Rennet

The biochemical process required in the production of cheese is the conversion of liquid milk into a soft gel structure, which usually means curds. Rennet and coagulants are preparations for the preparation of proteolytic enzymes that have been used in cheese production for thousands of years, and it seems. They are the oldest known use of enzymes. The term rennet/rennet is usually reserved for a functional enzyme preparation capable of forming a clot. Natural curd from ruminants is an asparagine protease of different molecular and genetic variants of chymosin. Rennet has different types such as animal, plant-based, microbial, etc. Also, curd can be in different forms, including:

1. Liquid curd: added directly to milk and does not need to be prepared.
2. Powdered curd: needs to be diluted with water before use.
3. Curd tablets: used for small volumes of milk and home use.

 The general characteristics of whey:

1. Physicochemical properties

The pH of rennet depends on the type, storage conditions, its production origin, and also the method of preparation.

The color of the rennet obtained from small ruminants is mainly dependent on the animal’s diet. So, it varies from white to yellow or green. Commercial cheeses are usually a light brown liquid with a distinctive odor. The color of the cheese may vary from category to category but does not affect the coagulation activity of the product.

2. Molecular and catalytic properties of milk coagulation enzymes

Chymosin and other milk coagulation enzymes belong to the group of aspartic acid proteinases (EC 3.4.23), which are characterized by high content of dicarboxylic and hydroxy amino acids and low levels of basic amino acids.

The third structure of aspartic proteinases shows high homology. This structure consists of a wide gap, which is the junction of the substrate and contains at least seven amino acids of k-casein.

Although milk coagulation enzymes have high activity at an almost neutral pH, the isoelectric point and optimal pH of all aspartic proteinases are in the acidic range.

3. Microbiology of commercial rennet

All coagulants must microbiologically meet the requirements of the Association of

Manufacturers of natural Animal-derived Food Enzymes (A.M.A.F.E). According to these rules

1. The coliform content should be up to 30 CFU / g, and the total mesophilic microflora should be up to 5 × 10⁴ CFU / g.
2. These products should contain no E. coli, Salmonella species, mycotoxins, and antibacterial agents.
3. The maximum levels of arsenic, lead, and heavy metals are 3, 10, and 40 ppm, respectively.
4. Milk Clotting Activity

The oldest definition of MCA is the definition given by Soxhlet in 1877, according to which MCA is defined as the volume of raw milk that can be coagulated by one unit volume of rennet in 40 minutes at 35 °C. MCA is one of the important technical features of a rennet because it affects all the properties of the clot.

However, this definition was unsatisfactory due to changes in the ability of raw milk to clot. In 1952, the British scientist NJ Berridge made progress in the analysis of milk clotting by proposing the use of standardized milk powder with 0.01 mol / l calcium chloride as a substrate.

The Berridge Unit or rennet unit (RU) is an activity that is capable of clotting 10 ml of standard milk (pH 6.3) in 100 seconds at 30 °C. By the 1990s, Soxhlet and Berridge units were widely used for almost all definitions of milk coagulation test. However, changes in the composition of commercial rennet and coagulants in today’s market have made the old definitions too uncertain and inaccurate.

Today, the total amount of rennet MCA is measured on standard milk according to the Relative Milk Clotting Activity Test of IDF (REMCAT method) and is obtained with IMCU (International Milk Clotting Units) per gram or ml of rennet or liquid is declared.

5. Proteolytic activity

Chymosin, the major proteinase in rennet, specifically hydrolyzes the Phenylalanine-Methionine bond, while pepsin lacks the specific ability to hydrolyze protein bonds including aromatic amino acids.

Proteolytic activity can be determined indirectly through cheese proteolysis. Since proteolytic activity is not equally affected by the type of rennet source and pepsin content, contradictory results will not be unexpected.

Compositional and Processing Factors that Affect Rennet Coagulation:

• Temperature:

The optimum temperature for the coagulation of milk by calf rennet at pH 6.6 is 45 ˚C, but this temperature varies depending on pH and rennet type. Prolonged cold storage of milk before renneting can result in a longer RCT and a weaker gel due mainly to the dissociation of β-casein from the micelles. This can be reversed to some extent by pasteurization of the milk before cheese making.

• Enzyme Concentration:

There have been many attempts to describe the influence of enzyme concentration on the coagulation time of milk; one of the most widely used is the Holter equation, which describes the well-known inverse relation between coagulation time and enzyme concentration:

Where CT is the clotting time, K and A are constants, and [E] is the enzyme concentration. Clotting time refers to the time taken by both the enzymatic and aggregation reactions. The constant A in the Holter equation reflects the time needed for the nonenzymatic phase of milk coagulation.

• pH:

The optimum pH for the action of chymosin in milk is 6.0, but the optimum pH is lower for isolated (purified) caseins or synthetic peptides. Lowering the pH of milk leads to a reduction in the RCT (due to reduced electrostatic repulsion) and a faster rate of increase in gel firmness.

• Calcium:

Generally, it is thought that calcium does not directly affect the enzymatic phase, although the addition of CaCl₂ does reduce milk pH, which accelerates the hydrolysis reaction.

• Heat Treatment of Milk:

Severe heating of milk (e.g., >70 °C) impairs its rennet coagulation properties.

6-  Total Solids:

As the casein concentration increases, the rate of aggregation increases. In milk with higher solid content, coagulation occurs at a lower degree of hydrolysis of қ-casein.

• Sodium Chloride:

Salt or ionic strength also affects rennet coagulation. As the addition of NaCl reduces milk pH, many of the effects depend on whether milk pH is kept constant. The addition of NaCl reduces the hydrolysis reaction, presumably by inhibiting the electrostatic interactions involved in the formation of the chymosin–қ-casein complex at the active site. Adding NaCl increases the RCT and reduces the initial rate of aggregation (even if the pH is kept constant).