阅读说明：本技术 一种改善凝乳酶热稳定性的保护剂及方法 (Protective agent and method for improving heat stability of chymosin ) 是由 武彬 于 2019-09-29 设计创作，主要内容包括：本发明属于酶工程技术领域,具体涉及一种改善凝乳酶热稳定性的保护剂及方法。所述保护剂,由以下质量配比的成分组成为葡萄糖：氯化钙：牛血清蛋白为40：0.5-1.0：0.1-0.25。本发明提供了一种改善凝乳酶热稳定性的保护剂,该保护剂各组份间彼此之间存在相互作用,提高凝乳酶热稳定性的作用效果显著。通过调节三种保护剂的比例,凝乳酶10-15min失活的温度从58℃提高到80℃,一共提高了22℃,远高于单一保护剂组分的65-71℃。(The invention belongs to the technical field of enzyme engineering, and particularly relates to a protective agent and a method for improving the thermal stability of chymosin. The protective agent comprises the following components in parts by mass: calcium chloride: bovine serum albumin is 40: 0.5-1.0: 0.1-0.25. The invention provides a protective agent for improving the thermal stability of rennin, and the components of the protective agent interact with each other, so that the effect of improving the thermal stability of the rennin is obvious. By adjusting the proportion of the three protective agents, the inactivation temperature of the rennin within 10-15min is increased from 58 ℃ to 80 ℃, the inactivation temperature is increased by 22 ℃ in total and is far higher than 65-71 ℃ of the single protective agent component.)

1. The protective agent for improving the thermal stability of chymosin is characterized by comprising the following components in parts by mass: calcium chloride: bovine serum albumin is 40: 0.5-1.0: 0.1-0.25.

2. A method for treating chymosin with the protecting agent of claim 1, which comprises: adding chymosin powder or chymosin solution into the protectant solution at 0-20 deg.C, stirring, and dissolving and mixing chymosin completely.

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to a protective agent and a method for improving the thermal stability of chymosin.

Background

Chymosin is an acidic protease, and is suitable for hydrolyzing proteins under acidic conditions to promote protein coagulation. The digestive system of young animals is not developed completely, the secretion of digestive enzymes is insufficient, and the absorption rate of nutrients in the feed is low. The protease preparation is added into the feed, so that the digestibility of the protein component in the feed for animals, particularly young animals, is improved, and the growth of the animals is promoted. The utilization rate of protein in the feed is improved, so that not only can feed resources be saved and the breeding cost be reduced, but also the ammonia nitrogen emission of animal wastes can be reduced, and the pollution of feeding to the environment is reduced.

The thermal stability and storage stability of the enzyme are key factors influencing the application of the enzyme in the fields of food and medicine. Therefore, it is necessary to develop a study for improving the thermal stability of chymosin. There are a number of ways to improve the thermostability of an enzyme, including: the method comprises the steps of immobilization, chemical modification, protein engineering and the like, wherein sodium alginate and other embedding agents are required to be introduced for immobilization, a large amount of impurities are introduced when the method is used for cheese production, and the method is not suitable for cheese production. Chemical modification can introduce chemical agents that are not edible. Protein engineering methods also present a number of difficulties. Firstly, the design and modification of the chymosin gene and the molecular structure make it very difficult to design enzyme molecules with high thermal stability; and secondly, improving the molecular structure of the stable enzyme may cause the reduction of the catalytic activity of the rennin and other problems. Thirdly, the chymosin is extracted from the abomasum of the calf to obtain a high molecular structure of the chymosin, and transgenic operation needs to be carried out on the bovine embryo. Therefore, it is difficult to improve the thermal stability of rennet for cheese production by immobilization, chemical modification and protein engineering. While the heat stability of protease is generally low, a large number of literatures show that the heat treatment temperature of chymosin inactivated for 10-15min is generally 55-70 ℃, and particularly that the heat resistance of the enzyme is difficult to be continuously improved on a certain basis.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the protective agent for improving the thermal stability of the chymosin, and the components of the protective agent interact with each other, so that the effect of improving the thermal stability of the chymosin is obvious.

In order to achieve the effect, the invention adopts the following technical scheme:

a protective agent for improving the thermal stability of rennin comprises the following components in parts by mass: 0.02Mol/LpH6.0 KH₂PO₄-K₂HPO₄Buffer solution: glucose: calcium chloride: bovine serum albumin is 58.75-59.4: 40: 0.5-1.0: 0.1-0.25. .

The method for treating chymosin by the protective agent comprises the following steps: at 0-20 deg.C, adding 1 part of chymosin powder or chymosin solution into more than 100 parts of protectant solution, and stirring to dissolve and mix chymosin completely.

Advantageous effects

The invention provides a protective agent for improving the thermal stability of rennin, and the components of the protective agent interact with each other, so that the effect of improving the thermal stability of the rennin is obvious. By adjusting the proportion of the three protective agents, the inactivation temperature of the rennin within 10-15min is increased from 58 ℃ to 80 ℃, the inactivation temperature is increased by 22 ℃ in total and is far higher than 65-71 ℃ of the single protective agent component.

Detailed Description

The present invention will be further illustrated by the following examples and comparative examples, but the present invention is not limited to the following examples.

