CN103173366A - Pectinase producing bacterial strain and application in preparation of peeled citrous complex enzyme - Google Patents
Pectinase producing bacterial strain and application in preparation of peeled citrous complex enzyme Download PDFInfo
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Abstract
The invention provides a pectinase producing bacterial strain, namely Asperillus niger WZW001, and an application in preparation of pectinase and peeled citrous complex enzyme. The strain is preserved in the CCTCC (china center for type culture collection) on Oct. 12, 2012, and the preservation No. is CCTCC No: M2012399. The peeled citrous complex enzyme prepared by the Asperillus niger WZW001 has the advantages of low production cost, specificity and efficiency, stable application and the like; during the application, as long as the Asperillus niger WZW001 strain fermentation liquor is used for preparing solid pectinase or is separated and purified to obtain PL, Exo-PG, and PE single enzyme elements, and one or more of the solid pectinase or the single enzyme elements is compounded with marketed cellulose without adding other enzymes, so that the orange capsule can be efficiently removed. According to the pectinase producing bacterial strain and the application, the problem of high cost of enzyme for an enzyme-method capsule removing process at present is solved, the replacement of the process to an acid-alkali chemical method is facilitated, and the energy-saving, emission-reduction, green and efficient production of the orange deep processing industry in the country has practical significance.
Description
(1) technical field
The present invention relates to a strain and produce polygalacturonase bacterial strain---aspergillus niger (Asperillus niger) WZW001, and the application in preparation polygalacturonase and citrus excystation clothing prozyme.
(2) background technology
China's mandarin orange planting area and output occupy first place, the world, and annual production has reached 2000 * 10
4More than t, China is also the place of production, center of world's mandarin orange can, and citrus is approximately ripe in 11～December more than 75%, adding the duration is only 2～3 months, the citrus deep processing can effectively solve the seasonal strong problem of citrus, promotes the development of China's citrus deep processing industry, brings huge economic and social benefit.Excystation clothing technique is the critical process of producing mandarin orange can and cyst, industrialization excystation clothing method generally adopts the runner type acid-alkali treatment method both at home and abroad at present, this method water loss is large, environmental pollution is serious, quality product is difficult to guarantee, easily causes the export trade of China's mandarin orange can and cyst to face technology and green barrier.And enzyme process excystation clothing art breading mild condition, constant product quality, safe and reliable alternative soda acid chemical method, but common food grade polygalacturonase and cellulase price cause the cost of enzyme process excystation clothing higher all at hundreds of unit/kg.
The main ingredient of citrus capsule clothing is pectin, a small amount of Mierocrystalline cellulose and hemicellulose.Polygalacturonase performance Main Function in citrus excystation clothing technique, polygalacturonase mainly comprises polygalacturonase (Exo-PG), Rohapect MPE (PE) and pectin lyase (PL), the food grade polygalacturonase is generally fermentation of Aspergillus niger production at present, generally also contains a small amount of cellulase and hemicellulase in polygalacturonase.Component concentration and the ratio differences such as Exo-PG, PE, PL in the polygalacturonase that different strains, different fermentation conditions are produced, cellulase and hemicellulase content are also different, cause different polygalacturonase Application Areas and effect different, also have different effects in the application of citrus excystation clothing.
Publication number is CN101095554A, patent name is in " method of the full fruit enzymatic excystation of citrus clothing ", single poplar, the people such as Li Gaoyang with polygalacturonase, cellulase and hemicellulase composite usage in the full fruit enzymatic excystation of citrus clothing technique, but this prozyme does not illustrate which kind of enzyme in polygalacturonase plays a major role and various enzyme between mutual synergy, and need three kinds of food grade commercial enzymes composite.Publication number is CN102078024A, patent name is " being used for liquid complex enzyme and complex process and the application of tangerine lobe excystation clothing ", the people such as Yang Xiaoying, Yuan Weiwei with polygalacturonase, cellulase, zytase and-the dextranase composite usage is in citrus excystation clothing technique, obtain good effect, but needed four kinds of composite costs of enzyme that cause equally of food grade commercial enzyme higher.
