Colloids and Surfaces A 546 (2018) 136–142

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Colloids and Surfaces A

journal homepage: www.elsevier.com/locate/colsurfa

Stability of bioactive compounds from Thai Riceberry bran extract T

encapsulated within gelatin matrix during in vitro gastrointestinal digestion
⁎
Methavee Peanparkdeea, Ryo Yamauchia,b, Satoshi Iwamotoa,b,
a
Division of Science of Biological Resources, United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan
b
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: Digestive stability of developed capsules of bioactive compounds from Riceberry bran extract was investigated
Antioxidants by applying simulated in vitro gastrointestinal digestion model to assess the recovery of bioactive compounds
Average particle size entrapped within two types of gelatin: acid-processed (type A) and alkaline-processed (type B) gelatin. The
High-performance liquid chromatography physicochemical properties, namely, net surface charge (ζ-potential) and average particle size (Z-average), of the
Riceberry bran
extract and gelatin in the solute state were used to explain the effects of gelatin type and concentration on the in
Simulated gastrointestinal digestion
Zeta potential
vitro digestion stability of produced capsules. Extracts encapsulated with positively charged type A gelatin ex-
hibited greater encapsulation efficiency and percent recovery of bioactive compounds (exhibiting a net negative
charge) and increased antioxidant activity as measured by ferric reducing antioxidant power assays during
simulated gastrointestinal digestion than those entrapped with negatively charged type B gelatin. This result
indicated that electrostatic interactions were the main force affecting gelatin-based capsule formation.
Moreover, the highest Z-average was found in 2% (w/v) type B gelatin (475.57 nm) due to the high rate of
intermolecular bond formation, resulting in higher viscosity than that obtained with a concentration of 1% (w/
v), which may block the interaction between gelatin and bioactive compounds. Therefore, extracts encapsulated
with 2% type B gelatin yielded the lowest percent recovery values for bioactive compounds. The overall results
showed that net surface charge and particle size of gelatin affected the in vitro gastrointestinal digestion stability
of bioactive compounds from Riceberry bran extract.

Abbreviations: FRAP, ferric reducing antioxidant power; HPLC, high-performance liquid chromatography; PDI, polydispersity index; RBE, Riceberry bran extract; TFC, total flavonoid
content; TPC, total phenolic content; Z-average, average particle size; ζ-potential, zeta potential
⁎
Corresponding author at: Division of Science of Biological Resources, United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
E-mail address: isatoshi@gifu-u.ac.jp (S. Iwamoto).

