CN101514336B - Method for producing renninum by solid-state fermentation of red yeast rice - Google Patents
Method for producing renninum by solid-state fermentation of red yeast rice Download PDFInfo
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- CN101514336B CN101514336B CN2009100202360A CN200910020236A CN101514336B CN 101514336 B CN101514336 B CN 101514336B CN 2009100202360 A CN2009100202360 A CN 2009100202360A CN 200910020236 A CN200910020236 A CN 200910020236A CN 101514336 B CN101514336 B CN 101514336B
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Abstract
The invention relates to a method for producing renninum by the solid-state fermentation of red yeast rice. Red yeast rice powder is inoculated to a solid-state fermentation culture medium to be cultured for 90 to 96 hours under the constant temperature of 28-30 DEG C; renninum solution is obtained by culturing, water extracting and concentrating; ethanol is added for alcohol precipitation twice; crude renninum is obtained by freezing, subliming and drying; and the crude renninum is purified to obtain the renninum. The invention combines the traditional fermentation technology with the modern biological preparation technology to produce the renninum, the invention has wide raw material source, low price and high yield, and the produced renninum has high activity.
Description
[technical field]
The invention belongs to the biotechnological formulation preparing technical field, relate to the method for producing renninum by solid-state fermentation of red yeast rice.
[background technology]
Rennin is the manufacturing a kind of important enzyme preparation of cheese, and milk is solidified.Mainly be to extract in the calf stomach that never weans, its composition is a kind of asparagine pepsin.Along with the needs that cheese production increases year by year, the demand of rennin is also increasing, and its output value accounts for about 15% of whole world zymin.More than 5,000 ten thousand of the annual veals in the whole world, the quantity of calf sharply reduces.Cheese in China's output seldom, and developed country nearly 1/3～1/2 raw dairy be used for cheese production, kind is nearly hundreds of, both at home and abroad the production gap is very huge.Although cheese can not become the principal item of China's milk-product in a short time, but in the long term, particularly under the overall background of economic globalization, this product will become one of staple product of following domestic dairy industry, to the research broad market prospect of rennin.
The existing report of the effect of microbial rennet in cheese is produced.Compare with the standard rennin, all do not have significant difference at milk-content utilization ratio, proteolysis rate and aspects such as cheese yield rate and aesthetic quality.Report that using the recombinant chymosin produced to reach clearly aspect acidifying, proteolysis, cheese ripening, the ultimate constituent and cheese yield and the cheese aesthetic quality in curdled milk time, curdy texture, milk ejection compares no significant difference with calf chymosin from intestinal bacteria.This explanation utilizes the rennin of microbial technique production can substitute the crude rennet that extracts fully from the calf abomasum.
Red kojic rice different name: red colouring agent for food, also used as a Chinese medicine; Red song; Red rice; Good fortune rice.Character: for red-brown to the mauve grain of rice.The source: with rice such as long-grained nonglutinous rice, japonica rice, glutinous rice is raw material, forms with the monascus ruber fermentation, is the red-brown or the red-purple grain of rice.Red kojic rice is the unique traditional food of China, history in thousand has been arranged apart from the present.
The Red kojic rice fermented liquid is that rennin in the fermented liquid is playing a role as milk coagulant, rennin in the Red kojic rice fermented liquid is microbial metabolites in the Red kojic rice fermented liquid in fact, belong to the microbial source rennin, because Red kojic rice is the traditional foodstuff additive of China, so rennin need not the non-enzyme meta-bolites of considering that Institute of Micro-biology produces from the Red kojic rice fermented liquid.
[summary of the invention]
At the deficiencies in the prior art, the invention provides a kind of method of producing renninum by solid-state fermentation of red yeast rice.
Technical scheme of the present invention is as follows:
A kind of Red kojic rice solid state fermentation prepares the method for rennin, and step is as follows:
1) red kojic rice powder is broken into powdery, and fineness 200-400 order is inoculated into red kojic rice powder on the solid-state fermentation culture medium, 28-30 ℃ of constant temperature culture 90-96h.The mass ratio 1 of red kojic rice powder and solid-state fermentation culture medium: 100-1: 150
2) add distilled water in above-mentioned culture, the distilled water volume is 2-2.5 a times of culture volume, and suction filtration is got filtrate, and Rotary Evaporators is concentrated into original volume 1/3-1/4, gets rennin solns.
3) add ethanol to above-mentioned rennin solns and carry out alcohol precipitation twice, the ethanol volume that uses in the ethanol precipitation twice all is 2-3 times of rennin solns volume, 4-6 ℃ of low-temperature centrifugation, and drying bu sublimation is the thick enzyme of rennin.
4) with above-mentioned steps 3) the thick enzyme of rennin that obtains is dissolved in pH and in 6.2 the phosphoric acid buffer, is the thick liquid of rennin.
5) use the thick liquid of Sephadex G-75 gel filtration chromatography purifying rennin, as elutriant, substep is collected with the phosphoric acid buffer of pH6.2, and the activity of liquid is collected in check, is associated with active elutriant.
