Aria Foods, Brabrand, Denmark
The production of cheese is a process of concentrating milk by the interaction of the milk, starter cultures, and, in most cases, rennet. Traditionally, cheese has been produced in small vats, but during the past 3 to 4 decades, processing has become increasingly industrialized. Before industrialization, little attention was directed toward the hygienic aspects of cheesemaking, partly because the batch sizes were small and partly because methods of analysis were not well developed; consumption of cheese would normally cause only a few of disease cases. In recent years, more attention is being given to the hygienic aspects for several reasons: methods for detecting pathogens have improved; more focus on emerging pathogens such as Listeria and E. coli H7:O157; and the larger batch sizes increase risk to larger numbers of consumers, if pathogens are present. Furthermore, because of the large batches, economic losses will be substantial if the quality is not acceptable. Finally, an unacceptable quality in just a few batches from a producer may lead to loss of market shares. All these reasons have led to a considerable increase in attention on the hygienic aspects of cheesemaking. These aspects include a range of factors such as hygiene, environmental and technological factors, interactions between microorganisms, and the setting up of control systems in order to prevent contamination or at least to inhibit the growth of pathogens.
Complications in doing this arise from the fact that there are many different cheese varieties, such as yellow (with or without surface ripening), fresh, blue-veined, white-molded, and cottage cheese, each with its own risks for the presence or growth of pathogens or spoilage microorganisms. In this chapter, the most important physical, chemical, and microbiological factors required for inhibiting or avoiding pathogens or spoilage microorganisms are described. The creation of a comprehensive control system is discussed.
Cheeses comprise a huge number of varieties and thus the composition also varies. The chemical composition of a cheese results from production under either high or low acidification, depending on the type of cheese, and the starter culture, which also plays a role in formation of the metabolic profile. As an example, the starter culture is able to form lactic acid as the major component, diacetyl, ethanol, acetic acid, benzoic acid, and bacteriocine (1).
Depending on the type of cheese, the water content varies from very low in Grana cheeses to very high in cottage cheese, and the pH may vary from very low in in blue-veined cheeses and Feta (4.6-4.8) to very high in Queso Fresco (6.2-6.5). As pH varies, so does the lactic acid content. The sodium chloride content varies from very high (4-6%) in some blue-veined cheeses and Feta to very low (0.8-1.0) in cottage cheese. The sodium chloride content, dry matter, and other salts in cheese are responsible for the water activity, which is a very important growth determinant for microorganisms.
Regarding growth of microorganisms on the surface of cheeses, the packaging conditions are very important because the oxygen barrier varies, depending on the packaging material. The choice of packaging material depends on the type of cheese to be packed. Curing times for some cheeses may vary from short (1-6 days) or longer (up to 2 years) time intervals. This factor challenges the hygienic conditions in the curing rooms in relation to the chemical composition of the cheeses. Another challenge in this respect is the variation in temperatures that may occur in curing rooms. The temperature may vary from very high (20-22°C), for example for some Swiss-type cheese varieties, to very low (2-5°C) for fresh cheeses or special varieties. During the curing time, temperature is elevated or lowered depending on the cheese type to be produced. Finally, the addition of nitrate or lysozyme to prevent growth of primarily Clostridia is an antimicrobial factor to be considered (2).
From a hygienic point of view, the cheese process in itself is a stabilizing factor. Starter culture is added to the cheese milk at 30 °C and, together with the action of rennet, the milk coagulates to form a gel. As pH drops through the formation of lactic acid from the starter culture, the water-binding capacity of the proteins drops. This, together with cutting of the formed gel, separates the milk into cheese and whey. After about 90 min, depending on type of cheese to be produced, the pH has dropped from 6.7 in fresh milk to about 6.0. After this initial cheese process, the cheese mass is pressed and anaerobic conditions are created. After pressing, the cheeses are left to complete acidification to the minimum pH (5.2), typically requiring 24 hr. Most cheese types are then cured in curing rooms at different temperatures, and they may be ripened with or without a surface ripening culture. Some cheeses are packed in different foils in the curing room. Some fresh types of cheeses, however, are packed directly, then stored at 5°C and consumed within a few weeks.
