CN104531646B - A kind of preparation method of distiller's yeast fermentation producing lab ferment - Google Patents

A kind of preparation method of distiller's yeast fermentation producing lab ferment Download PDF

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CN104531646B
CN104531646B CN201410799638.6A CN201410799638A CN104531646B CN 104531646 B CN104531646 B CN 104531646B CN 201410799638 A CN201410799638 A CN 201410799638A CN 104531646 B CN104531646 B CN 104531646B
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renin
distiller
glutinous rice
yeast
fermentation
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CN104531646A (en
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杨贞耐
赵笑
王辑
郑喆
赵爱梅
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Beijing Technology and Business University
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    • 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

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Abstract

The invention discloses a kind of preparation method of distiller's yeast fermentation producing lab ferment.The invention provides the method that kind prepares renin, comprise the following steps：Distiller's yeast, glutinous rice and water are mixed, fermentation, tunning is collected, that is, obtains renin；The condenser water level of the distiller's yeast is 4000Su/g, and the proteolysis vigor of the distiller's yeast is about 600U/g.The experiment proves that, the present invention has extracted a kind of renin to high temperature with relatively low tolerance, and the renin comes from glutinous rice wine food, has higher C/P and absolute security, while there is higher condenser water level at 35 DEG C, and temperature rise (>45 DEG C) condenser water level substantially loses.Caused by blanching enzyme deactivation is insufficient when this characteristic can avoid cheese making the problems such as cheesy flavor bitterness, so as to improve its actual application value in cheesemaking.

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, more particularly to a kind of preparation method 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, the effect of its curdled milk is by institute in glutinous rice wine Caused by the protease contained, belong to enzymic coagulation.Renin in glutinous rice wine is in glutinous rice wine fermentation process in leavening distiller's yeast The metabolite of microorganism, belongs to microbial rennet.
Renin is the key enzyme in cheese manufacturing process, is the important enzyme preparation in field of food, it is in curdled milk Main function be selectivity hydrolysis κ-casein polypeptide chain in peptide bond between Phe105-Met106 so that casein Coagulation occurs between particle.The milk-clotting activity of renin is by the reaction condition such as temperature of enzyme concentration, milk and pH value and calcium The influence of ion concentration etc..The main source of renin has 3 kinds, respectively animal origin, plant origin and microbe-derived.It is micro- The renin of biological source is not limited by region, time etc., and has the advantages that cost is low, yield is high, by increasing Pay attention to.
The content of the invention
It is an object of the present invention to provide a kind of method for preparing renin.
Method provided by the invention, comprises the following steps：Distiller's yeast, glutinous rice matrix and water are mixed, fermentation, collect fermentation production Thing, that is, obtain renin；
The condenser water level of the distiller's yeast is 4.90 ± 0.6Su/mL；
The condenser water level of the distiller's yeast is the rennet curdling vigor in its glutinous rice wine tunning.
The glutinous rice matrix is prepared as follows：Glutinous rice and water are mixed, heating, obtain glutinous rice matrix.
In the above method,
The proteolysis vigor of the distiller's yeast is 3.05 ± 0.81Su/mL；
The distiller's yeast and the mass ratio of the glutinous rice matrix are 3-6：100, the quality of the distiller's yeast and the glutinous rice matrix Than being specially 5.7：100.
In the above method, the fermentation time is 5-7 days, and the fermentation time is specially 72h.
In the above method, by the way of shaking table concussion, the rotating speed of the shaking table is 80-180rpm, described for the fermentation The rotating speed of shaking table is specially 120rpm, and its radius of turn is 13mm.
In the above method, 20-35 DEG C of the temperature of the fermentation, the temperature of the fermentation is specially 30 DEG C；
The system initial pH value of the fermentation is 4.5-6.5, and the system initial pH value of the fermentation is 5.0.
The water and the glutinous rice matrix mass ratio are 50:100；
Glutinous rice matrix glutinous rice as described in the glutinous rice and water according to 500g：The proportioning of 1L water is made.
