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Changes in Free Fatty Acids During Ripening of Idiazabal Cheese Manufactured at Different Times of the Year
Changes in Free Fatty Acids During Ripening of Idiazabal Cheese Manufactured at Different Times of the Year ´ VARRI,* M. ANGELES BUSTAMANTE,* FELISA CHA ´ N,† ARANTZA SANTISTEBAN,* MAILO VIRTO,* L.J.R. BARRO and MERTXE DE RENOBALES*,1 *Bioquı´mica y Biologı´a Molecular and de Alimentos, Facultad de Farmacia, Universidad del Paı´s Vasco/Euskal Herriko Unibertsitatea, Aptdo. 450, E-01080 Vitoria-Gasteiz, Spain †Tecnologı´a
ABSTRACT Lipolysis was studied in cheeses manufactured from raw ovine milk in winter, spring, and summer, with no starter culture added, up to 180 d of ripening. Total amounts of free fatty acids (FFA) after 180 d of ripening were significantly higher in winter than in spring or summer. The major FFA in winter were C10:0 (3363 mmoles/kg), C4:0 (3309 mmoles/kg), C18:1 (3187 mmoles/kg), and C16:0 (2602 mmoles/kg). The amounts of all FFA in cheese decreased as the cheesemaking period progressed from winter to summer with C10:0 exhibiting the largest decrease (72.1%) and C18:1 the smallest decrease (22.8%). The amounts of FFA shorter than C12:0 were significantly higher in winter than either in spring or summer. The percentage of volatile ( C 4 to C8) , medium ( C 10 to C14) , and long-chain FFA ( ≥C16) changed during ripening: in winter and spring volatile FFA increased from 15% ( d 1 ) to 30% ( d 90 and 180) of the total, while long-chain FFA decreased from 55% ( d 1 ) to 40% ( d 180) of the total. In contrast, in summer, the percentages of volatile and long-chain FFA after 180 ripening d were 25 and 50% of the total, respectively. We concluded that winter cheeses were different from summer cheeses. Acetic acid increased during the first 90 d of ripening to a final concentration of 26,500 mmoles/kg, the amount of which was independent of the time of the year. ( Key words: lipolysis, free fatty acids, ovine milk cheese, cheese ripening)
ranean countries ( 5 ) , and in the last 10 or 15 yr, a substantial increase in research efforts has been made to study and characterize these products. The composition and individual amounts of FFA are considered to influence the flavor of different types of cheeses ( 2 7 ) either directly as in Italian varieties ( 2 8 ) or as precursors for other compounds such as methyl ketones, lactones, alkanes, and esters (26). Thus, changes in FFA composition during ripening have been monitored for most cheese varieties produced in an attempt to relate them to ripening age, even though it appears to be a less useful ripening index than do proteolytic and glycolytic indicators ( 8 ) . Although the composition and total amount of fat in ovine milk varies during lactation ( 1 6 ) and ovine milk is usually not standardized with respect to either protein or fat content prior to cheesemaking, changes in FFA during ripening have seldom been studied at various times during lactation (18). Idiazabal cheese is a typical product of the Basque Country region of Northern Spain and is manufactured from raw ovine milk according to the specifications of its Denomination of Origin Regulatory Board ( 3 ) . The cheese-making season extends from January until June with most cheeses being commercialized between 90 (mild) and 180 (medium) d of ripening. In the present work we studied changes in FFA during ripening in cheeses made at three different times during lactation as part of an ongoing characterization of this cheese. MATERIALS AND METHODS
INTRODUCTION Materials The production of cheeses manufactured with ovine milk has been traditionally localized in the Mediter-
Received June 15, 1998. Accepted December 7, 1998. 1Corresponding author. 1999 J Dairy Sci 82:885–890
Free fatty acid standards were from Sigma-Aldrich Quı´mica, S.A. (Madrid, Spain). Aminopropyl-bonded columns (500 mg) were from Waters (Madrid, Spain). Solvents (Merck, Madrid, Spain) were of the highest grade available and were not redistilled before use.
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TABLE 1. Amounts of FFA (micromoles per kilogram of cheese) accumulated during ripening of cheeses made in winter, spring, or summer.1 Ripening
Winter X
1 d 90 d 180 d
2501a 11,215a 19,268a
Spring SD 1160 1790 4398
X 1209b 6230b 9989b
Summer SD 202 1864 2293
X 1218b 7723b 11,256b
SD 195 867 934
a,bDifferent superscripts for each ripening day represent significant differences ( P < 0.05) among times of the year. 1The mean ( ± SD) of the 12 values obtained for the three batches made at each time of the year on each ripening day.
