Obtaining a hydrolyzed milk fat fraction enriched in conjugated linoleic acid and trans-vaccenic acid

International Dairy Journal 36 (2014) 29e37

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Obtaining a hydrolyzed milk fat fraction enriched in conjugated linoleic acid and trans-vaccenic acid Sergio I. Martínez-Monteagudo, Mohamed Khan, Feral Temelli, Marleny D.A. Saldaña* Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 February 2013 Received in revised form 18 December 2013 Accepted 19 December 2013

Anhydrous milk fat (AMF) rich in conjugated linoleic acid (CLA) and trans-vaccenic acid (TVA) was enzymatically hydrolyzed and dry fractionated. Oxidation kinetic parameters of hydrolyzed AMF was determined by differential scanning calorimetry. Hydrolysis yielded 90.4% free fatty acids (FFAs) after 24 h at 50
C, using a ratio of 70 for water to fat (w/w) and a ratio of 40 for enzyme to fat (w/w). The oxidation of hydrolyzed AMF started at a lower temperature (108.3  4.1
C) compared with nonhydrolyzed AMF (155.7  3.4
C). Upon fractionation of hydrolyzed AMF, three fractions were obtained (high-, middle- and low-melting point FFA fractions). The middle fraction contained the highest concentration of CLA and TVA (64.8 and 249.3 mg g1 fat, respectively). The middle fraction shows great potential for fortification of dairy products but further optimization is needed to maximize the CLA and TVA contents, while minimizing the presence of the other fatty acids. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Milk fat is recovered during the production of skim milk and subsequently converted into anhydrous milk fat (AMF), which can be stored for months (Augustin & Versteeg, 2006). The increasing demand for fat-free dairy products due to health concerns associated with the consumption of milk fat have generated a large excess of milk fat stocks worldwide (Lubary, Hofland, & ter Horst, 2011). In addition, AMF presents important technological disadvantages due to its wide melting point range (40 to 40
C) and the relatively high content of saturated fatty acids (Lubary et al., 2011). Various researchers have highlighted the health benefits of some fatty acids naturally found in milk fat while searching for alternatives to add value to milk fat (Küllenberg, Taylor, Schneider, & Massing, 2012; Merrill et al., 1997; Molkentin, 2007). Among these, conjugated linoleic acid (CLA, C18:2) has been shown to have health benefits, such as reducing the risk of cancer and atherosclerosis, as well as weight control, and bone formation (Cook & Pariza, 1998; Fritsche et al., 1999; Lock & Bauman, 2004; Park, 2009). Another important fatty acid with health-promoting and disease-prevention properties is trans-vaccenic acid (TVA, C18:1 t11), a metabolic precursor of CLA (Jacome-Sosa et al., 2010; Wang et al., 2008). Although trans fatty acids naturally found in milk fat

* Corresponding author. Tel.: þ1 780 492 8018. E-mail addresses: [email protected], [email protected] (M.D.A. Saldaña). 0958-6946/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.idairyj.2013.12.010

are associated with these positive health benefits, trans fatty acids formed during the hydrogenation process for margarine production have been shown to have negative effects, increasing the risk of heart disease (Wang, Jacome-Sosa, & Proctor, 2012). CLA and TVA are minor components of milk fat, representing about 1 and 3% of the total milk fat, respectively (Creamer & MacGibbon, 1996). However, CLA and TVA can be isolated for further use as an ingredient to fortify food products (Lubary et al., 2011). Romero, Rizvi, Kelly, and Bauman (2000) fractionated nonhydrolyzed AMF using supercritical carbon dioxide and reported an increase of 89%, with CLA reaching a final concentration of 7.8 mg g1 fat. A 2.3-fold increase of CLA was reported in milk fat crystallized with urea (Kim & Liu, 1999). These authors showed that the long-chain saturated fatty acids formed a complex with urea, which was later eliminated by filtration. Consequently, a reduction in the concentration of saturated fatty acids was reported along with an increase in CLA content. O’Shea, Devery, Lawless, Keogh, and Stanton (2000) obtained a milk fat fraction enriched in CLA with a final concentration of 22 mg g1, representing a 63% increase compared with that in the parent fat. In addition, an increase of 36% of TVA was obtained in the same fraction. The fraction was obtained at a crystallization temperature of 10
C and a cooling rate of 0.58
C min1. Rehberger, Butikofer, Bisig, and Collomb (2008) concentrated CLA from anhydrous milk butter and highland butter using a two-step fractionation. The first fraction was obtained at 20
C, which was fractionated again at 12.5
C using a cooling rate of 0.2
C min1. After the second fractionation step, the concentration of CLA increased to 18.6% and 9.2% for anhydrous milk and

