CN1055118C - Bacillus subtilis and producing tech. for solid alkaline degumming enzyme - Google Patents
Bacillus subtilis and producing tech. for solid alkaline degumming enzyme Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明涉及一种枯草芽孢杆菌(Bacillus Subtilis)CGMCC No.0213以及用该菌，在高培养基浓度、适当碳、氮比条件下培养、发酵然后高温快速喷雾干燥，生产一种不含纤维素酶，以果胶酶为主，同时含有蛋白酶、淀粉酶、木聚糖酶的复合固体酶工艺。工艺简单，周期短，原料廉价、酶活性高，菌和酶无毒性，耐热、耐碱，不加防腐剂便可贮藏，且活性稳定，运输，使用皆方便，酶作用条件温和，可重复使用，废水无污染，用于苎麻脱胶回收率高，麻纤维结实，可节省4-5道工序，耗能低。The present invention relates to a kind of bacillus subtilis (Bacillus Subtilis) CGMCC No.0213 and use this bacterium to cultivate and ferment under the conditions of high medium concentration, appropriate carbon and nitrogen ratio, and then high-temperature rapid spray drying to produce a cellulose-free Enzymes, mainly pectinase, also contain protease, amylase, xylanase compound solid enzyme process. The process is simple, the cycle is short, the raw materials are cheap, the enzyme activity is high, the bacteria and enzyme are non-toxic, heat-resistant, alkali-resistant, can be stored without preservatives, and the activity is stable, convenient for transportation and use, and the enzyme action condition is mild and repeatable Use, no pollution to wastewater, high recovery rate for ramie degumming, strong hemp fiber, can save 4-5 processes, and low energy consumption.
Description
The present invention relates to the cultivation of a kind of genus bacillus (Bacillus), particularly subtilis (Bacillus Subtilis) bacterial strain and produce the solid alkaline degumming enzyme production technique with this bacterium.By separation, mutagenesis, filter out this bacterial strain, in high density with suitably under the condition of C.N ratio, cultivate and fermentation then, again through high temperature spray-drying, produce a kind of not cellulase, and contain polygalacturonase (being main), contain the composite solid zymin of proteolytic enzyme, amylase and zytase simultaneously.
Polygalacturonase is as the China grass degumming of degumming agent widespread use what textile industry, paper industry and other light industry.Being used for the China grass degumming aspect, to mainly contain the ramie enzyme (" ramie textile science and technology " the 12 total 53 phases of volume) that Denmark NOVO company produces be a kind of brown liquid enzyme, by aspergillus (belong to fungi) bacterial strain output, the mixed enzyme that generally contains polygalacturonase, cellulase and hemicellulase etc., slant acidity.Domestic also have a manufacturer production fungi acid pectase preparation.But above-mentioned enzyme easily decomposes fiber because of containing cellulase under acidity, cause fibre strength and row yielding to reduce, and numb factory keeps away and uses it.Hunan China normal university has applied for that Chinese patent CN85104284 and CN85104285 provide a kind of subtilis (Bacillus Subtilis) A2-5, produce the pectin mixed enzyme through industrial fermentation, though mentioning, these two patents produce the solid enzyme, but do not provide the production technique of solid enzyme in the specification sheets, do not speak of the application of solid enzyme yet, only speak of liquid enzymes production technique and concentrated enzyme when being strong basicity, adding millesimal benzoate again can anticorrosion guarantor's enzyme, preferably preserve half a year, otherwise liquid enzymes loses activity very soon, few person several days, many persons some months is with regard to inactivation, and general liquid enzymes be cannot say for sure to deposit.
The object of the invention is to overcome the many deficiencies of foregoing invention, a kind of subtilis (Bacillus Subtilis) CGMCC NO.0213 is provided and produces the solid alkaline degumming enzyme processing method with this bacterial strain, this kind of enzyme is a kind of not cellulase, and contain polygalacturonase (being main), contain the solid complex enzyme preparation of proteolytic enzyme, amylase and zytase simultaneously.Technology of the present invention is simple, cycle is short, the raw material cheapness, enzymic activity height, bacterium and enzyme nontoxicity, heat-resisting, alkaline-resisting, do not add any sanitas and can store yet and reach 1 year, and activity stabilized, solid enzyme transportation and easy to use, enzyme action condition gentleness, flaxen fiber intensity and row yielding height, can reuse again, thereby reduce cost, compare with traditional method, can save the 4-5 procedure, it is low to consume energy, and waste water is pollution-free, and can fully utilize, fiber crops factory just available this enzyme under existence conditions carries out retting, needn't increase new investment.
