CN102965358B - Method for extracting Beta-amylase from soybean - Google Patents
Method for extracting Beta-amylase from soybean Download PDFInfo
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- CN102965358B CN102965358B CN201210491225.2A CN201210491225A CN102965358B CN 102965358 B CN102965358 B CN 102965358B CN 201210491225 A CN201210491225 A CN 201210491225A CN 102965358 B CN102965358 B CN 102965358B
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Abstract
The invention discloses a method for extracting Beta-amylase from soybean. The method comprises the following steps in sequence: pre-treating raw materials, crushing tissues of the raw materials, extracting enzyme liquid, separating slag from slurry, precipitating to separate, draining and clarifying, ultra-filtering and concentrating, blending, and carrying out sterile subpackaging, so as to obtain the liquid Beta-amylase product with enzyme activity more than 500000 units. The Beta-amylase produced by the method disclosed by the invention is high in activity, purity and stability, and is widely applicable to the industrial production of maltose products according to the features. The separated slag charge is rich in protein, so the slag charge is a good feedstuff. The method has the highlights of being full in utilization of the raw materials, remarkable in product benefits, and good in market prospect. By adopting the method, higher content of crude enzyme can be obtained, the enzyme and the protein can be separated well, and the enzyme activity of the product can be improved. The method is specifically suitable for being used for processing beans, and is also convenient for further purifying the enzyme.
Description
Technical field
The present invention relates to a kind of diastatic method of extraction, particularly relate to a kind of method extracting beta-amylase from soya bean.
Background technology
Beta-amylase is extensively present in barley, wheat, soybean, contour the crossing in plant and some hydrolysis biologies of sweet potato.Barley and Wheat Species content the highest, sweet potato content is only about 1/2 of barley, and because the beta-amylase vigor of Production by Microorganism Fermentation is low, cost is high, and, be very difficult from bacterium scale operation β.The β-amylase of industrial use is all plant origin for this reason.
Beta-amylase is a kind of starch degrading enzyme, can hydrolyzing alpha-Isosorbide-5-Nitrae key.It is present in bacterium and plant, can be maltose from the non-reducing end of this starch chain by amylolysis.As contained abundant beta-amylase in grain, it is by the nutrition deposit in cereal when needed, and namely Starch Conversion is sugar.Starch in cereal mainly stores with the form of amylose starch and amylopectin.All amylose starchs are converted into maltose and the amylopectin of about 60% are converted into maltose and remaining is converted into dextrin by beta-amylase.
Beta-amylase is commercially important enzyme, as in starch industry, it can be used for producing maltose.Product containing maltose may be used for as candy and foodstuffs industry.People isolate beta-amylase from bacterium and plant.As obtained beta-amylase from bacillus (Bacillus) bacterium (US4970158 and JP60126080) and heat-resisting Clostridium (Clostridium) (US4647538).As obtained beta-amylase except being converted into except maltose by amylase from bacterium, also produce sizable trisaccharide maltose, and produce relatively many maltose from the beta-amylase of plant, be therefore more suitable for being used in object and obtain in method that is sweet as far as possible and/or fermentable product.And, be very difficult from bacterium scale operation beta-amylase.Industrial beta-amylase is plant origin, usual cereal, particularly barley or wheat, and soybean can be used as enzyme source.
Summary of the invention
The invention provides a kind of method extracting beta-amylase from soya bean, the method can obtain the crude enzyme liquid of high level and achieve enzyme and be separated with the better of protein, improves the enzyme activity of product.The method is particularly suitable for processing beans, facilitates again being further purified of enzyme simultaneously.
