ReviewLipolysis and free fatty acid catabolism in cheese: a review of current knowledge
Introduction
Lipolysis is an important biochemical event occurring during cheese ripening and has been studied quite extensively in varieties such as Blue and hard Italian cheeses where lipolysis reaches high levels and is a major pathway for flavour generation. However, in the case of cheeses such as Cheddar and Gouda, in which levels of lipolysis are moderate during ripening, the contribution of lipolytic end products to cheese quality and flavour has received relatively little attention. FFA are important precursors of catabolic reactions, which produce compounds that are volatile and contribute to flavour; however, these catabolic reactions are not well understood (McSweeney & Sousa, 2000). The aim of this work is to review the existing knowledge of the way in which lipolysis and FFA catabolism proceed during cheese ripening, how lipolysis may be measured and monitored and also how this biochemical event contributes to cheese flavour.
Bovine milk typically contains, ca. 3.5–5 g fat 100 mL−1 in the form of emulsified globules ranging from 0.1 to 10 μm in diameter (McPherson & Kitchen, 1983; Jensen, Ferris, & Lammi-Keefe, 1991). Milk may therefore be described as an oil-in-water emulsion with the fat globules dispersed in the continuous serum phase. Fat globules are surrounded by a thin membrane called the milk fat globule membrane (MFGM) and this interfacial layer lends stability to the fat globule (Brunner (1965), Brunner (1969); Huang & Kuksis, 1967; Prentice, 1969; Bauer, 1972; Anderson (1974), Anderson (1977); Anderson & Cawston, 1975; Magino & Brunner, 1975; Diaz-Maurino & Nieto, 1977; McPherson & Kitchen, 1983). Milk fat has a complex fatty acid composition, which is reflected in its melting behaviour. At room temperature (20°C), milk fat is a mixture of oil, semi-hard fat and hard fat. Melting begins at −30°C and is only complete at 40°C (Banks, 1991a; Boudreau & Arul, 1993). The range of fatty acid chain lengths and degree of unsaturation, as well as the stereospecific distribution of fatty acids, are responsible for the particular melting behaviour of milk fat (Boudreau & Arul, 1993). Ruminant milk fats contain a wide range of fatty acids and 437 distinct acids have been identified in bovine milk fats. The major FFA found in milk fat are butanoic (C4:0), hexanoic (C6:0), octanoic (C8:0), decanoic (C10:0), dodecanoic (C12:0), tetradecanoic (C14:0), hexadecanoic (C16:0), octadecanoic (C18:0), cis-9-octadecenoic (C18:1), cis, cis-9,12-octadecadienoic (C18:2), and 9,12,15-octadecatrienoic acids (C18:3) (Jensen, Gander, & Sampugna, 1962; Banks, 1991a; Jensen et al., 1991). Hexadecanoic and octadecanoic are the most abundant FFA (Banks, 1991b; Gunstone, Harwood, & Padley, 1994), comprising ∼25% and ∼27% of total lipids, respectively (Jensen et al., 1962). Some notable features of the fatty acid profiles of bovine milk lipids include the high level of butanoic acid and other short chain fatty acids, the low levels of polyunsaturated fatty acids and the fact that these lipids are rich in medium chain fatty acids (Oba & Wiltholt, 1994).
The principal lipids of milk are triacylglycerides, which may represent up to 98% of the total lipids (Christie, 1983; Jensen et al., 1991; Gunstone et al., 1994); the structure of triacylglycerides is illustrated in Fig. 1. Triacylglycerides have molecular weights ranging from 470 to 890 Da, corresponding to 24–54 acyl carbons (Boudreau & Arul, 1993; Balcao & Malcata, 1998). Triacylglycerides are esters of glycerol composed of a glycerol backbone with three fatty acids attached (Stryer, 1988). Positioning of fatty acids on the triacylglyceride is non-random; the sn-position of a fatty acid denotes its position on the triacylglyceride. Fatty acids may be esterified at positions 1, 2 or 3 as shown in Fig. 1. C4:0, and C6:0 are predominately located at the sn-3 position and the sn-1 and sn-3 positions, respectively. As chain length increases up to C16:0, an increasing proportion is esterified at the sn-2 position. C18:0 is generally located at the sn-1 position, while unsaturated fatty acids are esterified mainly at the sn-1 and sn-3 positions (Balcao & Malcata, 1998).
While phospholipids represent < 1% of total lipids, they play an important role in the MFGM. Phospholipids are amphipolar in nature and are strongly surface active. These properties enable them to stabilize both oil-in-water and water-in-oil emulsions (Banks, 1991a). On average, phospholipids contain longer and more unsaturated fatty acids than triacylglycerides (Banks, 1991a; Jensen et al., 1991). The principal phospholipids found in milk fat are phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin (Christie, 1983; Grummer, 1991; Gunstone et al., 1994). Trace amounts of other polar lipids have also been reported in milk fat, including ceramides, cerobrosides and gangliosides. Cholesterol is the dominant sterol of milk (>95% of total sterols) (Anderson & Cheesman, 1971; Christie, 1983; Jensen et al., 1991) and accounts for ca. 0.3% of total lipids. The MFGM itself consists of a complex mixture of proteins, phospholipids, glycoproteins, triacylglycerides, cholesterol, enzymes, and other minor components, and acts as a natural emulsifying agent enabling the fat to remain dispersed in the aqueous phase of milk (Anderson, Cheeseman, Knight, & Shipe, 1972; Kinsella, 1970; Mather & Keenan, 1975; Mather, 1978; Kanno, 1980; Keenan, Dylewski, Woodford, & Ford, 1983; McPherson & Kitchen, 1983).