Cheese Manufacture Cheeses were made in the pilot plant of Queserı´as Araia (Araia, Alava, Spain). Bulk ovine milk was from several local, commercial flocks of latxa sheep. The lactation period for an individual flock extends approximately 5 mo between November and July, depending on the altitude where the flock is located with flocks on lower elevations starting lactation earlier. Industrial cheesemaking (using mixed milk from different flocks) extends from December until the end of June. Thus, industrial milk in January contains a large percentage of milk from flocks in their early lactation stage, and industrial milk in June corresponds to the late lactation period of most flocks. In April, milk could be a mixture (of unknown composition) of milk from flocks in late, mid-, and early lactation. Milk had been refrigerated for up to 48 h prior to cheesemaking. Cheeses were made according to the traditional method for the industrial production of Idiazabal cheese approved by its Denomination of Origin ( 3 ) , using commercial calf rennet (Chr. Hansen, Madrid, Spain), in 200-L vats. Cheeses (1.0 to 1.2 kg and 13 cm in diameter) were made in 3 consecutive wk in winter (last week in January and first 2 wk in February), spring (last 3 wk in April), and summer (last 3 wk in June) (total of 9 batches). From each batch, two different cheeses were taken for analysis after 1, 90, and 180 d of ripening. In addition to these sampling days, cheeses from batches 2 (1st wk in February), 5 (3rd wk in April), and 8 (3rd wk in June) were also taken after 15, 30, 60, and 120 d of ripening. Cheese samples were wrapped in plastic film and aluminum foil and frozen at –80°C until analyzed. FFA Analysis Gas-liquid chromatographic analysis of each cheese sample was made in duplicate. The FFA were analyzed underivatized by gas-liquid chromatography as Journal of Dairy Science Vol. 82, No. 5, 1999
described previously ( 7 ) . Briefly, cheese (1.0 g ) was ground and extracted from an acidic medium with diethyl ether-heptane (1:1 vol/vol) after addition of an internal standard mixture consisting of pentanoic, nonanoic, and heptadecanoic acids. Triacylglycerols were separated from the FFA on aminopropyl-bonded phase columns (previously equilibrated with heptane) by elution with chloroform:isopropanol (2:1 vol/ vol). The FFA were eluted with 5.0 ml of diethyl ether containing 20 ml of formic acid/L. This fraction containing underivatized FFA was injected directly into the gas chromatograph (model 5890, series II, equipped with a flame ionization detector; Hewlett Packard, Mardrid, Spain), and FFA were separated on a fused silica capillary column (25 m × 0.32 mm) coated with FFA phase (cross-linked polyethylene glycol, 0.52-mm layer thickness). The carrier gas (helium) flow rate was 2 ml/min, and the temperature was raised from 65 to 240°C at 10 °C/min and then held at 240°C for 20 min. Statistical Analysis The BMPD statistical package ( 2 5 ) was used for statistical treatment of the results. Analysis of variance and multiple comparison tests were applied to determine the presence of significant differences in FFA contents on different days of ripening (1, 90, or 180) among the cheeses made at different times of the year (winter, spring, or summer). Simple linear regression analysis was applied to fit the total FFA content with the ripening time. RESULTS AND DISCUSSION Total levels of lipolysis after 1, 90, and 180 d of ripening varied considerably for all batches of cheeses made at different times of the year (Table 1). Statistical analysis indicated that the total amounts of FFA obtained for winter cheeses on the 3 ripening d studied were ( P < 0.05) higher than were those for
LIPOLYSIS IN IDIAZABAL CHEESE
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Figure 1. Total amounts of FFA accumulated during ripening of cheeses made with raw milk in the 1st wk of February ( o) , the 3rd wk of April ( ∫) , or the 3rd wk of June ( ◊) . Error bars represent the standard deviation of 4 measurements.
cheeses made either in spring or summer (which were not significantly different from each other). Differences in total lipolysis were not as large after 90 d of ripening. Total levels of lipolysis were lower than were those reported for Idiazabal cheese by Na´jera et al. (22), most likely because those cheeses had been manufactured with natural lamb rennet paste, which is known to increase lipolysis (23), and were about half of those reported for Manchego cheese ( 9 ) . Lipolysis was monitored at different times during ripening in the second batch made at each time of the year to assess the rate of FFA accumulation in the early ripening stages (Figure 1). The rate of FFA accumulation was linear during the 180 ripening d for the three batches of cheeses studied with the highest rate observed for the winter batch (67.5 mmoles· kg–1·day–1) , which was consistent with its higher levels of lipolysis. Lipoprotein lipase activity in milk was also highest in winter and decreased as the lactation period progressed ( 6 ) . This observed variability in total lipolysis after different ripening periods at different times of the year could account, among other factors, for the varied results reported in the literature (19, 22) for commercially obtained Idiazabal cheese samples. The amounts of individual FFA were different ( P < 0.05) for the three batches made at each of the three times of the year, indicating that each batch was independent of the other two. However, to compare times of the year (winter, spring, and summer), all three batches made in 3 consecutive wk at a given time were considered as triplicates. In this case, the amounts of fatty acids shorter than C12:0 (lauric
Figure 2. Individual FFA accumulated during ripening of cheeses made with raw milk in winter ( A ) , spring ( B ) , or summer ( C ) . Values reported are the mean ( ± SD) for the 3 batches made at each time of the year. Error bars represent the standard deviation of 12 measurements.
