Australian Journal of Basic and Applied Sciences, 1(1): 7-14, 2007

ISSN 1991-8178

Biosynthesis and Properties of Extracellular Amylase by

Encapsulation Bifidobatrium bifidum in Batch Culture

Reyed M. Reyed

Faculty of Biochemistry, Biophysics and Bioinformatics, National Centre for Biological Sciences,
Tata Institute of Fundamental Research, Bangalore, India.

Abstract: Cell encapsulation is likely to play a major role in cell and transplantation therapies in the
next decade. The capsules provide a special microenvironment in which cells always have different
behaviors compared with free non-encapsulated culture. In this work, the encapsulated cells of the
probiotic bacterium bifidobatrium bifidum No.1, 791 cultures on different carriers were investigated
for the production of amylase. Amylase production by encapsulation bifidobatrium bifidum No.1, 791
cultivated in liquid media containing soluble starch reached a maximum at 48h. The entrapped cells
of bifidobatrium bifidum No.1, 791 in calcium alginate showed the highest enzyme activity
(90 U mL -1 ). Encapsulation matrix was optimized. In repeated batch fermentation process, the
encapsulated cells preserve their ability to produce consistently over 21 cycles and the activity remain
between (90 and 95 U/ mL -1 ) throughout the cycles. Some properties of the crude enzyme produced
by entrapped cells of bifidobatrium bifidum No.1, 791 in repeated batch were studied. The enzyme
properties revealed that the optimum temperature for activity was 75C. The enzyme was stable for
2h at 60C, while at 60C, 70C and 90C, 4%, 13% and 38% of the original activities were lost,
respectively. The optimum pH of the enzyme was 7.5. After incubation of crude enzyme solution for
24h at pH 7.5, a decrease of about 5% of its original activity was observed. The enzyme was strongly
inhibited by Cu 2 + and Mg 2 + but less affected by Ca 2 + , Mn 2 + . The enzyme in 1M and 5M NaCl
solutions the enzyme retained 70% and 47% of the original activity after 24h of incubation at 4C,
respectively.

Key word: probiotics bifidobacteria; amylase, cell encapsulating

INTRODUCTION

Bifidobacteria are important constituents of the intestinal microflora of humans and animals (Molder, 1994).
These organisms are the predominant group of bacteria in the normal intestinal flora of healthy breast-fed
newborns where they constitute more than 95% of the total population(Yildirim et al., 1999). Recently it has
been reported that bifidobacteria the natural inhabitants of the human large intestinal tract, especially healthy
breast-fed infants, and exert many beneficial effects on human health(Benno and Mitsuoka, 1992). High
numbers of Bifidobacterium are reported to form a barrier pathogens by prohibiting colonization or by
controlling the intestinal pH through the release of acetic and lactic acids and stimulate the immune response
of the host, for these reasons they have been incorporated into various commercial products such as yogurts,
fermented milk, baby milk powder, pharmaceutical tablets and feed additives (Hughes and Hoover, 1991). The
human colon is described as a complex ecosystem, containing over 400 species of microbes, the majority of
which are anaerobic microorganisms. The distribution of amylolytic microorganisms in the human large
intestinal tract was investigated in various individuals of differing ages using anaerobic culture techniques. A
large percentage of the amylolytic microorganisms present belonged to the genus Bifidobacteria. The number
of Bifidobacteria increased significantly at 2 yr of age. Adults and children above 2 yr old carried ~ 0.8 10 9
- 2.0 10 1 0 colony forming units (CFU/g) of amylolytic Bifidobacteria (Ji et al., 1992).
Amylase production could be induced by starch in a stable form. W hen cells were grown on maltose or
glucose, amylase production was much lower than on starch and amylase activity disappeared after 24 h growth
on these media (Ji et al., 1992). Encapsulation has been effective in protecting sensitive bacteria from harsh

Corresponding Author: Reyed M. Reyed, Genetic Eng. & Biotechnology Research Institute, Mubarak City for
Scientific Research and Applied Technology, New Borg El-Arab, Post code 21934,
Alexandria, Egypt.
Tel: 03-4593421, Fax: 03459423 E-mail: rmrkater@mail.ru rmrkater@indiatimes.com

