These results demonstrate that whey proteins can be used as a convenient material for improving
L. fermetum 39-183 protection. Whey protein microcapsule has an excellent capacity to encapsulate
bioactive organisms that are sensitive to stomach circumstances, with concomitant controlled release at a
defined location. Whey protein encapsulation efficiently minimizes the bacteriocidal effects of the gastric
pH and maximizes the number of probiotics reaching the ileum and subsequently the colon. Thus, this
encapsulation technique may act as a platform technology for promoting targeted delivery of probiotics
with potential applications within the food and pharmaceutical industries.
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84
USE OF WHEY PROTEIN FOR ENCAPSULATION AND CONTROLED
RELEASE OF PROBITOIC FROM PROTEIN MICROENCAPSULATE IN
EX VIVO PORCINE GASTROINTESTINAL CONTENTS
Nguyen Thi My Le
*
, Nguyen Van Hieu, Pham Viet Nam
Ho Chi Minh City University of Food Industry
*
Email: mylethang81@yahoo.com
Received: 20/08/2017; Accepted: 30/08/2017
ABSTRACT
The aim of this study was to evaluate the efficacy of whey protein isolate (WPI) as an encapsulation
matrix for improvement of L. fermentum 39-183 viability to low pH and bile and releasing the
encapsulated bacteria in ex vivo porcine gastrointestinal (GI) contents. 1g of protein microcapsules (≈ 108
CFU of L. fermentum 39-183 or E. coli GFP+) were incubated in ex vivo porcine GI contents (9 mL)
under anaerobic conditions at 37 ºC. Results showed that there was higher than 86% cell survival for
encapsulated L. fermentum 39-183 after 3 h incubation in pH 2.0, whereas free cell experienced complete
viability loss. Encapsulated L. fermentum 39-183 showed only about 0.86 log reduction for all bile salt
levels tested (0.5 ÷ 2.0%), while 3.34 log decrease of free cell after 6 h of incubation. There was almost a
complete release (3.9 x 108 CFU) of microencapsulated bacteria in the ileal contents within 2 h, while
there was no significant release of encapsulated bacteria in the gastric contents even after 8 h of
incubation. This study led to the development and design of a protein capsulation for reinforced probiotic
protection during the stressful conditions of gastric and controlled release at a defined location.
Keywords: Protein capsules, ex-vivo porcine gastrointestinal contents, lactobacillus fermentum 39-183,
probiotics.
1. INTRODUCTION
Microencapsulation with respect to a food application involves reversible of active bio-molecules in
stable core and releasing it at desired sit (such as intestine or colon) in viable form. Probiotics, minerals,
vitamins, phytosterols, fatty acids, lycopene and antioxidants are some of the compounds which have
been delivered through microencapsulation techniques. Probiotics are defined as living microorganisms
that contribute to beneficial effects on human health upon ingested in adequate dose and have been
widely incorporated in various dairy products and marketed as functional foods [1]. However, there is a
considerable loss of viability as probiotic bacteria pass through the low pH of the stomach and high bile
salt conditions of the intestine. Choice of the capsule materials is a major element for successful
microencapsulation of probiotics and the use of microencapsulation probiotics in functional foods [2]. As
a biopolymer used for a coating agent of probiotic live bacteria, whey protein also appears as a potential
candidate because it is entirely biodegradable and frequently used in many types of food product. Whey
protein isolate have been shown to increase the survival of probiotics in SGJ by the addition of the isolate
to the bacterial culture or as a wall material for encapsulation. Bifidobacterium infantis was subjected to
SGJ at pH 2.0 for 3 h in the presence of 1 gL
-1
whey protein isolate [3]. The presence of whey protein
isolate significantly improved survivability when compared to bacteria incubated in SGJ without WPI.
Lactobacillus rhamnosus was encapsulated by extrusion using a 7:3 mixture of 12% WPI:bacteria. This
material was subjected to a dynamic gastrointestinal model which varied in pH from 4.4 to 2.0 over 90
min [4]. However, the survival level was strain-dependent and once the encapsulated, bacteria reach the
Use of whey protein for encapsulation and controled release of probitoic from protein...
