Formation, Stability, and Properties of an Algae Oil Emulsion for

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10 % algae oil model emulsion with 10 % OSA starch and. 40 % corn syrup ..... Moreover, pressure directly affected the breaking up process of droplet disrup-.
Formation, Stability, and Properties of an Algae Oil Emulsion for Application in UHT Milk Xing Huimin, Li Lin, Gui Shilin, Walid Elfalleh, He Shenghua, Sheng Qinghai & Ma Ying Food and Bioprocess Technology An International Journal ISSN 1935-5130 Food Bioprocess Technol DOI 10.1007/s11947-013-1054-3

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Author's personal copy Food Bioprocess Technol DOI 10.1007/s11947-013-1054-3

ORIGINAL PAPER

Formation, Stability, and Properties of an Algae Oil Emulsion for Application in UHT Milk Xing Huimin & Li Lin & Gui Shilin & Walid Elfalleh & He Shenghua & Sheng Qinghai & Ma Ying

Received: 8 October 2012 / Accepted: 14 January 2013 # Springer Science+Business Media New York 2013

Abstract This study aimed to develop a technology for producing ultrahigh temperature ultrahigh temperaturetreated (UHT) milk enriched with docosahexaenoic acid. Starch hydrophobically modified with octenyl succinic anhydride (OSA starch) was used as an emulsifier to make algae oil emulsion in UHT milk. In this study, the stability of oil-in-water emulsions was examined. The emulsification of 10 % algae oil model emulsion with 10 % OSA starch and 40 % corn syrup had small droplets and was completely stable. Milk enriched with unsaturated fatty acids was heated using an indirect UHT treatment, and the milk was then stored at different temperatures. The oxidative stability of fish oil-enriched milk was investigated by measuring peroxide value, measuring volatile secondary oxidation products, and carrying out sensory analysis. All of the milk samples were stable. In summary, fish oil-enriched milk is resistant to oxidation. Algae oil-enriched drinking milk is a stable product during 11 weeks of storage. Application of high storage temperature (40 °C) does not significantly increase the oxidation process. The present study suggested that stable algae oil emulsion can be formed by OSA starches with corn syrup, and a food formulation test confirmed the successful application of algae oil emulsion to extend the shelf life of milk.

X. Huimin : G. Shilin : S. Qinghai (*) R&D System Inner Mongolia Mengniu Dairy Industry (Group) CO., LTD, Hohhot 011500, China e-mail: [email protected] L. Lin : W. Elfalleh : H. Shenghua : M. Ying (*) School of Food Science and Engineering, Harbin Institute of Technology, Harbin 150090, China e-mail: [email protected]

Keywords Emulsions . Docosahexaenoic acid (DHA) . OSA starches . Stability . UHT milk

Introduction There is considerable interest in altering the composition of foods with the overall aim of improving the long-term health of consumers. During the last decades, marine lipids have received increasing attention because of their beneficial health effects. It has been recognized that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) provide extensive nutritional and human health benefits (Ruxton et al. 2004; Uauy-Dagach and Valenzuela 1996). Many studies have demonstrated that these fatty acids have a positive effect on myocardial infarction, immune functions, and eye functions (Werkman and Carlson 1996). For example, EPA and DHA have been recognized for contributing to the prevention of coronary heart disease, hypertension, type II diabetes, rheumatoid arthritis, Crohn’s disease, and obstructive pulmonary disease (Kinsella 1986; Simopoulos 1999). Recognition of the potential benefits of EPA and DHA has stimulated interest in the fortification of foods through these lipids. However, the inclusion of omega-3 fatty acids in food products gives rise to major formulation challenges. Many lipids are sensitive to heat, light, and oxygen, and lipids quickly undergo oxidative damage. Fatty acid oxidation is a major cause of food deterioration and can affect the flavor, aroma, texture, shelf life, and color of foods, which limits the use of marine oil for food fortification (Kolanowski et al. 1999, 2001). The main way of reducing oxidative damage is to encapsulate the oxidizable lipid to reduce its contact with oxygen, trace metals, and other substances that attack its double