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highland butter, respectively. These authors also reported the distribution of CLA isomers after the two-step fractionation. In the first step, only the concentration of the major isomer, cis-9/trans-11, increased by 6.5%. The second step yielded an increase of 10.2, 9.5 and 7.8% of the three major isomers, cis-9/trans-11, trans-7/cis-9 and trans-11/cis-13, respectively. A promising approach to improve the CLA concentration in milk fat is to feed oil- and oilseed-supplemented diets to dairy cattle (Bell, Griinari, & Kennelly, 2006; Bharathan et al., 2008; Ryhänen et al., 2005; Zheng et al., 2005). However, further CLA enrichment of milk fat is challenging because CLA is distributed throughout different triacylglycerols (TAGs), together with other fatty acids. Robinson and MacGibbon (2000) found that CLA is distributed in approximately 32 different types of TAG with different molecular weights and degrees of saturation. Another approach to expand milk fat utilization involves hydrolyzing the TAGs to free fatty acids (FFAs) and glycerol via enzymatic hydrolysis. Controlled hydrolysis of milk fat has been carried out to maximize the production of short-chain fatty acids (C4:0eC8:0), enhancing flavor development in dairy products (Lencki, Smink, Snelting, & Arul, 1998; Regado et al., 2007). Subsequently, the FFAs obtained can be separated based on their melting point by dry fractionation using vacuum or membrane filtration (Kontkanen et al., 2011). Removal of cholesterol from AMF has been carried out using supercritical ethane (Mohamed, Saldaña, Socantaype, & Kieckbusch, 2000) and supercritical CO2 (Mohamed, Neves, & Kieckbusch, 1998), but the CLA or TVA contents of the fractions obtained were not reported. An important characteristic often overlooked is the susceptibility of FFAs to oxidation compared with those esterified to the glycerol backbone. Enhancing the nutritional and physical properties of milk fat certainly has commercial implications; however, the hydrolysis and further fractionation of AMF to obtain fractions enriched in CLA and TVA has not been evaluated so far. The objective of this study was to obtain a lipid fraction enriched in CLA and TVA through enzymatic hydrolysis and dry fractionation of AMF already enriched in CLA and TVA by modification of cattle diet. In addition, the kinetics of non-isothermal oxidation of the hydrolyzed fraction rich in CLA and TVA was evaluated and compared with the oxidation kinetics of the non-hydrolyzed AMF enriched in CLA and TVA. 2. Materials and methods 2.1. Anhydrous milk fat rich in CLA and TVA Milk enriched in CLA and TVA was obtained from the Dairy Research and Technology Centre, University of Alberta, Canada, following the protocol reported earlier by Martínez-Monteagudo, Saldaña, Torres, and Kennelly (2012b). The diet provided to the dairy cattle for 21 days was modified to contain safflower oil. This milk enriched in CLA and TVA was then used to obtain anhydrous milk fat at Agri-Food Discovery Place, University of Alberta (Edmonton, AB, Canada) following the methodology described by Martínez-Monteagudo, Saldaña, and Kennelly (2012a). 2.2. Processing treatments 2.2.1. Enzymatic hydrolysis Initial screening experiments were carried out to identify the experimental variables and levels that significantly affect the enzymatic hydrolysis of AMF enriched in CLA and TVA. The tested variables were temperature (50e70
C), pH (6e8), reaction time (1e12 h), and enzyme load (0.2e20 g). An enzyme:fat weight ratio of 1:10 was kept constant during the screening experiments.

Enzymatic hydrolysis was carried out in a 25 mL conical flask immersed in a temperature controlled water bath. First, a representative sample of AMF enriched in CLA and TVA was melted at 40
C. Then, 2 mL of phosphate buffer (pH 6, 7 or 8) was added and mixed continuously using a magnetic stirrer to minimize any pH changes as the reaction proceeded. Once the desired temperature was reached (50, 60 or 70
C), different amounts of LipozymeÔ TL IM (sn-1,3 stereospecific lipases of Thermomyces lanuginosus immobilized on a granulated silica carrier; Novozymes A/S, Bagsvaerd, Denmark) were added slowly into the mixture. After a pre-determined reaction time (0, 1, 3, 6 and 12 h) had passed, samples (1 mL) were taken and frozen immediately at 18
C. The reaction pH was measured using a pH indicator paper, and the temperature was monitored using a thermocouple connected to an electronic temperature recorder. All experiments were carried out under nitrogen headspace to avoid oxidation. Prior to analysis, stored samples were thawed at 40
C and the hydrolyzed fat fraction was separated from the water and enzyme layers by centrifugation at 2500g for 5 min. The degree of fat hydrolysis was then estimated using the titration method reported by Leitgeb and Knez (1990). Based on the data obtained from the screening experiments, a factorial experimental design was developed with three variables: time (12e72 h), enzyme:fat weight ratio (E/F, 5e40), and water:fat molar ratio (W/F, 8.8e70.4). An additional set of experiments was carried out to test the impact of repeated use of LipozymeÔ TL IM, where the enzyme obtained after the centrifugation step was used again in the reaction until a substantial drop in the final FFA content was observed. 2.2.2. Fractionation protocol The fractionation protocol was designed based on the crystallization profile of the hydrolyzed AMF obtained by differential scanning calorimetry (DSC). General guidelines to obtain crystallization profiles of edible lipids using DSC can be found elsewhere (Humphrey, Moquin, & Narine, 2003). Samples of melted hydrolyzed AMF (11 mg) were pipetted into aluminum DSC pans, which were then hermetically sealed. An empty aluminum pan was used as a reference. The DSC pans were equilibrated at 90
C for 5 min and then cooled down to 40
C at a rate of 0.1
C min1. The crystallization profile was carried out in duplicate. The obtained thermograms were analyzed with TA Universal Analysis software (TA Instruments, New Castle, DE, USA) to locate the onset and peak maximum temperatures, and enthalpy of crystallization. These key parameters were determined using the first and the second derivative method (Bouzidi, Boodhoo, Humphrey, & Narine, 2005). Based on the results of the DSC crystallization profile obtained above, samples of hydrolyzed AMF (30 g) were heated to 70
C in a heating/cooling circulating system (Julabo F25 heating device, Allentown, PA, USA). After a holding period of 5 min at 70
C, the melted lipid samples were cooled down to 30
C at a rate of 0.1
C min1 under shear conditions (50 rpm) using a metallic mixer. The samples were kept at 30
C for 20 h. Then, the samples were centrifuged at 2500g for 5 min and two fractions were obtained, designated as S30 (solid fraction obtained at 30
C) and L30 (liquid fraction obtained at 30
C). The L30 fraction was heated again to 70
C for 5 min to erase the crystal memory and cooled down to 10
C at a rate of 0.1
C min1 under shear conditions (50 rpm). After 10 h at 10
C, the samples were centrifuged at 2500g for 5 min to obtain S10 and L10 fractions, solid and liquid fractions obtained at 10
C, respectively. The fatty acid composition of each fraction was determined by gas chromatography (GC).

