CN1314800C - Fixation method of microbiological lactase and its application - Google Patents
Fixation method of microbiological lactase and its application Download PDFInfo
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- CN1314800C CN1314800C CNB2005100804920A CN200510080492A CN1314800C CN 1314800 C CN1314800 C CN 1314800C CN B2005100804920 A CNB2005100804920 A CN B2005100804920A CN 200510080492 A CN200510080492 A CN 200510080492A CN 1314800 C CN1314800 C CN 1314800C
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Abstract
The present invention relates to a fixation method of lactase and the application of fixed lactase. The method uses resin obtained by the inventor through screening as a vector for adsorption and fixation, and the vector is fermented by recombinant enzymes to obtain free lactase outside cells in filtering clear liquid. The vector adsorbed with the enzymes is processed by a calcium alginate embedding method and a glutaral pentanedial crosslinking method to be prepared into fixed lactase with high bonding strength. The fixed lactase prepared by the method is used as biocatalyst to hydrolyze 5% of lactose solution, fixed enzymes with a dosage of 10% (w/v) to 100% (w/v) can continuously convert 52 batches of lactose solution after acting for 0.5 to 1 hour at 5 DEG C to 60 DEG C, and the hydrolysis rate of the lactose keeps higher than 75%. When the fixed lactase prepared by the method is used for processing fresh milk, the fixed lactase can continuously convert 30 batches of milk after acting for 0.2 to 8 hours at 5 DEG C to 60 DEG C, and the hydrolysis rate of the lactose keeps higher than 70%.
Description
Technical field
The present invention relates to a kind of process for fixation of enzyme, relate in particular to a kind of process for fixation of microorganism extracellular lactase and immobilized lactase and the application of this immobilized lactase in producing low-lactose milk for preparing by this method.
Background technology
(β-D-galactoside galactohydrolase EC3.2.1.23) is commonly called Sumylact L to beta-galactosidase enzymes.This enzyme can be semi-lactosi and glucose with lactose hydrolysis, and has the transferance of galactoside.
Lactose is formed by a part alpha-D-glucose and a part β-D-semi-lactosi condensation, is to be the β-D-galactoside of aglycon with glucose.Lactose is the main component in milk and the whey, and lactose accounts for 30% of milk dry-matter, influences local flavor.Many people are difficult to pegnin (different because of ethnic group, West Europe accounts for 2～8%, and Asia, Africa account for 60～90%) because of lacking Sumylact L in the body, behind the milk drink, lactose has arrived in the intestines, by the intestinal bacteria decomposing and fermenting, produce great amount of carbon dioxide gas, enteron aisle is expanded, and excited intestines peristalsis, make and shrink to strengthen, cause borborygmus and diarrhoea, this disease is called lactose intolerance, and (the GG uncle is strange etc., enzyme and food-processing, light industry press, 1991, p93～110).
Producing low-lactose milk and milk-product is the effective ways that solve lactose intolerance.The method of removing lactose or production low-lactose milk has three kinds: physical removal method, acid-hydrolysis method and enzyme hydrolysis method.
(1) physical removal method: adopt ultra-filtration technique to remove lactose, though most of lactose can be removed, VITAMIN and mineral substance be loss thereupon also, has to they be added after removing lactose again, and this has also caused the significant wastage of lactose.
(2) acid hydrolyzation be under extreme conditions (pH1～2,100～150 ℃ of temperature) with the lactose in free acid or the ion exchange resin hydrating solution.Although acid hydrolyzation can obtain high conversion at short notice, it can cause the albumen inactivation, can form some by products, can not be used for hydrolysed milk, and acid system needs corrosion resistant equipment, and is also unfavorable to environment.
(3) the enzyme hydrolysis method condition relatively relaxes (pH3.5～8, temperature 5-60 ℃).It is to use the external source Sumylact L the monose of lactose degradation for easily being absorbed by the body and utilizing.Adopt this method, product is simple, and mouthfeel is good, can not destroy other nutritive ingredient in the milk, is the good method that adopts in milk-product processing at present.
