CN104531646A - Preparation method for producing chymosin through distiller's yeast fermentation - Google Patents

Preparation method for producing chymosin through distiller's yeast fermentation Download PDF

Info

Publication number
CN104531646A
CN104531646A CN201410799638.6A CN201410799638A CN104531646A CN 104531646 A CN104531646 A CN 104531646A CN 201410799638 A CN201410799638 A CN 201410799638A CN 104531646 A CN104531646 A CN 104531646A
Authority
CN
China
Prior art keywords
distiller
rennin
glutinous rice
yeast
fermentation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201410799638.6A
Other languages
Chinese (zh)
Other versions
CN104531646B (en
Inventor
杨贞耐
赵笑
王辑
郑喆
赵爱梅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Technology and Business University
Original Assignee
Beijing Technology and Business University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Technology and Business University filed Critical Beijing Technology and Business University
Priority to CN201410799638.6A priority Critical patent/CN104531646B/en
Publication of CN104531646A publication Critical patent/CN104531646A/en
Application granted granted Critical
Publication of CN104531646B publication Critical patent/CN104531646B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23004—Chymosin (3.4.23.4), i.e. rennin

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a preparation method for producing chymosin through distiller's yeast fermentation, and discloses a method for preparing chymosin. The method comprises the following steps: uniformly mixing distiller's yeast, sticky rice and water, fermenting, collecting a fermentation product to obtain the chymosin, wherein the chymosin activity of the distiller's yeast is 4000Su/g, the protein hydrolytic activity of the distiller's yeast is about 600U/g. According to the experiments, the invention discloses chymosin with relatively low tolerance to a high temperature, wherein the chymosin is from fermented glutinous rice food, has relatively high C/P and absolute safety, relatively high curd activity at 35 DEG C, and curd activity loss is obvious when the temperature rises to higher than 45 DEG C. Therefore, the problems of bitter cheese flavor and the like caused by insufficient blanching enzyme deactivation during preparation of cheese can be avoided, and the practical application value of the preparation method in cheese production is increased.