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 Aust. J. of Basic and Appl. Sci., 1(1): 7-14, 2007

environments where they may not normally survive. Modification of biotechnology and processes, using
immobilized biocatalysts, has recently gained the attention of many biotechnologists. Application of
immobilized enzymes or whole cells is advantageous, because such biocatalysts display better operational
stability (Fortin and Vuillemard, 1990) and higher efficiency of catalysis (Ramakrishna et al., 1992) and they
are reusable.Microbial products are usually produced either by free or encapsulated cells. The use of capsulated
cells as industrial catalysts can be advantageous compared to batch fermentation process (Venkatasubramanian,
1979). W hole cell immobilization (Capsulation or entrapment form) has been a better choice over enzyme
immobilization(Kennedy et al., 1990; Ramkrishna and Prakasham, 1999) . W holecell encapsulation is a widely
used and simple technique. This paper reports the experimental results for extra-cellular amylase activities
production by immobilized Bifidobacterium Bifidum No., 1 791, also evaluated the using of the immobilized
cells of Bifidobacterium Bifidum No.1, 791 for extra-cellular amylase production in repeated batch according
to the productivity, and long-time operational stability. To the best of our knowledge, this is the first report
dealing with the capsulation of human intestinal tract Bifidobatrium bifidum No.1, 791 for biosynthesis of
amylases enzymes.

M ATERIALS AND M ETHODS

Microorganisms:
The bacterial strain Bifidobacterium Bifidum No.1, 791 used in the present work from department of
microbiology, Kazakh National University, Almaty, Kazakhstan.

Culture M edia and Growth Conditions:

The medium used for the cell biomass production for the cell encapsulation was Thioglycolate Broth with
Resazurine (Fluid Thioglycolate Medium, Thioglyolate Medium 90404, Fluka). The medium was inoculated
from 24 h old culture and was grown at 37 o C.

Extracellular Enzyme Am ylase Production:

The organism was found to produce amylase on culture medium composed of 1% (w/v) soluble starch.
The composition of Basal Medium (BM) contained (per liter) 30 g tryptone Pancreatic digest of casine (BD),
7 g yeast extract, 1 g M gSO 4 7H 2 O. After autoclaving BM was supplemented with 0.05% (wt/v) filter-
sterilized cysteine-HCl, and strains were grown at 37C under anaerobic conditions. The pH was adjusted to
6.0-6.5 with 1.0 M NaOH and this basal medium was sterilized by autoclaving at 121C for 15min. Yeast
extract, Bacto-tryptone and Soluble starch were sterilized separately and aseptically added to the flasks
containing the liquid medium, after cooling. The above medium (50 mL in 250 mL Erlenmeyer flasks) was
inoculated with 1 mL of an overnight culture and incubated at 37C, under anaerobic conditions. The turbidity
of the cultures was determined by measuring the optical density at 550 nm. Before assay, the cells were
separated by centrifugation at 10.000 rpm for 20 min and the clear supernatant was used as crude enzyme
preparation.

Am ylase Activity Assay:

The activity of amylase was assayed by incubating 0.3 mL enzyme with 0.5 mL Soluble starch (1%, w/v)
prepared in 0.05M Phosphate buffer, pH 6.5. After incubation at 90C for 10 min the reaction was stopped
and the reducing sugars released were assayed calorimetrically by the addition of 1 mL of 3-5- dinitrosalicylic
acid reagent (Miller 1959). An enzyme unit is defined as the amount of enzyme releasing 1 mmole of glucose
from the substrate in 1 min at 90C.

Encapsulation Procedures:
All the immobilization processes were performed under aseptic conditions. In separate experiments, the
cell pellets obtained from each culture (in the logarithmic phase of growth) were collected by centrifugation
(10.000 rpm, 20 min) in a refrigerated centrifuge (4 o C). Then, the wet cell pellets were suspended in 0.85%
sterile saline and used for the cell immobilization experiments.

