CHEESE, YOGURT, AND SOUR CREAM
5.6 LACTOBACTERIA AND FERMENTATION
making lactic acid), the milk becomes increasingly acidic and begins to curdle. You might be thinking, but isn’t all milk pasteurized? Doesn’t pasteurization kill all of the bacteria, lactobacteria, and other potentially harmful types? Pasteurization does kill harmful, disease‐causing bacteria like Salmonella, Listeria, and Escherichia coli, which are associated with many foodborne illnesses. It also destroys most of the lac-tobacteria that are native to the milk and critical for curd formation. Thus, for cheeses that are produced from the lactic acid generated through bacterial fermentation, one or more “starter bacteria” are added to the pasteurized milk during the cheesemaking process to ferment lactose and reduce the milk pH to induce curdling.
Broadly speaking, there are two groups of lactobacteria that are used to “start” or induce curdling: moderate‐temperature (i.e., mesophilic) lactococci and the heat‐
loving (i.e., thermophilic) lactobacilli (Fig. 5.14).
These lactobacteria are both homofermenters, as they only produce lactic acid as a product of fermentation and not ethanol, so that is beneficial from the perspective of cheese production. Moreover, some lactobacilli are beneficial to human health, as you have heard advertised for dairy products with “probiotic” bacteria.
The Lactococcus family is relatively small with only seven identified species, while the Lactobacillus species has approximately 50 members. What makes a cheese-maker choose a particular bacterial strain or type? The type of bacteria that is used depends upon the specific steps involved cheesemaking process for a particular cheese type (like temperature), as well as other characteristics of the bacteria that allow for the production of other molecules and by‐product that are distinct for a particular cheese type. If the milk is subjected to temperatures of up to approximately 70°C during curd formation, then lactococci will effectively ferment and reduce the pH of the milk adequately to induce the formation of the curd. Most cheeses are acidified by the lactococci. The mesophilic bacteria can survive and thrive under these conditions, fermenting the lactose to lactic acid. However, for the cheeses that undergo a high‐temperature cooking step, including mozzarella and the Italian hard cheeses, the thermophilic lactobacilli bacteria must be utilized since these bacteria
A disaccharide molecule of lactose (i.e., milk sugar)
This half of lactose is made of galactose
This half of lactose is made of glucose α-1,4-Glycosidic
HO OH
OH O H
H H
H H
H O
CH2 HO
OH
OH OH
OH O H
H CH2
FIGURE 5.14 Lactose.
thrive and survive under high‐temperature conditions. To almost all cheeses that are not prepared from milk that is curdled via direct addition of an acid (i.e., citric acid, lemon juice, etc.), a type or types of starter bacteria are added during the curdling phase of the cheesemaking process to acidify the milk, to induce the formation of the curd, and to contribute to the cheese ripening and aging process. However, also in the preparation of almost all cheeses, another agent is used to make the curd more robust, elastic, and strong—this agent is an enzyme that is called rennet.
5.6.1 Enzyme‐mediated Curd Formation
A second method used to induce curd formation and coagulation of casein proteins involves an enzyme called rennet. Rennet is a general name for an enzyme or enzyme mixture that contains protease enzymes. Proteases cleave or degrade proteins into smaller pieces by breaking dipeptide bonds (Fig. 5.15).
Proteases are very important in biological system efficiency and play a key role in the degradation of proteins that are no longer needed and in our ability to recycle amino acids. However, protease activity is not only important for proper function of a biological system but also relevant in many aspects of food chemistry and is the basis for the warning label on your gelatin box about the addition of fresh pineapple, kiwi, or papaya.
Traditional rennet is made from the fourth stomach of a milk‐fed calf. The effects of rennet on cheese production and curd formation are thought to have been discov-ered when ancient peoples stored their milk in pouches made from an animal stomach and discovered that the milk soured and coagulated more rapidly than stored in a different type of container. Through genetic engineering, a pure version of the calf enzyme that is critical to curd formation, called chymosin or rennin, is produced in a bacterium, mold, and a yeast and is purified from these model organisms.
Most cheese in the United States is made with these engineered rennets. Traditional FIGURE 5.15 Dry and fresh yeast.
rennet from a calf stomach is often required for traditional, artisanal European cheese production. An alternative enzyme is often used to avoid the animal origin of rennet. Vegetable rennet is actually closely related to rennet chymosin and isolated from thistle.
Some proteases are not very specific. They will break dipeptide bond links at any amino acid or at amino acids that have a certain property (i.e., positively charged, aromatic, etc.). These enzymes are very useful if you want to break a protein into its individual amino acid components during the process of biological protein degrada-tion. Other proteases fulfill a specific role and are very specific for the types of amino acids that they will recognize and proteins that they will cleave. The role of a protease called chymosin, one of the components of rennin, is critical in curd formation dur-ing the process of cheese production. Chymosin is a protease that catalyzes the cleavage of a single dipeptide bond between Phe‐105‐Met106 (phenylalanine and methionine amino acids at positions 105 and 106 in κ‐casein; Fig. 5.16).
