CN101570745A - Technology for preparing lactose enzyme - Google Patents
Technology for preparing lactose enzyme Download PDFInfo
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- CN101570745A CN101570745A CNA2008100368893A CN200810036889A CN101570745A CN 101570745 A CN101570745 A CN 101570745A CN A2008100368893 A CNA2008100368893 A CN A2008100368893A CN 200810036889 A CN200810036889 A CN 200810036889A CN 101570745 A CN101570745 A CN 101570745A
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Abstract
The invention provides a method for preparing lactose enzyme which is a novel high-efficient preparation. In the method, bacteria are processed by NTG with a certain concentration to produce high yield bacteria, solid fermentation is carried out under the condition which is suitable for the growth of microorganisms of the high yield bacteria, and industrial enzyme with high purity quotient and ideal yield coefficient is obtained by extracting and separating under a totally enclosed condition. The method has the advantages of soft preparing condition and little generation of three wastes.
Description
Technical field
The present invention relates to utilize microbial technique to prepare industrial enzymes, more specifically refer to adopt aspergillus oryzae to prepare Sumylact L through microbial technique.
Background technology
Zymin industry is knowledge-intensive high-tech industry, the Application Areas of zymin is very extensive, enzyme has efficiently, the characteristics of exclusive reaction mild condition, because it has so that characteristic can have selection, controlledly by specific mode, example hydrolysis, redox, cracking, chemical group transfer etc. make and are applied object by predetermined imagination generation chemical reaction, produce required purpose product.Enzyme is a kind of protein, self fully biodegradable.The biological catalyst that the enzyme conduct has specificity and high efficiency, the alternative traditional chemistry of enzyme method technique, physical technology, for example substituted chemistry reagent, minimizing process water, minimizing energy consumption, minimizing raw materials consumption, raising productive efficiency.The application of zymin for increasing economic efficiency, reduces the resource energy consumption, promotes the lifting of conventional industries, particularly helps the improvement of ecotope, meets the requirement of Green Chemistry.According to american chemical association statistics, about 3,000,000,000 dollars of industrial enzymes market, the annual whole world, zymin market, the world increases year by year with average 30% speed.Deep development along with China's reform economy, gratifying variation also takes place in zymin market, annual with 20% speed increment, and because present zymin research and development of China and production are in the state that comparatively falls behind, can not satisfy the production demand of every profession and trade, China needs a large amount of expensive industrial enzymes of import every year, mainly from national imports such as Denmark, Japan, the U.S..
New and effective zymin-Sumylact L is to be difficult for the lactose hydrolysis that is absorbed by the body in the milk-product and to become the semi-lactosi and the glucose that can be absorbed by the human body normal stool a kind of can the decomposition.Can fundamentally solve the problem of " lactose intolerance " that occur when the puzzlement consumers in general eat milk-product.
Show according to the recent survey data of relevant medical facility, the incidence of China adult " lactose intolerance " reaches about 75～92%, and for the crowd of vast lactose intolerance, the simplest, the most direct methods of treatment is when its EDIBLE LACTOSE food, supply with ectogenic lactase formulation and be used for hydrolysis, promote absorption of human body lactose.
As everyone knows, milk is brute force the most natural, the abundantest, the most cheap while of being healthy and strong food of easy absorption, and the example of " one glass of strong nationality of milk " is abroad arranged.Since the reform exploitation, the dairy industry production of China improves constantly and develops rapidly along with living standards of the people, the liquid state milk ultimate production was 6,300,000 tons in 2006, increased by 32% than the last year, according to world market consumption rule, low lactose milk calculates in 10% ratio of production, then need produce nearly 1,000,000 tons of low lactose milk in 2006, the consumption by 20001 calculates, and then needs 500 tons of Sumylact Ls, the consumption proportion of annual liquid state milk increases year by year in addition, so the Sumylact L market outlook are considerable.Simultaneously, this project product can superior cost performance be come into the world market.
Since the seventies, just engage in the research and development and the production of Sumylact L abroad, and the blank stage of genus is still produced in the research and development of China's Sumylact L, at present the domestic situation that is in complete dependence on import.The present invention produces the preparation Sumylact L and has the enzymic activity height, is swift in response, and the product purity height, the characteristic that storage stability is good, every index of product all reaches external like product, fully can the substituting import one Sumylact L.Succeeding in developing of this project not only will promote the technological competitiveness of China's zymin industry, and fill up domestic blank, quicken the development of national industry, and be that country's foreign exchange earning is contributed.
