CN101974503B - Method for preparing chymosin by mixed culture fermentation of bean dregs and waste material from fungal culture (WMFC) - Google Patents
Method for preparing chymosin by mixed culture fermentation of bean dregs and waste material from fungal culture (WMFC) Download PDFInfo
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- CN101974503B CN101974503B CN2010105319956A CN201010531995A CN101974503B CN 101974503 B CN101974503 B CN 101974503B CN 2010105319956 A CN2010105319956 A CN 2010105319956A CN 201010531995 A CN201010531995 A CN 201010531995A CN 101974503 B CN101974503 B CN 101974503B
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Abstract
The invention provides a method for preparing chymosin by mixed culture fermentation of bean dregs and waste material from fungal culture (WMFC). The method comprises the steps of: adding the WMFC into wet bean dregs, inoculating a tiny mucor strain for solid fermentation, and extracting the chymosin from a fermentation product. In the method, the respective characteristics of the bean dregs and the WMFC are skillfully utilized, sundried WMFC from edible fungi and the wet bean dregs are mixed to adjust the proportion of water content to dry materials in a solid fermentation medium, and thus, the wet bean dregs can be prevented from caking due to overhigh water content, a link of drying the bean dregs is omitted, and enzyme activity obtained through fermentation cannot be lowered due to the doping of the WMFC from the edible fungi. Above all, by mixing the wet bean dregs and the dry WMFC from the edible fungi to replace the wheat bran to carry out solid fermentation on the chymosin, wastes are turned into wealth, the production cost of the chymosin is reduced, and the technology is simple, convenient and easy to operate, is time-saving and economical and has higher enzyme activity.
Description
Technical field
The present invention relates to a kind of preparation method of microbial rennet, relate more specifically to the method that a kind of bean dregs and edible fungus bran mixed fermentation prepare rennin, belong to and utilize waste resource to carry out solid fermentation production rennin technical field.
Background technology
Bean dregs are main by products of soyabean processing, disposed as low value-added feed or fertilizer for many years, some in addition directly abandoned, not only economic benefit is low, the waste resource has also caused environmental pollution.Utilize modern biotechnology means such as fermentation technique, zymotechnic that bean dregs are fully utilized and process; Make its nutritive ingredient be able to full-scale development; Solve the environmental pollution that discarded bean dregs are caused, realize the cyclic utilization of waste, become the focus and the trend of current research.
The bacterium chaff is to utilize raw materials such as stalk, wood chip to carry out the edible mushrooms substituting stuff cultivation, and the substratum residuum after the results is commonly called as edible fungus culturing waste material, bacterium slag or clout; Be the residual body of hypha of edible fungus and through the edible mushrooms enzymolysis, the mixture of the compositions such as robust fibre of structure generation qualitative change.Culture medium of edible fungus is grouped into by one-tenth such as wood chip, wheat bran, cotton seed hullss usually.After edible fungus culturing finished, Mierocrystalline cellulose, semicellulose and xylogen were changed into mycelium by a large amount of the utilization, and protein contnt is improved accordingly, generally can reach more than 10%.At present, the edible mushrooms of some kinds is adopted factory culture, generally only goes out 1 damp mushroom; Therefore remaining nutritive substance is abundanter than general peasant household cultivation bacterium chaff in the factory culture bacterium chaff; Sanitary condition is also better, utilizes technology owing to lack the bacterium chaff, and edible fungus bran development and use research still is in the exploratory stage; Comprise as ruminant feed, culturing edible fungus, making fertilizer, be converted into biogas etc., edible fungus bran is also rare in the research of zymin Industrial Application.