https://doi.org/10.1016/j.colsurfa.2018.03.021
Received 27 November 2017; Received in revised form 23 January 2018; Accepted 7 March 2018
Available online 08 March 2018
0927-7757/ © 2018 Elsevier B.V. All rights reserved.
M. Peanparkdee et al.
1. Introduction
Thailand is one of the biggest agricultural producers and ex-
porters in the world, resulting in a large amount of agricultural
wastes and by-product of crop residues [1]. However, Thai farmers’
incomes are currently low compared to those in other Asian coun-
tries. Therefore, agricultural wastes and byproducts are being con-
sidered for development of novel products in order to expand the
utilization of these underrated materials as well as to increase Thai
farmer’s incomes.
Riceberry is a new variety of Thai purple rice cultivar which is
becoming popular in the world market due to its potential health
benefits. Thus, the Thailand government plans to encourage farmers
to grow Riceberry in order to meet the increasing market demand as
well as to increase farmers' incomes, since it has a higher price than
the normal white rice [2,3]. In commercial rice processing, rice is
usually milled and polished to remove husk and bran. Approximately
10% of the total weight of rice is rice bran, an undervalued by-pro-
duct obtained by the milling of whole grain rice [4,5]. In Thailand,
rice bran is still mainly used as animal feed [6], and the price is very
low.
Riceberry bran contains a large number of bioactive compounds,
including anthocyanins and some phenolic compounds. However, these
bioactive compounds are sensitive to environmental factors, such as pH,
temperature, light intensity, and oxygen [7,8], and have low stability
under human gastrointestinal conditions [9,10].
In our previous study [11], an encapsulation technique was used
to protect bioactive compounds from Riceberry bran. Various con-
ditions, including gelatin type and concentration and Riceberry bran
extract (RBE) concentration, were examined, and it was found that
1% (w/v) RBE provided capsules with good release behavior in water
at 37 °C and high stability under simulated gastric and intestinal
conditions. However, the effects of different types and concentrations
of gelatin on the stability of bioactive compounds from RBE under
gastrointestinal conditions have not yet been investigated or clar-
ified.
The digestive stability and bioaccessibility of antioxidant-rich cap-
sules are important considerations for ensuring appropriate bioactivity
of the target compounds when consumed. Therefore, the present study
aimed to develop a deep understanding of digestive stability using an in
vitro gastrointestinal model.
Gelatin, an animal protein, has been widely used as a polymer to
produce food and pharmaceutical products due to its biocompat-
ibility, biodegradability, good water solubility, low cost and nu-
merous available active groups to interact with targeting molecules
[12,13]. Additionally, it is considered as GRAS (Generally Recognized
as Safe) material by the United States Food and Drug Administration
(FDA) [13,14]. Gelatin can also improve the release properties of
drugs in the human digestive tract due to their structure with the high
number of different functional groups. These functional groups can
provide the opportunities to trap targeting-compounds which are
useful for developing targeted drug delivery systems [13,15]. In
general, gelatin is divided into two types based on the production
process, i.e., type A gelatin (via an acid process) and type B gelatin
(via an alkaline process), resulting in differences in the physico-
chemical properties, including the isoelectric point (pI) and pH, of
gelatin [16–18].
Accordingly, the purposes of this study were to (1) evaluate the
stability and functional properties of capsules from RBE under in vitro
gastrointestinal digestion with respect to the percent recovery of che-
mical properties and chemical compositions compared with un-
encapsulated crude extracts and (2) clarify the effects of gelatin type
and concentration on the stability of bioactive compounds using zeta
potential (ζ-potential) and average particle size data from gelatin so-
lutions and extracts.
137
Colloids and Surfaces A 546 (2018) 136–142
2. Materials and methods
2.1. Materials
Riceberry bran was collected from Kasetsart University
KamphaengSaen Campus, Nakhon Pathom, Thailand. RBE was pre-
pared by dissolving Riceberry bran in 65% (v/v) ethanol and sonicating
(43 kHz) at 51 °C for 40 min. The extract was filtered and vacuum dried
in a rotary evaporator at 40 °C [11].
Acid-processed (type A) gelatin and alkaline-processed (type B)
gelatin were obtained from Nitta Gelatin Inc., Osaka, Japan.
Protocatechuic acid, vanillic acid, p-coumaric acid, trans-ferulic acid,
sinapic acid, rutin, myricetin, quercetin 3-glucuronide, and anthocya-
nins, including cyanidin 3-glucoside and peonidin 3-glucoside, were of
high-performance liquid chromatography (HPLC) grade. Other reagents
and chemicals used were of analytical grade.
2.2. Preparation of capsules from RBE
According to the encapsulation procedures described in our pre-
vious study [11], capsules were produced by mixing 1% or 2% (w/v)
type A or type B gelatin solution with RBE at a gelatin:RBE weight ratio
of 4:1 at 37 °C. The mixture was then dropped on a Teflon-coated
stainless-steel plate and solidified in a freezer at −25 °C for 30 min. The
solidified particles were removed from the plate and dried using a
freeze dryer. The obtained capsules were kept at −25 °C until further
analysis.
2.3. ζ-Potential, average particle size (Z-average), and polydispersity index
(PDI)
A Zetasizer Nano ZS (Malvern Instruments, UK) was used to mea-
sure the ζ-potential of RBE and 1% and 2% (w/v) type A and type B
gelatin solutions. The calculation of ζ-potential values was based on the
Smoluchowski model.
The Z-average and PDI of RBE, 1% and 2% (w/v) type A and B
gelatin solutions, and 1 μg/mL of each standard chemical compound
were analyzed using dynamic light scattering. Measurements were
conducted at 37 °C.
2.4. Encapsulation efficiency
The entrapment efficiency of bioactive compounds from RBE in
gelatin matrix was determined by dissolving 50 mg of capsules in 1 mL
of methanol. The amount of each bioactive compounds was quantified
using HPLC, as described previously by Peanparkdee, Iwamoto and
Yamauchi [11]. The percentage of entrapment efficiency of anti-
oxidants was calculated as follows:
L
Encapsulation efficiency (%) = × 100
L0
where L is the amount of encapsulated bioactive compounds and L0 is
the initial amount of bioactive compounds used for capsules prepara-
tion.
2.5. Static in vitro digestion
A static model that simulated digestion in the mouth, stomach, and
intestines [19–21] was used, with modifications. For preparing simu-
lated saliva fluid, 2.38 g Na2HPO4, 0.19 g KH2PO4, and 8 g NaCl were
dissolved in 1 L distilled water, and the pH was adjusted to pH 6.75. α-
Amylase (EC 3.2.1.1) was added to the mixture to obtain 200 U enzyme
activity. To provide simulated gastric fluid, 0.31% pepsin (from porcine
gastric mucosa: pepsin A, EC 3.4.23.1) was mixed with 0.03 M NaCl,
and the pH of the mixture was adjusted to pH 1.2 with 1 M HCl. Bile
extract and pancreatin mixture was prepared by dissolving 0.05 g
M. Peanparkdee et al. Colloids and Surfaces A 546 (2018) 136–142