The chymosin activity was measured as in the following examples

The activity of the chymosin is measured by adopting an Arima method: chymosin was dissolved in 0.02Mol/L K2HPO4-KH2PO4 buffer pH6.0 (all solutions were tested in the following to use this buffer system). 0.5ml of chymosin liquid is preheated at 35 ℃ for 5min, added into 5ml of fully preheated 10% skim milk containing 1.0% CaCl2, immediately mixed evenly and counted until particles appear on the tube wall, and the curd time (T) is recorded. Taking the enzyme amount of 1ml of cow milk curded at 35 ℃ for 40min as an enzyme activity unit (1U), and the calculation formula of the chymosin activity is as follows:

in the formula: a is rennin activity; t is the curd time, and n is the dilution factor.

The protective agent for improving the thermal stability of chymosin in the following examples and comparative examples consists of glucose, calcium chloride and bovine serum albumin in the following mass percentages, and the rest is 0.02Mol/L pH6.0 KH₂PO₄-K₂HPO₄The buffer solution is added into 3mg of rennin, the starting enzyme activity is 600IU/ml, and the proportion of the protective agent used in each embodiment and the comparative example and the residual enzyme activity after the protective agent is subjected to water bath heat preservation at 75 ℃ for 15min are shown in Table 1.

TABLE 1

From the above table, it can be seen that: glucose concentration is 40%, calcium chloride concentration is 0.5% -1.0%, bovine serum concentration is 0.1-0.25%, and the three protective agents are used as protective agents of enzyme and have interaction with each other. The protective effect of the three protective agents on the rennin heat stabilizer does not increase linearly with the concentration of the rennin heat stabilizer, and the thermal protection effect is reduced when the concentration of the protective agent is increased under partial conditions, so that the combination of the protective agents is not easy to find.

Comparison of protective Effect of the combinations of protective Agents with the respective Individual Components

Because different protective agents have great influence on the thermal stability of the rennin, different heat preservation temperatures are selected, the inactivation time process curve of the enzyme is determined by controlling the temperature to be close to the heat treatment time (10-15min) as much as possible, the composite thermal protective agent has obvious improvement on the thermal stability of the rennin, and specific results are shown in tables 2, 3 and 4.

TABLE 2 influence of blank control group and bovine serum albumin group on heat stabilizer of chymosin

The heat protection effect of bovine serum albumin on chymosin is weak, according to the results in table 2, the heat protection effect of 0.25% bovine serum albumin solution on chymosin is weak, and the heat stability of chymosin can only be improved by about 2 ℃ under the concentration of 0.5-1.0%. Therefore, when screening for thermal stabilizers is generally performed, bovine serum albumin may not be selected as a protectant component. In particular, the results of the single factor experiment of bovine serum albumin show that concentrations above 0.5% are effective, while the results in Table 1 show that the optimal concentration range of bovine serum is 0.1% -0.25%, which makes bovine serum albumin neglected more easily. However, the results of comparative example 3 and example 1 in Table 1 show that bovine serum albumin is effective as a component of the heat stabilizer and the residual enzyme activity is improved by more than 20%.

TABLE 3 Effect of different concentrations of calcium chloride on the thermal stability of chymosin

The results in Table 3 show that the thermal protection of rennin by calcium chloride is not very relevant to its concentration, and that a calcium chloride solution with a concentration of 0.1% to 2% can raise the inactivation temperature of rennin from 58 ℃ to about 65 ℃. However, when calcium chloride is compounded with glucose and bovine serum albumin to form a protective agent, interaction exists between calcium ions and glucose and between calcium ions and bovine serum albumin, and the optimal concentration of the composite protective agent is obviously influenced. The results in table 1 show that the optimum concentration of calcium chloride in the composite protectant is 0.5% -1.0%; comparative examples 8, 11 and 16 in which the calcium chloride concentration was 1.5%, examples in which the residual enzyme activities after heat treatment were less than 0.5% and 1.0%. At calcium chloride concentrations of 0.1% and 0.25%, bovine serum albumin interacts with calcium chloride and is insufficient to provide adequate protection for rennin. When the formula of the composite protective agent is optimized, the protective agent with lower concentration is selected as much as possible under the condition of ensuring the protective effect. Therefore, the calcium chloride concentration will typically be selected to be 0.1% to 0.25%. As mentioned above, the optimization of the formula of the protective agent is not easy to find and select the bovine serum albumin, and the influence of the bovine serum albumin on the effective concentration of the calcium chloride is more difficult to find in the optimization process of the part, so that the calcium chloride with the concentration of 0.1-0.25% is likely to be directly selected, but not 0.5-1.0% required by the protective agent.

TABLE 4 Effect of glucose and thermal protector on chymosin thermal stability

The results in Table 4 show that at the inactivation temperature of rennin for 10-15min, the protective agent group reached 80 ℃ while the 40% glucose group was maintained at 71 ℃ for 7 min. According to the results in tables 2, 3 and 4, the protective agent has a very significant effect, greatly increases the inactivation temperature of rennin, and is difficult to compare favourably with the protective agent made of pure glucose or calcium chloride.

TABLE 565 ℃ residual enzyme Activity results for chymosin incubated in different solutions for different times

The pasteurization condition is usually 62-65 deg.C for 30min, or 75-90 deg.C for 15-16 s. At 65 ℃, chymosin is quickly inactivated in bovine serum solution and calcium chloride solution, and the activity loss of the chymosin is 43 percent in 40 percent glucose solution after heat preservation for 30min, but the activity loss of the chymosin in the protective agent is less than 20 percent under the same condition. The protective agent greatly improves the heat stability of the rennin and can completely meet the requirement of pasteurization on the heat stability. Meanwhile, because the granulating temperature of the feed is about 80 ℃ and the preparation time is within 1min, the heat stability of the rennin can meet the requirement of the feed granulation by using the protective agent, and the application space of the rennin on the pellet feed is greatly expanded.