http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, Early Online: 1–5 ! 2014 Informa UK Ltd. DOI: 10.3109/09637486.2014.917154
RESEARCH ARTICLE
Vitamins, fatty acids, and antioxidant capacity stability during storage of freeze-dried human milk Blanca Lozano1, Ana Isabel Castellote1,2, Rosa Montes1,2, and M. Carmen Lo´pez-Sabater1,2 Department of Nutrition and Food Science, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain and 2CIBER Physiopathology of Obesity and Nutrition (CIBEROBN), Institute of Health Carlos III, Madrid, Spain
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Abstract
Keywords
Although freezing is the most common method used to preserve human milk, nutritional and immunological components may be lost during storage. Freeze-drying could increase the shelf life of human milk, while preserving its original characteristics. Seventy-two samples of freezedried human milk were stored for different periods of time, up to a maximum of 3 months, at 4 C or 40 C. Vitamin C, tocopherols, antioxidant capacity, and fatty acids composition were analyzed. A new HILIC–UHPLC method improving vitamin C determination was also validated. Ascorbic acid and total vitamin C concentrations significantly decreased at both temperatures, while antioxidant capacity only decreased at 40 C. Fatty acids composition and both g-tocopherol and d-tocopherol contents remained unaltered. The stability after storage of freeze-dried milk was higher than that reported for frozen or fresh milk indicating that freezedrying is a promising option to improve the preservation of human milk in banks.
Ascorbic acid, antioxidant capacity, breast milk, fatty acids, freeze-drying, stability, tocopherols
Introduction According to health organizations, breastfeeding is the best way to nourish newborns until 6 months old (American Academy of Pediatrics, 2012; James et al., 2009). Human milk is a dynamic fluid whose composition changes depending on the length of gestation, the lactation cycle, and even on different days within a cycle, to meet the nutrient requirements of growing infants (Leaf & Winterson, 2009). Breast milk intake is linked to short-term and long-term child health including a lower risk of developing respiratory and gastrointestinal infections, acute otitis media, necrotizing enterocolitis, sudden infant death syndrome, atopic and celiac diseases, obesity in adolescence and adulthood, and type 1 and 2 diabetes mellitus (Akobeng et al., 2006; Duijts et al., 2010; Greer et al., 2008; Hauck et al., 2011; Ip et al., 2007, 2009; Owen et al., 2005, 2006; Sullivan et al., 2010). Breastfeeding is especially recommended in preterm infants, who are frequently exposed to oxidative stress caused by infections (Hanna et al., 2004). Since breast milk can suppress oxidative stress and DNA damage more effectively than infant formulae (Tijerina-Sa´enz et al., 2009), preterm infants should be fed human milk. When the mother’s own milk is unavailable, the American Academy of Pediatrics (2012) recommends using pasteurized donor milk. For this purpose, human milk banks have been created. These services are responsible for collecting, screening, processing, and distributing donated human milk to cover the specific medical requirements of the individuals for whom it is prescribed. Freezing at 20 C is the most common way to preserve donor milk. However, frozen storage can reduce Correspondence: M. Carmen Lo´pez-Sabater, Department of Nutrition and Food Science, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, E-08028 Barcelona, Spain. Tel: +34 934024512. Fax: +34 934035931. E-mail: [email protected]
History Received 30 January 2014 Revised 24 March 2014 Accepted 28 March 2014 Published online 19 May 2014
nutritional and immunological quality (Akinbi et al., 2010; Bank et al., 1985; Romeu-Nadal et al., 2008; Silvestre et al., 2010). Freeze-drying is commonly used to extend the shelf life of foods by preventing microbial growth and delaying fat oxidation (Shofian et al., 2011). It is particularly suitable for preserving heat-sensitive antioxidants, such as tocopherols and ascorbic acid (AA). This technology could reduce some of the limitations of the traditional freezing process by decreasing storage space, facilitating transport, and increasing shelf life, while preserving the flavor and the nutritional characteristics of milk (Ratti, 2001; Vega-Mercado et al., 2001). In summary, breastfeeding is essential to ensure optimal development of newborns, especially when they are preterm. Human milk bank treatments need to be improved to maintain the original characteristics of breast milk as long as possible. The aim of this study was to explore the suitability of the freeze-drying process for preserving human milk in banks focusing on changes in fatty acid composition, antioxidant capacity, and vitamin C and E contents of freeze-dried human milk during storage. In addition, a new method was developed to improve the chromatographic determination of vitamin C.
Methods Sample collection, treatment, and storage Human milk was obtained from eight healthy Spanish women by mechanical expression from both breasts using an Ameda breast pump (Ameda, Zug, Switzerland). Milk was pumped into sterile polypropylene bottles and transported to the laboratory at 4 C in less than 2 h. Then, all samples were homogenized by sonication in an ice bath (2 sets of 5 s pulses with 10 s cooling intervals on ice) using a Vibra Cell 75185 (Sonics, Newtown, CT) sonicator equipped with a 3 mm tip and set to 80% amplitude. After that,