Description

A kind of preparation method of distiller's yeast fermentation producing lab ferment
Technical field
The present invention relates to technical field of bioengineering, particularly relate to the preparation method of a kind of distiller's yeast fermentation producing lab ferment.
Background technology
Glutinous rice wine is a kind of traditional food of south China, has curdled milk effect, and its curdled milk effect is caused by proteolytic enzyme contained in glutinous rice wine, belongs to enzymatic coagulation.Rennin in glutinous rice wine is the meta-bolites of microorganism in starter distiller's yeast in glutinous rice wine fermenting process, belongs to microbial rennet.
Rennin is the key enzyme in cheese manufacturing process, it is the important enzyme preparation in field of food, its Main Function in curdled milk is the peptide bond in narrow spectrum hydrolysis κ-casein polypeptide chain between Phe105-Met106, thus makes, between casein particle, coagulation occurs.The milk-clotting activity of rennin is subject to the temperature of reaction conditions as enzyme concn, milk and the impact of pH value and calcium ion concn etc.The main source of rennin has 3 kinds, is respectively animal-origin, plant origin and microbe-derived.The rennin of microbial source by the restriction of region, time etc., and has that cost is low, output advantages of higher, is subject to increasing attention.
Summary of the invention
An object of the present invention is to provide a kind of method preparing rennin.
Method provided by the invention, comprise the steps: distiller's yeast, glutinous rice matrix and water to mix, fermentation, collects tunning, namely obtains rennin;
The condenser water level of described distiller's yeast is 4.90 ± 0.6Su/mL;
The condenser water level of described distiller's yeast is the rennet curdling vigor in its glutinous rice wine tunning.
Described glutinous rice matrix is prepared as follows: by glutinous rice and water mixing, heating, obtains glutinous rice matrix.
In aforesaid method,
The proteolysis vigor of described distiller's yeast is 3.05 ± 0.81Su/mL;
The mass ratio of described distiller's yeast and described glutinous rice matrix is 3-6:100, and the mass ratio of described distiller's yeast and described glutinous rice matrix is specially 5.7:100.
In aforesaid method, described fermentation time is 5-7 days, and described fermentation time is specially 72h.
In aforesaid method, described fermentation adopts the mode of shaking table concussion, and the rotating speed of described shaking table is 80-180rpm, and the rotating speed of described shaking table is specially 120rpm, and its rotation radius is 13mm.
In aforesaid method, the temperature 20-35 DEG C of described fermentation, the temperature of described fermentation is specially 30 DEG C;
The system initial pH value of described fermentation is 4.5-6.5, and the system initial pH value of described fermentation is 5.0.
Described water and described glutinous rice matrix mass ratio are 50:100;
Described glutinous rice matrix by described glutinous rice and water according to glutinous rice described in 500g: the proportioning of 1L water is made.
In aforesaid method, after described collection tunning, also comprise the steps:
1) filter described tunning, collect filtrate, obtain containing rennin filtrate;
2) obtaining mixed system A to described containing adding ethanol in rennin filtrate, making the volumn concentration of ethanol in described mixed system A be 50%, regathering supernatant liquor;
3) in described supernatant liquor, add ethanol and obtain mixed system B, make the volumn concentration of ethanol in described mixed system B be 60%, collected by centrifugation supernatant liquor;
4) to 3) process and add ethanol in the supernatant liquor that obtains and obtain mixed system C, make the volumn concentration of ethanol in described mixed system C be 70%, centrifugal collecting precipitation.
In aforesaid method, collection described precipitation after, also comprise the steps: by after described resolution of precipitate through gel filtration chromatography, collect elutriant, obtain rennin.
Solvent needed for described dissolving is the PBS solution (formula: NaCl 35g, KCl 1g, KOH1.2g, K of 0.05mol/L pH5.8 2hPO 49g, adds distilled water and is settled to 1L, with second acid for adjusting pH to 5.8).
In aforesaid method, the gel column medium of described gel filtration chromatography is Sephadex G75;
The elution buffer that described gel filtration chromatography adopts is concentration is 0.05mol/L, pH value is the PBS solution of 5.8;
Described collection elutriant is collection retention time is the elutriant of 100min-130min.
The rennin prepared by aforesaid method is also the scope of protection of the invention;
Or the milk-coagulating enzyme preparation to be prepared by described rennin.
Above-mentioned rennin has following 1)-3) middle at least one zymetology feature:
1) optimal reactive temperature of described rennin is 35 DEG C,
2) the optimal reaction pH value 5.4 of described rennin;
3) activity of described rennin can by Ca 2+promote.
