Advance Journal of Food Science and Technology 4(2): 112-117, 2012
ISSN: 2042-4876
© Maxwell Scientific Organization, 2012
Submitted: March 14, 2012
Accepted: March 26, 2012
Published: April 20, 2012
Production of Functional Kariesh Cheese by Microencapsulation of
Bifidobacterium adolescentis ATCC 15704
A.M. Abd-Elhamid
Department of Food and Dairy Science andTechnology, Faculty of Agriculture, Damanhour
University, Damanhour, 22516, Egypt
Abstract: Bifidobacterium adolescentis ATCC 15704 was microencapsulated with rennet gelation of milk
proteins. Encapsulated and free Bifidobacterium adolescentis ATCC 15704 under simulated gastrointestinal
conditions and bile tolerance were estimated. The survivability of encapsulated Bifidobacterium adolescentis
ATCC 15704 and the changes of organic acids in Kariesh cheese during 2 weeks of cold storage were
determined. The study demonstrated that microencapsulation using rennet gelation of milk proteins increased
the survival of Bifidobacterium adolescentis ATCC 15704 in simulated gastric conditions. Furthermore, the
results showed that, the survival of encapsulated Bifidobacterium adolescentis ATCC 15704 in bile solution
was significantly enhanced (p<0.05) compared to the free Bifidobacterium adolescentis ATCC 15704.
Microencapsulation protected Bifidobacterium adolescentis ATCC 15704 in Kariesh cheese during cold storage
as compared to free Bifidobacterium adolescentis ATCC 15704. In addition, the capsules made from rennet
gelation of milk proteins had a positive effect in organic acids during cold storage.
Key words: Bifidobacterium, bile tolerance, microencapsulation, organic acids, Kariesh cheese
products and in the gastrointestinal tract (Rao et al.,
1989). It is a process by which cells are retained within an
encapsulating matrix or membrane. The most widely used
encapsulating material is alginate, direct compression
encapsulation, and rennet-gelled protein encapsulation.
Rennet could be used to prepare water-insoluble
microcapsules based on milk-proteins without significant
loss of cell during the encapsulation process.
Microencapsulation protecting the cells during incubation
under simulated gastric conditions at low pH (Heidebach
et al., 2009).
The objectives of this work were to prepare
functional Kariesh cheese with encapsulated
Bifidobacterium adolescentis ATCC 15704 in capsules
made with rennet-gelled of milk proteins, to evaluate the
survival of encapsulated cultures under simulated
gastrointestinal conditions and to determined the changes
of encapsulated Bifidobacterium and organic acids in
Kariesh cheese during storage period.
INTRODUTION
Karish, Kariesh, or Kareish cheese is one of the most
popular types of soft cheese consumed in Egypt. It
contains most of the skim milk constituents including
16.70% protein, 3.98% sugar, 72.50% water and 0.1% fat.
Karish cheese is made from skimmed buffalo, s or cow’s
milk or a mixture of both. The increasing demand for it by
Egyptian consumers is mainly attributed to its low price
(Abd-El-Salam et al., 1984; Abou-Donia, 2008). Several
approaches have been proposed to improve therapeutic
effect of Kariesh cheese. One of the most common
approaches is to modify the manufacturing procedure in
order to increase the Bifidobacterium (El-Nemr et al.,
2007). The main therapeutic benefits bifidobacteria is:
immune enhancement; prevention of diarrhoeal
diseaseand colon cancer; enhancement of immunity
against intestinal infections; prevention of upper
gastrointestinal tract diseases; and hypercholesterolaemia;
improvement in lactose utilization; and stabilization of the
gut mucosal barrier (Kailasapathy and Chin, 2000).
The number of viable Bifidobacterium taken orally is
decreased due to action of processing and storage
conditions of the food containing them and to the action
of gastric juice and bile salts. Bifidobacterium has poor
survivability in dairy products as they do not tolerate
exposure to highly acidic and aerated media
(Muthukumarasamy et al., 2006). Microencapsulation
techniques have been investigated for improving the
viability of lactic acid bacteria when incorporated in dairy
MATERIALS AND METHODS
Strain: Bifidobacterium adolescentis ATCC 15704 was
obtained from the Department of Dairy Microbiology
(Animal Production Research Institute, Agriculture
Research Center, Dokki, Egypt).