Encapsulation in Ca-alginate:
Unless otherwise stated, the wet cell pellets obtained from 100 ml culture of each organism were mixed
with 10 ml of sodium alginate solution (Loba Chemie, Mumbai, India) in separate experiments. The alginate
entrapment of cells was performed according method of (Johnsen and Flink, 1986). Sodium alginate solution
was prepared by dissolving sodium alginate in 100 mL boiling water and autoclaved at 121C for 15 minutes.

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 Aust. J. of Basic and Appl. Sci., 1(1): 7-14, 2007

Encapsulation in Agar:
A definite quantity of agaragar (Hi-media, Mumbai, India) was dissolved in 18 mL of 0.9% sodium
chloride solution to get final concentration of 3 % and sterilized by autoclaving. The cell suspension (2 mL
equivalent to 0.03 g wCW ) was added to the molten agar-agar maintained at 40C, shaken well for few
seconds (without forming foam), poured into sterile flat bottom 4-inch-diameter petri plates and allowed to
solidify. The solidified agar block was cut into equal size cubes (4 mm 3 ), added to sterile 0.1 M phosphate
buffer (pH 7.0), and kept in the refrigerator (1 hour) for curing. After curing, phosphate buffer was decanted
and the cubes were washed with sterile distilled water 3 to 4 times (Veelken and Pape, 1982).

Encapsulation in Polyacrylam ide:

A cell suspension was prepared by adding 0.03 g cells to 10 mL chilled sterile distilled water. T o another
10 mL of 0.2 M sterile phosphate buffer (pH 7.0), the following chemicals were added: 2.85 g acrylamide
(Fluka, Buchs, Switzerland), 0.15 g bisacrylamide (Fluka), 10 mg ammonium persulphate, and 1 mL TEMED
(NNN 1 N 1 tetra methyl ethylene diamine). The cell suspension and the above phosphate buffer mixture was
mixed well and poured into sterile flat bottom 10 cm-diameter petriplates. After polymerization (solidification),
the acrylamide gel was cut into equal size cubes (4 mm 3 ), transferred to 0.2 M phosphate buffer (pH 7.0), and
kept in the refrigerator for 1 hour for curing. The cubes were washed 3 to 4 times with sterile distilled water
and stored in sterile distilled water at 4C until use.

Batch Process with Encapsulated Cells:

The batch experiments were performed in 250 ml Erlenmeyer flasks each containing 100 ml of BM. The
flasks were inoculated with the beads obtained from 10 ml gel with the calculated amounts of the immobilized
cells. Parallel experiments were carried out with equal amounts of free cells. The cultures were incubated for
48 h at 37 o C. All the experiments were carried out in triplicates.

Repeated Batch Process with Encapsulated Cells:

One of the advantages of using immobilized biocatalysts is that they can be used repeatedly and
continuously. Therefore, the reusability of Bifidobacterium Bifidum No.1, 791 cell immobilized in matrix was
examined. T his was done in 250 ml Erlenmeyer flasks each containing 100 ml of BM. Each flask was
inoculated with the beads obtained from 10 ml alginate gel comprising a cell loading of 1.08 g wet cells 100
ml / culture. After attaining the maximum production of amylase enzyme. the spent medium was replaced with
fresh production medium BM (100 mL) and at the end of each run, the gel particles were filtered and washed
with 25 ml of 0.05 M CaCl 2 and distilled water and the process was repeated for several batches until the
beads started disintegrating. The enzyme titers and cell leakage of each cycle were determined.

Cell Growth and Cell Leakage:

Both cell growth in freely suspended cultures and cells leaked from the gel matrix were determined as cell
wet weight by measuring the optical density at 600 nm. One absorbance unit was equivalent to 0.16 g/L (cell
wet / weight).

Effect of pH on Activity and Stability of Am ylase:

Effect of pH on the activity of amylase was measured by incubating 100mL of enzyme and 500mL of
buffers, adjusted to pH of 5.5 to 8.5, containing Soluble starch (0.5%). The buffers used were: sodium acetate
pH 5.5; phosphate pH 6.0 8.0; Tris-HCl pH 8.5. Stability of the enzyme at different pH values was also
studied by incubating the enzyme at various pH values ranging from 5.5 8.5 for 24h and then estimating
the residual activity.

