CN102229922B - Process for preparing high-stability liquid alpha-galactosidase - Google Patents
Process for preparing high-stability liquid alpha-galactosidase Download PDFInfo
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- CN102229922B CN102229922B CN 201110150313 CN201110150313A CN102229922B CN 102229922 B CN102229922 B CN 102229922B CN 201110150313 CN201110150313 CN 201110150313 CN 201110150313 A CN201110150313 A CN 201110150313A CN 102229922 B CN102229922 B CN 102229922B
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Abstract
The invention relates to a process for preparing high-stability liquid alpha-galactosidase, which comprises the following steps: 1, subjecting liquid coarse alpha-galactosidase product obtained after fermentation drawing off to high-speed centrifugation, subjecting supernate to microfiltration to remove bacteria, subjecting the supernate to ultrafiltration and concentration to obtain semi-finished high-concentration liquid alpha-galactosidase, and filling the semi-finished high-concentration liquid alpha-galactosidase into a stirring tank; 2, starting the stirring tank, slowly adding phosphoric acid or sodium hydroxide, and regulating the pH value of the liquid alpha-galactosidase to 4.6 to 5.0; 3, adding 0.15 to 0.40 mass percent of sodium benzoate and 0.35 to 0.55 mass percent of potassium sorbate; and 4, slowly adding 10 mass percent of stabilizer into the stirring tank, making the stabilizer fully dissolved till the solution is clear and transparent, standing the solution for 24 hours and filling. The process has the advantages that: (1) the high-stability liquid alpha-galactosidase is capable of suppressing growth of bacteria and has remarkable anticorrosion effect; (2) the anticorrosion effects of sodium benzoate and potassium sorbate can be further improved; and (3) the survival rate of the liquid alpha-galactosidase at a high temperature is increased effectively.
Description
Technical field
The present invention relates to the liquid enzyme formulation field, refer in particular to high stability liquid preparation method of alpha-galactosidase.
Background technology
Present liquid enzyme formulation especially liquid alpha-galactosidase has obtained using widely in feedstuff industry, food service industry, but, existing liquid stability of alpha-galactosidase is relatively poor, be exactly specifically when temperature higher especially when temperature more than 30 ℃ the time, its loss of activity rate is higher, existing liquid alpha-galactosidase preservative effect is poor simultaneously, is subjected to the pollution of bacterium easily, thereby is not easy to storage and transport.
Summary of the invention
The problem to be solved in the present invention is: a kind of high stability liquid preparation method of alpha-galactosidase is provided, the liquid alpha-galactosidase of making by this preparation method does not have muddiness, does not have precipitation, free from extraneous odour, has very high stability, namely than under the high atmospheric temperature, its activity can obviously not reduce, and its fungistatic effect and antiseptic property are strong simultaneously.
In order to solve the problems of the technologies described above, high stability liquid preparation method of alpha-galactosidase of the present invention may further comprise the steps:
The first step is put liquid crude enzyme product high speed centrifugation behind the jar with fermentation, and its supernatant liquor obtains high concentration liquid alpha-galactosidase work in-process behind micro-filtration degerming, ultrafiltration and concentration, put into stirred pot;
Second step, start stirred pot, slowly add phosphoric acid or sodium hydroxide, regulate the pH value of liquid alpha-galactosidase to 4.6-5.0; The 3rd step is according to Sodium Benzoate and the 0.35-0.55% potassium sorbate of mass percent adding 0.15-0.40%;
The 4th step slowly added 10% stablizer in stirred pot according to mass percent, made it fully to be dissolved to clear, left standstill can behind the 24h.
For the further stability that improves the liquid alpha-galactosidase, high stability liquid preparation method of alpha-galactosidase of the present invention, stablizer comprises the following component according to the mass percent proportioning:
Sodium-chlor 30-50%;
Sorbyl alcohol 40-60%;
Trehalose 10-30%.
The beneficial effect that the present invention obtains is: (1) Sodium Benzoate and potassium sorbate join in the liquid alpha-galactosidase as sanitas, have the function of bacteria growing inhibiting, favorable anti-corrosion effect; (2) put into phosphoric acid or sodium hydroxide, adjust the pH value of liquid alpha-galactosidase, pH value is stabilized between the 4.6-5.0, can further improve the preservative efficacy of Sodium Benzoate and potassium sorbate; (3) after sodium-chlor, sorbyl alcohol and trehalose are put into the liquid alpha-galactosidase as stablizer, can effectively improve the activity of liquid alpha-galactosidase under higher temperature and retain rate, make the liquid alpha-galactosidase after preserving six months under 40 ℃ of temperature, still can reach 85% activity.
Embodiment
During preparation high stability liquid alpha-galactosidase, adopt following steps:
The first step is put liquid crude enzyme product high speed centrifugation behind the jar with fermentation, and its supernatant liquor obtains high concentration liquid alpha-galactosidase work in-process behind micro-filtration degerming, ultrafiltration and concentration, put into stirred pot;
Second step, start stirred pot, slowly add phosphoric acid or sodium hydroxide, regulate the pH value to 4.8 of liquid alpha-galactosidase;
The 3rd step is according to Sodium Benzoate and 0.45% potassium sorbate of mass percent adding 0.25%;
The 4th step slowly added 10% stablizer in stirred pot according to mass percent, made it fully to be dissolved to clear, left standstill can behind the 24h.
