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International Dairy Journal 13 (2003) 841–866


Lipolysis and free fatty acid catabolism in cheese: a review of current knowledge
Yvonne F. Collinsa, Paul L.H. McSweeneyb, Martin G. Wilkinsonc,*
b a Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland Department of Food Science, Food Technology and Nutrition, University College, Cork, Ireland c Department of Life Sciences, University of Limerick, Castletroy, Limerick, Ireland

Received 2 January 2003; accepted 28 March 2003

Abstract The progress of lipolysis and its effect on flavour development during cheese ripening is reviewed. The review begins by describing the structure and composition of milk fat and thereafter discusses current knowledge regarding the role of various lipolytic agents and their influence on lipolysis in various cheese varieties. While free fatty acids (FFA) liberated during lipolysis directly affect cheese flavour, they are also metabolized to other highly flavoured compounds, including methyl ketones and lactones. The pathways of FFA catabolism and the effect of these catabolic products on cheese flavour are discussed. Finally, the current methods for the quantification of FFA in cheese are reviewed and compared. r 2003 Elsevier Ltd. All rights reserved.
Keywords: Lipolysis; Cheese ripening; Catabolic products; Flavour

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Milk fat: chemistry and structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agents of lipolysis in milk and cheese 2.1. Milk lipase . . . . . . . . . . . 2.2. Rennet paste . . . . . . . . . . 2.3. Microbial lipolytic enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 841 842 843 844 844 845 848 852 854 855 860 860

3. 4. 5. 6. 7.

Catabolism of fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contribution of lipolysis and metabolism of FFA to cheese flavour . . . . . . . . . . . . . . . . . . . . Patterns of lipolysis in various cheese varieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measurement of lipolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
*Corresponding author. Fax: +353-61-213440. E-mail address: (M.G. Wilkinson). 0958-6946/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0958-6946(03)00109-2

Lipolysis is an important biochemical event occurring during cheese ripening and has been studied quite

842 Y.F. Collins et al. / International Dairy Journal 13 (2003) 841–866

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. 1.1. Milk fat: chemistry and structure Bovine milk typically contains, ca. 3.5–5 g fat 100 mLÀ1 in the form of emulsified globules ranging from 0.1 to 10 mm 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, 1969; Huang & Kuksis, 1967; Prentice, 1969; Bauer, 1972; Anderson, 1974, 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 À30 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 B25% and B27% 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 o 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







[ R= (CH2)n-CH3]

Fig. 1. Triacylglyceride structure (Fox et al., 2000).

Mather. 2000). Polyunsaturated fatty acids are especially prone to oxidation. Hammond. El Soda. Lipids present in foods may undergo oxidative or hydrolytic degradation (McSweeney & Sousa. Microstructural and physico-chemical dynamics of fat globules in cheese also appear to influence the localization and retention of starter lactococci in cheese (Laloy. While this is a very interesting theory. Kanno. since they are activated only in the presence of a hydrophobic/hydrophilic interface. McSweeney & Sousa. 1983). McSweeney & Sousa. For all cheeses. Spectral changes of triacylglycerides. McPherson & Kitchen. which leads to the formation of various unsaturated aldehydes that are strongly flavoured and result in the flavour defect referred to as oxidative rancidity (Fox. .ARTICLE IN PRESS Y. no detailed scientific investigation has been undertaken to elucidate the mechanism of accessibility of fat in cheese for lipolysis. 1982. As ripening progressed. which are hydrolases that cleave the ester linkage between a fatty acid and the glycerol core of the triacylglyceride. Lipid oxidation does not occur to a significant extent in cheese. (2000) showed that fluorescence and infra-red spectroscopy could monitor and differentiate patterns of triacylglyceride phase transition in cheeses of varying composition. Dufour et al. enzymatic hydrolysis of triacylglycerides to fatty acids and glycerol. but at lowest fat content more globules were noted. producing FFA. Kinsella. However. Guinee. 2000). most globules were o2 mm in diameter. Using confocal laser scanning microscopy (CLSM). 1996). Collins et al. & Hotchkiss. whereby hydrolysis of the protein matrix may reduce pressure on the fat globule influencing starter localization within cheese (Laloy. & Simard. 1–21. lactococci became more intimately associated with the fat globule such that non-viable cells appeared to become integrated into the fat globule membrane. 1997). Lactococci. or milk in which milk fat had been replaced by other lipids. 2000). Auty. (2000) attributed the clumping and coalescence of globules to the destruction of the MFGM during processing and also to heating of curds during cheesemaking.. 21–82 days of ripening. indicating partial crystallization. & Shipe. Cogan. visualized using electron microscopy. & Ford. Gunasekaran and Ding (1999) examined the three dimensional characteristics of fat globules in one month old Cheddar of varying fat contents (B40– 340 g kgÀ1). vitamin E) (Fox & McSweeney. Guinee et al. Mather & Keenan. and were accompanied by an increase in viscosity. 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.. Fox et al. Knight. 1998). mono. were noted over ripening. 1978. & Simard. esterases have classical Michaelis–Menten type kinetics while lipases. Esterases hydrolyse soluble substrates in aqueous solutions while lipases hydrolyse emulsified substrates. Wijesundera. 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. were shown to be located on the periphery of the fat globule. while increasing fat content of cheese resulted in progressive clumping and coalescence of the globules. Keenan. Marchesseau. and Fenelon (2000) also examined the microstructure of Cheddar cheeses of fat contents. However. Full fat Cheddar cheese retained higher cell populations in the curd compared to 50% fat reduced Cheddar. & McSweeney. Unfortunately. Cheeseman. and. Gunasekaran. Muthuku-marappan. El-Safty & Isamil.. 2000. (2) physico-chemical nature of the substrate and (3) enzymatic kinetics. 2. 1970. The potential influence of proteolysis on this progressive association between starter cells and fat globule membrane was raised in this study. 1974. were evident in two distinct intervals. display interfacial Michaelis–Menten type kinetics (Chich. respectively. such cheeses did not develop correct flavour (Foda. Vuillemard. Reduction of fat content of cheese was accompanied by dispersion of discrete globules without clumping. 1975. the terms ‘‘esterases’’ and ‘‘lipases’’ are often used interchangeably in the scientific literature. Guinee. Lipolysis in cheese is due to the presence of lipolytic enzymes. These changes. and mono.g.or diacylglycerides (lipolysis) is essential to flavour development in some cheese varieties (McSweeney & Sousa. Esterases hydrolyse acyl ester chains between 2 and 8 carbon atoms in length. to date. Lipolytic enzymes may be classified as esterases or lipases. 1980. 1998). 2000. Dylewski. Fox et al. The enzymatic kinetics of esterases and lipases also differ. Woodford. probably because of its low redox potential (À250 mV) (Fox & Wallace. El Soda. The average globule size appeared to be inversely related to the total fat content of the cheese. Overall. Vuillemard. less circular globules. & Gripon. 1996). Microstructural studies of fat present in cheese indicate the presence of globules of varying size and shape. & Everett. its contribution to cheese flavour development is considered to be of little importance (Fox & Wallace. 2000) and the presence of natural antioxidants (e. increasing fat content in the cheese was reflected by the presence of larger.F. in the range B70– 300 g kgÀ1 using CLSM. 2000). 2000).and diacylglycerides (Deeth & Touch. globule size was smallest at lowest fat content. 1997. while lipases hydrolyse those acyl ester chains of 10 or more carbon atoms. which are distinguished according to three main characteristics: (1) length of the hydrolysed acyl ester chain. This was demonstrated in studies with cheeses made from skim milk. / International Dairy Journal 13 (2003) 841–866 843 phase of milk (Anderson. Reinbold. 1983. Drury. 1972. 1997.

. the differences noted in hydrolysis rates were attributed to higher concentrations of MCT present at the emulsion surface (Deckelbaum et al. C18:2. 1964). More recently. Rennet paste Commercial rennets are normally free from lipolytic activity. 1977).. the actual affinity of LPL was shown to be higher for LCT than for MCT emulsions which may reflect differences in the ‘‘quality’’ (composition and physical properties) of the enzyme-substrate interface.3-diacylglycerides and later to 2-monoacylglycerides.and medium-chain fatty acids are preferentially released by LPL. 1971. or C20:0 (Deckelbaum et al. this activity appeared to be associated with the extracted fat layer rather than the aqueous phase. inappropriate milking or milk-handling techniques.and 2.. 1995.2. Initially. Interestingly. 2. & Wallace. Virto. used in the manufacture of some hard Italian varieties (e.and triacylglycerides (Olivecrona. 2000). However.. under optimum conditions (37 C. (3) starter. McSweeney & Sousa. Fox et al. C18:0..1. it is still thought to contribute to lipolysis in pasteurized-milk cheese. pH 7) with addition of an apolipoprotein activator. 90% (w/v) LPL in milk is associated with the casein micelles and the fat. C8:0. hydrolysis of ewe milk fat yielded octanoic. foaming. due to agitation. lipolytic enzymes are specific for the outer ester bonds of tri. 1964. However. free butanoic acid (C4:0) occurs at a greater relative concentration in cheese than in milk fat (Bills & Day. Hydrolysis of as little as 1–2% (w/ v) of the milk triacylglycerides to fatty acids gives a rancid or ‘‘lipolysed’’ flavour to the milk (Olivecrona & Bengtsson-Olivecrona. Vilaro. 1990). / International Dairy Journal 13 (2003) 841–866 FFA are released upon lipolysis and contribute directly to cheese flavour. di. Romano). triacylglycerides are hydrolysed to 1. and de Renobles (1998) studied the enzymology of industrial raw and pasteurized ewes’ milk and Idiazabal cheese made from these milks. which normally never reaches its full activity in milk (Fox & Stepaniak. suggesting its selective release by lipases present in cheese or its synthesis by the cheese microflora (Bills & Day. 1977. LPL activity was assayed on the following substrates: trioctanoic. According to Deeth and Fitz-Gerald (1983). 1991). The enzyme is present in milk due to leakage through the mammary cell membrane from the blood where it is involved in the metabolism of plasma triacylglycerides. is located mainly at the sn-3 position and is preferentially released by lipolytic enzymes (Parodi. LPL has been shown to be relatively non-specific for fatty acid type. Overall. could theoretically release sufficient FFA acids within 10 s to cause perceptible hydrolytic rancidity. as 78 C Â 10 s is required for its complete inactivation (Driessen. 1993). ca.e. and hexadecanoic acids. these workers found that LPL activity in milks decreased significantly as lactation progressed. LPL is of more significance in rawmilk cheeses than in cheeses made from pasteurized milk. Lipases in cheese originate from six possible sources: (1) the milk. Suckling stimulates the secretion of PGE at the base of the tongue. homogenization. 2000). Deeth & Fitz-Gerald. Provolone. PGE is highly specific for short chain acids esterified at the sn-3 position (Nelson et al. apo-CII. since its activity is not reduced by pasteurization. This difference in rates of hydrolysis was attributed to the greater solubility and mobility of MCT in emulsion systems which allows a more rapid hydrolysis compared with LCT. contains the lipase.. 1993).g. 1993. Fox & Wallace. sn-1 and sn-3 positions) (Deeth & Touch. & Pitas. it is generally accepted that high-temperature short-time (HTST) treatment (72 C for 15 s) inactivates the enzyme very extensively. (5) non-starter bacteria and. 1997. 1964). Fox et al.2. trihexanoic acids. However. 1992).. Bovine milk contains 10–20 nm LÀ1 lipase which.F. Highest rates of hydrolysis were noted for trioctanoic acid. especially short. (6) their addition as exogenous lipases (Deeth & Fitz-Gerald. Santisteban. 1990). The proportions of free C6:0 to C18:3 in Cheddar cheese appear to be similar to those in milk fat.and intermediate-chain FFA (Bills & Day. 1989). However rennet paste. with pasteurization of milk causing an average 73–95% inactivation of LPL. Fox & Stepaniak. C18:3. as well as the other short. & Bengtsson-Olivecrona. but is specific for the sn-1 and sn-3 positions of mono-. Activity of LPL determined in aqueous cheese extracts during ripening was quite low and not very reproducible. tridodecanoic. 1978. occurring in globules. (4) adjunct starter. (2) rennet preparation (rennet paste). possibly.or diacylglycerides (i. e. Chavarri. Fox. decanoic. 2000. 2000).. LPL displays a preference for hydrolysis of medium-chain triacylglycerides (MCT) with a 2 fold increase in the rate of hydrolysis of MCT emulsions containing C6:0. McSweeney. Christie. is surrounded by a lipoprotein membrane (MFGM). Jensen. Hence. C10:0 or C12:0 esterified FA compared to long chain triacylglyceride (LCT) emulsions containing esterified C16:0. tridodecanoic and olive oil. In general. Law. with lower and comparable levels noted for tridecanoic. 2. butanoic.and medium-chain acids. . pregastric esterase (PGE) (Nelson. C18:1. tridecanoic. Milk lipase Milk contains a very potent indigenous lipoprotein lipase (LPL). 1978. short. Therefore.ARTICLE IN PRESS 844 Y. This does not occur under normal circumstances as LPL and fat are compartmentalized. McSweeney & Sousa. If the MFGM is damaged. and it is washed into the abomasa with the milk.g. 2000). 1995). significant lipolysis may occur resulting in off-flavours in cheese and other dairy products (Darling & Butcher. Collins et al. olive oil and ewe milk fat.