It is the relation of each component and its excystation clothing efficient with the enzyme enzyme that prior art is not all furtherd investigate the excystation clothing, untappedly go out citrus excystation clothing specific enzyme, and existing excystation clothing is composite with food grade commercial enzyme in the general needs 3～5 of enzyme, not only cause the use cost of enzyme higher, and the prozyme vigor is unstable, is unfavorable for industrial applications.
(3) summary of the invention
Deficiency for the prior art existence, the object of the invention is to provide citrus excystation clothing prozyme and the preparation method thereof that a kind of cost is low, efficient is high, application is stable, product safety is particularly suitable for the loose-skinned oranges such as Pon mandarin orange class, satsuma orange, to solve the higher problem of citrus enzyme process excystation clothing process costs.
The technical solution used in the present invention is:
One strain polygalacturonase Producing Strain---aspergillus niger (Asperillus niger) WZW001 is preserved in Chinese Typical Representative culture collection center, address: China on October 12nd, 2012, Wuhan, Wuhan University, postcode 430072, deposit number are CCTCC No:M2012399.
Bacterial strain of the present invention obtains according to the general method screening in this area, bacterial strain is aspergillus niger WZW001, preserving number is CCTCC M2012399, and the biological characteristics of this bacterial strain is: bacterium colony is grown on the PDA substratum rapidly, and 30 ℃ are cultivated 3 days diameters is 45～60 millimeters; Quality velvet shape or slightly be with cotton-shaped; The conidium structure is a large amount of, and brown-black is without transudate; The bacterium colony reverse side is slightly yellow.The strain culturing characteristic is: conidial head is spherical, 30～60 microns of diameters; Conidiophore sends out in matrix, conidiophore stem (100～400) micron * (4～8) micron, and wall is level and smooth; The top capsule is spherical or subsphaeroidal, 10～15 microns of diameters, and all surfaces can be educated; Conidial fructification is double-deck, metulae (1～2) micron * (0.4～0.7) micron, and bottle stalk (0.5～0.8) micron * (0.2 * 0.3) micron, conidium is spherical or subsphaeroidal, and 1.5～2 microns, wall is coarse.
The invention still further relates to the application of described aspergillus niger WZW001 in the preparation polygalacturonase.Bacterial strain of the present invention can obtain polygalacturonase through fermentation, and this polygalacturonase can be further used for preparing citrus excystation clothing prozyme.
The invention still further relates to the application of described aspergillus niger WZW001 in preparation citrus excystation clothing prozyme.
Concrete, described application is as follows: aspergillus niger WZW001 is seeded to the liquid fermentation medium that contains orange meal that routine is applicable to aspergillus niger, cultivated 72～96 hours for 30～35 ℃, fermented liquid after filtration, add appropriate Zulkovsky starch after membrane concentration and carry out spraying drying and make the solid pectin enzyme; Perhaps fermented liquid is through saltouing or organic solvent deposit, membrane sepn, ion exchange chromatography obtain respectively circumscribed-polygalacturonase (Exo-PG), Rohapect MPE (PE) and pectin lyase (PL) single enzyme component; With gained solid pectin enzyme, perhaps one or more in the single enzyme component and cellulase carry out compositely, namely get described citrus excystation clothing prozyme.
Preferably, described liquid fermentation medium is composed as follows: orange meal 16～24g/L, wheat bran 25～35g/L, ammonium sulfate 8～12g/L, soybean cake powder 8～12g/L, KH
2PO
42～6g/L, solvent are water, pH5.0～6.0.
Bacterial strain WZW001 of the present invention is in the upper growth of PDA substratum (inclined-plane and eggplant bottle inclined-plane) and preservation.
