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Journal of Applied Sciences Research, 9(2): 1097-1109, 2013

ORIGINAL ARTICLES
 
Effect of growth conditions on the production of exopolysaccharides by
microencapsulated Lactobacillus bulgaricus and use it to improve quality of Kareish
cheese
1
S.A. EL-Gizawy, 1Olfat, S. Barakat, 2O.M. Sharaf, 2Kawther EL-Shafei, 2Fatma A. Fathy and
2
Hoda S. EL-Sayed
1
Department of Agricultural Microbiology, Faculty of Agriculture, Cairo University.
2
Dairy Department (Dairy & Food Microbiology Lab) National Research Center, Dokki, Cairo, Egypt.

ABSTRACT

The purpose of this research is to select the optimal conditions for the exopolysaccharides production from
Lactobacillus bulgaricus. The effect of different incubation periods (6, 12, 18, 24 and 48h), temperature degrees
(25. 30, 37 and 40 ºC ), pH values (4.0, 4.5, 5.0, 5.5, 6.0, 6.5) and growth media (skim milk, skim milk +
5%sucrose, Elliker, Elliker + 5% sucrose, MRS and MRS + 5% sucrose) on the exopolysaccharides production
by encapsulated strain was studied. Microencapsulation with the sodium alginate (Extrusion technique) offered
greater production of exopolysaccharides especially on incubation period of 18h, temperature at 37 ºC and pH
value at 5.5 for encapsulated Lb. bulgaricus with sodium alginate. Also, Kareish cheese was manufactured by
inoculation with encapsulated strains using sodium alginate and the finished product was examined chemically,
bacteriologically and organoleptically at fresh and after 7, 15, 22 and 30 days of storage period at 7 ºC. The
count of encapsulated strain increased during the storage period and reached maximum after 15 days of storage
period compared with control. Moreover, the maximum production of exopolysaccharides observed after 22
days which reached to 570 mg/100g cheese. The use of exopolysaccharides producing encapsulated strain
improved the acceptable quality of Kareish cheese.

Key words: Microencapsulation, Exopolysaccharides, Lactobacillus bulgaricus, Kareish cheese

Introduction

Exopolysaccharides (EPS) are defined as long chain polysaccharides secreted by microbial cells (Laws &
Marshall, 2001). They are commonly associated with the ‘slimy’ or ‘ropy’ characteristic of some acidified milk
products. Exopolysaccharides produced by some strains of lactic acid bacteria (LAB) have been found to
improve textural properties and increase water retention in fermented dairy products (Ruas-Madiedo et al.,
2002). The presence of EPS seems to contribute to texture, mouthfeel, taste perception and stability of the final
products (Hassan et al., 1996; Folkenberg et al., 2005; Girard & Schaffer-Lequart, 2007). In addition to
processing functionality, the consumption of EPS has been associated with health benefits (Kitazawa et al.,
1998; Dal Bello et al., 2001 & Korakli et al., 2002). Therefore, the development of EPS produced by LAB as a
functional ingredient in foods has great potential.
The yield of EPS produced by different LAB is generally quite low. It has been reported to vary widely
from 50 to 2700 mg/L (Macedo et al., 2002). Several attempts have been made to control the fermentation
conditions to increase EPS production (Grobben et al., 1998; Macedo et al., 2002), but there are still many
unresolved questions. Because of their low production yields, very few studies have been carried out on the
application of EPS produced by LAB as a functional ingredient, and most research has focused on the
structuring effect of the polysaccharide in products, mainly for the manufacture of cheese and yogurt
(Hassan et al., 1996; Hassan et al., 2003a & Folkenberg et al., 2005). In these systems, fermentation time is
relatively short, and small amounts of EPS are present in the final product.
Multiple studies have reported an effect of the protein and carbohydrate composition of the
fermentation media on the production of EPS by LAB (Marshall et al., 1995; Grobben et al., 1997 & Petry et
al., 2003). EPS production seems to increase with increasing protein content in the medium (Grobben et al.,
1998).
Published efforts to optimize EPS production in lactic acid bacteria have included studies evaluating the
effects of such environmental conditions as temperature and pH (Garcia-Garibay & Marshall 1991; Cerning et
al., 1994; Grobben et al., 1995; Mozzi et al., 1995; Van den Berg et al., 1995 & Mozzi et al., 1996). A number
of these studies examined the effect of temperature by using various EPS producing strains of