Research and produce this zymin with microbial technique, and make this project industrialization, to improve China in this area research level, will produce economic results in society and to have influence, fully fundamentally solve and improve the present situation of Dairy Industry in China, promote the Application and Development of China's biotechnology in this field.Sumylact L has extensive potential Application Areas in industries such as medical industries, healthcare products simultaneously.
The application of industrial enzyme preparation, for raising the efficiency, cut down the consumption of energy and reduce blowdown unusual effect is arranged, it is typical Green Chemistry treatment process, the invention belongs to the new and effective industrial enzyme preparation of biocatalysis technology, be the research and development field that state key is supported, therefore have great far-reaching social and economic significance.
Summary of the invention
The object of the invention provides a kind of novel process for preparing Sumylact L.The present invention adopts and has used following gordian technique:
1) adopts novel triage techniques, the seed selection high-efficiency strain
Microbial fermentation is bionic important application technology, can produce the product that many chemical methods are difficult to produce.The selection of high-efficiency strain is to improving the microbial fermentation level, reduces production costs and to reduce blowdown flow rate significant.
It is to utilize the microorganism original strain that the present invention adopts novel triage techniques, handles by specific chemical process, and Screening and Identification goes out the production bacterial classification of high expression level.Culture propagation through screening is rapid, the fermentation activity height, and incubation time is short, the productivity effect height, it is rare to have solved traditional animal vegetable tissue extraction method raw material sources, and the extractive technique difficulty is difficult to realize the problem of industrialization.
2) adopt novel environment friendly fermentation raw material prescription, the protection natural resource
It is to utilize wide material sources that the present invention adopts novel environment friendly fermentation raw material prescription, agricultural-product wheat bran with low cost, foster base is joined in the main fermentation of conducts such as soybean cake powder, and the liquid fermentation method that at present general zymin manufacturing enterprise adopts, all adopt the smart raw material of higher-priced industrial chemicals and cereal, the cost high cost is big, consume natural resource, be unfavorable for environmental protection requirement, and the fermentation raw material that this project adopted mainly is with pair product material after the cereal processing, production cost is low, saves Biological resources, and environmental environmental protection is newly filled a prescription, the prescription that utilizes this uniqueness is produced the preparation Sumylact L under specific fermentation culture conditions.
3) adopt centrifugal ultra-filtration concentrated and purified, reduce the cleaner production new technology of blowdown flow rate
Zymin is extracted exquisite technology and is generally adopted ammonium sulfate folding and spray-dired traditional technology, adopt ammonium sulfate to roll over and contain big content ammonium sulfate composition in the zymin finished product of explained hereafter, do not meet the requirement of lactose processing industry, yield rate is low simultaneously, only be 50%, and adopting the zymin of drying process with atomizing production to produce large quantity of exhaust gas in production process, serious environment pollution is unfavorable for enterprise development and environmental requirement.And this problem is researched and developed and is adopted centrifugal ultra-filtration to concentrate novel extraction process for purification, makes the quality of product obtain upgrading, makes end product meet the requirement of market economic development.Simultaneously, avoid the pollution of environment, satisfy environmental requirement fully.This of this problem technology can make process recovery ratio be increased to 80%～95% from 50%.
4) adopt solid fermentation, improve utilization of resources degree
At present general zymin enterprise adopts liquid big jar to carry out fermentation culture, the investment goods cost is bigger, simultaneously in the production culturing process, consumption coal consumption power consumption vapour is comparatively serious, not only make enterprise's production cost high, simultaneously, consumption of natural resource, destroy environmental protection, seriously disturb enterprise's normal existence development.And the employing of this problem is novel original creation solid fermentation method, this kind fermentation culture method less investment, and cost consumption is low, in fermentation culture is produced, adopts the gentle means of natural lighting, ventilating and thermal insulating mostly.The energy consumption of coal electricity vapour is compared with the conventional liquid fermentation method, can save about 60%, and the fermentative activity height of this technology simultaneously equates with international zymin advanced production enterprise (Novozyme) fermentative activity, domestic genus top standard, and working condition is convenient to control.
5) adopt novel enzyme goods preservatives, product prolongs stationary phase.
Enzyme preparation product is a kind of biologically active substance that possesses, and it is comparatively responsive to temperature, pH, humidity, and domestic general enzyme preparation product was about about 3 months at present.This problem is according to biological enzyme particular organisms performance, by carrying out the screening operation that long-term a large amount of preservatives is filled a prescription, research and development have the biological nature of suitable Sumylact L, effectively stablize the biological activity of Sumylact L, compare with traditional preservatives preservation, preservation period can extend to 1 year.