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31

2.3. Analysis and characterization of products

2.4. Statistical analysis

2.3.1. Fatty acid composition The methodology for fatty acid profile determination using GC, including CLA and TVA, was the same as previously reported by Martínez-Monteagudo et al. (2012a,b). For hydrolyzed samples, the acid-catalyzed methylation of FFAs was carried out according to Prado, Khan, Saldaña, and Temelli (2012). Methyl heptadecanoate (1 mL, 1 mg mL1) was used as an internal standard (Fluka #51633 purity 99.5%, SigmaeAldrich, St. Louis, MO, USA). All the GC data were processed with the Galaxi software (version V1.19, Varian Inc, Walnut Creek, CA, USA) and the obtained peaks were identified with a milk fat reference standard (463-Nu Check Prep Inc, Elysian, MN, USA).

The hydrolysis, oxidation and fractionation experiments were carried out in triplicate. The mean values obtained after the enzymatic hydrolysis were compared using Tukey’s test (a ¼ 0.05). The statistical analysis was carried out using Sigmaplot software V11 (SPSS Inc., Chicago, IL, USA).

2.3.2. Determination of free fatty acids After AMF hydrolysis, the lipid mixture was centrifuged and the top layer was used for FFA analysis according to an acidebase titration method (Leitgeb & Knez, 1990; Prado et al., 2012). Samples (5 g) were diluted in ethanol and titrated with NaOH, using phenolphthalein as an indicator. The amount of FFAs was expressed as a percentage (w/w) using oleic acid as a reference. 2.3.3. Non-isothermal oxidation A modulated DSC Q100 (TA Instruments) was used to determine the oxidation kinetics of hydrolyzed AMF according to Saldaña and Martínez-Monteagudo (2013). Briefly, hydrolyzed AMF was melted at 70
C to erase its thermal memory. A liquid lipid sample was pipetted (1.5e3 mg) into an aluminum pan (TA Instruments), which was hermetically sealed with a pinhole lid. An empty DSC pan with a pinhole lid was used as an inert reference. Samples were then oxidized under non-isothermal conditions, following the heating program reported previously (Martínez-Monteagudo et al., 2012a). The protocol consisted of an equilibration period of 3 min at 100
C followed by a heating rate of 3, 6, 12, or 15
C min1. For each heating rate, the heat flow as a function of the heating temperature was recorded and analyzed to locate the start of oxidation temperature (Ts), the onset oxidation temperature (Ton), and the temperature of maximum heat flow (Tp). These oxidation parameters were determined using the first and second derivatives of the heat flow signal (Saldaña & Martínez-Monteagudo, 2013). The oxidation kinetics of hydrolyzed AMF was analyzed using the Arrhenius rate constant (k). Activation energy (Ea) and preexponential factor (z) were calculated using the OzawaeFlynne Wall method as reported previously (Martínez-Monteagudo et al., 2012a). In this method, Ts, Ton, and Tp were linearly correlated with the corresponding heating rate (b in K min1) and the Ea and z values were obtained from the slope and intercept, as follows:

log b ¼ a

1 þb T

a ¼ 0:4567

Ea R

  Ea b ¼ 2:315 þ log z R

(1)

(2)

(3)

where a and b are the slope and intercept from Eq. (1), respectively, and R is the universal gas constant (8.31 J mol1 K1).

3. Results and discussion 3.1. Fatty acid composition of AMF enriched in CLA and TVA Table 1 shows the fatty acid profiles of AMF and AMF enriched in CLA and TVA. The change in the diet of the animals increased the concentration of CLA in milk fat from 5.3  0.1 to 23.80  0.01 mg CLA g1 fat. In addition, the concentration of TVA in milk fat increased from 18.9  0.1 to 90.9  0.1 mg TVA g1 fat. Changing the diet of the cows substantially altered the composition of the major fatty acids. The levels of saturated fatty acids, such as C12:0, C14:0 and C16:0 were reduced on average by w42, 24 and 24% (w/w), respectively (Table 1). These saturated fatty acids have been associated with increased risk of cardiovascular disease (Givens, 2008; Noakes, Nestel, & Clifton, 1996) and their reduction can be considered as an additional benefit of the feeding regime. Another important consequence of the modified feeding regime is the increase in the ratio of unsaturated to saturated fatty acids compared with that in the non-enriched AMF (1.40  0.01 and 0.69  0.01, respectively; Table 1). Bell et al. (2006) used the Table 1 Fatty acid composition of anhydrous milk fat (AMF) and AMF enriched in conjugated linoleic acid (CLA; C18:2) and trans-vaccenic acid (TVA; C18:1 t11).a Fatty acid

