CN101182501A - High activity, high-purity and high temperature resistant Alpha-amylase production process - Google Patents
High activity, high-purity and high temperature resistant Alpha-amylase production process Download PDFInfo
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- CN101182501A CN101182501A CNA2007100361599A CN200710036159A CN101182501A CN 101182501 A CN101182501 A CN 101182501A CN A2007100361599 A CNA2007100361599 A CN A2007100361599A CN 200710036159 A CN200710036159 A CN 200710036159A CN 101182501 A CN101182501 A CN 101182501A
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Abstract
The invention provides a production process that bacteria strain (12 hours)to a first-class seed tank (28 hours)to big tank (about 100 hours, 12000u/ml draw off enzyme activity)to post treatment of flocculation (70 degree)-pressure filtration-cycle initial filtration-ultra-filtration to fine filtration (10 hours) to final product treatment (elevated temperature 80 degree) to precipitation (48 hours) to alpha-amylase production process with high activity, high purity and high temperature resistance. The XF-19 strain is selected; a secondary fermentation process, the addition of feeding material into the basic material, continuous feeding of ammonia and the application of cooked soybean meal as nitrogen source are applied during the production process; the cycle initial filtration and fine filtration are added in the post treatment process, which solves the shortcomings of old production process such as weak enzyme production ability per unit, decreasing pH value during auto-metabolism of the enzyme seed, little enzyme activity, poor temperature resistance, easy precipitation and high total bacteria count etc. The invention can reduce the production cost, great economic benefits and big popularization and application value.
Description
Technical field
The present invention relates to the production technique of the production technique of biotechnology zymin, particularly a kind of high activity, high-purity and high temperature resistant Alpha-amylase.
Background technology
To be Bacillus licheniformis cultivate and extraction process is made with extra care and formed through deep liquid high temperature resistant α-Dian Fenmei, has excellent heat resistance, and can be at the α in random hydrolysis starch, soluble dextrins and the oligose under the comparatively high temps-1.4 glucoside bond, the viscosity of gluey starch is reduced rapidly, and hydrolysis generates dextrin and a small amount of glucose and maltose.It is widely used in industries such as alcohol, beer, Dian Fentang, liquor, pharmacy and weaving, is the most a kind of enzyme of present industrial purposes.At present, the production technique of domestic high temperature resistant α-Dian Fenmei is: bacterial classification (24h) → first class seed pot (20h) → big jar of (about 80h, putting a jar enzyme activity 5000u/ml-9000u/m1) → aftertreatment flocculation (60 ℃) → press filtration → ultrafiltration → finished product handled (heating up 60 ℃) → precipitation (48h) → product.Its deficiency is: 1. can't guarantee rational carbon-nitrogen ratio, dissolved oxygen and osmotic pressure in the fermenting process, thereby be difficult to satisfy best metabolism of enzyme and condition of enzyme production, the enzymatic productivity of enzyme does not reach best effect.2. quality product is extremely unstable, color have dense have light, clear when dense during clarity, easily produce throw out, pH value instability, total plate count exceeds standard.
Summary of the invention
At the deficiency of above-mentioned high temperature resistant α-Dian Fenmei production technique, the objective of the invention is to improve and process innovation by bacterial screening renewal, fermentation culture, a kind of production technique of high activity, high-purity and high temperature resistant Alpha-amylase is provided.
Technical scheme of the present invention is:
1. select bacterial classification XF-19 (Jinshi New-type Fermentation Co., Ltd.'s preservation) for use.
2. new production technique is: bacterial classification (12h) → first class seed pot (28h) → big jar of (about 100h, putting a jar enzyme activity 12000u/ml) → aftertreatment flocculation (70 ℃) → press filtration → circulation filter → ultrafiltration just → essence filter (10h) → finished product handled (heating up 80 ℃) → precipitation (48h) → product.
3. technological condition for fermentation is to be nitrogenous source with ripe dregs of beans, and basestocks adds feed supplement, and ammonia is advanced in pulse, and leavening temperature is that 43 ℃, pH value are 6.8.
