CN102250862A - Method for producing chymosin by aspergillus niger solid fermentation - Google Patents
Method for producing chymosin by aspergillus niger solid fermentation Download PDFInfo
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- CN102250862A CN102250862A CN2010101784658A CN201010178465A CN102250862A CN 102250862 A CN102250862 A CN 102250862A CN 2010101784658 A CN2010101784658 A CN 2010101784658A CN 201010178465 A CN201010178465 A CN 201010178465A CN 102250862 A CN102250862 A CN 102250862A
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Abstract
The invention discloses a method for producing chymosin by aspergillus niger solid fermentation. The method adopts a solid basal medium and comprises the following steps of adding a nitrogen source and an acidic nutrient solution containing one or more inorganic salts and an amino acid into the solid basal medium to wet it, mixing well, carrying out a sterilization process at a temperature of 121 DEG C for 30 minutes, cooling, inoculating the sterilized solid basal medium with an aspergillus niger strain, carrying out solid fermentation at a fermentation temperature of 35 to 37 DEG C for 72 to 100 hours, carrying out solid-liquid extraction through adopting water as a solvent, carrying out centrifugation and extraction filtration, and carrying out the processes of vacuum rotation, evaporation, concentration and freeze-drying on supernate obtained by the previous step to obtain crude dry powder of chymosin. Chymosin which has the advantages of low added amount in raw milk such as milk and the like, rapid curd speed, specific catalytic site and low capacity for non-specific protein hydrolysis is produced through the method.
Description
Technical field
The present invention relates to a kind ofly utilize aspergillus niger (Aspergillus niger, CICC3.316) solid fermentation is produced the method for rennin, belongs to technical field of food biotechnology.
Background technology
Rennin belongs to aspartic protease, claims aspartate protease again, since being found beginning, promptly is widely used in diary industry.This enzyme is the key enzyme that in the cheese production milk is solidified and produce local flavor, and output value ratio shared in enzyme industry is up to 15%.The tradition source of rennin is the fourth stomach of calf lactation, and 5,000 ten thousand calves need be slaughtered every year approximately in the whole world at present, still can't satisfy the market demand growing to cheese product, and people turn one's attention to the surrogate of ox rennin gradually.The rennin source is very extensive, has in animals and plants and the microorganism.Nearly all proteolytic ferment can both make curdling solid under proper condition, therefore almost can both find the curdled milk enzyme in the biology of all kinds, but because the restriction of various conditions can realize that business-like enzyme is few.
In the rennin in various sources, animal rennet curdled milk vigor and proteolysis ability ratio are higher, are to make caseic first-selected enzyme.Separation and Extraction rennin from the calf abomasum (Chinese patent CN1379090) exists growth of animal slowly and cost an arm and a leg, inferior positions such as enterprise's production cost height, and animal system cheese is subjected to a lot of protection of animal ists, vegetarian's resistance.Plant-sourced rennin substitute wide material sources, the rennin that extracts from papaya (Chinese patent CN1415749) proteolytic activity is strong excessively, therefore the product special flavour that makes is bad, and the growth cycle of plant is long, it is very big to be subjected to influences such as region, season, weather simultaneously, can't guarantee the large-scale mass production rennin of stabilizing quality.And the microorganism growth cycle is short, required equipment is simple, be subjected to effects limit such as time region little, and rennin has been subjected to paying close attention to the most widely with the production technique of its outstanding curdled milk proterties and high efficiency, low cost.States such as English, U.S., lotus, day have taken the lead in beginning the correlative study of microorganism milk coagulant, China also begins to carry out correlative study at the beginning of the eighties, but also lack the rennin production technique that forms industrialization at present, produce the required rennin of cheese and mainly depend on import, the correlative study of therefore carrying out microbial rennet has higher economy and social effect.The subtilis of the high and stable yields producing lab ferment that one strain mutagenesis obtains (Chinese patent CN101665779) by the liquid state fermentation enzyme live only be 218.2u/mL, output cross low and liquid state fermentation microbiological contamination risk higher, be difficult to realize industrialization because of cost is too high.