AMF

C4:0 C6:0 C8:0 C9:0 C10:0 C11:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 t C16:1 c C18:0 C18:1 t9 C18:1 t11 (TVA) C18:1 n7 C19:0 C18:2 C20:0 C18:3 n6 C20:1 n12 C20:1 n15 C18:3 w3 C18:2 (CLA) C20:2 C22:0 C20:3 w6 C20:4 C20:5 C22:3 Unsat/sat

3.10 13.72 9.12 0.22 20.44 2.64 24.73 88.80 9.45 10.51 239.40 3.23 18.10 91.90 3.83 18.91 5.40 4.50 19.33 1.41 0.21 1.33 0.46 3.94 5.30 0.31 0.32 0.94 1.10 0.33 0.21 0.69

AMF enriched in CLA and TVA                                

0.10 0.01 0.01 0.01 0.03 0.01 0.03 0.10 0.01 0.02 0.20 0.01 0.10 0.10 0.02 0.04 0.10 0.20 0.01 0.01 0.01 0.01 0.01 0.01 0.10 0.01 0.01 0.01 0.01 0.01 0.01 0.02

2.11 6.63 4.90 0.21 7.90 2.50 14.41 67.40 6.92 4.30 183.10 4.50 11.20 95.30 171.50 90.90 5.50 3.90 23.90 0.91 2.11 0.75 2.71 1.90 23.80 0.21 0.31 0.78 0.59 0.44 0.31 1.40

                               

0.02 0.01 0.02 0.01 0.03 0.04 0.01 0.04 0.01 0.40 0.40 0.30 0.20 0.70 0.09 0.10 0.60 0.03 0.10 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01

a Values (mg fatty acid g1 fat) are means  standard deviation based on duplicates; t e trans fatty acids; c e cis fatty acids; unsat/sat, unsaturated to saturated fatty acids ratio. The values for unenriched AMF are those reported by MartínezMonteagudo et al. (2012a).

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biohydrogenation theory to explain the increase in the CLA content due to the change in the feeding regime.

100

3.2. Enzymatic hydrolysis

3.3. Repeated use of enzymes The effect of repeated use of the lipase on the hydrolysis of AMF enriched in CLA and TVA was evaluated for up to four cycles of 24 h each (Fig. 2). The amount of FFAs formed was evaluated after 24 h using E/F and W/F ratios of 40 and 70, respectively. The enzyme obtained after each hydrolysis cycle was separated by centrifugation and consecutively used four times. Repeated use of the enzyme reduced the percentage of FFAs in the final hydrolyzed product to 72.1, 52.5, and 32.6% (w/w) after the second, third and fourth cycle use, respectively. A possible reason for such a drop in conversion can be the accumulation of glycerol. The enzyme employed in this study, Lipozyme TL IM, is an immobilized enzyme and the glycerol generated during the reaction gradually accumulates in the beads, creating a barrier for TAG to reach the active site of the enzyme in the following cycle. Clean up protocols in between each cycle might reduce the gradual loss in conversion efficiency (Lozano, Bernal, & Navarro, 2012). The E/F ratio of 40 employed in this study is high considering potential industrial scale up. However, in terms of enzyme activity, 1 g of Lipozyme TL IM possesses an activity of 250 units, thus a ratio of 40 will provide 10,000 units of activity in one load of enzyme per unit load of feed material. Similarly, Kosugi, Takahashi, and Lopez (1995) employed a countercurrent fluidized-bed reactor, providing 16,000 units of enzyme activity for hydrolysis of sardine oil (95% of FFAs). The reactors used by Kosugi et al. (1995) had the capacity to process 200e300 tons of oil per month. Regardless, the

Free fatty acids (%)

90

b

b b b

80

b

b

a

a

a

70

60 0 16

20

24

Hydrolysis time (h) 100

(b)

Free fatty acids (%)

90

c b

b

80 a

a

b

b a

ab

70

60 0 16

20

24

Hydrolysis time (h) 100

(c) c

90 Free fatty acids (%)

Hydrolysis screening experiments indicated that increasing time, lipase enzyme load (E) and W/F ratio increased the extent of hydrolysis, but pH values of 6 and 8 and temperatures above 50
C had a negative impact, possibly due to enzyme inactivation under such conditions (data not shown). Values of pH in the range of 7.0e 7.5 were reported as optimal for lipase hydrolysis in different lipid systems (Han & Rhee, 1985; Tahoun, 1986). Chen and Pai (1991) reported an optimum temperature of 55
C during the enzymatic hydrolysis of micro-emulsions of milk fat using a lipase. Similarly, an optimum temperature of 55
C was reported for the enzymatic hydrolysis of AMF enriched in CLA in supercritical carbon dioxide (Prado et al., 2012). Therefore, further enzymatic hydrolysis experiments were carried out at pH 7.0 and 50
C. Results of the screening experiments showed that 30% of FFAs were hydrolyzed, and the enzyme load linearly affected the amount of FFAs, indicating that a plateau was not reached. Thus, increasing the hydrolysis time and enzyme load would result in higher amounts of FFAs. The percentage of FFAs obtained after 16, 20, and 24 h of enzymatic hydrolysis using different E/F and W/F ratios is shown in Fig. 1. For experiments carried out at a W/F ratio of 35 (Fig. 1a), the amount of FFAs increased significantly (P < 0.05) when the E/F ratio was increased from 20 to 30, but did not change (P > 0.05) with a further increase in the E/F ratio from 30 to 40, regardless of the hydrolysis time. Similar behavior was obtained when the hydrolysis was carried out at a W/F ratio of 50 (Fig. 1b). Finally, the highest percentage of FFAs (90.3  1.2%, Fig. 1c) was achieved using E/F and W/F ratios of 40 and 70, respectively, after 24 h of hydrolysis. These ratios were used to carry out additional experiments with prolonged hydrolysis time. After 48 and 72 h, the amounts of FFAs were not significantly different (88.3  5.0% and 89.7  3.4%, respectively) from that obtained at 24 h (90.3  1.2%), indicating that a plateau was reached (data not shown).

