Flavor Chemistry of Dairy Lipids - ACS Symposium Series (ACS

Chapter 14

Flavor Chemistry of Dairy Lipids Review of Free Fatty Acids

Downloaded by UNIV OF IOWA on June 26, 2016 | http://pubs.acs.org Publication Date: July 7, 1994 | doi: 10.1021/bk-1994-0558.ch014

I. J. Jeon Department of Animal Sciences and Industry, Kansas State University, Manhattan, KS 66506

Free fatty acids in dairy products, particularly in ultrahigh -temperature (UHT) processed milk and Cheddar cheese, are discussed. Although a fairly large amount of total free fatty acids is present in UHT milk, their influence on milk flavor is insignificant. This is because most fatty acids released from milk fat are long-chain fatty acids. Two different types of lipase apparently are involved in the lipolysis of UHT milk. Because patterns of releasing fatty acids are different from one lipase source to another, the source of lipase utilized is extremely important in cheese with reference to balancing flavor characteristics exerted by individual free fatty acids. Ratios and concentrations of these fatty acids could be significant parameters for determining cheese flavor characteristics.

Many excellent book chapters and review articles on the flavor chemistry of dairy lipids have appeared during the last three decades. Most works in the 1960's dealt extensively with lipids and their oxidation products (1-4). A number of excellent works have considered the mechanisms of the formation of chemical compounds from milk lipids, as well as their implications for the flavor of dairy products (2, 5). In addition, general reviews on off-flavors related to dairy lipids also have been published for milk (6, 7) and other dairy products (8). The purpose of this paper is to present some recentfindingson free fatty acids in milk and dairy products, particularly in ultrahigh-temperature (UHT) processed milk and Cheddar cheese. Influence of Free Fatty Acids on the Flavor of Dairy Products A unique feature of dairy lipids is that milk fat contains fairly large amounts of butyric, caproic, caprylic, capric, and lauric acids. Butyric acid residues alone comprise 8.5% of total fatty acid residues in milk fat triglycerides, and the total of 0097-6156/94/0558-0196$08.00/0 © 1994 American Chemical Society Ho and Hartman; Lipids in Food Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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these short-chain fatty acids amounts to 21% (9). Milk fat also contains small amounts of β-keto acids and hydroxy acids, which are known to be precursors of methyl ketones and lactones, respectively. It may be worthwhile to note that the distribution of these fatty acids in milk fat triglycerides is not random. For example, at position 3, the distribution of butyric acid is about 97% and that of caproic acid is 84%. These are strikingly high preference, and this difference in distribution is significant in regard to the hydrolysis of milk fat by the enzyme lipase. Normal milk contains a small amount of free fatty acids, and the significance of their contribution to the normal flavor of milk is unclear because their concentrations are well below their flavor threshold levels. However, it is well known that, in milk and other bland flavored dairy products, they cause lipolyzed flavor when their concentrations reach above usual levels (8). The literature suggests that no single fatty acid is a predominant contributor to the lipolyzed flavor in milk, although it is associated with short-chain fatty acids and, rarely, long-chain (C14 to C18) fatty acids (10, 11). On the other hand, Cheddar cheese and other aged cheese varieties contain a fairly large amount of free fatty acids, and these are considered to be essential flavor components (12, 13, 14). However, excess free fatty acids cause the lipolyzed off-flavor in cheese (15, 16, 17). In addition, concentrations of specific free fatty acids have an important influence on the flavor of aged cheese (13). Free Fatty Acids and UHT Milk Flavor Concentrations of total free fatty acids in UHT milk increase during aseptic storage. Research suggested that these concentrations as measured by acid degree value might exceed the lipolyzed flavor threshold levels recognized for raw and pasteurized milk (18, 19). However, no lipolyzed off-flavor was detected in these UHT milk samples. Recently, Choi and Jeon (20) found that the high concentrations of total free fatty acids observed were mostly due to the increase in long-chain fatty acids. The UHT milk samples that they utilized were indirectly processed in a commercial processing plant at 138°C for 10 s and aseptically packaged in 236 ml Brick Pak cartons. The samples were stored at 23 and 35°C and analyzed for free fatty acids by a solid phase extraction and gas chromatographic technique. As illustrated in Figure 1, few changes occurred in the concentrations of short-chain free fatty acids at 23 °C storage, although lauric acid (C12) showed a moderate increase (14%) after 12 wk. However, during storage at 35°C, concentrations of all short-chain free fatty acids showed small but steady increases ranging from 9% for caprylic (C8) to 45% for lauric (CI2) acid after 12 wk (Figure 2). However, actual concentrations of these fatty acids, except capric acid, in the UHT milk samples were well below the reported flavor threshold concentrations of 25.0,14.0,7.0, and 8.0 ppm for butyric (C4), caproic (C6), capric (C10), and lauric (C12) acids, respectively (2). Apparently, levels of individual short-chain free fatty acids at least four times higher than those found in these UHT milk samples are needed to produce moderately lipolyzed off-flavor in pasteurized milk. In contrast, long-chain free fatty acids, except oleic acid (CI8:1), showed