The present invention adopts aspergillus niger (Aspergillus niger, CICC3.316) as producing bacterial strain, produce rennin by the method for solid fermentation, its production cost is low, rennin output is high and have lower proteolysis activity, can remedy the defective of aforementioned production method.
Summary of the invention
The object of the present invention is to provide a kind of aspergillus niger (Aspergillus niger that utilizes, CICC3.316) solid fermentation is produced the method for rennin, can obtain the rennin that addition is low in raw dairy such as milk, curdled milk speed is fast, catalytic site is single-minded, nonspecific proteins matter hydrolysis ability is low by this method.
One strain can be to the bacterial strain of exocytosis rennin, and this bacterial strain is aspergillus niger (Aspergillus niger), buys from Research for Industrial Microbial Germ preservation administrative center (CICC), bacterium numbering 3.316.
Utilize this Aspergillus niger strain solid fermentation to produce the method for rennin, it is characterized in that: utilize the solid-based basal culture medium, add nitrogenous source and contain inorganic salt, amino acid whose acidotrophy liquid wetting, mixing and 121 ℃ sterilization 30 minutes, inoculate with Aspergillus niger strain after cooling, and carrying out solid fermentation, leavening temperature is 35～37 ℃, fermentation time 72～100 hours; Adopting water is that solvent carries out liquid-solid extraction, and behind centrifugal, the suction filtration, the supernatant liquor rotary evaporation in vacuo is concentrated and freeze-drying, obtain the coarse dry powder of rennin, wherein to account for the solid medium mass ratio be 1% to nitrogenous source, and every 10g solid medium is with 15ml acidotrophy liquid wetting.
The above-mentioned aspergillus niger solid fermentation that utilizes produces the method for rennin, described basic medium is a carbon source, be selected from the compound of wheat bran, rice bran and bran flour, wheat bran starch, rice bran ground rice, wherein composite mass ratio is 9: 1, with wheat bran and flour with 9: 1 (mass ratio) composite during as culture medium carbon source the vigor of fermentation producing lab ferment higher.
The above-mentioned aspergillus niger solid fermentation that utilizes produces the method for rennin, described nitrogenous source, be selected from ammonium nitrate, urea, skimmed milk powder, casein food grade and gelatin (nitrogenous source account for solid medium 1%), wherein, the vigor of fermentation producing lab ferment is higher during as nitrogenous source with 1% skimmed milk powder.
The above-mentioned aspergillus niger solid fermentation that utilizes produces the method for rennin, and in the described acidotrophy liquid, inorganic salt are selected from MgSO
4, FeSO
4In one or both, add 0.7gMgSO in the 100ml water
4, 0.9gFeSO
4In one or both, amino acid is selected from L-glutamic acid, leucine, proline(Pro), tryptophane, Histidine or tyrosine, 0.1g amino acid in every 100ml water, and regulate pH to 6.5～7.0 with 0.2N HCl.Experimental result shows that acidotrophy liquid optimum formula is to add 0.7gMgSO in every 100ml water
4, 0.9gFeSO
4Reach the 0.1g proline(Pro), and regulate pH to 6.5～7.0 with 0.2N HCl.
Through optimizing the solid bran mass proportioning and the culture condition that draw be: in the solid medium of unit weight, the mass ratio of wheat bran and flour is 9: 1, other adds 1% (mass ratio) skimmed milk powder, (adds 0.7gMgSO in every 100ml water with acid mineral ion nutritive medium
4, 0.9gFeSO
4Reach the 0.1g proline(Pro), and regulate pH to 6.5～7.0 with 0.2N HCl) soak into, every 10g solid uses 15ml acidotrophy liquid wetting, mixing and high-temperature sterilization, the back that cools is inoculated, and cultivates 98h for 37 ℃.
Beneficial effect of the present invention comprises: after optimizing, aspergillus niger can be the higher rennin of fermented substrate fermentative production enzymic activity with cheap raw materials such as wheat bran, flour, reduced production cost, this law can be used for preparing microbial food additive and industrial additive, the thick enzyme of rennin that makes by this law can be used for preparing cheese and casein food grade, shorten the time of raw dairy curdled milks such as cow's milk, can obtain the rennin that addition is low in raw dairy such as milk, curdled milk speed is fast, catalytic site is single-minded, nonspecific proteins matter hydrolysis ability is low by this method.