The present invention is achieved by the following technical programs:
From soya bean, extract a method for beta-amylase, the method comprises the following steps:
(1) raw materials pretreatment: soak after soya bean cleaning 6 ~ 8 hours, the water temperature of immersion water is 30 ~ 37 DEG C, be placed on after draining water and ventilate in moist environment, humidity keeps 70 ~ 85%, till sprouting, obtains bean sprouts;
(2) raw tissue is broken: refining equipment is put in bean sprouts, grinds while add waterside, the ratio of bean sprouts and water is 1: 1, obtains 180 ~ 220 object slurries;
(3) enzyme liquid extracts: the slurries obtained are added water by 1: 30, then adds extraction agent, temperature remains on 50 ~ 60 DEG C, is stirred and material is fully mixed;
(4) slag slurry is separated: by the mixture that obtains by 200 ~ 300 object filter pressure filtrations, bean dregs are separated with slurries;
(5) precipitate and separate: adding 0.15% calcium chloride by filtering the slurries obtained, stirring after 15 ~ 25 minutes, being left standstill 6 ~ 8 hours, after slurries precipitated and separated, collect supernatant liquor;
(6) drainage clarification: the supernatant liquor obtained is added flocculating aids, filters to obtain clear liquor by filter, the temperature of filtering the clear liquor obtained is 40 ~ 45 DEG C;
(7) ultrafiltration and concentration: make temperature be lowered to less than 40 DEG C by heat exchanger clear liquor, then carry out ultrafiltration and concentration by ultra-filtration membrane, obtain enzyme liquid;
(8) allocate: the enzyme liquid obtained is added stablizer and stirs;
(9) aseptic subpackaged: after the enzyme liquid obtained is carried out filtration sterilization by the microtubule filter that aperture is 0.15 ~ 0.25 μ, to carry out aseptic canning, namely obtain beta-amylase finished product.
Preferably, the extraction agent added described in step (3) comprises: 0.1% Sodium phosphate dibasic, 0.1% SODIUM PHOSPHATE, MONOBASIC and 0.2% Sodium Pyrosulfite.
Preferably, the flocculating aids described in step (6) comprises: tripoli and perlite.
Preferably, the ultra-filtration membrane carrying out ultrafiltration and concentration to clear liquor described in step (7) molecular weight that dams is 6000 ~ 10000K, and the enzyme activity of described enzyme liquid is more than 500,000 units.
Preferably, the stablizer described in step (8) comprises: 5 ~ 10% glycerol, 0.5% potassium sorbate and 1% trehalose.
The present invention has following beneficial effect:
The beta-amylase vigor that the present invention produces is high, purity is high, good stability, and product performance is widely used in the industrial production of maltose series products.Isolated slag charge rich in proteins in technological process is excellent feedstuff raw material.The present invention highlights making full use of raw material, product remarkable benefit, good market prospects.This production method can obtain the crude enzyme liquid of high level and achieve enzyme and be separated with the better of protein, improves the enzyme activity of product.The method is particularly suitable for processing beans, facilitates again being further purified of enzyme simultaneously.
Embodiment
Below in conjunction with tool embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to the scope that embodiment represents.
Embodiment 1
From soya bean, extract a method for beta-amylase, the method comprises the following steps:
(1) raw materials pretreatment: soak after soya bean cleaning 6 hours, the water temperature of immersion water is 30 DEG C, be placed on after draining water and ventilate in moist environment, humidity keeps 70%, till sprouting, obtains bean sprouts;
(2) raw tissue is broken: refining equipment is put in bean sprouts, grinds while add waterside, the ratio of bean sprouts and water is 1: 1, obtains 180 object slurries;
(3) enzyme liquid extracts: the slurries obtained are added water by 1: 30, then adds extraction agent, temperature remains on 50 DEG C, is stirred and material is fully mixed;
(4) slag slurry is separated: by the mixture that obtains by 200 object filter pressure filtrations, bean dregs are separated with slurries;
(5) precipitate and separate: adding 0.15% calcium chloride by filtering the slurries obtained, stirring after 15 minutes, being left standstill 6 hours, after slurries precipitated and separated, collect supernatant liquor;
(6) drainage clarification: the supernatant liquor obtained is added flocculating aids, filters to obtain clear liquor by filter, the temperature of filtering the clear liquor obtained is 40 DEG C;
(7) ultrafiltration and concentration: make temperature be lowered to 20 DEG C by heat exchanger clear liquor, then carry out ultrafiltration and concentration by ultra-filtration membrane, obtain enzyme liquid;
(8) allocate: the enzyme liquid obtained is added stablizer and stirs;
(9) aseptic subpackaged: after the enzyme liquid obtained is carried out filtration sterilization by the microtubule filter that aperture is 0.15 μ, to carry out aseptic canning, namely obtain beta-amylase finished product.