Encapsulation procedure: The encapsulation technique
was according to Heidebach et al. (2009).
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Adv. J. Food Sci. Technol., 4(2): 112-117, 2012
experiment. The artificial juice, like gastric juice, was
prepared with MRS broth without pepsin. The MRS broth
was prepared by adjusting to pH 1.5 and 2.5 with HCl
then sterilized by autoclaving for 15 min., at 121ºC. Fresh
aqueous microcapsule-slurry (2.0 g) was added to MRS
broth (pH 1.5 and 2.5) to a test-tube containing 18 mL of
MRS broth without pepsin and vortexed for a few
seconds. The test-tubes were incubated at 37ºC for 1, 2
and 3 h; respectively. The bacterial counts were
determined using MRS medium. Three replicates of
experiment were processed.
(A)
Enumeration of the free and encapsulated bacteria:
Numbers of Bifidobacterium adolescentis ATCC 15704
was determined by plate counting using MRS agar with
incubation at 37@C after 48 h; under anaerobic conditions
using GasPak® (Dection Dickinson Microbiology
System, Cocskeysville, MD). Encapsulated
Bifidobacterium was enumerated in freshly prepared or
previously acid-incubated capsules according Heidebach
et al. (2009), 0.5 g of freshly prepared capsules or 10 mL
of simulated gastric juice with capsules were diluted with
double-distilled water to a total volume of 500 mL and
homogenized by a stomacher at 7900 rpm for 2 min. Free
cells were not homogenized after simulated gastric
treatment. Each sample was assayed in triplicate.
(B)
Fig. 1: (A) Scanning electron photomicrograph
of
microcapsules. Bifidobacterium adolescentis ATCC
15704., (B) Section of milk protein-capsules showing:
Bifidobacterium adolescentis ATCC 15704.
Morphology examination of microcapsules: Samples of
probiotic cells containing microcapsules were taken from
the aqueous capsule-slurry and fixed for 1 h in 3%
glutaraldehyed and dehydrated in graded series of ethanol
(30-100). Sample were dried with acetone and
amylacetate and then, coated with a thin layer of gold in
a Fine coat JEOL (JFCL 1100E Ion Sputtering Device,
Tokyo, Japan) Sputter coater. Coated samples were
examined and photographed on JSM-5300, JEOL
Scanning Microscope, Tokyo, Japan, with an accelerating
voltage of 25 Kv at the Electron Microscopic Unit,
Faculty of Science and Alexandria University (Fig. 1).
Kariesh cheese preparation: Skimmed buffalo's milk (15
g fat/L) was obtained from Damanhour Agriculture
Secondary School, Damanhour, Egypt and a buffalo's
skim milk was pasteurization at 65±1ºC for 30 min, and
then cooled to 32±1ºC. The treated milk inoculated with
mesophilic lactic acid starter for cheese, which was added
in ratio 2% in milk at 32±1ºC until curding. The formed
curd was ladled into wooden frames lined with muslin
cloth, 1% salt was dispersed. After 6 h; resting of the
curd, the curd was divided into two portions.
Bifidobacterium adolescentis ATCC 15704 (1%W/V) was
added to the two portions as free (control) or
encapsulated, and the curd pressed by suitable weights.
Resultant cheese was stored at refrigerated temperature 9
±1ºC for 14 days (El-Nemr et al. 2007); and
Bifidobacterium strain was counted on MRS medium
supplemented with neomycin sulphate 100 mg/L,
naladixic acid 15 mg/L and lithium chloride 3 g/L (ElnasrPharmaceutical Chemicals Co., Abu Zaabal, Egypt.) , the
plates were incubated under anaerobic conditions using
GasPak® (Dection Dickinson Microbiology System,
Cocskeysville, MD) at 37±1ºC for 48 h; the selectivity of
the growth conditions was confirmed by morphology of
cells from single colonies under a microscope (Lapierre
et al., 1992). The Kariesh cheese making experiment was
repeated three times.