In the above method, after the collection tunning, also comprise the following steps：
1) tunning is filtered, filtrate is collected, obtains containing renin filtrate；
2) ethanol is added into the filtrate containing renin and obtains mixed system A, make ethanol in the mixed system A Volumn concentration be 50%, regather supernatant；
3) ethanol is added into the supernatant and obtains mixed system B, make volume hundred of the ethanol in the mixed system B It is 60% to divide content, and supernatant is collected by centrifugation；
4) add ethanol into the supernatant that 3) processing obtains and obtain mixed system C, make ethanol in the mixed system C Volumn concentration be 70%, precipitation is collected by centrifugation.
In the above method, after the precipitation is collected, also comprise the following steps:Through gel column layer after the precipitation is dissolved Analysis, eluent is collected, obtains renin.
PBS solution (the formula that solvent needed for the dissolving is 0.05mol/L pH5.8：NaCl 35g,KCl 1g, KOH1.2g,K2HPO49g, addition distilled water is settled to 1L, with second acid for adjusting pH to 5.8).
In the above method, the gel column medium of the gel filtration chromatography is Sephadex G75；
The elution buffer that the gel filtration chromatography uses is concentration is 0.05mol/L, pH value is 5.8 PBS solution；
The eluent of collecting is to collect the eluent that retention time is 100min-130min.
The renin prepared by the above method is also the scope of protection of the invention；
Or the milk-coagulating enzyme preparation prepared by the renin.
Above-mentioned renin has following 1) -3) at least one of zymetology feature：
1) optimal reactive temperature of the renin is 35 DEG C,
2) optimal reaction pH value 5.4 of the renin；
3) activity of the renin can be by Ca2+Promote.
The present invention is it is demonstrated experimentally that the present invention has extracted a kind of renin to high temperature with relatively low tolerance, the curdled milk Enzyme comes from glutinous rice wine food, has higher C/P and absolute security, while has higher condenser water level at 35 DEG C, And temperature rise (>45 DEG C) condenser water level substantially loses.Caused by blanching enzyme deactivation is insufficient when this characteristic can avoid cheese making The problems such as cheesy flavor bitterness, so as to improve its actual application value in cheesemaking.
Brief description of the drawings
Fig. 1 is influence of the different outbox conditions to distiller's yeast fermentation producing lab ferment
Fig. 2 is influence of the addition of different carbon source nitrogen source to distiller's yeast fermentation producing lab ferment
Fig. 3 be distiller's yeast ferment producing lab ferment response surface figure and it is high isogram(s)
Fig. 4 is ethanol precipitation SDS-PAGE
Fig. 5 is the column chromatography purification result and destination protein SDS-PAGE after purification of renin
Fig. 6 is renin zymetology properties study
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The preparation method of embodiment 1, distiller's yeast fermentation producing lab ferment
First, the screening of distiller's yeast
Distiller's yeast A-H purchased from different regions is fermented respectively under the conditions of 35 DEG C the glutinous rice wine hair for being fabricated to distiller's yeast A-H in 3 days Ferment product, the condenser water level (MCA) and proteolysis vigor (PA) of each glutinous rice wine tunning are determined respectively.Assay method is distinguished It is as follows：
Condenser water level assay method：Using the method for Arima (1970)：Taking 5mL100g/L skimmed milk, (fat content is about For 1.0%), 5min are incubated at 35 DEG C, add the glutinous rice wine tunning of 0.5mL distiller's yeasts, it is rapid well mixed, accurate recording from Prepare liquid is added to the curdling solid time.One Soxhlet is defined as with the enzyme amount needed for 40min solidifications 100g/L skimmed milk 1mL Unit (Soxhelt unit, Su).
SU=2400/T × 5/0.5 × D4
In formula:T is the curdled milk time (s)；D is extension rate.
Proteolysis vigour-testing method：Determined according to the Folin phenol reagent process employed in the documents such as Zhang Fuxin, specifically It is as follows：1ml enzyme liquids, under the conditions of certain temperature and pH value, 1min caseinhydrolysates produce lug tyrosine and are defined as 1 enzyme Active unit (U/m1)；
The preparation of casein solution：Casein 1.0009 (being accurate to 0.0019) is weighed, after a small amount of dense lactic acid moistening, is added Enter pH3.0 lactic acid buffer 80ml, heated while stirring in boiling water bath, until being completely dissolved, buffered after cooling with lactic acid Liquid is diluted to 100ml, as 10g/L casein solution.