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milk was pooled, vortex-mixed during 5 min, frozen at 80 C and freeze-dried for 24 h using a 4.5 L Freezone lyophilizer (Labconco, Kansas City, MO) with a vacuum of 103 mBar and a condensing plate temperature of 46 C. Finally, different aliquots were obtained to evaluate the storage stability. Nine aliquots were used for each storage conditions, as replicates. Thus, 27 freeze-dried human milk aliquots were stored at 4 C and maintained for three different periods (for 30, 60, and 90 d) and 36 aliquots were stored at 40 C (for 5, 15, 30, and 60 d). The 40 C conditions were considered an accelerated stability test. Another nine freeze-dried non-stored human milk aliquots were used as the control sample for each group. Overall 72 aliquots were obtained for further analysis. Each individual sample was packaged separately in a glass vial protected from air and light. All sample processing was carried out with protection from light, to prevent sample degradation. Water activity was determined using a Novasina Labswift-Aw (Novatron, West Sussex, UK) and remained below 0.3. The percentage of water removed by freeze-drying was 88.95%. Before the analysis, samples were reconstituted to the original volume with Milli-Q water (Millipore, Billerica, MA) and homogenized by inversion. Vitamin C analysis AA and total vitamin C (TVC) (AA plus dehydroascorbic acid (DHA)) concentrations were determined with a modified version of the procedure previously described by Romeu-Nadal et al. (2006). To improve AA retention, we introduced the use of hydrophilic interaction liquid chromatography (HILIC). Moreover, to decrease the time of analysis, ultra-high-pressure liquid chromatography (UHPLC) was used. Each sample was analyzed in triplicate for AA and TVC. Sample preparation To analyze AA, 100 mL of human milk, 300 mL of 75 mM oxalic acid, and 400 mL of acetonitrile were added to a 5 mL tube and vortex-mixed for 30 s. To determine TVC content, DHA was reduced to AA. About 100 mL of human milk and 275 mL of DL-dithiothreitol (DTT) 100 mM were added. After vigorous vortexing for 30 s, the tubes were left to react under dark conditions for 15 min. Then, 200 mL of 75 mM oxalic acid solution and 575 mL of acetonitrile were added to the tube, which was vortex-mixed for 30 s. For both determinations (AA and TVC), extracts were purified using Captiva NDlipids 96-well protein and lipid depletion plates (Agilent, Santa Clara, CA). Both solutions were transferred to the plates, and after the application of vacuum, the filtered solutions were collected in a 2 mL 96-well plate and covered with a cap mat. Finally, 3 mL of each extract were injected into the UHPLC system. Chromatographic conditions Chromatographic separation of AA was performed using a Waters Acquity ultra-pressure liquid chromatography system (UPLC) (Waters, Milford, MA), equipped with a binary solvent delivery module, an autosampler cooler, a column heater, and a 2996 photodiode array (PDA) detector. Optimum separation was achieved with an isocratic binary mobile phase, consisting of acetonitrile and 100 mM ammonium formate (80:20, v/v), adjusted to pH 4.0 at a constant flow rate of 0.6 mLmin1. Column and autosampler temperatures were maintained at 30 C and 4 C, respectively. All solvents were passed through a 0.22 mm pore diameter filter before use. Chromatographic separation was achieved with a Kinetex Hilic column (2.1 mm i.d. 50 mm, 1.7 mm particle size) (Phenomenex, Torrance, CA), protected with
Int J Food Sci Nutr, Early Online: 1–5
a Security Guard ULTRA Pre-column UHPLC Hilic (2.1 mm i.d. 2 mm, 1.7 mm particle size) (Phenomenex, Torrance, CA). The PDA wavelength selected for this measurement was 266 nm. Vitamin E analysis Alpha-, gamma-, and delta-tocopherol analyses were performed by UHPLC, following the method developed by Molto´-Puigmartı´ et al. (2009). Each sample was analyzed in duplicate. Antioxidant capacity Antioxidant capacity was measured following the Ferric Reducing Antioxidant Power (FRAP) method developed by Benzie & Strain (1996), with slight modifications. Before analysis, samples of reconstituted milk were diluted 1:10 with ultrapure water. FRAP reagent was prepared by mixing 25 mL of buffer acetate (pH ¼ 3.6), 2.5 mL of TPTZ (2,4,6-tripyridyl-s-triazine) 0.01 M in HCl, and 2.5 mL of FeCl36H2O. This solution is stable for approximately 12 h. Aqueous solutions of known Fe (II) concentration, in the range of 0.4–2.5 mM (FeSO47H2O), were used for calibration. A Fluostar Optima fluorometer (BMG LabTech, Ortenberg, Germany) and Nunc 96-microwell plates (Thermo Scientific, Waltham, FL) were used to perform the FRAP assay as follows: 190 mL of freshly prepared FRAP reagent was warmed to 37 C for 5 min and a reagent blank reading was taken at 590 nm. Ten microliters of milk sample were then added, along with 5 mL of water. Both water and sample were warmed to 37 C before addition. The final dilution of the sample in the reaction mixture was 1:19.5. The mixture was shaken for 60 s and absorbance was recorded every 35 s during the monitoring period. The mixture was shaken for 5 s before each absorbance reading. The differences between the absorbance values for each solution (measured after 6.6 min of reaction) and for the reagent blank were calculated for each sample and concentrations were determined by external calibration. Measurements were carried out in quadruplicate. Fatty acid analysis Fatty acids were separated and identified, following the method described by Molto´-Puigmartı´ et al. (2007). Thirty-five fatty acids, from C6:0 to C22:6n3, were identified and quantified. Each sample was analyzed in duplicate. Statistical analysis The statistical analysis was performed using SPSS statistical software (Version 18, SPSS Inc, Chicago, IL). Normality of all variables was evaluated with the Kolmogorov–Smirnov test. Differences during storage were estimated by a one-way ANOVA, using the Bonferroni post hoc test. The level of significance was set at p50.05.
Results and discussion Validation of the ultra-high-pressure liquid chromatographic method (UHPLC) for the analysis of vitamin C An UHPLC method based on HILIC was developed and validated for quantifying AA and TVC (ascorbic and DHAs) levels in human milk. Sample preparation was optimized by using oxalic acid as a stabilizer (Nova´kova´ et al., 2008). Chromatographic separation was achieved in less than 1.2 min and provided narrow peaks with good symmetry, which was a great improvement over other established methods (Chotyakul et al., 2013; Romeu-Nadal et al., 2006).
Stability of freeze-dried human milk
DOI: 10.3109/09637486.2014.917154
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Table 1. Quality parameters of the developed method for determination of AA and TVC. Sensitivity
Linearity R2 (0.5–40 mg mL1) 0.9999
Precision (RSD)
LOD
LOQ
(mg mL1) 0.13
(mg mL1) 0.44
Intra-day (n ¼ 10) AA 2.21
TVC 0.93
Inter-day (n ¼ 20)a AA 3.76
TVC 3.22
Recovery (%)b AA 96.84 ± 2.16
TVC 99.39 ± 4.93
a
Milk samples (n ¼ 5) processed in four different days. Mean values for three added concentrations 20, 50, and 80% over the expected concentration in human milk.