The extraction agent added described in above-mentioned steps (3) comprises: 0.1% Sodium phosphate dibasic, 0.1% SODIUM PHOSPHATE, MONOBASIC and 0.2% Sodium Pyrosulfite.
Flocculating aids described in above-mentioned steps (6) comprises: tripoli and perlite.
The ultra-filtration membrane carrying out ultrafiltration and concentration to clear liquor described in above-mentioned steps (7) molecular weight that dams is 6000K, and the enzyme activity of described enzyme liquid is more than 500,000 units.
Stablizer described in above-mentioned steps (8) comprises: 5% glycerol, 0.5% potassium sorbate and 1% trehalose.
Embodiment 2
From soya bean, extract a method for beta-amylase, the method comprises the following steps:
(1) raw materials pretreatment: soak after soya bean cleaning 7 hours, the water temperature of immersion water is 34 DEG C, be placed on after draining water and ventilate in moist environment, humidity keeps 80%, till sprouting, obtains bean sprouts;
(2) raw tissue is broken: refining equipment is put in bean sprouts, grinds while add waterside, the ratio of bean sprouts and water is 1: 1, obtains 200 object slurries;
(3) enzyme liquid extracts: the slurries obtained are added water by 1: 30, then adds extraction agent, temperature remains on 55 DEG C, is stirred and material is fully mixed;
(4) slag slurry is separated: by the mixture that obtains by 250 object filter pressure filtrations, bean dregs are separated with slurries;
(5) precipitate and separate: adding 0.15% calcium chloride by filtering the slurries obtained, stirring after 20 minutes, being left standstill 7 hours, after slurries precipitated and separated, collect supernatant liquor;
(6) drainage clarification: the supernatant liquor obtained is added flocculating aids, filters to obtain clear liquor by filter, the temperature of filtering the clear liquor obtained is 42 DEG C;
(7) ultrafiltration and concentration: make temperature be lowered to 25 DEG C by heat exchanger clear liquor, then carry out ultrafiltration and concentration by ultra-filtration membrane, obtain enzyme liquid;
(8) allocate: the enzyme liquid obtained is added stablizer and stirs;
(9) aseptic subpackaged: after the enzyme liquid obtained is carried out filtration sterilization by the microtubule filter that aperture is 0.20 μ, to carry out aseptic canning, namely obtain beta-amylase finished product.
The extraction agent added described in above-mentioned steps (3) comprises: 0.1% Sodium phosphate dibasic, 0.1% SODIUM PHOSPHATE, MONOBASIC and 0.2% Sodium Pyrosulfite.
Flocculating aids described in above-mentioned steps (6) comprises: tripoli and perlite.
The ultra-filtration membrane carrying out ultrafiltration and concentration to clear liquor described in above-mentioned steps (7) molecular weight that dams is 8000K, and the enzyme activity of described enzyme liquid is more than 500,000 units.
Stablizer described in above-mentioned steps (8) comprises: 7% glycerol, 0.5% potassium sorbate and 1% trehalose.
Embodiment 3
From soya bean, extract a method for beta-amylase, the method comprises the following steps:
(1) raw materials pretreatment: soak after soya bean cleaning 8 hours, the water temperature of immersion water is 37 DEG C, be placed on after draining water and ventilate in moist environment, humidity keeps 85%, till sprouting, obtains bean sprouts;
(2) raw tissue is broken: refining equipment is put in bean sprouts, grinds while add waterside, the ratio of bean sprouts and water is 1: 1, obtains 220 object slurries;
(3) enzyme liquid extracts: the slurries obtained are added water by 1: 30, then adds extraction agent, temperature remains on 60 DEG C, is stirred and material is fully mixed;
(4) slag slurry is separated: by the mixture that obtains by 300 object filter pressure filtrations, bean dregs are separated with slurries;
(5) precipitate and separate: adding 0.15% calcium chloride by filtering the slurries obtained, stirring after 25 minutes, being left standstill 8 hours, after slurries precipitated and separated, collect supernatant liquor;
(6) drainage clarification: the supernatant liquor obtained is added flocculating aids, filters to obtain clear liquor by filter, the temperature of filtering the clear liquor obtained is 45 DEG C;
(7) ultrafiltration and concentration: make temperature be lowered to 38 DEG C by heat exchanger clear liquor, then carry out ultrafiltration and concentration by ultra-filtration membrane, obtain enzyme liquid;
(8) allocate: the enzyme liquid obtained is added stablizer and stirs;
(9) aseptic subpackaged: after the enzyme liquid obtained is carried out filtration sterilization by the microtubule filter that aperture is 0.25 μ, to carry out aseptic canning, namely obtain beta-amylase finished product.
