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CHAPTER 3
MATERIALS AND METHODS
The present investigation was carried out in Biotechnology Research Laboratory,
Department of Food Engineering and Technology, Sant Longowal Institute of
Engineering and Technology, Longowal, Punjab (India). The materials and methods
used to fulfill the objectives of the study are given below:
3.1 Procurement of Microbial Cultures
The potential microbial strains, which can be used for -galactosidase
production and their subsequent use in lactulose production, were procured from
different culture collection centres. The details are given in Table 3.1.
Table 3.1 Sources of microbial cultures
Yeast strain Source
Kluyveromyces marxianus var marxianus MTCC
1388
Kluyveromyces marxianus var. lactis NCIM 3551
Kluyveromyces marxianus var. lactis NCIM 3566
Kluyveromyces marxianus NCIM 3465
Institute of Microbial
Technology, Chandigarh, India
National Chemical Laboratory,
Pune, India
do
do
3.2 Maintenance of Microbial Cultures
All the cultures were grown on agar slants containing (w/v) malt extract (0.3%),
yeast extract (0.3%), peptone (0.5%), glucose (1.0%) and agar-agar (2.0%), adjusted to
pH 5.0. The agar slants were incubated at 30 °C for 24 h. The cultures were maintained
by subculturing, aseptically at fortnight intervals and stored at 4 °C, until further use.
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3.3 Chemicals
All the reagents and chemicals used for experimental investigation were
analytical grade and procured from HiMedia Laboratories Pvt. Limited, Mumbai (India),
Merck India Ltd., Mumbai (India), Fluka Goldie Chemika-Biochemica, Mumbai (India)
and Sigma Aldrich (USA).
3.4 Collection of Samples for Yeast Isolation
For the isolation of yeast strains, whey samples from the following milk/dairy
plants of different states of India, i.e. Punjab, Haryana, Madhya Pradesh and Bihar were
collected:
Verka Milk Plant, Sangrur, Punjab (India)
Verka Milk Plant, Patiala, Punjab (India)
Verka Milk Plant, Ludhiana, Punjab (India)
Vita Milk Plant, Rohtak, Haryana (India)
Sanchi Milk Dairy Plant, Gwalior, Madhya Pradesh (India)
Darbhanga Dairy, Darbhanga, Bihar (India)
Sudha Dairy, Bhagalpur, Bihar (India)
3.5 Isolation of Yeast Cultures for β-Galactosidase Production
The enrichment of yeast cells in the whey samples was carried in Malt Extract
Broth (MEB) containing chloramphenicol (0.1 g/L) at 30 °C for 24 h under shaking
conditions at 100 rpm (Nahvi and Moeini, 2004). The yeast cultures were isolated on
spread agar plates containing (w/v) yeast extract (0.3%), peptone (0.5%), lactose
(2.0%), chloramphenicol (0.01%), and agar-agar (2.0%), after making serial dilutions.
The plates were incubated at 30 °C for 48 h. The colonies with distinct morphological
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differences were selected from different samples, were purified by streaking and were
examined using microscope. Approximately 50 yeast cultures were isolated which
displayed positive -galactosidase activity. However, cultures with higher enzyme
activity have been given in the present data.
3.6 Screening of Yeast Cultures for -Galactosidase Production
3.6.1 Preparation of Growth Media
All the yeast cultures were grown in 50 mL of media containing (w/v) malt
extract (0.3%), yeast extract (0.3%), peptone (0.5%), glucose (1.0%), and adjusted the
pH 5.0 in 250 mL capacity Erlenmeyer flask, and were sterilized by autoclaving.
3.6.2 Preparation of Fermentation Media
All the yeast cultures were grown in 50 mL of media containing (w/v) yeast
extract (0.3%), lactose (2.0%), and adjusted the pH 5.0 in 250 mL capacity of
Erlenmeyer flask. After sterilization, the flasks were inoculated with 6.0% culture from
growth media and incubated at 30 °C for 36 h, under shaking conditions at 100 rpm.
3.6.3 Measurement of β-Galactosidase Activity
All the standard yeast cultures namely Kluyveromyces marxianus MTCC 1388,
Kluyveromyces marxianus var. lactis NCIM 3551, Kluyveromyces marxianus var. lactis
3566, Kluyveromyces marxianus NCIM 3465 along with the isolated new yeast cultures
were screened for their -galactosidase production potential at shake flask level.
