CN101117628B - Method for producing phytase - Google Patents
Method for producing phytase Download PDFInfo
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- CN101117628B CN101117628B CN2006101037780A CN200610103778A CN101117628B CN 101117628 B CN101117628 B CN 101117628B CN 2006101037780 A CN2006101037780 A CN 2006101037780A CN 200610103778 A CN200610103778 A CN 200610103778A CN 101117628 B CN101117628 B CN 101117628B
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- phytase
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Abstract
The present invention discloses a method for producing microbial phytase. The present invention utilizes vinegar residue as a solid culture medium, and microbial strains can be inoculated to produce microbial phytase, then microbial phytase can be obtained after being fermented. The present invention has the following production technical processes: firstly, the vinegar residue culture medium is put into a container, then the water content of the culture medium is adjusted; secondly, the disinfection is carried on; thirdly, the inoculation is carried on under the condition of asepsis; fourthly, ferment is carried on. The method for producing microbial phytase in the present invention selects the waste -vinegar residue in vinegar production, thus changing waste into valuable, making the microbial phytase manufacturing cost greatly reduced, and resulting the reasonable reuse of the vinegar residue.
Description
Technical field
The present invention relates to a kind of method of producing phytase.
Background technology
Phosphorus is the necessary mineral element of animal body, in order to satisfy the demand of feed industry and aquaculture, has consumed the phosphate rock resource of a large amount of costlinesses.Have only very small portion phosphorus to deposit in the body and in the livestock product, thereby to environment, particularly during intensive farm, very serious to the pollution of underground water and soil earth.
Along with development of biology, it is found that phytase becomes the feed the most attractive approach of phosphorus problem that solves.By adding phytase, can improve the utilization ratio of phytate phosphorus in the feed (or phosphoric acid salt) in the monogastric animal feed, reduce the pollution of the discharge of phosphorus in the farm animal excrement environment and water.Along with after biotechnology particularly adopts the DNA recombinant technology, make phytase extensive application aborning become possibility.Microbial phytase commercialization production in large quantities as animal feedstuff additive.
In natural crop, as also containing phytase in maize germ, the soybean etc., but its content is low, and the growth cycle of crop is long.Extract phytase with this, cost is too high, the cycle is long, is not suitable for industrialization.
The production of existing phytase is to obtain by the strain fermentation with phytase generating characteristic, and two kinds of fermentation process are generally arranged: solid fermentation is cultivated and liquid fermentation and culture.Existing liquid fermentation and culture, for example: substratum adopts glucose: 30.0g; Zulkovsky starch: 30.0g; MgSO
47H
2O:0.5g; KCl:0.5g; Fe
2SO
4: 0.03g; MnSO
4: 0.03g; Wheat bran: 5.0g; NH
4NO
3: 5.0g; CaCO
3: after 5.0g mixes, formulated with acetic acid-sodium-acetate buffer of pH5.5.Insert aspergillus niger-903,29 ℃ fermentation culture 4d in the liquid nutrient medium.Existing solid fermentation cultured method: can adopt amount of water be 35% wheat bran as substratum, insert aspergillus niger AN00101,37 ℃, cultivate 114h.Therefore no matter be that solid medium or its cost of liquid nutrient medium are all very high, low being used to of a kind of cost method for preparing phytase becomes those skilled in the art's target.
Summary of the invention
Technical problem to be solved of the present invention is, a kind of production method of new phytase is provided, and the defective that it can overcome in the existing production method adopts the low solids component of a kind of cost to carry out the method for fermentation culture as substratum.
Above-mentioned purpose of the present invention is achieved through the following technical solutions: a kind of method of producing phytase is characterized in that: it is to utilize vinegar poor as solid medium, and inoculation can be produced the microbial strains of phytase, ferments, thereby obtains phytase; Wherein the microbial strains that is inoculated is preferred: Fructus Fici aspergillus (Aspergillus ficuum).
The method of production phytase of the present invention, its production process is:
(1) the poor substratum of vinegar is packed into container is adjusted the water content of substratum; (2) sterilize; (3) under aseptic condition, carry out the microbe inoculation bacterial classification; (4) fermentation.
The method of production phytase of the present invention, its inoculum size is: inoculation Fructus Fici aspergillus 1 * 10 during the vinegar of per 15 grams is poor
6～5 * 10
6Individual bacterium.
Its optimum inoculation amount is: inoculation Fructus Fici aspergillus 2 * 10 during the vinegar of per 15 grams is poor
6～4 * 10
6Individual bacterium.
