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

Abstract

The invention discloses a preparation method for producing chymosin by fermenting distiller's yeast. The invention provides a method for preparing rennet, comprising the following steps: uniformly mixing distiller's yeast, glutinous rice and water, fermenting, and collecting fermentation products to obtain rennet; the curdling activity of the distiller's yeast is 4000 Su/g, so The proteolytic activity of the distiller's yeast is about 600U/g. Experiments of the present invention prove that the present invention has extracted a kind of rennet that has lower tolerance to high temperature, and this rennet comes from glutinous rice wine food, has higher C/P and absolute safety, simultaneously in It has higher curdling activity at 35°C, but the curdling activity is obviously lost when the temperature rises (>45°C). This feature can avoid problems such as bitterness and bitterness of cheese flavor caused by insufficient blanching of enzymes during cheese making, thereby improving its practical application value in cheese production.

Description

A kind of preparation method of distiller's koji fermentation to produce chymosin

technical field

The invention relates to the technical field of bioengineering, in particular to a preparation method for producing chymosin by fermenting distiller's yeast.

Background technique

Rice wine is a kind of traditional food in southern my country, which has curdling effect, and its curdling effect is caused by the protease contained in rice wine, which belongs to enzyme coagulation. Rennet in glutinous rice wine is a metabolite of microorganisms in the starter koji during the fermentation of glutinous rice wine, and belongs to microbial rennet.

Rennet is a key enzyme in the cheese production process and an important enzyme preparation in the food field. Its main role in curdling is to specifically hydrolyze the peptide between Phe105-Met106 in the κ-casein polypeptide chain Bonds, so that coagulation occurs between casein particles. The milk-clotting activity of chymosin is affected by reaction conditions such as enzyme concentration, temperature and pH of milk, and calcium ion concentration. There are three main sources of chymosin, namely animal sources, plant sources and microbial sources. Microbial chymosin is not limited by region and time, and has the advantages of low cost and high yield, and has received more and more attention.

Contents of the invention

An object of the present invention is to provide a method for preparing chymosin.

The method provided by the invention comprises the following steps: uniformly mixing distiller's yeast, glutinous rice matrix and water, fermenting, and collecting fermentation products to obtain rennet;

The curdling activity of the distiller's yeast is 4.90±0.6Su/mL;

The curdling activity of the distiller's yeast is the curdling activity of rennet in the fermented product of glutinous rice wine.

The glutinous rice matrix is prepared according to the following method: mix glutinous rice and water, and heat to obtain the glutinous rice matrix.

In the above method,

The proteolytic activity of the distiller's yeast is 3.05±0.81Su/mL;

The mass ratio of the distiller's yeast to the glutinous rice base is 3-6:100, and the mass ratio of the distiller's yeast to the glutinous rice base is specifically 5.7:100.

In the above method, the fermentation time is 5-7 days, specifically 72 hours.

In the above method, the fermentation adopts a shaker vibration mode, the rotation speed of the shaker is 80-180 rpm, specifically 120 rpm, and the rotation radius of the shaker is 13 mm.

In the above method, the temperature of the fermentation is 20-35°C, and the temperature of the fermentation is specifically 30°C;

The initial pH value of the fermentation system is 4.5-6.5, and the initial pH value of the fermentation system is 5.0.

Described water and described glutinous rice matrix mass ratio are 50:100;

The glutinous rice matrix is made of the glutinous rice and water according to the ratio of 500 g of the glutinous rice: 1 L of water.

In the above method, after the fermentation product is collected, the following steps are also included:

1) filtering the fermentation product, collecting the filtrate to obtain a filtrate containing rennet;

2) adding ethanol to the filtrate containing rennet to obtain a mixed system A, so that the volume percentage of ethanol in the mixed system A is 50%, and then collecting the supernatant;

3) adding ethanol to the supernatant to obtain a mixed system B, so that the volume percentage of ethanol in the mixed system B is 60%, and centrifuging to collect the supernatant;

4) Add ethanol to the supernatant obtained in 3) to obtain a mixed system C, make the volume percentage of ethanol in the mixed system C be 70%, and collect the precipitate by centrifugation.