The extraction agent added described in above-mentioned steps (3) comprises: 0.1% Sodium phosphate dibasic, 0.1% SODIUM PHOSPHATE, MONOBASIC and 0.2% Sodium Pyrosulfite.
Flocculating aids described in above-mentioned steps (6) comprises: tripoli and perlite.
The ultra-filtration membrane carrying out ultrafiltration and concentration to clear liquor described in above-mentioned steps (7) molecular weight that dams is 10000K, and the enzyme activity of described enzyme liquid is more than 500,000 units.
Stablizer described in above-mentioned steps (8) comprises: 10% glycerol, 0.5% potassium sorbate and 1% trehalose.
Embodiment 4
From soya bean, extract a method for beta-amylase, the method comprises the following steps:
(1) raw materials pretreatment: soak after soya bean cleaning 6 hours, the water temperature of immersion water is 37 DEG C, be placed on after draining water and ventilate in moist environment, humidity keeps 80%, till sprouting, obtains bean sprouts;
(2) raw tissue is broken: refining equipment is put in bean sprouts, grinds while add waterside, the ratio of bean sprouts and water is 1: 1, obtains 220 object slurries;
(3) enzyme liquid extracts: the slurries obtained are added water by 1: 30, then adds extraction agent, temperature remains on 58 DEG C, is stirred and material is fully mixed;
(4) slag slurry is separated: by the mixture that obtains by 280 object filter pressure filtrations, bean dregs are separated with slurries;
(5) precipitate and separate: adding 0.15% calcium chloride by filtering the slurries obtained, stirring after 20 minutes, being left standstill 6 hours, after slurries precipitated and separated, collect supernatant liquor;
(6) drainage clarification: the supernatant liquor obtained is added flocculating aids, filters to obtain clear liquor by filter, the temperature of filtering the clear liquor obtained is 40 DEG C;
(7) ultrafiltration and concentration: make temperature be lowered to 35 DEG C by heat exchanger clear liquor, then carry out ultrafiltration and concentration by ultra-filtration membrane, obtain enzyme liquid;
(8) allocate: the enzyme liquid obtained is added stablizer and stirs;
(9) aseptic subpackaged: after the enzyme liquid obtained is carried out filtration sterilization by the microtubule filter that aperture is 0.18 μ, to carry out aseptic canning, namely obtain beta-amylase finished product.
The extraction agent added described in above-mentioned steps (3) comprises: 0.1% Sodium phosphate dibasic, 0.1% SODIUM PHOSPHATE, MONOBASIC and 0.2% Sodium Pyrosulfite.
Flocculating aids described in above-mentioned steps (6) comprises: tripoli and perlite.
The ultra-filtration membrane carrying out ultrafiltration and concentration to clear liquor described in above-mentioned steps (7) molecular weight that dams is 6500K, and the enzyme activity of described enzyme liquid is more than 500,000 units.
Stablizer described in above-mentioned steps (8) comprises: 6% glycerol, 0.5% potassium sorbate and 1% trehalose.