The preparation of standard liquid：Tyrosine 0.10009 (being accurate to 0.00029) is weighed, is dissolved with lmol/L hydrochloric acid 60ml Afterwards, 100ml, as 1mg/ml tyrosine standard liquid are settled to.1mg/m1 tyrosine standard liquid 10.00ml are drawn, then are used 0.lmol/L hydrochloric acid is settled to 100ml, produces 100ug/ml tyrosine standard liquids.
The formulation of standard curve：It is accurate to draw 100ug/m1 tyrosine standard liquid 0,1,2,3,4,5ml, move into 6 In 25ml test tubes, pure distilled water 10,9,8,7,6,5ml, as 0,20,20,30,40,50ug/ml tyrosine mark are separately added into Quasi- liquid.Titer 1.00ml is taken respectively, and each to add 0.4mol/L sodium carbonate liquor 5.00ml, Folin reagent uses liquid 1.00ml, is placed in 40 scholar, 0.2 DEG C of water-bath the 20min that develops the color, and takes out with 721 spectrophotometers in wavelength 680nm, 1cm cuvettes, Using 0 pipe without tyrosine as blank, its absorbance is determined respectively.Its regression equation is y=0.010x+5E-0.5 (r= 0.999), when absorbance be 1 when tyrosine amount K==99.5ug.
1.00ml enzyme liquids are taken in test tube, 2min is incubated in the case where 0.2 DEG C of 40 scholar, 10g/L casein 1.0ml is added, shakes up Afterwards, 10min is incubated at the same temperature, then adds 0.4mol/L trichloroacetic acid 2ml, shakes up stopped reaction, is taken out and is stood 10min, filtering, is measured by standard curve operating procedure.
Proteolytic activity (U/ml)=A*K*4/10*n
Wherein:A-sample absorbance
K-extinction constant
4-reaction reagent, cumulative volume
10-reaction time 10min
N- extension rates
Experimental result is as shown in table 1：
Table 1 is MCA, PA, C/P of separate sources distiller's yeast fermentation glutinous rice wine comparison
Mark：a-e：Same letter represents that difference is not notable in colleague, and different letters represent significant difference, P ＜ 0.5, table Bright significant difference.
Select condenser water level high, the relatively low distiller's yeast C of proteolysis vigor makes further research, desk study result table Bright, distiller's yeast C (is purchased from Suzhou oil and foodstuffs Co., Ltd, title：Honeybee board Suzhou honeybee koji) condenser water level be 4.90 ± 0.62Su/mL, proteolysis vigor are 3.05 ± 0.81Su/mL.
2nd, distiller's yeast fermentation prepares renin
1st, optimization culture conditions
The present invention have studied distiller's yeast addition (1%-6%), fermentation number of days (3-9 days), fermentation temperature (20-35 DEG C), hair Initial pH value (4.5-6.5), moisture addition (40%-70%), shaking speed (80-160rpm), the difference for adding 3% of ferment Carbon source (glucose, fructose, maltose, sucrose, lactose) and nitrogen source (ammonium citrate, ammonium sulfate, diammonium hydrogen phosphate, tryptone, Milk powder) influence to fermentation glutinous rice wine producing lab ferment vigor and proteolysis vigor, specific method is as follows：
1), distiller's yeast addition is groped
The preparation of glutinous rice matrix：Fresh glutinous rice 500g cleans immersion 3h, adds 1L distilled water, and normal pressure steams 0.5h, cooling, obtained It is 1000g to glutinous rice matrix；
The distiller's yeast C and water that above-mentioned one is obtained are added to the mixing of glutinous rice matrix, fermentation；
Distiller's yeast C is respectively 1%, 2%, 3%, 4%, 5% and 6% (distiller's yeast quality and glutinous rice base according to weight/mass percentage composition Matter mass percent) addition；
Water adds according to weight/mass percentage composition 50% (water 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；
The tunning of different distiller's yeast additions is collected, condenser water level MCA is detected according to method in above-mentioned one.