In the above method, after the precipitation is collected, the following steps are also included: after dissolving the precipitation, the gel column chromatography is used to collect the eluate to obtain chymosin.

The solvent required for the dissolution is 0.05mol/L PBS solution with pH 5.8 (recipe: NaCl 35g, KCl 1g, KOH 1.2g, K ₂ HPO ₄ 9g, add distilled water to 1L, adjust the pH to 5.8 with acetic acid ).

In the above method, the gel column medium of the gel column chromatography is Sephadex G75;

The elution buffer used in the gel column chromatography is a PBS solution with a concentration of 0.05mol/L and a pH value of 5.8;

The collected eluate is the eluate with a retention time of 100min-130min.

The chymosin prepared by the above method is also within the protection scope of the present invention;

Or a rennet preparation prepared from said rennet.

The above-mentioned chymosin has at least one enzymatic characteristic in the following 1)-3):

1) The optimal reaction temperature of the chymosin is 35°C,

2) The optimum reaction pH value of the rennet is 5.4;

3) The activity of the chymosin can be promoted by Ca ²⁺ .

Experiments of the present invention prove that the present invention has extracted a kind of rennet that has lower tolerance to high temperature, and this rennet comes from glutinous rice wine food, has higher C/P and absolute safety, simultaneously in 35 It has higher curdling activity at ℃, but the curdling activity is obviously lost when the temperature rises (>45℃). This feature can avoid problems such as bitterness and bitterness of cheese flavor caused by insufficient blanching of enzymes during cheese making, thereby improving its practical application value in cheese production.

Description of drawings

Figure 1 is the effect of different delivery conditions on the production of chymosin by fermentation of distiller's yeast

Figure 2 is the effect of the addition of different carbon and nitrogen sources on the production of chymosin by distiller's yeast fermentation

Figure 3 is the response surface diagram and high contour diagram of chymosin produced by fermentation of distiller's yeast

Figure 4 is the SDS-PAGE electrophoresis diagram of ethanol fractionation precipitation

Figure 5 is the column chromatography purification result of chymosin and the SDS-PAGE electrophoresis figure of the purified target protein

Figure 6 shows the enzymatic characteristics of chymosin

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the preparation method of distiller's koji fermentation producing chymosin

1. Screening of koji

The koji A-H purchased from different regions were fermented at 35°C for 3 days to produce the fermented rice wine of koji A-H, and the curdling activity (MCA) and proteolytic activity (PA) of each fermented rice wine were measured. The measurement methods are as follows:

Curd activity assay method: adopt the method of Arima (1970): take 5mL of 100g/L skimmed milk (fat content is about 1.0%), keep it warm at 35°C for 5min, add 0.5mL of rice wine fermentation product of distiller's yeast, mix quickly and evenly, Accurately record the time from adding the liquid to be tested to the milk coagulation. The amount of enzyme required to coagulate 1 mL of 100 g/L skim milk in 40 min is defined as a Soxhlet unit (Su).

SU=2400/T×5/0.5×D4

In the formula: T is the clotting time (s); D is the dilution factor.

Determination method of proteolytic activity: According to the Folin phenol reagent method used in the literature of Zhang Fuxin and others, the details are as follows: 1 ml of enzyme solution, under certain temperature and pH value conditions, 1 min of hydrolyzing casein to produce 1 ug of tyrosine is defined as 1 Enzyme activity unit (U/m1);

Preparation of casein solution: Weigh casein 1.0009 (accurate to 0.0019), wet it with a small amount of concentrated lactic acid, add 80ml of lactic acid buffer solution with pH 3.0, stir while heating in a boiling water bath until it is completely dissolved, and use it after cooling. Lactic acid buffer diluted to 100ml, that is, 10g/L casein solution.

Preparation of standard solution: Weigh tyrosine 0.10009 (accurate to 0.00029), dissolve it with 60 ml of 1mol/L hydrochloric acid, and set the volume to 100 ml, which is 1 mg/ml tyrosine standard solution. Draw 10.00ml of 1mg/ml tyrosine standard solution, and then dilute to 100ml with 0.1mol/L hydrochloric acid to obtain 100ug/ml tyrosine standard solution.