acid) on d 90 and 180 of the winter cheeses were higher ( P < 0.05) than were those of either spring or summer batches, and the latter two were not different from each other. Winter cheeses were also different from those made during the rest of the year. The amounts of individual FFA were observed to change from winter to summer fabrications (Figure 2). In winter, after 180 d of ripening, the major FFA were C4:0 (butyric acid) (3309 ± 608 mmoles/kg), C10:0 (capric acid) (3363 ± 1424 mmoles/kg), C16:0 (palmitic acid) (2602 ± 593 mmoles/kg) and C18:1 (oleic acid) (3187 ± 482 mmoles/kg). In contrast, in summer, C18:1 (2461 ± 302 mmoles/kg) was the major FFA with C4:0 (1679 ± 130 mmoles/kg), C16:0 (1933 ± Journal of Dairy Science Vol. 82, No. 5, 1999
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180 mmoles/kg), and C10:0 (937 ± 177 mmoles/kg) present at lower concentrations. The most prominent change in the amounts of FFA from winter to summer cheeses was, in fact, the 3.6-fold decrease in capric acid, which correlated with the decrease in this fatty acid present in milk triglycerides in June (L.J.R. Barron, 1997, unpublished observations) as well as with the lower concentrations of milk lipoprotein lipase at this time of the year ( 6 ) , which would indirectly support the role of milk lipoprotein lipase in the appearance of FFA during cheese ripening (13). The variability observed for the amount of C10:0 on d 180 of winter cheeses appeared to be due to the sample and not to analytical factors because the variability of this FFA observed in the rest of the spring and summer samples was comparable to that of other FFA. Palmitoleic acid ( C 16:1) was present in very low amounts at all times. Polyunsaturated fatty acids [C18:2 (linoleic acid) and C18:3 ( g-linoleic acid)] were present at very low concentrations, as reported for most cheeses but in sharp contrast to Serra cheese manufactured with ovine milk in which they appeared at levels comparable to those of C 18:1 (24). Other FFA such as C10:1 (undecenoic acid), C14:1 (myristoleic acid), and C15:0 (pentadecanoic acid) were detected in all cheeses at all times of the year in amounts below 1%, while FFA with 20 carbon atoms were detected in some of the cheeses at all times of the year, also at concentrations below 1% of the total. As depicted in Figure 2, the amounts of all individual FFA studied increased during ripening regardless of the total level of lipolysis attained, which has been reported for the great majority of cheeses. However, not all FFA increased by the same amounts, and, as a result, the FFA composition of the cheeses changed considerably over the 180 ripening d (Figure 3). The percentage of volatile FFA [C4:0 to C8:0 (caprylic acid)], which have a significant impact on the development of the characteristic aroma of the cheese, was observed to increase during ripening at all times of the year, while that of long-chain FFA ( ≥ C16:0) decreased. In contrast, the percentage of medium-chain FFA remained quite constant during ripening at all times of the year. These changes were comparable in winter and spring with short- and longchain FFA representing 30 and 40% of the total, respectively, after 180 d of ripening (Figure 3). In contrast, in summer cheeses, volatile FFA represented 25% of the total and long-chain FFA represented 50%. Thus, the composition of winter and spring cheeses was similar, but that of summer cheeses was quite different. Journal of Dairy Science Vol. 82, No. 5, 1999
Figure 3. Percentage of volatile, medium-, and long-chain FFA after 1, 90, and 180 d of ripening. Values correspond to the mean ( ± SD) of the three batches made at each time of the year. Error bars represent the standard deviation of 12 measurements.
The FFA composition during cheese ripening has not been reported as such to the best of our knowledge. Yet calculations made using published values of individual FFA increases that occur during ripening indicate that the FFA composition does vary in some cases. Thus, no changes were observed in the percentage of volatile FFA in some bovine milk cheeses, such as Parmesano and Mozzarella ( 2 8 ) or Afuega’l Pitu ( 1 ) , but in others there was either a slight increase, as observed in Ras cheese made with ultrafiltered milk (11), or a marked increase, as in Gruye`re cheese (29). Blue-type cheeses made with a mixture of bovine, ovine, and caprine milks, such as Cabrales ( 2 ) and Gamonedo (15), showed a substantial decrease in the percentage of volatile FFA. Caprine cheeses showed either a decrease in the percentage of volatile FFA ( 1 2 ) or no change (20). By contrast, Manchego cheese ( 1 0 ) manufactured with 70% ovine and 30% bovine milks and Portuguese ovine cheeses, such as Serra ( 1 8 ) and Picante cheese (14), exhibited similar increases in the percentage of volatile FFA to those reported in this work. At present, the possible significance is not known for the observed increase in the percentage of volatile FFA in some cheeses. In the sensory analysis, cheeses manufactured in January received the highest scores, and those manufactured at the end of June obtained the lowest values (21). The results presented in this paper indicate that winter cheeses had both highest levels of lipolysis and highest amounts of FFA shorter than C12:0, strongly suggesting that, at least in Idiazabal cheese, lipolytic events play an important role in overall sensory characteristics.
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LIPOLYSIS IN IDIAZABAL CHEESE
Figure 4. Accumulation of acetic acid in cheeses made in the 1st wk of February ( o) , the 3rd wk of April ( ∫) , or the 3rd wk of June ( ◊) . Error bars represent the standard deviation of 4 measurements.