Gobbetti. El-Wahab. LAB possess esterolytic/lipolytic enzymes capable of hydrolyzing a range of esters of FFA. casei L-14. helveticus.0–8. Activity of all lipases was stimulated by reduced glutathione and low (ca. 1997). lactis and Lb. Activity was detected using b-naphthyl butanoic acid (C4:0) as substrate and the purified enzyme was active on p-NP from C2 to C12 with pH and temperature optima of 7. Khalid and Marth (1990) reported the quantitative estimation of the lipolytic activity of Lb. plantarum strain isolated from Cheddar cheese. fermentum. 2000). 1990). lactis NCDO 763. However. Acinetobacter and Flavobacterium (Stadhouders & Veringa. 2001). Chaudhari and Richardson (1971) identified a second lipase.ARTICLE IN PRESS Y. casei L-7. According to Lee and Lee (1990). Lc. El-Soda. optimally active at 37 C and pH 7 to 8. delbrueckii subsp. Takayama. depending on the source of PGE (Nelson et al.0 and 55 C. termed gastric lipase. Kamaly. esterolytic and lipolytic enzymes were produced by cell lysis of Lb. plantarum L-34 and Lb. di. rather than in cheese made using starter culture (Reiter et al. LAB are considered likely to be responsible for the liberation of significant levels of FFA. Chich et al. cremoris showed the highest lipolytic activity of the strains studied on tributanoic acid and milk fat emulsions. lactis and Lb. cremoris (Piatkiewicz. the three emulsions were hydrolysed by the four strains of lactobacilli with the exception of Lb. acidophilus displayed the highest esterolytic activities. Holland. b-Naphthyl dodecanoic acid (C12:0) and b-naphthyl ethanoic acids (C2:0) were the substrates used for determination of lipase activity and esterase activity. Evidence for this comes from the very low levels of FFA in aseptic starter-free cheeses made using glucono acid-d-lactone. Esterase activity was higher than lipase activity in all strains. Maximum lipolytic activity was observed at pH 7.. Nelson et al. because of their presence in cheese at high numbers over an extended ripening period. Collins et al.. Castillo. citrate positive lactococci and Lc. Lb. 1973. 2 g 100 mLÀ1) concentrations of NaCl but inhibited by high concentrations of NaCl (ca. certain fungal lipases (e.2 and 37 C. 1987). Lb. lactis and Lc. To hydrolyse milk fat in milk and cheese. acidophilus. & Gobbetti. Lb. lactis subsp. kid and lamb PGEs which result in slight differences in flavour characteristics of the cheese. a lipase secreted by Rhizomucor miehei) may be acceptable alternatives (Fox. 1993).. Early studies on the role of LAB and lipolysis by Stadhouders and Veringa (1973) concluded that partially hydrolysed milk fat was a better substrate for lipolysis by starter bacteria than unhydrolysed milk fat. casei subsp. and Damiani (1996) reported the purification of an intracellular lipase from a Lb. Lb. in general. 1997. which is slurried in milk before being added to cheese milk (Fox & Stepaniak. lactis subsp.. are generally considered to be weakly lipolytic in comparison to species such as Pseudomonas. 2. olive oil and tributanoic acid emulsions were used as substrates. Liu. kids or lambs slaughtered after suckling. delbrueckii subsp. Ezzat. 1993. lactis. respectively.g. 1967). 1997. in an extract of cleaned gastric tissue and reported that a combination of calf gastric lipase and goat PGE resulted in Cheddar and Provolone of superior quality to cheese made with PGE alone. and Marth (1990) reported the presence of lipases in the cell-free extracts of a number of strains of Lc. Lb. helveticus L-53. especially Lactococcus and Lactobacillus spp.3. 1997). LAB. respectively. lactis subsp. lactis subsp. This enzyme had a molecular mass of 65 kDa . Smacchi. Fernandez de Palencia. None of the strains tested hydrolysed o. which failed to hydrolyse olive oil. Chich et al.. Lb. 1996. enzyme activity was inhibited by Ag+ and Hg2+ ions and stimulated by Mg2+ and Ca2+ (Lee & Lee. 2001). Most other lipases investigated are unsuitable for use in the manufacture of Italian varieties due to incorrect specificity. 20 g 100 mLÀ1). Fontecha. Fox. (1997) reported the presence of esterolytic activities in an intracellular extract of Lactococcus lactis subsp.F. All lactobacilli showed activity against substrates up to C5:0. 1999. Stepaniak. Fox & Wallace.. 1993).. Chich et al. a species found in the starter used in the manufacture of Parmesan cheese (Battistotti & Bosi. casei L-7. Liu et al. lipases/esterases of LAB appear to be exclusively intracellular and a number have been identified and characterized (Chich et al. delbrueckii subsp. (1977). Some interspecies differences in specificity have been reported for calf. and Ismail (1986) found intracellular esterolytic activities in four strains of lactobacilli: Lb. Despite the presence of these enzymes. casei LLG.. claimed it was doubtful whether the stomach wall secretes an intrinsic lipase and attributed gastric lipase activity to oral secretions or the regurgitation of intestinal contents containing pancreatic lipase. 1988). The presence of lipases and esterases has been demonstrated in nine strains of Lc. Milk fat. cremoris. / International Dairy Journal 13 (2003) 841–866 845 Rennet paste is prepared from the abomasa of calves. Desmazeaud. & Corsetti. & Crow. The abomasum is partially dried and ground into a paste. Requena. tri-. contains a cell surface-associated esterase specific for C4:0 which can hydrolyse b-naphtyl esters of fatty acids from C2:0 to C10:0 (Gobbetti. bulgaricus. 1997. However.5. 1977. and monoacylglyceride substrates (Holland & Coolbear. these lipases were.and p-nitrophenyl (p-NP) substrates containing fatty acids of the even numbered carbon atoms from 6 to 14. Fox et al. Microbial lipolytic enzymes Lipases and esterases of lactic acid bacteria (LAB) appear to be the principal lipolytic agents in Cheddar and Dutch-type cheeses made from pasteurized milk (Fox et al. To date. Fox & Stepaniak. Smacchi. lactis subsp. 1987)..