The establishment of the standard curve: Accurately draw 0, 1, 2, 3, 4, 5ml of 100ug/m1 tyrosine standard solution, transfer it into 6 25ml test tubes, add 10, 9, 8, 7, 6, 5ml of pure distilled water respectively, That is 0, 20, 20, 30, 40, 50ug/ml tyrosine standard solution. Take 1.00ml of the standard solution, add 5.00ml of 0.4mol/L sodium carbonate solution, and 1.00ml of Folin reagent solution, put it in a water bath at 40±0.2°C for 20min, take it out with a 721 spectrophotometer at a wavelength of 680nm, 1cm cuvette, with the 0 tube without tyrosine as the blank, measure its absorbance respectively. Its regression equation is y=0.010x+5E-0.5 (r=0.999), and when the absorbance is 1, the amount of tyrosine K=99.5ug.

Take 1.00ml of enzyme solution in a test tube, keep it warm at 40±0.2°C for 2min, add 1.0ml of 10g/L casein, shake well, keep warm at the same temperature for 10min, then add 2ml of 0.4mol/L trichloroacetic acid, Shake well to stop the reaction, take it out and let it stand for 10 minutes, filter, and measure according to the standard curve operation steps.

Proteolytic activity (U/ml) = A*K*4/10*n

Among them: A—sample absorbance

K—absorption constant

4—Reagents, total volume

10—response time 10min

n-dilution factor

The experimental results are shown in Table 1:

Table 1 is the comparison of MCA, PA and C/P of rice wine fermented from different sources of koji

Note: a-e: The same letter in the same row means no significant difference, different letters mean significant difference, P<0.5, indicating significant difference.

Select distiller koji C with high curd activity and relatively low proteolytic activity for further research. The preliminary exploration results show that the curd activity of distiller koji C (purchased from Suzhou Cereals, Oils and Foodstuffs Co., Ltd., name: Bee Brand Suzhou Bee Sweet Wine Koji) is 4.90 ±0.62Su/mL, proteolytic activity was 3.05±0.81Su/mL.

2. Fermentation of koji to prepare chymosin

1. Optimization of enzyme production conditions

The present invention has studied distiller's yeast addition (1%-6%), fermentation days (3-9 days), fermentation temperature (20-35 ℃), initial pH value of fermentation (4.5-6.5), moisture addition (40% -70%), shaker speed (80-160rpm), add 3% of different carbon sources (glucose, fructose, maltose, sucrose, lactose) and nitrogen sources (ammonium citrate, ammonium sulfate, diammonium phosphate, tryptone , milk powder) on fermented glutinous rice wine to produce the impact of rennet activity and proteolysis activity, concrete method is as follows:

1) Exploring the amount of distiller's yeast added

Preparation of glutinous rice matrix: 500 g of fresh glutinous rice was washed and soaked for 3 hours, added 1 L of distilled water, steamed at normal pressure for 0.5 h, and cooled to obtain 1000 g of glutinous rice matrix;

Add the distiller's koji C and water obtained in the above-mentioned step 1 to the glutinous rice base, mix evenly, and ferment;

Distiller's yeast C is added according to the mass percentage of 1%, 2%, 3%, 4%, 5% and 6% (distiller's yeast mass and glutinous rice matrix mass percentage);

Water is added according to mass percentage composition 50% (water quality and glutinous rice substrate mass percentage);

The above fermentation conditions are 30°C, 120rpm shaker speed, 3 days of fermentation, and the initial pH value of the fermentation is 5;

Fermentation products with different amounts of distiller's yeast were collected, and the milk curdling activity MCA was detected according to the above-mentioned method.

The results are shown in Figure 1A. It can be seen that when the amount of distiller's yeast added reaches 3%, the curdling activity is basically stable.