The extent of lipolysis (taken generally as the total amount of FFA present at any one time) is not considered as reliable an indicator of ripening age for most cheeses as are certain products of glycolysis and proteolysis ( 8 ) . Perhaps considering FFA in terms of their different chain lengths would be more meaningful. As reported herein, in Idiazabal cheese, the increase in percentage of volatile FFA with the concomitant decrease in the percentage of long-chain FFA appeared to correlate with the first 180 d of ripening throughout the cheese-making season, perhaps because FFA are the major components of the volatile fraction as determined by dynamic headspace (L.J.R. Barron, 1998, unpublished observations). Most analytical techniques frequently used to determine FFA concentrations in cheeses do not allow the simultaneous determination of acetic acid, and, thus, acetic acid concentrations are not usually included in reports of FFA during cheese ripening. Because acetic acid has a different biosynthetic origin than do the rest of the FFA (the appearance of butyric acid during ripening could also be due to a variety of processes), results are presented separately. The accumulation of acetic acid presented the same profile, at the same concentrations, and at all times of the year (Figure 4). After 90 d of ripening, acetic acid represented 66.5% of the total FFA in winter fabrications and 77.2% in summer cheeses. It is of interest that acetate is absent from the volatile fraction extracted by dynamic headspace at 20°C (L.J.R. Barron, 1998, unpublished observations). Yet, acetate is a major component of the FFA fraction when extracted from an acidic cheese slurry as seen previously. The pH of Idiazabal cheese during ripening (5.1 to 5.2) and the pka of acetic acid (4.76)
indicate that acetate must be present almost exclusively in the aqueous phase of the cheese matrix, where it should be mostly ionized, which would explain its absence from the volatile fraction in spite of its volatility. Other FFA, such as butyric, caproic, and caprylic, which comprise the majority of the volatile fraction compounds (L.J.R. Barron, 1998, unpublished observations) would partition between the aqueous and the organic phases of the cheese matrix. Thus, considering that the components of the aqueous phase contribute primarily to cheese taste (4, 13), the large amounts of acetate found in Idiazabal cheese are likely to play a significant role in the characteristic flavor of this cheese. High levels of acetic acid have been reported in Swiss cheese varieties (17, 29), in which it apparently makes a significant contribution to the sensory characteristics of these cheeses. Results presented in this paper clearly demonstrate that the time of the year has a marked influence, not only on the level of lipolysis at various ripening times but also on the amounts of short-chain FFA and the relative FFA composition of the cheeses. These differences could arise as a result of the different composition of the milk as the lactation period progresses. The concentration of protein or fat in ovine milk destined for Idiazabal cheese production is not standardized prior to cheesemaking. Thus, when milk is not standardized, the influence of time of the year or, rather, stage of lactation should be taken into consideration when studying the role of other variables. ACKNOWLEDGMENTS The authors thank M. A. Marquı´nez for excellent technical assistance, cheesemaster Arantza Pe´rez de Albe´niz for making the cheeses, and Queserı´as Araia (Araia, Alava, Spain) for the use of their pilot cheese manufacturing plant and ripening chambers. F. Cha´varri acknowledges a predoctoral fellowship from the Dep. of Industry, Agriculture and Fisheries of the Basque Government (Vitoria-Gasteiz, Spain). This work was supported by a grant from the Spanish Ministry of Education and Science (Madrid, Spain) (CICYT ALI93-0895-C02-02). REFERENCES 1 Alonso, L. 1993. Fat modification in Afuega’l Pitu cheese during ripening by capillary gas chromatography. J. Am. Oil Chem. Soc. 70:1035–1037. 2 Alonso, L., M. Jua´rez, M. Ramos, and P. J. Martı´n-Alvarez. 1987. Overall composition, nitrogen fractions and fat characteristics of Cabrales cheese during ripening. Z. Lebensm. Unters. Forsch. 185:481–486. Journal of Dairy Science Vol. 82, No. 5, 1999
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3 Basque Government (Dep. Agric. and Fisheries). 1986. Idiazabal cheese. Denomination of Origin Control Board. Boletin Oficial del Pais Vasco 252:6357–6373. 4 Bills, D. D., and E. A. Day. 1964. Determination of the major free fatty acids of Cheddar cheese. J. Dairy Sci. 47:733–738. 5 Biss, K. 1991. Sheep and goat cheese. J. Soc. Dairy Technol. 44: 104–106. 6 Cha´varri, F., A. Santisteban, M. Virto, and M. de Renobales. 1998. Alkaline phosphatase, acid phosphatase, lactoperoxidase and lipoprotein lipase activities in industrial ewe’s milk and cheese. J. Agric. Food Chem. 46:2926–2932. 7 Cha´varri, F., M. Virto, C. Martı´n, A. I. Na´jera, A. Santisteban, L.J.R. Barro´n, and M. de Renobales. 1997. Determination of free fatty acids in cheese: comparison of two analytical methods. J. Dairy Res. 64:445–452. 8 Farkye, N. Y., and P. F. Fox. 1990. Objective indices of cheese ripening. Trends Food Sci. Technol. August:37–40. 9 Ferna´ndez-Garcı´a, E., R. Lo´pez-Fandin˜o, and L. Alonso. 1994. Effect of a food-grade enzyme preparation from Aspergillus oryzae on free fatty acid release in Manchego-type cheese from ovine and bovine milk. Z. Lebensm. Unters. Forsch. 199: 262–264. 10 Ferna´ndez-Garcı´a, E., R. Lo´pez-Fandin˜o, L. Alonso, and M. Ramos. 1994. The use of lipolytic and proteolytic enzymes in the manufacture of Manchego type cheese from ovine and bovine milk. J. Dairy Sci. 77:2139–2149. 11 Fontecha, J., L. Amigo, M. A. de la Fuente, M. Jua´rez, M. Ramos, M. El-Shikh, and S. El-Shibiny. 1990. Ripening changes in Ras cheese prepared from ultrafiltered milk. Z. Lebensm. Untersuch. Forsch. 191:310–312. 12 Fontecha, J., C. Pela´ez, M. Jua´rez, T. Requena, C. Go´mez, and M. Ramos. 1990. Biochemical and microbiological characteristics of artisanal hard goat’s cheese. J. Dairy Sci. 73:1150–1157. 13 Fox, P. F., T. P. O’Connor, P.L.H. McSweeney, T. P. Guinee, and N. M. O’Brien. 1996. Cheese: physical, biochemical and nutritional aspects. Adv. Food Nutrition Res. 39:163–328. 14 Freitas, A. C., and F. X. Malcata. 1996. Influence of milk type, coagulant, salting procedure and ripening time on the final characteristics of Picante cheese. Int. Dairy J. 6:1099–1116. 15 Gonza´lez de Llano, D., M. Ramos, A. Rodrı´guez, A. Montilla, and M. Jua´rez. 1992. Microbiological and physicochemical characteristics of Gamonedo blue cheese during ripening. Int. Dairy J. 2:121–135.
