in that: press konjaku powder 1g: 80-120u enzyme unit alive drops into cellulase, hemicellulose enzyme mixture, and the enzyme ratio of living is 2.5-3.5: 1 in cellulase, the hemicellulose enzyme mixture; Enzymolysis adopts centrifuging, membrane filtration after stopping successively, obtains mannooligo saccharide mixed solution and filter residue; Thick slag is removed in centrifuging, and ultrafiltration membrance filter obtains the mannooligo saccharide mixed solution, removes macromole; The aperture of used filtering membrane is 2-10 microns.
Concrete production method of the present invention is:
Enzymolysis: in the enzymolysis device, in 100-140g: the 1000ml ratio adds konjaku powder and water, press konjaku powder 1g:80-120u enzyme unit alive and drop into cellulase, hemicellulose enzyme mixture, the enzyme ratio of living is 2.5-3.5: 1 in cellulase, the hemicellulose enzyme mixture, the pH value of control enzymolysis is 4.5-5.5,45 ℃ ± 2 ℃ of temperature, enzymolysis reaction after 5-7 hours reaction times;
Filter: enzymolysis adopts centrifuging, membrane filtration after stopping successively, obtains mannooligo saccharide mixed solution and filter residue; Thick slag is removed in centrifuging, and ultrafiltration membrance filter obtains the mannooligo saccharide mixed solution, removes macromole; The aperture of used filtering membrane is 2-10 microns;
Decolouring: the mannooligo saccharide mixed solution, adopt the column chromatography for separation coupling, the elutriant activated carbon decolorizing;
Concentrate: vacuum concentration, 60 ℃ of controlled temperature, vacuum tightness-0.8mPa obtains the mannooligo saccharide syrup;
Dry: as to adopt spraying drying, 75-80 ℃ of drying temperatures.
The present invention has the following advantages:
1, adopted non-specific commercial enzyme (cellulase and hemicellulase), cost is low, and the efficient height is fit to industrialization;
2, take the filter method of centrifuging, membrane filtration, optimize ultra-filtration membrane separation coupling purification technique and obtain mannooligo saccharide, purity height, the kanjak mannan oligosaccharides that obtains mainly are straight chain or the branched chain that 2-5 monose are formed by connecting by glycosidic link, and its molecular weight is 300-2000;
3, enzymolysis process is simple, and the yield height reaches 77.7%.
Description of drawings
Fig. 1 is the molecular structure of 6 kinds of products in the embodiments of the invention
Fig. 2 is thin layer chromatography analytical results figure.
Fig. 3, Fig. 4 are ion chromatography analysis figure as a result.
Concrete embodiment
One, Technology
(1) main raw:
Konjaku powder, cellulase, hemicellulase, sodium hydroxide (NaOH), industrial alcohol.
(2) main laboratory apparatus:
Water bath with thermostatic control, electric mixer, whizzer, ultrafiltration membrance filter device, activated carbon chromatography post, rotation concentration evaporator, spray-drier, silica gel vacuum drier.
(3) technical process:
Enzymolysis, filtration, decolouring, concentrated, dry.
(4) know-why of project:
The general name of the oligosaccharides little polymkeric substance that to be 2-10 monose be formed by connecting by the glycosidic link of certain position.Mannooligo saccharide is the zymolyte of mannosans (konjac glucomanna), extensively is present in konjaku, guar gum, sesbania gum and the multiple microorganism wall.The main component of konjaku is a Rhizoma amorphophalli glucomannan, and molecular formula is (C
6H
10O
5) n, the averagemolecular wt amount is between 20-200 ten thousand, be a kind of by D-glucose and D-seminose by the mol ratio of 2:3 or 1:1.6 by β-1,4 and the natural polysaccharide that is formed by connecting of β-1, glycosidic links such as 3, content is generally 44-65%, and the content of wherein white konjaku, elephant-foot yam kind can reach 50-65%.Rhizoma amorphophalli glucomannan and cellulosic structure are similar, all be to connect by β-1,4 glycosidic links, utilize cellulase and hemicellulase to carry out enzymolysis, be intended to utilize it to β-1,4 and the destruction of β-1, glycosidic links such as 3, Rhizoma amorphophalli glucomannan is cut into many short chains, be degraded to oligose.Why selected cellulase and hemicellulase blended biological enzyme can work to Rhizoma amorphophalli glucomannan, be because the substrate of Glycosylase (polysaccharide that β-1,4 glycosidic links connect) specificity, comprise the specificity of glycosyl one side, the α of glycosidic link-, β-different specificity.
Two, experimental technique:
1) enzymolysis:
In 1000 ml distilled waters, add 125 gram konjaku powders, stir, regulate its temperature to 45 ℃, and with NaOH (sodium hydroxide) the solution adjusting potential of hydrogen of 0.5M (volumetric molar concentration) to pH5.Add total enzyme live cellulase and hemicellulase (the enzyme ratio of living the is 3:1) mixture of 12500u (international unit), stirring reaction is 7 hours under 45 ℃, the condition of pH5.In the reaction process, regulate maintenance pH5 with the NaOH solution of 0.5M.After reaction finishes, boiling water bath deactivation in 15 minutes, standing over night under the room temperature.