Sumylact L is decomposed into glucose, the semi-lactosi that easily is absorbed by the body and utilizes with lactose, can solve the problem of lactose intolerance edible for patients milk-product.In addition, Sumylact L can also be improved the quality of milk-product and produce oligomeric galactose.Oligomeric galactose has the bifidus bacillus increment factor and relative physiological function as functional oligose: low sugariness is low in calories, and low carious tooth can be improved lipid metabolism, improves absorbing of mineral element.
Sumylact L extensively is present in plant, animal, the microorganism.Its potentiality in diary processing was known to the people at the beginning of last century.The sixties, commercially produce by the Sumylact L that extracts in the microorganism.At present, commercial Sumylact L is mainly derived from aspergillus niger, aspergillus oryzae, Kluyveromyces lactis and Crewe dimension saccharomyces fragilis etc.The preparation of free Sumylact L need be extracted Sumylact L usually in the prior art from cell, causes the unit output of Sumylact L lower.Biological Technology institute, Chinese Academy of Agricultural Sciences etc. have developed a kind of new lactase, from Aspergillus candidus CGMCC3.2919, be isolated and cloned into the Sumylact L encoding gene, utilize bio-reactor pichia spp (Pichia pastoris) to efficiently express, its expression product is secreted outside born of the same parents, and Sumylact L is lived and is 3600u/mL; It is proteic more than 90% that fermentative production Sumylact L albumen accounts in the fermented liquid total secretion, and foreign protein content seldom; Solved the defective that needs from cell, to extract Sumylact L in the prior art, improved the unit output of Sumylact L greatly, thereby (Chinese patent application CN 02108141.7, the open date: on October 8th, 2003) for a new way has been opened up in the production of Sumylact L Product industrialization.
When resolvase is used for foodstuffs industry, can cause foreign protein to pollute.Simultaneously, resolvase can't be recycled, and makes production cost improve.Compare with resolvase, immobilized enzyme has following advantage: (1) tool is physical strength preferably, can act under the mode that stirs or adorn post; (2) can be used for suitability for industrialized production, adapt to the requirement of serialization, automatic production; (3) product purity height, side reaction are few, so productive rate is higher; (4) can use repeatedly.Improve the utilization ratio of enzyme, reduced production cost.
The seventies in last century, Italy prepares immobilized lactase with cellulose acetate and has successfully realized the production of low-lactose milk (F.Morisi, Jouranl of dairy science, 1972,56 (9), 1123-1127; 57 (3), 269-272).But use chloroform and toluene in the immobilized enzyme preparation process, these two kinds of organic solvents are used for foodstuffs industry to make us and can't accept.The U.S. (United States Patent (USP) 4,748,121), Japan's (Japanese Patent 56051984) have also reported the immobilization of Sumylact L, but the preparing carriers complexity in the United States Patent (USP) needs in the Japanese technology Sumylact L in the extracellular microbial is extracted.People such as domestic Luo Cheng ancient term for country school, Qin Yan, king's oats orchid have carried out the trial that immobilized lactase is produced low-lactose milk, or with embedded immobilizations such as carrageenins, or with cotton fibre absorption glutaraldehyde cross-linking method, or, all be not suitable for the immobilization of microorganism extracellular lactase with permeability cell Sumylact L and tubular fibre biochemical reactor.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, and a kind of process for fixation of new microorganism extracellular lactase is provided.
Technical problem to be solved by this invention realizes by following technological approaches:
A kind of process for fixation of microorganism extracellular lactase may further comprise the steps:
(1) prepares the outer free milk xylanase of born of the same parents by art methods;
(2) free milk xylanase is adsorbed on the macroporous resin that pore diameter range is 80～200 ;
(3) macroporous resin that will adsorb enzyme soaks with sodium alginate soln, stirs, and leaches, and carries out embedding treatment again;
(4) through the macroporous resin cross-linking reagent of embedding treatment,, leach in will step (3), promptly get immobilized enzyme at 4～20 ℃ times crosslinked 1～4 hour.
In the said fixing method, wherein the outer free milk xylanase of the born of the same parents described in the step (1) can adopt that any one method prepares in the prior art, and can select application number as a reference for use is that the disclosed method of patent application of CN 02108141.7 prepares.Adsorption method described in the step (2) is: every 1g resin adds the Sumylact L liquid of 100～500 units, and adsorption temp is 10～50 ℃, and the vibration adsorption time is 1～4 hour.The aperture of the macroporous resin described in the step (2) is preferably 100～120 .The weight percent concentration of the sodium alginate soln described in the step (3) is preferably 1%～5%; Carry out the used reagent of embedding treatment and be selected from CaCl
2Solution, carrageenin or gelatin are preferably the CaCl that concentration is 0.1～1.0mol/L
2Solution.It is 0.25%～2.5% glutaraldehyde that cross-linking reagent described in the step (4) is preferably weight percent concentration.