increasing concentrations of the phospholipid substrate to levels favouring micelle . Liu et al. cremoris HP. (2000) confirmed the intracellular nature of a tributanoic acid esterase from Lc. Later work by Wilkinson. lactis subsp. O’Callaghan. McSweeney. Esterase I. Differences in substrate specificities between esterase I and II were noted. Cell free extracts prepared from both strains had generally similar levels of activity on lipase (tri cis-9-octadecenoic acid emulsion) or esterase (p-NP butanoic acid) substrates and these workers concluded that there was preliminary evidence for a relationship between autolysis of starter bacteria and lipolysis in cheese. Collins. This enzyme was not active on pnitrophenyl esters of fatty acids of chain length >C12:0. In common with other studies. as measured by the concentrations of FFA from C4:0 to C18:3 in Cheddar cheese made using either Lc. cremoris AM2 or Lc. Lowest activity was found against tetradecanoic acid.ARTICLE IN PRESS 846 Y. however. respectively. hydrolysed di. esterase activity was increased on elevation of NaCl concentration from 3.000 Da. cremoris HP as starters. few studies have been undertaken to establish whether a relationship exists between the extent of autolysis of LAB and lipolysis in various cheese varieties. However. with esterase I hydrolyzing p-nitrophenyl esters of short chain FFA C2 to C8 while esterase II hydrolysed C2–C6 p-nitrophenyl esters. b-monoacylglycerides (b-MAG) and b-naphtyl esters of C2:0 to C12:0 FA. (2001) identified three intracellular esterases in Streptococcus thermophilus.5–8.5 g 100 mLÀ1. was readily hydrolysed while activity was also detected against phospholipid substrates with medium chain fatty acid residues. Meyers. lactis subsp.F. an effect of which has not been previously reported. Cuppett. this gene did not encode for a signal sequence at the Nterminal required for extracellular secretion. Recently. Interestingly. These workers showed that the 744 base pair EstA gene encoded for a 258 amino acid protein of molecular mass B29. and Wilkinson (2003) examined the influence of starter autolysis on lipolysis during a 238 d ripening period. This finding indicated that starter strain properties may influence the levels of certain lipolytic end products in cheese. Cloning and up to 170-fold overproduction of this enzyme was possible using a nisin-controlled expression system which allow detailed characterization of enzyme specificity and kinetics.0. The genetic characterization of lipolytic enzymes of LAB by Fernandez et al. Profiles of b-MAG after hydrolysis by the purified lipase indicated that b-MAGs were generated with fatty acids C14:0 to C18:1 but those fatty acids of chain length shorter than C14:0 were not produced. less activity on tridodecanoic and trihexadecanoic acids and no activity on tri-cis-9-octadecenoic acid.7 to 7. As the location of most LAB esterase/lipase activities appears to be intracellular.and monoacylglycerides up to C14:0. cremoris HP. / International Dairy Journal 13 (2003) 841–866 with pH and temperature optima of 7. which was tested against a range of glyceride substrates. However to date.80. lactis subsp. lactis subsp. Guinee. with high activity also noted against. decreasing temperature in the range 25–37 C.99–0. Substrate specificity studies were carried out on triacylglycerides. However. hexadecanoic acid (C16:0). two of which were purified to homogeneity and designated esterase I and II with molecular masses of B34 and 60 kDa. ethanoic acid. and Fox (1994) demonstrated that strain AM2 was highly autolytic in comparison to strain HP and that secondary proteolysis was higher in Cheddar cheese made using strain AM2. These authors suggested that the microenvironment within whole cells may be more conducive to lipase activity. these workers did not monitor cell viability and autolysis of these strains in cheese during ripening. octanoic acid. However. and decreasing water activity (aw ) in the range 0. The impact of cheese compositional parameters on esterase I activity on p-NP butanoic acid substrate indicated that enzyme activity was reduced by decreasing pH in the range 5. cremoris AM2 developed higher total levels of odd-numbered C3–C15 methyl ketones compared with cheese made with Lc. Collins et al. The higher levels of secondary proteolysis in cheese made using this strain were ascribed to an early and more extensive release of intracellular peptidases on autolysis. hexanoic acid. hexadecanoic acid. tetradecanoic acid (C14:0). cremoris B1014. lactis subsp. The triacylglycerol substrate. This enzyme was relatively heat stable to a temperature of 65 C but was irreversibly inactivated on heating to 75 C for 2 min. lactis subsp. octadecanoic acid and cis-9-octadecenoic substrates. cremoris AM2 developed significantly higher levels of a number of FFA including octanoic acid (C8:0). decanoic acid. It is evident that they may require release into the cheese matrix through cell autolysis for maximum efficiency. The tributanoic acid esterase displayed highest activity on short chain p-NP esters of fatty acids with highest activity against p-NP hexanoic acid (C6:0).5 and 35 C. Both enzymes had maximum activity on p-NP butanoic acid. and octadecanoic acid (C18:0) during ripening compared with cheese made with the less autolytic strain Lc. and Hutkins (1996) found that release of FFA from either butter oil or a range of triacylglyceride substrates by incubation with whole cells of various LAB strains was higher than that found when the substrates were incubated with intracellular extracts prepared by sonication of the cells. tributanoic acid. Early work by Walker and Keen (1974) found that Cheddar cheeses made with Lc. lactis subsp. and dodecanoic acid substrates. Hydrolysis of triaclyglycerides indicated that the enzyme had highest activity on tributanoic acid. respectively. highest activity on b-naphtyl esterified fatty acids was found against butanoic acid. These workers found that Cheddar cheese made using the highly autolytic Lc.

Chandan. In mould-ripened cheeses such as Brie. 1982). under salt-induced stress conditions with 0. most of the lipase activity (80%) in Lc. Bistline. 1994). using tributyrin as a substrate. cremoris strains indicating the involvement of different extracellular proteinases in the processing of the lipase. Ehrlich. present in the microflora of Swiss-type cheese. Pintado. (2002) has provided a good insight into the complexity of the molecular processing and transmembrane secretion mechanisms required for the secretion of an active extracellular lipase of Staphylococcus hyicus which was cloned and overexpressed in Lc. / International Dairy Journal 13 (2003) 841–866 847 formation did not lead to an increase in enzyme activity through interfacial activation. Brevibacterium linens is a constituent of the flora of surface-ripened cheeses (e. McSweeney & Sousa. linens using tributanoic acid. hyicus consists of a signal peptide of 38 amino acids. The fact that deletion of PmpA did not exert an adverse effect on growth of Lc. lactis. (2002) subsequently identified a chromosomal gene pmpA coding for a PrsA-like lipoprotein (PLP) which has been shown to be involved in protein secretion in Bacillus subtilis.25 m NaCl in the medium. Interestingly. Ordal. Penicillium spp. Limburger) which are characterized by a significant level of lipolysis during ripening. possesses an . The authors suggest that various unidentified cell wall-associated proteinases may be responsible for this cleavage. growth rate was much reduced. Yoshioka. It is therefore important that research is carried out to establish the contribution to lipolysis of intact or autolysed LAB cells and whether any trans-membrane glyceride or fatty acid active transport system is involved in the formation of FFA and other lipolytic end products by LAB. however. hyicus when cloned and overexpressed in Lc. Secretion of a pro-protein of 86 kDa occurs followed by further cleavage by a metalloproteinase to generate the mature 46 kDa lipase. lipolytica. The lipase of S. shermanii. linens using emulsified olive oil as a substrate (San Clemente & Vadehra. Using emulsified tributanoic acid as a substrate. optimum pH and temperature for lipolytic activity were 7 and 37 C. lactis. lactis was possible up to 30% of total cellular proteins. the other with a more alkaline pH optimum (9–9. lactis. levels beyond this were toxic to Lc.g.5) (Morris & Jezeski. Niki. Oterholm. and Malcata. Camembert and Roquefort. 1953. these strains did not possess the PrtP enzyme. roqueforti possesses two lipases. lactis and Lc. one with a pH optimum of 7. indicating that PmpA may become a limiting factor in salt stress resistance factors in Lc. (1999) isolated four species of bacteria (Enterococcus faecium. further confirming the esterolytic nature of this enzyme. 1966. Welch Baillargeon.ARTICLE IN PRESS Y. Collins et al. Kman. and Witter (1970) showed that Propionibacterium freudenreichii subsp. lactis. Half of the intracellular activity was in the precursor pro-enzyme form anchored to the cell membrane while the remainder was trapped by the cell wall and cleaved at the N-terminal by cell wall associated proteinases.. & Shahani.5–8. Dupuis. Pintado. High lipolytic activity was reported for Y. and Renault (2000) and Drouault et al.0. lactis was concentrated intracellularly. S^rhaug and Ordal (1974) reported esterolytic and lipolytic activities in five strains of Br. 1976). The significance of this work for cheese ripening is not clear as the authors did not report whether the various truncated forms of the enzyme possess lipase activity. 1993. Emulsified oleic and palmitic acid esters were used as substrates. lactis. Freitas. Lipolytic activity has been demonstrated in Br. The major species present in Picante cheese throughout ripening are adventitious LAB and yeasts. as overproduction of PrsA reduced the production of intracellular truncated lipase fragments and increased the production of active correctly secreted prolipase of the extracellular lipase of S. 1974.F. several other truncated lipase forms were produced intracellularly and also secreted extracellularly. paracasei) and three species of yeasts (Debaryomyces hansenii. Lactobacillus plantarum and Lb. Picante cheese is a traditional cheese manufactured in Portugal from a mixture of ovine and caprine milks and is manufactured without deliberate addition of starter cultures. The work of Drouault. Yarrowia lipolytica and Cryptococcus laurentii) from Picante cheese and assayed each for proteolytic and lipolytic activities. camemberti produces an extracellular lipase optimally active on tributanoic acid at pH 9 and 35 C (Lamberet & Lenoir. and Sonnet (1989) reported lipase activity in three strains of Geotrichum candidum. 1966). faecalis. the other species studied released lower concentrations of FFA. PmpA and its gene product PLP also appears to be involved in protein folding and secretion in Lc. In addition to the production of pre-prolipase and prolipase. However. 2000). lactis in a rich medium suggests a limited role for this gene. a pro-peptide of 207 residues and a mature lipase of 396 amino acid residues. Despite the presence of the correct signal sequence from S. emulsified tributanoic acid and emulsified olive oil as substrates. P. P. 1967). are essential lipolytic agents (Gripon. The neutral lipase is more active on trihexanoic acid and the alkaline lipase is more active on tributanoic acid (Menassa & Lamberet. It is well recognized that propionic acid bacteria (PAB) are between 10 and 100 times more lipolytic compared to LAB (Knaut & Mazurek. These authors suggest that PmpA allows correct folding of the lipase which prevents its degradation by the surface protease HtrA. respectively. & Ahiko. Corthier. the kinetics of lipase degradation and cleavage specificities of cell wallassociated proteinases differed between Lc. hyicus and its replacement with a lactococcal leader peptide. Expression of this lipase in Lc. Drouault et al. E.