Author's personal copy Food Bioprocess Technol

bonds and other susceptible locations. For this purpose, oxidizable lipids have been combined with a number of other substances, including other oils, polysaccharides, and proteins. Starches have always been essential to human nutrition. Food enriched with starches acquires chemical modification allowing the increase of its process and storage stability. Furthermore, it is possible to add hydrophobic side chains to the original hydrophilic starch molecule, allowing starch to adsorb at oil/water (o/w) interfaces, thus stabilizing an emulsion. This type of emulsifying starch is an octenyl succinate (OSA) starch, which is made by the esterification of starch and anhydrous octenylsuccinic acid under alkaline conditions (Tescha et al. 2002). This modification has been approved by the Food and Drug Administration. OSA starches have also been approved for use as food additives in the European Union (E 1450). OSA starches have been used successfully for many years for encapsulation and beverage emulsions. An advantage of OSA starches is that they are almost colorless and tasteless in solution. When used in encapsulation processes, valuable ingredients are most favorably protected against oxidation. In the production of beverage emulsions, the high consistency of quality is remarkable, which is quite opposite to a complete natural ingredient, such as Gum Arabic. Oxidation in marine oil-enriched products can also be prevented by the addition of antioxidants. Vitamins, such as vitamins E and C, are known for their antioxidative effects (David 1997). Due to their healthy benefits and wide consumption, dairy products, such as milk, may be good vehicles for incorporation of marine oil (Gallaher et al. 2005; Nielsen et al. 2007). Therefore, the purpose of the study by Robertson (2002) was to make the stabile emulsion of marine oil and to evaluate the influence of marine oil fortification on the quality of ultrahigh temperature-treated (UHT) milk during storage. As UHT milk has a shelf life of several months, it is important to prevent both light transmission and oxygen permeation by choosing packaging material with barriers to light and oxygen. The purpose of this study was to establish the stable emulsion of marine oil and to evaluate the influence of marine oil fortification on the quality of UHT milk during storage. The first step was to evaluate the effect of emulsifier amount, droplet size, and oil concentration on the emulsion stability in a model o/w emulsion formulated with algae oil, octenyl-succinate starch, and high-fructose corn syrup. Thereafter, the second step was to investigate the stability of UHT milk enriched with algae oil for long-term storage. The oxidative stability was monitored during the storage period by means of lipid hydroperoxides, anisidine values and limited sensory analyses.

Materials and Methods Materials Algae oil (approximately 35 % ω-3 polyunsaturated fatty acid in the form of docosahexaenoic acid) was obtained from Martek DHA Algal Vegetable Oil (Martek Biociences Corporation, Columbia, MD, USA). OSA-modified waxy corn starches were obtained from National Starch and Chemicals (Shanghai, China). OSA starch, which is recommended as a good natural emulsifier, was of food and pharmaceutical grade. Corn syrup liquid [dextrose equivalent (DE) 42; molecular weight of 0.5 kDa; 71.20 wt% total solids (including 30.35 % fructose, 35.25 % glucose, and 5.58 amylose), 0.02 % sulfate ash, and 28.80 wt% water as determined by the manufacturer] was obtained from COFCO Shandong Peanut Imp. & Exp. Co., Ltd (Shandong, China). Liquid Emulsion Preparation The basic composition of the emulsions was algae oil, OSA starch, corn syrup (DE 42), and distilled water. To prevent an oxidation reaction before the test, 1.0 % vitamin E was added to the algae oil. The emulsion was made by 10 % algae oil, 10 % OSA starch, 10 % corn syrup, and 70 % water for the homogenization condition tests. The emulsion was made by 10 % algae oil, 5– 10 % OSA starch, 10–20 % corn syrup, and 60–80 % water for the composition of the emulsion tests. Continuous phase was formed by dissolving the required quantity of OSA starch and corn syrup in water at room temperature. An adequate amount of oil calculated on total emulsion mass was added in continuous phase to form an emulsion. To avoid microbiological contamination, 0.01 % sodium azide was added during the measurement of emulsion properties. Generally, after the dissolution of the water-soluble compounds, a coarse dispersion was prepared by an Ultra-Turrax T25 basic homogenizer equipped with an S 25 Ne18 G dispersing tool in an ice bath at 9,500 min−1. The total emulsifying time was 5 min. The emulsion was prepared by subsequent homogenization at various conditions in a high-pressure homogenizer (GEA Niro Soavi S.p.a., Parma, Italy). Prior to measurements, the prepared emulsions were stored at 4 °C. Impact of Homogenization Conditions To test the Homogenization Conditions, the Emulsion was Prepared According to the Experimental Plan (Table 1). Impact of the Composition of Emulsions To test the composition of the emulsions, the emulsion was prepared according to the experimental plan (Table 2). The