Content of the present invention is to use not cellulase-producing, nutritional condition is less demanding, heat-resisting, alkali proof bacillus subtilis mutant strain, preserving number are CGMCC NO.0213, with its inoculation (including small amounts of inorganic salt) in the Semen Maydis powder-soybean cake powder substratum of suitable C.N ratio, through cultivation and fermentation, high temperature spray-drying is made solid alkaline degumming enzyme then, this enzymic activity height, heat-resisting, alkaline-resisting, be suitable for come unstuck production, papermaking and other production in light industry of crudefiber crop and use.Its bacterial classification subtilis, in from numb soil, dividing from, again through ultraviolet ray (UV) and nitrosoguanidine (NG) mutagenesis, filter out and have hereditary mark streptomycin resistance (Sm
r) mutant, it mainly produces alkaline pectase, produces proteolytic enzyme simultaneously, amylase and zytase.On the meat soup flat board, bacterium colony by greyish white, thin, the limit bleaches gradually, thickens, complete then leucismus is thick, does not have concentric garden.Optimum growing condition: PH7-7.2,37 ℃; All fine growth on organic or synthetic inorganic medium, nutritional requirement is not high; During the static cultivation of liquid, the surface forms mycoderm, and following substratum is limpid; The bouillon agar stab culture, there is lawn on a surface, proves that growth produces gemma, the heat-resisting Sm that has
rThe heredity mark, favourable bacterium purifying, anti-assorted the preservation.This bacterium logical oxygen in corn-soybean cake powder substratum can be grown more vigorous, and itself is aerogenesis not.
Bacillus subtilis strain involved in the present invention what was delivered to China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (address: the Zhong Guan-cun, BeiJing, China), its classification name and preserving number thereof is: subtilis Bacillus Subtilis CGMCC NO.0213 on May 6th, 1994.
With above-mentioned bacillus subtilis strain CGMCC NO.0213, production solid alkaline degumming enzyme technological process is: bacterial classification → test tube meat soup inclined-plane → flat big inclined-plane → seeding tank → fermentor tank → high temperature fast spraying drying is made solid zymin → packing:
1. above-mentioned bacillus subtilis strain CGMCC NO.0213 is inserted on the test tube meat soup inclined-plane, the broth culture composition has extractum carnis, porcine haemoglobin peptone, NaCl, agar, pH value is 7.0-7.2, and sterilization is 30 minutes in high-pressure sterilizing pot (15 pounds), cultivates 16-24 hour under 37 ℃ of conditions;
2. be transferred on the flat bottle inclined-plane, cultivated 16-24 hour down for 37 ℃, the substratum composition gathers 1. with the above-mentioned step, adds a certain amount of meat soup afterwards, makes bacterium suspension;
3. change enlarged culturing in the seeding tank over to, the substratum composition has the Semen Maydis powder of certain carbon, nitrogen proportioning, soybean cake powder and MgSO in jar
47H
2O, KH
2PO
4, Na
2CO
3, K
2HPO
4, (NH
4)
2SO
4And an amount of soya-bean oil, 121-123 ℃ of down sterilization 40 minutes, PH is 8-9 before the sterilization, sterilization back PH reduces to about 6.5,37 ℃-40 ℃ and constantly stir under the logical oxygen condition, cultivates 10-12 hour;
4. be transferred to again in the fermentor tank, substantially the same step 3. of substratum composition and culture condition, but substratum concentration increases incubation time 24 hours-28 hours;
5. enzyme liquid in the fermentor tank is inserted the high-temperature spray tower, the fast spraying drying, about 160 ℃ of imports export about 80 ℃, make the solid zymin.
Measure the activity of various enzymes in the zymin with following method:
1. the mensuration of pectinase activity, measuring method are conventional DNS method, i.e. 3.5-dinitro an aromatic plant metioned in ancient books bigcatkin willow acid cut sugar method, and the liquid enzymes pectinase activity reaches hydrolysis of pectin as a result, produces galacturonic acid several thousand/μ g/mlmin or solid enzyme, reaches several ten thousand/μ g/g.
2. the mensuration of xylanase activity, with meat soup-xylan flat band method: bacterium is seeded on this flat board, cultivated about 24 hours for 37 ℃, the saturating circle of observations obviously prove the generation zytase.
3. the mensuration of amylase activity is measured with above-mentioned the 2nd method, and different is that substrate is used Zulkovsky starch instead, and the result has obvious transparent circle, and explanation can produce amylase.
4. the mensuration of protease activity is measured with Tolin-phenol method, and protease activity reaches hydrolyzed casein product die aromatischen Aminosaeuren several one hundred μ g/mlmin as a result.Measure with above-mentioned the 2nd method in addition, substrate is changed to casein, and the result has obvious transparent circle, proves to produce proteolytic enzyme.
5. cellulase is measured: collapse method with filter paper, filter paper does not disappear or is out of shape as a result, proves not cellulase-producing.
In a word: measurement result proof subtilis (Bacillus Subtilis) CGMCC NO.0213 can produce polygalacturonase (being main), and produce proteolytic enzyme, amylase and zytase except that cellulase-producing not.