Used macroporous resin all can be bought from market and obtain among the present invention, as long as pore diameter range is 80～200 , use of the present invention be can satisfy, macroporous resin D001, D001-CC, D061, D113, DA201, HPD600, HPD300, HPD100A, D202 of following model etc. for example can be selected for use.
The present invention as Sumylact L immobilization absorption carrier, obtains the immobilized lactase of excellent property with common commercial resins through simple embedding, crosslinking Treatment.The entire operation process does not need Sumylact L is carried out loaded down with trivial details extraction and purification step, makes production cost greatly descend.Simultaneously, the operational stability of immobilized enzyme is fine.Utilize preparation technology of the present invention, can realize effective immobilization of Sumylact L; After measured, the enzyme work of the immobilized lactase that process for fixation of the present invention is prepared is greater than 70～100u/g, and enzyme yield 50%～60% alive can be directly used in the production of low-lactose milk.
As biological catalyst hydrolysis 5% lactose solution, the immobilized enzyme consumption is 10%～100%, 30 ℃ of effect 0.5～10h down with the immobilized lactase of the inventive method preparation, and lactose hydrolysis ratio transforms more than 50 batches continuously more than 80%.Handle fresh milk (wherein lactose-content 3%～6%) with this immobilized lactase, 5～60 ℃ transform 0.2～8 hour down, and lactose hydrolysis ratio is 70～80%, and the production efficiency of unit immobilization enzyme-treated milk is 1.25L/gh.
Further illustrate beneficial effect of the present invention by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit the scope of the invention.
Embodiment
Illustrate: remove specified otherwise among the application, used solution is the aqueous solution.
The measuring method of work of Sumylact L enzyme and lactose-content:
Free lactose activity determination method: the Sumylact L enzyme liquid 1mL that gets suitable dilution, 30 ℃ are incubated 2 minutes, add 10mL5% (g/100mL) lactose solution, and 30 ℃ of reactions are after 15 minutes, the variation of lactose-content before and after 10 minutes termination reactions of boiling water bath, assaying reaction.Under 30 ℃, the required enzyme amount of Sumylact L per minute hydrolysis 1 μ mol lactose is an enzyme unit (u) alive.
The immobilized lactase activity determination method: get the 0.5g immobilized lactase, add 25mL5% (g/00mL) lactose solution, 30 ℃ of reactions extracted reaction solution and carry out 10 minutes termination reactions of boiling water bath after 15 minutes, the variation of lactose-content before and after the assaying reaction.Under 30 ℃, the required enzyme amount of Sumylact L per minute hydrolysis 1 μ mol lactose is an enzyme unit (u) alive.
Lactose content measuring method: HPLC measures lactose-content, equipment used is: Agillent 1100 high performance liquid chromatographs, differential refraction detector, stationary phase is Agilent Zorbax CarbohydrateAnalysis Column, moving phase is formed, acetonitrile: water=70: 30, flow velocity 0.5mL/min.The lactose appearance time is 18.098min.With the peak height is quantitative criterion.
Treatment process in the milk before the lactose content measuring: get 2mL milk in the 10mL volumetric flask, add 1mL 20% plumbic acetate solution, shake up; Add 1mL potassium oxalate-disodium phosphate soln (3g potassium oxalate and 7g Sodium phosphate dibasic are dissolved in the 100mL water formulated) again, shake up constant volume; Leave standstill after 30 minutes with dry filter paper and filter, 0.45 μ m filter membrane handles after lactose-content in the HPLC analytic sample.