As you recall from our discussion about κ‐casein earlier in this chapter, the nega-tively charged tail of κ‐casein (i.e., amino acid residues 116–169) assists in keeping the casein micelle soluble and small. The proteolysis cleavage event that is carried out by chymosin effectively removes the negatively charged tail of κ‐casein from the casein micelle (called the κ‐casein glycopeptide), essentially leading to a similar molecular outcome that was discussed in acid‐mediated curd formation. Without the negative charge, the casein micelles fragment, calcium is lost from the micelle, the individual casein proteins aggregate, and a rubbery, strong cheese curd is formed.
HN
HN
NH
NH
NH NH
HN H
N
HN O
O
O
O
O
Peptide or protein Site of proteolysis
O
O O–
+H3N
O
O
O
Products of proteolysis HN
HN
NH
NH
NH NH
HN H
N
HN
FIGURE 5.16 Hydrolysis of the protein backbone. Enzymes (proteases) or strong acids can break the protein backbone in a process generally called proteolysis.
The cheese curd formed by rennin is much stronger than that formed through the process of acid‐induced coagulation as the κ‐casein glycopeptide component that effectively prevented the micelles from aggregating is lost to the whey (which remains soluble in the water), so no structural pieces remain to prevent micelle aggregation. In acid‐induced coagulation, the aggregation can occur but still must overcome the structural presence of the κ‐casein glycopeptide.
As stated earlier, the rennet used in cheese production typically contains a mixture of proteolysis enzymes including chymosin, pepsin (an enzyme important in protein breakdown in the digestive system that is most effective at cleaving peptide bonds that involve a hydrophobic, aromatic amino acid), and lipase (an enzyme that cata-lyzes the hydrolysis of fats). The nonchymosin proteases cause degradation of pro-teins and other molecules during curd formation to a lesser and less specific degree than chymosin. In addition to its role in curd formation, this degradation is also important to the texture and flavor of the finished cheese product.
5.6.2 Temperature‐mediated Curd Formation
The final mechanism used to assist with curd formation in cheese production is temperature. By cooking the coagulating milk at a temperature in which the starter bacteria thrive, the microbes metabolize lactose and undergo fermentation more readily. Thus, at higher temperatures, more lactic acid is produced and the pH of the milk is decreased more rapidly. Proteins also denature at higher temperatures, caus-ing them to unfold and form nonnative structures. Denatured casein proteins will disperse from a micelle and begin to aggregate, causing them to precipitate and curdle out of solution. Thus, in addition to the supplementation with starter bacteria and rennet, increasing the temperature of pasteurized milk to between 78 and 104°F (26 and 40°C) promotes the coagulation of the milk and fermentation of the starter bacteria. For most cheeses, the optimal temperature lies between 86 and 95°F/30–
35°C; however some goat cheeses are made from goat milk that coagulates at 68–77°F/20–25°C, while Pecorino cheeses are coagulated from milk held at 95–104°F (35–40°C).
BOx 5.3 ACID COAGULATION TAKES MANY HOURS AND LEADS TO A CHEESE CURD THAT IS FRAGILE AND SOFT
In rennet coagulation, the cud forms in less than an hour and is firm enough to cut into pieces that are as small as a grain of wheat. Many cheeses use a combination of acid and rennet to yield a curd that is appropriate for the type of cheese being made. In the production of hard or semihard cheeses, such as Cheddar, Gouda, and Parmesan, rennet is the primary agent for coagulation. Cheeses of more moderate moisture content (which makes them softer) are curdled with a smaller amount of rennet in combination with acid.
During the process of acid‐mediated, enzyme‐mediated, or milk sitting on your kitchen counter on a hot summer day‐mediated curdling, the casein proteins clump together, forming a solid curd mass, while the whey and other solution proteins and peptides remain in solution. However, if the process stopped here, then most cheeses would resemble yogurt or cottage cheese: a curdled mass of protein sitting in a sea of whey, water, and other nonaggregated molecules (Fig. 5.17). In order to prepare a more solid and drier cheese mass, the solid curd must be separated from the liquid through the process of cutting, draining, and pressing.
NH
HN
O CH2 O
CH2 CH2 S CH3 Phe-105
Met-106 Site of rennin cleavage
Rennin
NH
O CH2
Amino acid residues 1–105 of κ-casein
O–
+H3N
O
CH2 CH2 S CH3 Amino acid residues 106–169 of κ-casein
FIGURE 5.17 Actions of rennin on protein. The proteolytic action by rennin on casein results in its digestion to peptides.
5.7 REMOVING MOISTURE FROM THE CHEESE