Corresponding Author: S.A. EL-Gizawy, Department of Agricultural Microbiology, Faculty of Agriculture, Cairo
University.
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Lactobacillus delbrueckii subsp. bulgaricus. (Garcia-Garibay & Marshall 1991) found that specific polymer
production (equivalent milligrams of dextran per cfu) by strain Leuconostoc mesenteriodes NCFB 2772
grown in skim milk was greater at a temperature (48 °C) than the optimum for growth (37 to 42 °C).
Greater specific polymer production [milligrams per gram of cells (dry weight)] at a higher temperature
(45 °C) was also found by (Grobben et al., 1995), who examined EPS production by the same strain in
defined medium. Mozzi et al., (1995) found a correlation between the optimum growth temperature (37 to 42
°C) and maximum polymer production by strain Lactobacillus bulgaricus CRL 870.
Different methods (physical entrapment in polymeric networks, attachment or adsorption to a performed
carrier, membrane entrapment, and microencapsulation) have been used for immobilizing lactic acid bacteria.
The purpose of these techniques is either to retain high cell concentration within the bioreactor, or to protect
cells from a hostile environment (Krasekoopt et al., 2003; Doleyres & Laeroix 2005). Other advantages of
immobilization cell technology are the reuse of cells, protection against shear damage and the high biological
stability during long-term continuous fermentation (Denkova et al., 2004 & Shene & Bravo, 2007).
Few studies used immobilization technique for producing polysaccharides such as (Bergmaier et al., 2003
& Bergmaier et al., 2005) which studied the production of exopolysaccharides by Lb. rhamnosus RW-9595M
during pH-controlled batch cultures with free cells and repeated-batch cultures with cells immobilized on solid
porous supports. The study clearly shows the high potential of the strain Lb. rhamnosus RW-9595M and
immobilized cell technology for production of EPS as a functional food ingredient.
The objective of the present study was to determine the optimum pH values, temperature degrees growth
media and incubation period for production of EPS by microencapsulated Lactobacillus dulbrueckii subsp.
bulgaricus, besides use of this strain to improve quality of Kareish cheese.

Materials and Methods

1. Strains:

Lactobacillus dulbrueckii subsp. bulgaricus was obtained from Dairy Microbiology Lab. National Research
Center, Dokki, Cairo, Egypt (EL-Shafei et al., 2011).

2. Cultivation and harvesting of Lb. bulgaricus cells:

MRS broth (Oxoid) was used to prepare the cell suspensions of Lb. bulgaricus. The medium was inoculated
with 2% active Lactobacilli cells and incubated at 37 °C for 48h. Cells were harvested by centrifugation at 5000
rpm for 15 min at 4 °C., and cells were washed twice with saline and used to prepare capsules.

3. Preparation of microencapsulated cells culture:

a. Extrusion technique:

1. Microencapsulation using alginate with NaCl:

A suspension of cells was mixed with an equal volume of sodium alginate (4%). The mixture was added
drop-wise into solution of sodium chloride (0.2 mol/L) and calcium chloride (0.5 mol/L) and magnetically
stirred at 200 rpm/min till alginate beads were formed according to Klinkenberg et al., 2001.

2. Microencapsulation using K- carrageenan:

Prepared by mixing 20 g cells (wet weight) in 1000 ml of a sterile solution of K-carrageenan (2%), then the
mixture was added drop-wise into KCl (3%) under agitation. K-carrageenan beads were formed within 10 min
according to Dinakar & Mistry 1994.

b. Emulsion technique:

1. Microencapsulation using sodium caseinate:

1.1. Preparation of the protein–cell mixture:

The protein suspensions were prepared by dispersing sodium caseinate in double distilled water to a
concentration of 15% (w/w). After 2 h of stirring, the pH of the casein suspension was adjusted to 7.0 and the

 
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solution was stirred overnight at 4 ˚C before further use. Tow g of strain concentrate was thawed and mixed
with 28 g of the casein dispersion, to create the protein–cell mixture.

1.2. Encapsulation process:

Transglutaminase enzyme (TGase) was added to the protein–cell mixture with an enzyme concentration of
10 U TGase per g substrate protein at 30 ˚C. Directly after TGase addition, 30 g of the protein–cell mixture
containing strain was added to 150 g of tempered (40 ˚C) sunflower oil in a 200 mL Erlenmeyer flask and
stirred at a constant speed of 900 rpm with a magnetic stirrer for 120 min. The temperature was maintained
during the process in a water bath controlled by thermostat. During the process the emulsified droplets of
protein–cell mixture were converted into gel particles according to Heidebach et al., 2009a.

2. Microencapsulation using skim milk:

2.1. Preparation of the milk-concentrate-cell-mixture:

Skim-milk-powder was dispersed in double-distilled water to obtain a 35% (w/w) solution and stirred
overnight at 10 ˚C. Tow g of strain concentrate was thawed and mixed with 28.0 g of the skim-milk-concentrate
to create a milk-concentrate cell-mixture.

2.2. Encapsulation process:

The 30 g milk-concentrate-cell-mixture was cooled to 5 ˚C, incubated with 400 ml rennet stock-solution,
and then kept at 5 ˚C to perform the cleavage of the k-casein. After 60 min incubation, 180 ml 10% (w/v) CaCl2
solution was added to the mixture and the encapsulation process was subsequently initiated. Fifteen g of the
cold-rennet mixture was added to 150 g of tempered (5 ˚C) vegetable oil in a 200 mL Erlenmayer flask and
magnetically stirred at 500 rpm for 5 min to emulsify the mixture into the oil. In preliminary experiments the
length of the emulsifying step at low temperature before warming was varied between 0 and 15 min, and 5 were
min found to be sufficient. Following the emulsifying process at 5 ˚C, the temperature was then raised to 40 ˚C
while the emulsion further agitated for another 15 min. When the temperature exceeded 18–20 ºC the emulsified
droplets turned into gel particles instantly. Subsequently, the gelatinized microcapsules were separated from the
oil by gentle centrifugation (500 g, 1 min) according to Heidebach et al., 2009b.