As a result as shown in Figure 1A, it can be seen that after distiller's yeast addition reaches 3%, condenser water level is basicly stable.
2), fermentation time is groped
The distiller's yeast C and water that above-mentioned one is obtained are added to the mixing of glutinous rice matrix, fermentation；
Distiller's yeast C adds according to weight/mass percentage composition for 3% (distiller's yeast quality and glutinous rice matrix mass percent)；
Water adds according to weight/mass percentage composition 50% (water 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；
Tunning is collected in the different fermentations time, condenser water level MCA is detected according to method in above-mentioned one.
As a result as shown in Figure 1B, it can be seen that for fermentation time at 5-7 days, condenser water level was higher, reached most at the 5th day It is high.
3), the condition of fermentation temperature is groped
The distiller's yeast C and water that above-mentioned one is obtained are added to the mixing of glutinous rice matrix, fermentation；
Distiller's yeast C adds according to weight/mass percentage composition for 3% (distiller's yeast quality and glutinous rice matrix mass percent)；
Water adds according to weight/mass percentage composition 50% (water 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；
The tunning at a temperature of different fermentations is collected, condenser water level MCA is detected according to method in above-mentioned one.
As a result as shown in Figure 1 C, it can be seen that for fermentation temperature at 30 DEG C, condenser water level reaches peak.
4), initial pH value is groped
The distiller's yeast C and water that above-mentioned one is obtained are added to the mixing of glutinous rice matrix, fermentation；
Distiller's yeast C adds according to weight/mass percentage composition for 3% (distiller's yeast quality and glutinous rice matrix mass percent)；
Water adds according to weight/mass percentage composition 50% (water quality and glutinous rice matrix mass percent)；
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm, and its radius of turn is 13mm, ferment 5 days, fermentation it is initial PH value 4.5-6.5；
The obtained tunning of different fermentations initial pH value is collected, condenser water level MCA is detected according to method in above-mentioned one.
As a result as shown in figure iD, it can be seen that the initial pH value 5 of fermentation, condenser water level reaches peak.
5), moisture addition
The distiller's yeast C and water that above-mentioned one is obtained are added to the mixing of glutinous rice matrix, fermentation；
Distiller's yeast C adds according to weight/mass percentage composition for 3% (distiller's yeast quality and glutinous rice matrix mass percent)；
Water adds according to weight/mass percentage composition 40%, 50%, 60%, 70% (water quality and glutinous rice matrix mass percent) Add；
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 5 days, the initial pH value 5 of fermentation；
The obtained tunning of different in moisture addition is collected, condenser water level MCA is detected according to method in above-mentioned one.
As a result as referring to figure 1E, it can be seen that when moisture addition is 40%, condenser water level reaches peak.
6), fermentation shaking speed
The distiller's yeast C and water that above-mentioned one is obtained are added to the mixing of glutinous rice matrix, fermentation；
Distiller's yeast C adds according to weight/mass percentage composition for 3% (distiller's yeast quality and glutinous rice matrix mass percent)；
Water adds according to weight/mass percentage composition 40% (water 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；
The tunning that different shaking speeds obtain is collected, condenser water level MCA is detected according to method in above-mentioned one.
As a result as shown in fig. 1F, it can be seen that shaking speed 120rpm, condenser water level reach peak.
7) different carbon source is groped
Distiller's yeast C, water and the carbon source (or nitrogen source) that above-mentioned one is obtained are added to the mixing of glutinous rice matrix, fermentation；
Distiller's yeast C adds according to weight/mass percentage composition for 3% (distiller's yeast quality and glutinous rice matrix mass percent)；
Water adds according to weight/mass percentage composition 40% (water quality and glutinous rice matrix mass percent)；
Carbon source (or nitrogen source) is according to weight/mass percentage composition 3% (carbon source (or nitrogen source) quality and glutinous rice matrix mass percent) Addition；
Carbon source is respectively glucose, fructose, maltose, sucrose, lactose；
Nitrogen source is respectively ammonium citrate：Ammonium sulfate：Diammonium hydrogen phosphate, tryptone, milk powder；
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 5 days, the initial pH value 5 of fermentation；
The tunning that different carbon source or nitrogen source obtain is collected, condenser water level MCA is detected according to method in above-mentioned one.
