Review Article
Factor affecting the properties of water-in-oil-in-water emulsions forencapsulation of minerals and vitamins
Nattapong Prichapan and Utai Klinkesorn*
Department of Food Science and Technology, Faculty of Agro-Industry,Kasetsart University, Chatuchak, Bangkok, 10900 Thailand.
Received: 15 November 2013; Accepted: 20 October 2014
Abstract
The direct fortification of minerals and vitamins into food may induce chemical degradation, change the level ofbioavailability or decrease the sensory quality of food products. The strategy to solve these problems is encapsulationtechnology. Numerous investigations described the use of water-in-oil-in-water (W/O/W) emulsions as encapsulation system.The properties and encapsulation efficiency of W/O/W emulsions are influenced by emulsion components, the emulsifica-tion processes, and environmental conditions. The recently published results of research done on the factors affecting theproperties of W/O/W emulsions for encapsulation of minerals and vitamins including form and concentration of corematerials, concentration of inner water phase and lipophilic emulsifier, type and concentration of oil phase, type and concen-tration of hydrophilic emulsifier and stabilizer and the pH of the outer water phase have been reviewed in this article.
Keywords: water-in-oil-in-water emulsions, encapsulation, minerals, vitamins
Songklanakarin J. Sci. Technol.36 (6), 651-661, Nov. - Dec. 2014
1. Introduction
Minerals and vitamins are very important to sustainbody functions and are essential for all parts of the body.Although minerals and vitamins are present in various foodsfrom both animal and plant sources, many people still havemineral and vitamin deficiencies. This does not only occurwith poor people in developing countries who do not haveenough food to eat, but also with other people around theworld. These mineral and vitamin deficiencies may appear insome groups of people such as the elderly, pregnant andlactating women, some people with limited consumptionchoices, such as vegetarians or vegans and may also comefrom changing food consumption behaviors to eating highprocessed food more than fresh whole foods (Bonnet et al.,2009; Guzun-Cojocaru et al., 2010; Kroner, 2011).
The direct fortification of minerals and vitamins intofood is not straightforward. Some micronutrients may causeadverse effects to the sensory quality of the food such ascolor changes, undesirable flavor, and a sandy texture, whilesome micronutrients may accelerate chemical reactionsleading to decreased nutritional quality of the food and mayalso lead to toxicants. Furthermore, some micronutrients arevery sensitive to high temperatures, exposure to light, changesin pH, high levels of oxygen, and moisture content, whichcan lead to degradation during preparation, processing, andstorage (Ball, 2006; Mehansho et al., 2006; Jimenez-Alvaradoet al., 2009; Liu, 2009; Lutz et al., 2009; Stevanovic andUskokovic, 2009; Guzun-Cojocaru et al., 2010; Lakkis et al.,2011; Giroux et al., 2013).
The strategy to solve these problems is encapsulationtechnology, a technique that entraps sensitive material, e.g.minerals and vitamins, in the carrier or coating material beforebeing added to food. Emulsion technology is one promisingtechnology for the encapsulation of such materials. Theadvantage of an emulsion system is it allows nutrients to bequite stable and easily dispersed in the medium.
* Corresponding author.Email address: [email protected]
http://www.sjst.psu.ac.th
N. Prichapan & U. Klinkesorn / Songklanakarin J. Sci. Technol. 36 (6), 651-661, 2014652
Usually, emulsion systems consist of two or moreimmiscible liquids, such as oil and water, where one of liquidsis dispersed as small spherical droplets in the other. Emulsionsystems are conventionally classified into two main groups;simple or single emulsions and multiple or double emulsions.Oil-in-water (O/W) emulsions consist of oil droplets dispersedin a continuous aqueous phase (Figure 1A), and water-in-oil(W/O) emulsions that consists of water droplets dispersed ina continuous oil phase (Figure 1B) are examples of a singleemulsion. Whereas, water-in-oil-in-water (W/O/W) emulsionscontaining W/O droplets dispersed in a continuous aqueousphase (Figure 1C), and oil-in-water-in-oil (O/W/O) emulsionswhere O/W droplets are dispersed in a continuous oil phase(Figure 1D) are classified as multiple emulsions.
W/O emulsions and W/O/W emulsions are normallyused to encapsulate water-soluble substances. The W/Oemulsions are suitable used for nutrient fortification inoil-based foods, while W/O/W emulsions are properly usedin water-based food products. O/W emulsions and O/W/Oemulsions are mostly used for encapsulation of oil-solublesubstances. The O/W emulsions encapsulate nutrients forfortification in water-based foods products, while O/W/Oemulsions are used for fortification in oil-based foods.Furthermore, W/O/W emulsions and O/W/O emulsions canbe used as a co-encapsulation system which is encapsula-tion together of both water-soluble and oil-solublesubstances. For example, W/O/W can entrap water-solublenutrients in the inner water phase and also entrap oil-solublesubstances in the oil phase of the same system (Benichouet al., 2002; Benichou et al., 2007; Bonnet et al., 2009; Lutzet al., 2009; Bonnet et al., 2010a; Bonnet et al., 2010b;O’Regan et al., 2010; Li et al., 2012; Giroux et al., 2013).