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16 Kalantzopoulos, G. C. 1993. Cheeses from ewes’ and goats’ milk. Pages 507–533 in Cheese: Chemistry, Physics and Microbiology. Vol. 2. P. F. Fox, ed. Chapman & Hall, London, United Kingdom. 17 Langler, J. E., and E. A. Day. 1966. Quantitative analysis of the major free fatty acids in Swiss cheese. J. Dairy Sci. 49:91–93. 18 Macedo, A. C., and F. X. Malcata. 1996. Changes in the major free fatty acids in Serra cheese throughout ripening. Int. Dairy J. 6:1087–1097. 19 Marcos, A., and M. A. Esteban. 1993. Iberian cheeses. Pages 173–219 in Cheese: Chemistry, Physics and Microbiology. Vol. 2. P. F. Fox, ed. Chapman & Hall, London, United Kingdom. 20 Martı´n-Herna´ndez, M. C., M. Jua´rez, and M. Ramos. 1992. Biochemical characteristics of three types of goat cheese. J. Dairy Sci. 75:1747–1752. 21 Mendı´a, C. 1998. Cambios proteolı´ticos y organole´pticos del Queso Idiazabal elaborado en diferentes e´pocas del an˜o: influencia de la pasterizacio´n de la leche y del cultivo iniciador. Ph.D. Diss., Univ. Pu´blica de Navarra, Pamplona, Spain. 22 Na´jera, A. I., L.J.R. Barro´n, and Y. Barcina. 1994. Changes in free fatty acids during ripening of Idiazabal cheese: influence of brining time and smoking. J. Dairy Res. 61:281–288. 23 Nelson, J. H., R. G. Jensen, and R. E. Pitas. 1977. Pregastric esterases and other oral lipases: a review. J. Dairy Sci. 60: 327–362. 24 Sousa, M. J., V. M. Balcao, and F. X. Malcata. 1997. Evolution of free fatty acids profile during ripening in cheeses manufactured from bovine, ovine and caprine milks with extracts of Cynara cardunculus as coagulant. Z. Lebensm. Unters. Forsch. A 205:104–107. 25 Statistical Software, Inc. 1983. Univ. California Press, Berkeley, CA. 26 Urbach, G. 1991. Butter flavor in food systems. Food Res. Q. 51: 50–54. 27 Woo, A. H., S. Kollodge, and R. C. Lindsay. 1984. Quantification of major free fatty acids in several cheese varieties. J. Dairy Sci. 67:874–878. 28 Woo, A. H., and R. C. Lindsay. 1984. Concentrations of major free fatty acids and flavor development in Italian cheese varieties. J. Dairy Sci. 67:960–968. 29 Zerfiridis, G. K., A. Vafopoulou-Mastrogiannaki, and E. Litopoulou-Tzanetaki. 1984. Changes during ripening of commercial Gruye`re cheese. J. Dairy Sci. 67:1397–1405.
ABSTRACT Lipolysis was studied in cheeses manufactured from raw ovine milk in winter, spring, and summer, with no starter culture added, up to 180 d of ripening. Total amounts of free fatty acids (FFA) after 180 d of ripening were significantly higher in winter than in spring or summer. The major FFA in winter were C10:0 (3363 mmoles/kg), C4:0 (3309 mmoles/kg), C18:1 (3187 mmoles/kg), and C16:0 (2602 mmoles/kg). The amounts of all FFA in cheese decreased as the cheesemaking period progressed from winter to summer with C10:0 exhibiting the largest decrease (72.1%) and C18:1 the smallest decrease (22.8%). The amounts of FFA shorter than C12:0 were significantly higher in winter than either in spring or summer. The percentage of volatile ( C 4 to C8) , medium ( C 10 to C14) , and long-chain FFA ( ≥C16) changed during ripening: in winter and spring volatile FFA increased from 15% ( d 1 ) to 30% ( d 90 and 180) of the total, while long-chain FFA decreased from 55% ( d 1 ) to 40% ( d 180) of the total. In contrast, in summer, the percentages of volatile and long-chain FFA after 180 ripening d were 25 and 50% of the total, respectively. We concluded that winter cheeses were different from summer cheeses. Acetic acid increased during the first 90 d of ripening to a final concentration of 26,500 mmoles/kg, the amount of which was independent of the time of the year. ( Key words: lipolysis, free fatty acids, ovine milk cheese, cheese ripening)
ranean countries ( 5 ) , and in the last 10 or 15 yr, a substantial increase in research efforts has been made to study and characterize these products. The composition and individual amounts of FFA are considered to influence the flavor of different types of cheeses ( 2 7 ) either directly as in Italian varieties ( 2 8 ) or as precursors for other compounds such as methyl ketones, lactones, alkanes, and esters (26). Thus, changes in FFA composition during ripening have been monitored for most cheese varieties produced in an attempt to relate them to ripening age, even though it appears to be a less useful ripening index than do proteolytic and glycolytic indicators ( 8 ) . Although the composition and total amount of fat in ovine milk varies during lactation ( 1 6 ) and ovine milk is usually not standardized with respect to either protein or fat content prior to cheesemaking, changes in FFA during ripening have seldom been studied at various times during lactation (18). Idiazabal cheese is a typical product of the Basque Country region of Northern Spain and is manufactured from raw ovine milk according to the specifications of its Denomination of Origin Regulatory Board ( 3 ) . The cheese-making season extends from January until June with most cheeses being commercialized between 90 (mild) and 180 (medium) d of ripening. In the present work we studied changes in FFA during ripening in cheeses made at three different times during lactation as part of an ongoing characterization of this cheese. MATERIALS AND METHODS
INTRODUCTION Materials The production of cheeses manufactured with ovine milk has been traditionally localized in the Mediter-
Received June 15, 1998. Accepted December 7, 1998. 1Corresponding author. 1999 J Dairy Sci 82:885–890
Free fatty acid standards were from Sigma-Aldrich Quı´mica, S.A. (Madrid, Spain). Aminopropyl-bonded columns (500 mg) were from Waters (Madrid, Spain). Solvents (Merck, Madrid, Spain) were of the highest grade available and were not redistilled before use.