The present invention finishes through the following steps:
A. the bacterial classification of the present invention's employing is aspergillus oryzae (Aspergillus oryzae) ATCC6061, it is that 100～1000 μ g/ml carry out immersion treatment with bacterial classification and NTG (N-methyl-N-Nitro-N-Nitro Soguanidine) concentration at first that bacterial classification is purchased in Japan Society For Culture Collections, time is 20～40 minutes, 4～8 times repeatedly.By the 500ml shake-flask culture, obtain high-yield strains.
B. bacterial classification inoculation is gone into and ferment in the disinfectant seed culture medium, seed culture medium is (weight %) glucose 2～5%, peptone 1～3%, yeastex 0.5～2.5%, ammonium phosphate salt 0.01～0.10%, adding water is made into volume required, 0.1MP sterilize 20 minutes, the seed culture temperature is 32～42 ℃, and incubation time is 16～40 hours.Bacterial classification with gained after the seed culture fermentation is inoculated into the solid-substrate fermentation cultivation by inoculum size 3～8%.
The bacterial classification inoculation of c, seed tank culture is in solid medium, this medium component is main raw material with the Testa Tritici, Testa Tritici content is 90～98%, soybean cake powder 2～10%, 0.1MP sterilize 40 minutes, adopt automatic solid fermentation machine, regulate moisture content to 45～65% moisture content, temperature is controlled at 28～38 ℃, pH4.5～6.8, fermentation culture were carried out solid fermentation in 62～68 hours and are cultivated.
D, be soaked in water by fermentation Hou De Qu, water temperature 25～35 degree, soak time 2～4 hours is used filter press then, after the coarse filtration, gets supernatant liquor through centrifuging and taking, centrifugal speed 3000～5000rpm.Be removed centrifugate behind all insolubless and carry out 2～6 times ultrafiltration and concentration, with the concentrated solution that obtains high density after 0.2um～0.4um film sterile filtration, become the pulvis finished product through air stream drying more at last, or obtain liquid end product by the cooperation of preservatives.
The mensuration of e, enzymic activity and method of calculation
I, by the optimal pH scope of enzyme reaction (scope 5.0～6.8 usually), preparation 5ppm sodium phosphate buffer, the matrix of 1～3% concentration (ONPG).
The optimum temperuture of ii, regulatory enzyme reaction is generally 30～40 ℃, institute's test sample product is joined carry out enzyme reaction in the response matrix, and the reaction times is 10～40 minutes.
Iii, after enzyme reaction is carried out 10～40 minutes, add enzyme deactivation agent 2N CCl
3The enzyme reaction of COOH stop.
Iv, use water as contrast, carry out the quantitative assay of enzyme activity at 400～600um with colorimetry, under these conditions, unit of enzyme activity is defined as per minute enzyme liquid hydrolysing substrate and generates the required enzyme amount of resolvent.
V, be benchmark, converse the unit vigor (international unit method, International Units Method) of enzyme with the concentration of standard solution curve
Beneficial effect of the present invention is as follows:
1, the present invention produces the Sumylact L of preparation as the unique biological catalyzer, be applied to diary industry, this product has the enzymic activity height, be swift in response, constant product quality becomes the lifting that diary industry promotes traditional technology, and technology simplifies the operation, improve product specification, the strong pillar that reduces production costs.
2, utilization new mushroom-seed culturing selection-breeding method, high-yield strains is cultivated in screening, to improving the microbial fermentation level, reduces production costs and to reduce blowdown flow rate significant.
3, the present invention adopts novel environment friendly fermentation raw material prescription, and the protection natural resource help environmental requirement.
4, it is concentrated and purified that the present invention adopts centrifugal ultra-filtration, reduces the cleaner production new technology of blowdown flow rate,
5, the present invention adopts solid fermentation, reduces cost of investment, reduces energy input, improves utilization of resources degree.
6, adopt novel enzyme goods preservatives, constant product quality, shelf life extension,
Description of drawings:
Fig. 1 is a process flow sheet of the present invention.
Embodiment
Below in conjunction with specific embodiment the present invention is done further narration, but do not limit the present invention.
1) implements the preparation Sumylact L
1-1 is that 100～1000 μ g/ml carry out immersion treatment from Aspergillusoryzae ATCC 6061 bacterial classifications that Japan Society For Culture Collections buys through chemical NTG concentration, and the time is 20～40 minutes, 4～8 times repeatedly.By carrying out the Sumylact L preparation after the processing of 500ml shake-flask culture.