Common, bacterial strain also needed before fermentation culture at liquid to cultivate and seed culture through conventional slant activation.Concrete, liquid-state fermentation technology of the present invention can be the inclined-plane to seed culture medium to the 5L fermentor tank.Bacterial strain grows to maturation in inclined-plane or eggplant bottle inclined-plane, ripeness standard is that spore is dense, is chocolate.Add stroke-physiological saline solution to make spore suspension (1～3 * 10
7Individual/as mL), to be inoculated in seed culture medium by 3%～5%(v/v) inoculum size, seed culture based formulas (100mL): orange meal 0.8～1.2g, wheat bran 0.6～1.4g, glucose 1.8～2.2g, ammonium sulfate 0.8～1.2g, MgSO
40.04～0.06g, before sterilization, adjust pH is 5.0～6.0,121 ℃ of sterilizations 20～35 minutes, culture condition: 30 ℃～35 ℃, and rotating speed 200r/min～250r/min, incubation time 24 hours.After seed liquor is grown well, be inoculated in the 5L fermentor tank by 6%～10%(v/v) inoculum size, fermentor tank liquid amount (v/v) 60%～70%, culture medium prescription (by 5L fermentor tank liquid amount 3L): orange meal 48～72g, wheat bran 75～105g, ammonium sulfate 24～36g, soybean cake powder 24～36g, KH
2PO
46～12g, before sterilization, adjust pH is 5.0～6.0, sterilized 20～35 minutes for 121 ℃, culture condition: pass into air pressure 0.1～0.15Mpa, control tank pressure 0.02～0.05Mpa, the fermented liquid of air velocity 0.5～1.5vvm(per minute unit volume passes into the volume number of air), 30 ℃～35 ℃ of temperature.Detect fermentation broth enzyme vigor, pH, thalli morphology every sampling in 6 hours, when enzyme activity is not rising or is being judged as fermentation termination during automyophagy.
After fermentation ends, filtering fermentating liquid, solid-liquid separation.Fermented liquid is with millipore Pellicon XL(PXB010A50 standard molecular weight 10000) concentrated 3～10 times of membrane filtration after to add quality be that the Zulkovsky starch spraying drying of concentrated solution quality 2%～5% makes the solid pectin enzyme.
The drying process with atomizing condition: 130 ℃～150 ℃ of intake air temperatures, 65 ℃～80 ℃ of exhaust outlet temperature, temperature 50 C in collector～70 ℃, the spraying gun rotating speed is 15000r/min～20000r/min.
Perhaps, the fermented liquid membrane concentration is by ammonium sulfate salting-out process or organic solvent deposit extracts and just separation, then utilize the further separation and purification of anion exchange chromatography, the elution fraction of collecting respectively under different elution requirements obtains Exo-PG, PE and PL single enzyme component.
Measure respectively enzyme activity and commercialization cellulase (in the FPA) enzyme activity of Exo-PG, PL and PE in the solid pectin enzyme, more respectively with solid pectin enzyme, PL, PL+Exo-PG, Exo-PG+PE and the cellulase composite citrus excystation clothing prozyme that makes four kinds of different componentss in proportion.
1. concrete, described citrus excystation clothing prozyme can be by described solid pectin enzyme and cellulase compounding and is got, and the ratio of the enzyme activity of circumscribed in prozyme-polygalacturonase, Rohapect MPE, pectin lyase and cellulase is: (4～8): (1/5～1/15): (1/50～1/150): 1.
2. concrete, but described citrus excystation clothing prozyme pectin lyase and cellulase are according to the ratio (1/40～1/120) of enzyme work: and 1 ratio is composite and get.
3. concrete, but described citrus excystation clothing prozyme pectin lyase, circumscribed-polygalacturonase and cellulase compounding and get, in prozyme, the ratio of pectin lyase, circumscribed-polygalacturonase and cellulose enzyme activity is (1/100～1/30): (1/10～1/5): 1.
4. concrete, described citrus excystation clothing prozyme can be got by circumscribed-polygalacturonase, Rohapect MPE and cellulase compounding, and the ratio of the enzyme work of circumscribed in prozyme-polygalacturonase, Rohapect MPE and cellulase is (1～4): (1/8～1/4): 1.