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TABLE 1. Amounts of FFA (micromoles per kilogram of cheese) accumulated during ripening of cheeses made in winter, spring, or summer.1 Ripening
Winter X
1 d 90 d 180 d
2501a 11,215a 19,268a
Spring SD 1160 1790 4398
X 1209b 6230b 9989b
Summer SD 202 1864 2293
X 1218b 7723b 11,256b
SD 195 867 934
a,bDifferent superscripts for each ripening day represent significant differences ( P < 0.05) among times of the year. 1The mean ( ± SD) of the 12 values obtained for the three batches made at each time of the year on each ripening day.
Cheese Manufacture Cheeses were made in the pilot plant of Queserı´as Araia (Araia, Alava, Spain). Bulk ovine milk was from several local, commercial flocks of latxa sheep. The lactation period for an individual flock extends approximately 5 mo between November and July, depending on the altitude where the flock is located with flocks on lower elevations starting lactation earlier. Industrial cheesemaking (using mixed milk from different flocks) extends from December until the end of June. Thus, industrial milk in January contains a large percentage of milk from flocks in their early lactation stage, and industrial milk in June corresponds to the late lactation period of most flocks. In April, milk could be a mixture (of unknown composition) of milk from flocks in late, mid-, and early lactation. Milk had been refrigerated for up to 48 h prior to cheesemaking. Cheeses were made according to the traditional method for the industrial production of Idiazabal cheese approved by its Denomination of Origin ( 3 ) , using commercial calf rennet (Chr. Hansen, Madrid, Spain), in 200-L vats. Cheeses (1.0 to 1.2 kg and 13 cm in diameter) were made in 3 consecutive wk in winter (last week in January and first 2 wk in February), spring (last 3 wk in April), and summer (last 3 wk in June) (total of 9 batches). From each batch, two different cheeses were taken for analysis after 1, 90, and 180 d of ripening. In addition to these sampling days, cheeses from batches 2 (1st wk in February), 5 (3rd wk in April), and 8 (3rd wk in June) were also taken after 15, 30, 60, and 120 d of ripening. Cheese samples were wrapped in plastic film and aluminum foil and frozen at –80°C until analyzed. FFA Analysis Gas-liquid chromatographic analysis of each cheese sample was made in duplicate. The FFA were analyzed underivatized by gas-liquid chromatography as Journal of Dairy Science Vol. 82, No. 5, 1999
described previously ( 7 ) . Briefly, cheese (1.0 g ) was ground and extracted from an acidic medium with diethyl ether-heptane (1:1 vol/vol) after addition of an internal standard mixture consisting of pentanoic, nonanoic, and heptadecanoic acids. Triacylglycerols were separated from the FFA on aminopropyl-bonded phase columns (previously equilibrated with heptane) by elution with chloroform:isopropanol (2:1 vol/ vol). The FFA were eluted with 5.0 ml of diethyl ether containing 20 ml of formic acid/L. This fraction containing underivatized FFA was injected directly into the gas chromatograph (model 5890, series II, equipped with a flame ionization detector; Hewlett Packard, Mardrid, Spain), and FFA were separated on a fused silica capillary column (25 m × 0.32 mm) coated with FFA phase (cross-linked polyethylene glycol, 0.52-mm layer thickness). The carrier gas (helium) flow rate was 2 ml/min, and the temperature was raised from 65 to 240°C at 10 °C/min and then held at 240°C for 20 min. Statistical Analysis The BMPD statistical package ( 2 5 ) was used for statistical treatment of the results. Analysis of variance and multiple comparison tests were applied to determine the presence of significant differences in FFA contents on different days of ripening (1, 90, or 180) among the cheeses made at different times of the year (winter, spring, or summer). Simple linear regression analysis was applied to fit the total FFA content with the ripening time. RESULTS AND DISCUSSION Total levels of lipolysis after 1, 90, and 180 d of ripening varied considerably for all batches of cheeses made at different times of the year (Table 1). Statistical analysis indicated that the total amounts of FFA obtained for winter cheeses on the 3 ripening d studied were ( P < 0.05) higher than were those for
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Figure 1. Total amounts of FFA accumulated during ripening of cheeses made with raw milk in the 1st wk of February ( o) , the 3rd wk of April ( ∫) , or the 3rd wk of June ( ◊) . Error bars represent the standard deviation of 4 measurements.