Effect of Temperature on Activity and Stability of Am ylase:

The effect of temperature on the enzyme activity was determined by performing the standard assay
procedure as mentioned earlier for 10 min at pH 6.5 within a temperature range of 40 110C. Thermostability
was determined by incubation of crude enzyme at temperatures ranging from 40-110C for 2h in a constant-
temperature water bath. After treatment the residual enzyme activities were assayed.

Effect of Various Metal Ions:

The effect of different metal ions on amylase activity was determined by the addition of the corresponding
ion at a final concentration of 1mM to the reaction mixture, and assayed under standard conditions. The
enzyme assay was carried out in the presence of Ca cl2 , Cu SO 4 . Fe Cl 3 , MnSo 4 , nacl and ZnSo 4 .

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 Aust. J. of Basic and Appl. Sci., 1(1): 7-14, 2007

RESULTS AND DISCUSSIONS

Secretion of Extracellular Am ylase with Encapsulated Cells in Various Matrices Techniques:

The suitability for Biosynthesis of amylase activity by the encapsulation entrapped cells of Bifidobacterium
Bifidum No.1, 791 in different encapsulated matrices was investigated in submerged culture. In another set of
experiments, the same amount of free cells from Bifidobacterium Bifidum N o.1, 791 culture was inoculated
along with the same amount of the encapsulated cells. The results (Table 1) indicated that, in all cases the
activity of the encapsulat cells was lower than the corresponding amount of free cells. The effectiveness factor
of the encapsulating cells, which is the ratio of the enzyme activity of the encapsulating cells to that of the
same amount of free cells under identical conditions, was in the order of 0.50.8. These values are similar to
those reported elsewhere for other encapsulated bacterial amylases (Yong et al., 1990; Jamuna and
Ramakrishna, 1992). The effectiveness factor of the encapsulated cells would always be less than one because
the encapsulated cells represent a heterogeneous catalysis fermentation in which the activity, or rather synthesis,
of primary or secondary metabolites is dependent upon the external and internal mass transport and adequate
oxygen supply (Jamuna and Ramakrishna, 1992), of all preparations, the cells of Bifidobacterium Bifidum No.1,
791 encapsulated in Ca-alginate showed the highest amylase activity (90.0 U ml -1 ) and the highest
specific productivity (60.0 U g wet cells h -1 ).

Table 1: Production of am ylase enzym es by free and encapsulated bacterial cultures.

Carrier for encapsulation Cell Biom ass Protein content am ylase enzym e Specific Effectiveness
loaded g wet (m g m l -1 ) productivity productivity factor
cell 100 m l (U m l -1 ) (U g wet cells -1 h -1 )
culture -1
Free cells 1.48 2.72 110.50 74.66 ------
Agar 1.48 0.62 53.64 36.24 0.60
Ca- alginate 1.48 0.93 90.0 60.81 0.81
Polyacrylamide 1.48 0.72 50.24 33.94 0.56

Optim um Conditions for Encapsulation of Bifidobacterium Bifidum No.1, 791 in Ca-alginate:

Different concentrations of sodium alginate (2%, w/v) were investigated for the immobilization process.
In all cases, a constant amount of cells was used (equivalent to 1.48 g wet cell pellets per 10 ml gel).
Inoculation of 100 ml BM was performed with the beads resulted from 10 ml Na alginate solution. The
results recorded in (Table 2) indicated that beads prepared from 2 % (w/v) alginate concentration were
much softer and showed the highest number of leaked cells (1.65 mg/ml) and therefore, they were
excluded. On the other hand the enzyme yield of the beads made of 6 % (w/v) was 70.8 less than that
recorded for the beads prepared from 4 % (w/v) alginate concentration. This is most likely because the
resulting beads had lower diffusion efficiency. Maximal enzyme yield (90.0 U) was obtained at 4% (w/v)
alginate concentration. This concentration was recommended for the production of amylase (Jamuna and
Ramakrishna, 1992) by encapsulated bacterial cells. Using the same conditions from the previous
experiment.