1-2 goes into bacterial classification inoculation and ferments in the disinfectant seed culture medium, seed culture medium is (weight %) glucose 2%, peptone 1%, yeastex 0.5%, ammonium phosphate salt 0.01%, adding water is made into volume required, 0.1MP sterilize 20 minutes, the seed culture temperature is 32～42 ℃, and incubation time is 16～40 hours.Seed culture liquid amount 200ml/500ml triangular flask, box reciprocal shaking table 200rpm, the bacterial classification with gained after the seed culture fermentation is inoculated into the solid-substrate fermentation cultivation by inoculum size.(inoculum size 3～8%)
The bacterial classification inoculation of 1-3 seed tank culture is in solid medium, this medium component is main raw material with the Testa Tritici, Testa Tritici content is 90～98%, soybean cake powder 2～10%, 0.1MP sterilize 40 minutes, adopt automatic solid fermentation machine, regulate moisture content to 45～65% moisture content, temperature is controlled at 28～38 ℃, pH4.5～6.8, fermentation culture were carried out solid fermentation in 62～68 hours and are cultivated.。
1-4 is soaked in water by fermentation Hou De Qu, 25～35 ℃ of water temperatures, and soak time 2～4 hours is used filter press then, after the coarse filtration, gets supernatant liquor through centrifuging and taking, centrifugal speed 3000-5000rpm.Be removed centrifugate behind all insolubless and carry out 2～6 times ultrafiltration and concentration, with the concentrated solution that obtains high density after 0.2um～0.4um film sterile filtration, become the pulvis finished product through air stream drying more at last, or obtain liquid end product by the cooperation of preservatives.
1-5 presses the measuring method of Sumylact L with finished product, uses water as contrast, carries out the quantitative assay of enzyme activity with colorimetry at 400～600um.
2) analytical procedure
2-1, thalli growth are measured: the fermentation soak solution dilutes 20-35 doubly with the sodium phosphate buffer of 0.5mol, and spectrophotometer 430nm measures the thalline optical density(OD).(spectrophotometer; Shanghai Spectrum Apparatus Co., Ltd.)
2-2, zymoprotein flow measurement: the fermentation soak solution dilutes 10-25 doubly with the sodium phosphate buffer of 0.5mol, and spectrophotometer 270nm measures protein content.(spectrophotometer; Shanghai Spectrum Apparatus Co., Ltd.)
2-3, pH measure: 818 type acidometers are measured.(acidometer; Shanghai one permanent Science and Technology Ltd.)
3) pilot-scale experiment
Table 1 traditional zymotic technology and zymotechnique of the present invention are relatively
| Project | Technology of the present invention | Traditional technology |
| Fermentation period (hour) | 62-68 | 80-120 |
| Mycelia body weight (g/ wheat bran) | 0.432-0.600 | 0.220-0.286 |
| Zymoprotein amount (%) | 7.8-8.8 | 3.6 |
Table 2 traditional extraction technique and extraction process of the present invention are relatively.
| Pollution rate (%) | Extracting cycle (hour) | Product yield (%) | Product percent of pass (%) | |
| Novel process | 1-5 | 62-72 | 80-95 | 98-99 |
| Traditional technology | 10-30 | 98-144 | 50-60 | 45-50 |
Table 3 seed fermentation is cultivated data
| Lot number | Cell density (430nm) | Incubation time (hrs) | pH |
| 1 | 0.453 | 25 | 5.67 |
| 1 | 0.443 | 22 | 5.65 |
| 2 | 0.434 | 28 | 5.75 |
| 2 | 0.435 | 22 | 5.70 |
| 3 | 0.438 | 21 | 5.69 |
| 3 | 0.440 | 20 | 5.68 |
Table 4200 square metre solid fermentation is cultivated data
| Lot number | Enzyme protein expression amount % | Incubation time hrs. | pH |
| 1 | 7.45 | 62 | 5.7 |
| 1 | 7.42 | 63 | 5.5 |
| 2 | 7.34 | 62 | 5.5 |
| 2 | 7.35 | 63 | 5.7 |
| 3 | 7.12 | 62 | 5.9 |
| 3 | 7.10 | 61 | 5.8 |
4) quality index
Sumylact L of the present invention has been obtained 200 square metres of fermentation culture, and finishes separation and purification purified technology, facts have proved this technology good reproducibility, constant product quality.
Table 5. Sumylact L product quality indicator of the present invention and relevant international zymin manufacturer G company are relatively
| External zymin manufacturer G company | Good meter (Shanghai) biological company limited | |
| Enzymic activity (μ/g) | 8,500 | 10,000 |
| Total plate count (individual/g) below | 2,000 | 1000 |
| Outward appearance | Dark powder or brown liquid | Pale yellow powder or brown liquid; |
| Below the heavy metal ppm | 100 | 5 |
| Pb | 100 | 5 |
| As | 50 | 5 |
。
Claims (5)
1, a kind ofly prepare the method for Sumylact L, it is characterized in that forming by the following step by strain fermentation:
A, a kind of be the bacterial classification aspergillus oryzae of 100-1000 μ g/ml solution soaking through NTG concentration;
B, seed fermentation;
C, solid culture fermentation;
D, refining separating enzyme.