Pectinase activity is specifically surveyed method: 0.4% import citrus pectin-Tris-HCl (PH7.0) 0.1ml+ enzyme liquid (or the enzyme liquid that diluted) 0.1ml → 50 ℃ 10 minutes → add DNS0.2ml → 50 ℃ 10 minutes → tartarize potassium sodium 3.6ml → with 721 spectrophotometers is in 575nm wavelength place's colorimetric estimation OD value, the contrast dead enzyme that boiled 15 minutes, other condition is identical.Find corresponding galacturonic acid content at mark on the curve, or press formula and calculate pectinase activity unit.
Pectinase activity X (μ g/mlmin)=[804.16065Y+23.495915] * Z
Wherein Y is the OD value, and Z is the enzyme extension rate
Bacterial classification can be given first heat treated (80 ℃ 10 minutes or 65 ℃ 30 minutes) or utilize anti-Sm
rCharacteristic, but purifying bacterial classification prevent to pollute, and in the process of producing enzyme, do not see contamination phenomenon, and microscopy or dull and stereotyped single bacterium colony inspection all prove very pure, the uncontaminated assorted bacterium of bacterium.
The CGMCC NO.0213 bacterial strain that the present invention is selected, nutritional requirement is not high, (including small amounts of inorganic salt) well-grown on the Semen Maydis powder-soybean cake powder substratum of cheapness, it is many, heat-resisting, alkaline-resisting to produce enzyme, produces gemma and has Sm
rGenetic marker is fit to high temperature spray-drying, suitability for industrialized production and help preventing and treating living contaminants purifying bacterial classification and prolonged preservation.The technology of producing the solid alkaline pectin compound enzyme with it is simple, and the cycle short (3 days) is so the productivity height, this prozyme is heat-resisting, alkaline-resisting, active high, contains polygalacturonase (being main), reaches proteolytic enzyme, amylase, zytase, the application but cellulase never, suitable crudefiber crop come unstuck; Above-mentioned solid pectin zymin, itself can be anticorrosion, do not add any sanitas, also can storage tolerance, room temperature storage 1 year, activity is very stable; And transportation and use all make things convenient for, and numb factory just can use having now under the part, needn't reinvest, and numb factory easily accepts; Through industrial scale test several times, proved that residual gum content, fibre strength etc. all can reach GB, enzyme can reuse, and reduces cost; Enzyme action condition gentleness, partially alkali and not cellulase again are to fibre-tendering, so fibre strength and rate of recovery height; Compare with traditional method, can economize the 4-5 procedure, it is low to consume energy, and waste water is pollution-free, also can fully utilize, and creates benefit again.Because adopt the high-concentration culturing base to ferment, bacteria growing is good, the production of enzyme height, the rate of recovery is also high, and the also favourable repeated use and the useless Water reuse of enzyme later on reduce cost.Owing to adopt suitable C, N proportioning, PH is unlikely to reduce too much influences the growth of bacterium and the output of enzyme, also helps the recycling and the useless Water reuse of enzyme.The enzyme meta-alkalescence is beneficial to China grass degumming.Owing to adopt the high temperature spray-drying method to make the solid zymin, because of drying not adding preservative agent still can preserve 1 year, and activity is still stable, transportation and use all convenient.Owing to select for use the product gemma to have Sm
rThe heredity mark, bacterial strain production zymin helps the bacterial classification purifying, prolonged preservation and prevent living contaminants.
Embodiment 1:
1) subtilis (Bacillus Subtilis) CGMCC NO.0213 is inserted on the test tube meat soup inclined-plane, broth culture composition and content are extractum carnis 1%, porcine haemoglobin peptone 1%, NaCl0.5%, agar is 1.5-2%, PH7.0-7.2 sterilized 30 minutes for 15 pounds, cultivated 16-24 hour for 37 ℃;
2) be transferred on two flat meat soup inclined-planes, enlarged culturing, its substratum composition and content are all with step 1, and every bottle of adding 100ml meat soup makes bacteria suspension afterwards;
3) bacterium liquid is transferred in the seeding tank, seeding tank is 1 ton of jar, interior dress 500kg substratum, and substratum composition and content are: Semen Maydis powder 2%, beans food cake powder 4%, MgSO
47H
2O0.02%, K
2HPO
40.4%, (NH
4)
2SO
40.4%, Na
2CO
3200g, KH
2PO
40.03%, soya-bean oil is an amount of, and 121-123 ℃ of sterilization 40 minutes is as cold as 37-40 ℃ and connects bacterium again, and PH is transferred to 9 before sterilization, drop to 6.3 after the sterilization, at 37-40 ℃, constantly stirs and logical oxygen reaches under the condition of 140 (air flowmeter high scale numbers) and cultivated 12 hours;
4) be transferred to continuation cultivation in the fermentor tank again, 10 tons of jars of fermentor tank, interior dress 500kg substratum, substratum composition and content are: Semen Maydis powder 3%, soybean cake powder 4%, remaining with step 3, cultivation is 28 hours under 37-40 ℃ of continuous stirring and logical oxygen condition;
5) with enzyme liquid in the high-temperature spray tower, spraying drying is made the solid zymin, enzyme rate of recovery 3-6%.