The present invention tests proof, the present invention has extracted a kind of rennin high temperature to lower tolerance, this rennin is from glutinous rice wine food, there is higher C/P and absolute security, at 35 DEG C, there is higher condenser water level, and temperature rising (>45 DEG C) condenser water level is obviously lost simultaneously.When this characteristic can avoid cheese making, blanching is gone out the problems such as the insufficient cheesy flavour caused of enzyme is pained, thus improves its actual application value in cheesemaking.
Accompanying drawing explanation
Fig. 1 is the impact of different outbox condition on distiller's yeast fermentation producing lab ferment
Fig. 2 is that the interpolation of different carbon source nitrogenous source is on the impact of distiller's yeast fermentation producing lab ferment
Fig. 3 be the distiller's yeast fermentation response surface figure of producing lab ferment and height isogram(s)
Fig. 4 is ethanol precipitation SDS-PAGE electrophorogram
Fig. 5 is the target protein SDS-PAGE electrophorogram after the column chromatography purification result of rennin and purifying
Fig. 6 is rennin zymetology properties study
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The preparation method of embodiment 1, distiller's yeast fermentation producing lab ferment
One, the screening of distiller's yeast
Distiller's yeast A-H purchased from different areas is fermented respectively under 35 DEG C of conditions and within 3 days, is made into the glutinous rice wine tunning of distiller's yeast A-H, measure condenser water level (MCA) and the proteolysis vigor (PA) of each glutinous rice wine tunning respectively.Measuring method is as follows respectively:
Condenser water level measuring method: the method adopting Arima (1970): the skimming milk (lipid content is about 1.0%) getting 5mL100g/L, at 35 DEG C of insulation 5min, add the glutinous rice wine tunning of 0.5mL distiller's yeast, mix rapidly, accurate recording is from adding liquid to be measured to curdling solid time.The enzyme amount needed for skimming milk 1mL of solidifying 100g/L with 40min is defined as a Soxhlet unit (Soxhelt unit, Su).
SU＝2400/T×5/0.5×D4
In formula: T is curdled milk time (s); D is extension rate.
Proteolysis vigour-testing method: measure according to the Folin phenol reagent process adopted in the documents such as Zhang Fuxin, specific as follows: 1ml enzyme liquid, under certain temperature and pH value condition, 1min caseinhydrolysate produces lug tyrosine and is defined as 1 unit of enzyme activity (U/m1);
The preparation of casein solution: take casein 1.0009 (being accurate to 0.0019), after moistening with a small amount of dense lactic acid, add the lactic acid buffer 80ml of pH3.0, heat while stirring in boiling water bath, until dissolve completely, be diluted to 100ml by lactic acid buffer after cooling, be the casein solution of 10g/L.
The preparation of standardized solution: take tyrosine 0.10009 (being accurate to 0.00029), after dissolving, is settled to 100ml, is 1mg/ml tyrosine standardized solution with lmol/L hydrochloric acid 60ml.Draw 1mg/m1 tyrosine standardized solution 10.00ml, then be settled to 100ml with 0.lmol/L hydrochloric acid, obtain 100ug/ml tyrosine standardized solution.
The formulation of typical curve: accurately draw 100ug/m1 tyrosine standardized solution 0,1,2,3,4,5ml, move in 6 25ml test tubes, add pure distilled water 10,9,8,7,6,5ml respectively, be the tyrosine reference liquid of 0,20,20,30,40,50ug/ml.Get this reference liquid 1.00ml respectively, respectively add 0.4mol/L sodium carbonate solution 5.00ml, Folin reagent uses liquid 1.00ml, be placed in 40 scholar, 0.2 DEG C of water-bath to develop the color 20min, take out with 721 spectrophotometers in wavelength 680nm, 1cm cuvette, not contain 0 pipe of tyrosine for blank, measures its absorbancy respectively.Its regression equation is y=0.010x+5E-0.5 (r=0.999), the amount K==99.5ug of tyrosine when absorbancy is 1.
Get 1.00ml enzyme liquid in test tube, 2min is incubated at 40 scholar 0.2 DEG C, add 10g/L casein 1.0ml, after shaking up, be incubated 10min at the same temperature, then add the trichoroacetic acid(TCA) 2ml of 0.4mol/L, shake up stopped reaction, take out and leave standstill 10min, filter, measure by typical curve operation steps.
Proteolytic activity (U/ml)=A*K*4/10*n
Wherein: A-sample absorbance
K-extinction constant
4-reaction reagent, cumulative volume
10-reaction times 10min
N-extension rate
Experimental result is as shown in table 1:
Table 1 is the comparison of MCA, PA, C/P of different sources distiller's yeast fermentation glutinous rice wine
Mark: a-e: in colleague, same letter represents that difference is not remarkable, and different letter representation significant difference, P < 0.5, shows significant difference.
Selection condenser water level is high, the relatively low distiller's yeast C of proteolysis vigor makes further research, desk study result shows, distiller's yeast C is (purchased from Suzhou oil and foodstuffs company limited, title: honeybee board Suzhou honeybee koji) condenser water level be 4.90 ± 0.62Su/mL, proteolysis vigor is 3.05 ± 0.81Su/mL.
Two, distiller's yeast fermentation is for rennin
1, optimization culture conditions