(a)

b

b

80 b

b

70

ab a

a

a

60 0 16

20

24

Hydrolysis time (h) Fig. 1. Free fatty acid content (%, w/w; values are means  standard deviations based on triplicates) after 16, 20, and 24 h of enzymatic hydrolysis at water:fat ratios of (a) 35, (b) 50 and (c) 70. Enzyme:fat ratios of bars are: ,, 20 enzyme/fat; , 30 enzyme/ fat; , 40 enzyme/fat; bars with different letters (aec) are significantly different (P < 0.05) within each histogram.

amount of enzyme used should be minimized to enhance economic viability of the process. It should also be noted that the reactor system used in this study is a batch process with important mass transfer limitations. Further improvements might include use of a packed bed continuous reactor, with prolonged times of use for each batch of enzyme, which was beyond the scope of this study.

S.I. Martínez-Monteagudo et al. / International Dairy Journal 36 (2014) 29e37

100

Free fatty acids (%)

80

60

40

20

0 1

2

3

4

Number of cycles Fig. 2. Effect of the number of cycles to evaluate the repeated use of enzyme on the amount of free fatty acids obtained from the hydrolysis of anhydrous milk fat enriched in conjugated linoleic acid and trans-vaccenic acid. The hydrolysis was carried out at 50
C for 24 h using enzyme:fat and water:fat ratios of 40 and 70, respectively. Mean  standard deviation based on triplicates.

3.4. Fractionation The crystallization profile of non-hydrolyzed and hydrolyzed AMF enriched in CLA and TVA obtained at 0.1
C min1 from 60 to 37.5
C is shown in Fig. 3. The difference in the crystallization profiles of non-hydrolyzed and hydrolyzed AMF is evident in Fig. 3. The crystallization behavior of non-hydrolyzed AMF is mainly determined by the melting points of the TAG molecules in the fat mixture, which is a function of the chain length, position, and number of unsaturated fatty acids esterified to the glycerol backbone (Deffense, 1993). The crystallization behavior of the hydrolyzed AMF is dictated by the melting points of the individual fatty acids. Moreover, the presence of partial (mono- and di-) acylglycerols in the hydrolysis product and its impact on the crystallization behavior should not be overlooked. In the case of non-hydrolyzed AMF, two exothermic peaks were recorded, represented by (1) and (2) in Fig. 3. The first

Heat flow (W g-1)

0.4 W g

(3)

(5) (4)

(2)

peak (1) started at 18.2  0.9
C and ended around 10.1  1.2
C, and the second exothermic peak started at 8.6  0.4
C followed by a broad shoulder, which continued to 37.5  0.4
C. Similar crystallization curves of AMF have been reported (Lopez & Ollivon, 2009; Lopez, Bourgaux, Lesieur, Riaublanc, & Ollivon, 2006; Reddy, 2010). The size and position of the peaks can vary with the thermal history and composition of the fat sample (Deffense, 1993). The thermal and structural behavior of AMF was studied extensively by Lopez et al. (2002), who crystallized AMF at a low cooling rate (0.15
C min1) and showed that AMF not only crystallizes in b0 and a polymorphic forms, coexisting simultaneously, but also that a polymorphic form can be transformed to a more stable form. Based on the melting behavior, milk fat can be classified into high-, middle- and low-melting point fractions (Lopez et al., 2006). In the case of non-hydrolyzed AMF, the peak represented by (1) in Fig. 3 is known as the stearin fraction (Lopez et al., 2002). The middle- and low-melting point fractions overlapped, which explains the broadness of the second peak (2) (Fig. 3). The stearin fraction is mainly composed of long-chain saturated fatty acids, including C14:0, C16:0 and C18:0 (Marangoni & Lencki, 1998). The olein fraction is comprised of short-chain and unsaturated fatty acids. Further separation of these fractions is difficult due to inclusion of liquid fat in the fat crystals. A comprehensive study on the crystallization behavior of stearin and olein fractions was reported by Lopez and Ollivon (2009). The crystallization profile obtained for hydrolyzed AMF showed three well-resolved exothermic peaks, (3)e(5) in Fig. 3. These peaks can be related to the existence of three types of fatty acids having high-, middle- and low-melting points. Long-chain and saturated fatty acids are concentrated in peak (3), including C15:0, C16:0, C18:0 and C20:0. Those fatty acids having a melting point spanning from w11 to 0
C were concentrated in the middle-melting point fraction. Such fatty acids include medium-chain saturated (C10:0, C12:0 and C14:0) and long-chain unsaturated fatty acids (C18:1 t11, C18:1 n9 and CLA). Finally, peak (5) consisted exclusively of shortchain fatty acids, C6:0 and C8:0. The enthalpy values of the different exothermic peaks are provided in Table 2. Enthalpy is the heat released per gram of sample and it is used to calculate the yield after fractionation. Marangoni and Lencki (1998) fractionated AMF with ethyl acetate and obtained yields of 12, 34 and 54% for high-, middle- and low-melting point fractions, respectively. Using the enthalpy ratios, yields of 59, 14, and 26% were obtained for high-, middle- and low-melting point fatty acids, respectively, in this study. Table 2 also shows the characterization of the exothermic peaks obtained from nonhydrolyzed and hydrolyzed AMF in terms of the full width at half maximum. The peaks (1) and (2) obtained for non-hydrolyzed AMF differed in their shape and broadness (Table 2). In contrast, in hydrolyzed AMF, there was no difference in the shape of the peaks, reflecting the uniformity of the FA profile within each fraction.