Advantage of the present invention is: 1. adopt flow feeding technology, overcome that carbon-nitrogen ratio height in the disposable charging technology, osmotic pressure are big, dissolved oxygen is poor, bacterial classification is grown under good envrionment conditions, the unit enzymatic productivity improves greatly; 2. adopt stream to add logical ammonia process, the problem that the pH value descends and enzyme activity does not increase that has produced when having solved owing to enzyme self metabolism can make the enzyme activity steady growth.3. change the soya-bean cake of continuing to use into ripe dregs of beans, reduced fat content, improved product enzyme environment, improved enzymatic productivity.4. product aftertreatment technology process has increased circulation filter just and smart filter, has solved product temperature tolerance poor, easily precipitation and the high problem of total plate count.5. the product of producing with the present invention detects the equal accord with Q B/T2306-1997 standard-required of every index through Hunan Province's food quality supervision check granting station.
Embodiment
Production technique of the present invention is: bacterial classification (12h) → first class seed pot (28h) → big jar of (about 100h, putting a jar enzyme activity 12000u/ml) → aftertreatment flocculation (70 ℃) → press filtration → circulation filter → ultrafiltration just → essence filter (10h) → finished product handled (heating up 80 ℃) → precipitation (48h) → product.
1, bacterial classification: after adopting the ultraviolet mutagenesis method and carrying out the genetic stability test of ten generations, obtain the bacterial classification (enzyme activity 12379u/ml, purity 98%, thermotolerance 96%, cycle 107hr) of called after XF-19.
2, fermentation adopts second order fermentation, basestocks to add that feed supplement, Continuous Flow add logical nitrogen, ripe dregs of beans is the technology of nitrogenous source, and leavening temperature is 43 ℃, and pH is 6.8.
1. seed tank culture: seeding tank volume 1m
3, standard ventilation machinery stirs; Culture medium prescription (volume 300L feeds intake): carbon source 10%～20%, nitrogenous source 2%～7%, dipotassium hydrogen phosphate 0.1%～0.5%, potassium primary phosphate 0.1%～0.5%, α-Dian Fenmei 0.05%, bubble enemy (PPE) 0.05%; With the substratum 0.5h that liquefies, sterilization 45min heats up; Inoculation when being cooled to 42 ℃, inoculum size 300ml kind mother obtains the mature seed that the pH value rises, mycelia is intensive, nothing is mixed bacterium.
2. fermentor cultivation: fermentor tank volume 25m
3, standard ventilation machinery stirs; Culture medium prescription: carbon source 30%～40%, nitrogenous source 2%～7%, vitamin H is an amount of, inorganic salt 1%～3%, high activity, high-purity and high temperature resistant Alpha-amylase 0.05%, pH value 6～7; With the substratum 0.5h that liquefies, sterilization 45min heats up; Inoculation when being cooled to 42 ℃, inoculum size is a seeding tank kind amount, an amount of simultaneously sterile air that feeds.
3. fermenting process stream adds logical ammonia adjusting pH to mycelial growth metabolism optimum value; Flow feeding can constantly replenish mycelial growth and produce the enzyme desired substance and improve the unit enzymatic productivity; Jar characteristic of stopping of fermentation ends is that the pH value rises, vigor no longer increases, the death of microscopy mycelia is many.
3, aftertreatment technology: jar feed liquid → aftertreatment flocculation (70 ℃) → press filtration → circulation filter → ultrafiltration just → essence filter (10h) → finished product is handled (heating up 80 ℃) → precipitation (48h) → product.
Contrast experiment embodiment:
1, zymotechnique
1. " basestocks adds feed supplement, Continuous Flow adds logical ammonia " continuous five batches of experimental results:
| Lot number | 32hr enzyme (u/ml) alive | 70hr enzyme (u/ml) alive | Stop jar enzyme (u/ml) alive | Volume (m 3) | Cycle (hr) | Single jar of output (T) |
| 020701 | 1760 | 5630 | 11347 | 15.7 | 103 | 17.36 |
| 020705 | 1650 | 6100 | 12050 | 15.5 | 114 | 18.67 |
| 020706 | 1896 | 6545 | 11900 | 15.5 | 105 | 18.45 |
| 020801 | 1530 | 5460 | 10870 | 15.1 | 108 | 16.41 |
| 020803 | 1640 | 5980 | 11236 | 15.0 | 101 | 16.85 |
Experimental result shows: this technology makes basic medium concentration low, and osmotic pressure is little, and dissolved oxygen is good, suitable mycelial growth, it is fast that produce enzyme early stage, and it is stable that the later stage is produced enzyme, finally put jar vigor and volume and all increase substantially, improved plant factor and reduced single jar of energy consumption and reduced production cost.