propanoic acid and butanoic acid esters and lipolytic activity against tributanoic acid substrates. Lamberet.and medium. 1991. McSweeney & Sousa. 1996). Dartley and Kinsella (1971) found the total concentration of methyl ketones in blue-veined cheese increased steadily up to 70 d of ripening and subsequently decreased. Penicillium roqueforti (Urbach. 2. Collins et al. & Le Quere. particularly in Blue cheese. However. and (5) aldehydes. Gripon. Catabolism of free fatty acids (Molimard & Spinnler. as well as vegetative mycelia. 1996). FFA also act as precursor molecules for a series of catabolic reactions leading to the production of flavour and aroma compounds. Monnet.2 and 47 C.or 5Hydroxyacids Unsaturated fatty acids Lactoperoxidase Hydroperoxidases Hydroperoxide lyase Aldehydes Secondary Alcohols Free Fatty Acids γ or δ Lactones Acids Alcohols Fig. Kalantzopoulos. Methyl ketones (alkan-2-ones) are important fatty acid catabolites. & Desmazeaud. 2 and catabolism will be dealt with under the following headings: (1) methyl ketones. Fox & Wallace. Lamberet. Hydrolysis of soluble substrates was small compared to hydrolysis of emulsified substrates and these workers therefore suggested that the enzyme be considered a lipase. Lamberet. Intracellular fractions derived from strains grown in liquid media showed both esterase and lipase activities. 1987.chain fatty acids directly contribute to cheese flavour.. trihexanoic acid (C6:0) and trioctanoic acid (C8:0). Concentrations of methyl ketones have also been shown to increase throughout the ripening period of Emmental cheese (Thierry. . Corre. FFA released as a result of lipolysis. Catabolism of fatty acids In cheese. In contrast to Dupuis et al. More recently. Auberger. 1982. & Lenoir. (4) lactones. or associated with the cell wall fractions. 1997). for the secretion of extracellular esterase and lipase activity during growth. especially short. Spores. followed in order by tributanoic acid (C4:0). 1997. however the extent to which this activity may have arisen as a result of cell lysis was not determined. in this study evidence was provided.g. (2000) was either cytoplasmic or cell membrane associated. for the first time. have been shown to produce methyl Triacylglyceride Lipase Free Fatty Acids β -oxidation β -Ketoacids β -oxidation 4. Maillard. Canteri. esters. Dupuis. alkanes and secondary alcohols (Gripon. & Lenoir. 3. / International Dairy Journal 13 (2003) 841–866 intracellular lipase with pH and temperature optima of 7. Pathways of fatty acid catabolism are outlined in Fig. and Tsakalidou (2000) purified and characterized an intracellular esterase from Propionibacterium freudenreichii subsp. Cerning. Kakariari. Georgalaki. respectively. (1993) esterase activity was not found in the cell-free medium. Esterase activity was assayed for in the cell-free growth medium. The enzyme purified by Kakariari et al. cell wall fractions and a sonicated intracellular extract. 2000). and Boyaval (1993) screened a number of strains of propionibacteria for both esterolytic activity against ethanoic acid. The maximum rate of hydrolysis on triacylglycerides was observed on tripropanoic acid (C3:0). such as methyl ketones. freudenreichii. lactones.F. 1999).ARTICLE IN PRESS 848 Y. 1966. Penicillium camemberti and Geotrichum candidum (Lawrence. (3) secondary alcohols. (2) esters. e. Molimard & Spinnler. Methyl ketones are formed in cheese due to the action of mould lipases.

Collins et al.2 and 193. It has been suggested that fatty acids are not the only methyl ketone precursors (Dartey et al. 1976a). high concentrations of FFA are toxic to P. the pathways involved are illustrated in Fig. Penicillium roqueforti spores have been shown to produce methyl ketones when long chain fatty acids. 1996). 3. Besson. 1976a). . Chalier and Crouzet (1998) studied the bioconversion of copra oil (rich in direct precursors of methyl ketones. 1994). Kinsella. respectively).cis-9. pH.. 1976b). In fact. 2000). Catabolism of fatty acids by Penicillium spp. involves four main steps and the pathway by which methyl ketones are formed is referred to as b-oxidation. e. respectively. 3.3–6. Larroche. & Gros. decarboxylation of keto acids to alkan-2-ones. & Kinsella. Metabolism of fatty acids by Penicillium spp. camemberti is more Saturated Fatty Acids (C2n) CoA-SH β-Oxidation. Demyttenaere. The steps include: release of FFA by lipases. dodecanoic and tetradecanoic acids) by two strains of Penicillium roqueforti spores. Without exogenous lipase action.1 mmol 100 gÀ1 of oil. The high concentrations of heptan-2one and nonan-2-one found in Blue and Camembert cheeses were not in proportion to the quantities of octanoic and decanoic acids present in milk fat (Kinsella & Hwang. & Kinsella. It has been shown that methyl ketones can be formed also by mould cultures from the ketoacids naturally present at low concentrations in milk fat or by oxidation of monounsaturated fatty acids (Kinsella & Hwang. The rate of production of methyl ketones in cheese is affected by temperature. in the presence or absence of exogenous lipase. octanoic. The final step is reversible under aerobic conditions (Kinsella & Hwang. roqueforti spores (Fan. the main fatty acid in milk fat is hexadecanoic acid (C16:0). 1976). Lipolysis of copra oil by Candida cylindracea lipase.ARTICLE IN PRESS Y. decanoic. Hwang. 1973.5 mmol 100 gÀ1 of oil. Strain dependant formation of methyl ketones resulted from the bioconversion of FFA present in the oil. are added to a culture medium (Chalier & Crouzet. methyl ketone production was low.. 1993). / International Dairy Journal 13 (2003) 841–866 849 ketones (Chalier & Crouzet.F. 1998) and spores have been used in the bioconversion of medium chain (C6 to C12) fatty acids (Dartey. 3. Growth of the mycelium of P. Konincks. & Meersman. 1966. oxidation of the released FFA to a-ketoacids. physiological state of the mould and the concentration of fatty acids. 1973.g. (McSweeney & Sousa. for both strains. resulted in a large increase in methyl ketone concentration (91. 1976a). Both resting spores and fungal mycelia are capable of producing alkan-2-ones at a rate that does not directly depend on the concentrations of FFA precursors. of less carbon atom. Kinsella & Hwang. hexadecanoic acid and cis. -2H2 + H2O Keto Acyl-CoA Thiohydrolase CoA-SH Thiolase CoA-SH +β-Ketonic acid Acetyl-CoA + Acyl-CoA (C2n-2) -Ketoacyldecarboxylase Krebs Cycle CO2 Methyl Ketone (C2n-1) + CO2 Reductase Secondary Alcohol (C2n-1) Fig. while the presence of glucose and amino acids have been known to stimulate methyl ketone formation by spores of Penicillium roqueforti or Aspergillus niger (Lawrence.12-octadecadienoic acid. followed by the reduction of alkan-2-ones to the corresponding alkan-2-ol.

Mycelia oxidize fatty acids over a wide pH range. branched-chain thioesters were not produced. their concentrations increased during the first part of the ripening process to a maximum at 60 d. & Belin. however. In full-fat Cheddar cheese. 1996). Cerf. When FFA are present at low concentrations in cheese. were identified. 1968). & Lee. Dekker. 1995). In another study. linens) and strains of L. Geotrichum candidum is also able to produce esters. a more recent study found that all of the fatty acid esters in Cheddar were ethyl derivatives (Arora. Pseudomonas fragi hydrolyses milk fat and esterifies certain of the lower fatty acids with ethanol. butanoic acid and pentanoic acid.6 mg 100 gÀ1. 1996). Edam. Esters are highly flavoured and are formed when FFA react with alcohols. de Jong. 1983). 2000). 1976). producing fruity flavours. Engels. A great diversity of esters is present in cheese (Molimard & Spinnler. Dirinck and De Winne (1999) reported levels of 2- heptanone and 2-nonanone ranging from 290. Gruyere. 1974. Lamberet. Concentrations of methyl ketones in low fat cheeses were found to be ca. heptan-2-one and undecan-2-one were found to increase steadily during ripening. with an optimum between pH 5 and 7.0 mg 100 gÀ1. and Visser (1997) compared the volatile compounds in the watersoluble fraction of 7 cheese varieties (Gouda. Heptan-2-one has been found in significant concentrations in Parmigiano-Reggiano cheese (Meinhart & Schreier. methyl. c). & Martinez Castro. butanoate. and Bergere (1997) compared the ability of various strains of coryneform bacteria. In a study of artisanal Blue cheese. & Juarez. Auberger. respectively (Villasen ˜ or. 1997). Cormier.3 to 321. Thioesters are formed when FFA react with free sulphydryl groups (Molimard & Spinnler. with mean levels of 73.1 to 196. levels of nonan-2-one. the synthesis of thioethyl-2-methylpropanoate. in Gouda cheese. Vayssier.F. Neeter. Similar esters have been identified in some lactic cultures used in the manufacture of Cheddar cheese (Molimard & Spinnler.7 and 36. butanoate and pentanoate was achieved via esterification of ethanethiol or butanethiol with . roqueforti have been found to oxidize fatty acids containing 2–12 carbon atoms with octanoic acid being the substrate which is most rapidly converted. 9 of which were Manchego cheese). Maasdam.0 mg 100 gÀ1 and 184.ARTICLE IN PRESS 850 Y. Parmesan and Cheddar). Methyl ketones are the most important flavour components present in Blue cheese and methyl ketones are present at the highest concentrations. some of which have a very pronounced melon odour (Jollivet. roqueforti. Roger. Chateaud. Methyl ketones with evennumbered chains appeared late in ripening and were never present in large amounts. Bensoussan. Collins et al. and are common components of cheese volatiles (Urbach. thiobutyl and thioexyl propanoic acid. nonan-2one and undecan-2-one increased for approximately 14 weeks and then decreased. similar to that of mature Blue cheese. to form S-methyl thioesters. camemberti catabolizes the individual fatty acid more rapidly (Fan et mediumchain fatty acids and the alcohols derived from lactose fermentation or from amino acid catabolism. after which time levels began to decrease (de Llano. Esters and thioesters are other products of fatty acid catabolism. Eleven methyl ketones were identified. 1996) as FFA are the principal methyl ketone precursors. A boxidative pathway has been indicated in mycelial metabolism of FFAs to methyl ketones (Lawrence & Hawke. 1986). 1986). Kinsella and Hwang (1976a) reported a positive correlation between free fatty acid level and the concentration of methyl ketones produced. Strains of Brevibacterium linens and Micrococcaceae were able to form branched and straight-chain thioesters. / International Dairy Journal 13 (2003) 841–866 sensitive to inhibition by fatty acids than that of P.8 mg 100 gÀ1. mostly methyl ketones. 1992). While ethyl. was found to inhibit lipolysis but to increase concentrations of methyl ketones (King & Clegg.2 and 359. pentan-2one and heptan-2-one were found to be the most abundant methyl ketones in aged ewes milk cheese (14 samples were analysed. propyl and butyl esters of even C2:0 to C10:0 fatty acids have been reported in various cheese varieties (Meinhart & Schreier.8 mg 100 gÀ1 and 176. & Miller. Sanz. Nine ketones. Gripon. 1979). 1996). Montilla. Coryneform bacteria (other than B. they are completely oxidized to CO2 and low amounts of methyl ketones are formed (Margalith. respectively. and Adda (1974b. Cavaille-Lefebvre and Combes (1997) demonstrated the ability of an immobilized lipase from Rhizomucor miehei to catalyse the synthesis of short-chain flavour thioesters such as thioethyl. even though P. Esterification reactions resulting in the production of esters occur between short. in Emmental. 1994). Proosdij. 1993). All strains synthesized S-methyl thioacetate. 25% of the levels observed in the full-fat cheese (Dimos. except in very mature cheeses. Valero. in which Blue cheese flavour was produced. Micrococcaceae and commercial starters of Lactococcus lactis and Leuconostoc spp. and Adda (1974a) and Dumont. Ramos. Urbach. 1981). Inhibition of lipolysis in low fat cheese was suggested as an explanation for this difference (Dimos. Ethyl esters arise from esterification of ethanol with acetylcoenzyme A (Yoshioka & Hashimoto. levels of heptan-2-one. lactis synthesized thioesters up to S-methyl thiobutyrate. 3-methylbutanoate. hexanoate and of thiobutyl propanoate. levels varied between 333. Addition of fatty acids to a slurry system. Roger. Spores of P. heptan-2-one and nonan-2-one were found to be the predominant methyl ketones.. respectively.8 and 198. In contrast. In a later study. mycelia are provided with optimum pH conditions for methyl ketone production in mature cheese (Dwivedi & Kinsella. Rodriguez. Hence. 1992). Methyl ketone formation in Camembert cheese has been studied by Dumont.