85
targeted organs, it is ideal for the microencapsulated matrix to release them in a controlled fashion. There
has little published data on the conditions and profile of release of probiotics from protein matrices in ex
vivo and in vivo GI condition. Such the objectives of this research were to assess acid and bile salt
resistance of whey microcapsule and investigate the release profile of Lactobacillus fermentum 39-183
from whey microcapsule in ex vitro porcine gastrointestinal contents.
2. MATERIALS AND METHODS
2.1. Bacterial strains and culture conditions
Lactobacillus fermentum 39-183 and green fluorescent tagged Escherichia coli (E. coli GFP
+
) were
used in this study. L. fermentum 39-183 sourced from our previous research [5].The stock culture was
maintained at -20 ºC in MRS broth (Merck Darmstadt, Germany) supplemented with 50% sterile glycerol.
Prior to use, the culture [1% (v/v)] was transferred twice to MRS broth and incubated at 37 ºC for
18÷20h.
E. coli GFP
+
K12 was supplied by the Department Biotechnology of Ho Chi Minh City University
of Science. Prior to use, the culture [1% (v/v)] was transferred twice to LB broth (Oxoid, Sydney,
Australia) containing ampicillin (100 mg/mL) and incubated at 37 ºC for 24 h.
2.2. Microencapsulation of microorganisms
The preparation of the whey microcapsule was carried out according to the method described by
O’Neill [6] with some modification. Whey protein isolate (WPI) powder (6% w/v) was rehydrated in
distilled water, agitating the solution for 1 hour at room temperature and then allowed to stand for 2 hours
to ensure complete hydration of the proteins. Sodium azide was added (0.02%) to the whey protein
solution and heated at 90
ºC for 30 minutes. The denatured WPI solution was then cooled and held at
room temperature for 2 hours. To form microcapsules, the encapsulation matrix (6% (w/v) WPI) and the
cell suspension mixture (7:3) was injected through in a 25G needle into a filter sterilized cross-linking
solution (5% (w/v) CaCl2 + 10% (w/v) Tween 80), which was stirred at 250 rpm by a magnetic stirrer.
The resulting capsules was allowed to harden in the cross – linking solution for 1 hour, and were then
collected by filtration using cheesecloth, which was sterilized in boiling water for 12 min ahead of use.
The microcapsules were then washed with distilled water and collected for the following tests.
2.3. Characterisation of microparticles
The shape and surface morphology of the microcapsules was observed with a scanning electron
microscope (SEM) and the average size of the microcapsules was evaluated by Particle Size Distribution
Analyzer LA-920 (HORIBA, JAPAN).
2.4. Determination of L. fermentum 39-183 Viability in Whey microcapsule
The viability of the encapsulated L. fermentum 39-183 in whey microcapsule was determined by
vigorously homogenizing 1g of the micro-bead in 9 mL of sterile phosphate buffer solution (PBS) pH 7.0
for in 10 min at room temperature. Viable cell (CFU/g or CFU/mL) was determined by plating on MRS
plates and incubating at 37 ºC for 48 hours.
2.5. Survival of free and encapsulated in simulated gastric juice (SGJ) and bile salts
The Simulated Gastric Juice (SGJ) were prepared by suspending of 3.5 g D-glucose, 2.05 g NaCl,
0.6 g KH2PO4, 0.11 g CaCl2, 0.37 g KCl, 0.05 g oxgall bile (MI, Sigma) and 13.3 g pepsin in 1000 mL
distilled water according to the method of Kim et al., (2007). The artificial gastric juice was adjusted to
different pH values (2, 4 and 7) used 1M HCl. MRS broth without addition of bile salt was used as a
control. Either wet whey microcapsule (1g) containing L. fermentum 39-183 or 1 mL of washed cell
suspension were added into the prepared tubes (9 mL prepared solutions/tube) and incubated 24 h at
37 ºC for 0, 6, 12, 18 and 24 h. The whey microcapsule was then removed and placed in 9 mL of sterile
phosphate buffer solution (PBS) pH 7.0 for in 10 min at room temperature. Total viable cells numbers
were determined by the plate count method. The resistance to bile salts was determined by inoculating
free and encapsulated cell in MRS broth containing 0.5%, 1.0%, 2.0% (w/v) Ox-bile (Biochemika, Fluka;
Nguyen Thi My Le, Nguyen Van Hieu, Pham Viet Nam
86
Sigma-Aldrich) after 6 h incubation at 37 ºC. All samples were treated in triplicates.