3. Production of EPS using microencapsulated and free strains:

Sterilized skim milk 11% w/v was inoculated by 2% of microencapsulated Lb. bulgaricus with different
methods and free strain to study the optimum conditions for the EPS production.

a. Effect of incubation periods on the production of EPS:

According to Zisu & Shah (2003) the inoculated milk was incubated at 35 ˚C for 6, 12, 18, 24 and 48h.

b. Effect of temperature on the production of EPS:

The inoculated milk with microencapsulated and free strain of Lb. bulgaricus was incubated at 25, 30, 37
and 40 ˚C for 18h, according to El-Shafei et al., 2011.

c. Effect of pH values on the production of EPS:

The pH of sterilized milk was adjusted to pH 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 by 10% sterilized lactic acid
(w/v) and was inoculated by microencapsulated and free strain of Lb. bulgaricus (2%) then incubated at 35 ˚C
for 18h to study the effect of pH, (El-Shafei et al., 2011).

d. Effect of growth media on the production of EPS:

Different sterilized growth media (skim milk, skim milk with 5% sucrose, Elliker, Elliker with 5% sucrose,
MSR and MRS with 5% sucrose) were inoculated by 2% of microencapsulated Lb. bulgaricus with different
methods and incubated at 35 ˚C for 24h for the production of EPS.

 
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4. Isolation and quantification of exopolysaccharides:

The culture samples were froze until measuring, 100 ml of the frozen sample was thawed over night at 4 ºC
to which 20 ml of 20% (w/v) the tricholoroacetic acid solution (Sigma) was added followed by centrifugation at
10,000 rpm for 20 min at 4 ºC to remove the precipitate and bacterial cells. The supernatant was neutralized to
pH 6.8 with 4 M NaOH and boiled for 30 min in a water bath and re-centrifuged at 10,000 rpm for 20 min at 4
˚C. An equal volume of chilled ethanol was added to the supernatant to precipitate the polysaccharide from the
supernatant and agitated at 100 rpm and 4 ºC overnight. The curd polysaccharide pellet was recovered by
centrifugation (10,000 rpm for 20 min at 4 ºC) according to Zisu & Shah (2003). The polysaccharide was
quantified using the phenol-sulfuric method (Bouzar et al., 1996).

5. The efficacy of selected microencapsulated strains with sodium alginate to improve the quality of Kareish
cheese:

The selected microencapsulated method using sodium alginate was prepared by culture beads were formed
through injection of the culture with sodium alginate solution using the Encapsulator instrument (model IE-50R
was purchased from Encap. Biosys., Switzerland) under sterile and specific conditions as follows: nozzle:
300µm, frequency 1700Hz, flow rate: 4 ml.min−1 and air pressure of 1 bar. The formed beads were received in
3% CaCl2 (w/v) and left in the hardening solution for up to 30 min and the beads were filtered.
Kareish cheese was made as described by Fahmi (1960) with some modification. Buffalo’s skim milk was
heated at 65 ºC for 30 min, cooled to 32 ºC, inoculated with 3% starter (Lactococcus lactis Subsp lactis) and
incubated at 32 ºC till reaching pH 5.5. The acidified milk was divided into tow portions. Calf rennet was added
to the first portion at a rate of 0.5 ml/10kg as control. Encapsulated strain of Lactobacillus dulbrueckii subsp.
bulgaricus with sodium alginate (108-109 cfu/ml) were added to the second portion at a rate of 1% before
renneting. Curd of each treatment was hoped to drainage at room temperature for 24h. The resultant cheese was
then cut into blocks. Cheeses were stored at 7 ºC for 30 days. Samples were taken at fresh, 7, 15, 22 and 30 days
for the chemical and microbiological analysis. Data were reported as the average of three independent trials.

a. Chemical analysis:

1. Moisture content:

Moisture content was determined according to the method described by Ling (1963).

2. Determination of pH values:

The pH of cheese was measured according to Ling (1963) using pH. Meter model Hanna HT-4817.

3. Determination of total nitrogen (TN) contents:

Total content were determined by semi-micro Kjeldahl method according to Ling (1963).