When the aqueous phase of Emmental cheese was studied by dynamic head space analysis. in freshly drawn milk are formed in the mammary gland by oxidation of fatty acids (Eriksen. Smith. The presence of disproportionate amounts of high molecular mass lactones has been reported in rancid Cheddar cheese. season. it was found that esters increased during the warm room stage of ripening and most esters showed a 4–20-fold increase between days 3 and 62 (Thierry et al. ethyl decanoate and methyl hexanoate being the most abundant. de Llano et al. dC12 and dC14 of 150. and Gripon (1988) reported that 2-phenylethyl acetate and 2-phenylethyl propanoate are qualitatively important in Camembert cheeses. resulting in the formation of an imide that can be decarboxylated. 80. 1997).. 1992). (1975) in Cheddar. 1993) formed by the intramolecular esterification of hydroxy fatty acids (Christie. C12:0 lactones may be formed by P. they have 5. from their precursor hydroxyacids (Eriksen. / International Dairy Journal 13 (2003) 841–866 851 short-chain fatty acids using a commercial immobilized Rhizomucor miehei lipase (Cavaille-Lefebvre.. & Berger.and dhydroxyacids following their release from triacylglycerides by lipolysis (Eriksen. 1975b). Shireman. butanal. Hydroxylation of fatty acids can also result from normal catabolism of fatty acids. Thirty eight esters were identified in ParmigianoReggiano cheese by Meinhart and Schreier (1986). Wilkins. g-dodecalactone and d-dodecalactone (Gallois & Langlois. 1983). the most significant lactone found was d-octalactone (Meinhart & Schreier. 1976). & Grosch. Basic studies of lactone formation in milk fat have illustrated that lactones are produced by heat.g. therefore. respectively (Eriksen. Several lactones have been identified in Parmigiano-Reggiano cheese. However some straight-chain aldehydes.and d-Lactones can also be formed spontaneously from the corresponding g. (1992) found 2-heptanol and 2-nonanol to be the main alcohols in artisanal Blue cheese. 1975). and concluded that the extensive lipolysis in Blue cheese influences the formation of lactones. 1990).ARTICLE IN PRESS Y. and the resultant formation of a ring structure (Molimard & Spinnler. Wong et al. ethyl octanoate. 490 and 890 mg 100 gÀ1.and 6sided rings. 1996). d-Dodecalactone and d-tetradecalactone were found to be the principal lactones in Blue cheese at 75 d of ripening (Jolly & Kosikowski. are responsible for the production of secondary alcohols (e. Latrasse. however. lactone levels increased most rapidly to a concentration well above their flavour threshold early in the ripening period (Jolly & Kosikowski. in the presence of water. 1986). through loss of water. 1989). It has been reported that lactones may be formed from keto acids after reduction to hydroxyacids (Wong. Schmidt. Collins et al. Combes. & Spinnler. 1983. Gruyere and Parmesan have high levels of lipolysis and contain high . 2000).. 1976). in Cheddar cheese at 14 months. however. Roger. 1993). It has been reported that the mammary glands of ruminants have an d-oxidation system for fatty acid catabolism (Fox et al. 1992). 2-heptanol and 2-nonanol) in blue-veined cheese due to reduction of methyl ketones (Martelli.. fatty acids or via glycolysis. quantitatively. precursors (alcohols) of these esters may be produced from amino acids. Lactones are cyclic compounds (Fox et al.. e. & Jezeski.F.. Fourteen different esters have been identified in Emmental cheese (Imhof & Bosset. 1976).and d-lactones are stable and have been identified in cheese. 2000). with ethyl ethanoate. 1998). The potential for (Dufosse lactone production depends on such factors as feed. a. Aldehydes may also be formed microbially.. It is also proposed that aldehydes are formed by Strecker degradation of amino acids (Keeney & Day. (1999) reported a 10–50-fold increase in the levels of secondary alcohols in the aqueous phase of Emmental during ripening. 1994). may be formed as a result of the boxidation of unsaturated fatty acids. Lactones may also be generated from unsaturated fatty acids by the action of lipoxygenases or hydratases ! . g. hydroxyacids. Ellis. stage of lactation and breed (Fox et al. 1986).g. & LaCroix. respectively. Jolly and Kosikowski (1975b) found that the concentration of lactones in Blue cheese was higher than the levels reported by Wong et al. d-decalactone. It has been reported that Streptococcus thermophilus and Lactobacillus delbrueckii subsp. 1975). the concentration of these lactones in cheese should. Secondary alcohols can be formed in cheeses by enzymatic reduction of methyl ketones (Engels et al. Rychlik. Rehbock. Production of 2-propanol from acetone and 2-butanol from butanone has been reported in Cheddar cheese (Urbach. The precursors of lactones. Aldehydes are formed from amino acids by transamination. (1975) reported levels of dC10. In Cheddar cheese. 1997). g. The sweet flavoured g-dodecanolactone and g-dodec-Z6-enolactone occur at much higher levels in milk from grain-fed cows than in milk from pasture fed cows (Urbach. bulgaricus possess the enzyme. Methyl and ethyl esters have been found in high proportions in artisanal Blue cheese (de Llano et al. while Thierry et al. which can catalyse the direct conversion of threonine and glycine to acetaldehyde (Marshall & Cole. Fatty acid ethyl esters and smaller quantities of methyl esters have been identified in Manchego cheese (Villasen ˜ or et al.. 2000). Degas. correlate with the extent of lipolysis. Warmke. 1994. threonine aldolase. 1997).and b-lactones are highly reactive and unstable in cheese (Fox & Wallace. 1957). 2-pentanol. heptanal and nonanal. 1997). gC12.. The Lactones found in Camembert cheese include g-decalactone. In contrast. 1976). Penicillium spp. 1975b). roqueforti spores and vegetative mycelium from long-chain saturated fatty acids (C18:1 and C18:2) (Chalier & Crouzet. which has led to the suggestion of other pathways for the formation of lactones (Wong et al.. 1999).

‘‘blue cheese’’ flavour note. for some cheese varieties.ARTICLE IN PRESS 852 Y. Short and intermediate-chain. volatile fatty acids can either contribute positively to the aroma of the cheese or to a rancidity defect. The flavour effect of FFA in cheese is regulated by pH. respectively. 1984. Barbosa. / International Dairy Journal 13 (2003) 841–866 concentrations of linear aldehydes. the flavour effect of fatty acids may be negated due to neutralization (Molimard & Spinnler. 1986). Roquefort cheeses). a specific class of compound is recognized as being the major contributor to flavour. Olson & Johnson. which may. Ha and Lindsay (1991. it has been proposed that the fat in cheese provides a fat– water–protein interface for flavour forming reactions to occur. 1984. The numerous compounds involved in cheese aroma and flavour are derived from three major metabolic pathways: catabolism of lactate. even-numbered fatty acids (C4:0–C12:0) have considerably lower perception thresholds and each gives a characteristic flavour note. surface bacterially ripened cheese. 1984). 1974. directly. little is known about the exact contribution of individual compounds to flavour (Wijesundera & Drury. 1984). Romano has the highest concentration of FFA (and the strongest FFAgenerated flavours). Ha. Hexanoic acid has a ‘‘pungent’’.1 mg kgÀ1 cheese in Parmesan cheese. Butanoic acid contributes ‘‘rancid’’ and ‘‘cheesy’’ flavours. 1996).4 mg kgÀ1 cheese. & Addeo. ‘‘goat’’. 1997). Free fatty acids. & Lindsay.0 and 189. the physical presence of fat in cheese is important for flavour development. semisoft cheeses) and sheep milk (Pyrenees.6 and 347. Depending on their concentration and perception threshold. contribute to cheese flavour and also serve as substrates for further reactions producing highly flavoured catabolic end products. Of these three Italian varieties. & Creamer. have been correlated with Swiss cheese flavour notes (Zerfiridis.g. (1997). FFA are significant contributors to the flavour (Woo & Lindsay. However. 1993). allowing their retention in cheese and release during consumption (Manning. When no such compound or class was found. 175. characteristic aroma of the cheese. 1996). 1993) reported that 4-ethyloctanoic acid contributed the characteristic goat. and Lindsay (1984) found high levels of butanoic and hexanoic acids in Limburger cheese which were related to the development of the strong.. Lipid hydrolysis results in the formation of FFA. Woo and Lindsay (1984) found butanoic acid. de la Feunte. FFA have been reported to play an important role in the flavour of Serra da Estrela cheese (Partidario. methyl ketones are important flavour contributors (Molimard & Spinnler. which is associated with its mild flavour (Woo & Lindsay. 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. while Provolone contains intermediate levels. enzymes from the milk. Lawrence. in Romano cheese. ‘‘soap’’. 1984). As well as being a source of flavour compounds. Brennand. 1996).5 and 122. enzymes from the rennet and accompanying lipases and secondary flora (Urbach. Etievant. which must be present at certain levels and in the correct balance to produce a flavour typical of a given variety. FFA are important flavour contributors in hard Italian varieties such as Romano.F. & Litopoulou-Tzanetaki. Vafopoulou-Mastrogiannaki. caused by the interaction of starter bacteria. Giles. Early research on Cheddar cheese flavour sought to identify a single compound or class of compounds responsible for characteristic flavours (see Aston & Dulley. protein and lipid (Molimard & Spinnler. 1993. The importance of butanoic acid in Camembert flavour was indicated by the generation of a Camembert-like flavour in a cheese base containing a mixture of butanoic acid. hexadecanoic acid and C18 congeners at levels of 175. The theory suggests that cheese flavour is made up of a balance of flavours contributed by a number of compounds. However. ‘‘rancid’’ and ‘‘fruity’’ note. ‘‘musty’’. Collins et al. Cheese flavour is very complex and differs from one cheese variety to another (Tomasini. Mozzarella cheese has a low FFA concentration. 78.0. Long-chain FFA (>12 carbon atoms) are considered to play a minor role in cheese flavour due to their high perception thresholds (Molimard & Spinnler. in particular butanoic. Wijesundera & Drury.6. Fonte! rez (1993) found levels of butanoic.. 1993). propanoic and ethanoic acids. Vangtal & Hammond. e. and Jua hexadecanoic and cis-9-octadecenoic acids of 100. Woo. respectively. The latter variety contains relatively high levels of butanoic and hexanoic acids (Woo & Lindsay. & Boas. 1982). 1999).and mutton-like flavours of cheeses manufactured from goat (fresh. For mould ripened cheeses. 1999). 389. 1990. Dekimpe. 1998). In cheeses with a high pH. Parmesan the lowest. cha. Butanoic and hexadecanoic acids and C18 congeners were reported at levels of 14. . the Component Balance Theory was proposed by Mulder (1952). respectively.5. Aldehydes were also detected in the water-solublefractions of all of the cheese varieties analysed by Engels et al. in the case of Cheddar cheese and similar varieties. Kollodge. 4.0 mg 100 mLÀ1 cheese. 1996). Fat also acts as a solvent for fat-soluble flavour compounds. octanoic acid has a ‘‘wax’’. 1989). & Lebeault. 1982). Bustillo. In the case of hard Italian cheeses. oct-1-en-3-ol and other compounds (Woo et al. in Parmesan cheese. In this respect. Parmesan and Provolone (Aston & Dulley. Straight-chain aldehydes are characterized by ‘‘green grass-like’’ aromas (Moio. methyl ketones.