Survival of encapsulated Bifidobacterium adolescentis
ATCC 15704 in bile solutions: The strain propagated
previously in MRS broth (Elnasr-Pharmaceutical
Chemicals Co., Abu Zaabal, Egypt) for 18 h; at their
optimum temperature were inoculated individually by 1%
into MRS broth supplemented with different
concentrations (0.15, 0.30 and 0.40%, respectively) of
bile salts (B8631, Sigma-Aldrich Chemical Co; St. Louis,
Missouri) in triplicate and incubated at their optimum
temperature. The growth was examined at zero time, after
3 and 6 h; by the changes in the total counts of the tested
strains (Kim and Worobo, 2000). Three replicates of
experiment were processed.
Survival of encapsulated Bifidobacterium adolescentis
ATCC 15704 in simulated gastric juices: An artificial
juice like intestinal as recommended by Kim and Worobo
(2000) at different pH values were used in this
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Adv. J. Food Sci. Technol., 4(2): 112-117, 2012
Determination of organic acids: Lactic and acetic acids
were determined using HPLC (Beckman) according
Akalin et al. (2002). For preparation of sample, 4 g of
cheese samples was added to 25 mL of 0.1 N H2SO4
homogenized and centrifuged at 5000×g for 10 min. the
supernatant was filtered through Whatman No 1 filter
paper and through a 0.20 :m membrane filter, and 2 mL
aliquots were stored in HPLC vials at-20ºC until HPLC
analysis. Operating conditions were: mobile phase,
aqueous 0.6% (w/v) (NH4)2HPO4 (0.038 M)-0.2% (v/v)
acetonitrile (0.049 M) adjusted to pH 2.24 with H3PO4;
flow rate 0.5 mL/min and ambient column temperature.
An ODS ultrasphere C18 (300×5mm) column (USA) was
used. The mobile phase was prepsred by dissolving
analytical-grade (NH4)2HPO4 in distilled water, HPLCgrade acetonitrile and H2SO4. The detector was set at 214
nm. HPLC-grade reagents were used as standards (Sigma
Chemical Co., St. Louis, MO/USA). Solvents were
degassed under vacuum. Both solvents and standard
solutions were filterd through a 0.20 :m membrane filter.
Quantitation was based on the external standard method
(Bevilacqua and Califano, 1989).
Log CFU g-1
Encapsulated bifidobacterium
adolescent is ATCC 15704 0.15%
Encapsulated bifidobacterium
adolescent is ATCC 15704 0.30%
Encapsulated bifidobacterium
adolescent is ATCC 15704 0.40%
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Free bifidobacterium adolescent is
ATCC 15704 0.15%
Free bifidobacterium adolescent is
ATCC 15704 0.30%
free bifidobacterium adolescent is
ATCC 15704 0.30%
Zero time
After 3 hrs.
After 6 hrs.
Fig. 2: Survival of encapsulated and free Bifidobacterium
adolescentis ATCC 15704 in different concentration of
high bile. Error bars give the standard
deviation(Average of 3 replicates).
resistance to bile effect was dependent on buffering
capacity of protein in addition to protecting the cells
during exposure to high concentrations of bile salt
(Heidebach et al., 2009). Ding and Shah (2009) suggested
that when free probiotic bacteria were exposed to
taurocholic acid, viability was reduced by 6.36 log
CFU/mL, whereas only 3.63, 3.27 and 4.12 log CFU/mL
was lost in probiotic organisms encapsulated in alginate,
xanthan gum and carrageenan gum, respectively. While,
encapsulation of Bifidobacterium using 2% alginate
enhanced the survival of Bifidobacterium by 16.6%
(Sridar and Kailasapathy, 2003).
Statistical analysis method: Data were analyzed using
the Statistical Analysis System software package (SAS,
2000). Analyses of variance were performed using
ANOVA procedures. Significant differences between
mean were determined using Duncan’s multiple range test.