Author's personal copy Food Bioprocess Technol Table 1 Experimental design for the homogenization conditions test sample

Homogenization pressure(MPa)

Cycle times

15 25 35 45

1~5 1~5 1~5 1~5

Ea1~5 Eb1~5 Ec1~5 Ed1~5

The composition of the emulsions was 10 % algae oil, 10 % OSA starch, 10 % corn syrup (DE 42) and 70 % distilled water

prepared conditions were followed according to the previously described conditions.

creaming index (H; in percent) was calculated using the following formula (1): Creaming indexðHÞ ¼ 100 

HC HE

ð1Þ

The results were expressed as the means of triplicate samples. Production of UHT Milk and Preparation of Samples for Analysis

where ni is the number of droplets of diameter di. The d43 value was used to monitor changes of the droplet-size distribution on emulsion storage.

The emulsion consisted of 10 % algae oil, 10 % OSA starch, 20 % corn syrup, and 60 % water, and it was homogenized at 35 MPa for three cycles. The emulsion (1.5 % by weight) was added to raw milk. Milk (200 Lh−1) was processed in a pilot plant as follows: homogenization at 160–180 bar and 60–90 °C; indirect UHT processing for 4 s at 137 ± 2 °C; and cooling to 25–30 °C (Process Pilot Plant; APV, Far East Ltd.). After processing, the milk was aseptically placed in three-layer high-density polyethylene boxes containing a carbon layer. The boxes were stored at 4, 20, and 40 °C for analysis. Samples were taken at time zero and every week for measurement of chemical composition, and oxidative stability was monitored at different time points spread over 11 weeks of storage. The entire procedure was replicated three times.

Creaming Stability Measurement

Oxidation Experiments

To monitor long-term stability, the prepared emulsion (20 g) was transferred into a test tube (internal diameter of 15 mm and height of 150 mm), which was then tightly sealed with a plastic cap. The tubes were then stored up to 3 weeks at room temperature (approximately 20 °C). Every 24 h, the emulsions became separated into an opaque layer at the top (cream layer) and a marginally turbid or transparent layer at the bottom (serum layer). The total height of the emulsions in the tubes (HE) and the height of the cream layer (HC) were measured (Klinkesorn et al. 2004; Zarena et al. 2012). The

Duplicate milk samples (10 mL) were taken periodically to determine the hydroperoxide and aldehyde levels present in the oil. Oil was extracted from the milk by adding isooctane/isopropanol (3:2, v/v), vortexing three times for 10 s each, and centrifuging for 5 min at 1,000 rpm. The clear upper layer was collected, and the solvent was evaporated under nitrogen. After solvent removal, the oil samples were weighed to accurately determine oxidation progression in each sample. Peroxide values (PVs) were determined using the International Dairy Foundation method described in detail by Shantha and Decker (1994). Anisidine values (AnVs) were determined according to the AOCS Official Method Cd 18-90 (AOCS 1998). The total oxidation (TOTOX) value was calculated as follows: TOTOX value = 2(PV) + AnV (Shahidi and Wanasundara 2002).

Particle Size Analysis Emulsion droplet-size distributions were measured using a Malvern Mastersizer MS2000 (Malvern Instruments Limited, UK, Mastersize 2000 Software version 3.20) laser light-scattering analyzer. The average droplet size was characterized by the mean diameter, d43, as defined by the following formula: .X X ni di 3 ; d43 ¼ ni di 4

Table 2 Experimental design for the composition of the emulsions test Sample

C1 C2 C3 C4 C5

Algae oil (%)

OSA starch (%)

Corn syrup (%)

Distilled water (%)

10 10 10 10 10

5 5 10 10 10

5 10 10 15 20

80 75 70 65 60

The homogenization pressure was 35 MPa with three cycles

Sensory Assessment To investigate the possible influence of DHA on the sensory quality of the milk, a triangle test was performed between the DHA-supplemented milk and control milk samples after 1 month of storage at different temperatures. Eight assessors

Author's personal copy were selected for the panel. All of the assessors had earlier experience in sensory evaluation of dairy products, but they were not particularly trained for the present experiment. Evaluations were conducted in a sensory cabinet equipped with individually partitioned booths. Milk samples were served at room temperature in an AAB, ABB, or ABA arrangement with A and B codes used for DHA-fortified milk and control milk, respectively. Water was provided to rinse the mouth between different samples. The triangle test was performed in duplicate for both DHA-fortified milk and control milk. The processing of the results was performed based on the tables of Roessler et al. (1978). Statistical Analyses All samples were analyzed in triplicate. Statistical analyses were performed using XLSTAT 2009 (www.xlstat.com). Data were expressed as the mean ± SD using ANOVA. Differences at p < 0.05 were considered statistically significant by Duncan’s new multiple range test.

