Food Bioprocess Technol (2017) 10:2005–2012
DOI 10.1007/s11947-017-1953-9
ORIGINAL PAPER
Microencapsulation of an Angiotensin I-Converting Enzyme
Inhibitory Peptide VLPVP by Membrane Emulsification
Guo-Qing Huang 1 & Jun-Xia Xiao 1 & Lu-Qing Hao 1,2 & Jian Yang 2
Received: 8 February 2017 / Accepted: 7 July 2017 / Published online: 2 August 2017
# Springer Science+Business Media, LLC 2017
Abstract The bioavailability of angiotensin I-converting en-
zyme inhibitory peptides (ACEIPs) plays a major role in their
practical applications, and microencapsulation has emerged as
an effective way to realize the targeted and controlled release
of bioactive peptides. In this work, sodium alginate–O-
carboxymethyl chitosan microspheres loaded with an
ACEIP Val-Leu-Pro-Val-Pro (VLPVP) were prepared by
Shirasu Porous Glass (SPG) membrane emulsification. The
effects of VLPVP addition amount and SPG membrane pore
size on microencapsulation were studied, and the impacts of
the microencapsulation process on the transport and in vitro
ACE inhibitory activity of VLPVP were investigated as well.
The results indicated that the microspheres could be saturated
by VLPVP and their particle size could be predicated by using
a linear equation when the SPG membrane pore size was
5.0 μm or higher. The membrane emulsification method was
more effective than the encapsulator and needle extrusion
methods in terms of microencapsulation efficiency, VLPVP
load, protection against gastric fluid, and controlled/
sustainable release without changing the transport behavior
or in vitro ACE inhibitory activity of VLPVP. Hence, SPG
membrane emulsification was a potential method for the
intestine-targeted delivery of ACEIPs.
* Jian Yang
jiany@szpt.edu.cn
1
College of Food Science and Engineering, Qingdao Agricultural
University, Qingdao 266109, China
2
School of Applied Chemistry and Biological Technology, Shenzhen
Polytechnic, Shenzhen 518055, China
Keywords Angiotensin-converting enzyme inhibitory
peptide . Val-Leu-Pro-Val-Pro . Membrane emulsification .
Microencapsulation . Controlled release
Introduction
Angiotensin I-converting enzyme (ACE) inhibitory peptides
are bioactive peptides with potential antihypertensive proper-
ties. As hypertension is a significant public health problem
worldwide, ACE inhibitory peptides (ACEIPs), as a part of
food products or nutraceuticals, may be of functional interest
in maintaining a healthy population. The bioavailability of
ACEIPs after oral administration plays a major role in their
practical applications. To exert in vivo physiological effects
after oral ingestion, it is of crucial importance that the peptides
remain active during gastrointestinal digestion and absorption
(Vermeirssen et al. 2007).
Several measures have been proposed to increase the bio-
availability of orally administered ACEIPs, including chemi-
cal modification (Mayo 2000) and delivery by genetically
engineered GRAS microorganisms (Krüger et al. 2002). In
recent years, microencapsulation has emerged as a strategy
to mask the bitter taste (Favaro-Trindade et al. 2010), reduce
the metabolism by lactic acid bacteria (Vaslin 2008), improve
the stability in food matrices (de Vos et al. 2010), and realize
the targeted and controlled release of bioactive peptides
(Champagne and Fustier 2007).
However, reports concerning the microencapsulation of
ACEIPs are still quite rare. One report revealed that microen-
capsulation into liposomes significantly prolonged the effective
duration of a tripeptide Leu-Lys-Pro in spontaneous hyperten-
sive rates (Chen et al. 2003); in another report, a protein hydro-
lysate with ACE inhibitory activity was microencapsulated into
2006
carboxymethylated gum/sodium alginate (SAL) beads and its
release behavior in simulated intestinal conditions was success-
fully controlled by varying the Ca2+-crosslinking degree (Ruiz
Ruiz et al. 2013). This limited information implied that micro-
encapsulation could be a potential way to improve the bio-
availability of ACEIPs.