4. Determination of EPS using encapsulated Lb. bulgaricus by sodium alginate:

The cheese samples were froze until measuring; 100 gm of the frozen cheese samples was thawed over
night at 4 ºC. The EPS determined according to Zisu & Shah (2003). The polysaccharide was quantified using
the phenol-sulfuric method (Bouzar et al., 1996) as mentioned before.

b. Microbiological examination:

1. Lactobacilli count:

Lactobacilli count was determined using MRS agar according to De Man et al., (1960).The plates were
incubated at 37 ºC for 48h under anaerobic condition.

c. Rheological properties of Kareish cheese made with encapsulated Lb. bulgaricus:

Textural properties of experimental cheeses were evaluated using texture analyzer. Cheese samples
presented to the instrument were 30 mm in diameter and 20 mm in height. Samples were allowed to equilibrate
at ambient temperature for approximately 30–45 min prior to testing. The following textural parameters were

 
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determined according to the method of International Dairy Ferderation (1991) Hardness, Springiness,
Cohesiveness, Gumminess and Chewiness.

d. Organoleptic assessment:

The Organoleptic properties of kareish cheese were assessed by a regular taste panel of the staff- members
of the dairy science department, National Research Center. Kareish cheese samples were evaluated for flavor
(50 points), body and texture (40 points) and appearance (10 points) according to Bodyfelt et al., (1988).

e. Statistical analysis:

The data analyzed according to Statistical Analysis System Users Guide (SAS, 1994). Separation among
means in three replicated was carried out by using Duncan multiple test.

Results and Discussion

a. Effect of incubation period on the production of EPS:

The optimal fermentation time for EPS production showed that the highest yield was collected at 18h for
all encapsulated specially the strain which encapsulated by sodium alginate (407 mg/l for encapsulated Lb.
bulgaricus compared with free strain Table (1). These results due to the high population counts of encapsulated
strains with sodium alginate, which the counts are 10.29 log cycle cfu/ml for encapsulated strain of Lb.
bulgaricus compared with free strain as Table (2). On other contrary, the lowest yield was collected at the same
time (18h) when Lb. bulgaricus encapsulated by whey protein (255 mg/l). The amount of EPS production are
declined significantly (p ≤ 0.05) after the first 18h. The decline in the EPS content was observed at 24h and at
48h which reached 381 and 324 mg/l respectively for encapsulated Lb. bulgaricus with sodium alginate
compared with encapsulated Lb. bulgaricus with other different materials and free strain. On contrary more
declines was observed with free cells and encapsulated Lb. bulgaricus with whey protein, which reached to 236
mg/l at 24h for encapsulated. Lb. bulgaricus with whey protein, and reached to 204 mg/l for the same strain
after 48h of fermentation. While, yield of EPS with free stain reached 211 mg/l after 24h and reached 186 mg/l
after 48h of fermentation for the same strain. These declined may be due to the enzymes degradation (Zisu &
Shah 2003). While Lin & Chien (2007) found that the highest EPS yield from Lb. helveticus BCRC14030 was
730 mg/l which observed at the less favorable fermentation condition of 60h with a total viable count, 109 x 107
cfu/ml. Gassem et al., (1995) observed a reduction of viscosity after 18h of fermentation for strains Str.
Salivarius subsp. thermophilus CH15, YB57 and YB58. Similar results were obtained by Macura &Townsley
(1984) they found a decrease in viscosity after 24h of cell growth. UI-Qader et al., (2001) studied the growth of
Leu. mesenteriodes PCSIR-3 and found that the maximum dextransucrase activity was obtained after 18h of
incubation time. Results obtained in the present study agree with those obtained by EL-Shafei et al., (2011)
which found the highest yield of EPS was collected at18h for microencapsulated Lb. bulgaricus which reached
to 276 mg/l for culture and 317 mg/l for encapsulated cells.

Table 1: Effect of Incubation periods on the production of exopolysaccharides by microencapsulated Lb. bulgaricus with different materials
and free cells (mg/l).
Incubation periods (h)
Encapsulated EPS (mg/l) Overall
methods 6 12 18 24 48 means
Na-alginate 323d 352c 407a 381b 324d 357a
K-carrageenan 271fg 319d 364c 335d 294e 316b
Skim milk 220hij 231h 285ef 267g 221hij 245c
Whey protein 209ijk 221hij 255g 236h 204jk 225d
Free cells 202k 223hi 258g 221hij 186l 218e
Overall means 245d 269c 314a 288b 246d
Data expressed as mean of 3 replicates. Means with the same letter are not significantly different (P≤ 0.05).

The cell growth shown in Table (2) was increased significantly (p ≤ 0.05) to the maximum concentration
after 18h especially with encapsulated Lb. bulgaricus with sodium alginate (10.29 log cfu/ml), followed by
encapsulated with K- carrageenan (9.46 log cfu/l for encapsulated strain of Lb. bulgaricus) compared with other
microencapsulated Lb. bulgaricus with different materials. In contrary, the lowest count of cell was observed
with encapsulated strain by whey protein which the viable count reached to 8.21 log cfu/ml at 18h. These results
are in harmony with those obtained by Champagne et al., (1989) who reported that the immobilized systems can
reach higher cell densities than classical free cell fermentation performed under the same conditions.
Furthermore, Hyndman et al., (1993) stated that after encapsulation, activity and viability of the immobilized

 
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culture were increased by cell growth within the capsules. Moreover, EL-Shafei et al., (2011) reported that the
cell growth of encapsulated Lb. bulgaricus with alginate was accelerated over the first 6h and reached maximum
concentration after 18h. They could reach 10.9 log cfu/l for encapsulated culture and 7.3 log cfu/ml for free
cells.