The present invention have studied the different carbon source (glucose of distiller's yeast addition (1%-6%), fermentation number of days (3-9 days), leavening temperature (20-35 DEG C), the initial pH value (4.5-6.5) of fermenting, moisture addition (40%-70%), shaking speed (80-160rpm), interpolation 3%, fructose, maltose, sucrose, lactose) and nitrogenous source (ammonium citrate, ammonium sulfate, Secondary ammonium phosphate, Tryptones, milk powder) on fermentation glutinous rice wine producing lab ferment vigor and the impact of proteolysis vigor, concrete grammar is as follows:
1), distiller's yeast addition is groped
The preparation of glutinous rice matrix: fresh glutinous rice 500g cleans and soaks 3h, adds 1L distilled water, and normal pressure steams 0.5h, cooling, and obtaining glutinous rice matrix is 1000g;
The distiller's yeast C obtained above-mentioned one and water add the mixing of glutinous rice matrix to, fermentation;
Distiller's yeast C respectively according to mass percentage be 1%, 2%, 3%, 4%, 5% and 6% (distiller's yeast quality and glutinous rice matrix mass percent) add;
Water adds according to mass percentage 50% (quality and glutinous rice matrix mass percent);
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 3 days, the initial pH value 5 of fermentation;
Collect the tunning of different distiller's yeast addition, detect condenser water level MCA according to method in above-mentioned.
Result as shown in Figure 1A, can be found out, when distiller's yeast addition reaches after 3%, condenser water level is basicly stable.
2), fermentation time is groped
The distiller's yeast C obtained above-mentioned one and water add the mixing of glutinous rice matrix to, fermentation;
Distiller's yeast C is that 3% (distiller's yeast quality and glutinous rice matrix mass percent) adds according to mass percentage;
Water adds according to mass percentage 50% (quality and glutinous rice matrix mass percent);
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 9 days, the initial pH value 5 of fermentation;
Collect tunning in the different fermentations time, detect condenser water level MCA according to method in above-mentioned.
Result as shown in Figure 1B, can be found out, fermentation time is when 5-7 days, and condenser water level is higher, reaches the highest at the 5th day.
3), the condition of leavening temperature is groped
The distiller's yeast C obtained above-mentioned one and water add the mixing of glutinous rice matrix to, fermentation;
Distiller's yeast C is that 3% (distiller's yeast quality and glutinous rice matrix mass percent) adds according to mass percentage;
Water adds according to mass percentage 50% (quality and glutinous rice matrix mass percent);
Above-mentioned fermentation condition is 20,25,30 and 35 DEG C, shaking speed 120rpm ferments 5 days, the initial pH value 5 of fermentation;
Collect the tunning at different fermentations temperature, detect condenser water level MCA according to method in above-mentioned.
Result as shown in Figure 1 C, can be found out, leavening temperature is at 30 DEG C, and condenser water level reaches maximum.
4), initial pH value is groped
The distiller's yeast C obtained above-mentioned one and water add the mixing of glutinous rice matrix to, fermentation;
Distiller's yeast C is that 3% (distiller's yeast quality and glutinous rice matrix mass percent) adds according to mass percentage;
Water adds according to mass percentage 50% (quality and glutinous rice matrix mass percent);
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm, and its rotation radius is 13mm, ferments 5 days, the initial pH value 4.5-6.5 of fermentation;
Collect the tunning that different fermentations initial pH value obtains, detect condenser water level MCA according to method in above-mentioned.
Result as shown in figure ip, can be found out, the initial pH value 5 of fermentation, condenser water level reaches maximum.
5), moisture addition
The distiller's yeast C obtained above-mentioned one and water add the mixing of glutinous rice matrix to, fermentation;
Distiller's yeast C is that 3% (distiller's yeast quality and glutinous rice matrix mass percent) adds according to mass percentage;
Water adds according to mass percentage 40%, 50%, 60%, 70% (quality and glutinous rice matrix mass percent);
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 5 days, the initial pH value 5 of fermentation;
Collect the tunning that different in moisture addition obtains, detect condenser water level MCA according to method in above-mentioned.
Result as referring to figure 1e, can be found out, when moisture addition is 40%, condenser water level reaches maximum.
6), fermentation shaking speed
The distiller's yeast C obtained above-mentioned one and water add the mixing of glutinous rice matrix to, fermentation;
Distiller's yeast C is that 3% (distiller's yeast quality and glutinous rice matrix mass percent) adds according to mass percentage;
Water adds according to mass percentage 40% (quality and glutinous rice matrix mass percent);
Above-mentioned fermentation condition is 30 DEG C, shaking speed 80-180rpm ferments 5 days, the initial pH value 5 of fermentation;
Collect the tunning that different shaking speed obtains, detect condenser water level MCA according to method in above-mentioned.
As shown in fig. 1f, can find out, shaking speed 120rpm, condenser water level reaches maximum to result.
7) different carbon source is groped