3.7 Identification of Isolated Yeast Culture
For the identification of isolate yeast, the following morphological,
physiological, biochemical and molecular characterization were studied.
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3.7.1 Morphological and Physiological Characteristics of Isolated Yeast Strain
Morphological analysis of the selected strain and culture characteristics were
performed for parameters like shape, size, presence of spore and type of budding of the
isolated yeast strain by standard methods. The isolate was inoculated in above
mentioned growth media at different pH ranging from 2-12, at different incubating
temperatures ranging between 10 - 60 °C followed by observation of growth.
3.7.2 Biochemical Characteristics
The isolate was inoculated in slants and in tubes containing various
carbohydrates and fermentation pattern was obtained by detection of gas production.
The selected strain was studied for different sugar fermentation, hydrolysis of starch,
acetic acid production and Diazonium Blue B reaction.
3.7.3 Molecular Characterization of Isolated Strain
The identification of a novel yeast isolate having maximum enzyme activity was
carried out with the help of partial 18S rRNA, ITS1, 5.8S rRNA, ITS2 and partial
28S rRNA gene analysis from Merck Specialities Private Limited GeNeiTM, Bangalore
(India). Sequence data was aligned by using the BLAST program, and analyzed the
closest homologs for the yeast. Phylogenic tree was made by using Neighbor Joining
method (Saitou and Nei, 1987).
3.8. Process Optimization for -galactosidase Production
The study on the optimization of media and process parameters has been carried
out to maximize the production of β-galactosidase from isolated yeast culture.
3.8.1 Optimization of Media Composition
The optimization of the fermentation media of isolated yeast culture having high
-galactosidase production was carried out to get maximum enzyme activity. Different
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concentration of lactose, nitrogen sources, salts and trace elements were supplemented
in the medium to enhance the -galactosidase activity.
3.8.1.1 Effect of Lactose Concentration
To optimize the concentration of the lactose to maximize the enzyme activity,
the medium was supplemented with different concentrations (1.0-6.0%, w/v) of lactose.
3.8.1.2 Effect of Nitrogen Source and its Concentration
To investigate the influence of different nitrogen sources on the -galactosidase
activity, the nitrogen sources such as ammonium nitrate, urea, ammonium sulphate,
sodium nitrate, L-aspartate and L-glutamate were supplemented individually at the
concentration equivalent to 0.042% N in the fermentation media to investigate their
influence on enzyme activity. Further, to optimize the concentration of the above
screened best nitrogen source (urea) to maximize the enzyme activity. For this, the
medium was supplemented with different concentrations of urea (0.05-0.3%, w/v).
3.8.1.3 Effect of Salt and its Concentration
Different salts like calcium chloride, magnesium sulphate heptahydrate and
potassium dihydrogen orthophosphate were supplemented individually in the
fermentation media, to study their effect on enzyme activity. Further, to optimize the
concentration of the above screened best salt source (magnesium sulphate heptahydrate)
to maximize the enzyme activity. For this, the medium was supplemented with different
concentrations of magnesium sulphate (0.025-0.2%, w/v).
3.8.2 Optimization of Process Parameters
The optimization of different process parameters like inoculum size, inoculum
age, agitation rate, pH, temperature and incubation time were carried out using the
above optimized fermentation media to further enhance the enzyme activity.
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3.8.2.1 Effect of Inoculum Size
Different inoculum size (4-12%, v/v) were added to the fermentation medium to
find out the optimum inoculum concentration.
3.8.2.2 Effect of Inoculum Age
To find the optimal inoculum age for the maximal β-galactosidase activity, the
fermentation medium inoculated with different age of inoculum (12-28 h).
3.8.2.3 Effect of pH
To study the effect the hydrogen ion concentration, different pH (4.0-6.0) of
fermentation medium was adjusted.
3.8.2.4 Effect of Agitation
To study the effect of agitation, the flasks were incubated under shaking
conditions at 60, 80, 100, 120 and 140 rpm on a rotary shaker.
3.8.2.5 Effect of Incubation Temperature
The fermentation of medium was carried out at 20-40 °C, to study the effect of
temperature on the enzyme activity.
3.8.2.6 Effect of Incubation Time
To find the optimal incubation time for the maximal β-galactosidase activity, the
fermentation medium inoculated with yeast culture was incubated for 36 h under the
optimized conditions.