Table 2: Effect of Incubation periods on the viability of microencapsulated Lb. bulgaricus with different materials and free cells (log
cfu/ml).
Incubation periods (h)
Encapsulated Total viable count (Log cfu/ml) Overall
methods 6 12 18 24 48 means
Na-alginate 8.25e 9.10d 10.29a 10.14b 9.09d 9.37a
ef e c
K-carrageenan 8.19 8.34 9.46 9.19d 8.33e 8.70b
l l e
Skim milk 6.72 7.79 8.32 8.00gh 7.18j 7.40c
Whey protein 6.20n 6.95k 8.21ef 7.89h 7.13j 7.27d
Free cells 6.17n 6.35m 8.07fg 7.68i 7.18j 7.09e
Overall means 7.11e 7.50d 8.87a 8.58b 7.78c
Data expressed as mean of 3 replicates. Means with the same letter are not significantly different (P≤ 0.05).

b. Effect of temperature on the production of EPS:

The maximum EPS production coincided with the optimal growing temperatures of most strains. As shown
in Table (3), the optimum temperature for the EPS production from Lb. bulgaricus which encapsulated with
different materials were 37 ˚C, especially for the strain encapsulated with sodium alginate which reached to 511
mg/l followed by encapsulated with K-carragenaan which reached to 443 mg/l compared with free cells (330
mg/l). Some workers found EPS production to be optimal at low temperatures than optimum growth (Gamar et
al., 1997), while others shown that EPS production to be favored at much higher temperatures than ptimum
growth (De Vuyst et al., 1998). Dupont et al., (2000) observed no significant difference in the quantity of EPS
production from Lb. rhamnosus R, 9595M and Lb. paracasei Type V, for the optimum growth temperature of
37˚ C and a suboptimal temperature of 32˚C. The authors reported that Lb. rhamnosus 9595M had the highest
EPS production 1275 mg/l and Lb. rhamnosus R gave a maximum EPS production of 601 mg/l. Moreover, EL-
Shaferi et al., (2011) found that the optimum temperature for EPS production by microencapsulated Lb.
bulgaricus at 35˚C, yielding 695 mg/l and 327 mg/l for free cells after 18h of fermentation, and the viscosity of
fermented milk increased at both 30 and 40 ºC. Lacroix et al., (2004) recorded that the microencapsulation
technique gave high cells density, very high volumetric productivity, high process stability (physical and
biological) over long fermentation periods, stimulation of production and secretion of secondary metabolites and
physical and chemical production of cells compared with free cells. Kimmel et al., (1998) found that the
optimum temperature and pH for the production of EPS from Lb. delbrueckii sub sp. bulgaricus RR was at 38
ºC and 5.0 respectively, with a predicted yield of 295 mg/l.

Table 3: Effect of temperature degrees on the production of exopolysaccharides by microencapsulated Lb. bulgaricus with different
materials and free cells (mg/l).
Temperature degrees (˚C)
Encapsulated EPS (mg/l) Overall
methods 25 30 37 40 means
Na-alginate 439c 485b 551a 498b 493a
K-carrageenan 379f 415d 443c 415d 413b
Skim milk 320hi 346g 397e 376f 360c
Whey protein 279ij 327h 365f 349g 337d
Free cells 285kl 300jk 330h 277l 298e
Overall means 347d 375c 417a 383b
Data expressed as mean of 3 replicates. Means with the same letter are not significantly different (P≤ 0.05).

c. Effect of pH values on the production of EPS:

In the present study, there appeared to be a relationship between pH and both biomass and EPS production.
At pH 5.5, EPS and biomass productions were favored by both microencapsulated and free cells Table (4).
Significantly the highest produced of EPS level was 496 mg/l produced by encapsulated Lb. bulgaricus with
sodium alginate, followed by encapsulated with K-carrageenan (455 mg/l) compared with the other
encapsulated methods and free cells. Generally the optimum pH for EPS production ranges between 5.0 and 7.0.
Even though in most cases the optimal pH conditions for EPS production coincide with those for optimal
growth. These results near to Svensson et al., (2005) who found that some strains from S. thermophilus,
produced more EPS than the other strains at pH 4.5 in enriched milk. In spite of this, the pH created unfavorable
growth condition for the Str. thermophilus. Vaningelgem et al., (2004) measured the viscosities for some strains
of Str. thermophilus at a spindle speed of 50 rpm. They observed that the apparent viscosity increased when the

 
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pH dropped below 5.75 during fermentation. Moreover, EL-Shafei et al., (2011) found that at pH 5.5, EPS
production was favored by Lb. bulgaricus yielding 366 mg/l by free culture and 503 mg/l by encapsulated
culture. Also, the highest viscosity was observed at pH for both encapsulated and free culture of Lb. bulgaricus
and found that at pH 5.5 the cell density in beads was yielding over 10.5 log cfu/ml, and count of cells culture
was over 8.6 log cfu/ml fermented milk.