2. " disposablely feed intake, intermittently logical ammonia " continuous five batches of experimental results:
| Lot number | 32hr enzyme (u/ml) alive | 70hr enzyme (u/ml) alive | Stop jar enzyme (u/ml) alive | Volume (m 3) | Cycle (hr) | Single jar of output (T) |
| 020104 | 1235 | 6130 | 7258 | 12.1 | 83 | 8.8 |
| 020105 | 1100 | 5900 | 7540 | 11.8 | 89 | 8.89 |
| 020106 | 1307 | 6245 | 7980 | 11.4 | 78 | 9.09 |
| 020107 | 1054 | 5800 | 7770 | 12.0 | 85 | 9.32 |
| 020108 | 1154 | 5532 | 8258 | 11.5 | 81 | 9.49 |
Experimental result shows: this technology is owing to base-material concentration height, and osmotic pressure is big, is unfavorable for the metabolism of mycelia raised growth, and it is slower to cause produce enzyme early stage, and the middle and later periods is because nutritive deficiency causes mycelia change too early to move back, so increased single jar of energy consumption and production cost.
2, quality product:
1. the continuous five batches of experimental results of the relevant quality index of product of the present invention:
| Lot number | Finished product enzyme (u/ml) alive | Volume (m 3) | Bacterium index (≤) | Have or not precipitation (96hr) |
| 020701 | 37612 | 3.5 | 100 | Do not have |
| 020705 | 43726 | 3.2 | 50 | Do not have |
| 020706 | 38339 | 3.8 | 500 | Do not have |
| 020801 | 39476 | 3.0 | 50 | Do not have |
| 020803 | 33620 | 4.5 | 100 | Do not have |
2. continuous five batches of experimental results of the relevant quality index of the product of former technology:
| Lot number | Finished product enzyme (u/ml) alive | Volume (m 3) | Bacterium index (≤) | Have or not precipitation (96hr) |
| 020104 | 26312 | 4.6 | 20000 | Small amount of precipitate is arranged |
| 020105 | 28900 | 4.5 | 40000 | Precipitation is unclear thorough than multienzyme liquid |
| 020106 | 23958 | 4.0 | 15000 | Precipitation is unclear thorough than multienzyme liquid |
| 020107 | 27690 | 4.1 | 30000 | Small amount of precipitate is arranged |
| 020108 | 24987 | 4.3 | 10000 | Precipitation is unclear thorough than multienzyme liquid |
Claims (3)
1. the production technique of a high activity, high-purity and high temperature resistant Alpha-amylase, the production technique that it is characterized in that it is: bacterial classification (12h) → first class seed pot (28h) → big jar of (about 100h, putting a jar enzyme activity 12000u/ml) → aftertreatment flocculation (70 ℃) → press filtration → circulation filter → ultrafiltration just → essence filter (10h) → finished product handled (heating up 80 ℃) → precipitation (48h) → product.
2. the production technique of a kind of high activity, high-purity and high temperature resistant Alpha-amylase according to claim 1, after it is characterized in that adopting the ultraviolet mutagenesis method and carrying out the genetic stability test of ten generations, obtain the bacterial classification (enzyme activity 12379u/ml, purity 98%, thermotolerance 96%, cycle 107hr) of called after XF-19.
3. the production technique of a kind of high activity, high-purity and high temperature resistant Alpha-amylase according to claim 1, it is characterized in that fermenting, to adopt that second order fermentation, basestocks add feed supplement, Continuous Flow adds logical ammonia, selects ripe dregs of beans for use be the technology of nitrogenous source, leavening temperature is 43 ℃, and pH is 6.8.
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| CNA2007100361599A CN101182501A (en) | 2007-11-15 | 2007-11-15 | High activity, high-purity and high temperature resistant Alpha-amylase production process |
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| CNA2007100361599A CN101182501A (en) | 2007-11-15 | 2007-11-15 | High activity, high-purity and high temperature resistant Alpha-amylase production process |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838635A (en) * | 2010-05-21 | 2010-09-22 | 长沙青出蓝科技有限公司 | Method for preparing high-temperature resistant amylase |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838635A (en) * | 2010-05-21 | 2010-09-22 | 长沙青出蓝科技有限公司 | Method for preparing high-temperature resistant amylase |
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