It is as follows that described citrus excystation clothing prozyme is used for citrus excystation clothing technique concrete steps:
A, citrus peeling, the distinguish that cleans up obtained citrus tangerine lobe;
B, the different enzyme amount enzyme liquid of water preparation, 2mol/L salt acid for adjusting pH value to 3.0～4.5;
C, enzyme liquid is mixed by 1～2:1(w/w) with the tangerine lobe, enzyme liquid submergence tangerine lobe, 40～60 ℃ of constant temperature enzymolysis, enzymolysis finishes after 0.5～4h, and filtered and recycled enzyme liquid is used for next batch to be processed, and the tangerine lobe is cleaned up with clear water to get full excystation clothing tangerine lobe;
D, will reclaim enzyme liquid and reconcile pH to 3.0～4.5 with 2M NaOH or 2mol/L hydrochloric acid, all the other operations as before, again add the tangerine lobe to carry out enzymolysis excystation clothing.
The present invention is in conjunction with modern separate analytical technique, on the basis of the relation that further investigation polygalacturonase component of enzyme system and cellulase and its excystation clothing are tired, find key enzyme, best of breed and ratio between searching enzyme system, and the recycling technique of the complex process of cellulose-binding enzyme and enzyme reduces the cost of enzyme process excystation clothing technique to improve excystation clothing and the service efficiency of enzyme.
Advantage of the present invention is as follows;
A. provide a strain new polygalacturonase Producing Strain aspergillus niger (Asperillus niger) WZW001, preserving number is CCTCC M2012399, and this bacterial strain has advantages of that yield of enzyme is high, enzyme is complete.
B. aspergillus niger WZW001 utilizes wheat bran, soybean cake powder, orange meal to carry out liquid state fermentation for main raw material, and raw material is the agricultural byproducts that the source is wide, price is lower, and has obtained culture medium prescription and the fermentation parameter optimized.
C. by research component of enzyme system Exo-PG, PE, PL and cellulase and its excystation clothing big or small relation of tiring, find that polygalacturonase component of enzyme system PL, PL and Exo-PG combination, Exo-PG and PE combination can remove citrus capsule clothing effectively, optimized accordingly enzyme and be the ratio between each component.And the relatively low cellulase of composite appropriate price on this basis, improved on the one hand the excystation clothing efficient of enzyme, can reduce on the other hand excystation clothing enzyme cost.Each component full synergism of citrus excystation clothing prozyme under this technique, and do not need additionally to add any other zymin and can remove efficiently citrus capsule clothing.
D. prozyme in citrus excystation clothing recycling technique enzyme activity comparatively vigor is stable, be fit to the industrialization continuous application.
The enzyme activity detection method that the present invention relates to is:
Circumscribed-polygalacturonase (Exo-PG): the mass concentration concentration of getting the preparation of 1.8mL pH3.5 phosphoric acid buffer is 1% pectin solution, adds 0.2mL dilution enzyme liquid, adds 2mL DNS termination reaction, boiling water bath 10min after 50 ℃ of insulation 30min.Cooling rear absorption 0.5mL is settled to 10mL, shakes up, and measures its light absorption value under 550nm.The enzyme amount that the depolymerized pectin substrate per hour produces the 1mg galacturonic acid is defined as the enzyme unit that lives.
Rohapect MPE (PE): the mass concentration of getting the configuration of 4.0mL pH3.5 distilled water is 1% pectin solution, adds 1.0mL dilution enzyme liquid, inactivator after 50 ℃ of insulation 30min.Carboxylic group with 0.01mol/LNaOH solution titration generation.The enzyme amount that depolymerized pectin substrate per minute discharges 1 mol carboxylic acid is defined as 1 enzyme activity unit.
Pectin lyase (PL): get the 4.0mL0.5% pectin solution in test tube, 50 ℃ of water-bath balance 5～10min add 1.0mL dilution enzyme liquid, 50 ℃ of insulation 10min.Get above-mentioned reaction mixture 0.5mL and mix termination reaction in 4.5mL0.0l mol/LHCl.Measure light absorption value at the 235nm place, the unsaturated double-bond molar absorptivity is 5200.The enzyme amount that depolymerized pectin substrate per minute produces the two keys of 1 mol is defined as the enzyme unit that lives.