III. ENVIRONMENTAL AND TECHNOLOGICAL FACTORS A. Organic Acids and pH
The starter culture consists of lactic acid bacteria (LAB), and within 24 hr, the minimum pH is usually achieved. The minimum pH may vary, but in most cheeses it is about 5.2 or lower; for Cheddar pH of 5.0 is normal, and in Feta the pH may be as low as 4.6. The buffer capacity of the cheeses is high due to the high protein content, but the amount of lactic acid formed in the cheeses is also very high, up to 1.5% for some cheese types. This amount of lactic acid, and the relatively low pH, achieves inhibition of many pathogens and spoilage microorganisms, especially gram-negatives. However, the gram-positives will also be inhibited under these conditions. Yeast and molds are only affected a little by the low pH and high amount of lactic acid. Depending on the type of starter culture, certain amounts of other organic compounds will also be formed (1). When gas-producing mesophilic LAB are used as starters, diacetyl is formed in amounts that are able to cause a little inhibition of pathogens and spoilage microorganisms. Due to the metabolism occurring in the cheeses, the starters will also form acetic acid; up to 250 ppm is normal. This amount is not enough to prevent the growth of pathogens or spoilage microorganisms and has little impact. Other organic compounds such as benzoic acid and ethanol may also have an impact on the growth of pathogens and spoilage microorganisms.
Although the amounts of organic compounds formed are difficult to control, it is easy to control pH and it is important to keep it as low as possible without altering the desirable organoleptic properties of the cheeses.
Temperature and curing duration are important variables from a technological and hygienic point of view. Although the curing temperature and time may improve the organo-leptic properties of the cheese, it may also possibly lead to microbial growth. At jC, given the right conditions, Listeria is able to grow (3), whereas others such as Clostridium tyro-butyricum are not able to grow below 8°C (4). Therefore, it is important to monitor the interaction between the curing temperature and time, in relation to the growth of selected microorganisms, and the cheese's organoleptic properties. From a hygienic point of view, the temperature should be kept as low as possible.
At a high NaCl content, and/or low water content, many microorganisms are prevented from growing (5). In such cheeses, Staphylococci, Listeria and yeast are chief concerns, because they are salt tolerant (6). In fresh cheeses of which the water activity is high and the NaCl content is about 0.8-1.0, the risk of growth is high: these are physiological conditions. It is not possible to lower the NaCl amount because it originates from the milk, and, in many cases, it is not possible to elevate the amount due to changes in the organoleptic properties. In these cases, other means must be used to prevent growth of pathogens and spoilage microorganisms.
Nitrate and lysozyme are often added to cheese milk in order to prevent late blowing from Clostridium tyrobutyricum (2-7). In most cases, these additives also inhibit the growth of other microorganisms. But it is worth noting that the activity of the starter may also be slightly inhibited, causing a slower decrease in pH during the fermentation process, resulting in less inhibition of pathogens and spoilage microorganisms during the acidification process.
IV. ANTAGONISTIC/SYMBIOTIC ACTIONS IN CHEESES
For several years, nisin, a bacteriocin produced during fermentation, has been recognized as preservative in a variety of cheeses. Nisin is produced by Lactococcus lactis subsp. lactis, one of the species used for acidification. The ability to produce nisin is strain dependent.
Nisin can be added to cheese milk or processed cheese as a powder for inhibiting grampositives. Use of a living nisin-producing Lactococcus in cheese production is not widespread because inhibition of the starter culture may be a problem. Other bacteriocins are known (8,9). The starter culture used in the production of surface-ripened cheeses consists of a mixture of yeast, Brevibacterium linens, other coryneform bacteria, Micrococcus, Staphylococcus (primarily equorum and xylosus), and gram-negatives in limited numbers (10). Bacteriocins from B. linens and Staphylococcus have been reported, and this is considered to be one way to control Listeria on surface-ripened cheeses (11-13). Enterococcus sp. has also been reported to produce bacteriocins, and this production may also have an impact on the control of harmful gram-positives on cheeses.
Apart from producing organic inhibitors and bacteriocins, the starter culture may also inhibit other microorganisms by direct competition for substrate. The starter culture ferments lactose into lactic acid/lactate, and thus inhibits the growth of harmful lactose fermenting microorganisms like coliforms or spoilage bacteria (e.g., heterofermentative lactobacilli). Other substrates converted by the starter culture during cheesemaking are citrate and protein fragments, which means that these compounds cannot serve as substrate for pathogens. The formation of lactic acid/lactate will in turn promote the growth of lactate-fermenting microorganisms (e.g., certain Clostridia). The best known is Clostridium tyrobutyricum, which causes late blowing in cheeses; however, there are means available to prevent this (see Sec. VI.A).
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