Results and Discussion Particle size analysis The particle size of each o/w emulsion prepared was evaluated by integrated light scattering (Figs. 1 and 2). According to the reports of Bock et al. (1994) and Betoret et al. (2009), the most important homogenization parameters for controlling particle size include homogenization pressure, temperature, and number of cycles. To prepare stable lipid emulsions, it was necessary to find the optimal operative conditions with regard to homogenization pressure and number of cycles when using a homogenizer. Regarding the piston-gap homogenizer, the homogenization was performed with one to five cycles at constant temperature (approximately 5 °C) with pressure ranging from 15 to 45 MPa. The efficiency of the emulsification process was evaluated by measuring particle diameter as shown in Table 3. 8

15MPa,three 25MPa,three 35MPa,three 45MPa,three

7 Volume(%)

6 5

cycles cycles cycles cycles

4 3 2 1 0 0.01

0.1

1

10 Diameter(µm)

100

1000

Fig. 1 Particle size distribution of emulsions made by different homogenization pressures. Data are the average of duplicate samples

Volume(%)

Food Bioprocess Technol 9 8 7 6 5 4 3 2 1 0 0.01

35MPa,one circle 35MPa,two circles 35MPa, three cicles

0.1

1

10 Diameter(µm)

100

1000

Fig. 2 Particle size distribution of emulsions made by different cycles. Data are the average of duplicate samples

Table 3 shows that the particle size decreased with increasing operation pressure and number of cycles. After three cycles, the particle size almost reached a plateau at 35 MPa, and additional cycles did not decrease the particle size. The droplet diameter decreased from 2.93 μm for a single cycle to a plateau of 1.62 μm after three cycles, and further processes had no notable effect. This observation was in agreement with findings reported by Trotta et al. (2002). For systems containing relatively high percentages of oil, increasing the operative pressure and longer emulsification times does not always lead to reduction of emulsion particle size. The higher pressure or longer time may lead to “over-processing” resulting in re-coalescence of emulsion droplets and an increase in particle size (Roessler et al. 1978). Therefore, to provide a stable and safe emulsion, variables concerning homogenization pressure and number of cycles should be optimized to 35 MPa and three cycles. Emulsion Stability The influences of individual factors on stability of the emulsions were obtained by observing the creaming stability of the emulsions for 15 days of storage. The creaming of emulsions was expressed through a creaming index. The term “creaming index” was used in this experiment to describe the stability of an emulsion over an observed time span. Lower creaming index indicates higher emulsion stability. Figure 3 shows the creaming profiles of homogenization pressure versus storage time (day). Discernible creaming took place within 24 h for the emulsions made at less than 25 MPa and one cycle. However, the separate layers were not clear. The upper part, which is known as the creaming layer, indicated the oil-rich phase, and the lower part, which is known as the serum layer, indicated the water-rich phase. These two boundaries were formed rapidly but remained constant during storage. With regard to oil droplet distribution, the creaming index of the emulsion made at 15 MPa, and one cycle was highest. In contrast, the other emulsions that were generated relatively the same had lower creaming indexes. Nevertheless, there was the presence of rapid creaming instability during storage in these systems, which suggested

Author's personal copy Food Bioprocess Technol Table 3 Effect of operative pressure and cycle numbers on particle size of emulsions (homogenization temperature was about 10 °C) Cycle No.