Made comparisons with not adding the blank of carbon source or nitrogen source.
As a result as shown in Fig. 21：Blank control 2：Glucose 3：Fructose 4：Maltose 5：Sucrose 6：Lactose 7：Ammonium citrate 8：Ammonium sulfate 9：Diammonium hydrogen phosphate 10：Tryptone 11：Milk powder, it can be seen that addition carbon source especially glucose, send out distiller's yeast Ferment producing lab ferment has certain facilitation；The addition of nitrogen source does not play promotion to the condenser water level of distiller's yeast fermentation producing lab ferment Effect, some nitrogen sources such as ammonium sulfate etc. can even reduce the milk-clotting activity of distiller's yeast producing enzyme.
The above results show that influence of the different factors to proteolysis vigor is not very big, and to the influence of condenser water level Obvious, with the change of each factor, proteolysis vigor is relatively low.Distiller's yeast addition increases and the extension of fermentation time, Condenser water level raises.
The influence of above single factor test is analyzed, wherein 3 distiller's yeast inoculum concentration, fermentation time and shaking table speed factors are lived to curdled milk The influence of power is the most notable, therefore chooses this 3 factors and carry out response surface analysises, such as Fig. 3, wherein, (A) be distiller's yeast inoculum concentration and The surface chart and height that fermentation time influences on condenser water level are isogram(s)；(B) it is distiller's yeast inoculum concentration and shaking table speed to condenser water level The surface chart and height of influence are isogram(s)；(C) surface chart and high line influenceed for fermentation time and shaking table speed on condenser water level Scheme (Fig. 3).
Response surface analysis obtains the regression equation using condenser water level as response：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*B2-5.33547E-003*C2.In formula, A-distiller's yeast inoculum concentration, %；B-fermentation Time, h；C-shaking table speed, rpm.
It is distiller's yeast inoculum concentration 5.7% (weight/mass percentage composition) to obtain optimal conditions of fermentation, fermentation time 72h shaking speeds 120rpm, the rennet curdling vigor of theoretical expectation values tunning is 52.29Su/mL；
Therefore, distiller's yeast inoculum concentration, fermentation time and shaking speed are most important optimizing factors.
2nd, optimum condition is fermented
The distiller's yeast C and water that above-mentioned one is obtained are added to the mixing of glutinous rice matrix, fermentation；
Distiller's yeast C adds according to weight/mass percentage composition for 5.7% (distiller's yeast quality and glutinous rice matrix mass percent)；
Water adds according to weight/mass percentage composition 40% (water quality and glutinous rice matrix mass percent)；
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 72h, the initial pH value 5 of fermentation；
Tunning is collected, detects condenser water level MCA according to method in above-mentioned one, as a result the renin in tunning coagulates Newborn vigor is 51.05Su/mL, is close with theoretical expectation values (52.29Su/mL).
Note：It is using fermentation time as variable during single factor analysis, is done under conditions of other factorses are constant, now distiller's yeast adds Dosage fixed bit is 3%, highest when fermentation time is 5 days, and response surface analysis is to analyze the mutual shadow between 3 variables simultaneously Ring, when distiller's yeast addition reaches 5.7%, fermentation time only needs 3 days to can reach highest.
The preparation of embodiment 2, renin
1st, ferment
The preparation of glutinous rice matrix：Fresh glutinous rice 500g cleans immersion 3h, adds 1L distilled water, and normal pressure steams 0.5h, cooling, obtained It is 1000g to glutinous rice matrix；
The distiller's yeast C and water that above-mentioned one is obtained are added to the mixing of glutinous rice matrix, fermentation；
Distiller's yeast C adds according to weight/mass percentage composition for 5.7% (distiller's yeast quality and glutinous rice matrix mass percent)；
Water adds according to weight/mass percentage composition 40% (water quality and glutinous rice matrix mass percent)；
Above-mentioned fermentation condition is 30 DEG C, shaking speed 120rpm ferments 72h, the initial pH value 5 of fermentation；
Tunning is collected, is filtered (4 layers of gauze), filtrate is collected, as containing renin filtrate, in 4 DEG C of refrigerator cold-storages It is standby.