2) Explore the fermentation time

Add the distiller's koji C and water obtained in the above-mentioned step 1 to the glutinous rice base, mix evenly, and ferment;

Distiller's koji C is added according to the mass percentage of 3% (distiller's yeast mass and glutinous rice matrix mass percentage);

Water is added according to mass percentage composition 50% (water quality and glutinous rice substrate mass percentage);

The above fermentation conditions are 30°C, 120rpm shaker speed, 9 days of fermentation, and the initial pH value of the fermentation is 5;

Fermentation products were collected at different fermentation times, and milk curdling activity MCA was detected according to one of the above methods.

The results are shown in Figure 1B. It can be seen that the curdling activity is higher when the fermentation time is 5-7 days, and reaches the highest on the 5th day.

3), exploring the conditions of fermentation temperature

Add the distiller's koji C and water obtained in the above-mentioned step 1 to the glutinous rice base, mix evenly, and ferment;

Distiller's koji C is added according to the mass percentage of 3% (distiller's yeast mass and glutinous rice matrix mass percentage);

Water is added according to mass percentage composition 50% (water quality and glutinous rice substrate mass percentage);

The above fermentation conditions were 20, 25, 30 and 35°C, 120rpm shaker speed, 5 days of fermentation, and the initial pH value of the fermentation was 5;

Fermentation products at different fermentation temperatures were collected, and curdling activity MCA was detected according to one of the above methods.

The results are shown in Figure 1C. It can be seen that the curdling activity reaches the highest value when the fermentation temperature is 30°C.

4), the initial pH value exploration

Add the distiller's koji C and water obtained in the above-mentioned step 1 to the glutinous rice base, mix evenly, and ferment;

Distiller's koji C is added according to the mass percentage of 3% (distiller's yeast mass and glutinous rice matrix mass percentage);

Water is added according to mass percentage composition 50% (water quality and glutinous rice substrate mass percentage);

The above fermentation conditions are 30°C, the rotating speed of the shaker is 120rpm, the rotation radius is 13mm, the fermentation is for 5 days, and the initial pH value of the fermentation is 4.5-6.5;

Fermentation products obtained at different initial pH values of fermentation were collected, and curdling activity MCA was detected according to one of the above-mentioned methods.

The results are shown in Fig. 1D. It can be seen that the initial pH value of the fermentation is 5, and the curdling activity reaches the highest value.

5), the amount of water added

Add the distiller's koji C and water obtained in the above-mentioned step 1 to the glutinous rice base, mix evenly, and ferment;

Distiller's koji C is added according to the mass percentage of 3% (distiller's yeast mass and glutinous rice matrix mass percentage);

Water is added according to mass percentage 40%, 50%, 60%, 70% (water quality and glutinous rice substrate mass percentage);

The above fermentation conditions are 30°C, 120rpm shaker speed, 5 days of fermentation, and the initial pH value of the fermentation is 5;

Fermentation products obtained with different water additions were collected, and the curdling activity MCA was detected according to the above-mentioned method.

The results are shown in Figure 1E. It can be seen that the curdling activity reaches the highest value when the water addition amount is 40%.

6), the rotation speed of the fermentation shaker

Add the distiller's koji C and water obtained in the above-mentioned step 1 to the glutinous rice base, mix evenly, and ferment;

Distiller's koji C is added according to the mass percentage of 3% (distiller's yeast mass and glutinous rice matrix mass percentage);

Water is added according to mass percentage composition 40% (water quality and glutinous rice substrate mass percentage);

The above fermentation conditions are 30°C, 80-180rpm shaker speed, 5 days of fermentation, and the initial pH value of the fermentation is 5;

The fermentation products obtained at different shaking table speeds were collected, and the curdling activity MCA was detected according to the above-mentioned method.

The results are shown in Figure 1F. It can be seen that the curdling activity reaches the highest value when the shaking table rotates at 120 rpm.

7) Explore different carbon sources

Add the distiller's koji C, water and carbon source (or nitrogen source) obtained in the above-mentioned one to the glutinous rice matrix, mix well, and ferment;

Distiller's koji C is added according to the mass percentage of 3% (distiller's yeast mass and glutinous rice matrix mass percentage);

Water is added according to mass percentage composition 40% (water quality and glutinous rice substrate mass percentage);

The carbon source (or nitrogen source) is added according to the mass percentage of 3% (the carbon source (or nitrogen source) mass and the glutinous rice substrate mass percentage);

The carbon sources are glucose, fructose, maltose, sucrose and lactose respectively;

Nitrogen sources are ammonium citrate: ammonium sulfate: diammonium hydrogen phosphate, tryptone, milk powder;

The above fermentation conditions are 30°C, 120rpm shaker speed, 5 days of fermentation, and the initial pH value of the fermentation is 5;

Fermentation products obtained from different carbon sources or nitrogen sources were collected, and milk curdling activity MCA was detected according to one of the above methods.