pancreatin and 0.3 g bile extract porcine in 35 mL of 0.1 M NaHCO3.

The pH of the mixture was adjusted to pH 6.0 with 0.1 M NaHCO3.

2.6. In vitro digestion of unencapsulated crude extract and encapsulated

RBE

A 0.1-mL volume of crude extract or 50 mg capsules was digested

[11]. The samples were transferred to test tubes, mixed with 10 mL
simulated saliva fluid, and incubated for 10 min. Next, 10 mL simulated
gastric fluid was added to the mixture, and the mixture was incubated
for 2 h. After gastric digestion, 7.5 mL of bile extract and pancreatin
mixture, 1.25 mL of 120 mM NaCl and 1.25 mL of 5 mM KCl were
added. The pH of the mixture was then increased to 7.0 with 1M NaOH. Fig. 1. Z-average and polydispersity index (PDI) of gelatin.

The mixtures were incubated again for 2 h. Incubation of all mixtures

was performed at 37 °C in a water bath equipped with horizontal agi-
tation.
The digested samples were analyzed for chemical properties (total
phenolic content [TPC], total flavonoid content [TFC], and ferric re-
ducing antioxidant power [FRAP]) and chemical compositions using
HPLC, as described previously by Peanparkdee, Iwamoto and Yamauchi
[11]. The results were calculated as the percent recovery as follow:
R the pI of type B gelatin and lower than that of type A gelatin. For this
Percent recovery (unencapsulated RBE) = × 100
R0
where R denotes the chemical properties of RBE after digestion, R0
denotes the chemical properties of RBE before digestion.
E
Percent recovery (encapsulated RBE) = × 100
E0
whereas 1% and 2% (w/v) alkaline-treated (type B) gelatin (-9.07 and
-8.64 mV, respectively) and RBE (−14.80 mV) exhibited a net negative
charge.
Type A gelatin had a pI of around 8.5–9.0, whereas that of type B
was around 4.5–5.1. During the encapsulation process, the pH value of
the mixtures of gelatin and RBE was around 5.8, which was higher than
reason, type A gelatin showed positive ζ-potential values, whereas type
B gelatin showed negative ζ-potential values. Additionally, positive and
negative charges on types A and B gelatin increased as the concentra-
tion of gelatin decreased.