2.6. Release profile of microencapsulated bacteria in porcine gastrointestinal contents (ex vivo)
Gastrointestinal contents (gastric, duodenum, jejunum, ileum and colon) from three different pigs (8
months old) were collected and used within 1-2 h after slaughtering. Wet whey microcapsule (1 g)
(containing ≈108 CFU L. fermentum 39-183 or E. coli GFP+/ 1g microcapsule) were incubated in different
sections of intestinal contents (9 mL) for 3 h at 37
ºC under anaerobic conditions. Samples of 1 mL were
collected at different time intervals (0, 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0 h), and enumerated for L. fermentum
39-183 or E. coli GFP
+
by spread plating on Lactitol-Lactobacillus-Vancomycin (LLV) agar or LB-
ampicillin/arabinose media, respectively. The fluorescent bacteria were plated on LB agar (Oxoid,
Australia) containing 100 mg/mL ampicillin and 1.2 mg/mL arabinose and enumerated after 24 h
incubation by observing green fluorescent colonies under a UV illuminator. All samples were treated in
triplicates.
2.7. Statistical analysis
Results of three independent assays are presented as mean values ± standard deviation (SD). Data
were analyzed by ANOVA and Turkey’s test. Statistical analysis was carried out with the Statgraphics
Centurion XV program (Statgraphics, USA). Results were considered significantly different at p < 0.05.
3. RESULTS AND DISCUSSION
3.1. Characteristics of Whey microcapsule
Extrusion is the oldest and most common technique used for microencapsulating probiotics in
hydrocolloid gel matrices. The size and shape of the capsules are influenced by many factors. In this
study, L. fermenrum 39-183 was microencapsulated with whey protein isolate by extrusion method and
the shape and surface morphologies of the whey protein microcapsule were investigated using SEM and
shown in Figure 1. Whey protein microcapsule exhibited a spherical shape with a wrinkled surface (a
round, flattened shape – not completely smooth – without visible cracks or pores on the surface).
Extrusion method has been used for producing capsules with 0.2 to 5 mm. Microcapsule size is an
important consideration since the microcapsules must have a high volume-to-surface ration for increasing
the protective effect and be sufficiently small to avoid a negative sensory impact [7]. In this study, the
mean whey protein microcapsule size was 311.9 µm, so formulation cannot be discriminated by the size
criterion.
Figure 1. Scanning electron microscopic (SEM) observation of whey microencapsule. Sympols: (A) –
shape morphology of whey microcapsule, (B) – surface morphology of whey microcapsule
3.2. Simulated gastric juice tolerance of whey protein microcapsule
One of the main barriers for oral probiotic bacteria is the stomach low pH, which is related to the
high hydrochloric acid concentration of the gastric acid. To test the performance of the encapsulated and
free cell L. fermentum 39-183 at different pH values, they were incubated in the artificial gastric juice
adjusted pH 2.0, 4.0 and 7.0 after 6, 12, 18 and 24 hours. Viability of L. fermentum 39-183 at pH 2.0 and
4.0 appeared to decrease as the incubation period increased (Fig 1). However, L. fermentum 39-183
showed growth over the incubation period at pH 7.0, with both encapsulated and free cell. Encapsulated
cell increased 2.18 log CFU/mL, while free cell increased 0.65 log CFU/mL. After 24 hours of exposure,
encapsulated cell was highly tolerant and retained their viability under acidic conditions at pH 4.0.
Use of whey protein for encapsulation and controled release of probitoic from protein...