Cellulase activity (in FPA): get 1ml dilution enzyme liquid, adding 1mL pH value is 3.5 damping fluids, then adds the filter paper bar of 50 ± 0.5mg(1cm * 6cm), is incubated enzyme digestion reactions 1 hour in 50 ℃.Add 2ml DNS termination reaction, boiling water bath 10min suitably measures its light absorption value under 550nm after dilution.The enzyme amount that degraded filter paper bar substrate per hour produces 1mg glucose is defined as an enzyme activity unit.
beneficial effect of the present invention is mainly reflected in: compared with prior art, utilize the citrus excystation clothing prozyme of aspergillus niger (AsperillUUs niger) WZW001 and method of the present invention preparation to have a production cost low, single-minded efficient, use the advantages such as stable, obtain PL after only needing during application aspergillus niger WZW001 bacterial strain fermentation liquor is prepared solid pectin enzyme or separation and purification, Exo-PG, PE single enzyme component, then with solid pectin enzyme or PL, PL+Exo-PG and Exo-PG+PE and commercially available cellulase are composite in proportion, the oranges and tangerines excystation clothing prozyme of four kinds of different componentss of this technique preparation does not need additionally to add other enzymes and can efficiently remove citrus capsule clothing.The invention solves present enzyme process excystation clothing technique with the higher problem of enzyme cost, promote this technique to the substituting of soda acid chemical method, have the meaning of reality for energy-saving and emission-reduction, the green high-efficient production of China's citrus deep processing industry
(4) description of drawings
Fig. 1 is ion exchange chromatography figure (A) and the SDS-PAGE albumin glue figure (B) of aspergillus niger WZW001 fermented liquid.
Fig. 2 is design sketch after citrus tangerine lobe excystation clothing.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
Bacterial strain of the present invention screens according to methods known in the art, obtains a strain and produces that pectinase activity is high, enzyme is complete aspergillus niger (Asperillus niger) WZW001, CCTCC No:M2012399.
Aspergillus niger WZW001 growth and preserving in PDA substratum (test tube slant and eggplant bottle inclined-plane), bacterial classification through slant medium go down to posterity 6 generation above enzymatic productivity stable.
Zymotechnique of the present invention is that the inclined-plane is to seed culture medium to the 5L fermentor tank.The bacterial classification 72 hours maturations of growing in the inclined-plane, ripe spore is dense, is chocolate.Add stroke-physiological saline solution to make spore suspension (1～3 * 10
7Individual/mL), and by 5%(v/v) inoculum size is inoculated in seed culture medium, seed culture based formulas (100mL): orange meal 1g, wheat bran 1g, glucose 2g, ammonium sulfate 1g, MgSO
40.05g before sterilization, adjust pH is 5.5,121 ℃ of sterilizations 20 minutes, culture condition: 30 ℃, rotating speed 200r/min, incubation time 24 hours.Until seed liquor grow good after, by 8%(v/v) inoculum size is inoculated in the 5L fermentor tank, fermentative medium formula (by 5L fermentor tank liquid amount 3L): orange meal 60g, wheat bran 90g, ammonium sulfate 30g, soybean cake powder 30g, MgSO
41.5g, KH
2PO
49g, pH6.0 uses 2mol/L hydrochloric acid with pH regulator to 5.5 before sterilization.
The present invention is conducive to the growth of thalline and produces enzyme, the culture condition of 5L fermentor tank is: fermentor tank liquid amount (v/v) 60%, pass into air pressure 0.1～0.15Mpa, control tank pressure 0.02～0.05Mpa, the fermented liquid of air velocity 1vvm(per minute unit volume passes into the volume number of air), 30 ℃ of temperature, sampling in every 6 hours detects fermentation broth enzyme vigor, pH, thalli morphology, when enzyme activity no longer rises or is judged as fermentation termination during automyophagy.
Fermentation of Aspergillus niger 84 as a child detected pectinase activity and no longer rose, and finding during microscopy has a large amount of self-dissolvings of mycelia, is judged as fermentation termination.
