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Calf rennet in foreign development and use of the earlier, but because of dry the casein production increased twelvemonth by twelvemonth, the increasing demand on the enzyme to be available.Calf after completing the production of meat, the diminishing figure of the current The research focuses on microbic rennet and recombinant rennin unfastened Fat use.

Cheese production in China is in its babyhood, cheese production Rennet chiefly rely on imports, and the monetary value is expensive, restricting my State cheese production development. Therefore, this survey relies on equal natural stuffs in China Resources, the usage of a slaughtered calf ( made serum ) Scraps – fourth stomach Raw stuffs, infusions of calf rennet, to replace imported rennet, with 10 Important practical significance.

Materials and Methods

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1.1 Materials

5 sucking optional 2-week-old calf, after butchering take fourth stomach, Clear contents, stripped of fat and connective tissue, hanging in a cool ventilated At prohibitionist. When used with a pulverizer pulverization into a pulverization, placed in a brown bottle

Set aside.

1.2 Extraction Method

Of dried fourth stomachs pulverization, plus 15 times of distilled H2O, with different concentrations of

Salt, intoxicant extracted at different pH 5 vitamin D, and so in the 3 000 R / min centrifugation 15 min, take drosss, with 1 mol / L Of HCl to set pH to 4.6, is activated at room temperature for 12 H, measured

Cloting activity.

1.3 Determination of Enzyme Activity

Ar ima method [ 5 ] , take 5 milliliters 100 g / L skim Milk, incubated at 35 a„? for 10 min, 5 milliliter of rennet solution,

Mixed quickly, accurately record from the enzyme solution was added to the milk curdling clip ( S ) , 40 min condensation of 1 milliliter of 100 g / L skimmed milk the sum of enzyme given

Defined a Soxhlet unit ( So xhlet unit, SU ) [ 6 ] .

Soxhlet unit ( SU ) = 2 400


– 50


– Calciferol

Wherein, T is the the curds clip ( s ) ; D is the dilution multiples.

2 Consequences and Analysis

The pick of salt concentration in the leaching solution of 2.1.Adding salt in the leaching solution, so that the mass concentration of 0, 20, 40, 60, 80, 100, 120 g / L, and the leaching consequences are shown in Table 1.

As can be seen from Table 1, the salt concentration of the extracted fourth stomach enzyme.The milk coagulating activity has a great influence. When the salt mass concentration is less than 40 g / L, along with the addition in the mass concentration of the rennet curd activity Increases ; seawater in the concentration of 40 ~ 60 g / L, rennet

Cloting activity of up to 1.01 to 1 12 SU after quality over salt

Ten [ Received Date ] 2001-07-12 ottle Set aside.

The volume and concentration, enzyme activity decreased bit by bit, which is chiefly concerned with rennet in a salt solution, the solubility of.

Table 1 salt concentration on the consequence of rennet infusion.

T able 1aˆˆInfluence of salt carbon monoxide ncentr ation on the consequence O degree Fahrenheit extr moving calf R ennet

e?Yc›?e??e‡??µ“a?¦/ ( g·L- 1 )

S Al T concent rat ion 0 20 40 60 80 100 120



E nzyme activity 0.30 0.81 1.12 1. 01 0. 70 0. 59 0. 55

Note: the figure of repeats n = 5. With the following tabular array.

2.2 Extracts the intoxicant concentration in the select

With anhydrous ethyl alcohol infusions dubbed volume fraction of 0, 3 % , Of 6 % , 9 % , 12 % , 15 % intoxicant solution fourth stomach extraction, Consequences

Consequences are shown in Table 2.

Table 2aˆˆInfluence of alcoho cubic decimeter carbon monoxide ncentr atio n O n the consequence of ex tr moving calf rennet

Alcohol concent rat ion 0 3 6 9 12 15

E nzyme act ivit y 0. 30 0. 28 0. 26 0. 26 0. 25 0. 24

Seen from Table 2, the intoxicant concentration on extraction of rennet curd Activity Little consequence, as the intoxicant concentration additions, extract abomasum enzyme activity A downward tendency. And compared with the control group ( without intoxicant ) , enzyme curd Decreased activity, consequences and Li Li Chiu et Al [ 7 ] reported consequences make non Consistent with Wu Yong thick [ 8 ] reported lamb fourth stomach enzyme extraction consequences.

2.3 The pick of the leaching solution pH

With 0.1 mol / L HCl extracts dubbed pH 1.0, 2.0, 3.0, 4.0, 5.0, 6. 0, 7.0 leaching trial. The consequences are shown in Table 3.

T able 3aˆˆInfluence of pH on the consequence of ex tr moving calf R ennet

pH 1. 0 2. 0 3. 0 4. 0 5. 0 6. 0 7. 0

E nzyme act ivit y 0. 79 1. 02 1. 20 0. 91 0. 42 0. 35 0. 33

Table 3 shows the the rennet coagulating activity of the leaching solution pH and extraction


When the pH of the leaching solution 3:00 extract rennet liquid rennet The activity of up to 12,000 SU ; infusions of pH less than 3, with the pH additions, the addition as enzyme curdling activity ; When the pH is greater than 3:00, Enzyme coagulating activity decreased quickly. Description extracted at lower pH fourth stomach Enzyme higher output can be obtained, but the pH should non be excessively low, otherwise it will Citation From the enzyme protein denaturation, loss of verve.