The aim of this work is to offer an overview of thehealth effects of some minerals and vitamins including iron,calcium, magnesium, vitamin C (ascorbic acid), vitamin B1(thiamine), vitamin B2 (riboflavin), and vitamin B12 (cobalamin).In addition, the use of W/O/W emulsions for encapsulationand the factors affecting the properties of W/O/W emulsionsincluding form and concentration of core material, concen-tration of inner water phase and lipophilic emulsifier, typeand concentration of oil phase, type and concentration ofhydrophilic emulsifier and stabilizer, and pH of outer waterphase are also highlighted.
2. Health Benefits, Deficiency and Fortification of Mineralsand Vitamins
Minerals and vitamins have different health benefitsand they are important for normal functions. Minerals andvitamins are essential for all parts of the body. Deficiency ofminerals and vitamins may cause dysfunction and lead toill-health. Thus, we gathered the health benefit, deficiencyproblem, and food source of some minerals and vitamins thathave the important health effect on our body including iron,calcium, magnesium, ascorbic acid, thiamine, riboflavin, andcobalami as shown in Table 1.
Fortification of minerals and vitamins in foods maysolve the problem of mineral and vitamin deficiency. However,direct fortification of some minerals and vitamins into foodsis not straightforward because it may not be stable or maycause adverse effects on the food products (Table 2). Inaddition, bioavailability of the nutrients is also an importantpoint and should be considered. Bioavailability is the amountof nutrients that can be absorbed and utilized by our body.There are three main factors that affect bioavailability,including personal factors, nutrient stability, and dissolutionor release of nutrients. The personal factors include age, sexand physiologic state (e.g. pregnancy) which affect thegastric residence time and permeability through the gastro-intestinal tract. Formulation (form of nutrients, pH andpresence of other nutrients), food processing, and storageconditions (temperature, light and oxygen) are factors thataffect nutrient stability.
The factors that influence dissolution or release ofnutrients are the encapsulation process and formulation suchas coatings or fillers excipient and surfactants. Thus, thedesign and utilizing the proper encapsulation technique maybe a promising way to enhance the fortification and bioavail-ability of nutrients in food products by protecting them untilthe time of consumption and delivery them to the target sitewithin the human body (Yetley, 2007; Fang and Bhandari,2010).
3. Encapsulation Technology
Generally, encapsulation technology is a techniquethat entraps sensitive materials, solid, liquid, or gas state, inwall material in small sealed capsules to protect them fromadverse environmental conditions such as high temperatures,high or low pH, exposure to UV light, and high levels ofdissolved oxygen. Moreover, the freeing of this substancefrom the encapsulated capsule can be controlled to releaseat certain rates and specific conditions. The size of theencapsulated particle may cover a range from sub-micron tomillimeters. Encapsulation technology can be also used toreduce the evaporation of aromatic compounds, mask un-desirable flavors and tastes of some substances or improvethe dispersion ability of some substances by making themdisperse more uniformly throughout the medium (Fang andBhandari, 2010; Abbas et al., 2012).
Figure 1. Schematic diagram of oil-in-water (O/W) emulsions (A),water-in-oil (W/O) emulsions (B), water-in-oil-in-water(W/O/W) emulsions (C), and oil-in-water-in-oil (O/W/O)emulsions (D) (not to scale).
653N. Prichapan & U. Klinkesorn / Songklanakarin J. Sci. Technol. 36 (6), 651-661, 2014
Tabl
e 1.
Sum
mar
y of f
ood
sour
ces,
heal
th b
enef
it, an
d de
ficie
ncy p
robl
em o
f som
e min
eral
s and
vita
min
s.
Nut
rient
sFo
od s
ourc
esH
ealth
bene
fitD
efic
ienc
y pro
blem
Refe
renc
es
Iron
Mea
ts, p
oultr
y, fis
h, sh
ellfi
sh,
Esse
ntia
l for
blo
od ci
rcul
ator
yA
nem
ia, h
igh
risk
of h
eart
atta
ck, s
low
Kro
ner,
2011
and
som
e pl
ants
syst
em, t
rans
ports
oxy
gen
deve
lopm
ent
in ch
ildre
n, an
d co
mpl
icat
ions
thro
ugho
ut t
he b
ody
of p
regn
ancy
Calci
umM
ilk, c
erea
ls, a
nd th
eir p
rodu
cts.