Particle size (μm, mean ± SD)

Cycle Cycle Cycle Cycle Cycle

15 MPa 5.40 ± 0.28 A 3.70 ± 0.21 A 3.57 ± 0.18 A 3.56 ± 0.16 A 3.49 ± 0.15 A

1 2 3 4 5

25 MPa 3.40 ± 020 B 3.06 ± 0.11 B 2.82 ± 0.17 B 2.80 ± 0.15 B 2.81 ± 0.13 B

35 MPa 2.93 ± 0.21 C 2.79 ± 0.17 B 1.62 ± 0.16 C 1.60 ± 0.17 C 1.58 ± 0.11 C

45 MPa 2.83 ± 0.21 C 2.76 ± 0.19 B 1.62 ± 0.16 C 1.57 ± 0.14 C 1.58 ± 0.13 C

Each value in the table is represented as mean ± SE (n = 3) Superscript letters with different letters in the same cycle, respectively, indicate significant difference (P < 0.05) analyzed by Duncan’s multiple range test

that lower homogenization pressure cannot create a kinetically stable emulsion. The combined effect of oil droplet size and formation has been shown to be correlated to the subsequent results of emulsion stability. The creaming indexes during 15 days of storage were plotted and are shown in Fig. 4. The emulsions made with 5 % OSA starch content started creaming within 24 h when stored at room temperature. The instability of this system was due to the formation of a large particle size, which, in turn, facilitated the coalescence process. In fact, emulsions made with 10 % OSA starch showed better emulsion stability. Conversely, the emulsion made with 10 % corn syrup started creaming after 5 days of storage at room temperature. The best result was found for the samples made with 20 % corn syrup because 20 % corn syrup delayed the creaming process and produced the finest oil droplet. Stability tests indicated that the emulsions with higher stability were the emulsions produced with a higher amount of wall materials, which was in agreement with the study by Ramakrishnan et al. (2013). These results demonstrated that homogenization at 35 MPa for three cycles is the appropriate process for making homogenized algae o/w emulsions. The present results also indicated that the emulsion composition affects its stability. Moreover, pressure directly affected the breaking up process of droplet disruption and lowered the covering of the surface active areas of emulsifiers. Corn syrup was added to the system as a stabilizer, and it increased the emulsion stability. Consequently,

the emulsion model of 10 % algae oil with OSA starch and corn syrup was successful. All emulsions had small droplets and were completely stable with the addition of the OSA starch stabilizer. For the tested 10 % OSA starch emulsions, the addition of a corn syrup was necessary to obtain the same stability. However, the tested OSA starch content of 15 % is rather high and may increase the costs of commercial DHA milk. Primary Oxidation of the Milk Fatty acid composition of DHA-enriched milk is shown in Table 4. Hydroperoxides were measured to determine the initial rate of oxidation because they are generally accepted as the first product formed by oxidation (Rossell 1986). It is well known that lipid oxidation is markedly accelerated by high temperature. The effect of temperature on lipid oxidation rates was confirmed in the present study. The peroxide values of milk enriched with the DHA emulsion increased during storage (Fig. 5).

Creaming index(%)

100 80 60 40 Ea1 Ec1

20 0

0

2

4

6

Eb1 Ed1

8 10 12 14 16 t(day)

Fig. 3 Time dependence of creaming index of the emulsion

Fig. 4 Time dependence of creaming index of emulsions 35 MPa homogenization for three cycles at different compositions and stored at room temperature

Author's personal copy Food Bioprocess Technol Table 4 Fatty acid composition (wt %) of DHA-enriched milk Fatty acids

Content

C8:0 C10:0 C12:0 C14:0 12mC14:0 13mC14:0 C15:0 C16:0

1.03 ± 3.16 ± 4.18 ± 10.34 ± 0.42 ± 0.23 ± 0.79 ± 33.83 ±

0.02 0.01 0.01 0.06 0.02 0.02 0.05 0.37

14mC16:0 15mC16:0 C17:0 C18:0 C20:0 C21:0 C22:0 C24:0 C14:1c9 C16:1c9 C17:1c9 C18:1c9 C18:1c11 C20:1c11 C16:1t9 C18:1t10 + t11 C18:2c9t11(CLA) C18:2t10c12(CLA) C18:2tt(CLA)

0.41 ± 0.23 ± 0.39 ± 8.28 ± 0.13 ± 0.02 ± 0.04 ± 0.03 ± 0.82 ± 2.11 ± 0.33 ± 18.22 ± 0.36 ± 0.01 ± 0.04 ± 1.62 ± 0.58 ± 0.08 ± 0.01 ±

0.02 0.03 0.03 0.13 0.00 0.01 0.01 0.01 0.03 0.03 0.01 0.12 0.01 0.00 0.00 0.03 0.02 0.01 0.00