The preparation of solid pectin enzyme
Ultra-filtration membrane with molecular weight cut-off＜10Kda after a, filtering fermentation liquor is concentrated into 700mL, concentrated enzyme liquid adds 3% Zulkovsky starch spraying drying, the drying process with atomizing condition is: 60 ℃ of feeding temperatures, 140 ℃ of intake air temperatures, 65 ℃ of air outlet temperature, air intake air pressure 0.2MPa, centrifugal turntable rotating speed 15000r/min, make solid pectin enzyme 20.7g, be light yellow, Powdered, uniform particles, exquisiteness, its enzyme activity is respectively: Exo-PG vigor 23000U/g, PE vigor 670U/g, PL:45U/g, cellulase activity 115U/g.
B. the cellulase (commodity) selected of this example is purchased from Novi's letter, cellulase activity (in FPA) 1000U/g, 1. solid pectin enzyme and cellulase compounding are made citrus excystation clothing prozyme, and in composite rear prozyme, PL:PE:Exo-PG:FPA enzyme activity ratio is 1/80:1/5.5:6.5:1.
1. be mixed with PL, PE, Exo-PG and FPA enzyme activity with the oranges and tangerines excystation clothing prozyme of above-mentioned preparation and be respectively 240U, 3500U, 1.2 * 10
5U and 1.8 * 10
4The enzyme liquid 7800mL of U is with 2mol/L salt acid for adjusting pH value to 3.5.Fresh Pon mandarin orange is cleaned, and peeling, distinguish obtain 5.2kg tangerine lobe, and the tangerine lobe is immersed in enzyme liquid fully, constant temperature enzymolysis under 50 ℃ of conditions.After 95min is found in research, enzymolysis finishes, and tangerine lobe surface tangerine pith and capsule clothing remove fully.The tangerine lobe is separated with enzyme liquid, the tangerine lobe cleans twice with clear water and removes residual enzyme liquid, enzyme liquid reclaims the excystation clothing that continues on for next batch and processes, and each enzyme activity of components of polygalacturonase and cellulase activity slow decreasing in the continuous recycling technique of enzyme liquid still can remove citrus capsule clothing efficiently.
Embodiment 2:
Select the citrus excystation clothing prozyme of preparation in example 1 in this example 1., the citrus of employing is the Wenzhou District of Zhejiang Province tangerine orange.1. be mixed with PL, PE, Exo-PG and FPA enzyme activity with oranges and tangerines excystation clothing prozyme and be respectively 170U, 2400U, 0.83 * 10
5U and 1.5 * 10
4The enzyme liquid 5400mL of U is with 2mol/L salt acid for adjusting pH value to 3.5.Fresh citrus unshiu Marcovitch cleans, and peeling, distinguish obtain 3.6kg tangerine lobe, and the tangerine lobe is immersed in enzyme liquid fully, the constant temperature enzymolysis.After 78min is found in research, enzymolysis finishes, and tangerine lobe surface tangerine pith and capsule clothing remove fully.The tangerine lobe is separated with enzyme liquid, the tangerine lobe cleans twice with clear water and removes residual enzyme liquid, enzyme liquid reclaims the excystation clothing that continues on for next batch and processes, and each enzyme activity of components of polygalacturonase and cellulase activity slow decreasing in the continuous recycling technique of enzyme liquid still can remove citrus capsule clothing efficiently.
Embodiment 3:
This example prepares aspergillus niger WZW001 fermented liquid according to example 1 method, and fermented liquid 10000r/min high speed centrifugation 7min after four layers of filtered through gauze obtains enzyme liquid 2000mL, obtains concentrated enzyme 450mL through the ultra-filtration membrane of molecular weight cut-off＜10Kda is concentrated.Concentrated enzyme liquid is collected 40%～90% deposited components through fraction precipitation of ammonium sulphate, 8000～14000Da dialysis band dialysed overnight desalination, loading under pH5.8 Lin acid hydrogen Er Na – citrate buffer solution condition.The chromatographic stuffing that this example adopts is DEAE Sepharose Fast Flow, and the substep linear elution is collected elutriant and obtained PE, Exo-PG, PL single enzyme component.Through SDS-PAGE electrophoresis (accompanying drawing 1) although and enzyme activity determination find that single enzyme component Exo-PG, PE, PL all also have the part heteroproteins, the three is all separately not impacts each other, can be respectively used to composite usage.