Esse
ntia
l for
bon
e an
d te
eth,
Cal
cium
rele
ase f
rom
bone
s, hi
gh ri
sk of
Theo
bald
et a
l., 2
005;
mus
cle c
ontra
ctio
n, d
iges
tion,
oste
opor
osis
, and
frac
ture
of b
ones
Saei
dy et
al.,
201
3an
d ne
urot
rans
mitt
er se
cret
ion
Mag
nesiu
mVe
geta
bles
, fru
its, a
nd p
ulse
sEs
sent
ial f
or p
rote
in sy
nthe
sis,
Car
diov
ascu
lar d
isea
ses,
hype
rtens
ion,
Bonn
et et
al.,
200
9; K
rone
r, 20
11en
zym
atic
reac
tions
, and
mus
cula
rm
uscu
lar w
eakn
ess,
and
diar
rhea
cont
ract
ion
Asc
orbi
c ac
idFr
uits
and
veg
etab
les
such
as
Deg
ener
ativ
e ch
roni
c di
seas
es,
Dry
and
split
ting
hair,
roug
h, d
ry an
dLi
u, 20
09; L
utz e
t al.,
200
9;(v
itam
in C
)ci
trus f
ruits
, gua
va, p
otat
oes,
invo
lved
in s
ynth
esis
of c
olla
gen
hem
orrh
age s
kin,
dec
reas
ed w
ound
Stev
anov
ic an
d U
skok
ovic
, 200
9;an
d to
mat
oes
and
epin
ephr
ine,
amin
o ac
id an
dhe
alin
g ra
te, w
eake
ned
toot
h en
amel
,K
rone
r, 20
11ch
oles
tero
l met
abol
ism, a
gain
stsc
urvy
, gin
givi
tis, a
nem
ia, a
nd h
igh
risk
ofox
idat
ive d
amag
e and
canc
ers
infe
ctio
nsTh
iam
ine
Mea
t, w
hole
gra
in ce
real
s, nu
ts,
Role
in ce
llula
r ene
rgy p
rodu
ctio
n,Sy
mpt
om to
two
syst
ems i
nclu
ding
Bate
s, 20
07; O
sieza
gha e
t al.,
2013
(vita
min
B1)
and
bean
san
d he
lps n
euro
n to
be n
orm
alca
rdio
vasc
ular
sys
tem
and
ner
vous
sys
tem
activ
esRi
bofla
vin
Dai
ry p
rodu
cts,
cere
als,
offa
l,Pr
otec
ting
agai
nst c
ance
rs,
Nig
ht b
lindn
ess,
high
risk
of ca
ncer
&Po
wer
s, 20
03(v
itam
in B
2)fis
h, a
nd d
ark-
gree
n ve
geta
bles
card
iova
scul
ar d
isea
ses,
and
play
sca
rdio
vasc
ular
dise
ase,
per
iphe
ral
a rol
e in
thyr
oxin
e met
abol
ismne
urop
athy
, neu
rode
gene
ratio
n, a
ndm
enta
l illn
ess
Coba
lam
inFo
und
in a
nim
al ti
ssue
sEs
sent
ial f
or p
rodu
ctio
n of
blo
odA
nem
ia, i
rrita
bilit
y, m
emor
y los
s, w
eakn
ess,
Kro
ner,
2011
(vita
min
B12
)ce
lls a
nd D
NA
, and
nec
essa
ry fo
rtre
mbl
ing
and
unst
able
mov
emen
ts, a
ndno
rmal
neu
rolo
gic f
unct
ion
psyc
hosi
s
N. Prichapan & U. Klinkesorn / Songklanakarin J. Sci. Technol. 36 (6), 651-661, 2014654Ta
ble 2
.Su
mm
ary o
f for
tific
atio
n fo
rm, r
ecom
men
ded
daily
dos
ages
and
dire
ct fo
rtific
atio
n pr
oble
m of
som
e min
eral
s and
vita
min
s.
Nut
rient
s
F
orm
for f
ortif
icat
ion
Reco
mm
ende
d da
ily d
osag
es *
D
irect
forti
ficat
ion
prob
lem
Iron
Ferr
ous s
ulph
ate,
ferr
ous g
luco
nate
, fer
ric1-
3 ye
ars 7
mg,
4-8
year
s 10
mg,
9-1
3 ye
ars 8
mg,
Sens
itive
to re
actio
n, co
lor c
hang
e, li
pid
amm
oniu
m ci
trate
, sod
ium
iron
ED
TA,
girls
14-
18 ye
ars 1
5 m
g, bo
ys 1
4-18
year
s 11
mg,
oxid
atio
n, p
reci
pita
tion
(Guz
un-C
ojoc
aru
ferr
ous b
isgl
ycin
ate,
ferr
ous f
umar
ate,
wom
en 1
9-50
year
s 18 m
g. P
regn
ancy
27
mg,
et a
l., 20
10; Z
imm
erm
ann
and W
indh
ab,
ferr
ic sa
ccha
rate
, fer
rous
citra
te, f
erric
lact
atio
n 9
mg,
afte
r 51
year
s 8 m
g 20
10)
citra
te, c
arbo
nyl i
ron,
and
ferr
icpy
roph
osph
ate (
Lync
h, 2
005;
Kro
ner,
2011
)Ca
lcium
Cal
cium
carb
onat
e, ca
lciu
m tr
ipho
spha
te,
0-6 m
onth
s 210
mg,
6-1
2 m
onth
s 270
mg,
1-3
year
sFl
occu
latio
n, sa
ndy
text
ure,
pre
cipi
tatio
nan
d ca
lciu
m m
alat
e (Th
eoba
ld, 2
005;
500
mg,
4-8
year
s 800
mg,
9-18
year
s 1,3
00 m
g,(L
akki
s et a
l., 2
011)
Kre
ssel
et a
l., 2
010;
Kro
ner,
2011
)19
-50
year
s 1,0
00 m
g, 50
year
s and
old
er 1,
200 m
g,pr
egna
ncy
and
lact
atio
n: 1
8 ye
ars o
r you
nger
1,30
0 mg,
19-5
0 yea
rs 1,
000 m
gM
agne
sium
Mag
nesi
um p
hosp
hate
, mag
nesi
um0-
6 m
onth
s 30
mg,
7-1
2 m
onth
s 75
mg,
1-3
year
sPr
otei
n ag
greg
atio
n, n
utrie
nts d
egra
da-
pyro
phos
phat
e, a
nd m
agne
sium
pot
assi
um80
mg,
4-8
year
s 130
mg,
9-1
3 ye
ars 2
40 m
g, m
en:
tion,
unf
avor
able
tast
e (B
onne
t et a
l.,ph
osph
ate (
Skel
cey e
t al.,
197
4)14
-18
year
s 410
mg,
19-
30 ye
ars 4
00 m
g, m
ore t
han
2009
)30
year
s 420
mg,
wom
en: 1
4-18
year
s 360
mg,
19-3
0 ye
ars 3
10 m
g, ov
er 3
0 ye
ars 3
20 m
g,pr
egna
ncy:
14-
18 ye
ars 4
00 m
g, 1
9 to
30
year
s35
0 m
g, ov
er 3
1 ye
ars 3
60 m
g, la
ctat
ion:
14-1
8 ye
ars
360
mg,
19
to 3
0 ye
ars 3
10 m
g, ov
er 3
1 ye
ars 3
20 m
gA
scor
bic a
cid
(vita
min
C)
Asc
orbi
c ac
id, a
scor
bate
salt,
and
Men
90
mg
and
wom
en 7
5 m
gVe
ry u
nsta
ble t
o hi
gh p
H, h
igh
tem
pera
-es
terif
ied
asco
rbat
etu
res,
UV
ligh
t, di
ssol
ved
oxyg
en (B
all,
(Liu
, 200
9; K
rone
r, 20
11)
2006
)Th
iam
ine (
vita
min
B1)
Thia
min
e hyd
roch
lorid
e9-
13 ye
ars 0
.9 m
g, bo
ys 1
4-18
year
s 1.2
mg,
girl
sU
nsta
ble t
o hi
gh p
H, h
igh
tem
pera
ture
s,(B
enic
hou
et a
l., 2
002)
14-1
8 ye
ars 1
.0 m
g, m
en 1.