The distiller's yeast C obtained above-mentioned one, water and carbon source (or nitrogenous source) add the mixing of glutinous rice matrix to, fermentation;
Distiller's yeast C is that 3% (distiller's yeast quality and glutinous rice matrix mass percent) adds according to mass percentage;
Water adds according to mass percentage 40% (quality and glutinous rice matrix mass percent);
Carbon source (or nitrogenous source) is added according to mass percentage 3% (carbon source (or nitrogenous source) quality and glutinous rice matrix mass percent);
Carbon source is respectively glucose, fructose, maltose, sucrose, lactose;
Nitrogenous source is respectively ammonium citrate: ammonium sulfate: Secondary ammonium phosphate, Tryptones, milk powder;
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 5 days, the initial pH value 5 of fermentation;
Collect the tunning that different carbon source or nitrogenous source obtain, detect condenser water level MCA according to method in above-mentioned.
Make comparisons with the blank of not adding carbon source or nitrogenous source.
Result as shown in Figure 2,1: blank 2: glucose 3: fructose 4: maltose 5: sucrose 6: lactose 7: ammonium citrate 8: ammonium sulfate 9: Secondary ammonium phosphate 10: Tryptones 11: milk powder, can find out, add carbon source especially glucose, have certain promoter action to distiller's yeast fermentation producing lab ferment; The condenser water level of interpolation to distiller's yeast fermentation producing lab ferment of nitrogenous source does not play promoter action, and some nitrogenous source such as ammonium sulfate etc. even can reduce the milk-clotting activity that distiller's yeast produces enzyme.
The above results shows, Different factor is not very large on the impact of proteolysis vigor, and the obvious effect to condenser water level, along with the change of each factor, proteolysis vigor is all lower.The prolongation of the increase of distiller's yeast addition and fermentation time, condenser water level raises.
Analyze above monofactorial impact, wherein distiller's yeast inoculum size, fermentation time and shaking table speed 3 factors are the most remarkable on the impact of condenser water level, therefore choose these 3 factors and carry out response surface analysis, as Fig. 3, wherein, (A) surface chart of affecting condenser water level for distiller's yeast inoculum size and fermentation time and height are isogram(s); (B) surface chart affected condenser water level for distiller's yeast inoculum size and shaking table speed and height are isogram(s); (C) surface chart affected condenser water level for fermentation time and shaking table speed and height are isogram(s) (Fig. 3).
Response surface analysis obtains taking condenser water level as the regression equation of response value: condenser water level (Su/mL)=-259.67706+32.44125*A+3.82846*B+1.36470*C+0.019479*A*B+0. 013781*A*C-1.92708E-003*B*C-3.17794*A2-0.025667*B2-5.335 47E-003*C2.In formula, A-distiller's yeast inoculum size, %; B-fermentation time, h; C-shaking table speed, rpm.
Obtaining optimal conditions of fermentation is distiller's yeast inoculum size 5.7% (mass percentage), fermentation time 72h shaking speed 120rpm, and the rennet curdling vigor of theoretical expectation values tunning is 52.29Su/mL;
Therefore, distiller's yeast inoculum size, fermentation time and shaking speed are most important optimizing factors.
2, top condition fermentation
The distiller's yeast C obtained above-mentioned one and water add the mixing of glutinous rice matrix to, fermentation;
Distiller's yeast C is that 5.7% (distiller's yeast quality and glutinous rice matrix mass percent) adds according to mass percentage;
Water adds according to mass percentage 40% (quality and glutinous rice matrix mass percent);
The initial pH value 5 that above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 72h, fermentation;
Collect tunning, detect condenser water level MCA according to method in above-mentioned, the rennet curdling vigor in result tunning is 51.05Su/mL, is close with theoretical expectation values (52.29Su/mL).
Note: be variable with fermentation time during single factor analysis, do under the condition that other factors is constant, now distiller's yeast addition fixed bit is 3%, fermentation time is the highest when being 5 days, simultaneously response surface analysis analyzes influencing each other between 3 variablees, when distiller's yeast addition reaches 5.7%, fermentation time only needs within 3 days, to reach the highest.
The preparation of embodiment 2, rennin
1, ferment
The preparation of glutinous rice matrix: fresh glutinous rice 500g cleans and soaks 3h, adds 1L distilled water, and normal pressure steams 0.5h, cooling, and obtaining glutinous rice matrix is 1000g;
The distiller's yeast C obtained above-mentioned one and water add the mixing of glutinous rice matrix to, fermentation;
Distiller's yeast C is that 5.7% (distiller's yeast quality and glutinous rice matrix mass percent) adds according to mass percentage;
Water adds according to mass percentage 40% (quality and glutinous rice matrix mass percent);
The initial pH value 5 that above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 72h, fermentation;
