Bitterness and accelerated ripening

For most cheeses, production of good aged cheese flavor and texture requires that there be significant hydrolysis of casein. At the same time, the main flavor defect in cheese is bitterness, which is also caused by casein hydrolysis and subsequent formation of bitter peptides. Thus, aging cheese is like walking a tightrope—

it requires a fine sense of preparation, balance, and proportion.A single misstep may lead to an unacceptable end. Accelerated ripening—obtaining good cheese flavor and texture in half or even a third of the time—is, to continue the analogy, like running across the tightrope. There is tremendous economic incentive, however, to decrease aging time, because the aging process adds about one cent per Kg per month to the cost of the cheese. Reduced fat cheese poses another set of flavor problems (Box 5-7). Not surprisingly, considerable research has been aimed at developing accelerated cheese aging programs.

Box 5—7. Reduced Fat Cheese—Taking the Fat (But Not the Flavor) Out of Cheese

Cheese, like other dairy products, is heavily regulated by the federal government, and standards of identity exist for most of the cheeses consumed in the United States (Figure 1). These standards (contained within the Code of Federal Regulations,Title 21, Part 133) not only describe the ingredients allowed in the manufacture of various cheeses, they specifically spell out the fat and compositional requirements for them. Importantly, the minimum amount of fat (on a dry or solids basis) and maximum amount of water are specified. Until relatively recently, Cheddar cheese that contained less than 50% fat on a dry basis (or in the dry matter or FDM) could not be called cheese. Similar standards exist for other cheeses, such that most contain at least 45% fat and derive more than 75% of their calories from fat.

Because milkfat was viewed, for hundreds of years, as a precious and desirable commodity, these standards were established to protect consumers and make sure they received full value for their money. Products labeled as cheese, but containing less than the required amount, were considered inferior and in violation of the law. This public perception of milkfat, however,

Reduced fat = 25% reduction in total fat per reference amount when compared to an appropriate reference food

Light = food that has 50% less fat per reference amount when compared to an appropriate reference food, when 50% or more of its calories are from fat, or in the case of food containing less than 50% of its calories from fat, less than one-third less calories from fat or less than 50% fat per reference amount when compared to an appropriate reference food.

Nonfat = Less than 0.5% fat per reference amount when compared to an appropriate reference food

Lowfat = Three grams or less fat per reference amount.

Figure 1. Reduced Fat Cheese Standards (Implemented 1996).

Box 5—7. Reduced Fat Cheese—Taking the Fat (But Not the Flavor) Out of Cheese (Continued)

changed in the 1960s and 70s, as consumers in the United States began to reduce their consumption of animal fats and to seek out lower-fat products. Sales of low-fat fluid milk products began to increase, and a market for other low-fat dairy products became evident. Although the cheese industry was well aware of the change in consumer attitudes and had been working on lower-fat versions, these new products could not be called cheese, due to existing standards. It was not until the 1990s that the U.S. government modified the definitions of cheese so that low-fat versions could still be labeled as cheese.These new standards for cheese follow the reduced fat, low-fat, and nonfat definitions used for other foods (see below).

The resolution of the labeling problem, however, did not lead to immediate consumer acceptance of reduced fat cheese. Making cheese with low-fat milk is technically no different than conventional cheese manufacture, but the products that were initially introduced suffered from serious flavor and body defects. This situation has slowly begun to change, and as of the year 2000, about two-thirds of consumers had purchased reduced or non-fat cheese. However, many of the problems that plagued the initial products have yet to be resolved and the market for these products is still relatively modest.

Making reduced fat cheese with flavor and texture properties comparable to full fat products, it turns out, is not easy, for two main reasons. First, when milkfat is taken out of cheese, it is essentially replaced by casein and water.Thus, the body becomes more firm and less smooth and pliable. Second, a higher moisture content means that the salt-in-moisture ratio decreases, resulting in an undesirable increase in microbial and enzymatic activities.All other things being equal, the cheese ages poorly, with potential for significant production of bitterness and other off-flavors. More moisture also results in more lactose in the curd, which may lead to excessive acid production. Not only does this affect flavor, but as the pH decreases, calcium is lost, which may cause other body defects.