2 mg,
wom
en 1.
1 mg,
high
moi
sture
cont
ent (
Ball
et a
l., 2
006)
preg
nanc
y & la
ctat
ion
1.4
mg
Ribo
flavi
n (v
itam
in B
2)Ri
bofla
vin,
and
ribof
lavi
ne-
1-3
year
s 0.5
mg,
4-8
year
s 0.6
mg,
9-1
3 ye
ars 0
.9U
nsta
ble t
o lig
ht an
d hi
gh p
H (B
all e
t al.,
5'-m
onop
hosp
hate
(Ow
usu
et a
l., 1
992)
mg,
boys
14-
18 ye
ars 1
.3 m
g, g
irls 1
4-18
year
s 1.0
2006
)m
g, m
en 1.
3 m
g, w
omen
1.1
mg,
pre
gnan
cy 1
.4 m
gCo
bala
min
(vita
min
B12
)C
obal
amin
and
aden
osyl
coba
lam
in1-
3 ye
ars 0
.9 µ
g, 4
-8 ye
ars 1
.2 µ
g, 9
-13
year
s 1.8
µg,
Uns
tabl
e to
Hig
h pH
(Bal
l et a
l., 2
006)
(Col
lins,
2003
)14
-18
year
s 2.4
µg,
men
& w
omen
2.4
µg,
pre
gnan
cy2.
6 µg
, lac
tatio
n 2.
8 µg
*(St
andi
ng C
omm
ittee
on
the
Scie
ntifi
c Ev
alua
tion
of D
ieta
ry R
efer
ence
Inta
kes
and
its P
anel
on
Fola
te, O
ther
B V
itam
ins,
and
Cho
line
and
Subc
omm
ittee
on
Upp
erRe
fere
nce L
evel
s of N
utrie
nts F
ood
and
Nut
ritio
n Bo
ard
Insti
tute
of M
edic
ine,
199
8; K
rone
r, 20
11; O
sieza
gha
et al
., 20
13)
655N. Prichapan & U. Klinkesorn / Songklanakarin J. Sci. Technol. 36 (6), 651-661, 2014
Many methods and techniques can be applied forencapsulation of nutrients including spray drying, fluid bedcoating, spray chilling or cooling, melt extrusion, coacerva-tion, liposome, inclusion encapsulation, cocrystallization,nanoencapsulation, freeze drying, yeast encapsulation andemulsions (Stevanovic and Uskokovic, 2009; Fang andBhandari, 2010; Zuidam and Shimoni, 2010; Abbas et al.,2012).
An emulsion encapsulation technique can be used toencapsulate both water-soluble and oil-soluble liquidsubstances. The size of the liquid droplets in emulsion isusually in the range from 0.1-5,000 µm. Preparation of multi-layer around the droplets in emulsions may obtain a morestable and effective encapsulated core. The liquid dropletsin emulsion, especially oil-soluble substances, can beproduced in a dry powder form by spraying or freeze dryingtheir emulsion. The advantage of this technique is that it isflexible because it can be used to encapsulate both hydro-philic and lipophilic substances (Fang and Bhandari, 2010;Zuidam and Shimoni, 2010; Abbas et al., 2012).
Abbas et al. (2012) mentioned that there are manymethods that can be applied for encapsulation, but there areno techniques that are effective for all food systems. Eachtechnique has different advantages and limitations. However,W/O/W emulsions are the one encapsulation technique thatis suitable to fortify nutrients in beverages and water-basedfoods because this technique can keep the nutrients to bequite stable and easily dispersed in an aqueous phase.Moreover, it is easily to be scaled up to industrial size(Krasaekoopt et al., 2003).