cheeses made either in spring or summer (which were not significantly different from each other). Differences in total lipolysis were not as large after 90 d of ripening. Total levels of lipolysis were lower than were those reported for Idiazabal cheese by Na´jera et al. (22), most likely because those cheeses had been manufactured with natural lamb rennet paste, which is known to increase lipolysis (23), and were about half of those reported for Manchego cheese ( 9 ) . Lipolysis was monitored at different times during ripening in the second batch made at each time of the year to assess the rate of FFA accumulation in the early ripening stages (Figure 1). The rate of FFA accumulation was linear during the 180 ripening d for the three batches of cheeses studied with the highest rate observed for the winter batch (67.5 mmoles· kg–1·day–1) , which was consistent with its higher levels of lipolysis. Lipoprotein lipase activity in milk was also highest in winter and decreased as the lactation period progressed ( 6 ) . This observed variability in total lipolysis after different ripening periods at different times of the year could account, among other factors, for the varied results reported in the literature (19, 22) for commercially obtained Idiazabal cheese samples. The amounts of individual FFA were different ( P < 0.05) for the three batches made at each of the three times of the year, indicating that each batch was independent of the other two. However, to compare times of the year (winter, spring, and summer), all three batches made in 3 consecutive wk at a given time were considered as triplicates. In this case, the amounts of fatty acids shorter than C12:0 (lauric
Figure 2. Individual FFA accumulated during ripening of cheeses made with raw milk in winter ( A ) , spring ( B ) , or summer ( C ) . Values reported are the mean ( ± SD) for the 3 batches made at each time of the year. Error bars represent the standard deviation of 12 measurements.
acid) on d 90 and 180 of the winter cheeses were higher ( P < 0.05) than were those of either spring or summer batches, and the latter two were not different from each other. Winter cheeses were also different from those made during the rest of the year. The amounts of individual FFA were observed to change from winter to summer fabrications (Figure 2). In winter, after 180 d of ripening, the major FFA were C4:0 (butyric acid) (3309 ± 608 mmoles/kg), C10:0 (capric acid) (3363 ± 1424 mmoles/kg), C16:0 (palmitic acid) (2602 ± 593 mmoles/kg) and C18:1 (oleic acid) (3187 ± 482 mmoles/kg). In contrast, in summer, C18:1 (2461 ± 302 mmoles/kg) was the major FFA with C4:0 (1679 ± 130 mmoles/kg), C16:0 (1933 ± Journal of Dairy Science Vol. 82, No. 5, 1999
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180 mmoles/kg), and C10:0 (937 ± 177 mmoles/kg) present at lower concentrations. The most prominent change in the amounts of FFA from winter to summer cheeses was, in fact, the 3.6-fold decrease in capric acid, which correlated with the decrease in this fatty acid present in milk triglycerides in June (L.J.R. Barron, 1997, unpublished observations) as well as with the lower concentrations of milk lipoprotein lipase at this time of the year ( 6 ) , which would indirectly support the role of milk lipoprotein lipase in the appearance of FFA during cheese ripening (13). The variability observed for the amount of C10:0 on d 180 of winter cheeses appeared to be due to the sample and not to analytical factors because the variability of this FFA observed in the rest of the spring and summer samples was comparable to that of other FFA. Palmitoleic acid ( C 16:1) was present in very low amounts at all times. Polyunsaturated fatty acids [C18:2 (linoleic acid) and C18:3 ( g-linoleic acid)] were present at very low concentrations, as reported for most cheeses but in sharp contrast to Serra cheese manufactured with ovine milk in which they appeared at levels comparable to those of C 18:1 (24). Other FFA such as C10:1 (undecenoic acid), C14:1 (myristoleic acid), and C15:0 (pentadecanoic acid) were detected in all cheeses at all times of the year in amounts below 1%, while FFA with 20 carbon atoms were detected in some of the cheeses at all times of the year, also at concentrations below 1% of the total. As depicted in Figure 2, the amounts of all individual FFA studied increased during ripening regardless of the total level of lipolysis attained, which has been reported for the great majority of cheeses. However, not all FFA increased by the same amounts, and, as a result, the FFA composition of the cheeses changed considerably over the 180 ripening d (Figure 3). The percentage of volatile FFA [C4:0 to C8:0 (caprylic acid)], which have a significant impact on the development of the characteristic aroma of the cheese, was observed to increase during ripening at all times of the year, while that of long-chain FFA ( ≥ C16:0) decreased. In contrast, the percentage of medium-chain FFA remained quite constant during ripening at all times of the year. These changes were comparable in winter and spring with short- and longchain FFA representing 30 and 40% of the total, respectively, after 180 d of ripening (Figure 3). In contrast, in summer cheeses, volatile FFA represented 25% of the total and long-chain FFA represented 50%. Thus, the composition of winter and spring cheeses was similar, but that of summer cheeses was quite different. Journal of Dairy Science Vol. 82, No. 5, 1999
Figure 3. Percentage of volatile, medium-, and long-chain FFA after 1, 90, and 180 d of ripening. Values correspond to the mean ( ± SD) of the three batches made at each time of the year. Error bars represent the standard deviation of 12 measurements.