2, preparation Sumylact L method according to claim 1 is characterized in that the concentration that bacterial classification and NTG handle is that 100～1000 (μ g/ml) carry out immersion treatment, and the time is 20～40 minutes, 4～8 times repeatedly.
3, preparation Sumylact L method according to claim 1 is characterized in that the seed fermentation culture medium prescription is (weight %)
Glucose 2～5%,
Peptone 1～3%,
Yeastex 0.5～2.5%,
Ammonium phosphate salt 0.01～0.10%,
The seed culture temperature is 32～42 ℃, and incubation time is 16～40 hours.
4, preparation Sumylact L method according to claim 1 is characterized in that the solid fermentation culture condition is a substratum;
Testa Tritici content is 90～98%,
Soybean cake powder 2～10%,
0.1MP sterilize 40 minutes, adopt automatic solid fermentation machine, regulate moisture content to 45～65% moisture content, temperature is controlled at 28～38 ℃, pH4.5～6.8, fermentation culture was carried out solid fermentation in 62～68 hours and is cultivated.
5, preparation Sumylact L method according to claim 1 is characterized in that being soaked in water by fermentation Hou De Qu, water temperature 25～35 degree, and soak time 2～4 hours.Be removed centrifugate behind all insolubless and carry out 2～6 times ultrafiltration and concentration, with the concentrated solution that obtains high density after 0.2um～0.4um film sterile filtration, become the pulvis finished product through air stream drying more at last, or obtain liquid end product by the cooperation of preservatives.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812435A (en) * | 2010-05-31 | 2010-08-25 | 鼎正动物药业（天津）有限公司 | Preparation and extraction process of extracellular lactase |
| CN102703405A (en) * | 2012-06-28 | 2012-10-03 | 甘肃农业大学 | Method for preparing lactase by taking bran as raw material |
| CN103555692A (en) * | 2013-10-29 | 2014-02-05 | 河北省微生物研究所 | Method for preparing aspergillus oryzae lactase fermentation starter by solid pot fermentation |
| CN109295037A (en) * | 2018-10-11 | 2019-02-01 | 山东隆科特酶制剂有限公司 | A kind of method and its institute's galactopoiesis carbohydrase using aspergillus oryzae fermenting and producing lactase |
| CN109321469A (en) * | 2018-10-11 | 2019-02-12 | 山东隆科特酶制剂有限公司 | The aspergillus oryzae and its enzymatic production method of one plant height galactopoiesis carbohydrase |
-
2008
- 2008-04-30 CN CNA2008100368893A patent/CN101570745A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812435A (en) * | 2010-05-31 | 2010-08-25 | 鼎正动物药业（天津）有限公司 | Preparation and extraction process of extracellular lactase |
| WO2011150754A1 (en) * | 2010-05-31 | 2011-12-08 | 鼎正动物药业（天津）有限公司 | Method for preparing and extracting extracellular lactase |
| CN102703405A (en) * | 2012-06-28 | 2012-10-03 | 甘肃农业大学 | Method for preparing lactase by taking bran as raw material |
| CN103555692A (en) * | 2013-10-29 | 2014-02-05 | 河北省微生物研究所 | Method for preparing aspergillus oryzae lactase fermentation starter by solid pot fermentation |
| CN103555692B (en) * | 2013-10-29 | 2015-09-30 | 河北省微生物研究所 | Adopt solid tank fermentation for the method for aspergillus oryzae lactase fermentation song |
| CN109295037A (en) * | 2018-10-11 | 2019-02-01 | 山东隆科特酶制剂有限公司 | A kind of method and its institute's galactopoiesis carbohydrase using aspergillus oryzae fermenting and producing lactase |
| CN109321469A (en) * | 2018-10-11 | 2019-02-12 | 山东隆科特酶制剂有限公司 | The aspergillus oryzae and its enzymatic production method of one plant height galactopoiesis carbohydrase |
| CN109295037B (en) * | 2018-10-11 | 2021-07-23 | 山东隆科特酶制剂有限公司 | Method for producing lactase by adopting aspergillus oryzae fermentation and produced lactase |
| CN109321469B (en) * | 2018-10-11 | 2021-07-23 | 山东隆科特酶制剂有限公司 | Aspergillus oryzae capable of producing lactase with high yield and fermentation enzyme production method thereof |
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