b
Table 2. Effects of storage at 4 C and 40 C on vitamin C, vitamin E, and antioxidant capacity content in freeze-dried human milk. Time (days)
Total vitamin Ca (mg 100 mL1)
Ascorbic acid (mg 100 mL1)
Total vitamin Eb (mg g fat1)
a-Tocopherol (mg g fat1)
g-Tocopherol (mg g fat1)
d-Tocopherol (mg g fat1)
Antioxidant capacity (mM Fe2+)
4
0 30 60 90
5.56 ± 0.32 5.21 ± 0.27* 5.04 ± 0.30* 4.95 ± 0.36*
5.34 ± 0.45 4.99 ± 0.52* 4.83 ± 0.46* 4.56 ± 0.41*
1.02 ± 0.16 0.95 ± 0.11 1.03 ± 0.04 1.03 ± 0.06
1.00 ± 0.16 0.94 ± 0.11 1.01 ± 0.04 1.01 ± 0.06
0.08 ± 0.01 0.08 ± 0.01 0.09 ± 0.01 0.09 ± 0.00
0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
0.79 ± 0.14 0.78 ± 0.16 0.71 ± 0.11 0.73 ± 0.15
40
0 5 15 30 60
5.56 ± 0.32 4.52 ± 0.32* 3.64 ± 0.24* 3.11 ± 0.23* 2.48 ± 0.11*
5.34 ± 0.45 4.22 ± 0.41* 3.40 ± 0.35* 2.83 ± 0.36* 2.17 ± 0.25*
1.02 ± 0.16 0.99 ± 0.19 1.14 ± 0.34 0.96 ± 0.27 0.77 ± 0.09*
1.00 ± 0.16 0.97 ± 0.19 1.12 ± 0.33 0.94 ± 0.27 0.75 ± 0.09*
0.08 ± 0.01 0.08 ± 0.02 0.10 ± 0.03 0.08 ± 0.01 0.07 ± 0.01
0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
0.79 ± 0.14 0.60 ± 0.13* 0.55 ± 0.16* 0.46 ± 0.14* 0.42 ± 0.14*
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Temperature ( C)
Mean ± standard deviation (n ¼ 9). *p50.05 versus zero time of storage. aTotal vitamin C: ascorbic acid + dehydroascorbic acid. Total vitamin E: a-Tocopherol + g-tocopherol*0.2 + d-tocopherol*0.1.
b
The method was validated for linearity, sensitivity, recovery, and intra- and inter-day precision (Table 1). Good linearity (0.5–40 mg mL1) and a high correlation coefficient (0.9999) were observed over the concentration range examined. The limits of detection (LOD) and quantification (LOQ) for AA were 0.013 mg 100 mL1 and 0.044 mg 100 mL1, respectively. These values were higher than those reported by other authors in cow milk (Chotyakul et al., 2013) but remain far below concentrations expected in human milk (Bank et al., 1985; Romeu-Nadal et al., 2008). Intra- and inter-day repeatabilities were evaluated and values were found to be below 4% for both AA and TVC. Recovery of the method was determined by adding amounts of AA to human milk at three levels, which were approximately equal to 20%, 50%, and 80% of the concentration expected in human milk. Recoveries were calculated on the basis of the difference between the total amounts observed in the non-spiked samples. All analyses were carried out in triplicate. The mean recoveries obtained, expressed as percentages, were above 96% (Table 1). Effect of storage on vitamin C Vitamin C is considered a biomarker of oxidative stability in human milk. In addition, it is one of the most important antioxidants in humans and plays an important role in disease prevention. L-AA is the main biologically active form, but DHA also exhibits biological activity, since it can be converted into AA in the human body. This reaction is reversible and is one of the main keys to AA antioxidant activity. Therefore, to measure TVC activity, we must measure both AA and DHA (Li & Franke, 2009; Lykkesfeldt, 2000; Romeu-Nadal et al., 2006). Table 2 shows TVC and AA levels throughout storage. During this period, AA and TVC concentration decreased significantly. However, losses after two months at 4 C (9%) were negligible compared with those found in the accelerated stability test at 40 C (55%, see Figure 1). In addition, degradation was slower than reported in the literature on AA and TVC losses during freezer storage, Romeu-Nadal et al. (2008) stored human fresh milk at 4 C for 4 d and found 63% losses. When freeze-dried milk
was stored at the same temperature, AA levels decreased by only 6%, even after 30 d. Furthermore, Buss et al. (2001) reported a greater reduction in TVC in refrigerated and frozen human milk, up to 40 and 60% after 24 h of refrigeration and 30 d of freezing, respectively. Thus, storage had a lower effect on vitamin C in freeze-dried samples than reported in the literature on fresh and frozen fresh milk. Effect of storage on alpha-, gamma-, delta-tocopherol, and total vitamin E Tocopherols are a set of isomers that have vitamin E activity. The most active form is alpha-tocopherol. Total vitamin E (TVE) was calculated following the formula used by Cha´vez-Servı´n et al. (2008): Total vitamin E ¼ -tocopherol þ -tocopherol 0:2þ -tocopherol 0:1
ð1Þ
Table 2 shows the total vitamin E and tocopherol levels at 4 C and 40 C during the 3 months of storage. Vitamin E content did not decrease in samples stored at 4 C. However, in the accelerated stability test at 40 C, TVE and alpha-tocopherol concentration decreased significantly after 2 months of storage (p50.05). In a recent study, Lacomba et al. (2012) found no significant vitamin E losses in milk frozen at 20 C for 30 d and no losses in the stability at 4 C after 48 h. However, they only analyzed samples after 48 h and this cannot be used to predict tocopherol losses for longer periods. Results reported by RomeuNadal et al. (2008) for the freezing process indicate that 25% of alpha-tocopherol and 30% of gamma-tocopherol were lost after 4 d at 4 C. In freeze-dried samples the concentration of tocopherols did not change, even after 90 d at 4 C. Thus, with respect to vitamin E, freeze-drying constitutes a better storage option than freezing. Effect of storage on antioxidant capacity As mentioned previously, tocopherols and vitamin C modulate antioxidant activity. However, other active compounds are
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involved in the antioxidant activity of breast milk, including carotenoids, vitamin A, or enzymes (Hanna et al., 2004). The antioxidant capacity remained practically constant in samples stored at 4 C (Table 2). However, losses of 24% were 1
4 ºC
60.0%
40 ºC
Effect of storage on fatty acids composition
50.0%
40.0%
Conclusions
30.0%
Freeze-drying human milk was shown in this study to be a good alternative to preserve breast milk in human milk banks. This technology can increase shelf life, facilitate transport, and preserve the nutritional characteristics of human milk. Vitamin C and vitamin E content and antioxidant capacity in freeze-dried human milk decreased to a much lesser extent and at lower rates than in frozen fresh milk. Fatty acids composition remained unaltered under the studied conditions. Nevertheless, further studies should be carried out to confirm these results. Despite the limited number of study samples, our results are consistent and this pilot study could be useful for future work in the field. AA and TVC had the highest significant variations under the studied conditions (refrigeration and accelerated stability test). Therefore, they could be proposed as the best biomarkers of human milk degradation. Future research should focus on the packaging of human milk and evaluate modified atmosphere and vacuum packaging. Moreover, immunological, bacteriological and virological parameters should be studied.
% of losses TVC
50.0%
Thirty-five fatty acids were analyzed in freeze-dried samples. Table 3 shows the percentages of some of the most important fatty acids in human milk and their evolution over time. No significant differences were found in composition during the study at both storage temperatures. This is extremely important for arachidonic acid (C20:4 n-6) and docosahexaenoic acid (C22:6 n-3). In newborns and especially preterm infants, the conversion rate is not high enough to obtain those LC-PUFA in the amounts required to ensure a normal development. Therefore, it is necessary to include these nutrients in the diet and extremely important to ensure their preservation in human milk (Huffman et al., 2011; Lauritzen & Carlson, 2011).