RESULTS AND DISCUSSION
Survival of encapsulated Bifidobacterium adolescentis
ATCC 15704 in bile solutions: The bile salts secreted
into the human small intestine consist of sodium
chenodeoxycholate, sodium deoxycholate and sodium
cholate. These substances can destabilize membrane
integrity in bacterial cell. The strains of probiotic bacteria
should be resistant to the action of bile salts and be able
to colonise the intestine surface. The in vivo bile
concentration in the intestine is lower, which could affect
lactic acid cultures (Olejnik et al., 2005).
As show in Fig. 2 the viability of free
Bifidobacterium adolescentis ATCC 15704 decreased
from 10.15 to 5.83 , from 10.15 to 5.19 and from 10.15 to
4.57 log CFU/g, respectivily at 0.15, 0.30 and 0.40% bile
salt concentration after 6 h; incubation at 37ºC.
Howevere, the viability of encapsulated Bifidobacterium
adolescentis ATCC 15704 was significantly better, with
regard to bile tolerance, than free Bifidobacterium
adolescentis ATCC 15704. The microcapsulation
increased the survival of Bifidobacterium adolescentis
ATCC 15704 in bile solution. In conclusion, the acquired
Survival of encapsulated Bifidobacterium adolescentis
ATCC 15704 in simulated gastric juices: Low pH
significantly decreased bacterial viability. Natural
concentration of HCl in stomach reaches 170 mM and the
minimum pH is about 1. After ingestion of a standard
meal the pH value in stomach could increase to 7, which
then declines to acidic values gradually, depending of the
buffer capacity of the meals (Russell et al., 1993).
Ekmekcioglu (2002) defined the time to decline one pH
value to be about 10-20 min; full gastric emptying lasts up
to 4 h; especially when meals are rich in fiber and fat.
As shown in Fig. 3 the viability of free
Bifidobacterium adolescentis ATCC 15704 significantly
decreased (at p<0.05) after incubation at pH 1.5. The
highest reduction of viability appeared after the first hour
of incubating, while the viability encapsulated
Bifidobacterium adolescentis ATCC 15704 attained the
highest tolerance to low pH and its viability after 3 h; of
incubation decreased to 6.78 and 7.14 log CFU/g at pH
1.5 and 2.5, respectively. Statistical analysis of results
114
Log CFU g-1
Adv. J. Food Sci. Technol., 4(2): 112-117, 2012
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
monitored over a storage period for 2 weeks for change in
viable cell count. Table 1 showed the Bifidobacterium
adolescentis ATCC 15704 count in the Kariesh cheese. In
the case of free Bifidobacterium adolescentis ATCC
15704, the cell numbers dropped substantially to 6.10 log
CFU/g by 2 weeks of storage at 9ºC. Furthermore the
encapsulated state of the same strain showed a decrease to
8.12 log CFU/g, which might be attributed to the
microencapsulation. There is a significant differences
(p<0.05) between the free and encapsulated state in
Kariesh cheese at the end of 2 weeks storage. From
Table 1; it can be further seen that for Bifidobacterium
adolescentis ATCC 15704 survival to a greater extent in
the encapsulated state compared to the free
Bifidobacterium adolescentis ATCC 15704 sample. This
might be ascribed to capsules that tend to be synergistic in
gelling and as a result may help in providing additional
protection to the entrapped bacterial cells (Sultana et al.,
2000; Jayalalitha et al., 2011).
Encapsulated bifidobacterium
adolescent is ATCC 15704 1.5
Encapsulated bifidobacterium
adolescent is ATCC 15704 2.5
free bifidobacterium adolescent is
ATCC 15704 1.5
Free bifidobacterium adolescent is
ATCC 15704 2.5
Zero time
After 1 hrs.
After 2 hrs.
After 3 hrs.
Fig. 3: Survival of encapsulated and free Bifidobacterium
adolescentis ATCC 15704 in over 6 h; of expsure to
different concentration of acid. Error bars give the
standard deviation (Average of 3 replicates).
obtained in this exeriment revealed significant differences
at p<0.05 between encapsulated and free Bifidobacterium
adolescentis ATCC 15704 viability. Encapsulation in
milk-protein-capsules greatly improved the survival rate
of Bifidobacterium lactis compared to free cells under
similar conditions at pH 2.5 (Heidebach et al., 2009).