2.4 The fourth stomach activity of extraction temperature of The fourth stomach at 15, 20, 25, 30, 35, 40, 45 a„?

50 g / L of the salt is leached, and the consequences are shown in Table 4.

Inf luence O f temperat ur vitamin E on the consequence O degree Fahrenheit ext racting calf R ennet a„?

T em perature 15 20 25 30 35 40 45


E nzyme act ivit y 1. 01 1. 05 1. 12 1. 21 1. 10 0. 93 0. 81

Can be seen from Table 4, when the extraction at 30 a„? abomasum enzyme activity The highest ; extraction temperature is below 30 & A ; deg ; C, with the extraction temperature increased

Due to the diffusion velocity of the enzyme molecule, the solubility of the enzyme is increased, so that The curdling activity improved extraction fourth stomach enzyme ; However, when the extraction temperature is greater than 30 a„? , enzyme activity decreased bit by bit, this may be with an enzyme for a long clip at higher Temperature under denaturing related.

2.5 extract proficient parametric quantities determine

On the footing of the above extraction trial, in order to analyze the multiple factorsCombined consequence of the salt mass concentration, intoxicant concentration, the pH of the leaching solution, The extraction temperature factors extract the the rennet coagulating activity to prove indexs, Four factors and three degrees of L9 ( 34 ) extraneous trial consequences are shown in Table 5.

The trial consequences ( Table 5 ) show that the extracted factors calf rennet Clotting activity had a important consequence, within the trial ranges, salt quality Concentration of really hapless up to 0.39, followed by the extraction temperature and alcoholic.The lower limit grades, pH value, the sequence is a salt concentration & A ; gt ; Alcohol concentration & A ; gt ; Extraction temperature and pH of the leaching solution.

To calf rennet pull outing more intuitive to detect a assortment of factors The consequence of experimental factors the abscissa, infusion of calf rennet The mean milk-clotting activity the ordinate as factors and trial indexs diagram.

Fig. 1aˆˆRelatio nship betw een ex tr actio n fact or s a nd calf R ennet act ivity

Graph losing see the paper

As can be seen from Figure 1, the calf rennet pull out the best proficient parametric quantities Number A2B3C2D1, extracts salt concentration of 50 g / L, pH 4, extraction temperature of 30 & A ; deg ; C, without intoxicant infusions

Infusion of calf rennet curd highest activity. To verify the consequences, 50 g / L saline, pH adjusted to 4.0 at 30 a„? calf wrinkled Stomach extraction, Get rennet coagulating activity of 16100 SU

Prove extract proficient parametric quantities is sensible and executable, and can be applied in the existent production.

3 Discussion

Infusions salt mass concentration is a major factor impacting the extraction consequence Prime 1. The rennet leaching consequence depends on its dissolution in the solution Solution, and the solubility of the size depending on the ionic strength of the solution, when immersed.Extract salt mass concentration of 50 g / L or less concentrated in the low-quality

Degrees impersonal salt solution, the charge of ions with the surface of the enzyme molecule interaction

With the formation of ionic bed, increases the solubility of the enzyme, but when the salt concentration.Is excessively high, the enzyme molecules and ions compete for H2O molecules, taking to the enzyme molecules tabular array

he surface of the hydration bed is destroyed, thereby cut downing the solubility of the enzyme, salting now appears

The elephant [ 9 ] . Therefore merely command the appropriate salt concentration conditions Greatest extent possible to fade out the enzyme, and to better the extraction efficiency.

About extract liquid intoxicant concentration in the extraction consequence Big contention. Some studies [ 10 ] , add intoxicant can pull out.Improve the output of rennet, but the consequences of this survey, along with intoxicant Tim.Plus increasing the sum of the extract enzyme curdling activity but reduced, which may be Is due to an organic dissolver to demobilize the enzyme caused. Kleiner [ 11 ] believe curd

The enzyme easy ethanol modified with ethyl alcohol as the organic dissolver precipitation rennet, Can for good inactivated enzyme. Friedenthal [ 12 ] that the organic dissolvers particular.Are more easy in a high concentration and high temperature conditions to demobilize the enzyme. enzyme.

The pH of the leaching solution is chiefly to impact the solubility and stableness of the enzyme. Enzyme molecule when the enzyme is at its isoelectric point the figure of net charge is zero, so the enzyme Lowest solubility, the solution is non easy to be leached out, but extract pH Besides should non be excessively low, otherwise the enzyme protein at low pH stableness is reduced, Laid, In the presence of NaCl under conditions prone self-digestion [ 12 ] , the enzyme activity. Reduced. Extraction rennet off when taking into history the enzyme isoelectric point, At the same clip, we should besides pay attending to the stableness of the enzyme at different pH values.Extraction temperature chiefly affects the rate of diffusion of the enzyme, when the extraction temperature Is high, the diffusion velocity of the enzyme to speed up the enzyme increased solubility, but extraction The temperature should non be excessively high, otherwise the enzyme under the conditions of high temperature for a long clip variableness Inactivation.