87
Encapsulated cell decreased 2.25 log less than free cell. At pH 2.0, statistical analysis showed a
significant difference (p < 0.05) between reductions obtained with encapsulated and free cells. The
encapsulated cell showed a 2.63 log reduction, while the free cell decreased by 4.78 log after 6 hour of
incubation. This suggested that whey protein isolated protected and significantly improved survivability
of L. fermentum 39-183 in the SGJ. According to Lundin et al., (2008), during digestion the microcapsule
could be influenced as follows: hydrolyzation by acid, proteolysis by pepsin, shearing forces by peristaltic
stomach movements and finally body temperature [8]. Gastric pepsin enzyme may cause the protein
hydrolysis into polypeptides, oligopeptides and some free amino acids. One of the reasons explaining the
good resistance of whey protein capsule could be that the cleavage sites were partially hidden in the
structure. In addition, it has been demonstrated that low pH had no effect on the composition and
structure of whey protein [9]. However, the survival both types of cell (encapsulated and free) rapidly
decreased and did not survive after 24 hours of incubation.
Figure 2. Survivability of encapsulated and free L. fermentum 39-183 over 24 hours of incubation at
different pH values. Symbols: a – free cell (pH 2); b – free cell (pH 4); c – free cell (pH 7); d –
encapsulated cell (pH 2); e – encapsulated cell (pH 4); f – encapsulated cell (pH 7); g – control.
3.3. Bile salt tolerance of whey microcapsule
After microorganisms pass through the stomach, they enter the upper intestinal tract where bile salts
are secreted into the gut. As a surface active compound, bile penetrates and reacts with lipophilic side of
bacterial cytoplasmic membrane causing a damage of membrane structure [10,11]. Table 1. Reduction in
viable counts of free and encapsulated L. fermentum 38-183 over 6 hours of incubation in bile salt
conditions.
Bile salt (%)
Free cell (log CFU/mL) Encapsulated cell (log CFU/mL)
Initial 6 h Reduction Initial 6 h Reduction
Mean
(SD)
Mean
(SD)
Mean
(SD)
Mean
(SD)
Mean
(SD)
Mean
(SD)
0.5
8.60
(0.17)
6.89
(0.17)
1.71
(0.03)
8.60
(0.17)
8.01
(0.18)
0.59
(0.03)
1.0
8.60
(0.17)
5.64
(0.11)
2.96
(0.22)
8.60
(0.17)
7.85
(0.15)
0.75
(0.19)
2.0
8.60
(0.17)
2.93
(0.14)
5.67
(0.15)
8.60
(0.17)
7.47
(0.21)
1.13
(0.21)
* Mean and standard deviation were obtained from triplicate samples
Bile also affects the structure and function of large macromolecules such as DNA and proteins leads
to the damage of molecule. To test bile salt tolerance, encapsulated and free cell L. fermentum 39-183
were exposed to solutions containing different levels of bile salts after 6 hours of incubation. Results
shown in Table 1 indicate that viable cells of encapsulated and free cells gradually decreased when the
concentration of bile salt was increased up to 2.0%. Encapsulated L. fermentum 39-183 with initial cell
load of 8.60 log CFU/g showed 0.58 log and 0.75 log reduction when exposed to 0.5% and 1.0% bile salt
broth, respectively, while the free cells decreased by 1.70 log and 2.97 log, respectively. Encapsulated L.
fermentum 39-183 was observed resistant to 2.0% bile salt, which remained survival rate higher than 50%
0,00
2,00
4,00
6,00
8,00
10,00
12,00
0 4 8 12 16 20 24
V
ia
b
le
c
el
ls
l
o
f
C
F
U
/m
L
Time (h)
Nguyen Thi My Le, Nguyen Van Hieu, Pham Viet Nam
88
after 6 hours of incubation, whereas free cell showed a decrease of 5.65 log. The results obtained also
showed that encapsulation provided protection for cells, since the survival of encapsulated cells was
significantly better (p < 0.05) than that of free cells. The results related to improve survivability of
encapsulated cells treated with bile salt obtained in this study are in accordance.