. These cheeses contained FFA concentrations 3–10 times higher than control cheeses Patton (1963) found that removing fatty acids from Cheddar cheese distillates had a significant effect on aroma. 1971). esters and secondary alcohols. It is also plausible that the interface between the lipid and aqueous phase in the cheese is important to flavour development. the flavour thresholds of methyl ketones are quite low ranging from 0. Paulin. 1999). 1993). 1976) rancidity was characterized by soapy off-flavour in cheeses manufactured from milk with added lipases from Pseudomonas fluorescens and Pseudomonas fragi. & Le Que . constitute some of the most important components in the aroma of surface-mould ripened cheese. however.0– 2.F. & Chapman.. 1977. & Dwivedi. The secondary metabolites resulting from lipolysis include: methyl ketones. as cheese made with milk containing vegetable fat was low in characteristic Cheddar flavour and in methyl ketones. 2000). Fatty acids and secondary alcohols are also major flavour components (Arnold. In another study (Law. FFA and flavour development. Butanoic acid appeared to be present at high enough concentrations to contribute to the flavours of these cheeses. the role of FFA in the flavour of Cheddar cheese and similar varieties is much less apparent than with the varieties already mentioned. Sharpe. Research concentrating on relating Cheddar flavour to levels of FFA is based on three approaches: (1) determination of FFA levels individually. Semon. from C3:0 to C15:0. or two commercial vegetable fats. Woo et al. Deeth and Fitz-Gerald (1975) reported that 3 month old Cheddar cheeses with an acid degree value >3 were described as unclean or butyric. Wijesundera and Watkins (2000) provided evidence of the significance of methyl ketones to Cheddar cheese flavour.0 mg 100 gÀ1 for propan-2-one ! re ! . 6 and 12 months of ripening and relationships between Cheddar flavour and chemical analyses were presented. 1974. However. Olson & Johnson. (2) addition of fatty acids to bland bases to determine if Cheddar flavour can be produced or improved.. Cheddar samples were presented to a taste panel at 1. may have important enzymes or other factors which play a role in the development of Cheddar flavour. Methyl ketones are responsible for the unique flavour of Blue cheese. 3. This supports the theory that FFA are important to Cheddar flavour (Tanaka & Obata. the flavour was unchanged. 1975. Foda et al. Singh and Kristoffersen (1970) reported a relationship between the formation of active sulphydryl groups.. 1997). In general. 1967). While methyl ketones are more important in relation to the flavour of Blue cheeses. e. Dimos et al. 1996). 1982). 1975a). The significance of methyl ketones to Cheddar flavour has not been established. when these fatty acids were removed by neutralization. (1984) reported low concentrations of FFA in Edam and Colby cheeses which had low-intensity. Cheese containing vegetable fats had very little Cheddar flavour. especially.5 mg kgÀ1 hexanoic acid. Best Cheddar flavour was associated with 4.5–5. However. 1974. In a later study. / International Dairy Journal 13 (2003) 841–866 853 Literature on the contribution of FFA to the flavour of cheese is vast and conflicting. collectively or as ratios in order to correlate with flavour development. The release of secondary metabolites is of great importance to cheese flavour.09 to 50. Wijesundera & Watkins. Molimard & in water (Moio. 1977). 1969. cheese containing mineral oil had a slight Cheddar flavour. Many studies have shown that reduced-fat Cheddar cheese lacks typical flavour and contains lower concentrations of FFA. (1974) examined flavour development in cheeses made from skim milk homogenized with milk fat.. Significant levels of pentan-2-one and heptan-2-one in the headspace of Cheddar has been attributed to mould contamination (Urbach. Shahani. this suggests that the milk fat globule membrane. In contrast to these findings. 1998). 1993. Tilsiter and Limburger (Dartley & Kinsella. 1966. flavour was still inferior to the whole-milk cheese. 1996. smooth flavours. To illustrate the importance of milk fat to Cheddar flavour.. they are also present in Camembert cheese at 25–60 mmol 100 gÀ1 of fat (Molimard & Spinnler. heptan-2-one and nonan-2one (Jolly & Kosikowski. FFA were indicated as important precursors of other flavour compounds in New Zealand Cheddar cheese (Lawrence. Gripon. Wijesundera et al. lactones. The homologous series of odd-chain methyl ketones. 1979). Given suitable conditions of maturation.09 mg 100 gÀ1 for heptan-2one in water to 4. these compounds will enhance the flavour complexity (Nicol & Robinson. Collins et al. King & Clegg. or selective removal of FFA from Cheddar extracts to detect any alteration in flavour. Foda et al.g. and (3) manufacture of reduced fat Cheddar cheese or cheeses with vegetable fat as a substitute for milkfat (Aston & Dulley. 1989). Cheese containing milk fat gave the best results. but a significant role for the other FFA was less apparent. mineral oil. Wijesundera and Drury (1999) reported no significant difference in Cheddar cheese flavour intensity between whole-milk cheese and cheeses made from skim-milk homogenized with cream or anhydrous milk fat. 1990. Manning & Price. Cheddar cheese manufactured with vegetable or mineral lipids has also been reported to develop atypical flavours (Foda et al. St.ARTICLE IN PRESS Y. Reddy & Marth.0 mg kgÀ1 butanoic acid and 2. The two major methyl ketones in Blue and Camembert cheeses are nonan-2one and heptan-2-one (Anderson & Day. Law and Sharpe (1977) reported that the ratio of FFA to H2S was found to be unrelated to Cheddar flavour (Law & Sharpe. 1994. It was found that Cheddar flavour rose and then declined as the concentrations of butanoic and hexanoic acids increased (Barlow et al. removed on homogenization of the milk fat.

particularly d-C14 (1. 2000). Dirinck and De Winne (1999). of all aromatic compounds in Camembert cheese. and their concentrations correlate with age and flavour intensity (Wong et al. Boset. decreases in the level of d-decalactone were associated with an improvement in Cheddar cheese flavour (Dimos. 1996). d-Lactones have low flavour thresholds compared to other volatile flavour compounds (O’Keefe. In a survey of various cheese varieties. undecan-2-one and tridecan-2-one are described as having ‘‘fruity’’. also resulting from lipolysis. 1995). e. (1997) found high concentrations of ethyl butanoate in cheeses with a ‘‘fruity’’ note such as Gruyere. Limburger and Havarti). Collins et al. which arises due to esters. (Dufosse d-C14. of the order of 2–7 g kgÀ1. Libbey. Groux. McSweeney & Sousa. Tilsit. 1994). Cheshire. 1993.. Arora et al.ARTICLE IN PRESS 854 Y.g.F. 1997. ‘‘floral’’ and ‘‘musty’’ notes. Parmesan and Proosdij. decan-2one. (1997). 1994). g-C14. mould-ripened..3 mg 100 gÀ1 cheese. 2000. It has been reported that esters are important contributors to the flavour of Parmigiano-Reggiano cheese (Meinhart & Schreier. In a later study.. This fruity flavour. 1993. were produced in rancid cheeses. Caerphilly) to extensive (e. S-methyl thioesters contribute a characteristic strong flavour to various smear-ripened soft cheeses (e.. / International Dairy Journal 13 (2003) 841–866 Spinnler. Although the aromas of lactones are not cheeselike. were attributed to high concentrations of lactones. Wong et al. Patterns of lipolysis in various cheese varieties Levels of lipolysis measured as release of FFA vary considerably between cheese varieties from moderate (e. Gruyere or Cheddar cheeses (Gripon. Cheddar. In low fat cheeses. and Horman (1975) found that heptan-2-ol and nonan-2-ol represented 10–20 and 5–10 g 100 gÀ1. normal cheeses contained B0. d-C12. Secondary alcohols. may contribute to cheese flavour (Arora et al. 1993). & Erhardt.and d-lactones. Engst. heptan-2one has a blue cheese note (Rothe. ‘‘apricot’’ and ‘‘coco! et al. Propan-2-ol. g-C16. 2000) and have been reported to contribute to a buttery character in cheese (Dirinck & De Winne. respectively. According to Eriksen (1976) lactones possess a strong flavour. 1998) and Law (1984) reported that thioesters have a ‘‘cheesy’’ aroma. thioesters formed by the reaction of esters of short-chain fatty acids with methional imparted the characteristic ‘‘cheesy’’ aroma to Cheddar cheese.. This would suggest that certain lactones are important in relation to Cheddar cheese flavour (Fox et al. d-C15.8 mg 100 gÀ1 cheese)... moderate levels of FFA. Fox & Wallace. nonan-2-one. Thresholds are relatively low for g-octalactone. d-C16. (1995) analysed the odour-active volatiles in Cheddar cheese headspace and found that most of the esters separated had a ‘‘buttery’’ to ‘‘fruity’’ aroma. 1980. Moinas. Fox et al. d-decalactone increased in full-fat Cheddar cheeses to a maximum at about 14 weeks and decreased to half the maximum value by 26 weeks. However. cheeses manufactured with vegetable fat which have a low Cheddar flavour have been shown to be deficient in g. 1997. garlic and some fruits (CavailleLefebvre et al. respectively. Thioesters often have characteristic aromas in many foods.. 1997). and has been suggested as one of the key compounds in the global aromatic note of Camembert cheese (Molimard & Spinnler. According to Lamberet et al. In a study of lactone levels in Cheddar cheese. Oct-1-en-3-ol has a raw mushroom odour with a perception threshold of 0. (1975) reported that greater quantities of higher molecular weight lactones. The level of lipolysis should not exceed 2% of triacylglycerides in Gouda. octan-2-ol and nonan-2-ol are encountered in most soft cheeses and are typical components of the flavour of Blue cheeses (Engels et al. 2000). & Lindsay. 1969).. butan-2-ol. g-C12. 1969) and are generally characterized by very pronounced. Fox & Wallace.001 mg 100 gÀ1.. 1993). 1982). 2002). McSweeney & Sousa. d-C10. According to Dimos (1992). Fox et al. Octan-2-one. Recent data for lipolysis in Emmental cheese manufactured using raw. in Gouda cheeses. suggesting their importance in relation to the flavour of this variety (Wijesundera & Drury. The mushroom and musty notes of methyl ketones are important contributors to the flavour of Camembert cheese (Molimard & Spinnler. 1993.. However.41 mg 100 gÀ1cheese) and d-C16 (B1. fruity notes (‘‘peach’’. gdecalactone and g-dodecalactone (0. hard Italian and surface bacterially ripened (smear) varieties (McSweeney & Fox. 1997. 1996). 5.g.7 mg 100 gÀ1 and B0. 1996). and microfiltered milk with and without various strains of propionibacteria .. Fox et al. and d-C18 lactones have been identified in Cheddar cheese (O’Keefe et al.1 mg 100 gÀ1 in water) and even lower for shorter chain lactones ! et al. 2000). 1975). Excessive lipolysis is considered undesirable and cheeses of the latter varieties containing a moderate level of FFA may be considered as rancid by some consumers (IDF.7–1. In Emmental cheese. the level of d-decalactone remained fairly constant throughout maturation. d-Decalactone and d-dodecalactone were more abundant in Gouda cheeses than in Emmental cheeses and the higher buttery notes. are liberated during ripening and make an important contribution to characteristic flavour and aroma in both raw and pasteurized milk cheese (Steffen.. Eberhard. Limited lipolysis is thought to be desirable in Dutch-type cheeses. 1996). 1999). they may contribute to overall cheese flavour (Fox et al. characterized the flavour of Gouda and Emmental cheeses. d-Lactones have generally nut’’) (Dufosse higher detection thresholds than those of g-lactones.. 1986). & Ruegg. Engels et al..g. Chamba & Perreard.g.. in onions. is considered undesirable in Cheddar cheese (Urbach. 1999).