4. Encapsulation of Minerals and Vitamins in Water-in-oil-In-water Emulsions
The water-in-oil-in-water or W/O/W emulsion system(Figure 1C) consists of small internal water dropletsembedded in larger oil droplets that are also dispersed inanother outer water continuous phase. This can be referredto as emulsions of emulsions that is to say water-in-oil-in-water emulsions consisting of water-in-oil emulsions (Figure1B) dispersed in a continuous water phase. The W/O/Wemulsions have two types of interfaces; these are innerwater-oil interface and oil-outer water interface, so it has touse oil-soluble emulsifiers to stabilize the inner water-oilinterface and water-soluble emulsifiers to stabilize the oil-outer water interface (Benichou et al., 2002; Dickinson, 2011;McClements, 2012).
For fortification of minerals and vitamins into foodproducts that have water as a continuous phase, W/O/Wemulsions are mostly used with different components assummarized in Table 3. Moreover, W/O/W emulsions mayalso be used for co-encapsulation, which is encapsulationfor both water-soluble substances and oil-soluble substancestogether by an encapsulating water-soluble substance in theinner water phase and encapsulating an oil-soluble substancein the oil phase of the same W/O/W emulsion system. For
example, Li et al. (2012) produced W/O/W emulsions for useas an encapsulation system consisting of riboflavin in theinner water phase and -tocopherol in the oil phase.
4.1 Preparation of W/O/W emulsions
W/O/W emulsions are usually produced using a two-step emulsification method (Figure 2). The first step is theproduction of W/O emulsions by homogenizing an oil phaseand the inner water phase together in the presence of a water-soluble core material at high pressure or high shear rate. Inthis step, a lipophilic emulsifier or oil-soluble emulsifier wasused to stabilize the water-oil interface. The second step isthe production of W/O/W emulsions by homogenizing theformer W/O emulsion and an outer water phase at a pressureor shear rate lower than that used in the first stage to avoidrupture or expulsion of inner droplets. To stabilize the oil-water interface, a hydrophilic emulsifier or water-solubleemulsifier was used in this step (Van der Graaf et al. 2005;McClements, 2012).
4.2 Factors affecting the properties of W/O/W emulsionsfor encapsulation
There are many factors that can affect the propertiesof W/O/W emulsions. In this review, we categorize them intofive groups: form and concentration of core material, con-centration of inner water phase and lipophilic emulsifier, typeand concentration of oil phase, type and concentration ofhydrophilic emulsifier and stabilizer and pH of outer waterphase.
4.2.1 Form and concentration of core material
The form and concentration of the core material affectsthe properties of W/O/W emulsions. For the form of the corematerial, different forms of core material lead to differencesin properties such as size, binding capacity, and hydrophilicproperty that all affect W/O/W emulsions characteristics.If the core material has the form of a bigger molecule, the
Figure 2. Schematic diagram of two-step procedure for preparationof water-in-oil-in-water (W/O/W) emulsions (not toscale).
N. Prichapan & U. Klinkesorn / Songklanakarin J. Sci. Technol. 36 (6), 651-661, 2014656
Tabl
e 3.
Exam
ples
of m
iner
als a
nd vi
tam
ins e
ncap
sula
ted
in w
ater
-in-o
il-in
-wat
er em
ulsio
ns.
Nut
rient
sH
ydro
phili
c em
ulsif
ier
and
stabi
lizer
Oil
phas
e;lip
ophi
lic em
ulsif
ier
Hyd
roph
ilic e
mul
sifie
r and
Refe
renc
esin
inne
r wat
er p
hase
stab
ilize
r in
oute
r wat
er p
hase
Iron
-W
hey
prot
ein
isol
ate (
WPI
)-
Cor
n oi
l; po
lygl
ycer
ol p
olyr
icin
olea
te-
Poly
oxye
thyl
ene
Sorb
itan
Choi
et a
l., 20
09-
Pano
dan
SDK
(est
ers o
fes
ter (
PGPR
)m
onol
aura
te (T
wee
n20)
mon
ogly
cerid
es a
nd d
igly
cerid
es-
Min
eral
oil;
PGPR
-W
hey
prot
ein
conc
entra
teof
dia
cety
l tar
taric
acid
)(W
PC) a
nd g
um ar
abic
orm
esqu
ite g
um or
low
met
hoxy
lpe
ctin
(LM
P)Jim
enez
-Alv
arad
o et a
l., 20
09Ca
lcium
--
Sunf
low
er oi
l; PG
PR-
Xan
than
gum
Mar
quez
and
Wag
ner,
2010
--
Can
ola o
il; g
lyce
rol m
onos
tear
ate
-G
elat
in an
d ag
arSa
eidy
et a
l., 2
013
(GM
S)M
agne
sium
--
Ole
in, m
igly
ol, o
live o
il or
rape
seed
oil;
-So
dium
case
inat
eBo
nnet
et a
l., 2
009
PGPR
--
Oliv
e oil;
PG
PR-
Sodi
um ca
sein
ate
Bonn
et et
al.,
201
0aA
scor
bic
acid
--
Med
ium
chai
n tri
glyc
erid
e or l
imon
ene;
-W
PI an
d m
odifi
ed p
ectin
Lutz
et a
l., 2
009
(vita
min
C)
-Pa
noda
n SD
K an
dgel
lan
gum
PGPR
-M
esqu
ite g
um, m
alto
dext
rinCa
rrill
o-N
avas
et a
l., 2
012
-Ch
ia es
sent
ial o
il; P
GPR
and
WPC
Thia
min
e-
-M
ediu
m ch
ain
trigl
ycer
ide;
PG
PR an
d-
WPI
and
xant
han
gum
Beni
chou
et a
l., 2
002
(vita
min
B1)
mon
ogly
cerid
e ole
ate
Ribo
flavi
n-
-So
y oil;
PG
PR-
WPI
and
LMP
or ê-
carr
agee
nan
Li et
al.,
2012
(vita
min
B2)
Coba
lam
in-
Gela
tin-
Med
ium
chai
n tri
glyc
erid
e; P
GPR
-So
dium
case
inat
e or s
odiu
mO
’Reg
an an
d M
ulvi
hill,
201
0(v
itam
in B
12)
case
inat
e-m
alto
dext
rin co
njug
ate
--
Butte
r oil;
PG
PR-
Skim
milk
or so
dium
case
inat
eG
iroux
et al
., 201
3
657N. Prichapan & U. Klinkesorn / Songklanakarin J. Sci. Technol. 36 (6), 651-661, 2014
encapsulation efficiency will be higher than for smaller forms.If the core material attaches to the molecule that has a higherbinding capacity, the encapsulation efficiency will be higher,because it will occur with a lower free form of the corematerial, so it is harder to release. Moreover, the higher hydro-philic property of the core material also leads to lower release,because higher hydrophilicity molecules are harder to diffusethrough the oil phase.