Manufacturers have adopted several strategies to produce more acceptable reduced fat cheeses. First, gums and other fat mimics can be added to give the body, texture, and mouth feel of full-fat cheese.To control acidity, the curds can be washed to remove excess lactose.Alterna-tively, researchers at the University of Wisconsin developed a process, based on enhancing the buffering capacity in the curd (by retaining calcium phosphate), that results in higher acid levels but moderate pH (Johnson et al., 2001).This also resulted in a lower level of calcium bound to casein and a softer body.An experimental centrifugation method in which the fat is removed after manufacture and aging was recently described (Nelson and Barbano, 2004).

Despite these improvements, flavor, either the lack thereof or the presence of undesirable flavors, remains a problem.According to some researchers, milkfat not only serves as an important

The main approach of most aging strategies is to promote proteolytic and other enzymatic activities.As described above and in Figure 59, the first step in the use of casein by the lactic starter culture is hydrolysis of casein by the cell wall anchored proteinase (PrtP). Of the hundred or more peptides that are produced, most contribute little to overall cheese flavor. However, several of these peptides have a bitter flavor. If the starter culture cannot degrade these bitter peptides, the cheese will be bitter. Starter culture strains that produce PrtP (or related proteinases), but that are otherwise un able to degrade the bitter peptides so produced, are referred to as bitter strains.

In general, bitter peptides are hydrophobic and contain proline, and are hydrolyzed only by specific intracellular aminopeptidases, including PepN and PepX. Bitter strains lack the ability to produce these enzymes. It is interesting that most commercial strains of L. lactis subsp. lactis, the most widely used organism in the cheese industry, are bitter. In contrast, most of the L. lactis subsp. cremoris strains are nonbitter. However, L. lactis subsp. lactis grows faster and, at least in unripened cheese (e.g.,

Box 5—7. Reduced Fat Cheese—Taking the Fat (But Not the Flavor) Out of Cheese (Continued)

flavor-generating substrate (i.e., fatty acids), it also performs as a solvent in cheese, since many of the important Cheddar cheese flavor compounds are found in the lipid phase (Midje et al., 2000).Although others contend that cheese flavor is associated with the water-soluble fraction (McGugan et al., 1979; Nelson and Barbano, 2004), it is clear that the quality of reduced fat cheese could be improved by increasing flavor intensity.

One popular approach to improve the flavor of reduced fat cheeses (and conventional cheese, as well) has been to add selected flavor-producing lactic acid bacteria to the milk, in the form of adjunct cultures (Fenelon et al., 2002; Midje et al., 2000). As described below (Box 8), adjunct cultures are added to the milk in much the same way as the starter culture. However, their function is to produce enzymes and flavor compounds, rather than lactic acid.Adjunct cultures typically contain strains of Lactococcus and Lactobacillus that produce aminopeptidases that specifically hydrolyze bitter peptides.Although bitter peptides are often present in full-fat cheese, they can be even more of a problem in reduced fat cheese, in part because they partition more in the non-lipid or serum phase,where they are more readily detected.Also,the lower salt-in-moisture ratio typical of reduced fat cheese may result in higher cell numbers and greater proteinase production and activity by the starter culture bacteria, which ultimately leads to bitter peptide accumulation.The use of non-bitter strains may, however, significantly reduce bitterness problems in these cheeses.

References

Fenelon, M.A.,T.P. Beresford, and T.P. Guinee. 2002. Comparison of different bacterial culture systems for the production of reduced-fat Cheddar cheese. Int.J. Dairy Technol. 55:194-203. Johnson, M.E., C.M. Chen, and J.J.Jaeggi. 2001. Effect of rennet coagulation time on composition, yield, and quality of reduced-fat Cheddar cheese. J. Dairy Sci. 84:1027-1033. McGugan W.A., D.B. Emmons, and E. Larmond. 1979. Influence of volatile and nonvolatile fractions on intensity of Cheddar cheese flavor. J. Dairy Sci. 62:398-403. Midje, D.L., E.D. Bastian, H.A. Morris, F.B. Martin,T. Bridgeman, and Z.M.Vickers. 2000. Flavor enhancement of reduced fat Cheddar cheese using an integrated culturing system. J. Agric. Food Chem. 48: 1630-1636.