the eluate is concentrated and FFA are directly quantified by GC. and Boudreau (1967). Partida Dulley and Grieve (1974) utilizes steam-distillation followed by GC to quantify volatile fatty acids. is homogenized and held before pasteurization thereby allowing lipolysis to proceed at a high rate (Nielsen. McNeill and Connolly (1989). Few studies have been performed to compare different methods of FFA quantification of the same sample. 1993). / International Dairy Journal 13 (2003) 841–866 855 indicated that FFA released during ripening originate from lipolytic activity of the added propionibacteria and that the extent of FFA developed in the cheese is strain specific (Chamba & Perreard. However. Martin-Hernandez & Juarez. Grana Padano. i. 1993).ARTICLE IN PRESS Y. The method of Partidario et al. Fontecha et al. Kid or lamb rennet paste is also used for coagulation of Provolone and Romano cheeses. Extensive lipolysis can be attained in mould-ripened cheeses without rancidity. used for the manufacture of the Danish blue cheese. 1993. The essential lipolytic agents in mould-ripened cheeses are the enzymes of Penicillium spp. and contains pregastric esterase (Battistotti & Corradini. thereby homogenization provides a larger lipid-serum interface for lipase activity. & Be or methylation to fatty acid methyl esters (FAME) (Ha & Lindsay. Collins et al. 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. and the high proportion of free oleic acid in Camembert has been attributed to the lipase of Geotrichum candidum. the flame ionization detector is robust with a wide and dynamic range enabling accurate FFA quantification. McSweeney & Sousa. 1993. resulting in the characteristic ‘‘piccante’’ flavour of these varieties (Fox et al.. followed by separation of FFAs by e. Woo & Lindsay. & Cabezas. Pe 1999). Limburger) (Woo et al.g. 1992.e. 5–10% of total triacylglycerides are hydrolysed in Camembert and up to 20% are hydrolysed in other blue-vein cheeses (Gripon et al. Ramos. Gripon. the third group of GC methods quoted in Table 1 involve solvent extraction of the lipid fraction in the cheese. Amundson. Organic solvents are used in the extraction procedures of the majority of the methods referenced. 1993). In general.g. Poveda. the high curd cooking temperatures used in the manufacture of ParmigianoReggiano. In a review of the determination of FFA in milk and milk products (IDF. this may be due to neutralization of fatty acids on elevation of pH (Gripon. 1993). FFA must be isolated from both the aqueous phase and the fat phase. This ‘‘piccante’’ flavour is due primarily to release of short chain FFAs. 1977). Lesage. Juarez.g. Provolone) which leads to higher levels of lipolysis in the ripened cheese due to the action of LPL. reduces fat globule size and increases the total fat globule surface area... The method of Woo and Lindsay (1982) is also included in this group. & Juarez (1986). The highest levels of lipolysis have been observed in traditional mould-ripened cheeses. Danablu. 1983. 1991.. Homogenization damages the MFGM. Snow. The method has since been used in other studies to quantify individual FFA in various cheeses (Woo & Lindsay. 2000). chromatographic columns (Deeth. This GC procedure enables rapid separation and quantification of FFA. Grana Padano reduces the activity of LPL during ripening. Martin-Hernandez.. The GC methods of Iyer. & Finner. 1991) methods for analysis of FFA by GC were grouped under two headings. surface bacterially ripened (smear) cheeses (e. Brevibacterium linens is a major constituent of the microflora of the smear of surface bacterially ripened cheeses and produces lipolytic enzymes (Reps.. 1990. Richardson. 1984). C4:0 to C10:0 (Nelson et al. Various methods are used to quantify FFA. (1990) belong to the second group of GC methods which involves the preliminary preparation of methyl esters. Romano and Provolone) (Bosset & Gauch.. Matthias. it is difficult to combine a good extraction of short chain FFA from the aqueous phase together with a good extraction from the fat phase. The first group permits direct analysis of FFA and includes the method of Nieuwenhof and Hup (1971). 2002). FFA are isolated on an alkaline silica gel column. 6. Cream. Many Italian varieties are manufactured from raw milk (e.. Arnold et al. Grana Padano. & MartinAlvarez. (1975) found a direct relationship between the flavour intensity of ripened Romano type cheese (an Italian variety) and its butyric acid content. 1993). 1996. According to IDF (1991). 1998. de la ! rez-Coello. Levels of 18–25% of total fatty acids as FFA have been reported for Danish Blue cheese (Anderson & Day. PGE is responsible for extensive lipolysis. 2000. 1984).g. isolation of FFA from the sample material is a vital step in any method. 1990. 1993).. this method involves removal of lactic acid by a partition pre-column followed by isolation of FFA on a modified silicic acid–potassium hydroxide arrestant column. & ! zard.. 1984). 1999). Parmigiano-Reggiano. Fitz-Gerald. According to IDF (1991). 1966).F. Voilley. the silicic acid column used by Nieuwenhof and Hup (1971) was later shown to induce hydrolysis of the fat. Extensive lipolysis is also characteristic of certain Italian varieties (e. Feunte et al. However. ! rio. Parmigiano-Reggiano. Lorient. Woo et al. FFA are then separated in formic acidmobilized elutes on a glass column packed with diethylene glycol succinate (DEGS-PS) by GC. Alanso. Kinderlerer.. Measurement of lipolysis The FFA compositions of some selected cheese varieties are shown in Table 1. 1984.. While these extraction methods commonly involve the use of internal standards of fatty acids . saponification (Martinez-Castro.

(1992) Poveda et al. (1993) Woo and Lindsay (1984) Romano RC. (1986) GC Bills and Day (1964) Marsili (1985) Dulley and Grieve (1974) McSweeney and Fox (1993) McNeill and Connolly (1989) de la Feunte et al.F. Woo and Lindsay (1982) de la Feunte et al.d 131 123 139 81 B130 168b 299b 169b 387b 365b 385b 1377b 172 61 2841 1476 667 635 413 206 238 175 111 9492 8254 4842 997 1028 6079d HPLC Kilcawley et al. Deeth et al. IB-R. (2001) GC Woo and Lindsay (1982) Woo et al. Collins et al. / International Dairy Journal 13 (2003) 841–866 476 1587 1270 952 952 794 15 111 308d 10 5 0 0 143 191 111 2 33 144d 24 16 25 6 B8 34 26 35 33 45 44 68 38 105 12 B128 175 159 111 6 38 185d 28 20 27 20 B12 22 16 18 25 29 29 73 41 13 6 B490 32 27 377 65 768 110 159 175 48 25 67 387d 571 619 238 37 68 225d 45 46 46 37 B45 18 25 13 30 35 35 94 81 15 952 746 397 103 183 423d 117 110 120 86 B70 38 61 27 59 76 74 175 218 48 1556 1253 619 285 397 1148d 309 270 299 231 B275 117 202 110 196 216 229 433 503 76 794 508 270 524b 131b 3259b. Juarez et al. EH. (1984) ARTICLE IN PRESS 503 524 496 200 B370 41 40 45 21 23 27 26 0 947 450 701 433 832 844 878 2506 GC McNeill and Connolly (1989) B25 16 29 16 55 22 22 134 49 12 11 B69 GC GC 37 43 45 47 56 60 142 115 69 43 Reddy and Marth (1993) Woo and Lindsay (1982) 865 467 209 69 36 40 10 Titration GC. (1999) .a Parmesan C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 Total FFA Method of FFA determination Reference 1055 140 451 106 843 243 84 328 440 158 942 439 181 428 1540 684 448 3896 1750 785 1171 1890b 1224b 3471 123 13 697 GC GC. (1993) B90 59 49 392 358 1169 202 B171 162 112 1007 667 2359 443 B295 394 275 2243 1553 6443 994 B299 132 94 620 372 1807 348 418 276 1438 1299 12 643 816 36 29 100 40 1069 12 8 8178 5577 32 404 Cheshire 8 0 505 285 2001 255 12 10 302 118 1030 133 Roncal Idiazabal Manchego 372 578 1642 318 GC GC. Martinez-Castro et al. (1983) GC GC. Rennet coagulated RC.856 Table 1 Free fatty acids (FFA) composition (mg kgÀ1 cheese) of some selected cheese varieties Cheese class FFA C2:0 1. IB-R.c Cheddar 1756 Y. H.