The FFA composition during cheese ripening has not been reported as such to the best of our knowledge. Yet calculations made using published values of individual FFA increases that occur during ripening indicate that the FFA composition does vary in some cases. Thus, no changes were observed in the percentage of volatile FFA in some bovine milk cheeses, such as Parmesano and Mozzarella ( 2 8 ) or Afuega’l Pitu ( 1 ) , but in others there was either a slight increase, as observed in Ras cheese made with ultrafiltered milk (11), or a marked increase, as in Gruye`re cheese (29). Blue-type cheeses made with a mixture of bovine, ovine, and caprine milks, such as Cabrales ( 2 ) and Gamonedo (15), showed a substantial decrease in the percentage of volatile FFA. Caprine cheeses showed either a decrease in the percentage of volatile FFA ( 1 2 ) or no change (20). By contrast, Manchego cheese ( 1 0 ) manufactured with 70% ovine and 30% bovine milks and Portuguese ovine cheeses, such as Serra ( 1 8 ) and Picante cheese (14), exhibited similar increases in the percentage of volatile FFA to those reported in this work. At present, the possible significance is not known for the observed increase in the percentage of volatile FFA in some cheeses. In the sensory analysis, cheeses manufactured in January received the highest scores, and those manufactured at the end of June obtained the lowest values (21). The results presented in this paper indicate that winter cheeses had both highest levels of lipolysis and highest amounts of FFA shorter than C12:0, strongly suggesting that, at least in Idiazabal cheese, lipolytic events play an important role in overall sensory characteristics.
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Figure 4. Accumulation of acetic acid in cheeses made in the 1st wk of February ( o) , the 3rd wk of April ( ∫) , or the 3rd wk of June ( ◊) . Error bars represent the standard deviation of 4 measurements.
The extent of lipolysis (taken generally as the total amount of FFA present at any one time) is not considered as reliable an indicator of ripening age for most cheeses as are certain products of glycolysis and proteolysis ( 8 ) . Perhaps considering FFA in terms of their different chain lengths would be more meaningful. As reported herein, in Idiazabal cheese, the increase in percentage of volatile FFA with the concomitant decrease in the percentage of long-chain FFA appeared to correlate with the first 180 d of ripening throughout the cheese-making season, perhaps because FFA are the major components of the volatile fraction as determined by dynamic headspace (L.J.R. Barron, 1998, unpublished observations). Most analytical techniques frequently used to determine FFA concentrations in cheeses do not allow the simultaneous determination of acetic acid, and, thus, acetic acid concentrations are not usually included in reports of FFA during cheese ripening. Because acetic acid has a different biosynthetic origin than do the rest of the FFA (the appearance of butyric acid during ripening could also be due to a variety of processes), results are presented separately. The accumulation of acetic acid presented the same profile, at the same concentrations, and at all times of the year (Figure 4). After 90 d of ripening, acetic acid represented 66.5% of the total FFA in winter fabrications and 77.2% in summer cheeses. It is of interest that acetate is absent from the volatile fraction extracted by dynamic headspace at 20°C (L.J.R. Barron, 1998, unpublished observations). Yet, acetate is a major component of the FFA fraction when extracted from an acidic cheese slurry as seen previously. The pH of Idiazabal cheese during ripening (5.1 to 5.2) and the pka of acetic acid (4.76)
indicate that acetate must be present almost exclusively in the aqueous phase of the cheese matrix, where it should be mostly ionized, which would explain its absence from the volatile fraction in spite of its volatility. Other FFA, such as butyric, caproic, and caprylic, which comprise the majority of the volatile fraction compounds (L.J.R. Barron, 1998, unpublished observations) would partition between the aqueous and the organic phases of the cheese matrix. Thus, considering that the components of the aqueous phase contribute primarily to cheese taste (4, 13), the large amounts of acetate found in Idiazabal cheese are likely to play a significant role in the characteristic flavor of this cheese. High levels of acetic acid have been reported in Swiss cheese varieties (17, 29), in which it apparently makes a significant contribution to the sensory characteristics of these cheeses. Results presented in this paper clearly demonstrate that the time of the year has a marked influence, not only on the level of lipolysis at various ripening times but also on the amounts of short-chain FFA and the relative FFA composition of the cheeses. These differences could arise as a result of the different composition of the milk as the lactation period progresses. The concentration of protein or fat in ovine milk destined for Idiazabal cheese production is not standardized prior to cheesemaking. Thus, when milk is not standardized, the influence of time of the year or, rather, stage of lactation should be taken into consideration when studying the role of other variables. ACKNOWLEDGMENTS The authors thank M. A. Marquı´nez for excellent technical assistance, cheesemaster Arantza Pe´rez de Albe´niz for making the cheeses, and Queserı´as Araia (Araia, Alava, Spain) for the use of their pilot cheese manufacturing plant and ripening chambers. F. Cha´varri acknowledges a predoctoral fellowship from the Dep. of Industry, Agriculture and Fisheries of the Basque Government (Vitoria-Gasteiz, Spain). This work was supported by a grant from the Spanish Ministry of Education and Science (Madrid, Spain) (CICYT ALI93-0895-C02-02). REFERENCES 1 Alonso, L. 1993. Fat modification in Afuega’l Pitu cheese during ripening by capillary gas chromatography. J. Am. Oil Chem. Soc. 70:1035–1037. 2 Alonso, L., M. Jua´rez, M. Ramos, and P. J. Martı´n-Alvarez. 1987. Overall composition, nitrogen fractions and fat characteristics of Cabrales cheese during ripening. Z. Lebensm. Unters. Forsch. 185:481–486. Journal of Dairy Science Vol. 82, No. 5, 1999
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