3.4. Release profile of microencapsulated bacteria in ex vivo porcine gastrointestinal contents
Figure 3 and 4 show the release profile of L. fermentum 39-183 and E. coli GFP
+
from protein
capsules in ex vivo porcine GI contents, respectively. The release profile of bacteria from whey protein
capsules varies in different GI conditions. A mount of 2.06 ± 0.11 CFU10 mL-1 was counted in the
duodenal contents (pH = 5.2), while there was greater amount of released bacteria in the jejunum contents
(pH = 6.5) around 4.50 ± 0.15 CFU10 mL-1 after 8 h of incubation. There was a complete release of L.
fermentum 39-183 or E. coli GFP
+
(8.60 ± 0.11 CFU10 mL-1) from whey protein capsules in ileum
(pH = ) after 2 h of incubation, while the cell count of both of strains gradually increased from 1.16 ±
0.15 CFU10 mL
-1
at 0.5 h to complete release (8.60 ± 0.10 CFU10 mL-1) after 6 h in colon. In contrast,
there was no significant release of L. fermentum 39-183 or E. coli GFP
+
in the gastric contents (pH =2.5)
after 8 h. This suggest that the bacteria either dead or trapped in the capsules. However, addition of
phosphate buffer (after 8 h) increased the viable counts of microencapsulated bacteria (L. fermentum 39-
183 and E. coli GFP
+
) to nearly 8.60 CFU10 mL
-1
with in 15 min in gastric, duodenal and jejunum
contents. This shows that the bacteria were alive but not released completely from capsules. However,
there was no significant decrease in the viable cell of E. coli GFP
+
in ileum and colon contents after a
complete release form microcapsules. This suggest that E. coli GFP
+
strain K12 is not a native gut
bacterium, therefore is not be able to survive in porcine gut contents. Our results are similar with Iyer et
al., (2005) who reported that L. casei Shirota was completely released from chitosan-coated alginate-
starch capsule in ileum and colon [12]. However, in our study, time release of L. fermentum from whey
protein capsule in in ileum and colon was shorter.
Figure 3. Release profiles of microencapsulated E. coli GFP
+
in porcine gastrointestinal contents. The
error bars represent standard deviation of mean (n=3).
Figure 4. Release profiles of microencapsulated L. fermentum 39-183 in porcine gastrointestinal
contents. The error bars represent standard deviation of mean (n=3).
4. CONCLUSIONS
These results demonstrate that whey proteins can be used as a convenient material for improving
L. fermetum 39-183 protection. Whey protein microcapsule has an excellent capacity to encapsulate
bioactive organisms that are sensitive to stomach circumstances, with concomitant controlled release at a
0
2
4
6
8
10
0 0,5 1 2 4 6 8
V
ia
b
le
c
el
ls
l
o
g
C
F
U
/m
L
Time (h)
Gastric
Duodenum
Jejum
Ilenum
Colon
0
2
4
6
8
10
0 0,5 1 2 4 6 8
V
ia
b
le
c
el
ls
l
o
g
C
F
U
/m
L
Time (h)
Gastric
Duodenu
m
Use of whey protein for encapsulation and controled release of probitoic from protein...
89
defined location. Whey protein encapsulation efficiently minimizes the bacteriocidal effects of the gastric
pH and maximizes the number of probiotics reaching the ileum and subsequently the colon. Thus, this
encapsulation technique may act as a platform technology for promoting targeted delivery of probiotics
with potential applications within the food and pharmaceutical industries.
Acknowledgement: We thank to Dr. Tran Van Hieu and his staff at Department Biotechnology of Ho
Chi Minh City University of Science for providing E. coli GFP
+
K12 and analyses of images of
encapsulated E. coli GFP
+
by fluorescence microscopy.
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1. FAO/WHO - Health and Nutritional Properties of Probiotics in Food Including Powder Milk with
Live Lactic Acid Bacteria, Report of a joint FAO/WHO Expert Consultation, 2001.
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vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and
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84 (1998) 759-768.
4. Ainsley Reid A., Vuillemard J. C., Britten M., Arcand Y., Farworth E. and Champagne C. P. -
Microentrapment of probiotic bacteria in a Ca2+-induced whey protein gel and effects on their
viability in a dynamic gastro-intestinal model, Journal of microencapsulation 22 (2005) 603-619.
5. Nguyen Thi My Le, Nguyen Thuy Huong, Pham Viet Nam - Probitoic properties of Lactobacilli
isolated from Vietnam traditional fermented foods, International Journal of Renewable Energy and
Environment Engineering 3 (2015) 06-12.