Table 4: Effect of pH values on the production of exopolysaccharides by microencapsulated Lb. bulgaricus with different materials and free
cells (mg/l).
pH values
Encapsulated EPS (mg/l) Overall means
methods 4.0 4.5 5.0 5.5 6.0 6.5
Na-alginate 331ghi 374f 376bc 496a 467b 429cd 424a
K-carrageenan 302jkl 339gh 416de 455b 422cd 394ef 388b
Skim milk 280lm 309ijk 338gh 382f 327ghij 306jkl 323c
no kl ghi g hijk kl
Whey protein 252 292 331 345 314 300 305d
Free cells 236o 267mn 292kl 308ijk 286klm 239o 271e
Overall means 280e 316d 365b 397a 363b 333c
Data expressed as mean of 3 replicates. Means with the same letter are not significantly different (P≤ 0.05).

d. Effect of different growth media on the production of EPS:

In the present study, remarkable differences in EPS production among the different microencapsulated
methods and the media are found in Table (5). Production of EPS from microencapsulated Lb. bulgaricus with
sodium alginate and followed by microencapsulated with K- carrageenan on Elliker especially with 5% sucrose
was generally significantly higher when compared to that produced from other microencapsulated methods or
free strains and on others media. These observations might be attributed to the higher concentration of sucrose
in Elliker medium compared with skim milk and MRS media. Production of EPS from encapsulated Lb.
bulgaricus by sodium alginate on Elliker medium with 5% sucrose was 593 mg/l, and followed by encapsulated
by K- carrageenan to the same strain which reached to 528 mg/l compared with other encapsulated methods and
free cells. Previous studies have shown that the production of EPS is influenced by medium composition
(Grobben et al., 2000), as it affects the amount of EPS produced from individual strains. In a study carried by
Zisu & Shah (2003), M17 medium used to specifically culture Str. thermophilus, although supported cell growth
failed to sustain EPS production. Gamar et al., (1997) reported that variation in carbon source in the growth
medium resulted in different yields of the EPS produced by a Lb. rhamnosus strain. Addition of glucose or
sucrose to the milk medium stimulated the EPS production and modified the sugar composition of the EPS
produced by Lb. casei spp. casei (Cerning et al., 1992). Dols et al., (1997) found that the growth of Leu.
mesenteriodes in sucrose medium was considerably more rapid than glucose, fructose or glucose-fructose
mixture. Behravan et al., (2003) stated that dextran is generally produced by growing culture of Leuconostoc in
sucrose-containing media. It also reported that dextransucrase which involved in the polymerization reaction of
dextran uses only sucrose as a substrate (Ricciardi & Clementi, 2000). On the other side, (Pham et al., (2000)
reported that when Lb. rhamnosus R was grown in batch cultures on lactose, the amount of EPS was comparable
(495 mg/l) to that produced by glucose-grown (438 mg/l). Also, Van Geed-Schutten et al., (1998) reported that
in liquid MRS medium with relatively high sugar concentration provides an excellent alternative, yielding a
large number of EPS-positive strains. The authors also reported that sucrose was the best substrate for EPS
synthesis and appeared to be the direct substrate for EPS synthesis by extracellular enzymes in Lb. reuteri LB
121 and LB 180. Also, Champagne et al., (1989) who reported that immobilized systems can reach higher cell
densities than classical free cell fermentations performed under the same conditions.

Table 5: Effect of different growth media on the production of exopolysaccharides by microencapsulated Lb. bulgaricus with different
materials and free cells (mg/l).
Growth media
Encapsulated EPS (mg/l) Overall
methods 1 2 3 4 5 6 means
Na-alginate 335gh 381e 508b 593a 410d 480c 451a
K-carrageenan 297ij 335gh 478c 528b 343fg 413d 399b
Skim milk 229l 260k 406d 466c 317hi 361ef 340c
Whey protein 220lm 238l 376e 424d 298ij 330gh 314d
Free cells 202m 221lm 399fgh 406d 280jk 320ghi 294e
Overall means 256f 287e 421b 483a 329d 381c
Data expressed as mean of 3 replicates. Means with the same letter are not significantly different (P≤ 0.05).1- Skim milk 2-Skim
milk+5% sucrose 3- Elliker 4- Elliker+5% sucrose 5- MRS 6-MRS+5%sucrose.

 
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e. The efficacy of selected microencapsulated strain with sodium alginate on the quality of Kareish cheese:

1. Chemical analysis:

a. Moisture content:

Moisture content in kareish cheese samples manufactured with microencapsulated and control strains
during storage period up to 30 days at 7 ˚C is presented in Table (6). The data show that there were significant
differences between the moisture content of control and all cheese treatments along the storage period. It was
significantly decreased with increasing the storage period in all treated samples. Total means of moisture
content indicated that kareish cheese made with microencapsulated Lb. bulgaricus had the highest moisture
content along the storage period. Whereas, lowest moisture content was recorded in the control. Generally, the
overall means of moisture content ranged at fresh significantly increased to at the end of storage period.
This is due to the ability of EPS producing cultures to produce cheese with high water holding capacity
(Hassan et al., 2003b). Perry et al., (1997, 1998) and Ricciardi & Clementi (2000) also, reported 2-4% increase
in the Mozzarella moisture when an EPS-producing culture was used. The increase in cheese moisture was
parallel to the increase in yield, indicating that the increase in yield was due to an increase in the moisture
content. Low et al., (1998) found that capsular EPS bound a sufficient amount of water to significantly increase
the moisture content of Mozzarella cheese. Also, Ahmed et al., (2005) found that Moisture and yield were about
2% higher in cheese made using EPS-producing culture than that made using the EPS-negative mutant, and
about 4% than the control.