Take the blank without adding carbon source or nitrogen source for comparison.

The results are shown in Figure 2, 1: 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, can It can be seen that the addition of carbon sources, especially glucose, has a certain promoting effect on the production of chymosin by fermentation of koji; the addition of nitrogen sources does not promote the curdling activity of chymosin produced by fermentation of koji, and some nitrogen sources such as ammonium sulfate etc. will even reduce the milk-clotting activity of distiller's yeast.

The above results indicated that different factors had little effect on the proteolytic activity, but had a more obvious effect on the curdling activity. With the change of each factor, the proteolytic activity was lower. With the increase of distiller's koji addition and the prolongation of fermentation time, the curdling activity increased.

Analyzing the influence of the above single factors, among them the three factors of distiller's koji inoculum, fermentation time and shaker speed have the most significant influence on the curdling activity, so these three factors are selected for response surface analysis, as shown in Figure 3, where (A) Surface plot and contour plot of the effect of koji inoculum amount and fermentation time on curd activity; (B) Surface plot and contour plot of the influence of koji inoculum amount and shaker speed on curd activity; (C) Fermentation time The surface plot and high contour plot of the influence of shaker speed on curdling activity (Figure 3).

Response surface analysis obtained a regression equation with curdling activity as the response value: curdling activity (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 the formula, A—inoculation amount of distiller’s yeast, %; B—fermentation time, h; C—shaker speed, rpm.

The optimum fermentation conditions obtained were 5.7% (mass percentage) of koji inoculum, 72 hours of fermentation time and 120 rpm of shaker speed, and the theoretically predicted rennet activity of the fermentation product was 52.29 Su/mL;

Therefore, the inoculum amount of koji, fermentation time and shaker speed are the most important optimization factors.

2. Fermentation under the best conditions

Add the distiller's koji C and water obtained in the above-mentioned step 1 to the glutinous rice base, mix evenly, and ferment;

Distiller's koji C is added according to the mass percentage of 5.7% (distiller's yeast quality and glutinous rice matrix mass percentage);

Water is added according to mass percentage composition 40% (water quality and glutinous rice substrate mass percentage);

The above fermentation conditions are 30°C, 120rpm shaker speed, 72h fermentation, and the initial pH value of fermentation is 5;

The fermentation product was collected, and the curdling activity MCA was detected according to the above-mentioned method. The result was that the curdling activity of chymosin in the fermentation product was 51.05 Su/mL, which was close to the theoretical prediction value (52.29 Su/mL).

Note: The single factor analysis is done with the fermentation time as the variable, and the other factors remain unchanged. At this time, the amount of distiller’s yeast added is fixed at 3%, and the fermentation time is the highest when the fermentation time is 5 days. The response surface analysis is to analyze 3 variables at the same time The interaction between them, when the amount of distiller's yeast added reaches 5.7%, the fermentation time only needs 3 days to reach the highest.

Embodiment 2, the preparation of chymosin

1. Fermentation

Preparation of glutinous rice matrix: 500 g of fresh glutinous rice was washed and soaked for 3 hours, added 1 L of distilled water, steamed at normal pressure for 0.5 h, and cooled to obtain 1000 g of glutinous rice matrix;

Add the distiller's koji C and water obtained in the above-mentioned step 1 to the glutinous rice base, mix evenly, and ferment;

Distiller's koji C is added according to the mass percentage of 5.7% (distiller's yeast quality and glutinous rice matrix mass percentage);

Water is added according to mass percentage composition 40% (water quality and glutinous rice substrate mass percentage);

The above fermentation conditions are 30°C, 120rpm shaker speed, 72h fermentation, and the initial pH value of fermentation is 5;

Collect the fermentation product, filter (4 layers of gauze), collect the filtrate, which is the filtrate containing rennet, and refrigerate at 4°C for later use.