where Ee denotes the chemical properties of encapsulated RBE after

digestion (without the chemical property values of gelatin), E0 denotes
the chemical properties of encapsulated RBE before digestion (without
the chemical property values of gelatin).
2.7. Statistical analysis
All determinations were performed in triplicate. The experimental
data were analyzed using analysis of variance, followed by Duncan’s
new multiple range test. Statistical significance was used to evaluate the
differences between means at a significance level of p < 0.05.
3. Results and discussion
3.1. ζ-Potential of the RBE and gelatin
Gelatin (types A and B) and bioactive compounds in RBE have an
electrical charge; therefore, electrostatic interactions may play an im-
portant role in determining their physicochemical properties and
binding abilities. The electrostatic interactions of gelatin and RBE can
be predicted by measuring the absolute values of ζ-potential, which is
an indicator of the surface charge of colloids or particles in solution
[22–24].
Table 1 demonstrates the absolute values of ζ-potential of the RBE
and gelatin. The data showed that 1% and 2% (w/v) acid-treated (type
A) gelatin had a net positive charge (8.03 and 6.12 mV, respectively),
3.2. Average particle sizes (Z-averages) and PDIs of gelatin, RBE, and
standard chemical compounds
Average particle sizes (Z-averages) and PDIs of types A and B gelatin
at concentrations of 1% and 2% (w/v) are presented in Fig. 1. Type B
gelatin had the largest particle size (475.57 nm) at a concentration of
2% (w/v), followed by 1% (w/v) type B gelatin (346.20 nm) and type A
gelatin; however, there were no significant differences between the Z-
averages of 1% and 2% (w/v) concentrations (245.90 and 278.33 nm,
respectively).
The size distribution of the particles in medium can be defined by
the PDI value. The PDI value range from 0.01 to 0.5 for monodispersed
particles, and PDI values greater than 0.7 are characteristic of poly-
dispersed particles with a broad size distribution [25]. From the results,
the PDI values of 1% and 2% type A gelatin and 1% and 2% (w/v) type
B gelatin were 0.33, 0.32, 0.34, and 0.41, respectively, which were all
lower than 0.5. These results revealed that all gelatin samples used in
this study were monodispersed with a narrow size distribution.
As shown in Table 2, the RBE exhibited a very large particle dia-
meter (2451 nm) with broad size distribution (PDI = 0.97) due to the
presence of various types of bioactive compounds, including phenolic
acids, flavonoids, and anthocyanins, which have different particle sizes.
The Z-averages of major compounds contained in RBE ranged from
388.23 to 741.47 nm, and the PDI values of all compounds were lower
than 0.5.

Table 1
ζ-Potential values and isoelectric points of RBE and gelatin.

Properties RBE Type A gelatin Type B gelatin

1% 2% 1% 2%

ζ-potential (mV) −14.80 ± 0.92 8.03 ± 0.21 6.12 ± 0.63 −9.07 ± 0.82 −8.64 ± 0.26
Isoelectric pointa NA 8.5–9.0 4.9–5.1

Values are expressed as means ± SDs (n = 3).

a
Data were provided by Nitta Gelatin Inc., Osaka, Japan.

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M. Peanparkdee et al. Colloids and Surfaces A 546 (2018) 136–142

Table 2
Z-average and polydispersity index (PDI) values of RBE and standard chemical com-
pounds.

Sample Z-average (d.nm) PDI

RBE 2451 ± 40.31 0.97 ± 0.04

Phenolic acids
Protocatechuic acid 741.47 ± 28.68 0.12 ± 0.02
Vanillic acid 474.20 ± 28.14 0.16 ± 0.01
p-Coumaric acid 511.20 ± 13.35 0.24 ± 0.02
Ferulic acid 422.27 ± 17.86 0.22 ± 0.01
Sinapic acid 556.10 ± 24.89 0.28 ± 0.03
Flavonoids
Rutin 562.55 ± 9.12 0.46 ± 0.07
Myricetin 564.95 ± 41.65 0.39 ± 0.01
Quercetin 3-glucuronide 388.43 ± 19.73 0.28 ± 0.01
Anthocyanins
Cyanidin 3-glucoside 476.65 ± 17.18 0.31 ± 0.01
Peonidin 3-glucoside 460.80 ± 8.94 0.23 ± 0.01

Values are expressed as means ± SDs (n = 3).

3.3. Encapsulation efficiency of bioactive compounds from RBE in gelatin

matrix

Table 3 shows that capsules produced using 1% (w/v) of type A

gelatin provided higher encapsulation efficiency of bioactive com-
pounds; approximately 25–99% of phenolic acids, 82–99% of flavo-
noids, and 96–97% of anthocyanins, than those produced using 2% (w/
v) of type A gelatin, and 1 and 2% (w/v) of type B gelatin. Moreover,
encapsulation efficiency of sinapic acid and myricetin within 2% (w/v)
of type B gelatin matrix could not be detectable.