Description of drawings
Fig. 1 embodiment 1 different sorts substratum is to the influence of enzymatic production;
Fig. 2 embodiment 2 different influences of adding nitrogenous source to enzymatic production;
Fig. 3 embodiment 3 mineral ions are to producing the influence of enzyme;
Fig. 4 embodiment 4 aminoacid addition are to producing the influence of enzyme.
Embodiment
Following example has been explained the present invention in more detail, but the present invention is not limited to following examples.
Analytical procedure:
Be that the solid culture of solvent after to abundant fermentation carries out solid-liquid contact extraction with water, the centrifugal and elimination impurity of extraction liquid low-temperature and high-speed, 40 ℃ of rotary evaporation in vacuo of supernatant liquor are condensed into crude enzyme liquid.
The rennin measuring method with reference to the improvement after the Arima method: get 5mL and contain skim-milk 10% (mass ratio), CaCl
2The separated milk of 0.1% (mass ratio) is put into plate and is incubated 10min down at 45 ℃, adds the above-mentioned crude enzyme liquid of 0.5mL, is agglutinating particle appears in the unit accurate recording to the plate surface from the adding crude enzyme liquid time with the second.Unit of enzyme is defined as: under 45 ℃ of constant temperature, 40min contains 0.1g skimming milk and 0.001gCaCl with 1mL
2The separated milk enzyme amount of solidifying be 1 Soxhlet unit (SU).Calculation formula is: SU=2400 * 5 * D/T * 0.5.
In the formula: T is the curdled milk time, and unit is second; D is an extension rate.
Described protease assay method is: the skimming milk solution of preparing quality 1% with the 0.1moL/L acetate buffer solution of pH5.5.Get this solution 5mL and 1mL crude enzyme liquid in 35 ℃ of reaction 10min, stop enzyme reaction, filter, get supernatant liquor 2mL and 5mL Na with the 2mL trichoroacetic acid(TCA)
2CO
3Solution mixes, and measures the OD value at wavelength 280nm.The OD value is big more represents that promptly the proteolysis vigor is more little.The R value is the rennin vigor and the ratio of proteolysis enzyme activity.
Embodiment 1
Choose several different basic mediums (9: 1 bran flours of wheat bran, rice bran, mass ratio are composite, wheat bran starch complex, rice bran ground rice composite) respectively and be carbon source, (nitrogenous source is a skimmed milk powder, and quality accounts for 1% of basic medium at its zymotechnique; Add 0.7gMgSO in every 100ml water
4, 0.9gFeSO
4And the 0.1g proline(Pro), and be acidotrophy liquid with 0.2N HCl adjusting pH to 6.5～7.0; Every 10g solid adds 15ml acidotrophy liquid; It is 35～37 ℃ that leavening temperature is selected the interval, the fermentation time interval is 72～100 hours) mixing and 121 ℃ of sterilizations 30 minutes under the identical situation, the back that cools is with the Aspergillus niger strain inoculation, and carries out solid fermentation, leavening temperature is 35～37 ℃, fermentation time 72～100 hours; Adopting water is that solvent carries out liquid-solid extraction, and behind centrifugal, the suction filtration, the supernatant liquor rotary evaporation in vacuo is concentrated and freeze-drying, obtains the coarse dry powder of rennin, and milk-curdling activity and protease activity are seen Fig. 1 in the aspergillus niger product.The result shows with wheat bran and flour (mass ratio 9: 1) is composite when being carbon source that milk-curdling activity is the highest.