Table 2 Characterization of the different crystallization peaks of hydrolyzed and nonhydrolyzed AMF enriched in conjugated linoleic acid and trans-vaccenic acid.a

(1)

Fractions Ton (
C)

-30

-15

0

15

33

30

45

60

Temperature ( C) Fig. 3. Crystallization profile of hydrolyzed (upper profile) and non-hydrolyzed (lower profile) anhydrous milk fat rich in conjugated linoleic acid and trans-vaccenic acid recorded at 0.1
C min1. The baseline is indicated by the dashed line; the scale bar is 0.4 W g1. Peaks are: 1, stearin fraction; 2, olein fraction; 3, high-melting point fraction; 4, middle-melting point fraction; 5, low-melting point fraction.

Stearin Olein HMP MMP LMP

17.2 7.6 24.7 11.5 27.3

    

Toff (
C)

Tmax (
C)

1.1 10.1  0.6 15.4 1.0 40.1  0.2 2.8 0.0 13.2  0.1 23.4 0.1 0.1  0.0 9.1 0.4 37.6  0.4 29.7

    

Enthalpy (J g1) FWHM (
C) 0.9 5.3  0.8 0.6 34.7  1.5 0.1 30.3  0.1 0.1 7.2  0.2 0.2 13.8  0.1

3.4 18.1 2.4 4.7 2.5

    

0.6 0.7 0.0 0.1 0.0

a Values are means  standard deviations based on triplicates. Abbreviations are HMP, high-melting point fraction; MMP, middle-melting point fraction; LMP, lowmelting point fraction; Ton, onset crystallization temperature; Toff, offset crystallization temperature; Tmax, maximum crystallization temperature; FWHM, full width at half maximum.

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The information presented in Table 2 was used to design the fractionation protocol for hydrolyzed AMF enriched in CLA and TVA, as described in Section 2.2.2. Our fractionation approach was to separate those fatty acids with a high melting point (S30) from those with a low-melting point (L10). Table 3 shows the fatty acid composition of the different lipid fractions obtained. The fractions 30, S10 and L10 corresponded to 37.1, 51.6, and 8.3% (w/w), respectively. A 3.7% (w/w) gap comprised a fraction between S30 and L30, which was not possible to recover. Similar gaps have been reported previously (Deffense, 1993) due to contamination between the different lipid fractions. The proportion of a gap varies depending upon the crystallization rate and sample composition. The L30 fraction was enriched in C14:0, C16:0, C18:0, as well as CLA and TVA, as only high melting point fatty acids (mainly C20:4 and C18:2 tt) were separated. The L30 fraction was enriched in TVA and CLA up to 98.3% and 72.7%, respectively, over the parent fat. In the S10 fraction, the concentrations of CLA and TVA were higher than those obtained in L30 and AMF enriched in CLA and TVA. The concentrations of TVA and CLA in S10 increased by up to 174.1 and 164.5% (w/w) over the parent fat, respectively. Additionally, the S10 fraction contained lower concentrations of C16:0 and C18:0 whereas the L10 fraction was enriched in the two low-melting point fatty acids (C6:0 and C8:0). All these approaches provide information for further CLA enrichment through dry fractionation of AMF. The CLA content reported previously in the milk fat fractions ranged from 8 to 25 mg g1 of fat, which is relatively low to be economically feasible (O’Shea et al., 2000; Rehberger et al., 2008; Romero et al., 2000). Our findings indicate that the final concentration of CLA in the S10 fraction was 63.0  0.1 mg g1 of fat, an 11-fold increase compared with that in normal AMF (untreated and non-enriched AMF). This concentration is remarkably higher than the concentration typically found in dairy products (1e16 mg g1), such as butter, yoghurt

Table 3 Fatty acid composition of the different lipid fractions obtained from hydrolyzed anhydrous milk fat (AMF) enriched in conjugated linoleic acid (CLA; C18:2) and trans-vaccenic acid (TVA; C18:1 t11).a Fatty acid

AMF enriched in L30 CLA and TVA

C6:0 6.60  0.01 C8:0 4.21  0.02 C10:0 7.90  0.03 C11:0 0.90  0.04 C12:0 10.91  0.01 C14:0 47.51  0.06 C14:1 5.20  0.04 C15:0 5.92  0.01 C16:0 135.90  0.4 C16:1t 4.73  0.02 C16:1c 7.20  0.01 C18:0 77.10  0.40 C18:1 t11 (TVA) 90.90  0.30 C18:1 n12 8.61  0.01 C18:1 n9 171.30  0.01 C18:1 n7 5.52  0.02 C18:2 16.90  0.10 C18:2 tt 0.21  0.01 C20:0 1.11  0.02 C20:1 n12 3.40  0.01 C20:1 n15 0.92  0.02 C18:3 w3 2.10  0.02 C18:2 (CLA) 23.80  0.20 C20:3 w6 0.34  0.01 C20:4 1.10  0.01

11.60 7.71 15.20 1.71 20.40 96.80 9.30 13.50 379.90 10.30 13.40 241.60 180.30 19.10 315.10 10.40 30.20 0.0 2.41 5.40 1.70 3.80 41.10 0.51 0.0