2nd, ethanol precipitation slightly carries renin
It is placed on 4 DEG C of above-mentioned 1 absolute ethyl alcohol stored containing renin filtrate and laboratory prepared to take out, by as follows Step operation：(1) absolute ethyl alcohol is added to containing in renin filtrate for above-mentioned 1 preparation, makes mixed volume fraction of ethanol For 50%, 4 DEG C, 5000r/min is centrifuged 30 minutes, collects supernatant precipitation, and albumen precipitation is dissolved with distilled water, is placed in 4 DEG C of ice Case is standby；(2) by the supernatant obtained in (1) continue add absolute ethyl alcohol make volume fraction of ethanol be 60%, 4 DEG C, 5000r/min Centrifugation 30 minutes, supernatant precipitation is collected, albumen precipitation dissolving, it is standby to be placed in 4 DEG C of refrigerators；(3) by the supernatant obtained in (2) after Continuous addition absolute ethyl alcohol makes volume fraction of ethanol be 70%, and 4 DEG C, 5000r/min is centrifuged 30 minutes, and albumen precipitation is dissolved, put It is standby in 4 DEG C of refrigerators；(4) by the supernatant obtained in (3) continue add absolute ethyl alcohol make volume fraction of ethanol be 80%, 4 DEG C, 5000r/min is centrifuged 30 minutes, and albumen precipitation is dissolved, it is standby to be placed in 4 DEG C of refrigerators；
The condenser water level of albumen precipitation in above-mentioned 4 steps is determined respectively.
As a result as shown in figure 4,1：Protein standard substance；2：50% ethanol precipitation albumen；3：60% ethanol precipitation albumen；4： 70% ethanol precipitation albumen；5：80% ethanol precipitation albumen, show the albumen that i.e. 70% ethanol precipitation obtains in only step (3) With obvious condenser water level.
SDS-PAGE electrophoretic analysis is carried out to the albumen of 50%, 60%, 70%, 80% ethanol precipitation respectively, as a result such as Fig. 4 Shown, 80% protein precipitation is more small molecular protein, and one and other albumen precipitations occurs in the albumen of 70% ethanol precipitation Different protein bands, molecular weight is between 34-43kD, it is more likely that is target protein.
It is above-mentioned it is demonstrated experimentally that the albumen of 70% ethanol precipitation is slightly carries renin, its vacuum freeze drying is solidifying into slightly carrying Galactenzyme dry powder.
3rd, Sephadex G75 purifying slightly carries renin
The 2 obtained thick solution for carrying rennin dry powder and being configured to 0.2g/mL by more than, solvent is 0.05mol/L pH5.8 PBS solution (formula：NaCl 35g,KCl 1g,KOH 1.2g,K2HPO49g, addition distilled water are settled to 1L, adjusted with acetic acid PH is to 5.8), through Sephadex G75 pillars (2.6 × 60cm), using 0.05mol/LpH5.8 PBS solution as elution buffer Eluted, applied sample amount 4mL, flow velocity 1mL/min, determine the condenser water level of each protein peak, collection has condenser water level Peak.
As a result as shown in Figure 5A, it can be seen that in retention time be 100~130min, obtain purpose peak.
Eluted products corresponding to purpose peak are collected, is freeze-dried into powder, enters after 10kD ultra-filtration centrifuge tube desalination and concentration Row SDS-PAGE electrophoretic analysis, 1 it is protein standard substance as a result as shown in Figure 5 B, 2 is purpose albumen, show, obtained albumen is Single band, it is about 39.7kD to calculate molecular weight, is purpose renin.
Embodiment 2, the research of renin enzymatic property
The purpose renin for being purified and being obtained with embodiment 1 is detected below.
1st, optimum temperature
Embodiment 1 is determined at 25,30,35,40,45,50,55 DEG C respectively and purifies obtained purpose rennet curdling work Power, method is with the one of embodiment 1, using condenser water level at 35 DEG C as 100%, calculates relative condenser water level.