3.4. Effects of simulated digestion on the chemical properties of

unencapsulated and encapsulated RBE

The percent recovery of chemical properties (TPC, TFC, and anti-

oxidant activity measured with FRAP assays) of the capsules under si-
mulated gastrointestinal conditions are shown in Fig. 2. RBE en-
capsulated with 1% (w/v) type A gelatin provided the highest percent Fig. 2. Percent recovery of chemical properties (TPC, TFC, and FRAP) of unencapsulated
recovery of TPC, TFC, and FRAP after simulated saliva digestion (55.80, (crude extract) and encapsulated RBE under simulated saliva (a), simulated gastric (b),
81.23, and 86.79, respectively), as presented in Fig. 2a. During in vitro and simulated intestinal (c) conditions.
gastric digestion (Fig. 2b), there was no significant difference between
percent recovery of FRAP for RBE encapsulated with 1% and 2% (w/v)

Table 3
Encapsulation efficiency of bioactive compounds in various types and concentrations of gelatin matrix.

Compounds Encapsulation efficiency (%)

Type A gelatin Type B gelatin

1% (w/v) 2% (w/v) 1% (w/v) 2% (w/v)

Phenolic acids
Protocatechuic acid 58.54a ± 0.06 51.47b ± 0.17 46.15c ± 0.17 45.09d ± 0.37
Vanillic acid 63.61b ± 0.44 65.90a ± 1.33 53.10c ± 0.23 52.54c ± 1.74
p-Coumaric acid (II) 34.68a ± 0.29 33.70b ± 0.16 22.83c ± 0.17 19.78d ± 0.33
Ferulic acid (II) 25.42b ± 0.11 28.63a ± 0.33 18.81d ± 0.35 21.32c ± 0.29
Sinapic acid 99.72a ± 3.14 ND 56.32b ± 1.09 ND
Flavonoids
Rutin 82.09a ± 0.23 69.63c ± 2.20 77.57b ± 0.43 62.58d ± 1.76
Myricetin 98.92a ± 0.62 ND 62.17b ± 1.13 ND
Quercetin 3-glucuronide 95.44a ± 1.24 85.66b ± 0.86 82.09c ± 2.01 66.84d ± 1.16
Anthocyanins
Cyanidin 3-glucoside 96.61a ± 0.46 87.27b ± 0.83 82.35c ± 0.70 74.45d ± 1.35
Peonidin 3-glucoside 97.24a ± 2.77 82.91c ± 2.74 89.39b ± 1.35 77.75d ± 1.57

Values followed by different letters are significantly different (p ≤ 0.05).

ND = not detectable.

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Table 4
Percent recovery of bioactive compounds contained in RBE and capsules under simulated saliva conditions.

Compounds Unencapsulated RBE Capsules

Type A gelatin Type B gelatin

1% (w/v) 2% (w/v) 1% (w/v) 2% (w/v)

Phenolic acids 　 　 　 　 　
Protocatechuic acid 22.72c ± 1.61 64.57a ± 4.59 60.49ab ± 4.30 54.12b ± 3.85 25.92c ± 1.85
Vanillic acid 25.12d ± 1.92 63.59c ± 4.85 81.09a ± 0.95 70.45b ± 0.83 26.83d ± 2.05
p-Coumaric acid 80.96b ± 5.78 9.53e ± 0.67 104.54a ± 2.22 56.04c ± 4.00 44.45d ± 3.18
Ferulic acid 20.85c ± 1.73 46.74a ± 3.84 18.19cd ± 1.50 31.87b ± 2.63 14.90d ± 0.01
Sinapic acid 23.89d ± 0.45 64.40b ± 1.21 70.41a ± 1.13 51.74c ± 1.03 ND
Total 25.01c ± 1.40 60.29a ± 3.36 58.48a ± 0.74 54.02b ± 0.69 24.88c ± 1.41
Flavonoids 　 　 　 　 　
Rutin 21.32c ± 1.73 61.88a ± 5.03 13.14d ± 1.07 12.84d ± 1.52 30.32b ± 2.46
Myricetin 68.17c ± 6.51 66.56c ± 0.10 101.08a ± 0.10 84.83b ± 1.00 32.66d ± 2.53
Quercetin 3-glucuronide 17.94d ± 0.81 51.22a ± 2.32 43.36b ± 1.97 46.37b ± 2.10 21.46c ± 0.97
Total 19.23d ± 1.62 56.77a ± 2.66 34.34b ± 1.62 33.89b ± 1.59 25.62c ± 1.20
Anthocyanins 　 　 　 　 　
Cyanidin 3-glucoside 12.56c ± 0.78 23.07b ± 1.43 36.71a ± 2.28 23.25b ± 1.44 ND
Peonidin 3-glucoside 21.16d ± 1.89 40.96c ± 3.65 73.88a ± 4.07 53.38b ± 2.94 ND
Total 17.48d ± 1.32 33.32c ± 2.52 62.41a ± 2.90 42.00b ± 3.18 ND

