CHEESE TECHNOLOGY

Module 7. Rennet preparation and properties

Lesson 16
ACTION OF RENNET ON MILK

16.1 Introduction

Casein and whey proteins are the two groups of proteins present in milk. Casein is mainly present in micellar form and whey proteins are present in soluble form. When rennet is added in milk, the enzymes present in rennet act on the casein micelle leading to its destabilization and thus coagulation takes place. So, to understand the basic chemistry behind this enzymatic coagulation of milk, it is important to understand the structure of casein and the forces that stabilize casein in milk.

The casein micelles are spherical colloidal particles, with a mean diameter of around 120 nm and a mean particle mass of about 108 Da. The micelles contain protein and non-protein species (calcium and phosphate), with smaller amounts of magnesium and citrate and traces of other metals. All these are collectively called as colloidal calcium phosphate (CCP). The microstructure of casein micelle and its stability has been a subject of research for long. Numerous models have been proposed such as sub-micelle model and dual-binding model. The sub-micelle model proposes that the micelle is built up from sub-micelles which are held together by CCP and surrounded and stabilized by a surface layer rich in κ-casein but with some of the other caseins exposed also. Further, it was proposed that hydrophilic C-terminal region of κ-casein protrudes from the surface, creating a hairy layer around the micelle and stabilizing it through a zeta potential of about -20 mV and steric stabilization. The dual-binding model of Horne proposes that individual casein molecules interact via hydrophobic regions in their primary structures, leaving the hydrophilic regions free and with the hydrophilic C-terminal region of κ-casein protruding into the aqueous phase.

Calcium salts of αs-casein and β-casein are almost insoluble in water, while those of κ-casein are readily soluble. Due to the dominating localization of κ-casein to the surface of the micelles, the solubility of calcium κ-caseinate prevails over the insolubility of the other two caseins in the micelles, and the whole micelle is soluble as a colloid. Here calcium has the role to integrate the sub-micelles. If calcium leaves the micelle, the micelle will disintegrate into sub-micelles. The structure of the casein micelle is shown in figures 16.1 and 16.2.

Fig. 16.1 Casein Micelle Structure

Fig. 16.2 Structure of casein sub-micelle

16.2 Enzymatic Coagulation of Milk

The enzymatic coagulation of milk is essentially a two stage process (Fig. 16.3). As discussed earlier, the casein micelle is stabilized by κ-casein layer on the surface of the micelle. The enzymes present in rennet (proteinases) hydrolyse κ-casein layer to form paracasein micelles which aggregate in presence of calcium and thus milk is coagulated. The hydrolysis of the κ-casein layer is called as the primary phase of rennet coagulation while the aggregation of paracasein micelles in presence of calcium is called the secondary phase of rennet coagulation of milk.

Fig. 16.3 Enzymatic hydrolysis of casein

The amino acid chain forming the κ-casein molecule consists of 169 amino acids. Rennet enzymes act specifically at 105 (phenyl alanine)-106 (methionine) bond of this amino acid, thereby splitting it into two parts. One part consists of amino acids from 1-105, called as para-κ-casein. This part is insoluble and remains in the curd together with αs and β-casein. The other part of amino acids from 106-169 is soluble part. These amino acids are dominated by polar amino acids and the carbohydrate, which gives this part its hydrophilic properties. This part of the κ-casein molecule is called the glycomacro-peptide and is released into the whey in cheesemaking.

The formation of the curd is due to the sudden removal of the hydrophilic macropeptides and the imbalance in intermolecular forces caused thereby. Bonds between hydrophobic sites start to develop and are enforced by calcium bonds which develop as the water molecules in the micelles start to leave the structure. This process is usually referred to as the phase of coagulation and syneresis. Hydrolysis of κ-casein during the primary phase of rennet action releases the highly charged, hydrophilic C-terminal segment of κ-casein (macropeptide), as a result of which the zeta potential of the casein micelles is reduced from -10/-20 to -5/-7 mV and the protruding peptides are removed from their surfaces, thus destroying the principal micelle-stabilizing factors (electrostatic and steric) and their colloidal stability. When roughly 85% of the total κ-casein has been hydrolyzed, the stability of the micelles is reduced to such an extent that when they collide, they remain in contact and eventually build into a three-dimensional network, referred to as a coagulum or gel.