S10                 

0.30 0.02 0.03 0.01 0.10 0.50 0.02 0.01 1.50 0.10 0.01 2.70 3.10 0.10 2.50 0.50 0.90

     

0.01 0.02 0.02 0.01 0.80 0.01

17.40 12.40 24.00 0.0 31.70 130.00 14.90 14.70 293.60 14.00 22.20 141.20 249.20 23.20 488.60 16.10 49.90 0.70 2.01 11.30 2.70 6.10 63.00 0.0 2.80

L10  0.40 20.00  0.70  0.30 32.50  1.90  0.90 0.0 0.0  1.50 0.0  2.00 0.0  0.80 0.0  0.40 0.0  2.40 0.0  1.00 0.0  0.80 0.0  1.60 0.0  4.60 0.0  1.10 0.0  5.70 0.0  0.80 0.0  0.90 0.0  0.01 0.0  0.02 0.0  0.70 0.0  0.06 0.0  0.05 0.0  0.70 0.0 0.0  0.10 0.0

Values (mg g1 hydrolyzed fat) are means  standard deviations based on triplicates. Abbreviations are t, trans fatty acids; c, cis fatty acids; L30, liquid fraction obtained at 30
C; S10, solid fraction obtained at 10
C; L10, liquid fraction obtained at 10
C. a

and some cheeses (O’Shea et al., 2000). The concentration of TVA in the S10 fraction was 249.2  1.1 mg g1 of fat, which is 19 times higher compared with that in normal AMF (untreated and nonenriched AMF). Other fractions obtained may have potential for different applications. For instance, L10 fraction is rich in C6:0 and C8:0, which is being used in some structured triglycerides with targeted fatty acid composition (Kaylegian, 1995). The potential value of each fraction needs to be further assessed. 3.5. Oxidation curves of hydrolyzed anhydrous milk fat The DSC oxidation curves of hydrolyzed AMF enriched in CLA and TVA obtained at different heating rates (3, 6, 12 and 15
C min1) in the temperature range of 100e350
C are shown in Fig. 4. The recorded heat flow values were due to oxidation rather than combustion, as the temperature range used in this study was below the self-ignition temperature of fats and oils (w350
C). In addition, hydrolyzed AMF heated at 3
C min1 under nitrogen flow revealed that neither exothermal nor endothermal events occurred (bottom line in Fig. 4). The values of Ts, Ton, and Tp increased with the heating rate. When hydrolyzed AMF is oxidized at high heating rates, the primary oxidation products are lost through evaporation as they are being formed, delaying further oxidation (Adhvaryu, Erhan, Lui, & Perez, 2000). In contrast, the primary oxidation products remain within the lipid sample at slow heating rates, further accelerating the oxidation (Saldaña & MartínezMonteagudo, 2013). Two maximum heat flow peaks were observed in the DSC spectra (Tp1 and Tp2) presented in Fig. 4. Similarly, Litwinienko, Daniluk, and Kasprzycka-Guttman (1999) and Litwinienko, Kasprzycka-Guttman, and Studzinski (1997) reported two maximum heat flow peaks for the non-isothermal oxidation of different edible oils. Interestingly, only the first peak is related to oxidation as demonstrated by Litwinienko et al. (1997). These authors oxidized corn and linseed oils with different peroxide numbers and found that the onset temperature of oxidation shifted to higher values as the peroxide number increased. In contrast, the first and second peak temperatures were not affected by an increase in the peroxide number (Litwinienko et al., 1997). A thermogravimetrical analysis of soy lecithin under nitrogen showed that within the temperature range of 100e250
C, the weight loss

Fig. 4. Differential scanning calorimetry oxidative profiles of hydrolyzed anhydrous milk fat enriched in conjugated linoleic acid and trans-vaccenic acid at different heating rates (A, 3
C min1; B, 6
C min1; C, 12
C min1; D, 15
C min1). The baseline is indicated by the dashed line, the nitrogen line is the lowest line; scale bar is 0.2 W g1.

S.I. Martínez-Monteagudo et al. / International Dairy Journal 36 (2014) 29e37

35

Table 4 Start, onset and maximum heat flow temperatures of conjugated linoleic acid- and trans-vaccenic acid-enriched hydrolyzed and non-hydrolyzed anhydrous milk fat (AMF) oxidation.a Parameter

1

b ( C min )


Ea (kJ mol1) z (min1) k120
C k200
C R2

Start temperature of oxidation (Ts)

3 6 12 15

Onset temperature of oxidation (Ton)

First maximum heat flow temperature (Tp)

Hydrolyzed AMF

Non-hydrolyzed AMFb

Hydrolyzed AMF

Non-hydrolyzed AMFb

Hydrolyzed AMF

Non-hydrolyzed AMFb

108.4  4.1 111.1  1.1 121.5  1.7 125.7  0.8 101.8 1.1  1011 0.004 0.79 0.92

155.7  3.4 164.1  5.3 174.9  1.6 177.9  3.4 87.6 6.3  107 0.0001 0.013 0.96

125.1  2.4 132.4  1.2 136.4  2.3 135.9  1.6 175.7 2.3  1020 0.0011 9.1 0.91

163.3  1.6 170.5  0.9 188.5  0.1 191.8  0.1 82.4 8.7  107 0.0009 0.069 0.94

178.8  0.5 188.8  2.5 198.6  2.1 204.1  2.4 110.9 6.9  109 0.00001 0.004 0.99

208.8  1.2 211.2  0.2 237.6  0.9 239.5  2.9 73.6 1.2  106 0.0002 0.008 0.94

a Values are means  standard deviations based on triplicates. Abbreviations are Ea, activation energy; z, pre-exponential factor; k, reaction rate constant; R2, determination coefficient; b, heating rate. b Values reported by Martínez-Monteagudo et al. (2012a).