Values followed by different letters are significantly different (p ≤ 0.05).

ND = not detectable.

type A gelatin (88.94% and 90.01%, respectively). In contrast, 1% (w/
v) type A gelatin gave the highest percent recovery of TPC (62.36%),
whereas those of TFC and FRAP were highest in RBE encapsulated with
2% (w/v) type A gelatin (101.46% and 70.43%, respectively) during
simulated intestinal digestion (Fig. 2c). These FRAP results provide
indirect evidence of structural inactivity of bioactive compounds after
simulated digestion of capsules. During simulated digestion process, the
structure of bioactive compounds might be changed from their original
forms into the inactive forms, resulting in a low antioxidant activity
value measured by FRAP assay. Therefore, capsules produced using 1%
(w/v) of type A gelatin which provided the greatest value of percentage
recovery of TPC, showed a lower value of percentage recovery of FRAP
than that obtained using 2% (w/v) of type A gelatin.
The chemical compositions of each capsule after in vitro gastro-
intestinal digestion were identified using HPLC. The results were cal-
culated as percent recovery of each compound (Tables 4–6). From the
results shown in Table 4, we found that 1% (w/v) type A gelatin had a
higher capacity to protect total flavonoids (56.77%), whereas 2% (w/v)
gelatin showed a higher efficiency to preserve total anthocyanins
(62.41%) from simulated saliva conditions.
In the case of simulated gastric digestion (Table 5), RBE en-
capsulated with 2% (w/v) type A gelatin provided higher percent re-
covery values for total phenolic acids (38.22%), flavonoids, and an-
thocyanins (59.93%) than other samples.
During in vitro intestinal digestion (Table 6), RBE encapsulated with
1% (w/v) type A gelatin showed greater stability of total phenolic acids
(30.12%) and anthocyanins (40.77%), whereas the highest percent re-
covery of total flavonoids (29.63%) could be observed in those en-
trapped with 2% (w/v) type A gelatin.
The overall results demonstrated that type A gelatin had a greater
ability to protect bioactive compounds from RBE compared with type B
gelatin. These results can be explained by the absolute values of ζ-po-
tential and particle size for RBE and gelatin, as described in Sections 3.1
and 3.2. The RBE had a negative surface charge because the major

Table 5
Percent recovery of bioactive compounds contained in RBE and capsules under simulated gastric conditions.

Compounds Unencapsulated RBE Capsules

Type A gelatin Type B gelatin

1% (w/v) 2% (w/v) 1% (w/v) 2% (w/v)