Collect tunning, filter (4 layers of gauze), collect filtrate, be containing rennin filtrate, for subsequent use in 4 DEG C of refrigerator cold-storages.
2, ethanol precipitation slightly carries rennin
The dehydrated alcohol containing rennin filtrate and laboratory storage of above-mentioned 1 preparation being placed in 4 DEG C is taken out, operate as follows: (1) adds dehydrated alcohol to above-mentioned 1 preparation containing in rennin filtrate, mixed volume fraction of ethanol is made to be 50%, 4 DEG C, centrifugal 30 minutes of 5000r/min, cleer and peaceful precipitation in collection, albumen precipitation distilled water dissolves, and is placed in 4 DEG C of refrigerators for subsequent use; (2) supernatant obtained in (1) being continued add dehydrated alcohol makes volume fraction of ethanol be 60%, 4 DEG C, centrifugal 30 minutes of 5000r/min, cleer and peaceful precipitation in collection, and albumen precipitation dissolves, and is placed in 4 DEG C of refrigerators for subsequent use; (3) supernatant obtained in (2) being continued add dehydrated alcohol makes volume fraction of ethanol be 70%, 4 DEG C, and centrifugal 30 minutes of 5000r/min, dissolves albumen precipitation, be placed in 4 DEG C of refrigerators for subsequent use; (4) supernatant obtained in (3) being continued add dehydrated alcohol makes volume fraction of ethanol be 80%, 4 DEG C, and centrifugal 30 minutes of 5000r/min, dissolves albumen precipitation, be placed in 4 DEG C of refrigerators for subsequent use;
Measure the condenser water level of albumen precipitation in above-mentioned 4 steps respectively.
Result as shown in Figure 4,1: protein standard substance; 2:50% alcohol settling albumen; 3:60% alcohol settling albumen; 4:70% alcohol settling albumen; 5:80% alcohol settling albumen, only shows that in steps the albumen that in (3), namely 70% alcohol settling obtains has obvious condenser water level.
Respectively SDS-PAGE electrophoretic analysis is carried out to the albumen of 50%, 60%, 70%, 80% alcohol settling, result as shown in Figure 4,80% protein precipitation is more small molecular protein, there is a protein band different from other albumen precipitations in the albumen of 70% alcohol settling, molecular weight, between 34-43kD, is probably target protein.
Above-mentioned experiment proves, its vacuum lyophilization, for slightly to carry rennin, becomes slightly to carry rennin dry powder by the albumen of 70% alcohol settling.
3, Sephadex G75 purification of crude carries rennin
What obtain above 2 slightly carries the solution that rennin dry powder is configured to 0.2g/mL, and solvent is the PBS solution (formula: NaCl 35g, KCl 1g, KOH 1.2g, K of 0.05mol/L pH5.8 2hPO 49g, add distilled water and be settled to 1L, with second acid for adjusting pH to 5.8), through Sephadex G75 pillar (2.6 × 60cm), with the PBS solution of 0.05mol/LpH5.8 for elution buffer carries out wash-out, applied sample amount is 4mL, and flow velocity is 1mL/min, measure the condenser water level of each protein peak, collect the peak with condenser water level.
Result as shown in Figure 5A, can be found out, is 100 ~ 130min in retention time, obtains object peak.
Collect the eluted products that object peak is corresponding, ultra-filtration centrifuge tube desalination and concentration postlyophilization through 10kD becomes powder, carry out SDS-PAGE electrophoretic analysis, result as shown in Figure 5 B, 1 is albumen for the purpose of protein standard substance, 2, shows, the albumen obtained is single band, calculate molecular weight be about 39.7kD, for the purpose of rennin.
Embodiment 2, rennin enzymatic property are studied
Detect below all with the object rennin that embodiment 1 purifying obtains.
1, optimum temperuture
At 25,30,35,40,45,50,55 DEG C, measure the object rennet curdling vigor that embodiment 1 purifying obtains respectively, method, with one of embodiment 1, with condenser water level when 35 DEG C for 100%, calculates relative condenser water level.
Result as shown in Figure 6A, shows that this rennin has good condenser water level at 35 DEG C, and temperature is lost substantially higher than 45 DEG C of later condenser water level.
2, optimum pH
The pH of skimming milk is adjusted with NaOH or HCl of 0.1mol/L, the object rennet curdling vigor that embodiment 1 purifying obtains is measured respectively when pH is 5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0, method is with one of embodiment 1, with condenser water level during pH5.8 for 100%, calculate relative condenser water level.
Result as shown in Figure 6B, shows that condenser water level raises along with the reduction of pH, and when pH5.4, condenser water level reaches the highest.
3, metal ion affects enzymic activity
In skimming milk, add NaCl, KCl, MgCl respectively 2, CaCl 2, FeCl 2, AlCl 3, CuCl 2, ZnCl 2, make its concentration be 10mmol/L, add the object rennet curdling vigor measuring embodiment 1 purifying and obtain, method, with one of embodiment 1, using the condenser water level of blank (BC) as 100%, calculates relative condenser water level.
As shown in Figure 6 C, result shows Na to result +, K +, Mg 2+, Ca 2+and Al 3+promoter action is had, especially Ca to this rennet curdling 2+effect the most obvious.
The rennin vacuum lyophilization of purifying is made zymin.