2. Rough extraction of chymosin by fractional precipitation with ethanol

Take out the rennet-containing filtrate prepared in the above 1 and the absolute ethanol stored in the laboratory at 4°C, and operate as follows: (1) Add absolute ethanol to the rennet-containing filtrate prepared in the above 1 to make The mixed ethanol volume fraction is 50%, 4°C, centrifuged at 5000r/min for 30 minutes, collect the supernatant and precipitate, dissolve the protein precipitate with distilled water, and put it in a 4°C refrigerator for later use; (2) The supernatant obtained in (1) Continue to add absolute ethanol so that the volume fraction of ethanol is 60%, centrifuge at 5000r/min at 4°C for 30 minutes, collect the supernatant and precipitate, dissolve the protein precipitate, and put it in a refrigerator at 4°C for later use; (3) the supernatant obtained in (2) Continue to add absolute ethanol to make the ethanol volume fraction 70%, centrifuge at 5000r/min at 4°C for 30 minutes, dissolve the protein precipitate, and put it in a refrigerator at 4°C for later use; (4) continue to add the supernatant obtained in (3) Make the volume fraction of ethanol to 80% with absolute ethanol, centrifuge at 5000r/min for 30 minutes at 4°C, dissolve the protein precipitate, and put it in a refrigerator at 4°C for later use;

The milk-clotting activity of protein precipitation in the above four steps was measured respectively.

The results are shown in Figure 4, 1: protein standard; 2: 50% ethanol precipitated protein; 3: 60% ethanol precipitated protein; 4: 70% ethanol precipitated protein; 5: 80% ethanol precipitated protein, indicating that only steps (3 ), that is, the protein obtained by precipitation with 70% ethanol has obvious curdling activity.

SDS-PAGE electrophoresis analysis was performed on the proteins precipitated by 50%, 60%, 70%, and 80% ethanol respectively. The results are shown in Figure 4. 80% of the precipitated proteins were more small molecular proteins, and 70% of the ethanol precipitated proteins appeared A protein band that is different from other protein precipitates, with a molecular weight between 34-43kD, is likely to be the target protein.

The above experiment proves that the protein precipitated by 70% ethanol is crude rennet, which is vacuum freeze-dried to obtain crude rennet dry powder.

3. Sephadex G75 purified crude chymosin

Prepare the crude rennet dry powder obtained in the above 2 into a 0.2g/mL solution, and the solvent is 0.05mol/L PBS solution with pH5.8 (recipe: NaCl 35g, KCl 1g, KOH 1.2g, K ₂ HPO ₄ 9g , add distilled water to 1L, adjust the pH to 5.8 with acetic acid), pass through a Sephadex G75 column (2.6×60cm), use 0.05mol/L PBS solution of pH 5.8 as the elution buffer for elution, and the sample volume is 4mL , with a flow rate of 1 mL/min, measure the curdling activity of each protein peak, and collect the peaks with curdling activity.

The results are shown in Fig. 5A. It can be seen that the target peak was obtained at the retention time of 100-130 min.

The eluted product corresponding to the target peak was collected, desalted and concentrated by a 10kD ultrafiltration centrifuge tube, freeze-dried into powder, and subjected to SDS-PAGE electrophoresis analysis. The results are shown in Figure 5B. 1 is the protein standard and 2 is the target protein, indicating that , the obtained protein is a single band with a calculated molecular weight of about 39.7kD, which is the target chymosin.

Embodiment 2, research on enzymatic characteristics of chymosin

The target chymosin obtained by purifying in Example 1 is used for detection below.

1. Optimum temperature

Measure the curdling activity of the purpose chymosin purified in Example 1 at 25, 30, 35, 40, 45, 50, and 55°C respectively. %, to calculate the relative curdling activity.

The results are shown in Figure 6A, indicating that the rennet has good curdling activity at 35°C, and the curdling activity is basically lost when the temperature is higher than 45°C.