Marquez and Wagner (2010) encapsulated differentcalcium salts in the inner water phase of W/O/W emulsionsusing sunflower oil as the oil phase, PGPR as a lipophilicemulsifier, soybean milk as the outer water phase, andxanthan gum as the stabilizer. They found that emulsionsthat used calcium lactate as the core material had lower Gand G and higher tan than emulsions that used calciumchloride as the core material. The lower G and G and thehigher tan values mean that the W/O/W emulsions have aweaker structure. This may due to the calcium lactate, whichis a bigger molecule, and has a lower release value thancalcium chloride, which occurs with a lower crosslink to thesoybean proteins and therefore has a weaker structure.
In the same way, Bonnet et al. (2010b) encapsulateddifferent magnesium salts in the inner aqueous phase ofW/O/W emulsions using olive oil as the oil phase, PGPR aslipophilic emulsifier, and sodium caseinate as the hydrophilicemulsifier. They found that the encapsulated MgCl2 had thehighest release rate and completed release in a 15 day-storage, since small molecules of magnesium can easilytransfer through the oil phase. The encapsulated gluconicacid hemimagnesium salt was found to have a lower releasevalue than MgCl2, but a higher release value than phosvitinwith MgCl2. This may due to the fact that gluconic acid has alower binding capacity than phosvitin and is quite a smallmolecule, so it can easily pass through the oil phase. Whereas,encapsulated phosvitin with MgCl2 was found to have thelowest release value because the hydrophilicity and highmolecular weight of phosvitin cause it to be unable to passthough the oil phase.
For the effect of concentration of core material onencapsulation efficiency of W/O/W emulsions, there wasreport that higher concentrations of the core material can leadto higher encapsulation efficiency. For example, Benichouet al. (2002) entrapped thiamine hydrochloride in the W/O/Wemulsions that used medium chain fatty acid triglycerides asthe oil phase, PGPR as the surfactant, monoglyceride oleatefor droplet size reduction and better stability, and wheyprotein isolate and xanthan gum as hydrophilic emulsifiers.They found that increasing vitamin B1 concentrations causeda decrease in vitamin B1 release. This may due to the fact thatthiamine hydrochloride has an amphiphilic structure, so ithas surface properties to reduce interfacial tension.
In a similar way, Lutz et al. (2009) encapsulatedsodium ascorbate in the W/O/W emulsions using PGPR asemulsifier, and WPI and modified pectin as a hydrophilicemulsifier. They found that fresh emulsions had a reducedrelease rate with increasing sodium ascorbate concentration,
but after 28 days there was no significant difference. Thedifference in release rate of fresh emulsions containing differ-ent core concentrations was not discussed by the authors.However, in our opinion, this may due to the effect of theosmotic gradient. The higher core material concentration ledto the higher osmotic gradient, resulting in emulsion dropletswelling. This occurs from diffusion of the water phase fromthe outer water phase to the inner water phase to balance theosmotic gradient (Dickinson, 2011). The intensity of waterdiffused from the outer phase to the inner phase was higherthan the diffusion of water from the inner phase to the outerphase resulting in a low release of sodium ascorbate in freshemulsions. However, for a longer time storage, the diffusion ofwater from the outer phase to the inner phase may cause somedroplets broken and release core material resulting in a highrelease of sodium ascorbate from emulsion droplets after 28days.
4.2.2 Concentration of inner water phase and lipophilicemulsifier
The concentration of the inner water phase and lipo-philic emulsifier affects the properties of W/O/W emulsions.Higher concentrations of the inner water phase lead to lowerrelease rates of the core material. In the same way, higherconcentrations of lipophilic emulsifier lead to lower releaserates of the core material. An example for the effect ofdispersed aqueous phase content was shown by Marquezand Wagner (2010). They found that higher dispersedaqueous phase content led to the formation of lower G andG and higher tan value emulsions. The lower G and Gand the higher tan meant that the W/O/W emulsions werethinner. This can explain why higher dispersed aqueousphase content reduced the calcium concentration leading toreduced osmotic gradient and calcium release, so there wasless crosslink with soybean proteins producing thinneremulsions. For the effect of lipophilic emulsifier concentra-tion, Marquez and Wagner (2010) found that higher PGPRconcentrations produced lower G and G and higher tan value emulsions. This may be due to increases in the PGPRconcentration which reduced the water droplet size of theW/O droplets, so reduced calcium release led to lesscrosslink and less elastic behavior of the emulsion.