Many methods are available for the microencapsulation of
peptides (Degim and Nevin 2007), but the membrane emulsi-
fication technique has received growing interests in recent
years as an alternative low-energy process for the preparation
of delivery systems with narrow particle size distribution
(Joseph and Bunjes 2014). The Shirasu Porous Glass (SPG)
membranes are the most commonly used in this technique and
have been applied in the microencapsulation of insulin, which
led to prolonged duration (Toorisaka et al. 2003) and con-
trolled release (Liu et al. 2006), but its utilization in the stabi-
lization of ACEIPs has not been reported yet.
The hydrogel beads formed by SAL and chitosan have
been extensively used as carriers to deliver protein and peptide
drugs to the intestine (George and Abraham 2006). However,
the SAL–chitosan microspheres prepared by spray drying,
coacervation, extrusion, or emulsification/solidification often
have large particle size and broad size distribution, which
could lead to possible side effects and poor repeatability dur-
ing practical applications (Wang et al. 2005). To overcome
these disadvantages, the membrane emulsification technique
was applied by Zhang et al. (2011) to prepare insulin-loaded
microspheres with narrow size distribution. Both in vivo and
in vitro experiments revealed that the loaded insulin was ef-
fectively protected from protease digestion, sustainably re-
leased to the blood, and exerted excellent hypoglycemic effect
for a longer time after oral administration to diabetic rats.
Nevertheless, chitosan is soluble only in acidic solutions,
which limits its application in the targeted delivery of acid-
susceptible compounds. Carboxymethyl chitosan has been
proven to be a good alternative to chitosan regarding this issue
(Mukhopadhyay et al. 2013; Yang et al. 2013).
Val-Leu-Pro-Val-Pro (VLPVP) is a promising ACEIP that
has been engineered into E. coli (Liu et al. 2007), and its
transport mechanism has been well elucidated (Lei et al.
2008). The purpose of this work was to prepare VLPVP-
loaded O-carboxymethyl chitosan (O-CMC)–SAL micro-
spheres by SPG emulsification to see if this novel technology
could be used for the targeted delivery of ACEIPs. The con-
ditions for the preparation of the microspheres were opti-
mized; the particle size distribution, morphology, and
VLPVP release behavior of the resultant microspheres in sim-
ulated gastrointestinal fluids were determined, and the effects
of the microencapsulation process on VLPVP transport and
in vitro ACE inhibitory activity were explored. This work is
expected to provide information for extending the application
of the SPG membrane emulsification technology in the micro-
encapsulation of ACEIPs.
Food Bioprocess Technol (2017) 10:2005–2012
Materials and Methods
Materials
SAL was purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Ningbo, China), and O-CMC with degree of substitution
of 0.35 was synthesized in the same method as in a previous
work (Huang et al. 2015). PO-500 (hexaglycerin penta ester)
was supplied by Sakamoto Yakuhin Kogyo Co., Ltd.
(Sakamoto, Japan). A high-speed mini kit KH-125 as the
membrane emulsification apparatus and SPG membranes
with pore sizes 0.1, 0.5, 5, 10, and 20 μm were obtained from
SPG Techno Co. Ltd. (Miyazaki, Japan). VLPVP with purity
of 98% was synthesized by Shanghai Science Peptide
Biological Technology Co., Ltd. (Shanghai, China). All the
materials used for VLPVP transport and in vitro ACE inhib-
itory activity evaluation were obtained from the same sources
as mentioned in the corresponding references (Cushman and
Cheung 1971; Lei et al. 2008).
Preparation of VLPVP-Loaded SAL–O-CMC
Microspheres by Membrane Emulsification
The VLPVP-loaded SAL–O-CMC microspheres were pre-
pared in the same method of Zhang et al. (2011) with minor
modifications. Acetate buffer of pH 4.2 containing 0.5% (w/v)
SAL and certain amount of VLPVP was used as the water
phase. The mixture of liquid paraffin and petroleum ether in
volume ratio 7:5 as well as 4% (w/w) PO-500 was used as the
oil phase. Under the pressure of nitrogen purge and room
temperature, 50 mL water phase was pressed through the
SPG membrane into 500 mL oil phase to form uniform-
sized W/O emulsion droplets. Then, CaCl2 emulsion prepared
by dispersing 1.5 mL of 0.5 mol/L CaCl2 solution into 10 mL
oil phase followed by sonication was added into the W/O
emulsion. After stirring at 300 r/min for 5 h, the solids were
collected and washed twice with petroleum ether and four
times with distilled water by centrifugation at 1000g and
25 °C for 5 min, followed by coating in 50 mL of 0.5% (w/
v) O-CMC solution for 1 h under 300 r/min stirring. The
resultant microspheres were washed twice with distilled water
and freeze-dried (Alpha 1–4 LDplus, Martin Christ, Germany)
for characterization.