Rennin is a kind of special proteolytic ferment; It is the key enzyme in the cheese production process; Its major function is 105～106 phenylalanine(Phe)s of hydrolysis Ruzhong, specificity ground casein polypeptied chain and the peptide bond between the methionine(Met); Form stable para-casein and hydrophilic PROVON 190, when casein is hydrolyzed to a certain degree, the newborn PROVON 190 that the calcium ion in the solution just can form grumeleuse or solidify through the chemical bond that forms at the casein glue intergranular.Rennin belongs to large zymin, and its output accounts for 15% of whole world zymin total amount, and its output occupies second of zymin industry.Traditional rennin derives from the calf abomasum; But simple dependence is slaughtered a large amount of calves and is obtained the needs that rennin still is difficult to satisfy world's cheese industry; Therefore must rely on microbial rennet to substitute, the microbial rennet share has accounted for the over half of rennin consumption at present.
In theory, all aspartic proteases have the curdled milk effect, but in fact, only have the proteolytic enzyme of minority microorganism secretion to be suitable as rennin.The ideal mikrobe should possess stronger curdled milk ability and less hydrolysis ability, and has biological safety.At present, food and drug administration (FDA) the approval mikrobe that is used to produce rennin has: the rice black wool is mould, Mucor pusillus and grain epidemic disease are mould.
At present, microbial rennet fermentation culture material will adopt starting material such as wheat bran, glucose, zein slurry usually.Although once reporting, domestic scholars Lu Fei etc. utilized graceful radiation Mucor (Actinomucor elegans) to carry out the research of bean dregs solid state fermentation; Its proteolyze enzyme activity can reach 600U/g; But its research does not relate to Mucor pusillus (Mucor pusillus); And research only limits to proteolytic ferment, does not relate to solid fermentation and produces the rennin technology.Utilizing wet bean dregs and dry mushroom bacterium chaff compound cultivation Mucor pusillus (Mucor pusillus) to carry out solid fermentation prepares the rennin technology and does not appear in the newspapers as yet at present.
Summary of the invention
The purpose of this invention is to provide the method that a kind of bean dregs and the mixed fermentation of bacterium chaff prepare rennin, be not merely the rennin suitability for industrialized production and find cheap raw material resources, also is that the development and use of bean dregs and edible fungus bran resource provide an effective way simultaneously.
Technical scheme of the present invention is following:
In wet bean dregs, add the bacterium chaff, inoculate the Mucor pusillus bacterial classification then and carry out solid fermentation, from tunning, extract rennin.
Said method specifically may further comprise the steps:
(1) bean dregs mechanical dehydration: with water cut be 80～85% wet bean dregs through the mechanical expression dehydration, make its water cut reduce to 60%～70%;
(2) preparation culture material: by the wet bean dregs after dewatering: the ratio of bacterium chaff=3～4:1 (w:w) mixes the two, and making solid fermentation substratum moisture content is 50～65% (w/w);
(3) charging sterilization: will mix mixed culture material and pack in triangular flask or the solid-state fermentation tank, the control charge, making bed thickness is 0.2～3cm, bottleneck is filled in tampon or sealing fermentor tank opening for feed, 121 ℃ of sterilizations 30 minutes;
(4) inoculation culture: the Mucor pusillus spore suspension after 5% (v/v) dilution is inserted by ordinary method in cooling back in culture material, 25～35 ℃ with 70%～90% relative humidity environment under cultivation 72～96h;
(5) extract rennin: fermentation ends, culture material is taken out from triangular flask or solid-state fermentation tank, add 4 ℃ of water of 10～20 times of weight, 0～4 ℃ of environment held 4～12 hours; Remove by filter insolubles, centrifugal 2～10 minutes of 2000～4000rpm collects supernatant, obtains the rennin crude extract.
Wherein said bacterium chaff dries through thorough washing in advance then, and water cut is 7～9%.
Said Mucor pusillus spore suspension is that female the kind with sterilized water of Mucor pusillus disperseed, and uses 4 layers of filtered through gauze then, the spore suspension concentration dilution to 10 in will filtrating with sterilized water again
6～10
7Individual/mL.
Said preparation culture material: in the dehydration after wet bean dregs: the ratio of bacterium chaff=3:1 (w:w) mixes the two, and making solid fermentation substratum moisture content is 50% (w/w).