Optimization of parameters by the conventional method involves changing one
independent variable while unchanging all others at a fixed level. This is extremely time
consuming and expensive for a large number of variables and also may result in wrong
conclusions. Thus, response surface methodology (RSM) can be applied for
optimization of -galactosidase production process to observe the main effects and
interactions of the factors.
51
3.8.3 Process Optimization for -Galactosidase Production Using Response Surface
Methodology
The optimization of media and process parameters for the of yeast cells has been
carried out using Response surface methodology (RSM).
3.8.3.1 Selection of Factor Levels
From the preliminary experiments, the low and high levels chosen for five
independent variables for yeast extract concentration, urea concentration, pH,
temperature and incubation time were 0.2-0.4% (w/v), 0.07-0.13% (w/v), 5.0-6.0,
25-35 °C and 24-28 h, respectively, to get maximum β-galactosidase activity.
3.8.3.2 Experimental Design and Statistical Analysis
The statistical analysis of the data was performed using Central Composite
Rotatable Design (CCRD) with five variables at five levels each. The design was
generated by Design Expert, Trial version 6.0 statistical software (Stat-Ease INC.,
Minneapolis, MN, USA). The levels of factors for β-galactosidase production used in
the experimental design are listed in Table 3.2.
Table 3.2 Levels of different process variables for β-galactosidase production
Factor Process parameter Levels
-2.378 -1 0 +1 +2.378
X1 Yeast extract (%, w/v) 0.06 0.2 0.3 0.4 0.54
X2 Urea (%, w/v) 0.03 0.07 0.1 0.13 0.17
X3 pH 4.3 5.0 5.5 6.0 6.69
X4 Temperature (°C) 18.11 25 30 35 41.89
X5 Incubation time (h) 18.49 24 28 32 37.51
The preliminary experiments were conducted for chosen the data of the factors.
The experimental plan in un-coded form of process variables was shown in Table 3.3.
The experiments were conducted randomly. Response surface methodology was fitted
to the response variables, i.e. β-galactosidase activity (IU/mgDW). The second order
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polynomial equation (Equation 3.1) was fitted to the experimental data of each
dependent variable as given below:
n
i
iiiji
n
i
n
ij
iji
n
i
ii xxxxY1
21
1 11
0 Equation 3.1
Where Yi = Response {Y = Enzyme activity (IU/mgDW)}
xi = Independent variables (x1= yeast extract concentration (%, w/v), x2 = urea
concentration (%, w/v), x3 = pH, x4 = temperature (°C), x5 = treatment time (min), βo is
the value of coefficient of fitted response at the central point of design, βi, βij, βii are the
linear, quadratic and cross product regression coefficients, respectively.
Total number of experiments = 2 No. of Variables
+ 2* No. of variables + Central Points
For five variables, total no. of experiments = 25
+ 2 × 5 + 8 = 50
Five different levels for each experiment in coded form are
- , -1, 0, +1, + , where, = 2 No. of variables / 4
= 2 5/4
=2.378
The actual level of each factor was calculated using the following equation
Equation 3.2 (Mayer and Montgomery, 1995)
The response surface was generated to study the interaction of any two
independent variables, while keeping the value of third variable as a constant. The
three-dimensional surface plot could be helpful to provide useful information about the
behavior of the system within the experimental design.
53
Table 3.3 Experimental design of process variables for the optimization of β-galactosidase
production
Yeast extract Conc.
(%, w/v)
Urea Conc.
(%, w/v) pH
Incubation temp.