40.0% 30.0% 20.0% 10.0% 0.0% 0
20
40
60
80
100
Days
% of losses AA
2 60.0%
20.0% 10.0% 0.0% 0
20
40
60
80
100
Days
3 60.0% % of losses anoxidant cap.
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found after 5 d of storage at 40 C (Figure 1). This decrease in antioxidant capacity was lower than that reported by other authors for frozen fresh human milk. Arun Mamachan et al. (2011) found a 25% reduction after a week at 8 C and Hanna et al. (2004) reported an even greater decrease of around 35% after a week at 20 C.
50.0% 40.0% 30.0% 20.0% 10.0% 0.0% 0
20
40
60
80
100
Days
Figure 1. Evolution of losses (given as percentage) of TVC, AA, and antioxidant capacity at different thermal conditions. (1) Total vitamin C, (2) ascorbic acid, (3) antioxidant capacity. Stability studies performed at 4 C (solid line) and 40 C (dotted line).
Acknowledgements The authors would like to thank Mr. Christopher Evans for the assistance with English correction. Special thanks go to the women who donated their milk.
Table 3. Effects of storage at 4 C and 40 C on fatty acid composition in freeze-dried human milk. Time (days)
C20:4 n-6a (%)
C22:6 n-3b (%)
C18:1 n-9c (%)
C18:2 n-6d (%)
C18:3 n-3e (%)
SFAf (%)
MUFAg (%)
PUFAh (%)
4
0 30 60 90
0.45 ± 0.03 0.45 ± 0.03 0.46 ± 0.04 0.45 ± 0.03
0.33 ± 0.03 0.33 ± 0.03 0.33 ± 0.03 0.33 ± 0.03
39.68 ± 1.30 39.63 ± 1.17 39.64 ± 1.25 39.70 ± 1.11
13.33 ± 0.64 13.38 ± 0.56 13.39 ± 0.54 13.41 ± 0.59
0.78 ± 0.05 0.79 ± 0.06 0.78 ± 0.05 0.78 ± 0.05
38.94 ± 0.74 38.96 ± 0.82 38.93 ± 0.90 38.85 ± 0.65
44.97 ± 1.24 44.87 ± 1.25 44.89 ± 1.33 44.96 ± 1.17
16.15 ± 0.52 16.21 ± 0.50 16.22 ± 0.44 16.24 ± 0.52
40
0 5 15 30 60
0.45 ± 0.03 0.45 ± 0.04 0.45 ± 0.03 0.45 ± 0.03 0.44 ± 0.03
0.33 ± 0.03 0.33 ± 0.03 0.32 ± 0.03 0.33 ± 0.03 0.32 ± 0.02
39.68 ± 1.30 39.69 ± 1.25 39.11 ± 1.14 39.59 ± 1.26 39.18 ± 1.02
13.33 ± 0.64 13.42 ± 0.51 13.51 ± 0.46 13.41 ± 0.58 13.29 ± 0.58
0.78 ± 0.05 0.78 ± 0.05 0.80 ± 0.05 0.79 ± 0.05 0.78 ± 0.07
38.94 ± 0.74 38.83 ± 0.92 39.37 ± 0.84 38.94 ± 0.86 39.55 ± 0.57
44.97 ± 1.24 44.96 ± 1.30 44.37 ± 1.20 44.87 ± 1.32 44.45 ± 1.11
16.15 ± 0.52 16.25 ± 0.41 16.30 ± 0.38 16.24 ± 0.48 16.04 ± 0.56
Temperature ( C)
Mean ± standard deviation (n¼9). *p50.05 versus zero time of storage. Arachidonic acid. bDocosahexaenoic acid. cOleic acid. dLinoleic acid. eLinolenic acid. fSaturated fatty acids. gMonounsaturated fatty acids. h Polyunsaturated fatty acids.
a
DOI: 10.3109/09637486.2014.917154
Declaration of interest The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported by the Spanish Ministry of Economy and Competitiveness, the Regional Government of Catalonia’s Department of Innovation, Universities and Enterprise and the former Spanish Ministry of Science and Innovation through the research projects BFU2012-40254-CO3-O2, 2009SGR-606, and AGL2009-09730/ALI, respectively. Blanca Lozano is grateful to the Faculty of Pharmacy (University of Barcelona) for her predoctoral grant and Rosa Montes thanks the Spanish Ministry of Economy and Competitiveness for her ‘‘Juan de la Cierva’’ post-doctoral contract.
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Ip S, Chung M, Raman G, Trikalinos TA, Lau J. 2009. A summary of the agency for healthcare research and quality’s evidence report on breastfeeding in developed countries. Breastfeed Med 4:S17–S30. James DC, Lessen R, American Dietetic Association. 2009. Position of the American Dietetic Association: promoting and supporting breastfeeding. J Am Diet Assoc 109:1926–1942. Lacomba R, Cilla A, Alegrı´a A, Barbera´ R, Silvestre D, Lagarda MJ. 2012. Stability of fatty acids and tocopherols during cold storage of human milk. Int Dairy J 27:22–26. Lauritzen L, Carlson SE. 2011. Maternal fatty acid status during pregnancy and lactation and relation to newborn and infant status. Matern Child Nutr 7:41–58. Leaf A, Winterson R. 2009. Breast milk banking: evidence of benefit. Paediatr Child Health 19:395–399. Li X, Franke AA. 2009. Fast HPLC-ECD analysis of ascorbic acid, dehydroascorbic acid and uric acid. J Chromatogr B 877:853–856. Lykkesfeldt J. 2000. Determination of ascorbic acid and dehydroascorbic acid in biological samples by high-performance liquid chromatography using subtraction methods: reliable reduction with Tris[2-carboxyethyl]phosphine hydrochloride. Anal Biochem 282: 89–93. Molto´-Puigmartı´ C, Castellote AI, Lo´pez-Sabater MC. 2007. Conjugated linoleic acid determination in human milk by fast-gas chromatography. Anal Chim Acta 602:122–130. Molto´-Puigmartı´ C, Castellote AI, Lo´pez-Sabater MC. 2009. UltraHigh-Pressure Liquid Chromatographic method for the analysis of tocopherols in human colostrum and milk. J Chromatogr A 1216: 4388–4394. Nova´kova´ L, Solichova´ D, Pavlovicova´ S, Solich P. 2008. Hydrophilic interaction liquid chromatography method for the determination of ascorbic acid. J Sep Sci 31:1634–1644. Owen CG, Martin RM, Whincup PH, Smith GD, Cook DG. 2005. Effect of infant feeding on the risk of obesity across the life course: a quantitative review of published evidence. Pediatrics 115: 1367–1377. Owen CG, Martin RM, Whincup PH, Smith GD, Cook DG. 2006. Does breastfeeding influence risk of type 2 diabetes in later life? A quantitative analysis of published evidence. Am J Clin Nutr 84: 1043–1054. Ratti C. 2001. Hot air and freeze-drying of high-value foods: a review. J Food Eng 49:311–319. Romeu-Nadal M, Morera-Pons S, Castellote AI, Lo´pez-Sabater MC. 2006. Rapid high-performance liquid chromatographic method for vitamin C determination in human milk versus an enzymatic method. J Chromatogr B 830:41–46. Romeu-Nadal M, Castellote AI, Lo´pez-Sabater MC. 2008. Effect of cold storage on vitamins C and E and fatty acids in human milk. Food Chem 106:65–70. Shofian NM, Hamid AA, Osman A, Saari N, Anwar F, Dek MSP, Hairuddin MR. 