- Email: [email protected]
Encapsulation of menhaden oil structured lipid oleogels in alginate microparticles
LWT - Food Science and Technology 116 (2019) 108566
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Encapsulation of menhaden oil structured lipid oleogels in alginate microparticles
T
Sarah A. Willett, Casimir C. Akoh∗ Department of Food Science and Technology, University of Georgia, Athens, GA, 30602-2610, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Menhaden oil Oleogel Structured lipid Microencapsulation Oxidative stability
Leaching of the internal phase during storage is a concern of microencapsulated products. This research proposes encapsulation of oleogels to reduce leaching and improve oxidative stability of the lipids. Oleogels were produced using a phytosterol blend of β-sitosterol/γ-oryzanol or a blend of sucrose stearate/ascorbyl palmitate (SSAP) as oleogelators, and menhaden oil or structured lipid (SL) prepared from menhaden oil and caprylic and/ or stearic acid as the lipid phase. The SL were produced enzymatically using the biocatalyst Lipozyme® 435, a recombinant lipase from Candida antarctica. Menhaden oil, SL, or respective phytosterol or SSAP oleogels, were encapsulated in alginate microparticles using a double emulsion method. Encapsulation efficiency (EE), morphology, oxidative stability, percent leaching, and other physicochemical properties were determined. Encapsulation of phytosterol or SSAP oleogels increased the EE for all lipids, from 89.6 to 91.3% for microcapsules containing only the lipid phases and from 95.7 to 99.2% for microcapsules containing the oleogels. Encapsulation of oleogels significantly reduced leaching, from 16.3-18.5% to 3.3–11.9%. Microencapsulated products had higher Oil Stability Index values (19.75–29.18 h) than the non-microencapsulated lipids (4.37–17.55 h), when measured at 80 °C. These microcapsules may find use as stable nutraceuticals or in fortification of food products with stabilized omega-3 fatty acids.
1. Introduction A structured lipid (SL) is any lipid that has undergone chemical or enzymatic modification from its natural biosynthetic form (Kim & Akoh, 2015). SL have a wide range of applications, though one main concern is their oxidative stability. Although enzymatic production of SL typically occurs under milder conditions than chemical modification, previous studies have found that purified SL have lower oxidative stability than the unmodified fat or oil (Decker, Warner, Richards, & Shahidi, 2005; Ifeduba, Martini, & Akoh, 2016; Pacheco, Crapiste, & Carrin, 2015; Zou & Akoh, 2013). Short-path distillation, a purification process necessary to remove free fatty acids and/or fatty acid ethyl esters from the SL, has been shown to reduce the tocopherols or antioxidants naturally present in the fat or oil (Zou & Akoh, 2013). Polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA) found in menhaden fish oil, are highly susceptible to oxidation due to the high amount of unsaturation. Further modification of menhaden oil to produce SL in previous studies was found to further reduce the oxidative stability (Jennings & Akoh, 2001; Willett & Akoh, 2018a). One way of improving oxidative stability of SL is by forming oleogels. Oleogels (also known as organogels), a lipid gel, have been shown to inhibit oil migration in chocolate, control the release of health beneficial sensitive compounds such as antioxidants, bioactive compounds, and PUFA, and increase oxidative stability of the oil (Co & Marangoni, 2012). However, oleogelation may not reduce the fishy off-flavors present in menhaden fish oil, limiting its use in food products. Another method that has been shown to improve the oxidative stability is by encapsulation of oil within polymeric microparticles. Microencapsulation is a method of coating tiny droplets (such as high PUFA-containing oils) to form small capsules and has
Abbreviations: DSC, differential scanning calorimeter; EE, encapsulation efficiency; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; GOED, Global Organization for EPA and DHA omega-3; M, menhaden oil; OSI, Oil Stability Index; p-AV, p-Anisidine value; PV, peroxide value; P-M, menhaden oil phytosterol oleogel; P-SL-C, SL-C phytosterol oleogel; P-SL-CS, SL-CS phytosterol oleogel; P-SL-S, SL-S phytosterol oleogel; PUFA, polyunsaturated fatty acids; SL, structured lipid; SL-S, SL-S SSAP oleogel; SL-C, structured lipid of menhaden oil and caprylic acid; SL-CS, structured lipid of menhaden oil and a blend of caprylic and stearic acids; SLS, structured lipid of menhaden oil and stearic acid; SSAP, sucrose stearate/ascorbyl palmitate; SSAP-M, menhaden oil SSAP oleogel; SSAP-SL-C, SL-C SSAP oleogel; SSAP-SL-CS, SL-CS SSAP oleogel; SSAP-SL-S, SL-S SSAP oleogel ∗ Corresponding author. E-mail address: [email protected] (C.C. Akoh). https://doi.org/10.1016/j.lwt.2019.108566 Received 28 March 2019; Received in revised form 9 August 2019; Accepted 27 August 2019 Available online 27 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
been found to mask fishy off-flavors (Bakry et al., 2016). Encapsulation of oil reduces its exposure to light, moisture, oxygen, and heat, all of which decrease the quality of the oil through oxidative deterioration, formation of undesirable flavor compounds, and production of free radicals (Sagiri et al., 2014). One disadvantage of microencapsulation is leaching of the internal oil phase during storage, which undoes the encapsulation and therefore reduces its efficiency (Sagiri et al., 2014). To reduce leaching, there are many approaches such as using blended polymers to encapsulate the oil or using a more complex method of producing the microcapsules such as coacervation (Ifeduba & Akoh, 2015; Sagiri et al., 2012). There is need for a simple method to reduce oil leaching. Previous literature has reported success at reducing leaching by encapsulating a sorbitan esterbased sunflower oil oleogel in alginate microparticles for drug delivery (Sagiri et al., 2014). However, the studies on encapsulation of oleogels did not report oxidative stability (Sagiri et al., 2012, 2014). It is of interest to determine the physicochemical properties, encapsulation efficiency, amount of leaching during storage, and oxidative stability of the alginate microparticles when different oleogelators and oil phases are used. The purpose of this research was to microencapsulate menhaden oil or SL oleogels in alginate microparticles to potentially reduce the amount of leaching during storage and improve the oxidative stability of the lipids. Two different oleogelator blends were compared in forming the microcapsules: a phytosterol blend of β-sitosterol and γoryzanol, and a blend of sucrose stearate/ascorbyl palmitate (SSAP). Oxidative stability, amount of leaching during storage, and other physicochemical properties of the microcapsules were studied. Physicochemical properties of the microcapsules will depend on the lipid phase encapsulated. These microencapsulated menhaden oil oleogels may have the potential for use in a wide range of food and nutraceutical applications.
USA). All other reagents and solvents were of analytical or HPLC grade and were purchased from Fisher Chemical (Fair Lawn, NJ, USA), SigmaAldrich Chemical Co (St. Louis, MO, USA), and J. T. Baker Chemical Co. (Phillipsburg, NJ, USA). 2.2. Microencapsulation of lipids and oleogels Menhaden oil (M), SL-C, SL-S, SL-CS, or respective phytosterol or SSAP oleogels, were microencapsulated in alginate microparticles. Table 2 shows the internal phase composition of each of the microcapsules studied. The formation and physicochemical characterization of the phytosterol and SSAP oleogels have been discussed in detail elsewhere (Willett & Akoh, 2018b). Phytosterol oleogels were formed for each lipid, and labeled P-M, P-SL-C, P-SL-CS, and P-SL-S. Briefly, a 1:1 M ratio blend of β-sitosterol and γ-oryzanol was dissolved at 8% (w/ w) in 10 g of lipid (menhaden oil, SL-C, SL-CS, or SL-S). The mixture was stirred for 10 min at 90 °C and cooled at 4 °C to set the gel. SSAP oleogels were formed for each lipid, and labeled SSAP-M, SSAP-SL-C, SSAP-SL-CS, and SSAP-SL-S. A 1:1 M ratio blend of sucrose stearate (HLB value of 2) and ascorbyl palmitate was dissolved at 12% (w/w) in 10 g of lipid (menhaden oil, SL-C, SL-CS, or SL-S). The mixture was stirred for 10 min at 110 °C and cooled at 4 °C to set the gel. The microcapsules were prepared by a modified double emulsion method, also known as an internal gelation method (Sagiri et al., 2014). First, an oil-in-water emulsion was prepared by mixing 0.5 g of sodium alginate and 20 g of water at 25 °C and 250 rpm. Then 0.4 g calcium carbonate was added, stirred at 250 rpm, and homogenized. After homogenization, 0.5 g of Span 80 and 5 g of internal phase (either menhaden oil, SL, or oleogel) was added and homogenized for 15 min. The resulting emulsion was used to form a double emulsion. The emulsion was homogenized further in an ice bath for 10 min to form a thick emulsion. This emulsion was added to 60 mL of ice-cold menhaden oil (external phase) and homogenized for 5 min at 250 rpm. 5 mL of acidified oil (4.5 mL of menhaden oil mixed with 0.5 mL glacial acetic acid) was added to the external phase while stirring to induce ionic crosslinking and gelation of the alginate layer to form the microcapsules. The formed microcapsules were washed with 0.5 M calcium chloride containing 1% tween 80, and then washed with water. Microcapsules were stored at 4 °C until further analysis.