Table 6: Chemical and microbial properties of Kareish cheese made with encapsulated Lb. bulgaricus during storage at 7 ˚C for 30 days.
Control Encapsulated Lb. bulgaricus
Storage period (days)
Fresh 7 15 22 30 Fresh 7 15 22 30
Moisture % 69.76c 69.21d 68.75e 68.21f 67.81f 74.78b 75.03b 75.88a 75.60a 75.10b
pH 5.27a 5.07b 4.91d 4.68f 4.55g 5.19a 5.03bc 4.95d 4.78e 4.66f
Total nitrogen 3.39bc 3.15b 3.80a 3.86a 3.78a 3.26cd 3.31cd 3.17de 3.08e 3.09e
Counts log cfu/g 8.34g 9.16e 9.25e 9.21e 8.95f 9.57d 10.36c 10.70a 10.65a 10.48b
Exopolysaccharides NDe NDe NDe NDe NDe 376.00d 554.67b 562.60ab 570.00a 544.66c
mg/100g
Data expressed as mean of 3 replicates. Means in the same row showing the same letters are not significantly different (P≤ 0.05).

b. pH values:

Effect of microencapsulated EPS producing strain on the pH values of kareish cheese during storage period
at 7 ºC are present in Table (6). Generally, the pH values significantly decreased in all cheese samples during
the storage period increased specially with control. pH values of fresh kareish cheese with microencapsulated
Lb. bulgaricus significantly decreased with the increase of storage period. Furthermore, kareish cheese samples
with microencapsulated Lb. bulgaricus lowest pH values compared with control. The obtained results are in
harmony with those obtained by Magdoub et al., (1995) who reported that the decrease in pH values may be due
to the converting of the residual lactose in cheese to lactic acid and free fatty acids which developed in the
cheese at the end of storage period. Also, Abou Dawood, (2002) reported that only, the cheese with non
encapsulated bifidobacteria and control showed a slight decreased in pH than the encapsulated treatment from
the time it was manufactured to the end of storage period. Moreover, El-Baz et al., (2011) found that the pH
value was not greatly affected by the applied treatments (Ras cheese made with non and EPS producing yoghurt
culture).

c. Total nitrogen content:

The data of total nitrogen content of kareish cheese with microencapsulated and control during storage
period at 7 ºC present in Table (6). Generally, there were significant differences between the control and
microencapsulated strain. Whereas, there were significant differences at the end of the storage period of all
kareish cheese samples. The lowest significant total nitrogen values were obtained in treatment with
microencapsulated Lb. bulgaricus in comparion with control. Total nitrogen content in the treated cheese with
microencapsulated strain ranged from 3.41 to 3.44 at the beginning and reached 3.18 to 3.07 at the end of the
ripening period.
The obtained results are in harmony with those obtained El-Baz et al., (2011) who found that the Ras
cheese made with EPS producing yoghurt cultures had almost the lowest protein and fat values compared with
control. Also, EL-Shafei et al., (2011) reported that slight change were observed in protein and lactose in labneh
cheese using EPS producing encapsulated Lb .bulgaricus.

 
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2. Viable counts of EPS producing encapsulated strain:

Differences in viable count of producing EPS encapsulated strains in kareish cheese during storage period
are present in Table (6). Viable count of Lb. bulgaricus in of all cheese samples are significantly increased till
15 days of storage followed by a slight decrease toward the end of the storage period especially with control.
This increase reveals the protective effect of microencapsulation on the viability of Lb. bulgaricus where, the
viable count in fresh sample was 9.57 log cfu/gm and reached to 10.48 log cfu/gm at the end of storage
compared with control. Champagne et al., (1989) reported that immobilized systems can reach higher cell
densities than classical free cell fermentation performed under the same conditions. Furthermore, EL-Shafei et
al., (2011) observed that the density of cells of Lb. bulgaricus in beads was stabile at approximately 8.6, 8.3 and
8.3 log cfu/ml in three treatments through storage of labneh cheese at refrigerator. Hyndman et al., (1993) stated
that after encapsulation, activity and viability of the immobilized cultures were increased by cell growth within
the capsules. Moreover, Jayalalitha et al., (2011) reported that the probiotic count of encapsulation treated
yoghurt was significantly higher with control yoghurt in every week interval of storage period. Extrusion
method of encapsulation using alginate + starch + gelatin as wall materials provided maximum viability (9 log
unit) for probiotics in yoghurt throughout the storage period of 21 days. In contrary, Champagene et al., (1992)
observed an increase free cell density as the beads were used for successive fermentation.