2011. Effect of freeze-drying on the antioxidant compounds and antioxidant activity of selected tropical fruits. Int J Mol Sci 12:4678–4692. Silvestre D, Miranda M, Muriach M, Almansa I, Jaren˜o E, Romero FJ. 2010. Frozen breast milk at 20 C and 80 C: a longitudinal study of glutathione peroxidase activity and malondialdehyde concentration. J Hum Lact 26:35–41. Sullivan S, Schanler RJ, Kim JH, Patel AL, Trawo¨ger R, Kiechl-Kohlendorfer U, Chan GM, et al. 2010. An exclusively human milk-based diet is associated with a lower rate of necrotizing enterocolitis than a diet of human milk and bovine milk-based products. J Pediatr 156:562–567. Tijerina-Sa´enz A, Innis SM, Kitts DD. 2009. Antioxidant capacity of human milk and its association with vitamins A and E and fatty acid composition. Acta Paediatr 98:1793–1798. Vega-Mercado H, Go´ngora-Nieto MM, Barbosa-Ca´novas GV. 2001. Advances in dehydration of foods. J Food Eng 49:271–289.
RESEARCH ARTICLE
Vitamins, fatty acids, and antioxidant capacity stability during storage of freeze-dried human milk Blanca Lozano1, Ana Isabel Castellote1,2, Rosa Montes1,2, and M. Carmen Lo´pez-Sabater1,2 Department of Nutrition and Food Science, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain and 2CIBER Physiopathology of Obesity and Nutrition (CIBEROBN), Institute of Health Carlos III, Madrid, Spain
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Abstract
Keywords
Although freezing is the most common method used to preserve human milk, nutritional and immunological components may be lost during storage. Freeze-drying could increase the shelf life of human milk, while preserving its original characteristics. Seventy-two samples of freezedried human milk were stored for different periods of time, up to a maximum of 3 months, at 4 C or 40 C. Vitamin C, tocopherols, antioxidant capacity, and fatty acids composition were analyzed. A new HILIC–UHPLC method improving vitamin C determination was also validated. Ascorbic acid and total vitamin C concentrations significantly decreased at both temperatures, while antioxidant capacity only decreased at 40 C. Fatty acids composition and both g-tocopherol and d-tocopherol contents remained unaltered. The stability after storage of freeze-dried milk was higher than that reported for frozen or fresh milk indicating that freezedrying is a promising option to improve the preservation of human milk in banks.
Ascorbic acid, antioxidant capacity, breast milk, fatty acids, freeze-drying, stability, tocopherols
Introduction According to health organizations, breastfeeding is the best way to nourish newborns until 6 months old (American Academy of Pediatrics, 2012; James et al., 2009). Human milk is a dynamic fluid whose composition changes depending on the length of gestation, the lactation cycle, and even on different days within a cycle, to meet the nutrient requirements of growing infants (Leaf & Winterson, 2009). Breast milk intake is linked to short-term and long-term child health including a lower risk of developing respiratory and gastrointestinal infections, acute otitis media, necrotizing enterocolitis, sudden infant death syndrome, atopic and celiac diseases, obesity in adolescence and adulthood, and type 1 and 2 diabetes mellitus (Akobeng et al., 2006; Duijts et al., 2010; Greer et al., 2008; Hauck et al., 2011; Ip et al., 2007, 2009; Owen et al., 2005, 2006; Sullivan et al., 2010). Breastfeeding is especially recommended in preterm infants, who are frequently exposed to oxidative stress caused by infections (Hanna et al., 2004). Since breast milk can suppress oxidative stress and DNA damage more effectively than infant formulae (Tijerina-Sa´enz et al., 2009), preterm infants should be fed human milk. When the mother’s own milk is unavailable, the American Academy of Pediatrics (2012) recommends using pasteurized donor milk. For this purpose, human milk banks have been created. These services are responsible for collecting, screening, processing, and distributing donated human milk to cover the specific medical requirements of the individuals for whom it is prescribed. Freezing at 20 C is the most common way to preserve donor milk. However, frozen storage can reduce Correspondence: M. Carmen Lo´pez-Sabater, Department of Nutrition and Food Science, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, E-08028 Barcelona, Spain. Tel: +34 934024512. Fax: +34 934035931. E-mail: [email protected]
History Received 30 January 2014 Revised 24 March 2014 Accepted 28 March 2014 Published online 19 May 2014
nutritional and immunological quality (Akinbi et al., 2010; Bank et al., 1985; Romeu-Nadal et al., 2008; Silvestre et al., 2010). Freeze-drying is commonly used to extend the shelf life of foods by preventing microbial growth and delaying fat oxidation (Shofian et al., 2011). It is particularly suitable for preserving heat-sensitive antioxidants, such as tocopherols and ascorbic acid (AA). This technology could reduce some of the limitations of the traditional freezing process by decreasing storage space, facilitating transport, and increasing shelf life, while preserving the flavor and the nutritional characteristics of milk (Ratti, 2001; Vega-Mercado et al., 2001). In summary, breastfeeding is essential to ensure optimal development of newborns, especially when they are preterm. Human milk bank treatments need to be improved to maintain the original characteristics of breast milk as long as possible. The aim of this study was to explore the suitability of the freeze-drying process for preserving human milk in banks focusing on changes in fatty acid composition, antioxidant capacity, and vitamin C and E contents of freeze-dried human milk during storage. In addition, a new method was developed to improve the chromatographic determination of vitamin C.