The method of production phytase of the present invention, the water content that its substratum vinegar is poor is 50%～75% by weight.
The water content that substratum vinegar is pickled with grains or in wine optimum range by weight is 55%～65%.
The method of production phytase of the present invention, the time of fermentation is 2～6 days.
The time of best fermentation is 2～4 days.
The method of production phytase of the present invention is characterized in that: the condition of described sterilization is: 121 ℃, 0.1MPa, and the time is 20 minutes; Described leavening temperature is 27 ℃.
Utilizing microbial fermentation to produce phytase is the unique channel that this enzyme is produced, the inventor is in the research through vinegar is pickled with grains or in wine, find that vinegar approximately contains 7%～15% coarse ash, 2%～9% crude fat, 9%～15% crude protein, 27%～36% robust fibre in poor, 0.20%～0.40% calcium, 0.03%～0.08% nutritive ingredients such as phosphorus, these nutritive ingredients can be utilized by Secondary Fermentation again.
In the research process that Secondary Fermentation utilizes, be surprised to find that can adopt vinegar to be pickled with grains or in wine produces phytase as substratum, its scientific and technological content is higher, and has originality.And compare with the production method of existing phytase, the cost of its substratum reduces significantly, and substratum of the present invention is to adopt the waste of producing vinegar---and vinegar is poor, turns waste into wealth, and poor having obtained of vinegar rationally utilized again.
Embodiment
The present invention describes content of the present invention in detail below in conjunction with specific embodiment, so that understand the present invention better.
At first, select for use vinegar poor, smoked unstrained spirits poor (unstrained spirits in the system vinegar through smoke and drench waste after the vinegar) is that the mixture with Chinese sorghum, corn and Chinese sorghum and corn is that three kinds of substratum of major ingredient are poor; Inoculation Fructus Fici aspergillus (Aspergillus ficuum), this bacterial classification is bought in Institute of Microorganism, Academia Sinica, and bacterium numbering is: 3.4322.Respectively different fermentation conditions is experimentized, thereby determines of the influence of different fermentation conditions phytase generating:
1, fermentation time is to the influence of phytase generating:
Fix two factors: the water content of substratum and inoculum size change fermentation time: 2d～6d;
Its experimentation is:
At first, the air-dry vinegar of 15 grams of packing in the 150ml triangular flask is poor;
Secondly, add entry 15 grams, the water content of adjusting substratum is 50%;
The 3rd, under 121 ℃, 0.1MPa condition, sterilized 20 minutes;
The 4th, under aseptic condition, inoculate inoculation Fructus Fici aspergillus 3 * 10
6Individual bacterium;
The 5th, be put in the incubator, at the suitable leavening temperature of Fructus Fici aspergillus: under 27 ℃, ferment, selected respectively 2d～6d of the time of fermentation (my god), the result is as shown in table 1 below: the measure unit of enzyme is in this test: IU/g (that is: international unit/every gram solid fermentation thing).
Table 1 fermentation time-phytase output
From top experiment as can be seen: Chinese sorghum group and combined group, its yield of enzyme is in rising trend before the fermentation 4d, and is on a declining curve after the 4d; The corn group, its yield of enzyme is in rising trend before the fermentation 3d, and is on a declining curve after the 3d.
2, the culture medium water content is to the influence of phytase generating:
Experimentation is identical with top experiment, and determine that fermentation time is: 4d, inoculum size is: 3 * 10
6Individual bacterium is adjusted water content 50%～75%, and test-results is as shown in table 2 below:
Table 2 water content-phytase output
The formation of solid fermentation matrix moisture content cell growth and enzyme plays an important role, and adds water the matrix bubble is risen, and promotes the utilization of microorganism to nutritive substance, above-mentioned test-results shows, water content all can be produced enzyme in 50%～75% scope, but the optimum moisture content scope is 55～65%, and water content of substrate is lower than 55%, mycelial growth is slow, the bent drying of enzyme, unfavorable product enzyme, water ratio are higher than 65% mycelia and nourish and grow vigorously, and meta-bolites (enzyme) accumulation is few, substrate consumption is many, and yield descends.