Mucor pusillus (Mucor pusillus) bacterial classification is buied by Chinese common micro-organisms culture presevation administrative center (being called for short CGMCC), and deposit number is CGMCC No.3.3445, is stored in Institute of Microorganism, Academia Sinica at present.
With wet bean dregs is single culture material when carrying out solid fermentation, because wet bean dregs water cut is big, agglomerating easily, is difficult to form the open structure that is similar to wheat bran, influence culture material heat radiation and gaseous interchange, so the vigor of the gained rennin that ferments is on the low side.For solving this difficult problem; The present invention adds in wet bean dregs culture material can increase culture material ventilation property exsiccant edible fungus bran; Both regulated substratum moisture; Increased the ventilation property of culture material, simultaneously the nutritive substance of bacterium chaff capable of using again ferments, its ferment effect near or surpass the fermentation of contrast wheat bran.It is simple that solid fermentation process has a processing units, and less investment is difficult for the advantage of bacteria infection, is adapted at promoting the use of in the medium-sized and small enterprises.
The solid fermentation step: will wet bean dregs and edible fungus bran mix with certain proportion, culture material are packed in triangular flask or the solid-state fermentation tank into the control charge; Making bed thickness is 0.2～3cm; Bottleneck is filled in tampon, or sealing fermentor tank opening for feed, sterilizes 30 minutes for 121 ℃.The Mucor pusillus bacterial classification is inserted in cooling back in culture material, at 25～35 ℃, 70%～90% relative humidity environment cultivation 72～96h down.After the fermentation ends, culture material is taken out from fermenting container, add the water of 10～20 times of weight, 0～10 ℃ of low temperature soaked 4～12 hours, obtained the enzyme crude extract after the filtration.Can also be through ultrafiltration, saltout, step such as lyophilize is refined into enzyme liquid concentrator or lyophilize powder with the rennin crude extract.
Remarkable advantage of the present invention:
Higher from the wet bean dregs water cut that bean product source mill comes out, even if also be difficult to reach 50% required moisture content of solid fermentation through mechanical expression dehydration moisture content, and easy conglomeration when big of bean dregs moisture content, influence the solid fermentation effect.If adopt through the dry bean dregs moisture that reduces, drying process not only consumes energy but also is apt to deteriorate smelly.In contrast, edible fungus bran is dry and not perishable easily, can pass through sun drying.The ingenious characteristics separately of having utilized bean dregs and bacterium chaff of the present invention; Drying in the sun edible fungus bran and wet bean dregs are mixed the moisture and the siccative ratio of regulating the solid fermentation substratum; So both can prevent because of the wet too high conglomeration of bean dregs moisture; Save the link of dry bean dregs, guaranteed that again fermentation gained enzyme activity does not reduce because of mixing of edible fungus bran.In a word, will wet bean dregs and dry mushroom bacterium chaff mix, and alternative wheat bran carries out the rennin solid fermentation, can turn waste into wealth, reduce the production cost of rennin, and be technological simple and easy to do, and timing-saving and economic, enzyme activity are higher.
Embodiment
(1) bean dregs mechanical dehydration: with water cut be 80～85% wet bean dregs through the mechanical expression dehydration, make its water cut reduce to 60%～70%;
(2) preparation culture material: by the wet bean dregs after dewatering: the ratio of bacterium chaff=3～4:1 (w:w) mixes the two, and making solid fermentation substratum moisture content is 50～65% (w/w); The bacterium chaff dries through thorough washing in advance then, and water cut is 7～9%;
(3) charging sterilization: will mix mixed culture material and pack in triangular flask or the solid-state fermentation tank, the control charge, making bed thickness is 0.2～3cm, bottleneck is filled in tampon or sealing fermentor tank opening for feed, 121 ℃ of sterilizations 30 minutes;
(4) inoculation culture: Mucor pusillus spore suspension after 5% (v/v) dilution is inserted by ordinary method in cooling back in culture material (said Mucor pusillus spore suspension is that female kind of Mucor pusillus disperseed with sterilized water; Use 4 layers of filtered through gauze then, the spore suspension concentration dilution to 10 in will filtrating with sterilized water again
6～10
7Individual/as mL), under 25～35 ℃ and 70%～90% relative humidity environment, to cultivate 72～96h;
(5) extract rennin: fermentation ends, culture material is taken out from triangular flask or solid-state fermentation tank, add 4 ℃ of water of 10～20 times of weight, 0～4 ℃ of environment held 4～12 hours; Remove by filter insolubles, centrifugal 2～10 minutes of 2000～4000rpm collects supernatant, obtains the rennin crude extract.