1.26 ± 0.02 ± 0.02 ± 0.1 ± 0.08 ± 0.37 ± 0.06 ± 0.09 ± 1.42 ±

0.03 0.00 0.00 0.01 0.01 0.02 0.01 0.01 0.02

C18:2c9c12 C18:3c6c9c12 C20:2c11c14 C18:3c6c9c12 C20:4c5,c8,c11,c14 C18:3c9c12c15 C20:5c5c8c11c14c17 C22:5c7c10c13c16c19 C22:6c3c7c10c13c16c19

Fig. 5 The effect of storage temperature on peroxides of DHAenriched milk over time

Secondary Oxidation The peroxides in oxidized oil are transitory intermediates that decompose into various types of carbonyl and other compounds (Rossell 1986). Measuring secondary oxidation products is essential when studying lipid oxidation in food products for human consumption because they are generally odor-active whereas primary oxidation products are colorless and flavorless. The AnV test was utilized to determine the level of aldehydes, principally 2-alkenals and 2,4-alkadienals, present in the emulsified oil (Shahidi and Wanasundara 2002). Significant correlations between AnV and flavor acceptability scores of soybean oil have been previously reported (List et al. 1974). Unlike hydroperoxides, aldehydes do not decompose rapidly, thus allowing the past history of an oil to be determined with the AnV (Shahidi and Wanasundara 2002).

Each value in the table is represented as mean ± SE (n = 3)

Conversely, after 4 weeks, there was no significant increase in peroxide levels. One possible cause of this stability may be that the peroxides decomposed. Therefore, we measured secondary oxidation to test if this occurred. It is recommended that PV levels should not exceed 30 meq peroxide/kg oil in an edible food product. The fish oil microcapsules were stable in yogurt with PV levels below 5 meq peroxide/kg oil even after 5 weeks (Naohiro and Shun 2006).

Fig. 6 The effect of storage temperature on the anisidine value over time

Author's personal copy Food Bioprocess Technol

Conclusion

Fig. 7 The effect of storage temperature on the TOTOX value over time

The AnV for OSA starch emulsions did not change significantly between 0 and 15 days at various temperatures (Fig. 6). The ability of OSA starch to inactivate peroxyl radicals in the emulsified oil, thus, preventing the development of secondary oxidation products may be responsible for the negligible changes in aldehydes measured during this study. Total Oxidation The TOTOX value combines evidence about the past history and present state of an oil and is used frequently in the food industry (Shahidi and Wanasundara 2002). Figure 7 illustrates the effect of storage temperature and time on the total oxidation of the DHA-enriched milk. Within 4 weeks, the TOTOX value reached the maximum. Cold storage may be necessary to slow oxidative deterioration in the milk fortified with DHA. After 4 weeks, time did not significantly affect the TOTOX values throughout the study. Sensory Analysis Sensory data were determined by a trained panel (eight members) that evaluated and scored fishy aroma in the algae oil-supplemented milk and control milk samples. When combing the results of the two conditions, only two (out of eight) panelists could discriminate between both samples. Based on the tables of Roessler et al. (1978), it can be concluded that the sensory panel was not able to discriminate between the algae oil-supplemented milk and control milk samples when using α = 0.05. The sensory testing analysis indicated that the algae oil-supplemented milk samples were liked as well as the control milk samples. These results showed that algae oils have potential use to fortify the quality of UHT milk.

Algae oil microcapsules stabilized with OSA starch and corn syrup were successfully prepared by a pre-emulsifying process followed by a high-pressure homogenization treatment. Stable solid particles were created in an aqueous solution after the homogenization process without the use of organic solvents or cross-linking reagents. The optimal conditions for microcapsule formation were 10 % OSA starch, a 1.0 oil/starch ratio, and added 20 % corn syrup liquid. For DHA-fortified milk, the present results demonstrated that algae oil-enriched drinking milk is a stable product during 11 weeks of storage. Even high storage temperature (40 °C) did not significantly promote oxidation. However, the present results indicated that the milk stored in high temperature (20 and 40 °C) have potential for long-term oxidation. Taken together, the data suggested that liquid milk may be a good vehicle for incorporation of DHA and that the product is best stored at lower temperatures for long-term storage. Acknowledgments The authors are grateful to anonymous reviewers and the editor for comments on the earlier version of this paper.

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