Table 2: Effect of alginate concentration on the production of am ylase by the encapsulated cells of Bifidobacterium Bifidum N o.1,
791.
Alginate concentration Cell Biom ass Protein content am ylase enzym e Specific Leak cell
(% ) loaded g wet (m g m l -1 ) productivity productivity (m g/m l)
-1 -1 -1
cell 100 m l (U m l ) (U g wet cells h )
culture -1
2 1.48 1.95 79.8 74.66 1.65
4 1.48 0.62 90.0 36.24 0.45
6 1.48 0.20 70.8 60.81 0.3

The Effect of Different Concentration of Starch on Productivity of Am ylases Enzyme:

Amylase is an inducible enzyme and is generally induced in the presence of starch. Using the optimum
conditions reached from the previous experiments (4 % w/v, alginate concentration, cell loading of 1.48
g wet cell pellets per 10 ml gel), the effect of different concentration of starch of productivity of amylases
enzyme (1.0 -6.0 ) was investigated for the production of amylase. Maximal enzyme activity (95 U / ml)
was attained at 5.0 % of starch concentration Fig. (1). The concentration of starch as substrate had
influences on the production of the enzyme. It was also observed that increase in the concentration of
starch in the culture medium enhanced Bifidobacterium Bifidum No.1, 791 growth and enzyme production.
Similarly strong amylase induction by starch in the case of A. oryzae DSM 63303 has been reported by

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 Aust. J. of Basic and Appl. Sci., 1(1): 7-14, 2007

Lachmund et al., 1993. Bacillus thermooleovorans is reported to prefer starch, glucose, non-conventional
substrates lactose, maltose and maltodextrins as carbon sources for a- amylase secretion(Narang and
Satyanarayana, 2001). In contrast, carbon sources such as glucose, maltose and starch did not enhance a-
amylase production by thermophilic B. coagulans in solid-state fermentation using wheat bran(Babu and
Satyanarayana, 1995). The carbon sources as glucose and maltose have been utilized for the production of
amylase. However, the use of starch remains promising and ubiquitous.

Fig. 1: Different concentration of starch (%).

Repeated Batch Operation with Imm obilized Cells:

The activity of encapsulated Bifidobacterium Bifidum No.1, 791 in cells for the production of amylase
continuously was investigated by using the cell- encapsulated beads, respectively, for several batches. The
medium was replaced every 48 h and the beads were washed thoroughly with 0.05 M CaCl2 , and distilled
water at the end of each cycle before reuse. The results (Fig. 2) show the activity of 11 cycles ( 21
Days). The activity of the encapsulated cells remain between 90 and 95.5 U / ml throughout the cycles.
These results are similar to those reported by Jamuna and Ramakrishna (Jamuna and Ramakrishna, 1992)
for the production of extracellular a- amylase by the immobilized cells of Bacillus sp. in repeated batch
fermentation.

Fig. 2: Repeated batch fermentation used encapsulated cells of Bifidobacterium Bifidum No.1, 791 for
amylase production.

Effect of Ph on Activity and Stability of Am ylase:

The effect of pH on amylase activity is shown in Fig.3. Optimum pH was found to be 7.5. The
enzyme activity at pH 5.5 and 10.0 were 73% and 55% of that at pH 7.5, respectively. After incubation
of crude enzyme solution for 24h at pH 5.010, a decrease of about 5% of its original activity at pH 7.5
was observed. At pH 10.0, the decrease was of 44%. Thus, amylase of Bifidobacterium bifidum No.1, 791
strain seems to be active in very broad pH range.

Effect of Temperature on Activity and Stability of Am ylase:

The supernatant amylase activity was assayed at different temperatures ranging from 40C - 110C at
a constant pH of 7.5 and a substrate concentration of 0.5% as shown in (Fig. 4). Enzyme activity increased
with temperature within the range of 40C to 70C. A reduction in enzyme activity was observed at values

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 Aust. J. of Basic and Appl. Sci., 1(1): 7-14, 2007

Fig. 3: Effect of pH on activity and stability of amylase.

above 70C. The optimum temperature of this amylase was 70C, which was higher or similar to that
described for other Bacillus amylases(Ara et al., 1993). The residual activity of crude amylase incubated
at different temperatures for a period of 2h and 24h was estimated at optimum temperature. The enzyme
was stable for 2h at temperatures ranging from 40-60C while at 70C and 90C, 13% and 38% of the
original activities were lost respectively.