Phenolic acids 　 　 　 　 　
Protocatechuic acid 20.23c ± 2.25 32.80b ± 2.33 13.81d ± 0.98 43.99a ± 3.13 23.35c ± 2.30
Vanillic acid 16.23d ± 1.24 41.71b ± 3.18 62.45a ± 4.77 15.10d ± 1.15 34.67c ± 0.43
p-Coumaric acid 39.58b ± 0.82 77.58a ± 5.61 27.37c ± 1.98 34.06b ± 2.43 27.78c ± 1.99
Ferulic acid 18.83b ± 1.55 18.87b ± 1.56 54.12a ± 4.47 16.71b ± 1.38 8.92c ± 0.74
Sinapic acid 43.29c ± 0.82 76.78a ± 1.47 62.41b ± 1.00 62.52b ± 1.25 31.04d ± 0.62
Total 19.56d ± 1.09 33.54b ± 0.44 38.22a ± 2.13 30.58b ± 1.71 24.14c ± 1.37
Flavonoids 　 　 　 　 　
Rutin 27.60c ± 2.24 45.84b ± 3.73 62.41a ± 5.07 25.33c ± 2.05 5.44d ± 0.44
Myricetin 65.06b ± 0.01 109.33a ± 1.00 48.34c ± 6.89 79.77b ± 1.06 20.73d ± 1.87
Quercetin 3-glucuronide 23.82d ± 3.69 52.15b ± 2.36 57.24a ± 2.60 48.14c ± 2.18 9.20e ± 0.41
Total 26.05d ± 1.22 53.07b ± 2.49 59.93a ± 2.81 40.21c ± 1.88 7.07e ± 1.01
Anthocyanins 　 　 　 　 　
Cyanidin 3-glucoside 45.57c ± 0.66 82.59a ± 5.11 65.13b ± 4.04 62.14b ± 3.85 29.19d ± 1.81
Peonidin 3-glucoside 44.80c ± 2.47 82.53a ± 7.35 87.69a ± 4.83 65.92b ± 5.87 30.14d ± 1.66
Total 44.05c ± 2.68 85.73a ± 3.97 83.63a ± 3.87 66.80b ± 3.09 30.57d ± 2.31

Values followed by different letters are significantly different (p ≤ 0.05).

ND = not detectable.

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Table 6
Percent recovery of bioactive compounds contained in RBE and capsules under simulated intestinal conditions.

Compounds Unencapsulated RBE Capsules

Type A gelatin Type B gelatin

1% (w/v) 2% (w/v) 1% (w/v) 2% (w/v)

Phenolic acids 　 　 　 　 　
Protocatechuic acid 23.80a ± 2.65 15.21b ± 1.08 5.42c ± 0.38 17.85b ± 1.27 4.26c ± 0.30
Vanillic acid 25.87c ± 1.97 53.84b ± 4.11 44.19b ± 3.37 5.86d ± 0.05 23.43c ± 1.79
p-Coumaric acid 77.96a ± 1.62 62.98a ± 4.52 14.35d ± 1.04 13.77d ± 0.80 21.74c ± 1.55
Ferulic acid 17.52b ± 0.01 23.31a ± 1.92 13.97c ± 1.16 6.78e ± 0.56 11.65d ± 0.01
Sinapic acid ND 53.95b ± 1.03 57.61a ± 0.92 8.19d ± 0.16 13.80c ± 0.28
Total 23.40b ± 0.30 30.12a ± 1.68 19.44c ± 1.08 12.16d ± 0.68 12.37d ± 0.70
Flavonoids 　 　 　 　 　
Rutin ND 16.47a ± 1.27 18.42a ± 1.50 10.28b ± 0.83 8.40b ± 0.68
Myricetin ND 40.82ab ± 5.71 48.92a ± 6.98 28.23bc ± 1.80 20.10c ± 1.78
Quercetin 3-glucuronide 19.12b ± 0.35 16.95c ± 0.76 39.87a ± 1.81 19.53b ± 0.89 20.13b ± 0.92
Total 9.56c ± 1.37 15.34b ± 2.38 29.63a ± 2.03 15.17b ± 1.46 15.63b ± 0.73
Anthocyanins 　 　 　 　 　
Cyanidin 3-glucoside 28.79a ± 1.78 24.38b ± 1.51 24.72b ± 1.53 ND ND
Peonidin 3-glucoside ND 50.32a ± 4.48 31.22b ± 2.78 ND ND
Total 12.29c ± 0.93 40.77a ± 1.89 28.43b ± 2.15 ND ND

Values followed by different letters are significantly different (p ≤ 0.05).

ND = not detectable.