Embodiment 5
From soya bean, extract a method for beta-amylase, the method comprises the following steps:
(1) raw materials pretreatment: soak after soya bean cleaning 6 hours, the water temperature of immersion water is 35 DEG C, be placed on after draining water and ventilate in moist environment, humidity keeps 78%, till sprouting, obtains bean sprouts;
(2) raw tissue is broken: refining equipment is put in bean sprouts, grinds while add waterside, the ratio of bean sprouts and water is 1: 1, obtains 200 object slurries;
(3) enzyme liquid extracts: the slurries obtained are added water by 1: 30, then adds extraction agent, temperature remains on 56 DEG C, is stirred and material is fully mixed;
(4) slag slurry is separated: by the mixture that obtains by 220 object filter pressure filtrations, bean dregs are separated with slurries;
(5) precipitate and separate: adding 0.15% calcium chloride by filtering the slurries obtained, stirring after 18 minutes, being left standstill 7.5 hours, after slurries precipitated and separated, collect supernatant liquor;
(6) drainage clarification: the supernatant liquor obtained is added flocculating aids, filters to obtain clear liquor by filter, the temperature of filtering the clear liquor obtained is 45 DEG C;
(7) ultrafiltration and concentration: make temperature be lowered to 40 DEG C by heat exchanger clear liquor, then carry out ultrafiltration and concentration by ultra-filtration membrane, obtain enzyme liquid;
(8) allocate: the enzyme liquid obtained is added stablizer and stirs;
(9) aseptic subpackaged: after the enzyme liquid obtained is carried out filtration sterilization by the microtubule filter that aperture is 0.22 μ, to carry out aseptic canning, namely obtain beta-amylase finished product.
The extraction agent added described in above-mentioned steps (3) comprises: 0.1% Sodium phosphate dibasic, 0.1% SODIUM PHOSPHATE, MONOBASIC and 0.2% Sodium Pyrosulfite.
Flocculating aids described in above-mentioned steps (6) comprises: tripoli and perlite.
The ultra-filtration membrane carrying out ultrafiltration and concentration to clear liquor described in above-mentioned steps (7) molecular weight that dams is 8000K, and the enzyme activity of described enzyme liquid is more than 500,000 units.
Stablizer described in above-mentioned steps (8) comprises: 7% glycerol, 0.5% potassium sorbate and 1% trehalose.
Embodiment 6
From soya bean, extract a method for beta-amylase, the method comprises the following steps:
(1) raw materials pretreatment: soak after soya bean cleaning 6.5 hours, the water temperature of immersion water is 35 DEG C, be placed on after draining water and ventilate in moist environment, humidity keeps 80%, till sprouting, obtains bean sprouts;
(2) raw tissue is broken: refining equipment is put in bean sprouts, grinds while add waterside, the ratio of bean sprouts and water is 1: 1, obtains 185 object slurries;
(3) enzyme liquid extracts: the slurries obtained are added water by 1: 30, then adds extraction agent, temperature remains on 50 DEG C, is stirred and material is fully mixed;
(4) slag slurry is separated: by the mixture that obtains by 270 object filter pressure filtrations, bean dregs are separated with slurries;
(5) precipitate and separate: adding 0.15% calcium chloride by filtering the slurries obtained, stirring after 23 minutes, being left standstill 8 hours, after slurries precipitated and separated, collect supernatant liquor;
(6) drainage clarification: the supernatant liquor obtained is added flocculating aids, filters to obtain clear liquor by filter, the temperature of filtering the clear liquor obtained is 40 DEG C;
(7) ultrafiltration and concentration: make temperature be lowered to 32 DEG C by heat exchanger clear liquor, then carry out ultrafiltration and concentration by ultra-filtration membrane, obtain enzyme liquid;
(8) allocate: the enzyme liquid obtained is added stablizer and stirs;
(9) aseptic subpackaged: after the enzyme liquid obtained is carried out filtration sterilization by the microtubule filter that aperture is 0.25 μ, to carry out aseptic canning, namely obtain beta-amylase finished product.
The extraction agent added described in above-mentioned steps (3) comprises: 0.1% Sodium phosphate dibasic, 0.1% SODIUM PHOSPHATE, MONOBASIC and 0.2% Sodium Pyrosulfite.
Flocculating aids described in above-mentioned steps (6) comprises: tripoli and perlite.