Claims (10)

1. prepare a method for rennin, comprise the steps: distiller's yeast, glutinous rice matrix and water to mix, fermentation, collects tunning, namely obtains rennin;
The condenser water level of described distiller's yeast is 4.90 ± 0.6Su/mL;
Described glutinous rice matrix is prepared as follows: by glutinous rice and water mixing, heating, obtains glutinous rice matrix.
2. method according to claim 1, is characterized in that:
The mass ratio of described distiller's yeast and described glutinous rice matrix is 3-6:100, and the mass ratio of described distiller's yeast and described glutinous rice matrix is specially 5.7:100.
3. method according to claim 1 and 2, is characterized in that: described fermentation time is 5-7 days, and described fermentation time is specially 72h.
4., according to described method arbitrary in claim 1-3, it is characterized in that: described fermentation adopts the mode of shaking table concussion, and the rotating speed of described shaking table is 80-180rpm, and the rotating speed of described shaking table is specially 120rpm.
5., according to described method arbitrary in claim 1-4, it is characterized in that:
The temperature 20-35 DEG C of described fermentation, the temperature of described fermentation is specially 30 DEG C;
The system initial pH value of described fermentation is 4.5-6.5, and the system initial pH value of described fermentation is 5.0.
Described water and described glutinous rice matrix mass ratio are 50:100;
Described glutinous rice matrix by described glutinous rice and water according to glutinous rice described in 500g: the proportioning of 1L water is made.
6., according to described method arbitrary in claim 1-5, it is characterized in that:
After described collection tunning, also comprise the steps:
1) filter described tunning, collect filtrate, obtain containing rennin filtrate;
2) obtaining mixed system A to described containing adding ethanol in rennin filtrate, making the volumn concentration of ethanol in described mixed system A be 50%, regathering supernatant liquor;
3) in described supernatant liquor, add ethanol and obtain mixed system B, make the volumn concentration of ethanol in described mixed system B be 60%, collected by centrifugation supernatant liquor;
4) to 3) process and add ethanol in the supernatant liquor that obtains and obtain mixed system C, make the volumn concentration of ethanol in described mixed system C be 70%, centrifugal collecting precipitation.
7. method according to claim 6, is characterized in that:
Collection described precipitation after, also comprise the steps: by after described resolution of precipitate through gel filtration chromatography, collect elutriant, obtain rennin.
8. method according to claim 7, is characterized in that:
The gel column medium of described gel filtration chromatography is Sephadex G75;
The elution buffer that described gel filtration chromatography adopts is concentration is 0.05mol/L, pH value is the PBS solution of 5.8.
9. the rennin prepared by described method arbitrary in claim 1-8;
Or the milk-coagulating enzyme preparation to be prepared by described rennin.
10. rennin according to claim 9, is characterized in that:
Described rennin has following 1)-3) middle at least one zymetology feature:
1) optimal reactive temperature of described rennin is 35 DEG C,
2) the optimal reaction pH value 5.4 of described rennin;
3) activity of described rennin can by Ca 2+promote.
CN201410799638.6A 2014-12-19 2014-12-19 A kind of preparation method of distiller's yeast fermentation producing lab ferment Active CN104531646B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410799638.6A CN104531646B (en) 2014-12-19 2014-12-19 A kind of preparation method of distiller's yeast fermentation producing lab ferment