Ho and Hartman; Lipids in Food Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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Figure 1. Percent changes in concentrations of individual short-chain free fatty acids in UHT milk during storage at 23°C. Thefreefatty acids C4, C6, C8, CIO, and C12 represent butyric, caproic, caprylic, capric, and lauric acids, respectively.

Figure 2. Percent changes in concentrations of individual short-chain free fatty acids in UHT milk during storage at 35°C. Thefreefatty acids C4, C6, C8, CIO, and C12 represent butyric, caproic, caprylic, capric, and lauric acids, respectively.


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moderate but significant increases (P < 0.05) at 23°C, ranging from 7 to 20% after 12 wk (Figure 3). At 35°C, the increases of these free fatty acids were much more rapid (Figure 4). For example, stearic acid showed the most increase (63%) after 12 wk, followed by myristic (50%), linoleic (40%), palmitic (38%), and oleic acids (26%). Cause for Increase of Free Fatty Acids in UHT milk The increase of free fatty acids in UHT milk during storage is believed to be caused by heat-resistant lipase (27, 22, 23). This is either an indigenous milk lipase (milk lipoprotein lipase) or a bacterial lipase produced by psychrotrophic bacteria during cold storage of raw milk (24). Most lipoprotein lipase can be inactivated at 77°C for 16 s (25) or 98°C for 1 s (9). However, bacterial lipases are much more heat-resistant. Several reports have indicated that some bacterial lipases of Pseudomonas fluorescens have survived in milk during UHT processing (22, 26, 27). Recently, Choi and Jeon (20) presented some evidence of residual lipase activities in commercial UHT milks, as well as in their centrifugal fractions (12,000 χ g for 30 min at 4°C). The cream fraction showed the highest lipase activities according to an agar diffusion method used, followed by the aqueous supernatant and casein precipitates. In addition, the cream fraction preferentially hydrolyzed long-chain fatty acids from milk fat, whereas the aqueous supernatant fraction hydrolyzed both short- and longchain fatty acids, indicating that more than one kind of lipase might be involved in the lipolysis of UHT milk during storage. Results of DEAE-cellulose chromatography indicated that protein isolates of the aqueous supernatant contained three lipase-active fractions, whereas milk fat globule membrane proteins (from the cream) exhibited only one lipase-active fraction. SDS-PAGE analysis revealed that the lipase-active fractions from the aqueous supernatant contained a major or minor κ-casein component as well as other casein and whey protein moieties. However, the lipase-active fraction of the milk fat globule membrane proteins was composed mainly of α-casein. Types of lipase Involved in UHT Milk Lipases associated with milk are known to be specific or nonspecific with regard to the liberation of fatty acids from milk fat (8). Generally, ratios of individual fatty acids released are used to determine the types of lipase involved in the lipolysis of milk fat (20, 28, 29). Choi and Jeon (20) reported no significant differences in the ratios offreefatty acids between commercial UHT milks stored at 23 and 35°C, suggesting that the same kind of mechanisms was involved in releasing fatty acids from milk fat at the two different storage temperatures. However, the ratios of the long-chain free fatty acids released were significantly higher than those of the fatty acids in milk fat, indicating the involvement of a rather specific lipase. Further examination of the milk samples revealed some evidence for the involvement of two different types of lipase in the lipolysis of UHT milk fat. According to the lipase-active fractions obtained by DEAE-cellulose chromatography, the lipase associated with the aqueous supernatant (centrifugal fraction) produced ratios of fatty acids similar to those in milk fat. However, lipase associated with the milk fat globule membrane proteins yielded ratios of stearic and oleic acids significantly higher than those in