4.2.3 Type and concentration of oil phase
The type and concentration of the oil phase affectsthe properties of W/O/W emulsions. Different types of oilphase lead to variations in properties such as viscosity, andsaturated fatty acid content affecting the encapsulationefficiency of W/O/W emulsions. An oil phase with higherviscosity values lead to higher encapsulation efficiencybecause the viscous oil phase retards diffusion and expulsionof the inner water phase with the core material inside. More-over, oil types with higher saturated fatty acid levels yieldhigher hydrophobicity or lower compatibility with water
N. Prichapan & U. Klinkesorn / Songklanakarin J. Sci. Technol. 36 (6), 651-661, 2014658
causing a faster expulsion of the inner water phase that hasthe core material inside.
For example, Bonnet et al. (2009) encapsulated MgCl2in W/O/W emulsions using olein, miglyol, olive oil orrapeseed oil as the oil phase, PGPR as a lipophilic emulsifier,sodium caseinate as a hydrophilic emulsifier, and lactose tomatch the osmotic pressure between the inner and outerwater phase to avoid water transfer. They found that doubleemulsions produced with miglyol had significantly fastermagnesium release than other oils. This can be explainedsince miglyol has the lowest viscosity and the highest satu-rated fatty acid of these four oils, so the lower viscosity leadsto easier material transfer and release, and higher saturatedfatty acid leads to higher hydrophobicity and lower com-patibility with the water causing faster magnesium release.In contrast, double emulsion produced with rapeseed oil,which has a quite high viscosity and the lowest saturatedfatty acid content, had the highest magnesium retention.
In the same way, Lutz et al. (2009) found that doubleemulsions produced from medium chain triglycerides had alower sodium ascorbate release rate than produced from R(+)-limonene. This can be explained since R(+)-limonene has alower viscosity which leads to easier release of the sodiumascorbate from the inner water phase to the outer waterphase.
For the effect of lipid phase content on property ofW/O/W emulsions, there was a report that higher lipid phasecontent lead to get more viscous W/O/W emulsions. Marquezand Wagner (2010) showed that increased lipid phase contentled to the formation of higher G and G and lower tan value emulsions. The higher G and G and the lower tan values mean that the W/O/W emulsions were more viscous.This can be explained in a way that the higher lipid phasecontent produced in the more water-in-oil droplets led totighter packing and a viscous systems.
4.2.4 Type and concentration of hydrophilic emulsifier andstabilizer
The type and concentration of the hydrophilicemulsifiers and stabilizers affect the properties of W/O/Wemulsions. Some hydrophilic emulsifiers and stabilizers havea synergistic effect on the stabilization of emulsion whenused together. Different types of hydrophilic emulsifiers andstabilizers, which have different properties such as molecularweight and charge, result in different effects on the propertiesof the W/O/W emulsions. Using two hydrophilic emulsifierstogether or hydrophilic emulsifiers with stabilizers mayincrease the encapsulation efficiency more than using eitheralone. For example, O’Regan and Mulvihill (2010) encapsu-lated vitamin B12 in W/O/W emulsions with gelatin to solidifythe inner water phase with medium chain triglyceride oil asthe oil phase, PGPR as the lipophilic emulsifier, sodiumcaseinate or sodium caseinate-maltodextrin conjugate as thehydrophilic emulsifier. They found that the encapsulationefficiency of double emulsion stabilized with sodium
caseinate-maltodextrin conjugate was significantly higherthan that of sodium caseinate, especially in the case of malto-dextrin with a dextrose equivalent (DE) of about 10. This wasbecause the protein-polysaccharide conjugate formed a morebulky polymeric layer at the interface and some parts of thepolysaccharide protruding toward the continuous phasecaused better steric stabilization against droplet flocculationand coalescence.
The greater molecular weight of polysaccharides leadto more effective protection of the core material than lowerones, for example, Jimenez-Alvarado et al. (2009) entrappedferrous bisglycinate in the W/O/W emulsions using mineraloil as the oil phase, and protein-polysaccharide complexes(whey protein concentrate (WPC) with gum arabic (GA) ormesquite gum (MG) or low methoxyl pectin (LMP)) as water-soluble surfactants in outer water phase. They found that theencapsulation yield and ferrous ion content of W/O/Wemulsions stabilized with 5 wt% total concentration of WPC:MG and 0.7 wt% total concentration of WPC:LMP weresignificantly highest at the initial time. This may be due toformation of a thick layer at the interface of the droplet, butonly ferrous ion content of water-in-oil-in-water emulsionstabilized with 5 wt% total concentration of WPC:MG weresignificantly higher at the end of storage time which may bedue to the molecular weight of the polysaccharides. Mesquitegum has a greater molecular weight than gum Arabic and lowmethoxyl pectin, respectively. Since mesquite gum has a highmolecular weight and can form a thick layer at the interface,therefore it was the most effective polysaccharide to protectagainst ferrous bisglycinate oxidation in between thesepolysaccharides.