2. Materials and methods 2.1. Materials Menhaden oil was obtained from Omega Protein Inc. (Reedville, VA, USA). Acyl donors, caprylic acid (≥98% purity) and stearic acid (≥98% purity), were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). These were used as substrates in acidolysis reactions to produce the different SL according to the method outlined previously (Willett, Martini, & Akoh, 2019). All acidolysis reactions were conducted in a 1L batch reactor for 24 h, with biocatalyst with Lipozyme® 435 lipase (Novozymes North America, Inc., Franklinton, NC, USA) at 10% (w/w) of total substrates and stirring at 250 rpm using a SL 2400 StedFast stirrer (Fisher Scientific Co., Fair Lawn, NJ, USA). For the SL containing 30 mol% of caprylic acid (SL-C), menhaden oil and caprylic acid at a 1:4.58 substrate molar ratio were incubated at 65 °C. For the SL containing 20 mol% of stearic acid (SL-S), menhaden oil and stearic acid at a 1:1.37 substrate molar ratio were incubated at 75 °C. For the SL containing about 14 mol% each of caprylic and stearic acid (SL-CS), menhaden oil, caprylic acid, and stearic acid, at a 1:3:1 substrate molar ratio were incubated at 75 °C. Table 1 shows the relative fatty acid composition of the menhaden oil and all SL (Willett et al., 2019). All SL had melting points between 25 and 35 °C. The detailed fatty acid and triacylglycerol (TAG) composition, oxidative stability, thermal behavior, and other physicochemical properties of the SL have been discussed in further detail elsewhere (Willett et al., 2019). Ryoto Sugar Ester (sucrose stearate) S-270 was obtained from Mitsubishi-Kagaku Foods Corporation (Tokyo, Japan). Ascorbyl palmitate, Tween 80, sodium alginate, Span 80, and β-sitosterol were obtained from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). γ-Oryzanol was obtained from TCI America (Portland, OR, USA). Calcium carbonate was obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA). Calcium chloride was obtained from VWR International, LLC (West Chester, PA,
2.3. Leaching of the internal phase Leaching of the internal phase was studied to determine the stability of the microcapsules. The higher the amount of leaching, the lower the stability of the microcapsule, as leaching of the internal phase into the coating and to the outside undoes the encapsulation. Microcapsules were wiped with filter paper to remove any traces of oil or moisture on the surface of microcapsules. 0.5 g of microcapsule was weighed and placed on clean filter paper. The microcapsules were then placed in the oven at 37 °C, and leakage was visually monitored for 2 h. To quantify the amount of leaching, another method was used (Sagiri et al., 2014; Bordenave, Janaswamy, & Yao, 2014). Briefly, 0.1 g of microcapsules was soaked in 1 mL of deionized (DI) water for 1 h at 37 °C. The mixture was centrifuged at 2000 rpm for 5 min. The supernatant was collected and dried at 55 °C for 48 h. The dried supernatant was weighed and leaching (%) was calculated as:
% leaching =
dried supernatant wt (g ) x 100 microcapsule initial wt (g )
Samples were analyzed in triplicate and average values were determined. 2.4. Encapsulation efficiency Encapsulation efficiency (EE) was determined as previously 2
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
Table 1 Relative fatty acid composition of menhaden oil and SL (Willett et al., 2019). Samplesa
TAG Position
Menhaden Oil
Total sn-2 sn-1,3c Total sn-2 sn-1,3 Total sn-2 sn-1,3 Total sn-2 sn-1,3
SL-C
SL-CS
SL-S
Fatty Acid (mol%)b C8:0
C16:0
C18:0
C20:5n3
ND ND ND 29.03 ± 1.37 11.72 ± 0.13 37.69 ± 0.75 16.24 ± 0.56 7.31 ± 1.01 20.71 ± 0.34 ND ND ND
10.20 ± 0.85 14.86 ± 0.74 7.87 ± 0.91 5.55 ± 0.06 16.94 ± 0.07 0.15 ± 0.05 4.73 ± 0.21 15.11 ± 0.06 0.46 ± 0.29 7.50 ± 0.05 4.29 ± 0.11 9.11 ± 0.02
3.08 ± 0.05 3.21 ± 0.37 3.02 ± 0.21 2.74 ± 0.10 2.97 ± 0.09 2.63 ± 0.10 13.04 ± 0.10 8.82 ± 0.55 15.14 ± 0.13 19.50 ± 0.23 10.07 ± 0.78 24.22 ± 0.51
16.90 16.42 17.14 13.61 12.22 14.31 13.87 10.69 15.46 14.16 11.85 16.21
± ± ± ± ± ± ± ± ± ± ± ±
C22:6n3 0.10 0.64 0.45 0.38 0.03 0.21 0.26 0.32 0.23 0.11 0.26 0.19
15.40 ± 0.16 29.37 ± 0.95 8.42 ± 0.37 10.42 ± 0.03 25.17 ± 0.46 3.05 ± 0.25 12.50 ± 0.20 16.66 ± 1.39 10.42 ± 0.40 14.62 ± 0.39 22.98 ± 0.18 10.44 ± 0.29
Mean ± STD, n = 3, ND = not detected. a SL-C: structured lipid (SL) of menhaden oil and 30 mol% caprylic acid, SL-S: structured lipid (SL) of menhaden oil and 20 mol% stearic acid, SL-CS: structured lipid (SL) of menhaden oil and 16 mol% caprylic acid and 13 mol% stearic acid. b Other fatty acids found at > 1 mol%: C14:0, C16:1n7, C17:1n7, C18:1n9, C18:2n6, C18:3n3, C20:0, C18:4n3, C22:5n3, and < 1 mol%: C13:0, C14:1n7, C15:0, C17:0, C19:0, C18:3n6, C20:1n7, C20:2n6, C22:0, C20:3n6, C20:3n3, C23:0, C22:2n6, C24:1n9, C22:4n6. c sn-1,3 mol% determined by equation: sn-1,3 (mol%) = [3xtotal mol%- sn-2 mol %]/2.
compressive analysis test via backward extrusion studies using a TAXTi2 texture analyzer (Stable Microsystems, Surrey, UK). Analysis was performed by moving the probe at a speed of 1 mm s−1 to a 10-mm distance within the emulsion and returned to the original position at the same speed. The experiment was performed in auto-force mode with a trigger force of 3 g. Samples were analyzed in triplicate and average values were determined.
Table 2 Internal phase composition of the microcapsules. Sample
Internal Phase
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS
Menhaden oil Phytosterol organogel containing menhaden oil SSAP organogel containing menhaden oil SL of menhaden oil containing 30 mol% caprylic acid Phytosterol organogel containing SL-C SSAP organogel containing SL-C SL of menhaden oil containing ~14 mol% each caprylic & stearic acid Phytosterol organogel containing SL-CS SSAP organogel containing SL-CS SL of menhaden oil containing 20 mol% stearic acid Phytosterol organogel containing SL-S SSAP organogel containing SL-S
P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
2.6. Rheological properties Rheological analyses were performed using an HR-3 Discovery Hybrid Rheometer (TA Instruments, New Castle, DE, USA). A rotational cone and plate (gap of 0.15 mm, cone angle 5.4°, plate diameter of 30 mm) was used for measurements. Analysis of the samples was conducted by varying the shear rate from 15 to 95 s−1 at 25 °C. Apparent viscosity of the primary emulsions were evaluated at 25 °C, at shear rates of 10, 50, and 100s−1 to mimic changes during common food processing conditions. Temperature was controlled with a Peltier Plate Temperature System (TA Instruments, New Castle, DE, USA). All experiments were conducted in triplicate. Data was analyzed using Trios software (TA Instruments, New Castle, DE, USA).
described (Ifeduba & Akoh, 2015). Briefly, both the solvent extractable and total oil of the microcapsules were determined and EE (%) was calculated as follows:
EE (%) = 100 − [(%Solvent extractable/ % Total oil) × 100] To determine solvent extractable oil, 0.25 g of microcapsules was added to 2.5 mL of hexane and vortexed for 15 min. The mixture was centrifuged for 5 min at 1000 rpm. The organic layer was collected, filtered through an anhydrous sodium sulfate, and then transferred to a pre-weighed round bottom flask. Solvent was removed at 60 °C using a rotary evaporator. Solvent extractable oil was calculated as w/w % of suspension. Samples were analyzed in triplicate and average values were determined. To determine total oil, 5 mL of 5 M HCl was added to 0.5 g of microcapsules and the mixture was agitated at 60 °C for 3 h and then cooled to room temperature. The mixture was transferred to a separatory funnel and extracted twice with 5 mL hexane. The extracted organic layer was filtered through an anhydrous sodium sulfate column and transferred to a pre-weighed round bottom flask. Solvent was removed at 60 °C using a rotary evaporator. Total oil was calculated at w/ w % of suspension. Samples were analyzed in triplicate and average values were reported.
2.7. Microscopy The microstructure of the microcapsules was observed under an upright bright-field microscope (Leica DMRXA2 Microscope, Leica Micro-systems Canada Inc., Richmond Hill, Canada). Slides were prepared by placing the microcapsules on a slide with a small amount of DI water. Samples of each microcapsule type, as shown in Table 2, were prepared in triplicate, and slides for each replicate were prepared in triplicate for a total of 9 slides analyzed for each microcapsule sample. Images were acquired using a charged coupled device (CCD) camera (QImaging Retiga, Burnaby, BC, Canada). 2.8. Thermal behavior Thermal behavior of microcapsules was analyzed using a 204 F-1 Phoenix differential scanning calorimeter (DSC) (Netzsch-Gerätebau GmbH, Selb, Germany). Microcapsules were wiped with filter paper to remove any traces of oil or moisture on the surface of microcapsules. Then microcapsule samples were heated from 22 to 120 °C at a rate of 20 °C/min. DSC analysis of peaks was determined using Proteus thermal analysis software (Netzsch-Gerätebau GmbH, Selb, Germany). Samples
2.5. Texture analysis Cohesiveness of the primary emulsions was determined by a 3
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
were analyzed in triplicate and average values reported.
Table 3 Percent leaching, encapsulation efficiency, and primary o/w emulsion cohesiveness of menhaden oil, SL, or organogel microcapsules.
2.9. Oxidative stability Oxidative stability of the microcapsules was determined by measuring peroxide (PV) and p-anisidine (p-AV) values over 1 month of storage at 4 °C. 5 g of sample was weighed into glass vials, closed, and placed in the refrigerator, held at 4 °C for 30 days. PV was determined using a method from the International Dairy foundation (Shantha & Decker, 1994). The p-AV was determined using AOCS Official Method Cd 18–90 (American Oil Chemists' Society, 2017). The lipid portion of the microcapsules was extracted with diethyl ether under the conditions specified by ICC Standard No. 136, with some modification (International Association for Cereal Science and Technology, 1984). 5 g of microcapsules was mixed with 4 mL of 95% ethanol and 1 mL of 7 M NaOH. Then, 5 mL of diethyl ether was added and shaken. After phase separation, 5 mL of hexane was added, mixed vigorously, and phase separation was allowed. The organic layer was collected, washed with 5 mL of 1 M HCl, followed with 5 mL of 0.5 M Na2SO4. The organic layer was transferred to a pre-weighed round bottom flask and solvent was evaporated under nitrogen. PV and p-AV were then measured. Oil Stability Index (OSI) of the microcapsules was also determined at 80 °C using an Oxidative Stability Instrument (Omnion Inc., Rockland, MA, USA) and AOCS Official Method Cd 12b-92 (American Oil Chemists’ Society, 2017). Samples were analyzed in triplicate and average values were reported.
Samplea
Leaching (%)
Encapsulation Efficiency (%)
Cohesiveness (kg s−1)
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
18.54 ± 1.13a 9.02 ± 0.60b 11.93 ± 0.78b 20.30 ± 1.08a 8.42 ± 1.71b 9.88 ± 0.11b 18.22 ± 1.03a 9.14 ± 0.74b 4.38 ± 0.33c 16.31 ± 0.85a 7.27 ± 0.56b 3.28 ± 0.34c
89.79 ± 0.77a 96.87 ± 0.61b 95.69 ± 0.72b 89.57 ± 0.32a 98.09 ± 0.77c 96.13 ± 0.67b 90.64 ± 0.95a 96.24 ± 0.76b 98.92 ± 0.45c 91.26 ± 0.89a 97.08 ± 0.96c 99.17 ± 0.59c
0.01 0.32 0.21 0.07 0.18 0.18 0.10 0.15 0.21 0.19 0.26 0.31
± ± ± ± ± ± ± ± ± ± ± ±
0.04a 0.06b 0.05b 0.01a 0.07b 0.02b 0.01a 0.02a,b 0.02b 0.04b 0.03b 0.07b
Mean ± STD, n = 3. Different letters in the same column are significantly different (α = 0.05). a Abbreviations are the same as described in Tables 1 and 2.
phase, but it was significantly less compared to the menhaden oil microcapsules. This is likely due to higher apparent viscosity and stabilization of the emulsion by the oleogels, which will be discussed in the rheological properties section (3.4). Table 3 shows the quantitative percent leaching for all microcapsule samples. The microcapsules that contained either the phytosterol oleogel or the SSAP oleogel all had significantly (p < 0.05) lower percentages of leaching compared to the microcapsules containing a non-gellated internal phase. Leaching of the internal phase was reduced from 16.3-18.5% to 3.3–11.9%. A previous study that compared encapsulating sunflower oil and sorbitan monostearate oleogel found 46.1 and 9.4% leaching, respectively (Sagiri et al., 2014). Amount of leaching can be attributed to internal phase composition (Sagiri et al., 2012). The SSAP and phytosterol oleogels showed a lower amount of leaching compared to the sorbitan monostearate oleogel and may be better oleogelators than sorbitan monostearate in terms of forming a stronger gel network that aids in preventing leaching.
2.10. Statistical analysis Statistical analysis was performed on the data using JMP® software (Version 10, SAS Institute Inc., Cary, NC, USA). Data was analyzed using ANOVA and significant differences (p < 0.05) were determined using the Tukey test. 3. Results and discussion 3.1. Leaching of the internal phase
3.2. Encapsulation efficiency
Fig. 1 shows a visual representation of internal phase leaching after 2 h in a 37 °C oven for M, P-M, and SSAP-M. The menhaden oil (M) microcapsules showed visual leakage compared to the P-M and SSAP-M. Both P-M and SSAP-M showed some visual leakage of the internal
Table 3 shows the EE for all microcapsule samples. A higher EE is preferred as that meant more lipid was encapsulated. The EE for all
Fig. 1. Visualization of microparticles before and after 2 h in 37 °C oven. Menhaden oil microparticles before (a) and after (d) 2 h, P-M microparticles before (b) and after (e) 2 h, and SSAP-M microparticles before (c) and after (f) 2 h. 4
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
samples ranged from 89.8 to 99.2%. The microcapsules that contained either the phytosterol oleogel or the SSAP oleogel had significantly (p < 0.05) higher EE compared to the M, SL-C, SL-CS, and SL-S microcapsules. The strong gel network of the phytosterol and SSAP oleogels were able to stabilize the o/w emulsions, allowing for a higher proportion of lipid to be encapsulated (Willett & Akoh, 2018b). These results show that oleogelation may be a suitable method for improving the stability of the microcapsules by reducing amount of leaching and improving EE.
viscosity. The increase in apparent viscosity for the phytosterol and SSAP oleogel samples was likely due to an increase in cohesiveness among the different components. These results were comparable to similar studies (Jeyakumari, Zynudheen, Parvathy, & Binsi, 2018; Sagiri et al., 2014). The results suggest that having an oleogel as the internal phase enhanced the viscosity and cohesiveness, which may have accounted for the reduction in leaching and increase in EE.
3.3. Texture analysis
Fig. 3 shows the microcapsules under the light microscope. The microcapsules all exhibited a spherical shape, though varied in size. Apart from P-M and SSAP-SL-C, the oleogel microcapsules had a larger particle size than M, SL-C, SL-CS, and SL-S (non-oleogel microcapsules). EE can be influenced by partition coefficient of target molecule in the solvents used in formulation, method used, and particle size (Piacentini, Poerio, Bazzarelli, & Giorno, 2016). The non-oleogel microcapsules (M, SL-C, SL-CS, and SL-S) all had mostly smaller particle sizes, and lower EE (section 3.2). The oleogel microcapsules also seemed to aggregate together more often than the non-oleogel microcapsules. This may be partly due to the oleogel primary emulsions having higher cohesiveness, as shown in Fig. 2 and Table 3. A previous study found that higher aggregation and particle size was seen with emulsions that exhibited higher cohesiveness and apparent viscosity (Jeyakumari et al., 2018; Sagiri et al., 2012).
3.5. Microscopy
Table 3 shows the cohesiveness of the primary emulsions for all samples. Generally, all phytosterol and SSAP oleogel samples had significantly (p < 0.05) higher cohesiveness values. The menhaden oil emulsion had a lower cohesiveness. SL-C and SL-CS primary emulsions had statistically similar (p > 0.05) cohesiveness compared to the menhaden oil emulsion. SL-S had a significantly higher cohesiveness than the menhaden oil, SL-C, and SL-CS. This was likely due to the higher stearic acid content and melting point, which aided in a stronger fat crystal network (Rogers, 2009). Addition of the oleogel enhanced the cohesiveness of the emulsion. Higher cohesiveness typically corresponds to a higher apparent viscosity and will be discussed in further detail in the rheological properties section (3.4). 3.4. Rheological properties
3.6. Thermal analysis Fig. 2 shows the apparent viscosity of the primary emulsions for all samples. Almost all the phytosterol and SSAP oleogel samples had a higher apparent viscosity. These results correspond well to the texture analysis. The menhaden oil microcapsules had a significantly lower apparent viscosity, lower cohesiveness, and lower EE (sections 3.2 and 3.3). The phytosterol and SSAP oleogel samples had higher cohesiveness and higher apparent viscosity, which led to the higher EE. The higher EE and reduced amount of leaching may be attributed to the gelation of the menhaden oil and SL. Increase in apparent viscosity has been shown to reduce leaching (Sagiri et al., 2012, 2014; Sharma, Madan, & Senshang, 2016). Previous studies have shown that fatty acyl oleogels, similar to the SSAP oleogels, have a tendency to accommodate water within the gel network, and may have resulted in the increase in viscosity of the emulsion (Behera, Sagiri, Pal, & Srivastava, 2013). There was no gel network in the menhaden oil sample, likely accounting for it having a lower apparent viscosity. Both the cohesiveness and apparent viscosity of the primary emulsions followed a similar trend, where a higher cohesiveness corresponds to a higher apparent
For all samples there was an endothermic peak between 35 and 110 °C (data not shown). Table 4 shows the values for peak temperature onset, completion, and enthalpy of all samples. The non-oleogel microcapsules (M, SL-C, and SL-CS) had a peak onset temperature at around 75 °C and peak completion temperature at around 92 °C. P-SLCS also had a statistically similar (p > 0.05) peak onset temperature (77.68 °C) but higher completion temperature (98.88 °C). In other samples, the oleogel microcapsules, had peak onset temperatures between 36.48 and 56.20 °C, and peak completion temperatures between 92.37 and 107.52 °C. In a previous study, we reported the gel to sol transition temperature of the phytosterol and SSAP oleogels to be between 40.1 and 53.1 °C. The broad peaks for the oleogel microcapsules may be explained by the simultaneous transition of the oleogel from gel to sol, and evaporation of water present in the oleogel. Previous studies have also shown this, where although the microcapsule samples were dried, it was difficult to remove bound water (associated with the alginate) present (Sagiri et al., 2014). The shift in the endothermic peak
Fig. 2. Apparent viscosity of primary emulsions. Abbreviations are the same as described in Tables 1 and 2. 5
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
Fig. 3. Light microscopy of microparticles produced with different lipid portion, where a) menhaden oil, b) P-M, c) SSAP-M, d) SL-C, e) P-SL-C, f) SSAP-SL-C, g) SLCS, h) P-SL-CS, i) SSAP-SL-CS, j) SL-S, k) P-SL-S, and l) SSAP-SL-S. Abbreviations for the different lipid portions are the same as described in Tables 1 and 2 Scale bars = 25 μm. Table 4 Thermal behavior of the menhaden oil, SL, and organogel microcapsules. Samplea
Onset (°C)
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
75.57 42.50 36.48 75.71 42.90 53.10 74.98 77.68 55.11 75.65 37.81 56.20
± ± ± ± ± ± ± ± ± ± ± ±
0.51a 0.64b 0.32c 0.95a 0.04b 0.30d 0.18a 0.16a 0.42d 0.37a 0.02c 0.15d
Completion (°C)
Peak Enthalpy (mW/mg)
92.03 ± 0.62a 107.52 ± 0.31b 92.37 ± 0.05a 92.12 ± 0.97a 95.01 ± 0.27c 93.87 ± 0.31c 97.17 ± 1.02d 98.88 ± 0.23d 105.53 ± 0.62b 94.01 ± 0.34c 96.81 ± 0.21d 106.51 ± 0.55b
1.08 4.05 1.57 1.12 1.09 3.28 7.65 1.23 5.81 1.83 4.36 6.63
± ± ± ± ± ± ± ± ± ± ± ±
Table 5 Oil stability index (OSI) values for menhaden oil, SL, or organogel microcapsules. Samplea
0.15a 0.03b 1.68a 0.16a 0.99a 0.17b 0.33c 0.61a 1.49b 0.37a 0.52b 0.19c
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
Mean ± STD, n = 3. Different letters in the same column are significantly different (α = 0.05). a Abbreviations are the same as described in Tables 1 and 2.
Non-Microencapsulated
Microencapsulated
OSI (h)
OSI (h) a
17.55 ± 1.21 20.88 ± 1.03b 24.92 ± 3.70c 4.37 ± 0.88d 7.24 ± 0.82e 12.72 ± 1.04f 11.70 ± 1.31f 15.48 ± 0.91a 22.31 ± 0.74b 9.13 ± 1.07e 13.66 ± 0.37f 18.42 ± 0.04a
21.13 28.80 29.18 19.75 25.67 26.15 20.23 26.72 26.78 20.35 28.53 27.63
± ± ± ± ± ± ± ± ± ± ± ±
0.21a 0.22b 0.08b 0.22a 0.23c 0.05c 0.10a 0.36c 0.15c 0.10a 0.21b 0.08b
Mean ± STD, n = 3. Different letters in the same column are significantly different (α = 0.05). a Abbreviations are the same as described in Tables 1 and 2 OSI conducted at 80 °C.
6
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
Table 6 Peroxide and p-Anisidine values of menhaden oil, SL, or organogel microcapsules after 30 days of storage at 4 °C. Samplea
Peroxide Value (mEq/kg) Day 0
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
p-Anisidine Value Day 30
a
7.59 ± 1.53 8.81 ± 1.56b 7.85 ± 1.77a 19.20 ± 3.25c 8.62 ± 1.21b 2.79 ± 0.48d 11.51 ± 1.34b 5.42 ± 0.63a 2.63 ± 0.51d 18.48 ± 1.45c 8.18 ± 0.93b 6.07 ± 0.65a
Day 0 a
Day 30 a
14.61 ± 2.53 5.73 ± 1.77b 3.01 ± 0.72c 24.37 ± 1.92d 11.03 ± 0.89e 3.01 ± 0.26c 38.61 ± 2.84e 15.73 ± 0.68a 13.08 ± 0.35a 22.27 ± 0.84d 9.69 ± 0.18e 8.58 ± 1.53e
9.16 ± 1.56 5.16 ± 0.30b 2.00 ± 0.15c 52.36 ± 0.75d 7.48 ± 0.66b 2.80 ± 0.52c 21.70 ± 0.87e 12.92 ± 0.35a 5.80 ± 0.12b 26.56 ± 1.14e 19.98 ± 1.24f 15.16 ± 1.24f
13.34 ± 1.01a 6.54 ± 0.22b 6.20 ± 0.35b 56.20 ± 1.42c 19.78 ± 0.82d 15.06 ± 0.75d 28.84 ± 1.11e 13.74 ± 0.26a 12.66 ± 0.60a 40.02 ± 0.85c 24.62 ± 1.32e 16.40 ± 0.64d
Mean ± STD, n = 3. Different letters in the same column are significantly different (α = 0.05). a Abbreviations are the same as described in Tables 1 and 2.
encapsulation process (Encina, Vergara, Gimenez, Oyarzun-Ampuero, & Robert, 2016). Our results showed that microencapsulation of oleogels is a good method for improving oxidative stability of SL and oils high in omega-3 fatty acids. The oils were protected from oxidation during the microencapsulation process.
suggests the lipids and respective phytosterol and SSAP oleogels were successfully encapsulated within the alginate microcapsules.
3.7. Oxidative stability Table 5 shows the OSI values for the microcapsules. All the microencapsulated products had significantly higher (p < 0.05) OSI values (19.75–29.18 h) than the non-microencapsulated samples (4.37–22.31 h). Also, the oleogel samples (either microencapsulated or non-microencapsulated) had significantly higher OSI values than the corresponding non-gelled lipid. These results show that oleogelation and formation of microcapsules with oleogels improved the oxidative stability of the lipids. A previous study also observed a similar trend in that both internal structuring (oleogel) and external coating (microencapsulation) led to improved oxidative stability, mostly due to protection against oxygen exposure which is a main factor in causing lipid oxidation (Lee, Tan, & Abbaspourrad, 2019). Table 6 shows the PV and p-AV of the microcapsules after 30 days of storage. The Global Organization for EPA and DHA omega-3 (GOED) sets voluntary limits for PV (5 meq/kg) and p-AV (20) of fish oil, which are much lower than the limits set for refined vegetable oils in most countries (5–10 meq/kg, and 15–30, respectively) (Ismail, Bannenberg, Rice, Schutt, & Mackay, 2016). Of the two values, p-AV is more indicative of the overall quality of the oil. The results in this study show that at day 0 of storage, all microencapsulated samples, except for SL-C, SL-CS, and SL-S, had PV of less than 10 meg/kg. During purification of SL by short-path distillation, endogenous antioxidants are lost, explaining the lower oxidative stability of the SL (Zou & Akoh, 2013). Only SSAP-SL-C had a PV less than 5 meq/kg, following the GOED guidelines. The oleogel microcapsules, except for P-M, all had significantly lower (p < 0.05) PV than the microcapsules containing the non-gelled lipids (M, SL-C, SL-CS, and SLS). After 30 days of storage, P-M, SSAP-M, SSAP-SL-C, P-SL-S, and SSAPSL-S had PV less than 10 meq/kg. Both SSAP-M and SSAP-SL-C had PV less than 5 meq/kg. Interestingly there was a decrease in PV for P-M and SSAP-M after 30 days of storage, which is likely due to the formation of secondary oxidation products from the hydroperoxides, as shown in the increase in p-AV from 0 to 30 days of storage. For the p-AV, all microcapsules, apart from SL-C, SL-CS, and SL-S, had p-AV less than 20, per GOED guidelines. The oleogel microcapsules also had significantly lower (p < 0.05) p-AV than the non-gelled lipid (M, SL-C, SL-CS, and SL-S) microcapsules. After 30 days of storage, all microcapsules, except for SL-C, SL-CS, and SL-S, had p-AV less than 20, showing that the quality of the oils was still acceptable after 30 days of storage. Previous studies have shown that microencapsulation may lead to a lower oxidative stability than the bulk lipid, due to the
4. Conclusion We showed that the leakage of menhaden oil or SL from the alginate microparticles were reduced by use of respective phytosterol or SSAP oleogels as internal phase. Encapsulation of the oleogels also led to increased EE, likely due to higher primary emulsion cohesiveness and apparent viscosity. Almost all microcapsules had acceptable oxidative stability, within the typical limits set by GOED and many countries. Because of the higher stability, these microcapsules should be acceptable for use in the fortification of food products with health beneficial omega-3 fatty acids and/or MLM-type SL. Declarations of interest None. Funding Research was supported in part by Food Science Research, University of Georgia. Structured lipids were produced through support by Agricultural and Food Research Initiative Grant No. 2017-6701726476 from the USDA National Institute of Food and Agriculture, Improving Food Quality-A1361. Conflicts of interest We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. Acknowledgments Special thanks to Omega Protein Inc. for providing the menhaden oil. References American Oil Chemists’ Society. (2017). Official methods and recommended practices of the AOCS (7th ed.). Champaign, IL: AOCS Press. Bakry, A. M., Abbas, S., Ali, B., Majeed, H., Abouelwafa, M. Y., Mousa, A., et al. (2016).
7
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
external coating in an oleogel-based delivery system for fish oil stabilization. Food Chemistry, 277, 213–221. https://doi.org/10.1016/j.foodchem.2018.10.112. Pacheco, C., Crapiste, G. H., & Carrín, M. E. (2015). Study of acyl migration during enzymatic interesterification of liquid and fully hydrogenated soybean oil. Journal of Molecular Catalysis B: Enzymatic, 122, 117–124. https://doi.org/10.1016/j.molcatb. 2015.08.023. Piacentini, E., Poerio, T., Bazzarelli, F., & Giorno, L. (2016). Microencapsulation by membrane emulsification of biophenols recovered from olive mill wastewaters. Membranes, 6, 25. https://doi.org/10.3390/membranes6020025. Rogers, M. A. (2009). Novel structuring strategies for unsaturated fats- meeting the zerotrans, zero-saturated fat challenge: A review. Food Research International, 42, 747–753. https://doi.org/10.1016/j.foodres.2009.02.024. Sagiri, S. S., Pal, K., Basak, P., Rana, U. A., Shakir, I., & Anis, A. (2014). Encapsulation of sorbitan ester-based organogels in alginate microparticles. Pharmaceutical Science and Technology, 15, 1197–1208. https://doi.org/10.1208/s12249-014-0147-2. Sagiri, S. S., Sethy, J., Pal, K., Banerjee, I., Pramanik, K., & Maiti, T. K. (2012). Encapsulation of vegetable organogels for controlled delivery applications. Designed Monomers & Polymers, 16, 366–376. https://doi.org/10.1080/15685551.2012. 747154. Shantha, N. C., & Decker, E. A. (1994). Rapid, sensitive, iron-based spectrophotometric methods for determination of peroxide values of food lipids. Journal of AOAC International, 77, 421–424. Sharma, N., Madan, P., & Senshang, L. (2016). Effect of process and formulation variables on the preparation of parenteral paclitaxel-loaded biodegradable polymeric nanoparticles: A cosurfactant study. Asian Journal of Pharmaceutical Sciences, 11, 404–416. https://doi.org/10.1016/j.ajps.2015.09.004. Willett, S. A., & Akoh, C. C. (2018a). Application of Taguchi method in the enzymatic modification of menhaden oil to incorporate capric acid. Journal of the American Oil Chemists’ Society, 95, 299–311. https://doi.org/10.1002/aocs.12043. Willett, S. A., & Akoh, C. C. (2018b). Physicochemical characterization of organogels prepared from menhaden oil or structured lipid with phytosterol blend or sucrose stearate/ascorbyl palmitate blend. Food and Function, 10, 180–190. https://doi.org/ 10.1039/C8FO01725E. Willett, S. A., Martini, S., & Akoh, C. C. (2019). Enzymatic modification of menhaden oil to incorporate caprylic and/or stearic acid. Journal of the American Oil Chemists’ Society. Early View https://doi.org/10.1002/aocs.12227. Zou, L., & Akoh, C. C. (2013). Identification of tocopherols, tocotreinols, and their fatty acid esters in residues and distillates of structured lipids purified by short-path distillation. Journal of Agricultural and Food Chemistry, 61, 238–246. https://doi.org/10. 1021/jf304441j.
Microencapsulation of oils: A comprehensive review of benefits, techniques, and applications. Comprehensive Reviews in Food Science and Food Safety, 15, 143–182. https://doi.org/10.1111/1541-4337.12179. Behera, B., Sagiri, S. S., Pal, K., & Srivastava, A. (2013). Modulating the physical properties of sunflower oil and sorbitan monopalmitate-based organogels. Journal of Applied Polymer Science, 127, 4910–4917. https://doi.org/10.1002/app.37506. Bordenave, N., Janaswamy, S., & Yao, Y. (2014). Influence of glucan structure on the swelling and leaching properties of starch microparticles. Carbohydrate Polymers, 103, 234–243. https://doi.org/10.1016/j.carbpol.2013.11.031. Co, E. D., & Marangoni, A. G. (2012). Organogels: An alternative edible oil-structuring method. Journal of the American Oil Chemists’ Society, 89, 749–780. https://doi.org/ 10.1007/s11746-012- 2049-3. Decker, E. A., Warner, K., Richards, M. P., & Shahidi, F. (2005). Measuring antioxidant effectiveness in food. Journal of Agricultural and Food Chemistry, 53, 4303–4310. https://doi.org/10.1021/jf058012x/. Encina, C., Vergara, C., Gimenez, B., Oyarzun-Ampuero, F., & Robert, P. (2016). Conventional spray-drying and future trends for the microencapsulation of fish oil. Trends in Food Science & Technology, 56, 46–60. https://doi.org/10.1016/j.tifs.2016. 07.014. Ifeduba, E. A., & Akoh, C. C. (2015). Microencapsulation of stearidonic acid soybean oil in complex coacervates modified for enhanced stability. Food Hydrocolloids, 51, 136–145. https://doi.org/10.1016/j.foodhyd.2015.05.008. Ifeduba, E. A., Martini, S., & Akoh, C. C. (2016). Enzymatic interesterification of high oleic sunflower oil and tripalmitin or tristearin. Journal of the American Oil Chemists’ Society, 93, 61–67. https://doi.org/10.1007/s11746-015-2756-7. International Association for Cereal Science and Technology. (1984). Determination of total fat content. ICC Standard No. 136. Ismail, A., Bannenberg, G., Rice, H. B., Schutt, E., & Mackay, D. (2016). Oxidation in EPAand DHA- rich oils: An overview. Lipid Technology, 28, 55–59. https://doi.org/10. 1002/lite.201600013. Jennings, B. H., & Akoh, C. C. (2001). Lipase catalyzed modification of fish oil to incorporated capric acid. Journal of Food Chemistry, 72, 273–278. https://doi.org/10. 1016/S0308-8146(00)00266-1. Jeyakumari, A., Zynudheen, A. A., Parvathy, U., & Binsi, P. K. (2018). Impact of chitosan and oregano extract on the physicochemical properties of microencapsulated fish oil stored at different temperature. International Journal of Food Properties, 21, 943–956. https://doi.org/10.1080/10942912.2018.1466319. Kim, B. H., & Akoh, C. C. (2015). Recent research trends on the enzymatic synthesis of structured lipids. Journal of Food Science, 80, 1713–1724. https://doi.org/10.1111/ 1750- 3841.12953. Lee, M. C., Tan, C., & Abbaspourrad, A. (2019). Combination of internal structuring and
8
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Encapsulation of menhaden oil structured lipid oleogels in alginate microparticles
T
Sarah A. Willett, Casimir C. Akoh∗ Department of Food Science and Technology, University of Georgia, Athens, GA, 30602-2610, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Menhaden oil Oleogel Structured lipid Microencapsulation Oxidative stability
Leaching of the internal phase during storage is a concern of microencapsulated products. This research proposes encapsulation of oleogels to reduce leaching and improve oxidative stability of the lipids. Oleogels were produced using a phytosterol blend of β-sitosterol/γ-oryzanol or a blend of sucrose stearate/ascorbyl palmitate (SSAP) as oleogelators, and menhaden oil or structured lipid (SL) prepared from menhaden oil and caprylic and/ or stearic acid as the lipid phase. The SL were produced enzymatically using the biocatalyst Lipozyme® 435, a recombinant lipase from Candida antarctica. Menhaden oil, SL, or respective phytosterol or SSAP oleogels, were encapsulated in alginate microparticles using a double emulsion method. Encapsulation efficiency (EE), morphology, oxidative stability, percent leaching, and other physicochemical properties were determined. Encapsulation of phytosterol or SSAP oleogels increased the EE for all lipids, from 89.6 to 91.3% for microcapsules containing only the lipid phases and from 95.7 to 99.2% for microcapsules containing the oleogels. Encapsulation of oleogels significantly reduced leaching, from 16.3-18.5% to 3.3–11.9%. Microencapsulated products had higher Oil Stability Index values (19.75–29.18 h) than the non-microencapsulated lipids (4.37–17.55 h), when measured at 80 °C. These microcapsules may find use as stable nutraceuticals or in fortification of food products with stabilized omega-3 fatty acids.
1. Introduction A structured lipid (SL) is any lipid that has undergone chemical or enzymatic modification from its natural biosynthetic form (Kim & Akoh, 2015). SL have a wide range of applications, though one main concern is their oxidative stability. Although enzymatic production of SL typically occurs under milder conditions than chemical modification, previous studies have found that purified SL have lower oxidative stability than the unmodified fat or oil (Decker, Warner, Richards, & Shahidi, 2005; Ifeduba, Martini, & Akoh, 2016; Pacheco, Crapiste, & Carrin, 2015; Zou & Akoh, 2013). Short-path distillation, a purification process necessary to remove free fatty acids and/or fatty acid ethyl esters from the SL, has been shown to reduce the tocopherols or antioxidants naturally present in the fat or oil (Zou & Akoh, 2013). Polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA) found in menhaden fish oil, are highly susceptible to oxidation due to the high amount of unsaturation. Further modification of menhaden oil to produce SL in previous studies was found to further reduce the oxidative stability (Jennings & Akoh, 2001; Willett & Akoh, 2018a). One way of improving oxidative stability of SL is by forming oleogels. Oleogels (also known as organogels), a lipid gel, have been shown to inhibit oil migration in chocolate, control the release of health beneficial sensitive compounds such as antioxidants, bioactive compounds, and PUFA, and increase oxidative stability of the oil (Co & Marangoni, 2012). However, oleogelation may not reduce the fishy off-flavors present in menhaden fish oil, limiting its use in food products. Another method that has been shown to improve the oxidative stability is by encapsulation of oil within polymeric microparticles. Microencapsulation is a method of coating tiny droplets (such as high PUFA-containing oils) to form small capsules and has
Abbreviations: DSC, differential scanning calorimeter; EE, encapsulation efficiency; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; GOED, Global Organization for EPA and DHA omega-3; M, menhaden oil; OSI, Oil Stability Index; p-AV, p-Anisidine value; PV, peroxide value; P-M, menhaden oil phytosterol oleogel; P-SL-C, SL-C phytosterol oleogel; P-SL-CS, SL-CS phytosterol oleogel; P-SL-S, SL-S phytosterol oleogel; PUFA, polyunsaturated fatty acids; SL, structured lipid; SL-S, SL-S SSAP oleogel; SL-C, structured lipid of menhaden oil and caprylic acid; SL-CS, structured lipid of menhaden oil and a blend of caprylic and stearic acids; SLS, structured lipid of menhaden oil and stearic acid; SSAP, sucrose stearate/ascorbyl palmitate; SSAP-M, menhaden oil SSAP oleogel; SSAP-SL-C, SL-C SSAP oleogel; SSAP-SL-CS, SL-CS SSAP oleogel; SSAP-SL-S, SL-S SSAP oleogel ∗ Corresponding author. E-mail address: [email protected] (C.C. Akoh). https://doi.org/10.1016/j.lwt.2019.108566 Received 28 March 2019; Received in revised form 9 August 2019; Accepted 27 August 2019 Available online 27 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
been found to mask fishy off-flavors (Bakry et al., 2016). Encapsulation of oil reduces its exposure to light, moisture, oxygen, and heat, all of which decrease the quality of the oil through oxidative deterioration, formation of undesirable flavor compounds, and production of free radicals (Sagiri et al., 2014). One disadvantage of microencapsulation is leaching of the internal oil phase during storage, which undoes the encapsulation and therefore reduces its efficiency (Sagiri et al., 2014). To reduce leaching, there are many approaches such as using blended polymers to encapsulate the oil or using a more complex method of producing the microcapsules such as coacervation (Ifeduba & Akoh, 2015; Sagiri et al., 2012). There is need for a simple method to reduce oil leaching. Previous literature has reported success at reducing leaching by encapsulating a sorbitan esterbased sunflower oil oleogel in alginate microparticles for drug delivery (Sagiri et al., 2014). However, the studies on encapsulation of oleogels did not report oxidative stability (Sagiri et al., 2012, 2014). It is of interest to determine the physicochemical properties, encapsulation efficiency, amount of leaching during storage, and oxidative stability of the alginate microparticles when different oleogelators and oil phases are used. The purpose of this research was to microencapsulate menhaden oil or SL oleogels in alginate microparticles to potentially reduce the amount of leaching during storage and improve the oxidative stability of the lipids. Two different oleogelator blends were compared in forming the microcapsules: a phytosterol blend of β-sitosterol and γoryzanol, and a blend of sucrose stearate/ascorbyl palmitate (SSAP). Oxidative stability, amount of leaching during storage, and other physicochemical properties of the microcapsules were studied. Physicochemical properties of the microcapsules will depend on the lipid phase encapsulated. These microencapsulated menhaden oil oleogels may have the potential for use in a wide range of food and nutraceutical applications.
USA). All other reagents and solvents were of analytical or HPLC grade and were purchased from Fisher Chemical (Fair Lawn, NJ, USA), SigmaAldrich Chemical Co (St. Louis, MO, USA), and J. T. Baker Chemical Co. (Phillipsburg, NJ, USA). 2.2. Microencapsulation of lipids and oleogels Menhaden oil (M), SL-C, SL-S, SL-CS, or respective phytosterol or SSAP oleogels, were microencapsulated in alginate microparticles. Table 2 shows the internal phase composition of each of the microcapsules studied. The formation and physicochemical characterization of the phytosterol and SSAP oleogels have been discussed in detail elsewhere (Willett & Akoh, 2018b). Phytosterol oleogels were formed for each lipid, and labeled P-M, P-SL-C, P-SL-CS, and P-SL-S. Briefly, a 1:1 M ratio blend of β-sitosterol and γ-oryzanol was dissolved at 8% (w/ w) in 10 g of lipid (menhaden oil, SL-C, SL-CS, or SL-S). The mixture was stirred for 10 min at 90 °C and cooled at 4 °C to set the gel. SSAP oleogels were formed for each lipid, and labeled SSAP-M, SSAP-SL-C, SSAP-SL-CS, and SSAP-SL-S. A 1:1 M ratio blend of sucrose stearate (HLB value of 2) and ascorbyl palmitate was dissolved at 12% (w/w) in 10 g of lipid (menhaden oil, SL-C, SL-CS, or SL-S). The mixture was stirred for 10 min at 110 °C and cooled at 4 °C to set the gel. The microcapsules were prepared by a modified double emulsion method, also known as an internal gelation method (Sagiri et al., 2014). First, an oil-in-water emulsion was prepared by mixing 0.5 g of sodium alginate and 20 g of water at 25 °C and 250 rpm. Then 0.4 g calcium carbonate was added, stirred at 250 rpm, and homogenized. After homogenization, 0.5 g of Span 80 and 5 g of internal phase (either menhaden oil, SL, or oleogel) was added and homogenized for 15 min. The resulting emulsion was used to form a double emulsion. The emulsion was homogenized further in an ice bath for 10 min to form a thick emulsion. This emulsion was added to 60 mL of ice-cold menhaden oil (external phase) and homogenized for 5 min at 250 rpm. 5 mL of acidified oil (4.5 mL of menhaden oil mixed with 0.5 mL glacial acetic acid) was added to the external phase while stirring to induce ionic crosslinking and gelation of the alginate layer to form the microcapsules. The formed microcapsules were washed with 0.5 M calcium chloride containing 1% tween 80, and then washed with water. Microcapsules were stored at 4 °C until further analysis.
2. Materials and methods 2.1. Materials Menhaden oil was obtained from Omega Protein Inc. (Reedville, VA, USA). Acyl donors, caprylic acid (≥98% purity) and stearic acid (≥98% purity), were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). These were used as substrates in acidolysis reactions to produce the different SL according to the method outlined previously (Willett, Martini, & Akoh, 2019). All acidolysis reactions were conducted in a 1L batch reactor for 24 h, with biocatalyst with Lipozyme® 435 lipase (Novozymes North America, Inc., Franklinton, NC, USA) at 10% (w/w) of total substrates and stirring at 250 rpm using a SL 2400 StedFast stirrer (Fisher Scientific Co., Fair Lawn, NJ, USA). For the SL containing 30 mol% of caprylic acid (SL-C), menhaden oil and caprylic acid at a 1:4.58 substrate molar ratio were incubated at 65 °C. For the SL containing 20 mol% of stearic acid (SL-S), menhaden oil and stearic acid at a 1:1.37 substrate molar ratio were incubated at 75 °C. For the SL containing about 14 mol% each of caprylic and stearic acid (SL-CS), menhaden oil, caprylic acid, and stearic acid, at a 1:3:1 substrate molar ratio were incubated at 75 °C. Table 1 shows the relative fatty acid composition of the menhaden oil and all SL (Willett et al., 2019). All SL had melting points between 25 and 35 °C. The detailed fatty acid and triacylglycerol (TAG) composition, oxidative stability, thermal behavior, and other physicochemical properties of the SL have been discussed in further detail elsewhere (Willett et al., 2019). Ryoto Sugar Ester (sucrose stearate) S-270 was obtained from Mitsubishi-Kagaku Foods Corporation (Tokyo, Japan). Ascorbyl palmitate, Tween 80, sodium alginate, Span 80, and β-sitosterol were obtained from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). γ-Oryzanol was obtained from TCI America (Portland, OR, USA). Calcium carbonate was obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA). Calcium chloride was obtained from VWR International, LLC (West Chester, PA,
2.3. Leaching of the internal phase Leaching of the internal phase was studied to determine the stability of the microcapsules. The higher the amount of leaching, the lower the stability of the microcapsule, as leaching of the internal phase into the coating and to the outside undoes the encapsulation. Microcapsules were wiped with filter paper to remove any traces of oil or moisture on the surface of microcapsules. 0.5 g of microcapsule was weighed and placed on clean filter paper. The microcapsules were then placed in the oven at 37 °C, and leakage was visually monitored for 2 h. To quantify the amount of leaching, another method was used (Sagiri et al., 2014; Bordenave, Janaswamy, & Yao, 2014). Briefly, 0.1 g of microcapsules was soaked in 1 mL of deionized (DI) water for 1 h at 37 °C. The mixture was centrifuged at 2000 rpm for 5 min. The supernatant was collected and dried at 55 °C for 48 h. The dried supernatant was weighed and leaching (%) was calculated as:
% leaching =
dried supernatant wt (g ) x 100 microcapsule initial wt (g )
Samples were analyzed in triplicate and average values were determined. 2.4. Encapsulation efficiency Encapsulation efficiency (EE) was determined as previously 2
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
Table 1 Relative fatty acid composition of menhaden oil and SL (Willett et al., 2019). Samplesa
TAG Position
Menhaden Oil
Total sn-2 sn-1,3c Total sn-2 sn-1,3 Total sn-2 sn-1,3 Total sn-2 sn-1,3
SL-C
SL-CS
SL-S
Fatty Acid (mol%)b C8:0
C16:0
C18:0
C20:5n3
ND ND ND 29.03 ± 1.37 11.72 ± 0.13 37.69 ± 0.75 16.24 ± 0.56 7.31 ± 1.01 20.71 ± 0.34 ND ND ND
10.20 ± 0.85 14.86 ± 0.74 7.87 ± 0.91 5.55 ± 0.06 16.94 ± 0.07 0.15 ± 0.05 4.73 ± 0.21 15.11 ± 0.06 0.46 ± 0.29 7.50 ± 0.05 4.29 ± 0.11 9.11 ± 0.02
3.08 ± 0.05 3.21 ± 0.37 3.02 ± 0.21 2.74 ± 0.10 2.97 ± 0.09 2.63 ± 0.10 13.04 ± 0.10 8.82 ± 0.55 15.14 ± 0.13 19.50 ± 0.23 10.07 ± 0.78 24.22 ± 0.51
16.90 16.42 17.14 13.61 12.22 14.31 13.87 10.69 15.46 14.16 11.85 16.21
± ± ± ± ± ± ± ± ± ± ± ±
C22:6n3 0.10 0.64 0.45 0.38 0.03 0.21 0.26 0.32 0.23 0.11 0.26 0.19
15.40 ± 0.16 29.37 ± 0.95 8.42 ± 0.37 10.42 ± 0.03 25.17 ± 0.46 3.05 ± 0.25 12.50 ± 0.20 16.66 ± 1.39 10.42 ± 0.40 14.62 ± 0.39 22.98 ± 0.18 10.44 ± 0.29
Mean ± STD, n = 3, ND = not detected. a SL-C: structured lipid (SL) of menhaden oil and 30 mol% caprylic acid, SL-S: structured lipid (SL) of menhaden oil and 20 mol% stearic acid, SL-CS: structured lipid (SL) of menhaden oil and 16 mol% caprylic acid and 13 mol% stearic acid. b Other fatty acids found at > 1 mol%: C14:0, C16:1n7, C17:1n7, C18:1n9, C18:2n6, C18:3n3, C20:0, C18:4n3, C22:5n3, and < 1 mol%: C13:0, C14:1n7, C15:0, C17:0, C19:0, C18:3n6, C20:1n7, C20:2n6, C22:0, C20:3n6, C20:3n3, C23:0, C22:2n6, C24:1n9, C22:4n6. c sn-1,3 mol% determined by equation: sn-1,3 (mol%) = [3xtotal mol%- sn-2 mol %]/2.
compressive analysis test via backward extrusion studies using a TAXTi2 texture analyzer (Stable Microsystems, Surrey, UK). Analysis was performed by moving the probe at a speed of 1 mm s−1 to a 10-mm distance within the emulsion and returned to the original position at the same speed. The experiment was performed in auto-force mode with a trigger force of 3 g. Samples were analyzed in triplicate and average values were determined.
Table 2 Internal phase composition of the microcapsules. Sample
Internal Phase
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS
Menhaden oil Phytosterol organogel containing menhaden oil SSAP organogel containing menhaden oil SL of menhaden oil containing 30 mol% caprylic acid Phytosterol organogel containing SL-C SSAP organogel containing SL-C SL of menhaden oil containing ~14 mol% each caprylic & stearic acid Phytosterol organogel containing SL-CS SSAP organogel containing SL-CS SL of menhaden oil containing 20 mol% stearic acid Phytosterol organogel containing SL-S SSAP organogel containing SL-S
P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
2.6. Rheological properties Rheological analyses were performed using an HR-3 Discovery Hybrid Rheometer (TA Instruments, New Castle, DE, USA). A rotational cone and plate (gap of 0.15 mm, cone angle 5.4°, plate diameter of 30 mm) was used for measurements. Analysis of the samples was conducted by varying the shear rate from 15 to 95 s−1 at 25 °C. Apparent viscosity of the primary emulsions were evaluated at 25 °C, at shear rates of 10, 50, and 100s−1 to mimic changes during common food processing conditions. Temperature was controlled with a Peltier Plate Temperature System (TA Instruments, New Castle, DE, USA). All experiments were conducted in triplicate. Data was analyzed using Trios software (TA Instruments, New Castle, DE, USA).
described (Ifeduba & Akoh, 2015). Briefly, both the solvent extractable and total oil of the microcapsules were determined and EE (%) was calculated as follows:
EE (%) = 100 − [(%Solvent extractable/ % Total oil) × 100] To determine solvent extractable oil, 0.25 g of microcapsules was added to 2.5 mL of hexane and vortexed for 15 min. The mixture was centrifuged for 5 min at 1000 rpm. The organic layer was collected, filtered through an anhydrous sodium sulfate, and then transferred to a pre-weighed round bottom flask. Solvent was removed at 60 °C using a rotary evaporator. Solvent extractable oil was calculated as w/w % of suspension. Samples were analyzed in triplicate and average values were determined. To determine total oil, 5 mL of 5 M HCl was added to 0.5 g of microcapsules and the mixture was agitated at 60 °C for 3 h and then cooled to room temperature. The mixture was transferred to a separatory funnel and extracted twice with 5 mL hexane. The extracted organic layer was filtered through an anhydrous sodium sulfate column and transferred to a pre-weighed round bottom flask. Solvent was removed at 60 °C using a rotary evaporator. Total oil was calculated at w/ w % of suspension. Samples were analyzed in triplicate and average values were reported.
2.7. Microscopy The microstructure of the microcapsules was observed under an upright bright-field microscope (Leica DMRXA2 Microscope, Leica Micro-systems Canada Inc., Richmond Hill, Canada). Slides were prepared by placing the microcapsules on a slide with a small amount of DI water. Samples of each microcapsule type, as shown in Table 2, were prepared in triplicate, and slides for each replicate were prepared in triplicate for a total of 9 slides analyzed for each microcapsule sample. Images were acquired using a charged coupled device (CCD) camera (QImaging Retiga, Burnaby, BC, Canada). 2.8. Thermal behavior Thermal behavior of microcapsules was analyzed using a 204 F-1 Phoenix differential scanning calorimeter (DSC) (Netzsch-Gerätebau GmbH, Selb, Germany). Microcapsules were wiped with filter paper to remove any traces of oil or moisture on the surface of microcapsules. Then microcapsule samples were heated from 22 to 120 °C at a rate of 20 °C/min. DSC analysis of peaks was determined using Proteus thermal analysis software (Netzsch-Gerätebau GmbH, Selb, Germany). Samples
2.5. Texture analysis Cohesiveness of the primary emulsions was determined by a 3
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
were analyzed in triplicate and average values reported.
Table 3 Percent leaching, encapsulation efficiency, and primary o/w emulsion cohesiveness of menhaden oil, SL, or organogel microcapsules.
2.9. Oxidative stability Oxidative stability of the microcapsules was determined by measuring peroxide (PV) and p-anisidine (p-AV) values over 1 month of storage at 4 °C. 5 g of sample was weighed into glass vials, closed, and placed in the refrigerator, held at 4 °C for 30 days. PV was determined using a method from the International Dairy foundation (Shantha & Decker, 1994). The p-AV was determined using AOCS Official Method Cd 18–90 (American Oil Chemists' Society, 2017). The lipid portion of the microcapsules was extracted with diethyl ether under the conditions specified by ICC Standard No. 136, with some modification (International Association for Cereal Science and Technology, 1984). 5 g of microcapsules was mixed with 4 mL of 95% ethanol and 1 mL of 7 M NaOH. Then, 5 mL of diethyl ether was added and shaken. After phase separation, 5 mL of hexane was added, mixed vigorously, and phase separation was allowed. The organic layer was collected, washed with 5 mL of 1 M HCl, followed with 5 mL of 0.5 M Na2SO4. The organic layer was transferred to a pre-weighed round bottom flask and solvent was evaporated under nitrogen. PV and p-AV were then measured. Oil Stability Index (OSI) of the microcapsules was also determined at 80 °C using an Oxidative Stability Instrument (Omnion Inc., Rockland, MA, USA) and AOCS Official Method Cd 12b-92 (American Oil Chemists’ Society, 2017). Samples were analyzed in triplicate and average values were reported.
Samplea
Leaching (%)
Encapsulation Efficiency (%)
Cohesiveness (kg s−1)
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
18.54 ± 1.13a 9.02 ± 0.60b 11.93 ± 0.78b 20.30 ± 1.08a 8.42 ± 1.71b 9.88 ± 0.11b 18.22 ± 1.03a 9.14 ± 0.74b 4.38 ± 0.33c 16.31 ± 0.85a 7.27 ± 0.56b 3.28 ± 0.34c
89.79 ± 0.77a 96.87 ± 0.61b 95.69 ± 0.72b 89.57 ± 0.32a 98.09 ± 0.77c 96.13 ± 0.67b 90.64 ± 0.95a 96.24 ± 0.76b 98.92 ± 0.45c 91.26 ± 0.89a 97.08 ± 0.96c 99.17 ± 0.59c
0.01 0.32 0.21 0.07 0.18 0.18 0.10 0.15 0.21 0.19 0.26 0.31
± ± ± ± ± ± ± ± ± ± ± ±
0.04a 0.06b 0.05b 0.01a 0.07b 0.02b 0.01a 0.02a,b 0.02b 0.04b 0.03b 0.07b
Mean ± STD, n = 3. Different letters in the same column are significantly different (α = 0.05). a Abbreviations are the same as described in Tables 1 and 2.
phase, but it was significantly less compared to the menhaden oil microcapsules. This is likely due to higher apparent viscosity and stabilization of the emulsion by the oleogels, which will be discussed in the rheological properties section (3.4). Table 3 shows the quantitative percent leaching for all microcapsule samples. The microcapsules that contained either the phytosterol oleogel or the SSAP oleogel all had significantly (p < 0.05) lower percentages of leaching compared to the microcapsules containing a non-gellated internal phase. Leaching of the internal phase was reduced from 16.3-18.5% to 3.3–11.9%. A previous study that compared encapsulating sunflower oil and sorbitan monostearate oleogel found 46.1 and 9.4% leaching, respectively (Sagiri et al., 2014). Amount of leaching can be attributed to internal phase composition (Sagiri et al., 2012). The SSAP and phytosterol oleogels showed a lower amount of leaching compared to the sorbitan monostearate oleogel and may be better oleogelators than sorbitan monostearate in terms of forming a stronger gel network that aids in preventing leaching.
2.10. Statistical analysis Statistical analysis was performed on the data using JMP® software (Version 10, SAS Institute Inc., Cary, NC, USA). Data was analyzed using ANOVA and significant differences (p < 0.05) were determined using the Tukey test. 3. Results and discussion 3.1. Leaching of the internal phase
3.2. Encapsulation efficiency
Fig. 1 shows a visual representation of internal phase leaching after 2 h in a 37 °C oven for M, P-M, and SSAP-M. The menhaden oil (M) microcapsules showed visual leakage compared to the P-M and SSAP-M. Both P-M and SSAP-M showed some visual leakage of the internal
Table 3 shows the EE for all microcapsule samples. A higher EE is preferred as that meant more lipid was encapsulated. The EE for all
Fig. 1. Visualization of microparticles before and after 2 h in 37 °C oven. Menhaden oil microparticles before (a) and after (d) 2 h, P-M microparticles before (b) and after (e) 2 h, and SSAP-M microparticles before (c) and after (f) 2 h. 4
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
samples ranged from 89.8 to 99.2%. The microcapsules that contained either the phytosterol oleogel or the SSAP oleogel had significantly (p < 0.05) higher EE compared to the M, SL-C, SL-CS, and SL-S microcapsules. The strong gel network of the phytosterol and SSAP oleogels were able to stabilize the o/w emulsions, allowing for a higher proportion of lipid to be encapsulated (Willett & Akoh, 2018b). These results show that oleogelation may be a suitable method for improving the stability of the microcapsules by reducing amount of leaching and improving EE.
viscosity. The increase in apparent viscosity for the phytosterol and SSAP oleogel samples was likely due to an increase in cohesiveness among the different components. These results were comparable to similar studies (Jeyakumari, Zynudheen, Parvathy, & Binsi, 2018; Sagiri et al., 2014). The results suggest that having an oleogel as the internal phase enhanced the viscosity and cohesiveness, which may have accounted for the reduction in leaching and increase in EE.
3.3. Texture analysis
Fig. 3 shows the microcapsules under the light microscope. The microcapsules all exhibited a spherical shape, though varied in size. Apart from P-M and SSAP-SL-C, the oleogel microcapsules had a larger particle size than M, SL-C, SL-CS, and SL-S (non-oleogel microcapsules). EE can be influenced by partition coefficient of target molecule in the solvents used in formulation, method used, and particle size (Piacentini, Poerio, Bazzarelli, & Giorno, 2016). The non-oleogel microcapsules (M, SL-C, SL-CS, and SL-S) all had mostly smaller particle sizes, and lower EE (section 3.2). The oleogel microcapsules also seemed to aggregate together more often than the non-oleogel microcapsules. This may be partly due to the oleogel primary emulsions having higher cohesiveness, as shown in Fig. 2 and Table 3. A previous study found that higher aggregation and particle size was seen with emulsions that exhibited higher cohesiveness and apparent viscosity (Jeyakumari et al., 2018; Sagiri et al., 2012).
3.5. Microscopy
Table 3 shows the cohesiveness of the primary emulsions for all samples. Generally, all phytosterol and SSAP oleogel samples had significantly (p < 0.05) higher cohesiveness values. The menhaden oil emulsion had a lower cohesiveness. SL-C and SL-CS primary emulsions had statistically similar (p > 0.05) cohesiveness compared to the menhaden oil emulsion. SL-S had a significantly higher cohesiveness than the menhaden oil, SL-C, and SL-CS. This was likely due to the higher stearic acid content and melting point, which aided in a stronger fat crystal network (Rogers, 2009). Addition of the oleogel enhanced the cohesiveness of the emulsion. Higher cohesiveness typically corresponds to a higher apparent viscosity and will be discussed in further detail in the rheological properties section (3.4). 3.4. Rheological properties
3.6. Thermal analysis Fig. 2 shows the apparent viscosity of the primary emulsions for all samples. Almost all the phytosterol and SSAP oleogel samples had a higher apparent viscosity. These results correspond well to the texture analysis. The menhaden oil microcapsules had a significantly lower apparent viscosity, lower cohesiveness, and lower EE (sections 3.2 and 3.3). The phytosterol and SSAP oleogel samples had higher cohesiveness and higher apparent viscosity, which led to the higher EE. The higher EE and reduced amount of leaching may be attributed to the gelation of the menhaden oil and SL. Increase in apparent viscosity has been shown to reduce leaching (Sagiri et al., 2012, 2014; Sharma, Madan, & Senshang, 2016). Previous studies have shown that fatty acyl oleogels, similar to the SSAP oleogels, have a tendency to accommodate water within the gel network, and may have resulted in the increase in viscosity of the emulsion (Behera, Sagiri, Pal, & Srivastava, 2013). There was no gel network in the menhaden oil sample, likely accounting for it having a lower apparent viscosity. Both the cohesiveness and apparent viscosity of the primary emulsions followed a similar trend, where a higher cohesiveness corresponds to a higher apparent
For all samples there was an endothermic peak between 35 and 110 °C (data not shown). Table 4 shows the values for peak temperature onset, completion, and enthalpy of all samples. The non-oleogel microcapsules (M, SL-C, and SL-CS) had a peak onset temperature at around 75 °C and peak completion temperature at around 92 °C. P-SLCS also had a statistically similar (p > 0.05) peak onset temperature (77.68 °C) but higher completion temperature (98.88 °C). In other samples, the oleogel microcapsules, had peak onset temperatures between 36.48 and 56.20 °C, and peak completion temperatures between 92.37 and 107.52 °C. In a previous study, we reported the gel to sol transition temperature of the phytosterol and SSAP oleogels to be between 40.1 and 53.1 °C. The broad peaks for the oleogel microcapsules may be explained by the simultaneous transition of the oleogel from gel to sol, and evaporation of water present in the oleogel. Previous studies have also shown this, where although the microcapsule samples were dried, it was difficult to remove bound water (associated with the alginate) present (Sagiri et al., 2014). The shift in the endothermic peak
Fig. 2. Apparent viscosity of primary emulsions. Abbreviations are the same as described in Tables 1 and 2. 5
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
Fig. 3. Light microscopy of microparticles produced with different lipid portion, where a) menhaden oil, b) P-M, c) SSAP-M, d) SL-C, e) P-SL-C, f) SSAP-SL-C, g) SLCS, h) P-SL-CS, i) SSAP-SL-CS, j) SL-S, k) P-SL-S, and l) SSAP-SL-S. Abbreviations for the different lipid portions are the same as described in Tables 1 and 2 Scale bars = 25 μm. Table 4 Thermal behavior of the menhaden oil, SL, and organogel microcapsules. Samplea
Onset (°C)
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
75.57 42.50 36.48 75.71 42.90 53.10 74.98 77.68 55.11 75.65 37.81 56.20
± ± ± ± ± ± ± ± ± ± ± ±
0.51a 0.64b 0.32c 0.95a 0.04b 0.30d 0.18a 0.16a 0.42d 0.37a 0.02c 0.15d
Completion (°C)
Peak Enthalpy (mW/mg)
92.03 ± 0.62a 107.52 ± 0.31b 92.37 ± 0.05a 92.12 ± 0.97a 95.01 ± 0.27c 93.87 ± 0.31c 97.17 ± 1.02d 98.88 ± 0.23d 105.53 ± 0.62b 94.01 ± 0.34c 96.81 ± 0.21d 106.51 ± 0.55b
1.08 4.05 1.57 1.12 1.09 3.28 7.65 1.23 5.81 1.83 4.36 6.63
± ± ± ± ± ± ± ± ± ± ± ±
Table 5 Oil stability index (OSI) values for menhaden oil, SL, or organogel microcapsules. Samplea
0.15a 0.03b 1.68a 0.16a 0.99a 0.17b 0.33c 0.61a 1.49b 0.37a 0.52b 0.19c
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
Mean ± STD, n = 3. Different letters in the same column are significantly different (α = 0.05). a Abbreviations are the same as described in Tables 1 and 2.
Non-Microencapsulated
Microencapsulated
OSI (h)
OSI (h) a
17.55 ± 1.21 20.88 ± 1.03b 24.92 ± 3.70c 4.37 ± 0.88d 7.24 ± 0.82e 12.72 ± 1.04f 11.70 ± 1.31f 15.48 ± 0.91a 22.31 ± 0.74b 9.13 ± 1.07e 13.66 ± 0.37f 18.42 ± 0.04a
21.13 28.80 29.18 19.75 25.67 26.15 20.23 26.72 26.78 20.35 28.53 27.63
± ± ± ± ± ± ± ± ± ± ± ±
0.21a 0.22b 0.08b 0.22a 0.23c 0.05c 0.10a 0.36c 0.15c 0.10a 0.21b 0.08b
Mean ± STD, n = 3. Different letters in the same column are significantly different (α = 0.05). a Abbreviations are the same as described in Tables 1 and 2 OSI conducted at 80 °C.
6
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
Table 6 Peroxide and p-Anisidine values of menhaden oil, SL, or organogel microcapsules after 30 days of storage at 4 °C. Samplea
Peroxide Value (mEq/kg) Day 0
M P-M SSAP-M SL-C P-SL-C SSAP-SL-C SL-CS P-SL-CS SSAP-SL-CS SL-S P-SL-S SSAP-SL-S
p-Anisidine Value Day 30
a
7.59 ± 1.53 8.81 ± 1.56b 7.85 ± 1.77a 19.20 ± 3.25c 8.62 ± 1.21b 2.79 ± 0.48d 11.51 ± 1.34b 5.42 ± 0.63a 2.63 ± 0.51d 18.48 ± 1.45c 8.18 ± 0.93b 6.07 ± 0.65a
Day 0 a
Day 30 a
14.61 ± 2.53 5.73 ± 1.77b 3.01 ± 0.72c 24.37 ± 1.92d 11.03 ± 0.89e 3.01 ± 0.26c 38.61 ± 2.84e 15.73 ± 0.68a 13.08 ± 0.35a 22.27 ± 0.84d 9.69 ± 0.18e 8.58 ± 1.53e
9.16 ± 1.56 5.16 ± 0.30b 2.00 ± 0.15c 52.36 ± 0.75d 7.48 ± 0.66b 2.80 ± 0.52c 21.70 ± 0.87e 12.92 ± 0.35a 5.80 ± 0.12b 26.56 ± 1.14e 19.98 ± 1.24f 15.16 ± 1.24f
13.34 ± 1.01a 6.54 ± 0.22b 6.20 ± 0.35b 56.20 ± 1.42c 19.78 ± 0.82d 15.06 ± 0.75d 28.84 ± 1.11e 13.74 ± 0.26a 12.66 ± 0.60a 40.02 ± 0.85c 24.62 ± 1.32e 16.40 ± 0.64d
Mean ± STD, n = 3. Different letters in the same column are significantly different (α = 0.05). a Abbreviations are the same as described in Tables 1 and 2.
encapsulation process (Encina, Vergara, Gimenez, Oyarzun-Ampuero, & Robert, 2016). Our results showed that microencapsulation of oleogels is a good method for improving oxidative stability of SL and oils high in omega-3 fatty acids. The oils were protected from oxidation during the microencapsulation process.
suggests the lipids and respective phytosterol and SSAP oleogels were successfully encapsulated within the alginate microcapsules.
3.7. Oxidative stability Table 5 shows the OSI values for the microcapsules. All the microencapsulated products had significantly higher (p < 0.05) OSI values (19.75–29.18 h) than the non-microencapsulated samples (4.37–22.31 h). Also, the oleogel samples (either microencapsulated or non-microencapsulated) had significantly higher OSI values than the corresponding non-gelled lipid. These results show that oleogelation and formation of microcapsules with oleogels improved the oxidative stability of the lipids. A previous study also observed a similar trend in that both internal structuring (oleogel) and external coating (microencapsulation) led to improved oxidative stability, mostly due to protection against oxygen exposure which is a main factor in causing lipid oxidation (Lee, Tan, & Abbaspourrad, 2019). Table 6 shows the PV and p-AV of the microcapsules after 30 days of storage. The Global Organization for EPA and DHA omega-3 (GOED) sets voluntary limits for PV (5 meq/kg) and p-AV (20) of fish oil, which are much lower than the limits set for refined vegetable oils in most countries (5–10 meq/kg, and 15–30, respectively) (Ismail, Bannenberg, Rice, Schutt, & Mackay, 2016). Of the two values, p-AV is more indicative of the overall quality of the oil. The results in this study show that at day 0 of storage, all microencapsulated samples, except for SL-C, SL-CS, and SL-S, had PV of less than 10 meg/kg. During purification of SL by short-path distillation, endogenous antioxidants are lost, explaining the lower oxidative stability of the SL (Zou & Akoh, 2013). Only SSAP-SL-C had a PV less than 5 meq/kg, following the GOED guidelines. The oleogel microcapsules, except for P-M, all had significantly lower (p < 0.05) PV than the microcapsules containing the non-gelled lipids (M, SL-C, SL-CS, and SLS). After 30 days of storage, P-M, SSAP-M, SSAP-SL-C, P-SL-S, and SSAPSL-S had PV less than 10 meq/kg. Both SSAP-M and SSAP-SL-C had PV less than 5 meq/kg. Interestingly there was a decrease in PV for P-M and SSAP-M after 30 days of storage, which is likely due to the formation of secondary oxidation products from the hydroperoxides, as shown in the increase in p-AV from 0 to 30 days of storage. For the p-AV, all microcapsules, apart from SL-C, SL-CS, and SL-S, had p-AV less than 20, per GOED guidelines. The oleogel microcapsules also had significantly lower (p < 0.05) p-AV than the non-gelled lipid (M, SL-C, SL-CS, and SL-S) microcapsules. After 30 days of storage, all microcapsules, except for SL-C, SL-CS, and SL-S, had p-AV less than 20, showing that the quality of the oils was still acceptable after 30 days of storage. Previous studies have shown that microencapsulation may lead to a lower oxidative stability than the bulk lipid, due to the
4. Conclusion We showed that the leakage of menhaden oil or SL from the alginate microparticles were reduced by use of respective phytosterol or SSAP oleogels as internal phase. Encapsulation of the oleogels also led to increased EE, likely due to higher primary emulsion cohesiveness and apparent viscosity. Almost all microcapsules had acceptable oxidative stability, within the typical limits set by GOED and many countries. Because of the higher stability, these microcapsules should be acceptable for use in the fortification of food products with health beneficial omega-3 fatty acids and/or MLM-type SL. Declarations of interest None. Funding Research was supported in part by Food Science Research, University of Georgia. Structured lipids were produced through support by Agricultural and Food Research Initiative Grant No. 2017-6701726476 from the USDA National Institute of Food and Agriculture, Improving Food Quality-A1361. Conflicts of interest We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. Acknowledgments Special thanks to Omega Protein Inc. for providing the menhaden oil. References American Oil Chemists’ Society. (2017). Official methods and recommended practices of the AOCS (7th ed.). Champaign, IL: AOCS Press. Bakry, A. M., Abbas, S., Ali, B., Majeed, H., Abouelwafa, M. Y., Mousa, A., et al. (2016).
7
LWT - Food Science and Technology 116 (2019) 108566
S.A. Willett and C.C. Akoh
external coating in an oleogel-based delivery system for fish oil stabilization. Food Chemistry, 277, 213–221. https://doi.org/10.1016/j.foodchem.2018.10.112. Pacheco, C., Crapiste, G. H., & Carrín, M. E. (2015). Study of acyl migration during enzymatic interesterification of liquid and fully hydrogenated soybean oil. Journal of Molecular Catalysis B: Enzymatic, 122, 117–124. https://doi.org/10.1016/j.molcatb. 2015.08.023. Piacentini, E., Poerio, T., Bazzarelli, F., & Giorno, L. (2016). Microencapsulation by membrane emulsification of biophenols recovered from olive mill wastewaters. Membranes, 6, 25. https://doi.org/10.3390/membranes6020025. Rogers, M. A. (2009). Novel structuring strategies for unsaturated fats- meeting the zerotrans, zero-saturated fat challenge: A review. Food Research International, 42, 747–753. https://doi.org/10.1016/j.foodres.2009.02.024. Sagiri, S. S., Pal, K., Basak, P., Rana, U. A., Shakir, I., & Anis, A. (2014). Encapsulation of sorbitan ester-based organogels in alginate microparticles. Pharmaceutical Science and Technology, 15, 1197–1208. https://doi.org/10.1208/s12249-014-0147-2. Sagiri, S. S., Sethy, J., Pal, K., Banerjee, I., Pramanik, K., & Maiti, T. K. (2012). Encapsulation of vegetable organogels for controlled delivery applications. Designed Monomers & Polymers, 16, 366–376. https://doi.org/10.1080/15685551.2012. 747154. Shantha, N. C., & Decker, E. A. (1994). Rapid, sensitive, iron-based spectrophotometric methods for determination of peroxide values of food lipids. Journal of AOAC International, 77, 421–424. Sharma, N., Madan, P., & Senshang, L. (2016). Effect of process and formulation variables on the preparation of parenteral paclitaxel-loaded biodegradable polymeric nanoparticles: A cosurfactant study. Asian Journal of Pharmaceutical Sciences, 11, 404–416. https://doi.org/10.1016/j.ajps.2015.09.004. Willett, S. A., & Akoh, C. C. (2018a). Application of Taguchi method in the enzymatic modification of menhaden oil to incorporate capric acid. Journal of the American Oil Chemists’ Society, 95, 299–311. https://doi.org/10.1002/aocs.12043. Willett, S. A., & Akoh, C. C. (2018b). Physicochemical characterization of organogels prepared from menhaden oil or structured lipid with phytosterol blend or sucrose stearate/ascorbyl palmitate blend. Food and Function, 10, 180–190. https://doi.org/ 10.1039/C8FO01725E. Willett, S. A., Martini, S., & Akoh, C. C. (2019). Enzymatic modification of menhaden oil to incorporate caprylic and/or stearic acid. Journal of the American Oil Chemists’ Society. Early View https://doi.org/10.1002/aocs.12227. Zou, L., & Akoh, C. C. (2013). Identification of tocopherols, tocotreinols, and their fatty acid esters in residues and distillates of structured lipids purified by short-path distillation. Journal of Agricultural and Food Chemistry, 61, 238–246. https://doi.org/10. 1021/jf304441j.
Microencapsulation of oils: A comprehensive review of benefits, techniques, and applications. Comprehensive Reviews in Food Science and Food Safety, 15, 143–182. https://doi.org/10.1111/1541-4337.12179. Behera, B., Sagiri, S. S., Pal, K., & Srivastava, A. (2013). Modulating the physical properties of sunflower oil and sorbitan monopalmitate-based organogels. Journal of Applied Polymer Science, 127, 4910–4917. https://doi.org/10.1002/app.37506. Bordenave, N., Janaswamy, S., & Yao, Y. (2014). Influence of glucan structure on the swelling and leaching properties of starch microparticles. Carbohydrate Polymers, 103, 234–243. https://doi.org/10.1016/j.carbpol.2013.11.031. Co, E. D., & Marangoni, A. G. (2012). Organogels: An alternative edible oil-structuring method. Journal of the American Oil Chemists’ Society, 89, 749–780. https://doi.org/ 10.1007/s11746-012- 2049-3. Decker, E. A., Warner, K., Richards, M. P., & Shahidi, F. (2005). Measuring antioxidant effectiveness in food. Journal of Agricultural and Food Chemistry, 53, 4303–4310. https://doi.org/10.1021/jf058012x/. Encina, C., Vergara, C., Gimenez, B., Oyarzun-Ampuero, F., & Robert, P. (2016). Conventional spray-drying and future trends for the microencapsulation of fish oil. Trends in Food Science & Technology, 56, 46–60. https://doi.org/10.1016/j.tifs.2016. 07.014. Ifeduba, E. A., & Akoh, C. C. (2015). Microencapsulation of stearidonic acid soybean oil in complex coacervates modified for enhanced stability. Food Hydrocolloids, 51, 136–145. https://doi.org/10.1016/j.foodhyd.2015.05.008. Ifeduba, E. A., Martini, S., & Akoh, C. C. (2016). Enzymatic interesterification of high oleic sunflower oil and tripalmitin or tristearin. Journal of the American Oil Chemists’ Society, 93, 61–67. https://doi.org/10.1007/s11746-015-2756-7. International Association for Cereal Science and Technology. (1984). Determination of total fat content. ICC Standard No. 136. Ismail, A., Bannenberg, G., Rice, H. B., Schutt, E., & Mackay, D. (2016). Oxidation in EPAand DHA- rich oils: An overview. Lipid Technology, 28, 55–59. https://doi.org/10. 1002/lite.201600013. Jennings, B. H., & Akoh, C. C. (2001). Lipase catalyzed modification of fish oil to incorporated capric acid. Journal of Food Chemistry, 72, 273–278. https://doi.org/10. 1016/S0308-8146(00)00266-1. Jeyakumari, A., Zynudheen, A. A., Parvathy, U., & Binsi, P. K. (2018). Impact of chitosan and oregano extract on the physicochemical properties of microencapsulated fish oil stored at different temperature. International Journal of Food Properties, 21, 943–956. https://doi.org/10.1080/10942912.2018.1466319. Kim, B. H., & Akoh, C. C. (2015). Recent research trends on the enzymatic synthesis of structured lipids. Journal of Food Science, 80, 1713–1724. https://doi.org/10.1111/ 1750- 3841.12953. Lee, M. C., Tan, C., & Abbaspourrad, A. (2019). Combination of internal structuring and
8