6) utilizing 52 pairs of above-mentioned elutriants of DEAE-Mierocrystalline cellulose to be further purified, is initial damping fluid with the phosphate buffered saline buffer of pH7.2, adopts the NaCl gradient elution, and substep is collected, and detection of active is associated with active eluant, concentrated freeze-driedly obtains highly purified rennin.
Above-mentioned steps 1) solid-state fermentation culture medium in is: (pressing the mass percent of dry-matter) wheat bran 40%-50% and dregs of beans 50%-60%, the glucose of adding wheat bran and dregs of beans total mass 0.5%-1% adds water and makes solid-to-liquid ratio 1: 1.5-1: 2 mass ratio.
Above-mentioned steps 3) alcohol concn that use in the alcohol precipitation first time in is 72%wt, and the alcohol concn that uses in the alcohol precipitation is 95%wt for the second time.
Above-mentioned steps 6) utilize the damping fluid of the preferred 0.15mol/L of NaCl gradient elution, 0.25mol/L, 0.35mol/L, 0.45mol/L and 0.55mol/L NaCl concentration to carry out gradient elution described in.
But the various raw materials that relate in the inventive method are all buied in market.The processing of fermentation residue is simple, through behind the High Temperature Sterilization, can be used as the superior being feed.
More than the gained rennin vigor 4300U/mg of the present invention.
The rennin vigour-testing method: utilize the Arima method, getting the 5mL mass concentration is the skimming milk solution of 100g/L, and 40 ℃ of insulation 5min are standby.The enzyme liquid of getting after 0.5mL handles adds the separated milk that is incubated, and mixes on the vortex mixer, and accurate recording is from adding enzyme liquid to the solid time of curdling.
Unit of enzyme is defined as: 40min is that the enzyme amount that the skimming milk of 100g/L solidifies is 1 unit (U) with the 1mL mass concentration.
Calculation formula is U=2400 * 5 * D/ (T * 0.5)
In the formula: T is the curdled milk time; D is an extension rate.
The present invention is a kind of method of producing rennin with Red kojic rice as microbial source, utilize the traditional foodstuff additive Red kojic rice of China, and adopt solid-state fermentation technology to carry out the production of rennin, adopt the secondary ethanol precipitation from fermented liquid, to extract rennin, further use Sephadex G-75 and DEAE-52 purifying, obtain highly purified rennin.
Beneficial effect of the present invention is as follows:
1. the present invention utilizes the traditional zymotic technology production rennin that combines with modern biologic product technology, the productive rate height, and the processing of fermentation residue is simple.
2. make a living yield wheat powder and the butyrous byproduct of beans--wheat bran and dregs of beans of solid-state fermentation culture medium raw material, wide material sources, cheap.
3. by guaranteeing to obtain highly purified rennin, more than the rennin vigor 4300U/mg to the optimum combination of extraction and purification process.
[embodiment]
Below in conjunction with embodiment method of the present invention is described further, but is not limited thereto.
The prosperous road of the Red kojic rice Accessories during Binzhou of using among embodiment commerce and trade company limited sells, and look valency 1000-3000u/g, moisture be less than 8%, fineness 160-200 order, and other indexs meet the requirement of standard GB 4926-1985.But wheat bran and dregs of beans market are buied.Sea cowry base bio tech ltd is on sale on the Sephadex G-75 gel column, the magnificent Sheng Ke in DEAE-Mierocrystalline cellulose 52 Beijing Bioisystech Co., Ltd is on sale.
Embodiment 1:
Red kojic rice grinds to the 200-300 order through mortar, is inoculated on the solid-state fermentation culture medium 30 ℃ of constant temperature culture 96h at 1: 100 by the mass ratio of red kojic rice powder and solid-state fermentation culture medium.Solid-state fermentation culture medium is composed as follows: 45 parts in (pressing the weight part of dry-matter) wheat bran, and 55 parts of dregs of beans, 0.6 part of glucose adds water to 1: 1.6 mass ratio of solid-to-liquid ratio.
Add 2 times of volume distilled water in above-mentioned culture, suction filtration is got filtrate, and Rotary Evaporators is concentrated into original volume 1/4, gets rennin solns.The concentration that slowly adds 3 times of volumes to rennin solns is 72% ethanol again, 4 ℃ leave standstill 2h after, with the centrifugal 15min of 7000r/min, get supernatant liquor, concentrate with Rotary Evaporators, 95% ethanol with 3 times of rennin solns volumes precipitates once more again, and 4 ℃ leave standstill 24h promptly with the centrifugal 15min of 8000r/min, lyophilize is the thick enzyme of rennin; Be dissolved in pH and be in 6.2 the phosphoric acid buffer, be the thick liquid of rennin.
The thick liquid of gained rennin at first uses Sephadex G-75 gel filtration chromatography purifying, and as elutriant, substep is collected with the phosphoric acid buffer of pH6.2, and the activity of liquid is collected in check, is associated with active elutriant.