The cellulase that this example is selected is purchased from Novi letter, and 2. cellulase activity (in FPA) 1000U/g is that 1/60:1 is composite with pectin lyase (PL) single enzyme component and cellulase in the ratio of PL:FPA enzyme activity makes citrus excystation clothing prozyme.2. prepare with oranges and tangerines excystation clothing prozyme the enzyme liquid 5400mL that PL and FPA enzyme activity are respectively 150U and 9000U, with 2mol/L salt acid for adjusting pH value to 3.5.Fresh citrus unshiu Marcovitch cleans, and peeling, distinguish obtain 3.6kg tangerine lobe, and the tangerine lobe is immersed in enzyme liquid fully, the constant temperature enzymolysis.After 105min is found in research, enzymolysis finishes, and tangerine lobe surface tangerine pith and capsule clothing remove fully.The tangerine lobe is separated with enzyme liquid, the tangerine lobe cleans twice with clear water and removes residual enzyme liquid, enzyme liquid reclaims the excystation clothing that continues on for next batch and processes, and each enzyme activity of components of polygalacturonase and cellulase activity slow decreasing in the continuous recycling technique of enzyme liquid still can remove citrus capsule clothing efficiently.
Embodiment 4:
The cellulase that this example is selected is purchased from Novi's letter, 3. cellulase activity (in FPA) 1000U/g is that 1/60:1/6:1 is composite with the pectin lyase (PL) of embodiment 3 preparation, circumscribed-polygalacturonase (Exo-PG) and cellulase in the ratio of PL:Exo-PG:FPA enzyme activity makes citrus excystation clothing prozyme.3. be mixed with oranges and tangerines excystation clothing prozyme the enzyme liquid 5400mL that PL, Exo-PG and FPA enzyme activity are respectively 150U, 1450U and 9000U, with 2mol/L salt acid for adjusting pH value to 3.5.Fresh citrus unshiu Marcovitch cleans, and peeling, distinguish obtain 3.6kg tangerine lobe, and the tangerine lobe is immersed in enzyme liquid fully, the constant temperature enzymolysis.After 70min is found in research, enzymolysis finishes, and tangerine lobe surface tangerine pith and capsule clothing remove fully.The tangerine lobe is separated with enzyme liquid, the tangerine lobe cleans twice with clear water and removes residual enzyme liquid, enzyme liquid reclaims the excystation clothing that continues on for next batch and processes, and each enzyme activity of components of polygalacturonase and cellulase activity slow decreasing in the continuous recycling technique of enzyme liquid still can remove citrus capsule clothing efficiently.
Embodiment 5:
The cellulase that this example is selected is purchased from Novi's letter, cellulase activity (in FPA) 1000U/g, with embodiment 3 preparation circumscribed-4. be that 2:1/6:1 is composite in the ratio of Exo-PG:PE:FPA enzyme activity make citrus excystation clothing prozyme for polygalacturonase (Exo-PG), Rohapect MPE (PE) and cellulase.4. be mixed with Exo-PG, PE and the FPA enzyme activity is respectively 3.0 * 10 with oranges and tangerines excystation clothing prozyme
4U, 2500U and 1.5 * 10
4The enzyme liquid 5400mL of U is with 2mol/L salt acid for adjusting pH value to 3.5.Fresh citrus unshiu Marcovitch cleans, and peeling, distinguish obtain 3.6kg tangerine lobe, and the tangerine lobe is immersed in enzyme liquid fully, the constant temperature enzymolysis.After 183min is found in research, enzymolysis finishes, and tangerine lobe surface tangerine pith and capsule clothing remove fully.The tangerine lobe is separated with enzyme liquid, the tangerine lobe cleans twice with clear water and removes residual enzyme liquid, enzyme liquid reclaims the excystation clothing that continues on for next batch and processes, and each enzyme activity of components of polygalacturonase and cellulase activity slow decreasing in the continuous recycling technique of enzyme liquid still can remove citrus capsule clothing efficiently.