components were phenolic acids, flavonoids, and anthocyanins, which
consist of negatively charged carboxyl and hydroxyl groups [26].
Therefore, RBE could show stronger electrostatic interactions with po-
sitively charged (type A) gelatin compared with negatively charged
(type B) gelatin. These results provide evidence that electrostatic in-
teractions played an active role in mediating the interaction between
gelatin and bioactive compounds in RBE. Furthermore, gelatin can also
interact with bioactive compounds through other bonds, namely hy-
drogen bonds, and hydrophobic interactions [12,15,27]. Haroun and El
Toumy [27] studied the effects of polyphenol extracts from Acacia ni-
lotica bark on the physicochemical properties of crosslinked gelatin-
based polymeric biocomposites. They characterized the prepared films
using Fourier-transform infrared spectroscopy ((FT-IR)) and reported
that hydrogen bonding and hydrophobic interactions were the major
forces involved in the stabilization of gelatin-based polymeric bio-
composite film. Quiroz-Reyes, Ronquillo-de Jesús, Duran-Caballero and
Aguilar-Méndez [12] investigated the interactions among the gelatin
protein matrix and polyphenolic extracts from cocoa using (FT-IR).
They found that the C]O group of gelatin interacted with the OeH
groups of polyphenolic extracts via hydrogen bonding. Moreover, the
percent recoveries of chemical properties and chemical compounds of
the RBE encapsulated with type A gelatin were also higher than those of
unencapsulated RBE.
Concentration has an influence on the formation of intra- and in-
termolecular crosslinks of gelatin. Intermolecular crosslink is the in-
teraction between functional group of two or more gelatin molecules,
whereas intramolecular crosslink is the interaction within one molecule
[28,29]. At low concentrations, intramolecular crosslinks are formed,
whereas both intra- and intermolecular crosslinks occur at high con-
centrations [28,30], yielding a gelatin solution with high viscosity.
These phenomena may affect the average particle size obtained by
dynamic light scattering as well as the efficiency of capsules. The results
of the average particle size show that 2% (w/v) type B gelatin was
significantly larger than that of 1% (w/v) type B gelatin which might be
due to the formation of intermolecular crosslinks. However, the con-
centration seemed to have no effect on the particle size of type A ge-
latin. These results were also observed in previous studies. Fruhner and
Kretzschmar [31] explained that the viscosity of gelatin solution de-
pends on the size and conformational changes of the molecules. In this
study, the pH value applied during the encapsulation process was
around 5.8, which was close to the pI of type B gelatin. Thus, gelatin
may promote intra- and intermolecular crosslink formation and ag-
gregation, resulting in the increase in PDI and viscosity.
In addition, the high viscosity of the solution may prevent the in-
ternal interactions between gelatin and bioactive compounds from RBE,
resulting in large and thick-walled capsules [11,32–34]. For this reason,
2% (w/v) type B gelatin had reduced ability to entrap and protect
bioactive compounds from RBE than 1% (w/v) type B gelatin.
4. Conclusions
The acid-processed (type A) gelatin had high ability to entrap and
protect bioactive compounds from RBE. RBE encapsulated with type A
gelatin showed an obvious decrease in the loss of bioactive compounds,
namely, phenolic acids, flavonoids, and anthocyanins, and in their an-
tioxidant activity, as measured by FRAP assays during simulated in vitro
gastrointestinal digestion. Moreover, it was also found that the ζ-po-
tential and average particle size values of gelatin played important roles
in determining the efficiency of produced capsules. The positively
charged type A gelatin could effectively interact with negatively
charged RBE by electrostatic interactions. However, alkaline-processed
(type B) gelatin, a negatively charged polymer, exhibited a low capacity
for preserving bioactive compounds from RBE due to its surface charge
and large particle size. In this study, the average particle size of type B
gelatin increased with increasing concentration of gelatin, thereby re-
ducing the ability of 2% (w/v) gelatin. Nevertheless, the concentration
had no effect on the physical properties of type A gelatin.
In this study, the stability of capsules of bioactive compounds from
RBE under simulated gastrointestinal digestion was investigated.
Nevertheless, the stability of capsules under various storage conditions,
including differences in light, temperature, and oxygen, is also an im-
portant parameter in evaluation of the efficiency of capsules and should
be examined in further studies in order to facilitate the development of
novel products in the food and pharmaceutical industries, such as food
additives, food supplements, and medicines.
Conflicts of interest
None.

141
M. Peanparkdee et al.
Acknowledgements
The authors would like to thank Associate Professor Apichart
Vanavichit (Kasetsart University, Thailand) for kindly providing the
Riceberry bran sample, Associate Professor Emiko Yanase (Gifu
University, Japan) for the suggestions and advices, and Nitta Gelatin,
Inc. for providing the gelatin samples. This work was partly support by
JSPS KAKENHI (grant number JP26450364).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfa.2018.03.021.
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