changed only slightly, whereas at higher temperatures (>250
C), the weight decreased rapidly (Ulkowski, Musialik, & Litwinienko, 2005). This indicates that the exothermal signal detected by the DSC in the range of 100e250
C corresponds to oxidation. Table 4 summarizes the calculated Ts, Ton, and Tp for the oxidation of hydrolyzed AMF, which starts at a lower temperature (w108
C) compared with the start temperature of oxidation reported for non-hydrolyzed AMF (w155
C) (Martínez-Monteagudo et al., 2012b). The same tendency was observed for onset and maximum heat flow temperatures, which confirms that the FFA oxidizes faster than those fatty acids attached to the TAG molecule. 3.6. Kinetic parameters Table 4 also shows the values of the effective activation energy (Ea), pre-exponential factor (z), and reaction rate (k) for nonisothermal oxidation of hydrolyzed AMF enriched in CLA and TVA. The values of Ea, calculated from Ts, Ton, and Tp, were 101.8, 175.8, and 110.9 kJ mol1, respectively. In general, these Ea values were within the range of those values reported for edible lipids of animal and plant sources, recently reviewed by Saldaña and Martínez-Monteagudo (2013). The Ea values calculated with Ts, Ton and Tp were higher in the hydrolyzed AMF compared with those for the non-hydrolyzed AMF reported previously by Martínez-Monteagudo et al. (2012a) (Table 4). This indicates that the oxidation of free fatty acids occurred more rapidly at higher temperatures compared with that of fatty acids in the TAG form. The different Ea values shown in Table 4 suggest that the calculated Ea values are the cumulative effect of all the Ea values associated with the different components present in the lipid matrix during the non-isothermal oxidation, including Ea values of intermediate compounds that have their own kinetic values. As oxidation proceeds, several reactions occur simultaneously at different rates. Therefore, the calculated Ea values should not be used as a single parameter to compare and rank the oxidative stability of different lipid systems. This has been exemplified in the non-isothermal oxidation of AMF with different ratios of unsaturated to saturated fatty acids (Martínez-Monteagudo et al., 2012a) and blends of soybean and AMF (Thurgood, Ward, & Martini, 2007). In these investigations, the Ton shifted to lower values as the amount of unsaturated fatty acids increased, and the only kinetic parameter that exhibited the same pattern was the oxidation rate constant. Therefore, it is recommended to use the reaction rate constant (k) to compare and rank the oxidative stability of edible lipids. Table 4 summarizes the k values from Ts, Ton, and Tp calculated at 120
C and 200
C. As expected, the k values increased as

the temperature increased from 120
C to 200
C, regardless of the selected reference point (Ts, Ton, and Tp). This increase was remarkable for k values obtained from Ton where an increase from 0.0011 at 120
C to 9.1 at 200
C was observed. A comparison between oxidation parameters obtained for hydrolyzed and non-hydrolyzed AMF samples is also provided in Table 4. For non-hydrolyzed AMF, where the fatty acids are esterified to the glycerol backbone, the Ts, Ton and Tp values were considerably higher than those of hydrolyzed AMF where fatty acids are in the free form. Therefore, the kinetic parameters calculated from the Ts, Ton, and Tp values were lower for nonhydrolyzed AMF. For example, the Ea values calculated from Ton were 175.7 and 82.4 kJ mol1 for hydrolyzed and non-hydrolyzed AMF enriched in CLA and TVA, respectively. Although the fatty acid composition was the same for both samples, FFAs (hydrolyzed sample) oxidized faster than those fatty acids in the TAG form (nonhydrolyzed sample). Indeed, Tsuzuki et al. (2004) compared the oxidative stability of CLA in FFA and TAG forms at 37
C and found that CLA in the TAG form was 10 times more stable than CLA in the FFA form. These authors hypothesized that the oxidative stability of CLA in TAG form is remarkably improved because the carboxylic group of CLA is protected through esterification in the TAG form, which might delay the formation of primary oxidation products. 4. Conclusions A two-step process consisting of hydrolysis and fractionation of AMF enriched in CLA and TVA was studied. Enzymatic hydrolysis of AMF enriched in CLA and TVA resulted in 90.3  1.2% of free fatty acids after 24 h of hydrolysis using enzyme/fat and water/fat ratios of 40 and 70, respectively. The repeated use of Lipozyme TL IM enzyme reduced the amount of FFAs in the final hydrolyzed product. The DSC oxidation kinetics confirmed that FFAs are more susceptible to oxidation than fatty acids attached to the TAG molecule. Samples obtained after enzymatic hydrolysis were dryfractionated, and the highest CLA and TVA contents were found in the medium melting point fraction (64.9 and 249.2 mg g1, respectively). Further optimization of the fractionation process is needed to maximize the CLA and TVA contents while minimizing the other fatty acids. The S10 fraction can be used for fortification of dairy products. The L10 fraction can be used for synthesis of structured triglycerides with targeted fatty acid composition as C6:0 and C8:0 were the major fatty acids in this fraction based on GC analysis. The effect of fortifying dairy products with FFA fraction enriched in CLA and TVA on the sensory properties needs to be assessed for each specific product. Hydrolysis and further dry fractionation of AMF enriched in CLA and TVA yielded a lipid

36

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