Embodiment 1
According to number of patent application is that the disclosed method of patent application of CN 02108141.7 prepares the extracellular free milk xylanase; With pore diameter range is that the macroporous resin D113 of 100～120 is the Sumylact L absorption carrier, immobilization technology is: resin demand is the outer free milk xylanase enzyme liquid of born of the same parents that every 1g resin adds 200 units, 10 ℃ of adsorption temps, vibration adsorption time 4 hours, leach the resin of adsorptive enzyme, add concentration and be in 2% the sodium alginate soln and stir, leach, place 0.1mol/L CaCl
2Soaked in the solution 5 minutes, and leached resin, add again in the glutaraldehyde of concentration 2.5%, under 4 ℃ of temperature, left standstill crosslinked 1 hour.Measure the immobilized enzyme 91u/g of being alive, enzyme yield alive is 56.88%.
Embodiment 2
According to number of patent application is that the disclosed method of patent application of CN 02108141.7 prepares the extracellular free milk xylanase; With pore diameter range is that the macroporous resin D001-CC of 80～120 is the Sumylact L absorption carrier, immobilization technology is: resin demand is the outer free milk xylanase enzyme liquid of born of the same parents that every 1g resin adds 100 units, 35 ℃ of adsorption temps, vibration adsorption time 2 hours, leach the resin of adsorptive enzyme, add concentration and be in 5% the sodium alginate soln and stir, leach, place 1mol/L CaCl
2Soaked in the solution 5 minutes, and leached resin, add concentration again and be in 0.25% the glutaraldehyde, under 20 ℃ of temperature, left standstill crosslinked 3 hours.Measure the immobilized enzyme 94u/g of being alive, enzyme yield alive is 58.98%.
Embodiment 3
According to number of patent application is that the disclosed method of patent application of CN 02108141.7 prepares the extracellular free milk xylanase; With pore diameter range is that the macroporous resin HPD300 of 100～120 is the Sumylact L absorption carrier, immobilization technology is: resin demand is the outer free milk xylanase enzyme liquid of born of the same parents that every 1g resin adds 500 units, 50 ℃ of adsorption temps, vibration adsorption time 1 hour, leach the resin of adsorptive enzyme, add concentration and be in 1% the sodium alginate soln and stir, leach, place 0.6mol/L CaCl
2Soaked in the solution 5 minutes, and leached resin, add concentration again and be in 1% the glutaraldehyde, under 10 ℃ of temperature, left standstill crosslinked 4 hours.Measure the immobilized enzyme 93u/g of being alive, enzyme yield alive is 57.76%.
Test example 1 resin shaker test
Prepare the extracellular free milk xylanase according to Chinese patent application number for the disclosed method of patent application of CN 02108141.7; With different models such as D001, D001-CC, D061, D113, DA201, HPD600, HPD300, HPD100A, D202, pore diameter range is each 3g of macroporous resin of 80～200 , the Sumylact L liquid 3mL that adds 280.89u/mL respectively, under 20 ℃, vibration absorption 4 hours, measuring resin absorption fixed Sumylact L lives, calculate enzyme yield alive, the results are shown in Table 1.
Table 1 resin The selection result
Above-mentioned The selection result shows that the employing pore diameter range is that the adsorbed fixed Sumylact L of macroporous resin of 80～200 all has higher enzyme yield alive, and wherein preferred pore diameter range is the macroporous resin of 100～120 .
Test example 2 immobilized lactase catalysis lactose hydrolysis effect tests of the present invention
One, test materials
1, for test agent: the immobilized lactase that the embodiment of the invention 1 is prepared.
2, lactose solution: concentration is 50g/L.
Two, test method and result
Will be for the test agent lactose solution that is 50g/L in 30 ℃ of following conversion of substrate, the immobilized enzyme amount is 10% (g/100mL) of substrate solution, the transformation time curve sees Table 2.
Table 2 transformation time curve
Determine that transformation time is 1 hour, transform 52 batches repeatedly in batches, the results are shown in Table 3.