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Figure 3. Percent changes in concentrations of individual long-chain free fatty acids in UHT milk during storage at 23°C. The free fatty acids C14, C16, C18:0, C18.1, and C18:2 represent myristic, palmitic, stearic, oleic, and linoleic acids, respectively.

Figure 4. Percent changes in concentrations of individual long-chain free fatty acids in UHT milk during storage at 35°C. The free fatty acids C14, C16, C18:0, C18:l, and CI8:2 represent myristic, palmitic, stearic, oleic, and linoleic acids, respectively.


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milk fat. Therefore, these results suggested that the lipase activity associated with protein isolates of the aqueous supernatant is of a nonspecific lipase, perhaps milk lipoprotein lipase, whereas that associated with the milk fat globule membrane proteins is of a microbial origin, because it is more preferential toward some longchain fatty acids.


Free Fatty Acids and Cheddar Cheese Flavor As indicated previously, short-chain free fatty acids play a significant role in the flavor of Cheddar cheese (72, 13, 30), whereas long-chain fatty acids may contribute little to the direct cheese flavor but influence an overall cheese background flavor (75, 31). Arbige et al. (32) suggested that the total concentration of C4, C6, and C8 free fatty acids is an important factor for flavor development during cheeseripening.Lin and Jeon (33) suggested the total concentration of C4 and C6 free fatty acids as a good indicator of this flavor development. On the other hand, Marsili (34) reported that the best indicator of lipolytic age of Cheddar cheese was the combination of the CIO, CI2, CI4, and CI6 free fatty acids. Concentrations of free fatty acids in Cheddar cheese are quite high, although considerable variations have been observed by different investigators (Table I). For example, in 3- to 4-month aged cheese, butyric acid content ranges from 13 to 76 ppm and caproic acid content from 8 to 29 ppm. Medium and sharp cheeses also show quite a variation in concentrations. This large variability is quite understandable because many factors, such as source of milk, manufacturing practices, and curing conditions, will affect the degree of lipolysis. In addition, the method of analysis used also would affect the quantitative results. For example, Bills and Day (75) used a silicic acid column to isolate free fatty acids from Cheddar cheese. The results of Woo and Lindsay (16) were obtained using a silicic acid-KOH arrestant column. Lin and Jeon (33) and Marsili (34) used neutral alumina for the extraction of free fatty acids (35).

Table I. The free fatty acid content of Cheddar cheese at various ages Age (Mo) 3-4 4 3 3 6 6 7-12 10 12 12

C4

76 69 20 13 8 15 127 23 46 111

C6 29 31 12 8 8 2 33 18 50 33

Free Fatty Acids (ppm) C8 CIO CM C14 C16 Clé:Ô C l 8:1 Reference 36 55 87 191 516 104 319 6 37 232 1027 11 69 88 211 542 28 8 292 30 46 139 371 116 360 29

Flavor Chemistry of Dairy Lipids - ACS Symposium Series (ACS

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