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Calf rennet in foreign development and utilization of the earlier, but because of dry the casein production increased year by year, the increasing demand on the enzyme to be available .Calf after finishing the production of meat, the decreasing number of the current The research focuses on microbial rennet and recombinant chymosin open Fat utilization.
Cheese production in China is in its infancy, cheese production Rennet mainly rely on imports, and the price is expensive, limiting my State cheese production development. Therefore, this study relies on adequate raw materials in China Resources, the use of a slaughtered calf (made serum) Scraps - abomasum Raw materials, extracts of calf rennet, to replace imported rennet, with 10 Important practical significance.
Materials and Methods
5 sucking optional 2-week-old calf, after slaughtering remove abomasum, Clear contents, stripped of fat and connective tissue, hanging in a cool ventilated At dry. When used with a pulverizer powder into a powder, placed in a brown bottle
1.2 Extraction Method
Of dried abomasums powder, plus 15 times of distilled water, with different concentrations of
Salt, alcohol extracted at different pH 5 d, and then in the 3 000 r / min centrifugation 15 min, remove impurities, with 1 mol / L Of HCl to adjust pH to 4.6, is activated at room temperature for 12 h, measured
1.3 Determination of Enzyme Activity
Ar ima method , take 5 mL 100 g / L skim Milk, incubated at 35 â„ƒ for 10 min, 5 mL of rennet solution,
Mixed rapidly, accurately record from the enzyme solution was added to the milk coagulation time (S), 40 min condensation of 1 mL of 100 g / L skimmed milk the amount of enzyme given
Defined a Soxhlet unit (So xhlet unit, SU) .
Soxhlet unit (SU) = 2 400
Wherein, T is the the curds time (s); D is the dilution multiples.
2 Results and Analysis
The choice 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 results are shown in Table 1.
As can be seen from Table 1, the salt concentration of the extracted abomasum enzyme .The milk clotting activity has a great influence. When the salt mass concentration is less than 40 g / L, along with the increase in the mass concentration of the rennet curd activity Increases; brine in the concentration of 40 ~ 60 g / L, rennet
Clotting activity of up to 1.01 to 1 12 SU after quality over salt
X [Received Date] 2001-07-12 ottle Set aside.
The volume and concentration, enzyme activity decreased gradually, which is mainly concerned with rennet in a salt solution, the solubility of.
Table 1 salt concentration on the effect of rennet extract.
T able 1ã€€Influence of salt co ncentr ation on the effect o f extr acting calf r ennet
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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 number of repetitions n = 5. With the following table.
2.2 Extracts the alcohol concentration in the select
With anhydrous ethanol extracts dubbed volume fraction of 0, 3%,Of 6%, 9%, 12%, 15% alcohol solution abomasum extraction, Results
Results are shown in Table 2.
Table 2ã€€Influence of alcoho l co ncentr atio n o n the effect of ex tr acting 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 alcohol concentration on extraction of rennet curd Activity Little effect, as the alcohol concentration increases, extract abomasum enzyme activity A downward trend. And compared with the control group (without alcohol), enzyme curd Decreased activity, results and Li Li Chiu et al  reported results do not Consistent with Wu Yong thick  reported lamb abomasum enzyme extraction results.
2.3 The choice 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 test. The results are shown in Table 3.
T able 3ã€€Influence of pH on the effect of ex tr acting 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 clotting 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; extracts of pH less than 3, with the pH increases, the increase as enzyme clotting activity; When the pH is greater than 3:00, Enzyme clotting activity decreased rapidly. Description extracted at lower pH abomasum Enzyme higher yield can be obtained, but the pH should not be too low, otherwise it will Citation From the enzyme protein denaturation, loss of vitality.
2.4 The abomasum activity of extraction temperature of The abomasum at 15, 20, 25, 30, 35, 40, 45 â„ƒ
50 g / L of the salt is leached, and the results are shown in Table 4.