High stability liquid preparation method of alpha-galactosidase of the present invention, stablizer comprises the following component according to the mass percent proportioning:
Sodium-chlor 40%;
Sorbyl alcohol 45%;
Trehalose 15%.
Test 1
In order to verify the activity of high stability liquid alpha-galactosidase, do following test:
Get the 500ml sample respectively and preserved in 4 ℃, room temperature, 40 ℃ 1-12 month, the activity that table 1 is the 1st, 3,6, detect December retains rate.
Table 1 high stability liquid alpha-galactosidase activity retains rate
As can be seen from Table 1, the product of being prepared by the present invention be under (4 ℃), the room temperature condition under the refrigerated condition, or hot conditions under (40 ℃) its stability all good, even preserving 6 months activity under hot conditions retains rate and still can reach more than 85%, and the product appearance quality does not change, show that product stability can be higher, be applicable to that normal temperature is preserved and long-distance transport.
Test 2
In order to verify the bacteriostasis of high stability liquid alpha-galactosidase, do following test:
Get the 100ml sample respectively and in 4 ℃, room temperature, 40 ℃, preserved 0-12 month, the assorted bacterial content that table 2 is the 0th, 1,3,6, detect December, wherein assorted bacterial content is with the CFU plate count.
Table 2 liquid alpha-galactosidase bacterium amount (individual/mL)
As can be seen from Table 2, this product no matter be under refrigerated condition under (4 ℃), the room temperature condition, or hot conditions under the sanitas of (40 ℃) its interpolation, mainly be Sodium Benzoate and potassium sorbate, its fungistatic effect good (the food grade products GB require＜50000/ml), can not make that product produces flatulence, goes mouldy in the preservation process, fermentation and peculiar smell, (4 ℃) preservation effect is better under refrigerated condition.
Claims (1)
1. high stability liquid preparation method of alpha-galactosidase is characterized in that may further comprise the steps:
1) fermentation is put liquid crude enzyme product high speed centrifugation behind the jar, its supernatant liquor obtains high concentration liquid alpha-galactosidase work in-process behind micro-filtration degerming, ultrafiltration and concentration, put into stirred pot;
2) start stirred pot, slowly add phosphoric acid or sodium hydroxide, regulate the pH value of liquid alpha-galactosidase to 4.6-5.0;
3) add 0.25% Sodium Benzoate and 0.45% potassium sorbate according to mass percent;
4) slowly add 10% stablizer in stirred pot according to mass percent, make it fully to be dissolved to clear, leave standstill can behind the 24h; Described stablizer comprises the following component according to the mass percent proportioning:
Sodium-chlor 40%;
Sorbyl alcohol 45%;
Trehalose 15%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201110150313 CN102229922B (en) | 2011-06-07 | 2011-06-07 | Process for preparing high-stability liquid alpha-galactosidase |
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| CN 201110150313 CN102229922B (en) | 2011-06-07 | 2011-06-07 | Process for preparing high-stability liquid alpha-galactosidase |
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| CN102229922A CN102229922A (en) | 2011-11-02 |
| CN102229922B true CN102229922B (en) | 2013-08-07 |
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| CN103773751A (en) * | 2012-10-22 | 2014-05-07 | 丰益(上海)生物技术研发中心有限公司 | Lipase stabilizing agent |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1189502A (en) * | 1996-11-20 | 1998-08-05 | 株式会社绿十字 | Method for producing antithrombin-III, method for purifying it, and preparation containing it |
| CN101016539A (en) * | 2007-01-23 | 2007-08-15 | 浙江大学 | Protective agent for increasing stability of alpha-galactosidase prepared by aspergillus foetidus fermentation and application thereof |
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2011
- 2011-06-07 CN CN 201110150313 patent/CN102229922B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1189502A (en) * | 1996-11-20 | 1998-08-05 | 株式会社绿十字 | Method for producing antithrombin-III, method for purifying it, and preparation containing it |
| CN101016539A (en) * | 2007-01-23 | 2007-08-15 | 浙江大学 | Protective agent for increasing stability of alpha-galactosidase prepared by aspergillus foetidus fermentation and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| α-半乳糖苷酶的液体发酵条件和酶学性质研究;刘彩琴 等;《中国食品学报》;20080215;第8卷(第1期);30 * |
| β-甘露聚糖酶基因测序、克隆表达及其工程菌的代谢通量研究;武伟娜;《中国优秀硕士学位论文全文数据库基础科学辑》;20090815;第2009卷(第8期);14-15 * |
| 刘彩琴 等.α-半乳糖苷酶的液体发酵条件和酶学性质研究.《中国食品学报》.2008,第8卷(第1期),27-32. |
| 武伟娜.β-甘露聚糖酶基因测序、克隆表达及其工程菌的代谢通量研究.《中国优秀硕士学位论文全文数据库基础科学辑》.2009,第2009卷(第8期),A006-107. |
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Effective date of registration: 20190724 Address after: 266000 East China Road 777, Qingdao High-tech Zone, Shandong Province Patentee after: QINGDAO SHANGDE BIOTECHNOLOGY CO., LTD. Address before: 256219 Baiyun Six Road, Haosheng Town, Zouping County, Binzhou City, Shandong Province Co-patentee before: Shi Kunjing Patentee before: Shandong Sinuo Baite Biological Technology Co.,Ltd. |