| Transformation time (h) | 0.5 | 1 | 1.5 | 2 | 6 | 9 | 24 |
| Residue lactose-content (g/L) | 18.14 | 8.11 | 4.15 | 2.30 | 0.36 | 0.27 | 0.15 |
| Lactose hydrolysis ratio (%) | 58.69 | 81.53 | 90.49 | 94.75 | 99.18 | 99.39 | 99.66 |
Table 3 immobilized enzyme of the present invention transforms the test-results of lactose solution repeatedly in batches
| Use batch | Residue lactose-content (g/L) | Lactose hydrolysis ratio (%) | Use batch | Residue lactose-content (g/L) | Lactose hydrolysis ratio (%) |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 | 4.29 5.70 6.80 5.93 9.22 7.17 7.68 7.l0 7.50 7.74 7.54 8.35 11.97 7.70 8.14 8.73 7.37 9.67 7.98 7.74 7.34 8.38 7.73 8.06 9.02 8.10 | 91.29 88.42 86.18 87.95 81.26 85.43 84.38 85.56 84.76 84.27 84.68 83.02 75.67 84.34 83.46 82.26 85.02 80.35 83.78 84.27 85.08 82.98 84.30 83.61 81.67 83.54 | 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 | 8.52 8.86 8.71 8.51 9.83 14.24 7.57 9.62 9.01 10.23 9.69 9.36 9.61 9.98 11.43 9.65 6.41 10.33 8.81 10.39 9.90 6.49 9.34 9.62 10.04 11.74 | 82.69 81.99 82.29 82.71 80.01 71.06 84.60 80.45 81.68 76.69 77.92 78.69 78.10 77.27 73.96 78.03 85.40 76.48 79.94 76.33 77.45 85.21 78.72 78.09 77.14 73.26 |
Above-mentioned test-results shows that the immobilized lactase enzymic activity height that the present invention is prepared has higher hydrolyzes lactose efficient.
Test example 3
The immobilized lactase that embodiment 2 is prepared, 30 ℃ transform down milk (commercially available, the Bright brand fresh milk, down together), the immobilized enzyme amount is 10% (g/100mL) of milk consumption, lactose-content in different time sampling and measuring milk sample, and the transformation time curve sees Table 4.
Table 4 immobilized lactase transforms lactose-content variation in the milk process
| Transformation time (h) | 0 | 2 | 4 | 6 | 8 | 10 | 20 |
| Residue lactose-content (g/L) | 38.24 | 20.57 | 15.05 | 11.06 | 6.92 | 5.61 | 3.04 |
| Lactose hydrolysis ratio (%) | - | 46.59 | 60.92 | 71.29 | 82.03 | 85.43 | 92.12 |
Be 8 hours action time.Transform 30 batches repeatedly in batches, the results are shown in Table 5.
Table 5 immobilized lactase hydrolysed milk of the present invention 1-30 criticizes test-results
| Utilize batch | Lactose-content (g/L) | Lactose hydrolysis ratio (%) | Utilize batch | Lactose-content (g/L) | Lactose hydrolysis ratio (%) |
| 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 | 40.52 1.81 5.47 9.54 12.90 3.93 12.27 18.65 10.89 11.56 10.38 15.00 10.51 14.72 11.95 15.84 | - 95.55 86.51 76.45 68.16 90.30 69.72 53.98 73.14 71.48 74.39 62.99 74.07 63.67 70.50 60.90 | 0 * 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 | 63.23 9.31 11.11 5.88 10.50 10.53 11.47 13.28 12.42 18.13 15.49 13.20 13.29 13.06 12.96 18.45 | 85.28 82.42 90.70 83.39 83.35 81.86 79.00 80.36 71.33 75.50 79.12 78.98 79.35 79.50 70.83 |
*Wherein the 16th batch begins to use instead the milk of newly purchasing.
Above-mentioned test-results shows that the immobilized lactase enzymic activity height that the present invention is prepared has higher percent hydrolysis for lactose in milk.
Test example 4
The immobilized lactase that embodiment 3 is prepared, 60 ℃ transform milk (commercially available, Bright brand fresh milk) down, and the immobilized enzyme amount is 100% (g/100mL) of milk consumption, and transformation time is 15 minutes, the results are shown in Table 6.
60 ℃ of following immobilized enzyme of table 6 transform milk
| Use batch | Lactose-content (g/L) | Lactose hydrolysis ratio (%) |
| 0 1 2 3 4 5 6 7 8 9 10 | 63.23 7.07 7.28 15.77 13.86 15.81 19.16 21.36 21.00 28.32 25.00 | - 88.81 88.49 75.06 78.08 74.99 69.69 66.21 66.79 55.22 60.45 |
Test example 5
The method of embodiment 1 is prepared immobilized lactase, transform milk (commercially available, Bright brand fresh milk) under 5 ℃～7 ℃, the immobilized enzyme amount is 100% (g/100mL) of milk consumption, and transformation time is 30 minutes, the results are shown in Table 7.