(°C)
Incubation
Time (h)
0.2 0.07 5.0 25 24
0.4 0.07 5.0 25 24
0.2 0.13 5.0 25 24
0.4 0.13 5.0 25 24
0.2 0.07 6.0 25 24
0.4 0.07 6.0 25 24
0.2 0.13 6.0 25 24
0.4 0.13 6.0 25 24
0.2 0.07 5.0. 35 24
0.4 0.07 5.0 35 24
0.2 0.13 5.0 35 24 0.4 0.13 5.0 35 24
0.2 0.07 6.0 35 24
0.4 0.07 6.0 35 24
0.2 0.13 6.0 35 24
0.4 0.13 6.0 35 24
0.2 0.07 5.0 25 32
0.4 0.07 5.0 25 32
0.2 0.13 5.0 25 32
0.4 0.13 5.0 25 32
0.2 0.07 6.0 25 32
0.4 0.07 6.0 25 32
0.2 0.13 6.0 25 32 0.4 0.13 6.0 25 32
0.2 0.07 5.0 35 32
0.4 0.07 5.0 35 32
0.2 0.13 5.0 35 32
0.4 0.13 5.0 35 32
0.2 0.07 6.0 35 32
0.4 0.07 6.0 35 32
0.2 0.13 6.0 35 32
0.4 0.13 6.0 35 32
0.062 0.1 5.5 30 28
0.537 0.1 5.5 30 28 0.3 0.028 5.5 30 28
0.3 0.171 5.5 30 28
0.3 0.1 4.31 30 28
0.3 0.1 6.69 30 28
0.3 0.1 5.5 18.10 28
0.3 0.1 5.5 41.89 28
0.3 0.1 5.5 30 18.48
0.3 0.1 5.5 30 37.51
0.3 0.1 5.5 30 28
0.3 0.1 5.5 30 28
0.3 0.1 5.5 30 28
0.3 0.1 5.5 30 28 0.3 0.1 5.5 30 28
0.3 0.1 5.5 30 28
0.3 0.1 5.5 30 28
0.3 0.1 5.5 30 28
54
3.9 Permeabilization of Yeast Cells for -galactosidase Activity
The permeabilization of yeast cells for β-galactosidase activity was carried out
followed the method of Panesar et al. (2007). The cells were harvested from 5 mL of
broth by centrifugation (5000 rpm × 5 min at 4 °C) and washed twice with phosphate
buffer (0.1M, pH 7.0). Different permeabilization agents were added to the yeast
biomass and the final volume was made 5 mL using the same buffer. The contents were
mixed on a vortex mixture and incubated for 10 min under shaking conditions. After
this, the cells were recentrifuged and washed twice with the phosphate buffer, and
analyzed for enzyme activity.
3.9.1 Screening of Permeabilization Agents
The permeabilization of yeast cells was carried out by using various chemical
agents and its different concentration; n-butanol (5-40%, v/v), n-propanol (5-40%, v/v),
iso-propanol (10-45%, v/v), acetone (20-50%, v/v), ethanol (20-70%, v/v), benzene
(5-40%, v/v), tritonX-100 (5-40%, v/v) and toluene (10-45%, v/v) for β-galactosidase
activity. The mixtures of permeabilizing agents in which ethanol (50%, v/v) was
combined with other organic solvents like acetone (30%, v/v), n-propanol (20%, v/v),
iso-propanol (40%, v/v), toluene (25%, v/v), n-butanol (10%, v/v) in 1:1 ratio were also
tested for β-galactosidase activity. Further, the optimization of concentration of above
best permeabilizing agent (a mixture of toluene and ethanol) has also been carried out
using different ratio of toluene and ethanol (10: 90- 60: 40%, v/v).
3.9.2 Optimization of Permeabilization Conditions
The above screened permeabilizing agents were used for the optimization of
temperature and incubation time for the permeabilization of yeast cells to get maximum
β-galactosidase activity.
55
3.9.2.1 Effect of Treatment Temperature
The yeast cells and permeabilizing agent mixture was incubated at different
temperature (10-40 °C), to find out the optimum temperature for the permeabilization of
yeast cells.
3.9.2.2 Effect of Treatment Time
To find out the optimal treatment time for the permeabilization of yeast cells,
the yeast cells and permeabilizing agent mixture was incubated at 25 °C for 5-30 min.
Optimization of parameters by the conventional method involves changing one
independent variable while unchanging all others at a fixed level. This is extremely
time-consuming and expensive for a large number of variables and also may result in
wrong conclusions. Thus, response surface methodology (RSM) can be applied for the
optimization of permeabilization process to observe the main effects and interactions of
the factors.
3.9.3 Process Optimization for Permeabilization of Yeast Cells Using Response
Surface Methodology
The optimization of process parameters for the permeabilization of yeast cells
has been carried out using response surface methodology (RSM).
3.9.3.1 Selection of Factor Levels
From the preliminary experiments, the low and high levels chosen for three
independent variables for toluene: ethanol ratio, treatment time and temperature were
30:70-50:50, 10-20 min, and 20-30 ºC, respectively to get maximum β-galactosidase
activity.
3.9.3.2 Experimental Design for the Process Optimization
For the optimization of permeabilization process, the experiments were
conducted according to Central Composite Rotatable Design with three variables
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(toluene: ethanol, temperature and treatment time) at five level each. The experimental
design was generated by using Design Expert statistical software (Trial version 6.0,
Stat-Ease Inc., Minneapolis, MN, USA).