Embodiment 2
Composite with 9: 1 bran flour of mass ratio is the basic medium carbon source, select different nitrogenous sources (ammonium nitrate, urea, skimmed milk powder, casein food grade and gelatin respectively, the nitrogenous source quality account for the solid medium quality 1%), (add 0.7gMgSO in every 100ml water at its zymotechnique
4, 0.9gFeSO
4And the 0.1g proline(Pro), and to regulate pH to 6.5～7.0 with 0.2NHCl be acidotrophy liquid; Every 10g solid adds 15ml acidotrophy liquid) mixing and 121 ℃ of sterilizations 30 minutes under the identical situation, the back that cools is with the Aspergillus niger strain inoculation, and carries out solid fermentation, and leavening temperature is 35～37 ℃, fermentation time 72～100 hours; Adopting water is that solvent carries out liquid-solid extraction, and behind centrifugal, the suction filtration, the supernatant liquor rotary evaporation in vacuo is concentrated and freeze-drying, obtains the coarse dry powder of rennin, and milk-curdling activity and protease activity are seen Fig. 2 in the aspergillus niger product.Experimental result shows, 1% skimmed milk powder is during as nitrogenous source, and the activity of rennin is the highest in the product.
Embodiment 3
To composite with 9: 1 bran flour of mass ratio be the basic medium carbon source, add 1% skimmed milk powder and be in the solid medium of nitrogenous source preparation, add the acidotrophy liquid that contains inorganic salt: every 100ml water adds 0.7gFeSO respectively
4Or 0.9gMgSO
4, adding the 0.1g proline(Pro) simultaneously, and regulate pH to 6.5～7.0 with the HCl of 0.2N, every 10g solid adds 15ml acidotrophy liquid.Mixing and 121 ℃ sterilization 30 minutes, the back that cools be with the Aspergillus niger strain inoculation, and carry out solid fermentation, and leavening temperature is 35～37 ℃, fermentation time 72～100 hours; Adopting water is that solvent carries out liquid-solid extraction, and behind centrifugal, the suction filtration, the supernatant liquor rotary evaporation in vacuo is concentrated and freeze-drying, obtains the coarse dry powder of rennin, and milk-curdling activity and protease activity are seen Fig. 3 in the aspergillus niger product.Experimental result shows, Fe
2+Ion and Mg
2+There is the activity that can obviously improve rennin in the tunning in ionic, selects to add 0.7gMgSO in every 100ml water
4, 0.9gFeSO
4Acidotrophy liquid.
Embodiment 4
To composite with 9: 1 bran flour of mass ratio be the basic medium carbon source, add 1% skimmed milk powder and be in the solid medium of nitrogenous source preparation, add the acidotrophy liquid that contains inorganic salt, every 100ml water contains 0.7gFeSO
4, 0.9gMgSO
4Add 0.1g L-glutamic acid, leucine, proline(Pro), tryptophane, Histidine or tyrosine simultaneously respectively, and regulate pH to 6.5～7.0 with the HCl of 0.2N, every 10g solid adds 15ml acidotrophy liquid, mixing and 121 ℃ sterilization 30 minutes, the back that cools be with the Aspergillus niger strain inoculation, and carry out solid fermentation, leavening temperature is 35～37 ℃, fermentation time 72～100 hours; Adopting water is that solvent carries out liquid-solid extraction, and behind centrifugal, the suction filtration, the supernatant liquor rotary evaporation in vacuo is concentrated and freeze-drying, obtains the coarse dry powder of rennin, and milk-curdling activity and protease activity are seen Fig. 4 in the aspergillus niger product.Experimental result shows that in each seed amino acid, proline(Pro) has the greatest impact to the raising of rennin production, and the rennin work the when specific activity of rennin does not have aminoacid addition in the interpolation proline(Pro) after product has improved 60.82%.
Embodiment 5
Aspergillus niger produces enzyme in Erlenmeyer flask, every 250mL Erlenmeyer flask packing 9g wheat bran, 1g flour, 0.1g skimmed milk powder (are pressed mass volume ratio preparation 0.7%MgSO with the acid mineral ion nutritive medium of 15mL
47H
2O, 0.9%FeSO
47H
2O, 0.1% proline(Pro) adds 0.2N HCl and regulates pH to 6.61) soak into, mixing and 121 ℃ of sterilizations 30 minutes, the back inoculated aspergillus niger spore suspension 1.1mL that cools cultivates 98h for 37 ℃.Because the product rennin is unstable in water, after fully fermenting promptly is solvent with water, uses the anti-phase contact extraction process of three step solid-liquids to extract tunning from solid medium, through after the centrifuging, mode by rotary evaporation in vacuo concentrates crude enzyme liquid rapidly, is lyophilized into thick enzyme powder.Thick enzyme powder rennin work can reach 21000SU/g.