Nelson, B.K., and D.M. Barbano. 2004. Reduced-fat Cheddar cheese manufactured using a novel fat removal process. J. Dairy Sci. 87:841-853.

cheese destined for the process kettle), its inability to degrade bitter peptides is not an issue.

Even cheese made with non-bitter strains, if aged long enough, can eventually accumulate enough bitter peptides to develop bitter flavor. Bitter peptide production is further increased at higher ripening temperatures (i.e., >12°C), which is perhaps the most common way to accelerate cheese ripening. Although some bitterness is unavoidable, the addition of adjunct cultures, or strains capable of producing aminopeptidases, has been promoted as a means of accelerating the aging process (Box 5-8). Adjunct cultures, which typically contain selected strains of Lactobacillus and Lactococcus, can be added directly as part of the starter culture. However, because excess acid production is to be avoided, variants that are unable to ferment lactose are preferred.

The observation that some strains of lacto-cocci and streptococci were autolytic led to another innovative means for accelerating the ripening process. As described previously, ripening depends largely on the presence of enzymes released by bacteria as they lyse during the aging process. Ordinarily, cell lysis occurs only when the cells are no longer able to maintain cell wall integrity, due to the lack of energy sources and the generally inhospitable conditions (i.e., low pH, high salt, etc.) present within the cheese. In contrast, if lysis and enzyme release were to occur during the early

Box 5-8. Adding Flavor With Adjunct Cultures

The development of cheese flavor and texture is a complicated process, involving numerous enzyme-catalyzed reactions.The enzymes involved in these reactions originate from three main sources: the coagulant, the milk, and bacteria.The latter are either normally present in milk due to unavoidable contamination or are deliberately added.And although it is certainly possible to make cheese without bacteria (i.e., aseptic cheese), the finished product would contain few, if any of the flavor or texture characteristics one might expect.This would be especially true if the cheese were aged, since aged flavor and texture would simply not develop.

While the coagulant and natural milk enzymes certainly contribute to cheese flavor and texture development, their roles are considered to be rather limited, whereas the presence of bacteria is essential (El Soda et al., 2000). Moreover, it is not just the lactic starter culture bacteria that are responsible for the desirable changes that occur during cheese ripening—bacteria present as part of the natural milk flora also serve as a vital reservoir of enzymes that are necessary for proper cheese maturation. If, however, the milk is pasteurized or if it is produced and handled under strict hygienic conditions, the natural microflora will be rather limited, both in number and diversity.The finished cheese will ripen slowly and, in some cases, never achieve the desired quality expectations.

The realization that the natural milk flora is important for cheese ripening led investigators first to identify the relevant organisms, and then to consider the possibility of adding them directly to the milk to boost or enhance flavor development (Reiter et al., 1967).These studies revealed that the bacteria that were most frequently associated with good cheese flavor were lactic acid bacteria, but not the same species or strains used as the starter culture.Therefore, as a group, they are referred to as non-starter lactic acid bacteria (NSLAB). Cheese flavor is enhanced when the correct NSLAB, which generally consist of species of Lactococcus and Lactobacillus, are added to cheese milk. It also appears that the presence of these NSLAB may out-compete "wild"NSLAB that might otherwise cause flavor defects.They are now commercially available in the form of adjunct cultures, and are widely used for accelerated ripening programs as well as for reduced fat cheese, which often suffer from flavor problems (Box 5-7).

When adjunct cultures were first considered for modifying cheese flavor, cells attenuated by freeze or heat shocking, physical-chemical treatment, or genetic means were used (El Soda et al., 2000).This was done to make the cells nonviable and to minimize subsequent acid production and metabolism. However, as noted above, adjunct cultures are now commercially available and are added directly to the cheese milk, albeit at much lower inoculation rates compared to the starter culture (Fenelon et al., 2002).

Among the species currently used commercially are strains of Lactobacillus helveticus, Lac-tobacillus casei, and Lactobacillus paracasei. In particular, the specific NSLAB used as adjunct cultures are selected based largely on their ability to produce aminopeptidases and to lyse dur-

stages of aging, the ripening process could be shortened.

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