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410799638.6A CN104531646B (en) 2014-12-19 2014-12-19 A kind of preparation method of distiller's yeast fermentation producing lab ferment

Publications (2)

Publication Number Publication Date
CN104531646A true CN104531646A (en) 2015-04-22
CN104531646B CN104531646B (en) 2017-12-19

Family

ID=52847280

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410799638.6A Active CN104531646B (en) 2014-12-19 2014-12-19 A kind of preparation method of distiller's yeast fermentation producing lab ferment

Country Status (1)

Country Link
CN (1) CN104531646B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4141791A (en) * 1976-12-27 1979-02-27 Alessandro Martini Milk coagulating microbial enzyme
CA2491463A1 (en) * 2002-07-02 2004-01-15 Mahoroba Co., Ltd. Milk-coagulating enzyme originating in bacterium and process for producing cheese using the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4141791A (en) * 1976-12-27 1979-02-27 Alessandro Martini Milk coagulating microbial enzyme
CA2491463A1 (en) * 2002-07-02 2004-01-15 Mahoroba Co., Ltd. Milk-coagulating enzyme originating in bacterium and process for producing cheese using the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
滕国新: "酒曲中根霉凝乳酶性质及对扣碗酪凝乳质地影响的研究", 《中国博士论文数据库工程科技1辑》 *
蒋晓雪: "江米酒酒醪中凝乳酶酶学性质的初步研究", 《中国优秀硕士学位论文全文数据库工程科技1辑》 *

Also Published As

Publication number Publication date
CN104531646B (en) 2017-12-19

Similar Documents

Publication Publication Date Title
Kumar et al. Extracellular acid protease from Rhizopus oryzae: purification and characterization
Yegin et al. Aspartic proteinases from Mucor spp. in cheese manufacturing
CN106754508B (en) Bacillus subtilis strain and application thereof in soy sauce fermentation and aroma enhancement
CN104673773B (en) A kind of preparation method of direct putting type milk-coagulating enzyme preparation and its product and application
CN110408663B (en) Lactobacillus with high extracellular polysaccharide yield, preparation method and application thereof
CN110358712B (en) Lactic acid bacteria composition and application thereof
JPH07505770A (en) A novel ketoester-reductase, its production method, and its use in enzymatic redox reactions
CN101508984B (en) Method for producing renninum with red yeast liquid state fermentation
CN112175845B (en) Saccharomyces cerevisiae suitable for brewing tea wine and application thereof
JP2019509025A (en) Pseudomonas aeruginosa strains and their application to protease production
Li et al. Production, purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1
CN109971657B (en) Rhizopus oryzae capable of producing saccharifying enzyme at high yield and application of rhizopus oryzae
CN108949617B (en) Bacillus subtilis, application thereof and enzyme preparation
CN104450655A (en) Preparation method and product of paenibacillus chymosin
CN113528359A (en) Candida planiformis and isolated culture method and application thereof
CN117247861A (en) Lactococcus garvieae Y3 for high yield of lipase, auxiliary starter and application thereof in cheese
CN104531646A (en) Preparation method for producing chymosin through distiller&#39;s yeast fermentation
CN105647815B (en) A method of improving aspergillus oryzae kojic acid yield
SALJI et al. Proteolysis and Lipolysis in Ripening Cheddar Cheese Made with Conventional Bulk Starter and with Frozen Concentrated Direct‐to‐the‐Vat Starter Culture
Alessandro et al. Partial purification and characterization of a yeast extracellular acid protease
CN103305495B (en) Method for preparing glutamate decarboxylase (GAD)
CN105200102B (en) Method for extracting glutathione from candida utilis fermentation liquor
CN105670936A (en) Trametes hirsute strain, application of trametes hirsute strain and method for processing Pu&#39;er tea
KR102125358B1 (en) Novel Aspergillus oryzae IF18-2 with high saccharogenic power and high liquerying activity and drying method thereof
CN107384898B (en) Method for producing high-temperature-resistant rennin by fermenting bacillus methanolicus in distiller&#39;s yeast

Legal Events

Date Code Title Description
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant