Food Chemistry 237 (2017) 532–537

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Encapsulation of ferulic acid ethyl ester in caseinate to suppress

off-flavor formation in UHT milk
Yongguang Guan, Qixin Zhong ⇑
Department of Food Science, The University of Tennessee, Knoxville, TN 37996, USA

a r t i c l e i n f o a b s t r a c t

Article history: Phenolic compounds can principally suppress the off-flavor development in ultrahigh temperature (UHT)
Received 27 January 2017 treated milk, but little has been studied for lipophilic phenolic compounds that are to be encapsulated for
Received in revised form 27 May 2017 even distribution in milk. The objective of this work was to study physicochemical properties of ferulic
Accepted 29 May 2017
acid ethyl ester (FAEE) encapsulated in sodium caseinate and the inhibition of volatile formation after
Available online 30 May 2017
UHT processing. The capsules had an average hydrodynamic diameter of 246.2 ± 10.9 nm, a polydisper-
sity index of 0.26 ± 0.01, and a zeta-potential of
31.72 ± 0.74 mV. The capsules and the encapsulated
Keywords:
FAEE were stable after heating at 138 °C for 16 min and UV radiation at 365 nm for 32 h. The encapsu-
Ferulic acid ethyl ester
Casein
lated FAEE at a level of 0.18–1.42 mg/mL suppressed the formation of 2-acetyl-2-thiazoline in model
Encapsulation UHT milk by 32.8–63.2% after 30-day storage at 30 °C. Therefore, FAEE encapsulated in caseinate can
Physicochemical properties be potentially used to improve the quality of UHT milk.
Off-flavor formation Ó 2017 Elsevier Ltd. All rights reserved.
UHT milk

1. Introduction
One of the major challenges limiting the acceptability of ultra-
high temperature (UHT) sterilized milk products in certain regimes
of consumers such as those in the United States is the formation of
off-flavor compounds (Ferretti, Edmondson, & Douglas, 1974;
Hutton & Patton, 1952; Josephson, 1954; Patton, 1958). Early stud-
ies found that a series of reactions such as the Strecker degradation
(Schonberg & Moubacher, 1952) originating from the Maillard
reaction between milk proteins and reducing lactose were the pri-
mary cause of off-flavor formation (Ferretti et al., 1974). The reac-
tions can continue during ambient storage of UHT milk to further
deteriorate milk quality. Therefore, there is a need of novel solu-
tions to improve the quality of UHT milk, which can reduce storage
costs and increase the consumption of milk.
Natural antioxidants such as phenolic compounds have shown
positive effects on suppressing the Maillard reaction (Colahan-
Sederstrom & Peterson, 2005; Kokkinidou & Peterson, 2014;
Voziyan & Hudson, 2005). Epicatechin was observed to quench
the formation of C2, C3, and C4 sugar fragments in the Maillard
reaction involving glucose and glycine (Totlani & Peterson, 2005).
Hydrophilic phenolic acids such as vanillic acid, sinapic acid and
ferulic acid can also suppress the Maillard reaction but may have
⇑ Corresponding author at: Department of Food Science, The University of
Tennessee, 2510 River Drive, Knoxville, TN 37996, USA.
E-mail address: qzhong@utk.edu (Q. Zhong).
flavors not native to milk (Heiniö et al., 2008). Hydrophobic pheno-
lic compounds may not be identified by taste buds (Noble,
Philbrick, & Boulton, 1986; Norris, Noble, & Pangborn, 1984) to
impact sensory properties of foods and therefore may be more fea-
sible in improving the quality of UHT milk. Ferulic acid ethyl ester
(FAEE) is a natural colorless hydrophobic phenolic compound
(Warner & Laszlo, 2005b) that has shown excellent radical scav-
enging properties in vivo (Joshi et al., 2006) and in vitro (Perluigi
et al., 2006). However, FAEE has not been studied as a lipophilic
antioxidant to improve the quality of UHT milk.
Encapsulation is a common approach to incorporate lipophilic
phenolic compounds in aqueous systems (Lupo, Maestro, Porras,
Gutiérrez, & González, 2014). For dairy applications, dairy proteins
are a rational choice of encapsulation materials. Casein micelles
have been used to encapsulate lipophilic bioactive substances with
low water solubility using various principles. One of the scalable
technologies is to dissolve both lipophilic compounds and caseins
in warm aqueous ethanol that can dissociate casein micelles above
ca. 65 °C to enhance the interactions (O’Connell, Kelly, Auty, Fox, &
de Kruif, 2001). This principle has been used to encapsulate lipo-
philic curcumin (Pan, Zhong, & Baek, 2013) and bixin (Zhang &
Zhong, 2013) by spray drying the warm aqueous ethanol disper-
sions with sodium caseinate (NaCas). Although NaCas and casein
micelles are structurally different, the dissociated casein molecules
in warm aqueous ethanol can interact with hydrophobic com-
pounds by hydrophobic interactions to enable encapsulation after
spray-drying. The dispersions hydrated from spray-dried powder

http://dx.doi.org/10.1016/j.foodchem.2017.05.140
0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.
 Y. Guan, Q. Zhong / Food Chemistry 237 (2017) 532–537
at 6–7 mg/mL were observed to be transparent and stable at neu-
tral pH during storage in both studies.
The first objective of the present study was to encapsulate FAEE
in NaCas to prepare stable dispersions and characterize physical
and chemical properties of dispersions. The second objective was
to evaluate the effectiveness of encapsulated FAEE in inhibiting
the off-flavor formation in milk heated at simulated UHT condi-
tions and stored at 30 °C for 30 days. The chosen storage conditions
following a literature study were used as accelerated testing equiv-
alent to an estimated duration of 2–3 months at ambient condi-
tions (Kokkinidou & Peterson, 2014).
2. Materials and methods
2.1. Chemicals
FAEE (99%), ethyl acetate (analytical pure), ethanol (analytical
pure) and hexane (analytical pure) were purchased from Fisher Sci-
entific (Pittsburgh, PA, USA). 2-acetyl-2-thiazoline (2A2T, 96%),
2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammo-
nium salt (ABTS, >98%), potassium persulfate (99%), potassium fer-
ricyanide (99%), trichloroacetic acid (99%) and ferric chloride
(>97%) were purchased from Sigma-Aldrich Corp. (St. Louis, MO,
USA). Sodium caseinate (NaCas, 93% protein, dry basis) was pro-
cured from the American Casein Co. (Burlington, NJ, USA). Pasteur-
ized whole milk with a labeled fat content of 3.25% in a 1 gallon
(3.785 L) jug was purchased from a local supermarket (Knoxville,
TN, USA) and was used directly in experiments. The milk product
had 3.33% fat, 5.00% lactose, and 3.33% protein according to the
label.
2.2. Encapsulation of FAEE
The method in our previous studies (Pan et al., 2013; Zhang &
Zhong, 2013) was used to encapsulate FAEE, with some modifica-
tions. Briefly, 0.5 g FAEE was dissolved in 35 mL ethanol and mixed
with 65 mL of an aqueous solution with 3.08% w/v NaCas. After
stirring at 60 °C for 30 min, 200 mL deionized water was slowly
added. Then, the mixture was stirred at 500 rpm for another
30 min on a stir plate, followed by spray drying at an inlet
temperature of 160 °C and a pump rate of 15% using a B-290 mini
spray-dryer (Büchi Labortechnik AG, Flawil, Switzerland). The
spray-dried powder was collected and stored at 21 °C in dark
before use within 48 h.
2.3. Determination of FAEE content in spray-dried powder
To estimate the amount of unencapsulated FAEE after spray
drying, 0.1 g of a powder sample was mixed with 10 mL ethyl acet-
ate at 500 rpm for 5 min on a stir plate. After centrifugation at
12,955g for 5 min (MiniSpin plus, Eppendorf, Germany) at ambient
temperature (21 °C), the organic phase was filtrated through a
0.45 mm polyvinylidene difluoride (PVDF) syringe membrane filter
(Fisher Scientific, Pittsburgh, PA) to collect the permeate for HPLC
analysis. The total amount of FAEE in a spray-dried sample was
determined similarly, except with a mixing time of about 12 h.
Because the yield of lab-scale spray drying was low, the encapsu-
lation efficiency was not estimated.
The Agilent 1200 Series HPLC system (Agilent Technologies Inc.,
Palo Alto, CA, USA) equipped with a DAD plus detector and a 5 lm
Zorbax Eclipse Plus C18 150  4.6 mm column (Agilent Co., USA)
was used to quantify FAEE. The column temperature was main-
tained at 21 °C. The sample injection volume was 10 lL. The
mobile phase was run at 1.0 mL/min using a linear gradient from
a binary solvent mixture with water and methanol at a volume
533
ratio of 70:30 (solvent A) to a volume ratio of 40:60 (solvent B)
in the first 30 min, another liner gradient from solvent B to solvent
A from 30 to 32 min, and an isocratic step of solvent A from 32 to
40 min. The detector wavelength was set at 320 nm. The standard
curve was established from solutions with 0.0016–0.8 mg/mL FAEE
dissolved in ethyl acetate.
2.4. Preparation of samples for characterization of physical and
chemical properties
The aqueous dispersions with FAEE were prepared by hydrating
spray-dried powder at a concentration of 0.5 mg/mL in deionized
water at ambient temperature of 21 °C and stirring at 700 rpm
for 1 h. The dispersions as prepared had a pH of 6.6. A dispersion
control was prepared by hydrating the same amount of NaCas pro-
cessed at conditions in Section 2.2 except without FAEE. A solution
control of FAEE was prepared by dissolving FAEE in ethyl acetate
directly at a concentration of 0.1 mg/mL.
2.5. Physical properties of aqueous dispersions
The turbidity of aqueous dispersions was measured for absor-
bance at 600 nm using an UV/vis spectrophotometer (model Evolu-
tion 201, Thermo Scientific Co., Waltham, MA, USA). The
hydrodynamic diameter (dh) and polydispersity index (PDI) of 4-
fold diluted dispersions were determined at 21 °C using a DelsaTM
Nano-Zeta Potential and Submicron Particle Size Analyzer (Beck-
man Coulter Inc., Brea, CA, USA). The zeta-potential of the diluted
samples was measured at 21 °C using a Malvern Zetasizer Nano
ZS instrument (Malvern Instruments Ltd, Worcestershire, UK).
Two independent dispersion replicates were measured. Each dis-
persion was measured 4 times for particle size and PDI and 3 times
for zeta-potential.
Atomic force microscopy (AFM) was used to further character-
ize capsules in aqueous dispersions, as in our recent work (Liu &
Zhong, 2013; Pan & Zhong, 2013). Briefly, dispersions were diluted
20 times using deionized water. Five microliters of the diluted
sample was spread on a freshly cleaved mica sheet with an area
of 1.8 cm2 and dried at ambient temperature (21 °C) overnight
(12 h) in a fume hood. Topography images were collected at
the tapping mode using a nano-probe cantilever tip (BrukerNanop-
robe, Camarillo, CA, USA) at a frequency from 50 to 100 kHz on a
Multimode VIII microscope (Bruker Corporation, Billerica, MA,
USA). Images were analyzed using the AFM instrument software
(Nanoscope Analysis version 1.50, Bruker Corporation, Billerica,
MA, USA).
2.6. Thermal and UV stability of dispersions and FAEE
The 1 mL aqueous dispersion with encapsulated FAEE and the
FAEE control solution were contained in 4 mL scintillated glass
vials and heated at 138 °C in a glycerol bath for up to 16 min, fol-
lowed by cooling in an ice-water bath immediately. The same sam-
ples were contained in transparent glass vials and placed at 15 cm
under a 15 W UV lamp (UVP, LLC, Upland, CA, USA) at 365 nm for
up to 32 h. The treated samples were stored in dark before follow-
ing analyses. The turbidity and dh of dispersions were measured as
in the previous section. To quantify the FAEE concentration, 2 mL
of an aqueous dispersion was extracted using 5 mL ethyl acetate
by stirring at 500 rpm overnight (12 h) in dark. The organic phase
was filtrated through a 0.45 mm PVDF syringe membrane filter
(Fisher Scientific, Pittsburgh, PA, USA), and the permeate was used
for HPLC analysis using the protocol detailed in Section 2.3.
534 Y. Guan, Q. Zhong / Food Chemistry 237 (2017) 532–537

2.7. ABTS+ radical-scavenging capacity of free and encapsulated FAEE for 1 h. To simulate UHT processing that requires heating at 138 °C
for 8 s (McGarrahan, 1982), 1 mL of a milk sample was pipetted in
The antioxidant property of free and encapsulated FAEE was a 4 mL scintillated glass vial and heated at 138 °C for 1 min in a
estimated using the ABTS method (Re et al., 1999). The fresh ABTS glycerol bath to ensure sufficient thermal treatment throughout
stock solution (7 mM in distilled water) was mixed with 2.45 mM the vial. The heated samples were immediately cooled in an ice-
potassium persulfate and stored at 21 °C in dark for 16 h to gener- water bath and stored at 30 °C for 30 days in an oven (model Pre-
ate ABTS+ radicals. The ABTS+ stock solution was diluted 10 times cision 6958, Thermo Scientific Inc., Waltham, MA). The content of
in deionized water as a working solution. The 0.1 mL of a sample 2A2T in model UHT milk was monitored before and after storage
was mixed with 3 mL of the ABTS+ working solution. After incuba- because it is one of the major off-flavor compounds (Colahan-
tion at 30 °C in dark for 10 min, the absorbance was measured at Sederstrom & Peterson, 2005; Kokkinidou & Peterson, 2014).
734 nm using the above UV–vis spectrophotometer. Distilled water Gas chromatography (GC) analysis of 2A2T followed a literature
was used to prepare a blank by substituting a sample in the ABTS method (Kokkinidou & Peterson, 2014). To extract 2A2T, 2-methyl-
assay. The ABTS+ radical scavenging activity (%) was calculated pentanoic acid was added as an internal standard at a final concen-
using Eq. (1). tration of 15.6 lg/mL into a milk sample, followed by addition of
2 mL of a binary hexane-ethyl acetate solvent (1:1, v/v). After 30-
Radical scavenging activity ð%Þ ¼ ð1
AE =AB Þ  100 ð1Þ s vortexing, samples were stored in dark at 21 °C for 12 h and then
where AE and AB are the respective absorbance values of a sample centrifuged at 10,000g for 30 min. The organic phase was filtered
and the blank, respectively. through a PVDF filter with 0.45 lm pore size (Fisher Scientific,
Pittsburgh, PA, USA). The permeate was analyzed with an HP
6890 Series system (Agilent Technologies Inc., Palo Alto, CA, USA)
2.8. Quantitation of 2A2T in model UHT milk supplemented with
with a DB-WAX column (30 m*0.250 mm*0.25 lm) (Agilent Tech-
encapsulated FAEE
nologies Inc., Santa Clara, CA, USA). The column heating profile was
set at holding at 40 °C for 2 min, ramping to 160 °C at 3 °C/min,
Spray-dried powder was hydrated in the full fat milk at 1.25 to
holding at 160 °C for 2 min, ramping to 240 °C at 30 °C/min, and
10.0 mg/mL in a 20-mL transparent glass vial with stirring at 21 °C
holding at 240 °C for 35 min. The detection limit of 2A2T/2-
Table 1
methyl-pentanoic acid was determined to be 0.015625 mg/mL in
Hydrodynamic diameter, polydispersity index, and zeta-potential of dispersions with preliminary studies. The inhibition ratio of 2A2T (%) was calculated
NaCas only and capsules with encapsulated FAEE at pH 6.6.* using Eq. (2).
Dispersions Hydrodynamic diameter Polydispersity Zeta-potential
(nm) index (mV)
Inhibition ratio of 2A2T ð%Þ ¼ ð1
C E =C B Þ  100 ð2Þ
NaCas only 174.3 ± 4.2b 0.20 ± 0.02b
30.70 ± 3.35a where CE and CB are the respective concentrations of 2A2T in model
Capsules 246.2 ± 10.9a 0.26 ± 0.01a
31.72 ± 0.74a
UHT milk after 30-day storage at 30 °C with encapsulated FAEE and
*
Numbers are mean ± standard deviation (n = 2). Different superscript letters a control sample added with the same amount of NaCas but without
represent significant differences in the mean of the same parameter (p < 0.05). FAEE.

Fig. 1. AFM topography images of dispersions with NaCas only (A) and encapsulated FAEE (B) at pH 6.6. Plots show the heights of particles along the drawn lines. Bar = 1 lm.
 Y. Guan, Q. Zhong / Food Chemistry 237 (2017) 532–537 535

2.9. Statistical analysis 3. Results and discussions

All experiments were carried out at least twice. Means and
standard deviations of the data were calculated for each treatment.
The Independent-Samples t Test was used to determine any signif-
icant differences (p < 0.05) of dh, PDI, and zeta-potential between
fresh NaCas and FAEE-NaCas capsule dispersions. Analysis of vari-
ance (ANOVA) followed by multiple post hoc comparisons with
Duncan method was carried out to determine significant differ-
ences between treatments (p < 0.05) using SPSS 16.0 software
(SPSS Inc., Chicago, IL, USA).
101
A also significantly (p < 0.05) higher than that of NaCas (0.26 ± 0.01
3.1. Physical properties of aqueous dispersions with encapsulated FAEE
The spray-dried powder had an FAEE loading of
14.20 ± 0.85 wt% (n = 6). About 43.0% of FAEE (6.11 wt% of powder
mass) was detected after extracting powder for 5 min using ethyl
acetate. The dh, PDI, and zeta-potential at pH 6.6 are presented in
Table 1 for NaCas and dispersions hydrated with spray-dried
powder with FAEE. The dh of FAEE-loaded capsules was
246.2 ± 10.9 nm, which was significantly (p < 0.05) larger than that
of NaCas (174.3 ± 4.2 nm). The PDI of the dispersion with FAEE was

100 120
FAEE retention ratio (%)

A
100

FAEE retention ratio (%)

99
80

98 60
Free FAEE

NaCas-FAEE 40

97
0 4 8 12 16 20 Free FAEE
Heating time (min) NaCas-FAEE
0
0.3 0 4 8 12 16 20 24 28 32
B NaCas-FAEE
Radiation time (h)

NaCas only 0.2

B NaCas-FAEE

0.2 NaCas only

0.15
Abs600

Abs600

0.1
0.1

0.05

0
0 4 8 12 16
0
Heating time (min)
0 4 8 12 16 20 24 28 32
300 Radiation time (h)
C NaCas-FAEE
300
NaCas only C
Hydrodynamic diameter (nm)

250
250
Hydrodynamic diameter (nm)

200
200

150

150 100

NaCas-FAEE
50
NaCas only
100
0 4 8 12 16
0
Heating time (min) 0 4 8 12 16 20 24 28 32
Radiation time (h)
Fig. 2. Degradation of encapsulated (NaCas-FAEE) and free FAEE (A) and changes of
absorbance at 600 nm (B) and hydrodynamic diameter (C) of dispersions after Fig. 3. Degradation of encapsulated (NaCas-FAEE) and free FAEE (A) and changes of
heating at 138 °C for up to 16 min. The concentrations of FAEE and NaCas in all absorbance at 600 nm (B) and hydrodynamic diameter (C) after radiation at 365 nm
treatments were 0.1 and 0.4 mg/mL, respectively. Error bars are standard deviations and 21 °C for up to 32 h. The respective concentrations of FAEE and NaCas were 0.1
(n = 2). and 0.4 mg/mL in all treatments. Error bars are standard deviations (n = 2).
536 Y. Guan, Q. Zhong / Food Chemistry 237 (2017) 532–537

vs. 0.20 ± 0.02). The PDIs of both dispersions were lower than 0.3,
suggesting relative homogeneity in the particle dimension
(Verma, Verma, Blume, & Fahr, 2003). Increases in dh and PDI have
also been reported after encapsulation of bixin (Zhang & Zhong,
2013) and curcumin (Pan et al., 2013) in NaCas. The zeta-
potentials of FAEE-loaded capsules and NaCas at pH 6.6 were

31.72 ± 0.74 and
30.70 ± 3.35 mV, respectively, which were
similar to values reported in a previous study (Pan et al., 2013).
AFM was used to study the morphology of particles. NaCas par-
ticles were mostly spherical and had a narrow height distribution.
The FAEE-loaded capsules showed the increases in both width and
height when compared with NaCas processed at identical condi-
tions (Fig. 1), which suggests the encapsulation of FAEE.
3.2. Degradation of FAEE and physical stability of dispersions after
thermal treatment
Degradation of FAEE after heating at 138 °C for up to 16 min is
shown in Fig. 2A. FAEE showed an excellent thermal stability with
and without encapsulation in NaCas, corresponding to only
0.3 ± 0.1% and 1 ± 0.1% loss, respectively, after 16-min heating at
138 °C. The excellent thermal stability of FAEE has been reported
for about 20% degradation after 5-h thermal treatment at 180 °C
(Warner & Laszlo, 2005a). Because most phenolic compounds can
be easily oxidized during thermal treatment (Rice-Evans, Miller,
& Paganga, 1997), the good thermal stability of FAEE is an impor-
3.4. Inhibition of 2A2T formation in model UHT milk supplemented
with encapsulated FAEE
There was no detectable 2A2T in fresh UHT milk at the studied
conditions. As shown in Fig. 4, after 30-day storage at 30 °C, the
content of 2A2T in model UHT milk with 0.18 mg/mL of encapsu-
lated FAEE was about 32.8 ± 9.2% lower than the control with an
equivalent amount of NaCas. A greater extent of 2A2T inhibition
was observed with a higher content of FAEE in the model UHT milk,
reaching about 63.2 ± 4.1% inhibition for the treatment with
1.42 mg/mL of encapsulated FAEE.
To understand if the inhibition of 2A2T formation in the model
UHT milk was related to the radical scavenging activity of FAEE
(Joshi et al., 2006), the capacity of scavenging ABTS+ radicals
was estimated for 1.25–10.0 mg/mL capsules, corresponding to
those in UHT milk (Fig. 4), in comparison with same amounts of
free FAEE and NaCas (Fig. 5). NaCas alone was able to scavenge
up to 24.1 ± 3.4% of ABTS+. The capsule dispersion was able to
scavenge up to 80.7 ± 6.3% of ABTS+, which indicates the antioxi-
dant activity is mostly due to FAEE. In contrast, free FAEE dissolved
in ethyl acetate showed the highest activity (97.0 ± 0.4%) in scav-
enging radicals.
80
Inhibition ratio of 2A2T (%)

tant feature to prevent off-flavor generation due to degradation

of antioxidants. 60
As shown in Fig. 2B, the turbidity of dispersions with FAEE after
heating at 138 °C for up to 16 min did not change significantly
(p > 0.05), which is similar to the NaCas dispersion. The dh of NaCas 40
and the dispersion with encapsulated FAEE decreased in the first
2 min of heating and remained constant (p > 0.05) at a longer heat-
ing time (Fig. 2C). The reduction of dh is due to the improved
20
hydration of NaCas during heating (O’Connell, Kelly, Auty, Fox, &
de Kruif, 2001). Caseins are amorphous proteins with little higher
order structures, and the electrostatic repulsion, resulting from
its high magnitude of zeta-potential, and steric repulsion provide 0
good stability of caseins against aggregation at neutral pH (Fox, 0.18 0.36 0.71 1.42

2003). The long-range electrostatic repulsion is significant to the
thermal stability of caseins, as insignificant changes in zeta-
potential have been reported after heating milk at 90 °C for
30 min and at 135 °C for 50 min (Darling & Dickson, 1979).
Net content of encapsulated FAEE in milk (mg/mL)
Fig. 4. The inhibition ratio of 2A2T in model UHT milk supplemented with 1.25–
10 mg/mL spray-dried powder, corresponding to net FAEE content of 0.18–1.42 mg/
mL, after storage at 30 °C for 30 days. Error bars are standard deviations (n = 2).

3.3. Degradation of FAEE and physical stability of dispersions after UV

radiation 120 Capsules
Equivalent free FAEE
ABTS radical scavenging rate (%)

The degradation of FAEE after radiation at 365 nm for up to 32 h 100 Equivalent NaCas
is shown in Fig. 3A. The gradual reduction of FAEE dissolved in
ethyl acetate to 6.0 ± 2.5% was observed after 32 h, whereas
80
82 ± 1.9% FAEE was retained for the encapsulation treatment. UV
radiation can generate radicals and promote the photolytic oxida-
60
tion of organic substances (Glaze, Lay, & Kang, 1995). A previous
study reported the generation of hydroxyl radicals after UV radia-
tion of phenolic compounds (Benitez, Beltran-Heredia, Acero, & 40
Pinilla, 1997), which is also responsible to the first-order UV degra-
dation kinetics of FAEE (Wu et al., 2007). Because caseins contain 20
5.48% tyrosine and 1.62% tryptophan (Goodwin & Morton, 1946),
the p-p conjugated structures of these amino acid residues absorb 0
UV radiation to reduce the degradation of FAEE. 0 2 4 6 8 10
The turbidity and dh of dispersions are shown in Fig. 3B and C, Capsule content in dispersions (mg/mL)
respectively. Insignificant (p > 0.05) changes were found for both
Fig. 5. ABTS+free radical scavenging rate of dispersion hydrated with 1.25–10 mg/
parameters after UV radiation. The results indicated that UV radi- mL capsules, in comparison to equivalent amounts of NaCas (1.07–8.58 mg/mL) and
ation at the studied conditions was insufficient to induce changes free FAEE (0.18–1.42 mg/mL, dissolved in ethanol) that are not plotted according to
of particle structures. the scale. Error bars are standard deviations (n = 4).
 Y. Guan, Q. Zhong / Food Chemistry 237 (2017) 532–537
Caseins contain amino acids enabling the antioxidant activities
detected in common assays (Rival, Boeriu, & Wichers, 2001). The
ability of caseins to scavenge ABTS+ radicals is much weaker when
compared to FAEE (Fig. 5). The high antioxidant activity of FAEE in
Fig. 5 results from its phenolic hydroxyl group reacting with catio-
nic ABTS+ radicals (Graf, 1992) to change ABTS to its reduced state
(Liu, Zhou, Wu, Sun, & Chen, 2015). Encapsulation of FAEE in NaCas
reduces the contact between FAEE and ABTS+ due to steric hin-
drance, which lowers the radical scavenging capacity in the assay
when comparing with free FAEE (Fig. 5).
The retained antioxidant activity of FAEE (Fig. 5) resulted in the
suppression of 2A2T formation during storage of the model UHT
milk (Fig. 4). Phenolic compounds have been reported to reduce
the formation of Maillard-type off-flavors in UHT milk by elec-
trophilic aromatic substitution reactions of flavan-3-ols (the main
antioxidant phenolic hydroxyl group of A-ring) functioning as trap-
ping agents on the transient Maillard-type reactive carbonyl spe-
cies (Colahan-Sederstrom & Peterson, 2005; Totlani & Peterson,
2005). Addition of 1.7 mM catechin, genistein, or daidzein was
reported to reduce 2A2T formation in the model UHT milk by
47–58% after 30-day storage at 30 °C, which was speculated due
to the formation of phenolic-Maillard-type reactive carbonyl spe-
cies adducts (Kokkinidou & Peterson, 2014). This mechanism can
also be the case of encapsulated FAEE suppressing the formation
of 2A2T in UHT milk in the present study.
4. Conclusions
In conclusion, FAEE was successfully encapsulated in NaCas by
the direct spray-drying method. The capsules after rehydration
maintained their dimension after thermal treatment and UV radi-
ation. Encapsulation significantly improved the stability of FAEE
after UV radiation and retained most of the ability of FAEE scaveng-
ing free radicals. The dispersion of encapsulated FAEE in the model
UHT milk enabled the suppression of 2A2T generation after 30-day
storage at 30 °C. These observations suggest the potential of encap-
sulating thermal stable lipophilic antioxidants such as FAEE in
NaCas to improve the quality of UHT milk. Future work verifying
sensory properties of UHT milk with encapsulated FAEE will be
needed for practical applications.
Acknowledgements
This work was supported by the University of Tennessee and
the USDA National Institute of Food and Agriculture Hatch Project
223984.
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