Sultana et al. (2000) reported that survival cells of
Lactobacillus casei entrapped in alginate-starch beads
decreased 2 log CFU/g after their incubation in simulated
high acid gastric conditions and high bile during 3 h; at
37ºC. The binary blends of sodium alginate : amidated
low-methoxyl pectin (1:2, 1:4, 1:6 ratios) help in
providing additional protection to entrapped Lactobacillus
casei cells against simulated gastriotesinal conditions
(Sandoval-Castilla et al., 2010).
Changes in free and encapsulated oraganic acids of
kariesh cheese during cold storage: Organic acids
formed as result of bacterial metabolism and hydrolysis of
fat lactose, milk proteins and citrate during manufacture
and storage of cheese (Ong and Shah, 2009).
The initial acetic and lactic acids of Kariesh cheese
made with free and encapsulated Bifidobacterium
adolescentis ATCC 15704 were 0.63 and 3.94 g/kg,
respictivly while at the end of storage (after 2 weeks)
were 1.39 and 5.71 g/kg, respectively (Table 1). This
might be due to utilization of lactose by
Bifidobacterium,which can ferment the lactose result in 3
moL of acetic acid and 2 moL of lactic acid per 2 moL of
glucose (Scardovi and Trovatelli, 1965). Compared with
encapsulated Bifidobacterium adolescentis ATCC 15704,
the acetic and lactic acids were 1.10 and 4.77 g/kg,
respictivly after 2 weeks of cold storage. This might be
due to the microcapsulation, which effectively protected
the Bifidobacterium from acidic environmental in Kariesh
cheese during cold storage. The goal of encapsulation was
to create a micro-environment, in which the bacteria will
survive during processing and storage and be released at
Survival of encapsulated Bifidobacterium adolescentis
ATCC 15704 in kariesh cheese during cold storage:
Encapsulation is often defined as a way to protect bacteria
against several enviromental factors (Anal and Singh,
2007; Champagne and Fustier, 2007).
In this study, free or encapsulated Bifidobacterium
adolescentis ATCC 15704 were used separately with the
Kariesh cheese starter culture. Kariesh cheese was
Table 1: Changes of chemical composition in kariesh cheeses made with encapsulated and free Bifidobacterium adolescentis ATCC 15704 during
cold storage
Variables
Storage period
Bifidobacterium (Log
at 9@C (days)
pH value
CFU/g) adolescentis
Acetic acid (g/kg)
Lactic acid (g/kg)
free Bifidobacterium adolescentis
0
4.67c
7.96d
0.49e
3.10f
ATCC 15704 (control)
7
4.58d
7.01e
0.62d
4.00d
15
4.53e
6.10f
1.10b
4.77b
encapsulated Bifidobacterium
0
4.79a
9.12a
0.63d
3.94e
adolescentis ATCC 15704
7
4.71b
8.73b
0.93c
4.61c
15
4.61c
8.12c
1.39a
5.71a
Means followed by the same superscript are not significantly different at p<0.05
115
Adv. J. Food Sci. Technol., 4(2): 112-117, 2012
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appropriate sites (e.g., small intestine) in the digestive
tract (Anal and Singh, 2007).
CONCLUSION
Microencapsulation in protein milk micro particles
successfully improved the survival of Bifidobacteriu
adolescentis ATCC 15704 in bile solutions and protected
the Bifidobacterium adolescentis ATCC 15704 in the
gastrointestinal tract. In addition, the microencapsulation
significantly increased the survival of Bifidobacterium
adolescentis ATCC 15704 in Kariesh cheese during cold
storage. The numbers of Bifidobacterium adolescentis
ATCC 15704 in encapsulated state were 8.12 log CFU/g
at the end of two weeks of cold storage. This viable cell
number was higher than that recommended by the
International Dairy Federation (107 CFU/g), indicating
that the high initial number of probiotic could provide the
recommmended number in the final product. Moreover, it
could be concluded that the use of milk-proteins in
microcapsulation of Bifidobacterium adolescentis ATCC
15704 could increase the acetic and lactic acids during
storage.
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