The ultra-filtration membrane carrying out ultrafiltration and concentration to clear liquor described in above-mentioned steps (7) molecular weight that dams is 9000K, and the enzyme activity of described enzyme liquid is more than 500,000 units.
Stablizer described in above-mentioned steps (8) comprises: 10% glycerol, 0.5% potassium sorbate and 1% trehalose.
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and example as described herein.
Claims (4)
1. from soya bean, extract a method for beta-amylase, it is characterized in that, the method comprises the following steps:
(1) raw materials pretreatment: soak after soya bean cleaning 6 ~ 8 hours, the water temperature of immersion water is 30 ~ 37 DEG C, be placed on after draining water and ventilate in moist environment, humidity keeps 70 ~ 85%, till sprouting, obtains bean sprouts;
(2) raw tissue is broken: refining equipment is put in bean sprouts, grinds while add waterside, the ratio of bean sprouts and water is 1:1, obtains 180 ~ 220 object slurries;
(3) enzyme liquid extracts: the slurries obtained are added water by 1:30, then adds extraction agent, temperature remains on 50 ~ 60 DEG C, is stirred and material is fully mixed;
(4) slag slurry is separated: by the mixture that obtains by 200 ~ 300 object filter pressure filtrations, bean dregs are separated with slurries;
(5) precipitate and separate: adding 0.15% calcium chloride by filtering the slurries obtained, stirring after 15 ~ 25 minutes, being left standstill 6 ~ 8 hours, after slurries precipitated and separated, collect supernatant liquor;
(6) drainage clarification: the supernatant liquor obtained is added flocculating aids, filters to obtain clear liquor by filter, the temperature of filtering the clear liquor obtained is 40 ~ 45 DEG C;
(7) ultrafiltration and concentration: make temperature be lowered to less than 40 DEG C by heat exchanger clear liquor, then carry out ultrafiltration and concentration by ultra-filtration membrane, obtain enzyme liquid;
(8) allocate: the enzyme liquid obtained is added stablizer and stirs;
(9) aseptic subpackaged: after the enzyme liquid obtained is carried out filtration sterilization by the microtubule filter that aperture is 0.15 ~ 0.25 μ, to carry out aseptic canning, namely obtain beta-amylase finished product.
2. the method extracting beta-amylase from soya bean according to claim 1, is characterized in that, the flocculating aids described in step (6) comprises: tripoli and perlite.
3. the method extracting beta-amylase from soya bean according to claim 1, it is characterized in that, the ultra-filtration membrane carrying out ultrafiltration and concentration to clear liquor described in step (7) molecular weight that dams is 6000 ~ 10000K, and the enzyme activity of described enzyme liquid is more than 500,000 units.
4. the method extracting beta-amylase from soya bean according to claim 1, is characterized in that, the stablizer described in step (8) comprises: 5 ~ 10% glycerol, 0.5% potassium sorbate and 1% trehalose.
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| FR3022257B1 (en) | 2014-06-16 | 2018-03-30 | Roquette Freres | PROCESS FOR PRODUCING A STABLE AQUEOUS SOLUTION OF BETA-AMYLASE, AQUEOUS SOLUTION OBTAINED AND USES THEREOF |
| FR3038618B1 (en) | 2015-07-06 | 2017-08-25 | Roquette Freres | PROCESS FOR PRODUCING MALTITOL HAVING IMPROVED PERFORMANCE |
| CN109480185A (en) * | 2018-10-31 | 2019-03-19 | 刘敏 | A kind of production method of vegetables various grains noodle |
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| CN102399763A (en) * | 2011-09-18 | 2012-04-04 | 淮北市三和诺生物工程有限责任公司 | Novel method for producing food grade beta-amylase with ultrahigh activity |
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| CN102399763A (en) * | 2011-09-18 | 2012-04-04 | 淮北市三和诺生物工程有限责任公司 | Novel method for producing food grade beta-amylase with ultrahigh activity |
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| Title |
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| 发芽黄豆β-淀粉酶的提取研究;靳利娥 等;《太原理工大学学报》;20040531;第35卷(第3期);第338页右边第4段，第339页左边第3段 * |
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