Methods Sample collection, treatment, and storage Human milk was obtained from eight healthy Spanish women by mechanical expression from both breasts using an Ameda breast pump (Ameda, Zug, Switzerland). Milk was pumped into sterile polypropylene bottles and transported to the laboratory at 4 C in less than 2 h. Then, all samples were homogenized by sonication in an ice bath (2 sets of 5 s pulses with 10 s cooling intervals on ice) using a Vibra Cell 75185 (Sonics, Newtown, CT) sonicator equipped with a 3 mm tip and set to 80% amplitude. After that,
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milk was pooled, vortex-mixed during 5 min, frozen at 80 C and freeze-dried for 24 h using a 4.5 L Freezone lyophilizer (Labconco, Kansas City, MO) with a vacuum of 103 mBar and a condensing plate temperature of 46 C. Finally, different aliquots were obtained to evaluate the storage stability. Nine aliquots were used for each storage conditions, as replicates. Thus, 27 freeze-dried human milk aliquots were stored at 4 C and maintained for three different periods (for 30, 60, and 90 d) and 36 aliquots were stored at 40 C (for 5, 15, 30, and 60 d). The 40 C conditions were considered an accelerated stability test. Another nine freeze-dried non-stored human milk aliquots were used as the control sample for each group. Overall 72 aliquots were obtained for further analysis. Each individual sample was packaged separately in a glass vial protected from air and light. All sample processing was carried out with protection from light, to prevent sample degradation. Water activity was determined using a Novasina Labswift-Aw (Novatron, West Sussex, UK) and remained below 0.3. The percentage of water removed by freeze-drying was 88.95%. Before the analysis, samples were reconstituted to the original volume with Milli-Q water (Millipore, Billerica, MA) and homogenized by inversion. Vitamin C analysis AA and total vitamin C (TVC) (AA plus dehydroascorbic acid (DHA)) concentrations were determined with a modified version of the procedure previously described by Romeu-Nadal et al. (2006). To improve AA retention, we introduced the use of hydrophilic interaction liquid chromatography (HILIC). Moreover, to decrease the time of analysis, ultra-high-pressure liquid chromatography (UHPLC) was used. Each sample was analyzed in triplicate for AA and TVC. Sample preparation To analyze AA, 100 mL of human milk, 300 mL of 75 mM oxalic acid, and 400 mL of acetonitrile were added to a 5 mL tube and vortex-mixed for 30 s. To determine TVC content, DHA was reduced to AA. About 100 mL of human milk and 275 mL of DL-dithiothreitol (DTT) 100 mM were added. After vigorous vortexing for 30 s, the tubes were left to react under dark conditions for 15 min. Then, 200 mL of 75 mM oxalic acid solution and 575 mL of acetonitrile were added to the tube, which was vortex-mixed for 30 s. For both determinations (AA and TVC), extracts were purified using Captiva NDlipids 96-well protein and lipid depletion plates (Agilent, Santa Clara, CA). Both solutions were transferred to the plates, and after the application of vacuum, the filtered solutions were collected in a 2 mL 96-well plate and covered with a cap mat. Finally, 3 mL of each extract were injected into the UHPLC system. Chromatographic conditions Chromatographic separation of AA was performed using a Waters Acquity ultra-pressure liquid chromatography system (UPLC) (Waters, Milford, MA), equipped with a binary solvent delivery module, an autosampler cooler, a column heater, and a 2996 photodiode array (PDA) detector. Optimum separation was achieved with an isocratic binary mobile phase, consisting of acetonitrile and 100 mM ammonium formate (80:20, v/v), adjusted to pH 4.0 at a constant flow rate of 0.6 mLmin1. Column and autosampler temperatures were maintained at 30 C and 4 C, respectively. All solvents were passed through a 0.22 mm pore diameter filter before use. Chromatographic separation was achieved with a Kinetex Hilic column (2.1 mm i.d. 50 mm, 1.7 mm particle size) (Phenomenex, Torrance, CA), protected with
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a Security Guard ULTRA Pre-column UHPLC Hilic (2.1 mm i.d. 2 mm, 1.7 mm particle size) (Phenomenex, Torrance, CA). The PDA wavelength selected for this measurement was 266 nm. Vitamin E analysis Alpha-, gamma-, and delta-tocopherol analyses were performed by UHPLC, following the method developed by Molto´-Puigmartı´ et al. (2009). Each sample was analyzed in duplicate. Antioxidant capacity Antioxidant capacity was measured following the Ferric Reducing Antioxidant Power (FRAP) method developed by Benzie & Strain (1996), with slight modifications. Before analysis, samples of reconstituted milk were diluted 1:10 with ultrapure water. FRAP reagent was prepared by mixing 25 mL of buffer acetate (pH ¼ 3.6), 2.5 mL of TPTZ (2,4,6-tripyridyl-s-triazine) 0.01 M in HCl, and 2.5 mL of FeCl36H2O. This solution is stable for approximately 12 h. Aqueous solutions of known Fe (II) concentration, in the range of 0.4–2.5 mM (FeSO47H2O), were used for calibration. A Fluostar Optima fluorometer (BMG LabTech, Ortenberg, Germany) and Nunc 96-microwell plates (Thermo Scientific, Waltham, FL) were used to perform the FRAP assay as follows: 190 mL of freshly prepared FRAP reagent was warmed to 37 C for 5 min and a reagent blank reading was taken at 590 nm. Ten microliters of milk sample were then added, along with 5 mL of water. Both water and sample were warmed to 37 C before addition. The final dilution of the sample in the reaction mixture was 1:19.5. The mixture was shaken for 60 s and absorbance was recorded every 35 s during the monitoring period. The mixture was shaken for 5 s before each absorbance reading. The differences between the absorbance values for each solution (measured after 6.6 min of reaction) and for the reagent blank were calculated for each sample and concentrations were determined by external calibration. Measurements were carried out in quadruplicate. Fatty acid analysis Fatty acids were separated and identified, following the method described by Molto´-Puigmartı´ et al. (2007). Thirty-five fatty acids, from C6:0 to C22:6n3, were identified and quantified. Each sample was analyzed in duplicate. Statistical analysis The statistical analysis was performed using SPSS statistical software (Version 18, SPSS Inc, Chicago, IL). Normality of all variables was evaluated with the Kolmogorov–Smirnov test. Differences during storage were estimated by a one-way ANOVA, using the Bonferroni post hoc test. The level of significance was set at p50.05.
Results and discussion Validation of the ultra-high-pressure liquid chromatographic method (UHPLC) for the analysis of vitamin C An UHPLC method based on HILIC was developed and validated for quantifying AA and TVC (ascorbic and DHAs) levels in human milk. Sample preparation was optimized by using oxalic acid as a stabilizer (Nova´kova´ et al., 2008). Chromatographic separation was achieved in less than 1.2 min and provided narrow peaks with good symmetry, which was a great improvement over other established methods (Chotyakul et al., 2013; Romeu-Nadal et al., 2006).