Detect enzyme activity: (1) liquid enzymes is cultivated and was reached 4969 μ g/mlmin in 28 hours
(2) the solid enzyme activity is 28000 μ g/g, for light yellow
Embodiment 2:
All processing steps are with implementing 1, with dress 500Kg in 1 ton of jar
The substratum composition: Semen Maydis powder 2%, soybean cake powder 4%, other inorganic salt are the same, before the PH sterilization is 7.4, and the sterilization back is PH6, and its result cultivated 28 hours for 37 ℃-40 ℃, liquid enzymes reaches 4697.948 μ g/mlmin, makes the solid zymin through high temperature fast spraying drying, the rate of recovery 5%.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94105708A CN1055118C (en) | 1994-05-24 | 1994-05-24 | Bacillus subtilis and producing tech. for solid alkaline degumming enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94105708A CN1055118C (en) | 1994-05-24 | 1994-05-24 | Bacillus subtilis and producing tech. for solid alkaline degumming enzyme |
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| CN1113951A CN1113951A (en) | 1995-12-27 |
| CN1055118C true CN1055118C (en) | 2000-08-02 |
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| CN94105708A Expired - Fee Related CN1055118C (en) | 1994-05-24 | 1994-05-24 | Bacillus subtilis and producing tech. for solid alkaline degumming enzyme |
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Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100344751C (en) * | 2001-10-09 | 2007-10-24 | 华东师范大学 | Method for batch-culturing bacillus subtilis variant strain by using solid fermentation |
| EP1759052A4 (en) * | 2004-06-15 | 2009-12-30 | Novozymes North America Inc | SIMULTANEOUS DISPOSAL AND WASHING PROCESS |
| CN102021157B (en) * | 2009-09-23 | 2012-08-08 | 中国科学院微生物研究所 | Pectinase and coding gene thereof |
| CN101948788B (en) * | 2010-09-03 | 2011-11-09 | 湖南润久科技有限公司 | A kind of screening method of degumming bacterial strain and the degumming bacterial strain that screens out |
| CN102559638B (en) * | 2010-12-17 | 2013-06-05 | 武汉新华扬生物股份有限公司 | Alkaline pectinase poly lactic acid (PLA) and gene and application thereof |
| CN102899299B (en) * | 2012-09-06 | 2015-07-15 | 青岛蔚蓝生物集团有限公司 | Alkaline pectinase mutant and recombinant expression engineering bacteria thereof |
| CN104911120A (en) * | 2015-03-11 | 2015-09-16 | 浙江理工大学 | Bacillus subtilis fermentation production technology |
| CN106047745A (en) * | 2016-05-10 | 2016-10-26 | 西北农林科技大学 | A microorganism and applications thereof |
| CN106222154A (en) * | 2016-08-22 | 2016-12-14 | 安徽省华龙麻业有限公司 | A kind of compound enzymic preparation improving China grass degumming efficiency and preparation method thereof |
| CN106047841A (en) * | 2016-08-22 | 2016-10-26 | 安徽省华龙麻业有限公司 | Composite enzyme preparation for ramie boiling degumming and preparation method thereof |
| CN106434476A (en) * | 2016-11-02 | 2017-02-22 | 青岛蔚蓝生物集团有限公司 | High-yield strain for alkaline pectinase and application of high-yield strain |
| CN110885806B (en) * | 2019-12-06 | 2021-06-01 | 鹤山市东古调味食品有限公司 | Method for producing pectinase by using pickling brine and troxerutin brine |
| CN113005058A (en) * | 2021-02-25 | 2021-06-22 | 阿勒泰戈宝茶股份有限公司 | Apocynum degumming biological preparation and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1003304B (en) * | 1985-03-28 | 1989-02-15 | 罗纳·布朗克公司化学部 | Flexible industrial copolyetheramides at low temperatures |
| CN1102671A (en) * | 1993-11-10 | 1995-05-17 | 福州洪山生化实业有限公司 | Granular pectase preparation and its prodn. method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1003304B (en) * | 1985-03-28 | 1989-02-15 | 罗纳·布朗克公司化学部 | Flexible industrial copolyetheramides at low temperatures |
| CN1102671A (en) * | 1993-11-10 | 1995-05-17 | 福州洪山生化实业有限公司 | Granular pectase preparation and its prodn. method |
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