Microstructural studies of fat present in cheese indicate the presence of globules of varying size and shape. Using confocal laser scanning microscopy (CLSM), Gunasekaran and Ding (1999) examined the three dimensional characteristics of fat globules in one month old Cheddar of varying fat contents (∼40–340 g kg−1). For all cheeses, most globules were <2 μm in diameter. However, globule size was smallest at lowest fat content, but at lowest fat content more globules were noted. The average globule size appeared to be inversely related to the total fat content of the cheese. Overall, increasing fat content in the cheese was reflected by the presence of larger, less circular globules. These workers noted that the nature of protein matrix in low fat cheese may influence fat globules by preventing changes in their size and shape. Guinee, Auty, and Fenelon (2000) also examined the microstructure of Cheddar cheeses of fat contents, in the range ∼70–300 g kg−1 using CLSM. Reduction of fat content of cheese was accompanied by dispersion of discrete globules without clumping, while increasing fat content of cheese resulted in progressive clumping and coalescence of the globules. Guinee et al. (2000) attributed the clumping and coalescence of globules to the destruction of the MFGM during processing and also to heating of curds during cheesemaking, respectively. Dufour et al. (2000) showed that fluorescence and infra-red spectroscopy could monitor and differentiate patterns of triacylglyceride phase transition in cheeses of varying composition. Spectral changes of triacylglycerides, indicating partial crystallization, were noted over ripening. These changes, were evident in two distinct intervals, 1–21, and, 21–82 days of ripening, and were accompanied by an increase in viscosity. Microstructural and physico-chemical dynamics of fat globules in cheese also appear to influence the localization and retention of starter lactococci in cheese (Laloy, Vuillemard, El Soda, & Simard, 1996). Full fat Cheddar cheese retained higher cell populations in the curd compared to 50% fat reduced Cheddar. Lactococci, visualized using electron microscopy, were shown to be located on the periphery of the fat globule. As ripening progressed, lactococci became more intimately associated with the fat globule such that non-viable cells appeared to become integrated into the fat globule membrane. The potential influence of proteolysis on this progressive association between starter cells and fat globule membrane was raised in this study; whereby hydrolysis of the protein matrix may reduce pressure on the fat globule influencing starter localization within cheese (Laloy, Vuillemard, El Soda, & Simard, 1996). While this is a very interesting theory, to date, no detailed scientific investigation has been undertaken to elucidate the mechanism of accessibility of fat in cheese for lipolysis.
Section snippets
Agents of lipolysis in milk and cheese
It is well established that milk fat is essential for the development of correct flavour in cheese during ripening. This was demonstrated in studies with cheeses made from skim milk, or milk in which milk fat had been replaced by other lipids; such cheeses did not develop correct flavour (Foda, Hammond, Reinbold, & Hotchkiss, 1974; El-Safty & Isamil, 1982; Wijesundera, Drury, Muthuku-marappan, Gunasekaran, & Everett, 1998).
Lipids present in foods may undergo oxidative or hydrolytic degradation (
Catabolism of fatty acids
In cheese, FFA released as a result of lipolysis, especially short- and medium- chain fatty acids directly contribute to cheese flavour. FFA also act as precursor molecules for a series of catabolic reactions leading to the production of flavour and aroma compounds, such as methyl ketones, lactones, esters, alkanes and secondary alcohols (Gripon, Monnet, Lamberet, & Desmazeaud, 1991; Fox & Wallace, 1997; McSweeney & Sousa, 2000). Pathways of fatty acid catabolism are outlined in Fig. 2 and
Contribution of lipolysis and metabolism of FFA to cheese flavour
The flavour of mature cheese is the result of a series of biochemical changes that occur in the curd during ripening, caused by the interaction of starter bacteria, enzymes from the milk, enzymes from the rennet and accompanying lipases and secondary flora (Urbach, 1997). The numerous compounds involved in cheese aroma and flavour are derived from three major metabolic pathways: catabolism of lactate, protein and lipid (Molimard & Spinnler, 1996). Lipid hydrolysis results in the formation of
Patterns of lipolysis in various cheese varieties
Levels of lipolysis measured as release of FFA vary considerably between cheese varieties from moderate (e.g., Cheddar, Cheshire, Caerphilly) to extensive (e.g., mould-ripened, hard Italian and surface bacterially ripened (smear) varieties (McSweeney & Fox, 1993; Fox & Wallace, 1997; Fox et al., 2000; McSweeney & Sousa, 2000). The level of lipolysis should not exceed 2% of triacylglycerides in Gouda, Gruyere or Cheddar cheeses (Gripon, 1993). Excessive lipolysis is considered undesirable and
Measurement of lipolysis
The FFA compositions of some selected cheese varieties are shown in Table 1. Various methods are used to quantify FFA. Gas chromatography (GC) has been the method most commonly used to quantify levels of individual FFAs in cheese and is the dominant technique for the routine analysis of FFA; the flame ionization detector is robust with a wide and dynamic range enabling accurate FFA quantification. In a review of the determination of FFA in milk and milk products (IDF, 1991) methods for analysis
Conclusion
While the existing knowledge of how lipolysis impacts on the flavour of certain cheese varieties, e.g., Blue and hard Italian varieties, is extensive, additional research would undoubtedly be of benefit for varieties such as Cheddar, which is of major economic importance in many countries. The outcomes of this research should allow a better understanding of the catabolic reactions resulting from the release of FFA during cheese ripening as well as more detailed knowledge of how the catabolic
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