Claims (4)
1. method of utilizing the Aspergillus niger strain solid fermentation to produce rennin, it is characterized in that: utilize the solid-based basal culture medium, add nitrogenous source and contain inorganic salt, amino acid whose acidotrophy liquid wetting, mixing and 121 ℃ sterilization 30 minutes, inoculate with Aspergillus niger strain after cooling, and carrying out solid fermentation, leavening temperature is 35～37 ℃, fermentation time 72～100 hours; Adopting water is that solvent carries out liquid-solid extraction, and behind centrifugal, the suction filtration, the supernatant liquor rotary evaporation in vacuo is concentrated and freeze-drying, obtain the coarse dry powder of rennin, wherein to account for the solid medium mass ratio be 1% to nitrogenous source, and every 10g solid medium is with 15ml acidotrophy liquid wetting;
Above-mentioned described basic medium is a carbon source, is selected from the compound of wheat bran, rice bran and bran flour, wheat bran starch, rice bran ground rice, and wherein composite mass ratio is 9: 1;
Above-mentioned described nitrogenous source is selected from ammonium nitrate, urea, skimmed milk powder, casein food grade and gelatin;
In the above-mentioned described acidotrophy liquid, inorganic salt are selected from MgSO
4, FeSO
4In one or both, add 0.7gMgSO in the 100ml water
4, 0.9gFeSO
4In one or both, amino acid is selected from L-glutamic acid, leucine, proline(Pro), tryptophane, Histidine or tyrosine, 0.1g amino acid in every 100ml water, and regulate pH to 6.5～7.0 with 0.2N HCl.
2. claim 1 a kind of utilizes the Aspergillus niger strain solid fermentation to produce the method for rennin, and it is characterized in that: inorganic salt are elected MgSO as in the described acidotrophy liquid
4And FeSO
4, add 0.7gMgSO in the 100ml water
4And 0.9gFeSO
4, amino acid is proline(Pro).
3. claim 1 a kind of utilizes the Aspergillus niger strain solid fermentation to produce the method for rennin, and it is characterized in that: described nitrogenous source is a skimmed milk powder.
4. claim 1 a kind of utilizes the Aspergillus niger strain solid fermentation to produce the method for rennin, it is characterized in that: in the solid medium of unit weight, the mass ratio of wheat bran and flour is 9: 1, other adds 1% (mass ratio) skimmed milk powder, with acid mineral ion nutritive medium be: add 0.7gMgSO4,0.9gFeSO4 and 0.1g proline(Pro) in every 100ml water, and regulate pH to 6.5～7.0 with 0.2N HCl and soak into, every 10g solid uses 15ml acidotrophy liquid wetting, mixing and high-temperature sterilization, inoculation after cooling is cultivated 98h for 37 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532296A (en) * | 2011-12-27 | 2012-07-04 | 甘肃华羚酪蛋白股份有限公司 | Method for converting Qula acid-method casein into chymosin casein |
| CN103421696A (en) * | 2013-08-28 | 2013-12-04 | 光明乳业股份有限公司 | Interwoven acremonium implicatum for producing rennin and application thereof |
-
2010
- 2010-05-21 CN CN2010101784658A patent/CN102250862A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532296A (en) * | 2011-12-27 | 2012-07-04 | 甘肃华羚酪蛋白股份有限公司 | Method for converting Qula acid-method casein into chymosin casein |
| CN102532296B (en) * | 2011-12-27 | 2014-05-14 | 甘肃华羚酪蛋白股份有限公司 | Method for converting Qula acid-method casein into chymosin casein |
| CN103421696A (en) * | 2013-08-28 | 2013-12-04 | 光明乳业股份有限公司 | Interwoven acremonium implicatum for producing rennin and application thereof |
| CN103421696B (en) * | 2013-08-28 | 2015-09-16 | 光明乳业股份有限公司 | A kind of intertexture top spore of producing lab ferment is mould and apply |
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Application publication date: 20111123 |