Fig. 4: Effect of temperature on activity and stability of amylase.

Effect of Metal Ions on the Activity of Am ylase Enzym e:

The effect of some metal ions at the concentration of 1 mM in the activity of amylase was
investigated. As can be observed in Fig. (5) , the amylase did not require any specific ion for catalytic
activity. A slight activity inhibition was produced in the activity by Ca Cl 2 , and MnSO 4 , and a stronger
inhibitory effect was observed in the presence of Cu 2 + some amylases are metallo enzymes, containing a
metal ion for catalytic activity. The inhibition of Bacillus subtilis JS-2004 a - amylase by Co 2 +, Cu 2 +, and

Fig. 5: Effect of metal ions on the activity of amylase enzyme.

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 Aust. J. of Basic and Appl. Sci., 1(1): 7-14, 2007

Hg 2 + ions but less affected by Mg 2 +, Zn 2 +, Ni 2 +, Fe 2 +, and Mn 2 could be due to competition between the
exogenous cations and the protein-associated cation, resulting in decreased metalloenzyme activity(Laderman
et al., 1993). There is also report where Ca + 2 did not have any effect on the enzyme (Asgher et al., 2007).
Kundu et al., (1973) reported that Ca 2 + was inhibitory to amylase production by A. oryzae EI 212. The
enzyme activity in NaCl 1m M solution retained 70% of the original activity. Dutta et al.,(2006) had earlier
reported that amylase produced by Heliodiaptomus viduus was retained of its activity at 60% in 2 M NaCl
after 24 h incubation, while full activity was found in 0.5 M NaCl for the same duration of incubation.

Conclusion:
It is known that starches are a major carbohydrate source in the human colon. Some starches escape
complete digestion due to their size and molecular conformation during passage through the human small
intestine; these compounds arrive in the colon as fermentable carbohydrate sources for intestinal bacteria
. The approach used in this study was to use encapsulation technology as a means to protect a recognized
probiotic organism from the harsh environment during fermentation . A variety of wall materials including
alginate has been used for the successful encapsulation of probiotics (Doleyres and Lacroix, 2005). Alginate
wall materials were chosen in the present study as they were found to provide the best protection for
Bifidobacterium bifidum No.1, 791 against external condition (M uthukumarasamy et al., 2006). Increased
bacterial stability following encapsulation in alginate is reported in the literature and has met with varying
levels of success (Iyer and Kailasapathy, 2005). The alginate matrix was found to be superior to the other
matrices studied in this work . In addition, the alginate carrier is not expensive, nontoxic, and preparation
of biocatalyst involves mild conditions, which is an added advantage. The results show that calcium alginate
shows potential method for microencapsulating of Bifidobacterium bifidum No.1, 791 for amylases
production. amylases production by biomicroencapsulating is superior to that of free cells because it leads
to enzyme productivity within the same time of fermentation. Specific advantages of this technique such
as long life-term stability 21 days (11 cycles), reusablility, and possibility of regeneration to be adaptable
also to scale-up the obtained data. In addition, the experiments with repeated batches of alginate
encapsulated bacterial growth by introducing fresh nutrients every 48 hours leads to activity of the
encapsulated cells continue between 90 and 95.5 U / ml, throughout the cycles. To our knowledge the
present work is the first study investigating the potential use of microencapsulation technology to produce
amylases from bifidobacteria. The results showed that microencapsulated Bifidobacterium bifidum No.1, 791
potential strain for amylase production using fermentation with soluble starch as substrate. Interesting
observation was that it showed and retained 95 % enzyme activity at 70 C. Furthermore, the enzyme was
found to show active in very broad pH range.

ACKNOW LEDGM ENT

I am very much obliged to Dr. M ukund Thatti National Center for Biological Sciences for supporting
me with fund and all facilities to do this research, interacting with Scientists of N ational Center for
Biological Sciences, Bangalore, India and other research institutes in different states of India. I would like
to thank S. V ivek Raj for his technical assistance. Funding to pay the Open Access publication charges
for this article was provided by the National Center for Biological Sciences, Bangalore, India. I am very
grateful to Indian National Science Academy for supporting me with Fellowship.

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