Preparation of VLPVP-Loaded SAL–O-CMC
Microspheres by Encapsulator B-390 and Needle
Extrusion
VLPVP-loaded SAL–CMC microspheres were also prepared
by using a Buchi B-390 encapsulator (Flawil, Switzerland)
and needle extrusion for the comparison between different
microencapsulation techniques.
Food Bioprocess Technol (2017) 10:2005–2012
Due to the high viscosity of the SAL solution, a 300-μm
nozzle was selected for the encapsulator. Fifty milliliters of
1.5% (w/v) SAL solution that was dissolved in pH 4.2 acetate
buffer and contained 250 mg VLPVP was extruded through
the nozzle to 50 mL pH 6.0 CaCl2 solution. In the needle
extrusion method, the same SAL solution was extruded to
50 mL 2% CaCl2 (w/v) solution of pH 6.0 at a speed of
30~40 drops per minute by using a G20 needle. Both the
solutions were then left for 5 h under 300 r/min stirring, and
the solids were collected and coated by O-CMC in the same
way as mentioned in microsphere preparation by SPG mem-
brane emulsification. All the resultant microspheres were
rinsed thoroughly with distilled water to remove unabsorbed
O-CMC and freeze-dried for characterization.
Determination of Microencapsulation Efficiency
and VLPVP Load
The VLPVP content was determined using an Agilent 1200
system (Agilent Technologies Inc., California, USA)
equipped with an Eclipse XDB-C18 column (4.6 × 250 mm,
5 μm). After 20-μL sample was injected, the column was
eluted isocratically with a mobile phase consisting of acetoni-
trile and distilled water in volume ratio 17:83 as well as 0.05%
trifluoroacetic acid at 1.0 mL/min and 30 °C. The wavelength
of the UV detector was set at 199 nm, and the content of
VLPVP was determined by comparing the peak area with
the standard curve.
The content of VLPVP on microsphere surface (Wex) was
determined as follows: Freeze-dried microspheres of a certain
weight (Wm) were dispersed in 1 mL distilled water for 10 min
and then separated by centrifugation at 1000g for 5 min. The
supernatant was collected, filtered through a 0.22-μm filter,
and subjected to VLPVP content determination.
The content of total VLPVP in the microspheres (Wtot) was
determined as follows: Freeze-dried microspheres of a certain
weight (Wm) were dispersed in 1 mL pH 7.4 phosphate buffer
and sonicated for 30 min to allow the complete liberation of
VLPVP. The release medium was then centrifuged at 1000g
for 5 min, and the VLPVP content in the supernatant (Wtot)
was measured. The microencapsulation efficiency (ME) and
VLPVP load were calculated according to the following equa-
tions:
W tot −W ex
ME ¼
W tot
W tot
VLPVP load ¼
Wm
Particle Size Determination
The size distribution was analyzed by a Mastersizer 2000 laser
particle analyzer (Malvern Instruments Ltd., Malvern, UK),
2007
and the average particle size was calculated by the accompa-
nying software. The polydispersity of particle size distribution
was determined by the SPAN factor expressed as follows:
Dðv; 90Þ−Dðv; 10Þ
SPAN ¼
Dðv; 50Þ
where D(v, 90), D(v, 10), and D(v, 50) are volume size diam-
eters at 90, 10, and 50% of the cumulative volume,
respectively.
Morphology Observation
The freeze-dried and LVPVP-loaded SAL–O-CMC micro-
spheres prepared with SPG membrane of pore sizes 0.1, 0.5,
5, 10, and 20 μm were mounted on circular aluminum stubs
with double-sided sticky tape, coated with gold, then exam-
ined, and photographed in a scanning electron microscope
(JSM-7001F, Jeol, Japan) at an accelerating voltage of 5 kV.