Immobilized enzyme transforms milk under table 7 low temperature
| Use batch | Lactose-content (g/L) | Lactose hydrolysis ratio (%) |
| 0 1 2 3 4 5 6 7 8 9 10 | 29.87 7.63 9.87 11.08 10.89 8.86 10.27 12.91 9.15 10.89 10.05 | - 74.46 66.95 62.91 63.53 70.33 65.63 56.77 69.35 63.55 66.34 |
Claims (6)
1. fixing method of free milk xylanase may further comprise the steps:
(1) prepares free milk xylanase by art methods;
(2) free milk xylanase is adsorbed on the macroporous resin that pore diameter range is 80-200 ;
(3) macroporous resin that will adsorb free milk xylanase soaks with sodium alginate soln, stirs, and leaches, and carries out embedding treatment with embedding reagent again;
(4) macroporous resin that passes through embedding treatment in the step (3) was descended crosslinked 1-4 hour at 4-20 ℃ with cross-linking reagent, leach, promptly get immobilized lactase.
2. according to the method for claim 1, it is characterized in that in the step (2) free milk xylanase being adsorbed on the macroporous resin that pore diameter range is 80-200 according to following condition: every 1g resin adds the Sumylact L liquid of 100-500 unit, adsorption temp is 10-50 ℃, and the vibration adsorption time is 1-4 hour.
3. according to the method for claim 1, the aperture that it is characterized in that the macroporous resin described in the step (2) is 100-120 .
4. according to the method for claim 1, the weight percent concentration that it is characterized in that the sodium alginate soln described in the step (3) is 1%-5%; Described embedding reagent is selected from CaCl
2Solution, carrageenin or gelatin.
5. according to the method for claim 4, it is characterized in that described embedding reagent is that concentration is the CaCl of 0.1-1.0mol/L
2Solution.
6. according to the method for claim 1, it is characterized in that the cross-linking reagent described in the step (4) is that concentration is the glutaraldehyde of 0.25%-2.5%.
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| CN100366737C (en) * | 2006-03-01 | 2008-02-06 | 哈尔滨美华生物技术股份有限公司 | Production of curing lactase |
| CN101575135B (en) * | 2009-06-05 | 2011-12-21 | 上海大学 | Method for separating out phenols substances in water by catalytic oxidation with enzyme-carrying silicon-based mesoporous molecular sieve SBA-15 |
| CN108064936A (en) * | 2016-11-11 | 2018-05-25 | 内蒙古伊利实业集团股份有限公司 | A kind of preparation method for the liquid diary product for reducing Mei Lade products |
| CN111763666A (en) * | 2020-07-29 | 2020-10-13 | 安徽大学 | Immobilization method of beta-galactosidase and application of beta-galactosidase in preparation of low-lactose milk |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0037667A2 (en) * | 1980-04-04 | 1981-10-14 | Sumitomo Chemical Company, Limited | Immobilized lactase, its preparation and use |
| US4465772A (en) * | 1980-10-06 | 1984-08-14 | Sumitomo Chemical Company, Limited | Method for disinfecting and washing of immobilized lactase |
| CN2362866Y (en) * | 1999-02-11 | 2000-02-09 | 中国牧工商(集团)总公司 | Hyaluronic cell lactase hollow fibre biochemical reactor |
-
2005
- 2005-07-06 CN CNB2005100804920A patent/CN1314800C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0037667A2 (en) * | 1980-04-04 | 1981-10-14 | Sumitomo Chemical Company, Limited | Immobilized lactase, its preparation and use |
| US4465772A (en) * | 1980-10-06 | 1984-08-14 | Sumitomo Chemical Company, Limited | Method for disinfecting and washing of immobilized lactase |
| CN2362866Y (en) * | 1999-02-11 | 2000-02-09 | 中国牧工商(集团)总公司 | Hyaluronic cell lactase hollow fibre biochemical reactor |
Non-Patent Citations (1)
| Title |
|---|
| 微生物乳糖酶的特性和发展现状 王敏等，河北农业大学学报，第26卷 2003 * |
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