The rennin vigor adopts the Arima method usually, promptly uses 0.01mol/L CaC1
2Solution is prepared 10% skim-milk liquid, gets 5mL 10 % skim-milk liquid in 35 ℃ of insulation 10min, adds the suitably enzyme liquid of dilution (enzyme liquid in advance also should in 35 ℃ of insulations) of 5mL, shakes up immediately, and picking up counting, (the curdled milk time generally is controlled at 40s-90s.), and test tube inclination 45
oMore than, along the rotation of test tube direction of principal axis, beginning to occur the aggegation small-particle on the observation tube wall is terminal point, the record curdled milk time.Under these conditions, the 40min enzyme amount of coagulating l mL 10% skim-milk is defined as a Soxhlet unit (SU).
Enzyme activity (SU)=(supplying examination emulsion volume/rennin volume) * D * 2400/t
In the formula: D is an enzyme liquid extension rate; T is the reaction times.
Embodiment 1
The bean dregs (water cut is 80～85%) that will wet are through the mechanical expression dehydration; Its water cut is reduced to about 65%; Mixing and stirring edible fungus bran with weight ratio 3:1 ratio again (dries through thorough washing in advance then; Water cut 8%), making solid fermentation substratum moisture content is that 50% (w/w) and bean dregs and bacterium chaff butt weight ratio are about 1:1.The substratum that mixes and stirs is packed in triangular flask or the solid-state fermentation tank, and control charge size makes bed thickness be not more than 3cm.Bottleneck is filled in tampon, or sealing fermentor tank opening for feed, sterilizes 30 minutes for 121 ℃, and the Mucor pusillus spore suspension is inserted with 5% volume in the cooling back in culture material (concentration is 10
7Individual/as mL), under 35 ℃ and 70% relative humidity environment, to cultivate 72h.Fermentation ends is taken out culture material from fermenting container, add 4 ℃ of water of 10 times of weight, and 4 ℃ of environment held 12 hours remove by filter insolubles, and centrifugal 2 minutes of the 4000rpm that will filtrate collects supernatant, acquisition rennin crude extract.
Through detecting, the wet bean dregs solid fermentation enzyme work of water cut 50% is 2295 U/g, contrast wheat bran solid fermentation rennin productive rate 9081 U/g, and the vigor of bean dregs and bacterium chaff blended solid working up curd enzyme is 9412 U/g.
Embodiment 2
Bean dregs of the present invention and the mixed fermentation of bacterium chaff prepare the method for rennin, and it is characterized in that: said method specifically may further comprise the steps:
(1) bean dregs mechanical dehydration: with water cut be 80～85% wet bean dregs through the mechanical expression dehydration, make its water cut reduce to 60%～70%;
(2) preparation culture material: by the wet bean dregs after dewatering: the ratio of bacterium chaff=4:1 (w:w) mixes the two, and making solid fermentation substratum moisture content is 65% (w/w), and bean dregs and bacterium chaff butt weight ratio are about 1:0.8; The bacterium chaff dries through thorough washing in advance then, and water cut is 7%;
(3) charging sterilization: will mix mixed culture material and pack in the triangular flask, the control charge, making bed thickness is 0.2cm, bottleneck is filled in tampon, 121 ℃ of sterilizations 30 minutes;
(4) inoculation culture: Mucor pusillus spore suspension after 5% (v/v) dilution is inserted by ordinary method in cooling back in culture material (concentration is 10
6Individual/as mL), under 25 ℃ and 90% relative humidity environment, to cultivate 96h;
(5) extract rennin: fermentation ends, culture material is taken out from triangular flask or solid-state fermentation tank, add 4 ℃ of water of 20 times of weight, 0 ℃ of environment held 4 hours; Remove by filter insolubles, centrifugal 10 minutes of 2000rpm collects supernatant, obtains the rennin crude extract.