Claims (8)
1. a strain polygalacturonase Producing Strain---aspergillus niger (Asperillus niger) WZW001 is preserved in Chinese Typical Representative culture collection center on October 12nd, 2012, and deposit number is CCTCC No:M2012399.
2. the application of aspergillus niger WZW001 as claimed in claim 1 in preparation polygalacturonase and citrus excystation clothing prozyme.
3. application as claimed in claim 2, it is characterized in that described application is as follows: aspergillus niger WZW001 is seeded to the liquid fermentation medium that contains orange meal, cultivated 72～96 hours for 30～35 ℃, fermented liquid after filtration, add Zulkovsky starch after membrane concentration and carry out spraying drying and make the solid pectin enzyme; Perhaps fermented liquid through membrane sepn, saltout or organic solvent deposit, ion exchange chromatography obtain respectively circumscribed-polygalacturonase (Exo-PG), Rohapect MPE (PE) and pectin lyase (PL) single enzyme component; With gained solid pectin enzyme, perhaps one or more in the single enzyme component and cellulase carry out compositely, namely get described citrus excystation clothing prozyme.
4. application as claimed in claim 3 is characterized in that described liquid fermentation medium is composed as follows: orange meal 16～24g/L, wheat bran 25～35g/L, ammonium sulfate 8～12g/L, soybean cake powder 8～12g/L, KH
2PO
42～6g/L, solvent are water, pH5.0～6.0.
5. application as claimed in claim 3, it is characterized in that: described citrus excystation clothing prozyme is by described solid pectin enzyme and cellulase compounding and get, and the ratio of the enzyme activity of circumscribed in prozyme-polygalacturonase, Rohapect MPE, pectin lyase and cellulase is: (4～8): (1/5～1/15): (1/50～1/150): 1.
6. application as claimed in claim 3 is characterized in that: described citrus excystation clothing prozyme is by pectin lyase and the cellulase ratio (1/40～1/120) according to enzyme work: 1 ratio is composite and get.
7. application as claimed in claim 3, it is characterized in that: described citrus excystation clothing prozyme is got by pectin lyase, circumscribed-polygalacturonase and cellulase compounding, and in prozyme, the ratio of pectin lyase, circumscribed-polygalacturonase and cellulose enzyme activity is (1/100～1/30): (1/10～1/5): 1.
8. application as claimed in claim 3, it is characterized in that: described citrus excystation clothing prozyme is got by circumscribed-polygalacturonase, Rohapect MPE and cellulase compounding, and the ratio of the enzyme work of circumscribed in prozyme-polygalacturonase, Rohapect MPE and cellulase is (1～4): (1/8～1/4): 1.
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| CN105112396A (en) * | 2015-09-24 | 2015-12-02 | 南京天凯生物技术股份有限公司 | Aspergillus niger pectin lyase mutant and viscosity reducer for syrup during corn ethanol fermentation production |
| CN107151631A (en) * | 2017-03-16 | 2017-09-12 | 西北农林科技大学 | A kind of high yield pectase bacterial strain and its application |
| CN111004788A (en) * | 2019-12-17 | 2020-04-14 | 中国农业科学院麻类研究所 | Pectin esterase and preparation method and application thereof |
| CN114698800A (en) * | 2022-03-29 | 2022-07-05 | 北京勘迪科技有限公司 | Enzyme method sectioning and capsule coat removing process for orange processing |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105112396A (en) * | 2015-09-24 | 2015-12-02 | 南京天凯生物技术股份有限公司 | Aspergillus niger pectin lyase mutant and viscosity reducer for syrup during corn ethanol fermentation production |
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| CN107151631A (en) * | 2017-03-16 | 2017-09-12 | 西北农林科技大学 | A kind of high yield pectase bacterial strain and its application |
| CN111004788A (en) * | 2019-12-17 | 2020-04-14 | 中国农业科学院麻类研究所 | Pectin esterase and preparation method and application thereof |
| CN114698800A (en) * | 2022-03-29 | 2022-07-05 | 北京勘迪科技有限公司 | Enzyme method sectioning and capsule coat removing process for orange processing |
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| CN103173366B (en) | 2015-05-27 |
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