Release Behavior in Simulated Gastrointestinal Fluids
The freeze-dried VLPVP-loaded microspheres prepared using
the 20 μm SPG membrane and VLPVP concentration in the
water phase 5 g/L as well as those prepared by the B-390
encapsulator and needle extrusion were subjected to total
VLPVP content (ctot) determination, weighed (m), and im-
mersed in 50 mL simulated gastric fluid (pH 1.2 HCl solu-
tion). After incubation at 37 °C for 2.0 h, the microspheres
were separated by centrifugation at 4000g for 5 min and
dipped in 50 mL simulated intestinal fluid (pH 6.8 phosphate
buffer) for another 4 h. At an interval of 0.5 h, 0.2 mL release
medium was taken out and fresh release medium was added to
maintain a constant volume. The sampled solutions were cen-
trifuged at 5000g for 5 min, and the content of VLPVP in the
supernatant (cres) was determined in the HPLC method men-
tioned above. The cumulative release of VLPVP, which re-
ferred to the ratio of released to total VLPVP in the micro-
spheres, was calculated using the following equation:
Cumulative VLPVP release ð%Þ
cres  50  10−3
¼  100%
m  ctot
Transport of VLPVP by Caco-2 Monolayer
Freeze-dried VLPVP microspheres of 2.5 and 5.0 mg pre-
pared with SPG membrane pore size 20 μm and VLPVP
content in the water phase 5 g/L were dispersed in 10 mL
pH 7.4 Hank’s balanced salt solution (HBSS) and sonicated
for 1 h. Then, the lysate was clarified through a 0.22-μm filter
2008
and the transport of VLPVP by the Caco-2 monolayer was
evaluated in exactly the same method of Lei et al. (2008).
In Vitro ACE Inhibitory Activity
Freeze-dried VLPVP microspheres of certain weight prepared
with SPG membrane pore size 20 μm and VLPVP content in
the water phase 5 g/L were dispersed in 0.1 mol/L pH 8.3
borate buffer and sonicated for 1 h. The content of VLPVP
in the lysate was determined by HPLC and diluted to 1 mg/mL
with the same borate buffer. The in intro ACE inhibitory ac-
tivities of free and released VLPVP were then determined
spectrophotometrically in the method described by Cushman
and Cheung (1971).
Statistics
All the experiments were done in at least triplicate. Statistical
differences between the treatments were determined using a
one-way analysis of variance (ANOVA) test. Probability
values of p < 0.05 were considered to be statistically
significant.
Results and Discussion
Effect of VLPVP Concentration in the Water Phase
on Microencapsulation
The concentration of drug in the water phase is a critical factor
that affects its encapsulation by SPG membrane emulsifica-
tion (Liu et al. 2006). The SPG membrane pore size 20 μm
was selected to explore the effects of VLPVP concentration in
the water phase on its microencapsulation.
As illustrated in Fig. 1a, when the VLPVP concentration in
the water phase increased from 1.25 to 3.75 g/L, the ME
increased significantly from 64.6 to 87.8%, as the VLPVP
concentration further increased to 5 g/L and higher, no signif-
icant changes were observed (p > 0.05). A higher ME implied
more VLPVP entrapped inside the microspheres. Hence, this
parameter could be used as an indicator of stability against the
gastric fluid and controlled/sustainable release potency of the
microspheres. The results in Fig. 1a revealed that too low a
VLPVP concentration in the water phase was not beneficial
for its stability or controlled release in gastrointestinal fluids.
A similar trend was also found for the VLPVP load in the
SAL–O-CMC microspheres. As the VLPVP concentration in
the water phase grew from 1.25 to 5 g/L, its content in the
microspheres increased significantly with values of 0.7, 1.1,
7.1, and 7.8%, respectively, for the four concentrations select-
ed; when the VLPVP content further raised to 6.25 g/L, the
peptide load did not further increase (Fig. 1b). This result
Food Bioprocess Technol (2017) 10:2005–2012
Fig. 1 Effect of VLPVP concentration in the water phase on the
microencapsulation efficiency (ME, a) and VLPVP load of the SAL–O-
CMC microspheres (b). The pore size of the SPG membrane used was
20 μm. Bars with different letters indicated significant difference at
p < 0.05, similarly hereinafter
indicated that too high a VLPVP concentration in the water
phase could lead to great drug loss during microencapsulation.
