Rennin

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Rennin is an aspartic protease first discovered in the stomach of unweaned calves. It can specifically cleave the peptide bond between Phe105-Met106 of κ-casein in milk and destroy the casein micelles. Milk coagulation, rennet's curdling ability and protein hydrolysis ability make it a key enzyme for forming texture and special flavor in cheese production, and is widely used in the production of cheese and yogurt. There are rennets in the gastric juice of mammals, which can cause the proteins in milk to coagulate into cheese, which is easily digested by various protein enzymes. Rennet only improves the efficiency of the enzyme, not actually counted as an enzyme. Animals other than mammals do not eat milk, so rennet is rarely present. Rennet is an indispensable preparation for the production of cheese, and its output value accounts for 15.5% of the total output value of the entire enzyme preparation. The main source is the gastric mucosa of the unweaned calf.

Figure 1. Protein structure of Rennin.

Source

• Animal-derived rennet: rennet was originally derived from the fourth gastric mucosa of calves, and rennet was subsequently extracted from the stomachs of other animals. However, due to the instability of animal-derived rennet, and for consideration of ethnicity and belief, researchers have turned the research direction to rennet of microbial and plant origin.
• Plant-derived rennet: Many plants contain a wide range of proteases that can make milk coagulate. At present, there are several researches, including papain, fig protease, bromelain, ginger protease, albizin, artichoke protease, etc.
• Microbial Rennin: Microorganisms have the advantages of short growth cycle, high yield, limited time and space, low production cost, easy extraction, and high economic benefits. They are currently the most promising development direction of rennet sources. At present, more than 40 kinds of microorganisms have been found to produce certain viable rennet, mainly fungi, actinomycetes, bacteria, etc.

Properties

The solubility of rennet is affected by pH, temperature and ionic strength of the solution. It is soluble in NaCl solution with a pH of 5.5 and a concentration of 1 mol/L. The crystal structure of rennet is relatively stable at 25℃, and the solubility increases with the increase of ionic strength. The isoelectric point of rennet is pH 4.5, which is the most stable at pH 5.3~6.3, but when pH is 3-4, the activity will decrease due to self-degradation. Under alkaline conditions, an irreversible conformational change will occur and the activity will be reduced.

States of rennet

There are three states of rennet: liquid, powder and tablet. Traditionally, rennet has been extracted from the abomasum of the fourth stomach of calves. With the progress of rennet research, its source is continuously expanding. Today, the classification includes three categories: animal rennet, derived from cow stomach and pig Stomach and goat's stomach; plant rennet, derived from fig sap and pineapple fruit; microbial rennet, derived from mold and yeast. The rennet of mold from Danish cheese production is widely used.

Mechanism

The curd action of rennet is divided into two steps: the first step is to enzymatically hydrolyze the peptide bond between Phe105-Met106 of the κ-casein polypeptide chain in the milk to form stable para-κ-casein and pro Aqueous glycomacropeptide; the second step is when the total kappa-casein is hydrolyzed by about 80%, in the presence of calcium ions through the chemical bonds formed between the casein micelles to form a clot or coagulated milk. This is because κ-casein molecules are located on the surface of casein micelles, and the subunits are connected together by a hydrophobic bond and colloidal calcium phosphate interaction.

Influencing factors

• PH: Rennin activity is strongest in an acidic environment, and any small changes in the acidity of raw milk can significantly affect the activity of rennet. Most of the Rennin activity comes from trypsin, and a small part comes from bovine pepsin (however, the active ingredient in porcine rennet is porcine pepsin). The optimal pH of trypsin is 5.4, while the optimal pH of pepsin is lower than that of trypsin.
• Temperature: The optimal temperature of rennet is 42℃. (At 55-60°C, the enzyme itself is destroyed) Because milk temperature significantly affects the coagulation rate. When the milk temperature is 30℃, the coagulation time of raw milk is 2-3 times of 42℃. However, in actual cheese production, the milk temperature is usually maintained at 30-33 ℃, one is to consider the optimal temperature of lactic acid bacteria (for example, the optimal temperature of Streptococcus is around 30 ℃, the highest cannot exceed 40 ℃); the second is higher milk, the clot hardening speed is too fast at temperature, so that the subsequent cutting is more difficult.
• Ca²⁺ concentration: Casein converted by rennet can be coagulated only when free calcium ions are present in the raw milk. Therefore, calcium ion concentration will affect the curd time, clot hardness and whey discharge.