Inf luence o f temperat ur e on the effect o f ext racting calf r ennet â„ƒ
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 â„ƒ abomasum enzyme activity The highest; extraction temperature is below 30 ° C, with the extraction temperature increased
Due to the diffusion speed of the enzyme molecule, the solubility of the enzyme is increased, so that The clotting activity improved extraction abomasum enzyme; However, when the extraction temperature is greater than 30 â„ƒ, enzyme activity decreased gradually, this may be with an enzyme for a long time at higher Temperature under denaturing related.
2.5 extract technical parameters determine
On the basis of the above extraction test, in order to examine the multiple factorsCombined effect of the salt mass concentration, alcohol concentration, the pH of the leaching solution,The extraction temperature factors extract the the rennet clotting activity to test indicators,Four factors and three levels of L9 (34) orthogonal test results are shown in Table 5.
The test results (Table 5) show that the extracted factors calf rennet Clotting activity had a significant effect, within the test ranges, salt quality Concentration of very poor up to 0.39, followed by the extraction temperature and alcoholic .The minimum degrees, pH value, the sequence is a salt concentration> Alcohol concentration>Extraction temperature and pH of the leaching solution.
To calf rennet extracting more intuitive to observe a variety of factors The effect of experimental factors the abscissa, extract of calf rennet The average milk-clotting activity the ordinate as factors and test indicators diagram.
Fig . 1ã€€Relatio nship betw een ex tr actio n fact or s a nd calf r ennet act ivity
Graph missing see the paper
As can be seen from Figure 1, the calf rennet extract the best technical parameters Number A2B3C2D1, extracts salt concentration of 50 g / L, pH 4, extraction temperature of 30 ° C, without alcohol extracts
Extract of calf rennet curd highest activity. To verify the results, 50 g / L saline, pH adjusted to 4.0 at 30 â„ƒ calf wrinkled Stomach extraction, Get rennet clotting activity of 16100 SU
Prove extract technical parameters is reasonable and feasible, and can be applied in the actual production.
Extracts salt mass concentration is a major factor affecting the extraction effect Prime one. The rennet leaching effect depends on its dissolving 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 neutral salt solution, the charge of ions with the surface of the enzyme molecule interaction
With the formation of ionic layer, increases the solubility of the enzyme, but when the salt concentration .Is too high, the enzyme molecules and ions compete for water molecules, leading to the enzyme molecules table
he surface of the hydration layer is destroyed, thereby reducing the solubility of the enzyme, salting now appears
The elephant . Therefore only control the appropriate salt concentration conditions Greatest extent possible to dissolve the enzyme, and to improve the extraction efficiency.
About extract liquid alcohol concentration in the extraction effect Big controversy. Some reports , add alcohol can extract .Improve the yield of rennet, but the results of this study, along with alcohol Tim .Plus increasing the amount of the extract enzyme clotting activity but reduced, which may be Is due to an organic solvent to inactivate the enzyme caused. Kleiner  believe curd
The enzyme easily ethanol modified with ethanol as the organic solvent precipitation rennet, Can permanently inactivated enzyme. Friedenthal  that the organic solvents special .Are more easily in a high concentration and high temperature conditions to inactivate the enzyme. enzyme.
The pH of the leaching solution is mainly to affect the solubility and stability of the enzyme. Enzyme molecule when the enzyme is at its isoelectric point the number of net charge is zero, then the enzyme Lowest solubility, the solution is not easy to be leached out, but extract pH Also should not be too low, otherwise the enzyme protein at low pH stability is reduced, Laid , In the presence of NaCl under conditions prone autolysis , the enzyme activity. Reduced. Extraction rennet away when taking into account the enzyme isoelectric point,At the same time, we should also pay attention to the stability of the enzyme at different pH values.Extraction temperature mainly affects the rate of diffusion of the enzyme, when the extraction temperature Is high, the diffusion speed of the enzyme to accelerate the enzyme increased solubility, but extraction The temperature should not be too high, otherwise the enzyme under the conditions of high temperature for a long time variability Inactivation.