It should be noted that the VLPVP load in the microspheres
was not always proportional to its concentration in the water
phase. When the concentration of VLPVP in the water phase
increased from 1.25 to 2.5 g/L, the peptide load in the micro-
spheres increased by only 0.57-fold, as the concentration in-
creased to 3.75 g/L, the drug load increased tremendously by
up to 9.14-fold, but after the VLPVP concentration further
increased to 5 and 6.25 g/L, the drug load increased only
slightly (Fig. 1b). This result was similar to the insulin-
loaded SAL–chitosan microspheres, in which the percentages
of loaded insulin decreased markedly from 86.93 to 43.89%
when the insulin content in the water phase increased from 1.0
to 4.0% (Liu et al. 2006), and the bovine serum albumin
(BSA)-loaded chitosan microspheres prepared by membrane
emulsification, in which the BSA content in the microspheres
did not further increase after the BSA content in the water
phase exceeded 4 mg/mL (Wang et al. 2005), but was contrary
to the lysosome-loaded chitosan microspheres prepared by
SPG membrane emulsification, in which the enzyme content
in the microspheres increased proportionally as the enzyme
concentration in the aqueous phase increased from 1 to
1.5 g/L (Akamatsu et al. 2012).
The variation patterns of ME and VLPVP load could be
possibly ascribed to the interaction with SAL and diffusion of
the pentapeptide. The pka of SAL is in the range 3.4 to 4.4
(Shinde and Nagarsenker 2009), and the theoretical pI of
VLPVP is 5.49 according to the website http://web.expasy.
org/compute_pi/. Since the pH of the water phase was 4.2,
VLPVP and SAL were oppositely charged and electrostatic
attraction occurred between them. When the concentration of
VLPVP in the water phase was below 2.5 g/L, SAL was in
excess and a balance was reached between VLPVP adsorption
Food Bioprocess Technol (2017) 10:2005–2012
by SAL and diffusion, and consequently, the ME and VLPVP
load in the microspheres increased with the amount of VLPVP
added. When the VLPVP concentration in the aqueous phase
increased to 3.75 g/L and higher, the negative charges carried
by SAL were nearly completely neutralized by VLPVP and
the excessive peptides diffused quickly to the outside water
phase, leading to only slight changes in ME and VLPVP load
in the microspheres.
Effect of SPG Membrane Size on Microsphere Size
Distribution
It has been proposed that there exists a linear relationship
between the sizes of microspheres and the corresponding
SPG membrane pore and the microsphere diameter can be
accurately controlled by choosing a proper membrane
(Nakashima et al. 2000). The finding of this work was partial-
ly consistent with this proposal. As shown in Table 1, in SPG
membrane pore size ranging from 5.0 to 20.0 μm, the resultant
microsphere diameter (y) was approximately two times of the
corresponding SPG membrane pore size (x) and a linear equa-
tion of y = 16.97 × x − 8.7967 with correlation coefficient of
0.9709 could be extrapolated to describe the relationship, in-
dicating the controllability of microsphere size and conse-
quently the release behavior of VLPVP from the micro-
spheres. This relationship was similar to the poly(lactic-co-
glycolic acid) (PLGA) microspheres prepared with SPG mem-
brane of pore sizes 1.00, 1.95, 2.60, 3.63, and 5.25 μm (Ito
et al. 2008), but differed from the lysozyme-loaded chitosan
microspheres, whose particle diameter was nearly the same as
the selected SPG membrane pore size of 1.1, 5.5, and 12 μm
(Akamatsu et al. 2012).
However, such relationship was not observed in the case of
membrane pore sizes 0.1 and 0.5 μm, which were much
higher than the expected value. The possible reason was that
small particles aggregated readily due to their high specific
surface energy (Wang et al. 2005) and/or the formation and
chokage of solid monomer crystals in the pores or at the end of
the pores of the SPG membrane (Chu et al. 2003). The varia-
tion pattern of the SPAN value agreed with the pore size
changes very well. As the membrane pore size increased, the
aggregation phenomenon nearly disappeared and the unifor-
mity of the microspheres increased markedly.