The higher charge of polysaccharides has both posi-tive and negative effects on the properties of W/O/W emul-sions. For the positive effect, Lutz et al. (2009) encapsulatedsodium ascorbate in W/O/W emulsions that used differentmodified pectin types U63 and C63, the modified pectin typesU63 and C63 are 63% degree of esterification, but U63 hasa more negative zeta potential and more intermolecularinteractions than C63. They found that double emulsionstabilized with the WPI/U63 complex had lower sodiumascorbate release rates than found in a double emulsion stabi-lized with the WPI/C63 complex. They explained that U63pectin have more charges which leads to more interactionand causes more elastic and a greater stiffness complex withWPI. Thus, the WPI/U63 complex was more effective toprevent the release of sodium ascorbate than the WPI/C63complex.
In contrast, Li et al. (2012) encapsulated riboflavin inthe W/O/W emulsions with hydrophilic emulsifiers and stabi-lizers of whey protein isolate and low methoxyl pectin (LMP)or -carrageenan (KCG), which has a higher minus chargethan low methoxyl pectin. They found that the encapsulationefficiency of the WPI-LMP complex stabilized emulsion washigher than that of the WPI-KCG complex when the WPI: PSratio was 5:0.5. They explained that the low charge poly-saccharide chain has a flexible worm-like form that easily to
659N. Prichapan & U. Klinkesorn / Songklanakarin J. Sci. Technol. 36 (6), 651-661, 2014
curvature and interact with protein molecule, while the highcharge polysaccharide chain has a rigid stick-like form, solow methoxyl pectin, which has lower charge, has a moreflexible chain, and can more easier bind with proteins to createa dense and seal interface leading to more encapsulationefficiency than that of the WPI-KCG complex.
For the effect of hydrophilic emulsifier or stabilizerconcentration, the higher hydrophilic emulsifier or stabilizerconcentration should have higher encapsulation efficiencybecause increased hydrophilic emulsifier levels lead tostronger interfacial films against core material release. Forexample, Li et al. (2012) found that increasing the polysac-charide concentration caused the encapsulation efficiency toincrease. In the same way, Benichou et al. (2002) found thatincreases in the xanthan gum ratio reduced the vitamin B1release, because more xanthan gum content led to the pro-duction of a more synergistic and rigid film around thedroplet.
However, increasing the concentration of some hydro-philic emulsifiers can lead to decreases in encapsulationefficiency. For example, Bonnet et al. (2010a) encapsulatedmagnesium in the W/O/W emulsions by using olive oil as anoil phase, PGPR as a lipophilic emulsifier, and sodiumcaseinate as a hydrophilic emulsifier. Surprisingly, the highersodium caseinate concentrations led to higher magnesiumrelease. This may due to increased levels of sodium caseinatemeaning more sodium ions leading to more osmotic pressurein the external water phase. When the external osmoticpressure is higher than the internal osmotic pressure, thewater may migrate from the internal to the external waterphase.
4.2.5 pH of outer water phase
When two ionic hydrophilic emulsifiers or stabilizersare used together, the pH of outer water phase will affect theencapsulation efficiency of W/O/W emulsions. The pH thatshould be used is one that allows two ionic hydrophilicemulsifiers or stabilizers to have different charges in order tolet them exhibit electrostatic attraction. For example, whenprotein and anionic polysaccharide are used as a hydrophilicemulsifier and stabilizer, respectively, the pH of the systemshould be lower than pI (isoelectric point) of protein in orderto obtain the cationic structure of protein that can exhibitelectrostatic attraction with an anionic polysaccharidestructure (Figure 3A). The larger difference in charge inten-sity between the ionic hydrophilic emulsifiers or stabilizerslead to increased electrostatic attraction and provides athicker and denser interfacial layer.
Benichou et al. (2007) entrapped thiamine hydro-chloride in W/O/W emulsions by using medium chain fattyacid triglyceride as the oil phase, PGPR as the lipophilicemulsifier, monoglyceride oleate and glycerol for droplet sizereduction and better stability. Whey protein isolate andxanthan gum was used as hydrophilic emulsifier and stabili-zer, respectively. They found that lower pH levels led to
lower vitamin B1 release, but at a pH of 3.5 vitamin B1 wasreleased less than at pH 2 because at pH values more thanabout 4.5 (above the isoelectric point of the whey proteinisolate) this caused the whey protein isolate to have a minuszeta potential as xanthan gum, so the whey protein isolateand xanthan gum did not interact (Figure 3B) which led to aless sealed interface and higher vitamin B1 release. On theother hand, at pH 2 xanthan gum had less minus zeta potentialand a reduced different of zeta potential between wheyprotein isolate and xanthan gum, which led to an interactionbetween them being less than at pH 3.5. This had a greaterdifference in zeta potential between the whey protein isolateand xanthan gum, so the complex interface at pH 3.5 wasmore sealed and had lower vitamin B1 release.
5. Conclusions
W/O/W emulsions can be used as an entrapping anddelivering system for minerals and vitamins. However, thereare many factors that affect the stability and efficiency ofthese emulsion systems that have to be considered such asvolume and properties of oil and inner or outer water phase,concentration and form of core material, type and concentra-tion of emulsifiers and stabilizers, and also process condi-tions. For encapsulation of minerals and vitamins, W/O/Wemulsions should have both high encapsulation efficiencyand physicochemical stability, including being stable toenvironmental stress and chemical reactions. Moreover, itshould still be stable when it is applied to food products andshould not decrease the quality of those products.
Acknowledgements
The authors would like to thank the Thailand ResearchFund under The Royal Golden Jubilee Ph.D. Program (PHD/0001/2555: 2.F.KU/55/I.1.R.03) for their financial support.Special thanks to S. Watmough for his assistance in writingthe manuscript.
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