3, inoculum size is to the influence of phytase generating:
Experimentation is identical with top experiment, determines that fermentation time is: 4d, and the water content of substratum is: 65%; Adjust the inoculum size of bacterium cake: inoculation 1 * 10
6～5 * 10
6Individual bacterium experimentizes, and the result is as shown in table 3 below:
Table 3 inoculum size-phytase output
Test-results shows that the vinegar of per 15 grams is poor, microbe inoculation bacterium cake 1 * 10
6～5 * 10
6Individually all can produce enzyme, producing enzyme optimum inoculation amount scope is 2 * 10
6～4 * 10
6Individual bacterium, inoculum size is less than 2 * 10
6Mycelial growth is slow during individual bacterium, unfavorable product enzyme, and inoculum size is more than 4 * 10
6During individual bacterium, mycelia nourishes and grows vigorous, and meta-bolites (enzyme) accumulation is few, and substrate consumption is many, and yield descends.
By top three groups of experiments, as can be seen: the fermentation condition of phytase is: fermentation time 1d～6d, and best fermentation time is: 2d～4d; The water content of culture medium is 50%～75%, and optimum range is: 55%～65%; Inoculum size is 1 * 10
6～5 * 10
6Individual bacterium/15g vinegar is pickled with grains or in wine (weight that does not add water), and optimum inoculation amount is 2 * 10
6～4 * 10
6Individual bacterium.
Below by concrete most preferred embodiment the production method of phytase of the present invention is described:
Embodiment 1-9, referring to table 4, the technological process of its fermentation is with top experimentation, equally, selecting air-dry is the poor 15g of vinegar of major ingredient with the Chinese sorghum, adds water respectively, the water content of adjusting matrix is 55%, 60%, 65%, sterilize, the temperature, pressure of sterilization is with top experiment, and the time is 20 minutes; Under aseptic condition, inoculation bacterium cake quantity is respectively 2 * 10
6, 3 * 10
6, 4 * 10
6Individual bacterium, fermentation time were respectively 2,3,4 days, and the result is as shown in the table:
Table 4 Chinese sorghum group orthogonal test phytase output
| Project | Fermentation time (d) | Water content (%) | Inoculum size (* 10 6Individual) | Phytase amount (IU/g) |
| 1 | 2 | 55 | 2 | 7.88 |
| 2 | 2 | 60 | 3 | 8.41 |
| 3 | 2 | 65 | 4 | 9.19 |
| 4 | 3 | 55 | 4 | 10.22 |
| 5 | 3 | 60 | 2 | 8.86 |
| 6 | 3 | 65 | 3 | 8.08 |
| 7 | 4 | 55 | 3 | 8.95 |
| 8 | 4 | 60 | 4 | 8.12 |
| 9 | 4 | 65 | 2 | 9.44 |
Embodiment 10-18, it is that the vinegar of major ingredient is poor that base starting material is changed into the corn, and other carries out orthogonal test with above-mentioned embodiment to the best fermentation condition scope, and the result is as shown in the table:
Table 5 corn group orthogonal test phytase output
| Project | Fermentation time (d) | Water content (%) | Inoculum size (* 10 6Individual) | Phytase amount (IU/g) |
| 10 | 2 | 55 | 2 | 8.62 |
| 11 | 2 | 60 | 3 | 7.01 |
| 12 | 2 | 65 | 4 | 7.75 |
| 13 | 3 | 55 | 4 | 8.74 |
| 14 | 3 | 60 | 2 | 7.38 |
| 15 | 3 | 65 | 3 | 5.70 |
| 16 | 4 | 55 | 3 | 7.63 |
| 17 | 4 | 60 | 4 | 7.05 |
| 18 | 4 | 65 | 2 | 7.18 |
The top condition combination that can be drawn Chinese sorghum group and corn group phytase generating by table 4 and table 5 is a water content 55%, inoculum size 4 * 10
6Individual bacterium, fermentation time 3d.
Embodiment 19-27, adopt with Chinese sorghum and corn mixture be the vinegar of major ingredient poor be substratum, two groups of embodiment above the set of variations contract of other technological process and parameter, the result is as shown in the table:
Table 6 Chinese sorghum corn combined group orthogonal test phytase output
| Project | Fermentation time (d) | Water content (%) | Inoculum size (* 10 6Individual) | Phytase amount (IU/g) |
| 19 | 2 | 55 | 2 | 7.34 |
| 20 | 2 | 60 | 3 | 7.22 |
| 21 | 2 | 65 | 4 | 14.34 |
| 22 | 3 | 55 | 4 | 8.37 |
| 23 | 3 | 60 | 2 | 6.23 |
| 24 | 3 | 65 | 3 | 7.79 |
| 25 | 4 | 55 | 3 | 13.64 |
| 26 | 4 | 60 | 4 | 10.63 |
| 27 | 4 | 65 | 2 | 8 |
The top condition combination that can be drawn the combined group phytase generating by table 6 is an inoculum size 4 * 10
6Individual bacterium, fermentation time 4d, water content 65%.