Utilize 52 pairs of above-mentioned activated elutriants of DEAE-Mierocrystalline cellulose to be further purified, phosphate buffered saline buffer with pH7.2 is initial damping fluid, carry out gradient elution with the damping fluid that contains 0.15mol/L, 0.25mol/L, 0.35mol/L, 0.45mol/L and 0.55mol/L NaCl concentration, substep is collected, detection of active, be associated with active eluant, concentrated freeze-driedly obtain highly purified rennin.Freeze-drying obtains rennin behind the purifying, and the rennin vigor reaches 4462.1U/mg.
Embodiment 2:
Red kojic rice grinds to the 300-400 order through mortar, is inoculated on the solid-state fermentation culture medium 29 ℃ of constant temperature culture 94h at 1: 140 by the mass ratio of red kojic rice powder and solid-state fermentation culture medium.Solid-state fermentation culture medium is composed as follows: 50 parts in (pressing the weight part of dry-matter) wheat bran, and 50 parts of dregs of beans, 0.7 part of glucose adds water to 1: 1.5 mass ratio of solid-to-liquid ratio.
Add 2 times of volume distilled water in culture, suction filtration is got filtrate, and Rotary Evaporators is concentrated into original volume 1/3.
Be 72% ethanol again to the concentration that wherein slowly adds 3 times of volumes, 5 ℃ leave standstill 2h after, with the centrifugal 10min of 7000r/min, get supernatant liquor, concentrate with Rotary Evaporators, the ethanol with 3 times of volumes 95% precipitates once more again, and 5 ℃ leave standstill 20h promptly with the centrifugal 10min of 7500r/min, lyophilize is the thick enzyme of rennin; Be dissolved in pH and be in 6.2 the phosphoric acid buffer, be the thick liquid of rennin.At first utilize to use the thick liquid of Sephadex G-75 gel filtration chromatography purifying rennin, as elutriant, substep is collected with the phosphoric acid buffer of pH6.2, and the activity of liquid is collected in check, is associated with active elutriant.Utilize DEAE-Mierocrystalline cellulose 52 to be further purified, phosphate buffered saline buffer with pH7.2 is initial damping fluid, carry out gradient elution with the damping fluid that contains 0.15mol/L, 0.25mol/L, 0.35mol/L, 0.45mol/L and 0.55mol/L NaCl concentration, distribute and collect, detection of active, be associated with active eluant, concentrated freeze-driedly obtain highly purified rennin.Freeze-drying obtains rennin behind the purifying, and the rennin vigor reaches 4326.7U/mg.
Claims (1)
1. a Red kojic rice solid state fermentation prepares the method for rennin, and step is as follows:
1) red kojic rice powder is broken into powdery, and fineness 200-400 order is inoculated into red kojic rice powder on the solid-state fermentation culture medium, 28-30 ℃ of constant temperature culture 90-96h;
Described solid-state fermentation culture medium is: press the mass percent wheat bran 40%-50% and the dregs of beans 50%-60% of dry-matter, add the glucose of wheat bran and dregs of beans total mass 0.5%-1%, add water and make solid-to-liquid ratio 1: 1.5-1: 2 mass ratioes;
2) add distilled water in the culture that above-mentioned step 1) obtains, the distilled water volume is 2-2.5 a times of culture volume, and suction filtration is got filtrate, and Rotary Evaporators is concentrated into original volume 1/3-1/4, gets rennin solns;
3) to above-mentioned steps 2) rennin solns that obtains adds ethanol and carries out alcohol precipitation twice, the ethanol volume that uses in the ethanol precipitation twice all be the rennin solns volume 2-3 doubly, 4-6 ℃ of low-temperature centrifugation, drying bu sublimation is the thick enzyme of rennin;
The alcohol concn that uses in the alcohol precipitation is 72%wt for the first time, and the alcohol concn that uses in the alcohol precipitation is 95%wt for the second time;
4) with above-mentioned steps 3) the thick enzyme of rennin that obtains is dissolved in pH and in 6.2 the phosphoric acid buffer, is the thick liquid of rennin;
5) use Sephadex G-75 gel filtration chromatography purification step 4) the thick liquid of rennin that obtains, as elutriant, substep is collected with the phosphoric acid buffer of pH6.2, and the activity of liquid is collected in check, is associated with active elutriant;
6) utilizing 52 pairs of above-mentioned steps 5 of DEAE-Mierocrystalline cellulose) elutriant that obtains is further purified, and is initial damping fluid with the phosphate buffered saline buffer of pH7.2, adopts the NaCl gradient elution, and substep is collected, and detection of active is associated with active eluant, concentrated freeze-dried rennin;
Described employing NaCl gradient elution is that the damping fluid with 0.15mol/L, 0.25mol/L, 0.35mol/L, 0.45mol/L and 0.55mol/LNaCl concentration carries out gradient elution.
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| CN101870967B (en) * | 2010-07-22 | 2012-05-23 | 安泰生物工程股份有限公司 | Method for producing microbial rennet by semicontinuous fermentation |
| CN101974503B (en) * | 2010-11-04 | 2012-04-11 | 福建省农业科学院农业工程技术研究所 | Method for preparing chymosin by mixed culture fermentation of bean dregs and waste material from fungal culture (WMFC) |
| CN103355421B (en) * | 2013-07-29 | 2015-03-11 | 上海理工大学 | Preparation method of soft white mould cheese |
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