Effect of SPG Membrane Pore Size
on Microencapsulation
The effect of SPG membrane pore size on the ME and VLPVP
load of the microspheres is shown in Fig. 2. When the mem-
brane pore size increased from 0.1 to 5.0 μm, both the param-
eters increased significantly; however, as the membrane pore
size further increased to 10.0 and 20.0 μm, no significant
changes were observed. A similar trend has also been reported
2009
Table 1 Effect of SPG membrane pore size on the average particle
diameter and SPAN value of VLPVP-loaded SAL–O-CMC microspheres
SPG membrane pore size (μm) Average particle diameter (μm) SPAN
0.1 5.39 2.43
0.5 7.88 1.90
5.0 9.87 1.35
10.0 21.75 0.88
20.0 43.81 0.51
for the insulin-loaded polylactic acid (PLA)/PLGA microcap-
sules prepared by membrane emulsification, in which the ME
decreased and the drug release increased significantly with
decreasing SPG membrane pore size (Liu et al. 2006). This
could be explained by the fact that the microspheres prepared
with SPG membrane of smaller pore sizes had relatively larger
specific surface area, allowing the rapid diffusion of VLPVP
to the outside of the microspheres during crosslinking with
Ca2+ and coating with O-CMC.
Morphology of Microspheres
The SEM micrographs of the VLPVP-loaded microspheres
prepared with SPG membranes of different pore sizes are
shown in Fig. 3. All the microspheres were spherical, and their
diameters increased in larger SPG membrane pore sizes,
which was consistent with the measurements revealed in
Table 1. The surfaces of the microspheres yielded with mem-
brane of pore sizes 0.1 μm (Fig. 3a) and 0.5 μm (Fig. 3b) were
rough, as the membrane pore size increased to 5 μm (Fig. 3c)
and higher (Fig. 3d, e), the microsphere surfaces became
much smoother.
Fig. 2 Effect of SPG membrane pore size on the microencapsulation
efficiency (ME, a) and VLPVP load of the SAL–O-CMC microspheres
(b). The concentration of VLPVP in the water phase was 5 g/L
2010 Food Bioprocess Technol (2017) 10:2005–2012

Fig. 3 SEM micrographs of the VLPVP-loaded SAL–O-CMC microspheres prepared with SPG membrane pore sizes 0.1 μm (a), 0.5 μm (b), 5 μm (c),
10 μm (d), and 20 μm (e). The concentration of VLPVP in the water phase was 5 g/L

Comparisons with Other Microencapsulation Methods released the drug more sustainably, and the highest cumulative
release occurred on the fourth hour of the entire incubation
The Buchi B-390 encapsulator has been used to produce mi- process and reached 77.80%, as the incubation further elon-
crospheres with desired sizes and uniform size distribution for gated, the encapsulated VLPVP could no longer be liberated.
various bioactive compounds (Zhang et al. 2016), and needle While for the microspheres prepared by SPG membrane emul-
extrusion is a classic method for the preparation of SAL-based sification, the drugs were released more sustainably and the
microspheres (Martins et al. 2007). The comparisons of the cumulative release increased gradually with incubation elon-
SPG membrane emulsification method with these two tech- gation. At the end of 5-h incubation, the accumulative release
niques are illustrated in Figs. 4 and 5.
Among the three methods and under selected conditions,
the SPG membrane emulsification method had the highest
ME and VLPVP load. As for ME, the SPG membrane emul-
sification method caused a 1-fold higher value than the needle
extrusion method (Fig. 4a), while for VLPVP load, microen-
capsulation by membrane emulsification contributed to a 0.8-
fold higher peptide content than the encapsulator (Fig. 4b).
These comparisons revealed that the membrane emulsification
method was an efficient method for the microencapsulation of
ACEIPs.
The release behaviors of VLPVP from the microspheres in
simulated gastrointestinal fluids are given in Fig. 5. After in-
cubation in the simulated gastric fluid for 2 h, the micro-
spheres prepared by SPG membrane emulsification released
only 9.59% of loaded peptide, while those prepared by the Fig. 4 Effect of preparation methods on encapsulation efficiency (ME, a)
encapsulator and needle extrusion reached up to 19.35 and and VLPVP load (b) of the SAL–O-CMC microspheres. The pore size
23.74%, respectively. After transfer to the simulated intestinal used in the membrane emulsification method was 20 μm, and the
concentration of VLPVP in the water phase was 5 g/L. The nozzle size
fluid, the microsphere prepared by the encapsulator released
of the B-390 encapsulator was set to 300 μm. In the needle extrusion
nearly all the loaded drugs (98.53%) at the end of 1.5-h incu- method, a G20 needle was used and the extrusion speed was 30~40 drops
bation. The microspheres prepared by needle extrusion per minute
Food Bioprocess Technol (2017) 10:2005–2012
Fig. 5 Release behavior in the simulated gastrointestinal fluids of
VLPVP from the SAL–O-CMC microspheres prepared by SPG
membrane emulsification, encapsulator B-390, and needle extrusion.