2. The optimum pH value

Adjust the pH of the skim milk with NaOH or HCl of 0.1mol/L, measure and measure the target rennet coagulation obtained by purifying in Example 1 when the pH is 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 respectively Milk activity, the method is the same as that of Example 1, with the milk curd activity at pH 5.8 being 100%, the relative milk curd activity is calculated.

The results are shown in Fig. 6B, indicating that the curdling activity increases with the decrease of pH, and the curdling activity reaches the highest at pH 5.4.

3. Effect of metal ions on enzyme activity

In skim milk, add NaCl, KCl, MgCl ₂ , CaCl ₂ , FeCl ₂ , AlCl ₃ , CuCl ₂ , ZnCl ₂ respectively to make the concentration 10 mmol/L, and add the target chymosin clotting obtained from purification in Example 1 for determination. Milk vigor, the method is the same as that of Example 1, with the curdling vigor of the blank control (BC) as 100%, the relative curdling vigor is calculated.

The results are shown in Fig. 6C. The results showed that Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ and Al ³⁺ could promote the rennet curd, especially the effect of Ca ²⁺ was the most obvious.

Purified chymosin was vacuum freeze-dried to make enzyme preparation.

Claims (10)

1. A method for preparing rennet, comprising the steps of: mixing distiller’s yeast, glutinous rice substrate and water, fermenting, and collecting fermentation products to obtain rennet; The curdling activity of the distiller's yeast is 4.90±0.6Su/mL; The glutinous rice matrix is prepared according to the following method: mix glutinous rice and water, and heat to obtain the glutinous rice matrix. 2. The method according to claim 1, characterized in that: The mass ratio of the distiller's yeast to the glutinous rice base is 3-6:100, and the mass ratio of the distiller's yeast to the glutinous rice base is specifically 5.7:100. 3. The method according to claim 1 or 2, characterized in that: the fermentation time is 5-7 days, specifically 72 hours. 4. according to the method described in any one in claim 1-3, it is characterized in that: described fermentation adopts the mode of shaking table vibration, and the rotating speed of described shaking table is 80-180rpm, and the rotating speed of described shaking table is specifically 120rpm . 5. The method according to any one of claims 1-4, characterized in that: The temperature of the fermentation is 20-35°C, and the temperature of the fermentation is specifically 30°C; The initial pH value of the fermentation system is 4.5-6.5, and the initial pH value of the fermentation system is 5.0. Described water and described glutinous rice matrix mass ratio are 50:100; The glutinous rice matrix is made of the glutinous rice and water according to the ratio of 500 g of the glutinous rice: 1 L of water. 6. The method according to any one of claims 1-5, characterized in that: After the fermentation product is collected, the following steps are also included: 1) filtering the fermentation product, collecting the filtrate to obtain a filtrate containing rennet; 2) adding ethanol to the filtrate containing rennet to obtain a mixed system A, so that the volume percentage of ethanol in the mixed system A is 50%, and then collecting the supernatant; 3) adding ethanol to the supernatant to obtain a mixed system B, so that the volume percentage of ethanol in the mixed system B is 60%, and centrifuging to collect the supernatant; 4) Add ethanol to the supernatant obtained in 3) to obtain a mixed system C, make the volume percentage of ethanol in the mixed system C be 70%, and collect the precipitate by centrifugation. 7. The method according to claim 6, characterized in that: After the precipitation is collected, the following steps are also included: dissolving the precipitation and performing gel column chromatography to collect the eluate to obtain chymosin. 8. The method of claim 7, wherein: The gel column medium of the gel column chromatography is Sephadex G75; The elution buffer used in the gel column chromatography is a PBS solution with a concentration of 0.05 mol/L and a pH value of 5.8. 9. The chymosin prepared by the method according to any one of claims 1-8; Or a rennet preparation prepared from said rennet. 10. Chymosin according to claim 9, characterized in that: The chymosin has at least one enzymatic characteristic in the following 1)-3): 1) The optimal reaction temperature of the chymosin is 35°C, 2) The optimum reaction pH value of the rennet is 5.4; 3) The activity of the chymosin can be promoted by Ca ²⁺ .