Through detecting, the enzyme activity of rennin crude extract is 548 U/ml.
Claims (3)
1. bean dregs and the mixed fermentation of bacterium chaff prepare the method for rennin, it is characterized in that: in wet bean dregs, add the bacterium chaff, inoculate the Mucor pusillus bacterial classification then and carry out solid fermentation, from tunning, extract rennin;
Said method specifically may further comprise the steps:
(1) bean dregs mechanical dehydration: with water cut be 80～85% wet bean dregs through the mechanical expression dehydration, make its water cut reduce to 60%～70%;
(2) preparation culture material: press the wet bean dregs after dewatering: the mass ratio of bacterium chaff=3～4:1 mixes the two, and the mass percent that makes solid fermentation substratum moisture content is 50～65%;
(3) charging sterilization: will mix mixed culture material and pack in triangular flask or the solid-state fermentation tank, the control charge, making bed thickness is 0.2～3cm, bottleneck is filled in tampon or sealing fermentor tank opening for feed, 121 ℃ of sterilizations 30 minutes;
(4) inoculation culture: the Mucor pusillus spore suspension after volume ratio 5% dilution is inserted by ordinary method in cooling back in culture material, cultivation 72～96h under 25～35 ℃ and 70%～90% relative humidity environment; Mucor pusillus spore suspension after the said dilution is that female the kind with sterilized water of Mucor pusillus disperseed, and uses 4 layers of filtered through gauze then, the spore suspension concentration dilution to 10 in will filtrating with sterilized water again
6～10
7Individual/mL;
(5) extract rennin: fermentation ends, culture material is taken out from triangular flask or solid-state fermentation tank, add 4 ℃ of water of 10～20 times of weight, 0～4 ℃ of environment held 4～12 hours; Remove by filter insolubles, centrifugal 2～10 minutes of 2000～4000rpm collects supernatant, obtains the rennin crude extract.
2. bean dregs according to claim 1 and the mixed fermentation of bacterium chaff prepare the method for rennin, it is characterized in that: said bacterium chaff dries through thorough washing in advance then, and water cut is 7～9%.
3. bean dregs according to claim 1 and the mixed fermentation of bacterium chaff prepare the method for rennin; It is characterized in that: said preparation culture material: press the wet bean dregs after dewatering: the mass ratio of bacterium chaff=3:1 mixes the two, and the mass percent that makes solid fermentation substratum moisture content is 50%.
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| CN109306346B (en) * | 2018-10-11 | 2021-09-07 | 山东隆科特酶制剂有限公司 | Method for producing chymosin with high yield and chymosin produced by same |
| CN109207376B (en) * | 2018-10-11 | 2021-09-07 | 山东隆科特酶制剂有限公司 | Mucor minutissima strain for producing chymosin with high yield and method for producing chymosin |
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| CN101514336A (en) * | 2009-03-30 | 2009-08-26 | 山东轻工业学院 | Method for producing renninum by solid-state fermentation of red yeast rice |
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| CN101514336A (en) * | 2009-03-30 | 2009-08-26 | 山东轻工业学院 | Method for producing renninum by solid-state fermentation of red yeast rice |
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| 侯立娟等.食用菌菌糠再利用研究概述.《中国食用菌》.2008,第27卷(第3期), * |
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