2) separate the coupling purifying:
With the product of last step, under 3600 rev/mins of conditions, removed precipitation in centrifugal 10 minutes.Can be used as feed after this drying precipitate, also can drop into enzymolysis once more.Centrifuge mother liquor is the ultrafiltration membrance filter by 2-10 micron pore size further, returns enzymolysis step next time after the filter residue and drying, and filtrate is separated the mixture solution that coupling obtains the mannooligo saccharide behind the purifying by the activated carbon column chromatography method.Before carrying out column chromatography, with 8 liters of distillation washing posts, use the linear tonsure wash-out of ethanol of 40L0-30% (W/W) then, elution speed is 500 milliliters/hour, collects and numbers and preserve with automatic distribution collector.Oligosaccharide content in the separated product and composition use phenol-sulfuric acid process and TLC method to detect respectively.According to the detected result merging filtrate.
3) concentrate, decolouring, drying
The mannooligo saccharide mixture solution that previous step is rapid is used activated carbon decolorizing, and the supernatant liquor after the filtration in temperature is below 60 ℃, carry out vacuum concentration under the condition of vacuum tightness-0.8mPa, obtains the mannooligo saccharide syrup.Syrup obtains 97.2g mannooligo saccharide powder-like product, yield 77.7% through spraying drying.In this way, the mannooligo saccharide that obtains has six kinds of compositions, is followed successively by according to the elution order of activated carbon column: I (A), II (B), III (C), IV (D), V (E), VI (F).(see figure 1)
Embodiment 2
1) enzymolysis:
In 1000 ml distilled waters, add 110g konjaku powder, 15 gram centrifugation filter residues, 3 gram ultra-filtration membranes separation filter residues, stir, add cellulase and hemicellulose enzyme mixture (cellulose enzyme activity: hemicellulase enzyme work=3:1, total enzyme 12500u alive), fully mixing, regulate its temperature to 50 ℃, and regulate pH to 5 with sodium hydroxide (NaOH) solution of 0.5M (volumetric molar concentration), stirring reaction is 7 hours under 50 ℃, the condition of pH5, enzymolysis reaction.In reaction process, regulate maintenance pH5 with the NaOH solution of 0.5M.Behind the reaction terminating, boiling water bath 15min deactivation, standing over night under the room temperature.
2) separate the coupling purifying:
With the product of last step, under 3600 rev/mins of conditions, removed precipitation in centrifugal 10 minutes.Can be used as feed after this drying precipitate, drop into enzymolysis after also can handling in proportion once more.Centrifuge mother liquor is the ultrafiltration membrance filter by 2-10 micron pore size further, returns enzymolysis step next time after the filter residue and drying, and filtrate is separated the mixture solution that coupling obtains the mannooligo saccharide behind the purifying by the activated carbon column chromatography method.Before carrying out column chromatography, with 8 liters of distillation washing posts, use the linear tonsure wash-out of ethanol of 40L0-30% (W/W) then, elution speed is 500 milliliters/hour, collects and numbers and preserve with automatic distribution collector.Oligosaccharide content in the separated product and composition use phenol-sulfuric acid process and TLC method to detect respectively.According to the detected result merging filtrate.
3) concentrate, decolouring, drying
The filtrate that previous step is rapid is used activated carbon decolorizing, and the supernatant liquor after the filtration in temperature is below 60 ℃, carry out vacuum concentration under the condition of vacuum tightness-0.8mPa, obtains the mannooligo saccharide syrup.Syrup obtains 88.6g mannooligo saccharide powder-like product through spraying drying, relatively (konjaku powder charging capacity) yield 80.5%, definitely yield (total charging capacity) 69.2%.In this way, the mannooligo saccharide that obtains has six kinds of compositions, is followed successively by according to the elution order of activated carbon column: I (A), II (B), III (C), IV (D), V (E), VI (F).The relative yield 80.5% of (see figure 1), absolute yield 69.2%
Three, interpretation of result
1, analytical procedure:
(1) chromatography of ions (HPAEC) method:
Instrument: chromatography of ions IC2500, adopt ED50 detector, GP50 pump, CarboPac chromatographic column.
Condition: mobile phase A: NaOH (A.R.) 100mM, B: water;
Gradient 40mM-70mM drip washing 30 minutes; 30 ℃;
(2) thin-layer chromatography (TLC) method:
Developping agent: acetonitrile: water=8:2 (V/V); Launch number of times: three times
Developer: the methanol solution of 5% H2SO4 (V/V)
Color condition: 110 ℃, 10 minutes
2, detected result:
(1) thin-layer chromatography (TLC) method: the results are shown in Figure 2.
(2) chromatography of ions (HPAEC) method: the results are shown in Figure 3, Fig. 4.
Claims (1)
1. the production method of a konjak mannose using cellulase, its technical process is: enzymolysis, filtration, decolouring, concentrated, dry, it is characterized in that: press konjaku powder 1g:80-120u enzyme unit alive and drop into cellulase, hemicellulose enzyme mixture, the enzyme ratio of living is 2.5-3.5:1 in cellulase, the hemicellulose enzyme mixture; Enzymolysis adopts centrifuging, ultrafiltration membrance filter after stopping successively, obtains mannooligo saccharide mixed solution and filter residue; Thick slag is removed in centrifuging, and ultrafiltration membrance filter obtains the mannooligo saccharide mixed solution, removes macromole; The aperture of used ultra-filtration membrane is 2-10 microns.
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