6. O’Neill G. J., Egan T., Jacquier J. C., O’Sullivan M., and O’Riordan E. D. - Whey microbeads as a
matrix for the encapsulation and immobilisation of riboflavin and peptides, Food Chemistry 160
(2014) 46-52.
7. Anal A. K., and Singh H. - Recent advances in microencapsulation of probiotics for industrial
applications and targeted delivery, Trends in Food Science and Technology 18 (2007) 240-251.
8. Lundin A., Bok C. M., Aronsson L., Bjorkholm B., Gustafsson J. A., Pott S., Arulampalam V.,
Hibberd M., Rafter J. and Pettersson S. - Gut flora, Toll-like receptors and nuclear receptors: a
tripartite communication that tunes innate immunity in large intestine, Cell. Microbiol. 10 (2008)
1093-1103.
9. Gbassi G. K., Vandamme T., Ennahar S. and Marchioni E. - Microencapuslation of Lactobacillus
plantarum spp in alginate matrix coated with whey proteins, International Journal of Food
Microbiology 129 (2009) 103-105.
10. Kim P., Young Jung M., Hyo Chang Y., Kim S., Jae-Kim S., Ha Park Y. - Probiotic properties of
Lactobacillus and Bifidobacterium strains isolated from porcine gastrointestinal tract, Applied
Microbiology and Biotechnology 74 (2007)1103-1111.
11. Favier C., Neut C., Mizon C., Cortot A., Clombel J. F., Mizon J. - Differentiation and identification
of human faecal anaerobic bacteria producing beta-galactosidase (a new methodology), Journal of
Microbiology Methods 27 (1996) 25-31.
12. Iyer C., Phillips M., Kailasapathy K. – Release studies of Lactobacillus casei strain Shirota from
chitosan – coated alginate – starch microcapsules in ex vivo porcine gastrointestinal contents,
Letters in Applied microbiology 41 (2005) 493 - 497.
Nguyen Thi My Le, Nguyen Van Hieu, Pham Viet Nam
90
TÓM TẮT
SỬ DỤNG WHEY PROTEIN ĐỂ VI GÓI VÀ ĐIỀU KHIỂN SỰ GIẢI PHÓNG PROBIOTIC RA
KHỎI VI HẠT TRONG ĐIỀU KIỆN ĐƯỜNG TIÊU HÓA LỢN MÔ HÌNH EX VIVO
Nguyễn Thị Mỹ Lệ*, Phạm Viết Nam, Nguyễn Văn Hiếu
Trường Đại họ ng nghiệp Th c ph m TP. H h inh
*
Email: mylethang81@yahoo.com
Mục tiêu của nghiên cứu là đánh giá tỷ lệ sống vi khuẩn L. fermentum 39-183 đ c vi g i trong điều
kiện pH thấp, muối mật và sự giải ph ng probiotic ra khỏi vi hạt trong dịch đ ờng tiêu h a l n mô hình
ex vivo. 1g vi hạt protein (≈ 108 CFU L. fermentum hoặc E. coli GFP+) đ c ủ trong 9 mL dịch tiêu h a
l n (ex vivo) ở 37 ºC. Các kết quả cho thấy, ở pH 2,0 trong thời gian 3 giờ L. fermentum đ c vi g i c tỷ
lệ sống cao h n 86% trong khi tế bào tự do mất hoàn toàn khả năng sống. Sau 6 giờ ủ trong muối mật
(0,5-2,0%), L. fermentum đ c vi g i với whey protein chỉ giảm 0,86 log trong khi probiotic tự do giảm
đến 3,34 log. Trong dịch hồi tràng sau 2 giờ, L. fermentum đ c giải ph ng hoàn toàn ra khỏi vi hạt,
trong khi sự giải ph ng không đáng kể đ c quan sát thấy trong dịch dạ dày sau 8 giờ ủ. Nghiên cứu này
cho thấy whey protein c thể đ c phát triển nh một vật liệu bao g i giúp bảo vệ probiotic trong các
điều kiện cực đoan và điều khiển giải ph ng vi khuẩn ra khỏi vi hạt tại vị trí mong muốn.
Từ khóa: Vi hạt protein, dịch đ ờng tiêu h a l n mô hình ex vivo, Lactobacillus fermentum 39-183,
Probiotics.
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