Stability of freeze-dried human milk
DOI: 10.3109/09637486.2014.917154
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Table 1. Quality parameters of the developed method for determination of AA and TVC. Sensitivity
Linearity R2 (0.5–40 mg mL1) 0.9999
Precision (RSD)
LOD
LOQ
(mg mL1) 0.13
(mg mL1) 0.44
Intra-day (n ¼ 10) AA 2.21
TVC 0.93
Inter-day (n ¼ 20)a AA 3.76
TVC 3.22
Recovery (%)b AA 96.84 ± 2.16
TVC 99.39 ± 4.93
a
Milk samples (n ¼ 5) processed in four different days. Mean values for three added concentrations 20, 50, and 80% over the expected concentration in human milk.
b
Table 2. Effects of storage at 4 C and 40 C on vitamin C, vitamin E, and antioxidant capacity content in freeze-dried human milk. Time (days)
Total vitamin Ca (mg 100 mL1)
Ascorbic acid (mg 100 mL1)
Total vitamin Eb (mg g fat1)
a-Tocopherol (mg g fat1)
g-Tocopherol (mg g fat1)
d-Tocopherol (mg g fat1)
Antioxidant capacity (mM Fe2+)
4
0 30 60 90
5.56 ± 0.32 5.21 ± 0.27* 5.04 ± 0.30* 4.95 ± 0.36*
5.34 ± 0.45 4.99 ± 0.52* 4.83 ± 0.46* 4.56 ± 0.41*
1.02 ± 0.16 0.95 ± 0.11 1.03 ± 0.04 1.03 ± 0.06
1.00 ± 0.16 0.94 ± 0.11 1.01 ± 0.04 1.01 ± 0.06
0.08 ± 0.01 0.08 ± 0.01 0.09 ± 0.01 0.09 ± 0.00
0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
0.79 ± 0.14 0.78 ± 0.16 0.71 ± 0.11 0.73 ± 0.15
40
0 5 15 30 60
5.56 ± 0.32 4.52 ± 0.32* 3.64 ± 0.24* 3.11 ± 0.23* 2.48 ± 0.11*
5.34 ± 0.45 4.22 ± 0.41* 3.40 ± 0.35* 2.83 ± 0.36* 2.17 ± 0.25*
1.02 ± 0.16 0.99 ± 0.19 1.14 ± 0.34 0.96 ± 0.27 0.77 ± 0.09*
1.00 ± 0.16 0.97 ± 0.19 1.12 ± 0.33 0.94 ± 0.27 0.75 ± 0.09*
0.08 ± 0.01 0.08 ± 0.02 0.10 ± 0.03 0.08 ± 0.01 0.07 ± 0.01
0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
0.79 ± 0.14 0.60 ± 0.13* 0.55 ± 0.16* 0.46 ± 0.14* 0.42 ± 0.14*
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Temperature ( C)
Mean ± standard deviation (n ¼ 9). *p50.05 versus zero time of storage. aTotal vitamin C: ascorbic acid + dehydroascorbic acid. Total vitamin E: a-Tocopherol + g-tocopherol*0.2 + d-tocopherol*0.1.
b
The method was validated for linearity, sensitivity, recovery, and intra- and inter-day precision (Table 1). Good linearity (0.5–40 mg mL1) and a high correlation coefficient (0.9999) were observed over the concentration range examined. The limits of detection (LOD) and quantification (LOQ) for AA were 0.013 mg 100 mL1 and 0.044 mg 100 mL1, respectively. These values were higher than those reported by other authors in cow milk (Chotyakul et al., 2013) but remain far below concentrations expected in human milk (Bank et al., 1985; Romeu-Nadal et al., 2008). Intra- and inter-day repeatabilities were evaluated and values were found to be below 4% for both AA and TVC. Recovery of the method was determined by adding amounts of AA to human milk at three levels, which were approximately equal to 20%, 50%, and 80% of the concentration expected in human milk. Recoveries were calculated on the basis of the difference between the total amounts observed in the non-spiked samples. All analyses were carried out in triplicate. The mean recoveries obtained, expressed as percentages, were above 96% (Table 1). Effect of storage on vitamin C Vitamin C is considered a biomarker of oxidative stability in human milk. In addition, it is one of the most important antioxidants in humans and plays an important role in disease prevention. L-AA is the main biologically active form, but DHA also exhibits biological activity, since it can be converted into AA in the human body. This reaction is reversible and is one of the main keys to AA antioxidant activity. Therefore, to measure TVC activity, we must measure both AA and DHA (Li & Franke, 2009; Lykkesfeldt, 2000; Romeu-Nadal et al., 2006). Table 2 shows TVC and AA levels throughout storage. During this period, AA and TVC concentration decreased significantly. However, losses after two months at 4 C (9%) were negligible compared with those found in the accelerated stability test at 40 C (55%, see Figure 1). In addition, degradation was slower than reported in the literature on AA and TVC losses during freezer storage, Romeu-Nadal et al. (2008) stored human fresh milk at 4 C for 4 d and found 63% losses. When freeze-dried milk
was stored at the same temperature, AA levels decreased by only 6%, even after 30 d. Furthermore, Buss et al. (2001) reported a greater reduction in TVC in refrigerated and frozen human milk, up to 40 and 60% after 24 h of refrigeration and 30 d of freezing, respectively. Thus, storage had a lower effect on vitamin C in freeze-dried samples than reported in the literature on fresh and frozen fresh milk. Effect of storage on alpha-, gamma-, delta-tocopherol, and total vitamin E Tocopherols are a set of isomers that have vitamin E activity. The most active form is alpha-tocopherol. Total vitamin E (TVE) was calculated following the formula used by Cha´vez-Servı´n et al. (2008): Total vitamin E ¼ -tocopherol þ -tocopherol 0:2þ -tocopherol 0:1
ð1Þ
Table 2 shows the total vitamin E and tocopherol levels at 4 C and 40 C during the 3 months of storage. Vitamin E content did not decrease in samples stored at 4 C. However, in the accelerated stability test at 40 C, TVE and alpha-tocopherol concentration decreased significantly after 2 months of storage (p50.05). In a recent study, Lacomba et al. (2012) found no significant vitamin E losses in milk frozen at 20 C for 30 d and no losses in the stability at 4 C after 48 h. However, they only analyzed samples after 48 h and this cannot be used to predict tocopherol losses for longer periods. Results reported by RomeuNadal et al. (2008) for the freezing process indicate that 25% of alpha-tocopherol and 30% of gamma-tocopherol were lost after 4 d at 4 C. In freeze-dried samples the concentration of tocopherols did not change, even after 90 d at 4 C. Thus, with respect to vitamin E, freeze-drying constitutes a better storage option than freezing. Effect of storage on antioxidant capacity As mentioned previously, tocopherols and vitamin C modulate antioxidant activity. However, other active compounds are
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involved in the antioxidant activity of breast milk, including carotenoids, vitamin A, or enzymes (Hanna et al., 2004). The antioxidant capacity remained practically constant in samples stored at 4 C (Table 2). However, losses of 24% were 1
4 ºC
60.0%
40 ºC
Effect of storage on fatty acids composition
50.0%
40.0%
Conclusions
30.0%
Freeze-drying human milk was shown in this study to be a good alternative to preserve breast milk in human milk banks. This technology can increase shelf life, facilitate transport, and preserve the nutritional characteristics of human milk. Vitamin C and vitamin E content and antioxidant capacity in freeze-dried human milk decreased to a much lesser extent and at lower rates than in frozen fresh milk. Fatty acids composition remained unaltered under the studied conditions. Nevertheless, further studies should be carried out to confirm these results. Despite the limited number of study samples, our results are consistent and this pilot study could be useful for future work in the field. AA and TVC had the highest significant variations under the studied conditions (refrigeration and accelerated stability test). Therefore, they could be proposed as the best biomarkers of human milk degradation. Future research should focus on the packaging of human milk and evaluate modified atmosphere and vacuum packaging. Moreover, immunological, bacteriological and virological parameters should be studied.