16.3 Factors Affecting Rennet Coagulation

The primary and secondary phase of rennet coagulation are affected by some of the compositional and environmental factors like milk composition, temperature, pH, calcium content, pre-heating of milk, rennet concentration etc. The effects of all these factors are summarized here.

16.3.1 Composition of milk

Variation in the composition of milk mainly affects the rate of coagulation and the curd firmness. Fast coagulation results in firmer curd. The rate of clotting is largely dependent on the nature of the casein micelles and the equilibrium with the calcium phosphate and calcium ions. The firmness of the curd are affected by pH value, calcium concentration, temperature, fat content and the ratio of rennin to casein. The rennet coagulation time (RCT) is markedly affected by the protein content in milk. RCT decreases with protein content in the range of 2-3%. Further increase in milk protein level i.e. more than 3% result in a slight increase in gelation time. This is due to decrease in rennet:casein ratio, which necessitates an increase in the time required to generate sufficient hydrolysis of κ-casein to induce aggregation of paracasein micelles. A minimum protein content of 2.5-3.0 is necessary to obtain gel in about 30-40 minutes during cheesemaking. Increase in fat content also results in decreased RCT but the effect is lower than that of the protein content.

16.3.2 Heat treatment of milk

Heating milk to pasteurization temperature has beneficial effect on rennet coagulation due to heat induced precipitation of calcium phosphate and a concomitant decrease in pH. But heating further to higher temperatures causes other effects which in combination dominate the positive effects of heating to pasteurization. Some such effects are:

• Whey protein denaturation and the interaction of denatured β-lactoglobulin with micellar κ-casein

• The deposition of heat-induced insoluble calcium phosphate leading to reduction in the concentration of native micellar calcium phosphate. This micellar calcium phosphate is important for cross linking para-κ-casein micelles and their aggregation during gel formation.

16.3.3 Set temperature

The principal effect of set temperature is on the secondary phase of enzymatic coagulation, which does not occur at temperatures below around 18°C. Above this temperature, the coagulation time decreases to a broad minimum at 40-45°C and then increases again, as the enzyme becomes denatured. In cheesemaking, rennet coagulation normally occurs at around 31°C. This is necessary to optimize the growth of starter bacteria which will not survive the temperature more than 40°C. In addition, the structure of the coagulum is improved at the lower temperature, which is therefore used even for cheeses made using thermophilic cultures.

16.3.4 Rennet concentration

The rate of enzymatic coagulation is directly related to the concentration of enzyme. Increase in concentration of rennet decrease RCT. During cheesemaking, rennet is added in such a concentration so as to coagulate the milk 30-40 minutes. More rennet concentrations can be used to shorten the coagulation time but it leads to retention of more rennet in the curd which has pronounced effect in ripening of the cheese, particularly proteolysis. Some studies also suggest that using increased concentration of rennet may jeopardize the curd firming rate and curd firmness.

16.3.5 Concentration of calcium ions

The concentration of calcium ions mainly affect the secondary phase of enzymatic coagulation. Increased calcium concentration is beneficial for coagulation of milk. For this reason, sometimes CaCl₂ is added to milk prior to cheesemaking. This promotes cheesemaking via three beneficial changes, viz. an increase in calcium ion concentration, an increase in the concentration of colloidal calcium phosphate and a concomitant decrease in pH (the addition of CaCl₂ to 0.02%, i.e. 1.8 mM Ca, reduces the pH by ~ 0.05-0.1 units, depending on protein level).