As a result as shown in Figure 6A, show the renin has a preferable condenser water level at 35 DEG C, temperature higher than 45 DEG C with Condenser water level is lost substantially afterwards.
2nd, optimum pH
With 0.1mol/L NaOH or HCl adjustment skimmed milk pH, respectively pH be 5.4,5.6,5.8,6.0,6.2, 6.4th, 6.6,6.8,7.0 when measure measure embodiment 1 purify obtained purpose rennet curdling vigor, method is the same as embodiment 1 One, using condenser water level during pH5.8 as 100%, calculate relative condenser water level.
As a result as shown in Figure 6B, show that condenser water level raises with pH reduction, condenser water level reaches most in pH5.4 It is high.
3rd, metal ion influences on enzymatic activity
In skimmed milk, NaCl, KCl, MgCl are added respectively2、CaCl2、FeCl2、AlCl3、CuCl2、ZnCl2, make its dense Spend for 10mmol/L, add measure embodiment 1 and purify obtained purpose rennet curdling vigor, method with the one of embodiment 1, with The condenser water level of blank control (BC) calculates relative condenser water level as 100%.
As a result as shown in Figure 6 C, the results showed that Na+, K+, Mg2+, Ca2+And Al3+There is facilitation to the rennet curdling, especially It is Ca2+Effect it is most obvious.
Enzyme preparation is made in the renin vacuum freeze drying of purifying.

Claims (3)

1. a kind of method for preparing renin, comprises the following steps：Distiller's yeast, glutinous rice matrix and water are mixed, fermentation, collect fermentation Product, that is, obtain renin；
The condenser water level of the distiller's yeast is 4.90 ± 0.6Su/mL；
The proteolysis vigor of the distiller's yeast is 3.05 ± 0.81U/mL；
The glutinous rice matrix is prepared as follows：Glutinous rice and water are mixed, heating, obtain glutinous rice matrix；
The distiller's yeast and the mass ratio of the glutinous rice matrix are 5.7：100；The fermentation time is 72h；The fermentation, which uses, shakes The mode of bed concussion, the rotating speed of the shaking table is 120rpm；
The temperature of the fermentation is 30 DEG C；
The system initial pH value of the fermentation is 5.0；
The water and the glutinous rice matrix mass ratio are 50:100；
Glutinous rice matrix glutinous rice as described in the glutinous rice and water according to 500g：The proportioning of 1L water is made；
After the collection tunning, also comprise the following steps：
1) tunning is filtered, filtrate is collected, obtains containing renin filtrate；
2) ethanol is added into the filtrate containing renin and obtains mixed system A, make body of the ethanol in the mixed system A Product percentage composition is 50%, regathers supernatant；
3) ethanol is added into the supernatant and obtains mixed system B, contain volume basis of the ethanol in the mixed system B Measure as 60%, supernatant is collected by centrifugation；
4) add ethanol into the supernatant that 3) processing obtains and obtain mixed system C, make body of the ethanol in the mixed system C Product percentage composition is 70%, and precipitation is collected by centrifugation；
After the precipitation is collected, also comprise the following steps:, through gel filtration chromatography, eluent will be collected after the precipitation dissolving, Obtain renin；
The gel column medium of the gel filtration chromatography is Sephadex G75；
The elution buffer that the gel filtration chromatography uses is concentration is 0.05mol/L, pH value is 5.8 PBS solution.
2. the renin prepared by claim 1 methods described；
Or the milk-coagulating enzyme preparation prepared by the renin.
3. renin according to claim 2, it is characterised in that：
The renin has following 1) -3) at least one of zymetology feature：
1) optimal reactive temperature of the renin is 35 DEG C,
2) optimal reaction pH value 5.4 of the renin；
3) activity of the renin can be by Ca2+Promote.
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US4141791A (en) * 1976-12-27 1979-02-27 Alessandro Martini Milk coagulating microbial enzyme
JP4067349B2 (en) * 2002-07-02 2008-03-26 北海道 Bacteria-derived milk-clotting enzyme and production of cheese using the enzyme

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