Above embodiment makes up best fermentation condition, and its zymogenic rate all is higher than the yield that single condition is selected optimum value for use.These embodiment; it only is explanation to a better embodiment of the present invention; be not the restriction to protection scope of the present invention, the present invention aims to provide a kind of new, substratum that cost is low, phytase, thereby a kind of production method of new phytase is provided.The selection of the present invention's Fructus Fici aspergillus bacterial classification in the above-described embodiments; be in the microorganism that can produce phytase, select a kind of; obviously can select other microorganism that can produce phytase to substitute Fructus Fici aspergillus for those skilled in the art; for example: aspergillus niger, methanol yeast, subtilis, candida krusei etc., these should be all in protection scope of the present invention.
Claims (9)
1. method of producing phytase is characterized in that: it is to utilize vinegar poor as solid medium, and inoculation can be produced the microbial strains of phytase, ferments, thereby obtains phytase; The microbial strains that is inoculated is: Fructus Fici aspergillus (Aspergillus ficuum).
2. the method for production phytase according to claim 1 is characterized in that: described production process is:
(1) the poor substratum of vinegar is packed into container is adjusted the water content of substratum; (2) sterilize; (3) under aseptic condition, carry out the microbe inoculation bacterial classification; (4) fermentation.
3. the method for production phytase according to claim 2 is characterized in that: the Fructus Fici aspergillar inoculum size during the vinegar of per 15 grams is poor is 1 * 10
6～5 * 10
6Individual bacterium.
4. the method for production phytase according to claim 3 is characterized in that: the Fructus Fici aspergillar inoculum size during the vinegar of per 15 grams is poor is 2 * 10
6～4 * 10
6Individual bacterium.
5. the method for production phytase according to claim 2 is characterized in that: the water content of the poor substratum of vinegar is 50%～75% by weight.
6. the method for production phytase according to claim 5 is characterized in that: the water content of the poor substratum of vinegar is 55%～65% by weight.
7. the method for production phytase according to claim 2 is characterized in that: the time of fermentation is 2～6 days.
8. the method for production phytase according to claim 7 is characterized in that: the time of fermentation is 2～4 days.
9. the method for production phytase according to claim 2 is characterized in that: the condition of described sterilization is: 121 ℃, 0.1MPa, and the time is 20 minutes; Described leavening temperature is 27 ℃.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101037780A CN101117628B (en) | 2006-08-01 | 2006-08-01 | Method for producing phytase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101037780A CN101117628B (en) | 2006-08-01 | 2006-08-01 | Method for producing phytase |
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| Publication Number | Publication Date |
|---|---|
| CN101117628A CN101117628A (en) | 2008-02-06 |
| CN101117628B true CN101117628B (en) | 2010-12-08 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1374402A (en) * | 2002-02-26 | 2002-10-16 | 东莞市豪发生物工程开发有限公司 | Once Aspergillus niger fermentation process of obtaining complex enzyme of several kinds of enzyme and with high enzyme activity |
| US6667066B2 (en) * | 1999-01-25 | 2003-12-23 | Gie Agro Industrie | Multi-enzyme product with glucoamylase, proteolytic and xylanase activities and method for producing same by solid state fermentation of wheat bran with Aspergillus niger |
-
2006
- 2006-08-01 CN CN2006101037780A patent/CN101117628B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6667066B2 (en) * | 1999-01-25 | 2003-12-23 | Gie Agro Industrie | Multi-enzyme product with glucoamylase, proteolytic and xylanase activities and method for producing same by solid state fermentation of wheat bran with Aspergillus niger |
| CN1374402A (en) * | 2002-02-26 | 2002-10-16 | 东莞市豪发生物工程开发有限公司 | Once Aspergillus niger fermentation process of obtaining complex enzyme of several kinds of enzyme and with high enzyme activity |
Non-Patent Citations (2)
| Title |
|---|
| 卜春文，等，.秸秆氨化后生物技术处理的工艺.无锡轻工大学学报21 6.2002,21(6),648-650，具体参见1.1, 1.3.4, 表3. |
| 卜春文等.秸秆氨化后生物技术处理的工艺.无锡轻工大学学报21 6.2002,21(6),648-650,具体参见1.1, 1.3.4, 表3. * |
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