The pore size used in the membrane emulsification method was 20 μm,
and the concentration of VLPVP in the water phase was 5 g/L
reached 87.63%. These comparisons revealed that the SPG
membrane emulsification method was more suitable for pre-
paring pH-sensitive intestine-targeted SAL–O-CMC micro-
spheres due to its better stability against the gastric environ-
ment and sustainable release potency.
Effect of Microencapsulation on the Transport
and In Vitro ACE Inhibitory Activity of VLPVP
Lei et al. (2008) had studied the transport mechanism of
VLPVP by using human intestinal Caco-2 monolayers as the
model. It was found that the transport of this peptide was
bidirectional and involved an active process. In this work,
the same method was used to explore whether the microen-
capsulation process could affect the transport of encapsulated
VLPVP and the result is given in Fig. 6a. It could be seen that
the peptide was transported bidirectionally in the
concentration-dependent manner and the apical to basolateral
transport rate was significantly higher than the basolateral to
apical transport rate, which was the same as the peptide in its
free form (Lei et al. 2008). Hence, the microencapsulation
process did not destroy the transport of VLPVP.
For the same reason, the in vitro ACE inhibitory activity of
the VLPVP liberated from the SAL–O-CMC microspheres
was also determined. The IC50 of native VLPVP was reported
to be 1.7 μmol/L, corresponding to 1 mg/mL (Lei et al. 2008).
Hence, native VLPVP as well as that extracted from the mi-
crosphere in concentration 1 mg/mL was subjected to in vitro
ACE inhibitory activity determination. As shown in Fig. 6b,
native VLPVP exerted an inhibitory ratio of 48.2%, which
was quite close to its reported percentage (Lei et al. 2008).
2011
Fig. 6 Cumulative transepithelial flux across the Caco-2 cell monolayer
(a) and in vitro ACE inhibitory activity of VLPVP (b) extracted from the
SAL–O-CMC microspheres. AP-BL indicates flux from apical to
basolateral side, and BL-AP indicates flux from basolateral to apical
side. Comparisons were carried out between data of the same series
In the same concentration, the VLPVP extracted from the
SAL–O-CMC microspheres showed an inhibitory ratio of
45.2% that was not significantly different from native
VLPVP, indicating that the microencapsulation process had
no adverse effect on the ACE inhibitory activity of VLPVP.
A similar result was also found for the alginate–chitosan mi-
crospheres prepared by membrane emulsification, in which
the activity of insulin was nearly completely retained (Zhang
et al. 2011), but was contrary to the chitosan microspheres, in
which only half of the activity of the encapsulated lysozymes
was maintained when employing the SPG membrane emulsi-
fication technique (Akamatsu et al. 2012). Hence, the SPG
membrane emulsification method could be used for the mi-
croencapsulation of ACEIPs.
Conclusions
The SPG membrane emulsification method was used to pre-
pare ACEIP-loaded SAL–O-CMC microspheres by using
VLPVP as a model. The microspheres could be saturated by
VLPVP, and their particle size could be predicated with a
linear equation when the pore size of the SPG membrane
was 5.0 μm or higher. This method was more efficient than
the B-390 encapsulator and needle extrusion in terms of ME,
VLPVP load, protection against simulated gastric fluid, and
controlled release, and the microencapsulation process did not
destroy the transport behavior or in vitro ACE inhibitory ac-
tivity of the pentapeptide. Hence, the SPG membrane emulsi-
fication was an effective way to microencapsulate ACEIPs.
2012
Acknowledgements We greatly thank the financial support from
t h e S c i e n c e a n d Te c h n o l o g y P l a n P r o j e c t o f S h e n z h e n
(JCYJ20170413155047512) and the National Natural Science
Foundation of China (31571890).
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
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