% of losses TVC
50.0%
Thirty-five fatty acids were analyzed in freeze-dried samples. Table 3 shows the percentages of some of the most important fatty acids in human milk and their evolution over time. No significant differences were found in composition during the study at both storage temperatures. This is extremely important for arachidonic acid (C20:4 n-6) and docosahexaenoic acid (C22:6 n-3). In newborns and especially preterm infants, the conversion rate is not high enough to obtain those LC-PUFA in the amounts required to ensure a normal development. Therefore, it is necessary to include these nutrients in the diet and extremely important to ensure their preservation in human milk (Huffman et al., 2011; Lauritzen & Carlson, 2011).
40.0% 30.0% 20.0% 10.0% 0.0% 0
20
40
60
80
100
Days
% of losses AA
2 60.0%
20.0% 10.0% 0.0% 0
20
40
60
80
100
Days
3 60.0% % of losses anoxidant cap.
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found after 5 d of storage at 40 C (Figure 1). This decrease in antioxidant capacity was lower than that reported by other authors for frozen fresh human milk. Arun Mamachan et al. (2011) found a 25% reduction after a week at 8 C and Hanna et al. (2004) reported an even greater decrease of around 35% after a week at 20 C.
50.0% 40.0% 30.0% 20.0% 10.0% 0.0% 0
20
40
60
80
100
Days
Figure 1. Evolution of losses (given as percentage) of TVC, AA, and antioxidant capacity at different thermal conditions. (1) Total vitamin C, (2) ascorbic acid, (3) antioxidant capacity. Stability studies performed at 4 C (solid line) and 40 C (dotted line).
Acknowledgements The authors would like to thank Mr. Christopher Evans for the assistance with English correction. Special thanks go to the women who donated their milk.
Table 3. Effects of storage at 4 C and 40 C on fatty acid composition in freeze-dried human milk. Time (days)
C20:4 n-6a (%)
C22:6 n-3b (%)
C18:1 n-9c (%)
C18:2 n-6d (%)
C18:3 n-3e (%)
SFAf (%)
MUFAg (%)
PUFAh (%)
4
0 30 60 90
0.45 ± 0.03 0.45 ± 0.03 0.46 ± 0.04 0.45 ± 0.03
0.33 ± 0.03 0.33 ± 0.03 0.33 ± 0.03 0.33 ± 0.03
39.68 ± 1.30 39.63 ± 1.17 39.64 ± 1.25 39.70 ± 1.11
13.33 ± 0.64 13.38 ± 0.56 13.39 ± 0.54 13.41 ± 0.59
0.78 ± 0.05 0.79 ± 0.06 0.78 ± 0.05 0.78 ± 0.05
38.94 ± 0.74 38.96 ± 0.82 38.93 ± 0.90 38.85 ± 0.65
44.97 ± 1.24 44.87 ± 1.25 44.89 ± 1.33 44.96 ± 1.17
16.15 ± 0.52 16.21 ± 0.50 16.22 ± 0.44 16.24 ± 0.52
40
0 5 15 30 60
0.45 ± 0.03 0.45 ± 0.04 0.45 ± 0.03 0.45 ± 0.03 0.44 ± 0.03
0.33 ± 0.03 0.33 ± 0.03 0.32 ± 0.03 0.33 ± 0.03 0.32 ± 0.02
39.68 ± 1.30 39.69 ± 1.25 39.11 ± 1.14 39.59 ± 1.26 39.18 ± 1.02
13.33 ± 0.64 13.42 ± 0.51 13.51 ± 0.46 13.41 ± 0.58 13.29 ± 0.58
0.78 ± 0.05 0.78 ± 0.05 0.80 ± 0.05 0.79 ± 0.05 0.78 ± 0.07
38.94 ± 0.74 38.83 ± 0.92 39.37 ± 0.84 38.94 ± 0.86 39.55 ± 0.57
44.97 ± 1.24 44.96 ± 1.30 44.37 ± 1.20 44.87 ± 1.32 44.45 ± 1.11
16.15 ± 0.52 16.25 ± 0.41 16.30 ± 0.38 16.24 ± 0.48 16.04 ± 0.56
Temperature ( C)
Mean ± standard deviation (n¼9). *p50.05 versus zero time of storage. Arachidonic acid. bDocosahexaenoic acid. cOleic acid. dLinoleic acid. eLinolenic acid. fSaturated fatty acids. gMonounsaturated fatty acids. h Polyunsaturated fatty acids.
a
DOI: 10.3109/09637486.2014.917154
Declaration of interest The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported by the Spanish Ministry of Economy and Competitiveness, the Regional Government of Catalonia’s Department of Innovation, Universities and Enterprise and the former Spanish Ministry of Science and Innovation through the research projects BFU2012-40254-CO3-O2, 2009SGR-606, and AGL2009-09730/ALI, respectively. Blanca Lozano is grateful to the Faculty of Pharmacy (University of Barcelona) for her predoctoral grant and Rosa Montes thanks the Spanish Ministry of Economy and Competitiveness for her ‘‘Juan de la Cierva’’ post-doctoral contract.
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