MartinHernandez et al. (1993) Martin-Hernandez and Juarez (1992) RC.876 704 426 398 340 608 1925 508 1384 106 7285 GC.d 256 75b tr 14 50g 4277 2926 9843d 842 59 31 1481 GC. (1967) 16 44 McNeill and Connolly (1989) Woo et al. CWE.F. Woo and Lindsay (1982) 2671 Woo and Lindsay (1984) RC. Alonsa.e Caerphilly Mahon Colby Monterey Jack Majorero 18 383 81 93 431 216 15 377 9 3 400 250 31 274 33 10 513 225 394 49 37 1964 723 57 307 22 20 1378 364 150 810 67 110 2327 663 387 2107 196 252 5877 1745 127 902 93b 211 1327 b 419 2235 36 82 13 8743 356 736 20 794 6144 GC GC GC. / International Dairy Journal 13 (2003) 841–866 4921 1400 886 528 de la Feunte et al. SH. and Ramos (1994) RC.f Swiss ARTICLE IN PRESS 170 345 392d 112 1037 288 120 90 21 135d 45 83 26 45 25 150d 71 6 21 122 53 381d 208 88 522d 68 13 51 311 267 1337d 207 58 160 1904 930 4262d 515 193 475 1427b 1197b 2664b. (1984) Zerfiridis et al. Woo and Lindsay (1982) GC Martin-Hernandez et al. IB-R. IB-R. DT. Woo and Lindsay (1982) Woo et al. (1984) Y. (1984) RC. (1984) 857 . Woo and Lindsay (1982) Brie Woo and Lindsay (1984) Lesage et al. IB-R. Iyer et al. (1993) Woo et al. ST. Lopez Fand# ıno. (1984) Emmental Gruyere GC GC. Woo and Lindsay (1982) Woo et al. (1993) Woo et al. Collins et al. IB-R. (1990) McNeill and Connolly (1989) de la Feunte et al. SM-R. P-FC.i Mozzarella Provolone 54 782 127 j 7 308 63 1 81 65 120 172 141 12 122 104 27 120 210 76 199 537 66b 334 1424b b GC. Woo and Lindsay (1982) GC. CWE. (1993) de la Feunte et al. (1988) Fernandez-Garcia. Camembert 35 208 101 361 124 5 58 287 9 14 35 43 69 448 622 255 270 1028 1442 839 210b 1421 1043 681 160 19 225 44 298 74 303 1314b 5066 2678 GC. (1984) RC. M-R. Woo and Lindsay (1982) GC GC GC. Nieuwenhof and Hup (1971).h Edam 60 8 9 14 47 39 122 57b 356 GC. Dulley and Grieve (1974).

/ International Dairy Journal 13 (2003) 841–866 937 7850 19 768 9157 30 434 75 685 GC. c Rennet coagulated. internal bacterially ripened. Whey cheese Gjetost a 106 35 31 180 49 170 456 631b 4558 GC. f Rennet coagulated. Partidario (1998) GC. (1984) Rennet coagulated. Woo and Lindsay (1982) Woo et al. Ingham. Jaeggi. (1992) RC. (1993) Woo et al. Wendorff. l Limit of detection for method=10 mg kgÀ1 cheese.7 163 4 688 2 27 232d 34 34 102 8 24 8 68 446d 33 35 66 54 50 35 193 1067d 89 97 154 33 92 22 153 613d 46 52 206 86 602 63 357 1805d 98 102 704 275 565 161 952 2835d 198 201 2057 199b 709b 39b 538b 5977b. mould-ripened. internal bacterially ripened. internal bacterially ripened. (1984) Total FFA Method of FFA determination Reference 105 700 620 804 815 160 1040 2135 235 3366 4460 185 2329 Kinderler et al. semi-hard. b . j Rennet coagulated. Pasta-Filata cheeses.k Roquefort C4:0 992 961 Blue Blue d’ Auvergne Blue (with conidia) White (no conidia) Gamonedo blue 1146 C6:0 751 626 777 C8:0 715 707 546 C10:0 2104 2280 1275 C12:0 1403 1295 1835 C14:0 2632 3185 4147 C16:0 6452 6230 11 416 C18:0 17404b 2241 14 088b 6282 896 C18:1 C18:2 C18:3 32 453 25 969 35 230 GC. C18:0 congeners. (1984) ARTICLE IN PRESS 402 2350 13 970d 220 208 1412 12 11 59 7 3 504 GC FAME. internal bacterially ripened. surface mould-ripened. IM-R. cheeses with eyes.F. Woo and Lindsay (1982) Woo et al.858 Table 1 (Continued) Cheese class FFA C2:0 RC. hard. internal bacterially ripened.d 106 103 833 700 GC. g C18:2+C18:3. i Rennet coagulated. Ha and Lindsay (1990) Partidario et al. e Rennet coagulated. internal bacterially ripened.m Port Salut Limburger Brick 41 1475 72 62 995d 37 35. Woo and Lindsay (1982) GC GC. S-R. surface-ripened. h Rennet coagulated. Dutch-type. and Houck (2000) Serra de Esterla Munster 2. m Rennet coagulated. (1990) de Llano et al. internal mould-ripened. extra hard. (1984) de la Feunte et al. k Rennet coagulated. M-R. Woo and Lindsay (1982) GCl Woo et al. Swiss-type. Collins et al. mould-ripened. cheeses with eyes. (1998) ! rio (1999) Partida de Leon-Gonzalez. d commercial samples exhibiting distinct flavour defects. Fontecha et al. (1996) Y.

because the FFA were injected in the form of soaps. Three gas chromatographic methods were compared . For FFA analysis the bottom layer is separated. Classification of . The ‘‘copper soaps’’ method is a classical colorimetric method which enables the determination of total levels of FFA (IDF. the eluate of each FFA must be titrated separately and the method is not as accurate as more sophisticated GC or HPLC methods. they were lower than those reported by McNeill and Connolly (1989) and Deeth et al. / International Dairy Journal 13 (2003) 841–866 859 dissolved in an organic solvent. Accuracy was reported to have increased substantially for the cheese with the higher FFA content and there was an improvement in the overall coefficient of variation reported by MartinHernandez. 1985). The fraction containing FFA was then injected directly onto the gas chromatograph. the intensity of which is related to the concentration of copper salts. This involves diethyl ether extraction of fat followed by methylation with tetramethylammonium hydroxide. 2000). this does not guarantee the completeness of the extraction procedure. C3:0 and C4:0 were recovered by steam-distillation and quantified by ligand-exchange. In the method of Kilcawley. which results in an upper layer containing methyl esters from glycerides and a lower layer which holds the tetramethylammonium soaps of FFA. Percentage recoveries ranged from 91% for butanoic acid (C4:0) to 103% for octadecanoic acid (C18:0). . Collins et al. In method 1. The method described by de Jong and Badings (1990) is now a commonly used procedure for the quantification of FFA in cheese. It was later shown that the ion-exchange resin used induced fat hydrolysis (McNeill & Connolly. Acid degree value (ADV) is defined as the number of milliequivalents of alkali required to neutralize the FFA in 100 g of fat. C2:0. The methyl esters are then analysed by programmed GC as described by Juarez. a coloured complex is then formed between the copper salts and sodium diethyl dithiocarbamate. In this method the air stream was freed from CO2 by bubbling through 20% KOH.ARTICLE IN PRESS Y. The derivatization technique tested resulted in no loss of the volatile components. N2 or He were used as carrier gases. using phenolphthalein as an indicator. and Polychroniadou (1999) for the analysis of by Ardo free fatty acids in cheese. In the work of Chavarri et al. ion-exclusion HPLC. When using a titration method to quantify FFA. Accurate and rapid determination of FFA from C2:0 to C20:0 is achieved by direct separation of underivatized FFA using capillary GC.F. The upper layer may be used to analyse the fatty acid composition of glycerides. after fat extraction. FFA were separated from triacylglycerides by aminopropyl-bonded phase chromatography. the method involves FFA extraction using ether/heptane from a cheese paste containing anhydrous sodium sulphate and sulphuric acid. Alonso. HPLC can operate at ambient temperature ensuring relatively little risk to sensitive functional groups (Christie. extracted fat was treated with tetramethylammonium hydroxide and the methyl ester derivatives were then formed in the injector. The accuracy and recovery rates are reported for the first method only. 1991). 1989). Cheddar with an ADV >3. two methods of sample preparation were compared for the determination of FFA in ewes’ milk cheese by GC. While the method is sensitive and rapid (IDF. Recovery rates were examined by adding standard mixtures of FFA to a sample from a cheese of known FFA content. and IDF (1991) recommend titration under nitrogen. and Fontecha (1992). Removal of CO2 from the air stream when titrating is vital to maintain the stability of the end point. (1997). Method 1 included the preparation and methylation procedures described by Martinez-Castro et al. Extracted FFA are then isolated using alumina or an anionexchange method (aminopropyl columns) and separation of FFA (C2:0 to C20:0 ) is achieved by capillary GC. 1991). de la Feunte. particularly for samples in which a minor fraction of the triacylglycerides have been hydrolysed to FFA. and Polychroniadou (1999) reported the accuArdo racy of the above method using two types of cheese. In brief. (1986). Copper salts of FFA are selectively transferred to an organic phase. and Fox (2001). It was concluded that the FFA fraction should be separated from the triacylglyceride fraction before derivatization and chromatographic analysis. washed with ethyl ether and adjusted to pH 9. Injecting samples at 300 C gave the best quantitative results. only a global estimation of FFA levels may be obtained. and Fontecha (1988). 50% cyanopropyl as the stationary phase. In method 2. Bills and Day (1964) used an ion-exchange silicic acid column to separate FFA. Wilkinson. (1983) (GC methods) also for Cheddar cheese.0 meq 100 gÀ1 fat is usually classed as rancid (Dairy Research and Development Corporation. however. Reproducibility values for the fresh cheese were comparable to those reported by Woo and Lindsay (1982) (GC method) for Cheddar cheese. one was a fresh cheese with a low FFA content and the other a cheese that had undergone moderate lipolysis. It was concluded that this technique provides a fast and reliable method of FFA analysis in cheeses with low FFA contents and particularly in those with high FFA contents. Juarez. which were then quantified by titration with potassium hydroxide. 50% phenyl. C6:0–C18:3 are derivatized to bromophenacyl esters following solvent extraction and overall separation is achieved by reversed-phase HPLC. HPLC should not be ignored as a method for FFA analysis (Marsili. the column was a silica capillary column with silicone. 1997).

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Banks. evaluation and effect of certain microorganisms on flavour components of Blue cheese. Bulletin 260 (pp. & Polychroniadou. Mangia. B. which is of major economic importance in many countries. M. M. 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 products impact on final cheese flavour. Miranda. K. & Virgili. & Dulley. Milk lipids.. A. Balcao. is extensive. Careri. 43. and semi-quantitative composition was determined by GC/MS profiling. D. and processing of the semi-quantitative data by multivariate (pattern recognition) statistics. additional research would undoubtedly be of benefit for varieties such as Cheddar. 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