3. Production of EPS using microencapsulated Lb. bulgaricus during storage period:

Differences in the production of EPS using encapsulated Lb. bulgaricus in kareish cheese during storage are
present in Table (6). Significantly the highest yield was collected after 22 days of storage which reached 570
mg/100g, the amount of EPS slightly declined to reach to 544.66 mg/100g at 30 days and not detected in the
control during storage. The results may due to enzymes degradation (Zisu & Shah 2003). Also, Dabour et al.,
(2005) highlighted the positive effect of EPS producing culture on the physical and functional properties of
reduced fat cheese. Moreover, El-Ahwany et al., (2007) who observed a 2.6 fold increase in xylanase produced
by B. pumilus with immobilized cells more than free cells at optimized medium condition.

4. Rheological properties of Kareish cheese made with encapsulated Lb. bulgaricus:

Several textural parameters were determined in cheese made with the EPS-producing strain and control in
Table (7). The textural properties of Kareish cheese were significantly (P ≤ 0.05) influenced by using the EPS
producing culture. Values of Hardness, Springiness, Cohesiveness, Gumminess and Chewiness were
significantly lower in cheese made with the EPS-producing culture than those in control cheese. The higher
values of Hardness, Springiness, Cohesiveness, Gumminess and Chewiness in control could be related to its
lower moisture content and compact structure previously observed by Hassan et al., (2003b). Beal and Mittal
(2000) suggested that high moisture weakens the protein network resulting in a less firm cheese. In addition to
producing curd of higher moisture content, the EPS can interfere with protein–- protein interactions than can
result in a soft cheese curd (Hassan et al., 2003a). Also, El-Baz et al ., ( 2011) found that using EPS-producing
culture improved most of the rheological properties tested including hardness, adhesiveness, springiness,
cohesiveness, gumminess and chewiness, whereas a great improvement was recorded for the sensory properties
including a much better flavor. Moreover, Ahmed et al., (2005) showed that the textural characteristics
(hardness, consistency, adhesiveness, chewiness, relaxation and modulus) were significantly lower in Kareish
cheese made using EPS-producing culture.

Table 7: Rheological properties of Kareish cheese made with encapsulated Lb. bulgaricus.
Treatments
Storage period (days) Control Encapsulated Lb. bulgaricus
Hardness (N)
Zero 8.00a 4.00b
30 7.00a 3.50b
Cohesiveness
Zero 0.497a 0.650a
30 0.465a 0.586a
Springiness (mm)
Zero 0.832a 0.796a
30 0.852a 0.650b
Chewiness (N/mm)
Zero 4.14a 1.66bc
30 3.49ab 1.06c
Gumminess (N)
Zero 5.20a 1.99b
30 4.10a 1.63b
Data expressed as mean of 3 replicates. Means in the same row showing the same letters are not significantly different (P≤ 0.05).

 
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5. Organoleptic assessment:

The result of the organoleptic assessment of kareish cheese stored at 7 ˚C for 30 days are given in Table (8).
The scores for flavor, texture and appearance were affected by the type of culture. Cheese made using
encapsulated Lb. bulgaricus received high scores than control which reflected the ability of EPS to improve the
texture of kareish cheese. Moreover, the highest organoleptic scores were recorded on 15 days of storage period.
Flavor, body & texture scores indicate that all kareish cheese treatments contained microencapsulated strain had
higher significant scores of flavor, body & texture when compared with control cheese samples. These results
are consistent with those of other workers who used EPS-producing lactic starter cultures to improve the sensory
attributes of various reduced and low fat cheese varieties (Perry et al., 1997, 1998). Also, Ahmed et al., (2005)
found that the sensory analysis confirmed the ability of EPS producing culture to improve kareish cheese
texture, as it received higher body and texture scores after aging for 7 and 15 days. Moreover, El-Shafei et al.,
(2011) noted that the use of exopolysaccharides producing encapsulated Lb. bulgaricus and probiotic bacteria
can provide acceptable quality to functional labneh. This product can be used as therapeutic and diabetic milk
product with highly acceptable qualities.
Table 8: Organoleptic scores of encapsulated Lb. bulgaricus.
control Encapsulated Lb. bulgaricus
Storage period (days)
Fresh 7 15 22 30 fresh 7 15 22 30
Flavor (50) 36cde 40bc 39bcd 34de 32e 38bcd 43ab 46a 41abc 40bc
ab ab a b b a a a
Body and texture (40) 30 31 32 31a 27 33 34 34 33a 32a
b b b c c b ab a
Appearance (10) 7.33 7.30 7.10 5.70 5.30 7.50 7.66 9.00 8.40b 7.50b
Data expressed as mean of 3 replicates. Means in the same row showing the same letters are not significantly different (P≤ 0.05).

Conclusion:

This study has shown that microencapsulation can be used for the enhancement of the production of EPS by
increasing the viable count of the strain. Also, EPS-producing culture has a positive impact on the textural
characteristics of Kareish cheese. Sensory evaluation indicated that Kareish cheese made using EPS-producing
culture was smoother and softer than control so it could improve quality and consumer acceptability of this
product.

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