Three factors and Central Composite Rotatable design (CCCD) with 20 design
points having 14 combinations with 6 replications of the central point were adapted in
this study. The independent variables and their levels are presented in Table 3.4.
Table 3.4 Level of different process variables for permeabilization of yeast cells
Factor Process parameter Level
1.682 -1 0 +1 +1.682
X1 Toluene and ethanol (%, v/v) 23.18:76.82 30:70 40:60 50:50 56.82:43.18
X2 Treatment time (min) 6.59 10 15 20 23.40
X3 Temperature (°C) 16.59 20 25 30 33.41
The highest and lowest levels of the interested range for each variable were
coded as plus and minus one, respectively, and the center point of the range was coded
to be zero.
Five different levels for each experiment in coded form are -α, -1, 0, +1, +α,
Where a = [2] (No. of variables/4)
= [2]3/4
= 1.682
The relationship between the coded and uncoded form of the variables is:
Equation 3.3
Where Xi is the actual setting in the uncoded units of the i th factor, i is the
average of the low and high settings for the ith
factor, and Ri is the range between the
low and high settings. Based on our preliminary investigation, the low and high levels
chosen for three independent variables for toluene: ethanol ratio, temperature and
57
treatment time were 30:70-50:50, 20-30 °C and 10-20 min respectively. The
experimental design of process variables for optimization of enzyme activity was shown
in Table 3.5.
Table 3.5 Experimental designs of process variables for the optimization of
permeabilization process
Std.
Conc. of toluene in ethanol
(%,v/v) Treatment time (min) Temperature (°C)
1 30 10 20
2 50 10 20
3 30 20 20
4 50 20 20
5 30 10 30
6 50 10 30
7 30 20 30
8 50 20 30
9 23.18 15 25
10 56.82 15 25
11 40 6.59 25
12 40 23.4 25
13 40 15 16.59
14 40 15 33.41
15 40 15 25
16 40 15 25
17 40 15 25
18 40 15 25
19 40 15 25
20 40 15 25
3.10 Biotransformation of Lactose to Lactulose Using Permeabilized
Yeast Cells
The optimization of various parameters such as lactose: fructose ratio, yeast
biomass, temperature, pH, and reaction time have been carried out to get maximum
yield of lactulose.
3.10.1 Effect of Biomass Concentration
Lactulose production was investigated using lactose (40%, w/v) and fructose
(20%, w/v) mixture in sodium phosphate buffer (50 mM, pH 7.0) containing NaCl
58
(10 mM) and MgCl2 (1mM) by employing different concentrations (1-10 gDW/L) of
biomass at temperature of 40 °C for different reaction periods (2-6 h). The reaction was
stopped by boiling the mixture for 5 min (Lee et al., 2004). After that, samples were
dried in freeze dryer. The gas-chromatography (GC) analysis of freeze dried sample was
carried out by following the method of Montilla et al. (2005a) with slight modifications.
The synthesis of oligosaccharides was confirmed with LC-MS (Finnigan Mat, LCQ,
US). Further, lactulose production was also confirmed by Gas Chromatography-Mass
Spectroscopy (GC 2010, Shimadzu, Japan) with the help of GC-MS profile and MS
spectra fragmentation pattern.
3.10.2 Effect of Lactose: Fructose Ratio
Lactulose production was investigated using different ratio of lactose: fructose
(50:10, 45:15, 40:20, 35:25, 30:30) in the reaction mixture, to find the optimal ratio of
lactose: fructose.
3.10.3 Effect of pH
The effect of hydrogen ion (pH) concentration on lactulose production was
monitored by using different pH (5.5-7.5) of the reaction mixture.
3.10.4 Effect of Reaction Temperature
The flasks containing the reaction mixture were incubated at different
temperature (30-70 °C) under shaking conditions, to find out the optimal temperature
for lactulose production.
3.10.5 Effect of Reaction Time
The influence of reaction time on the production of lactulose was analyzed for
different time intervals (1-6 h) and the samples were drawn at the time interval of every
1 h.
59
3.11 Biotransformation of Lactose to Lactulose using Immobilized Cell
System
The following parameters were investigated to optimize the lactulose production
using immobilized yeast cells.
3.11.1 Screening of Matrices for the Immobilization of Permeabilized Yeast Cells
Immobilization of Yeast Cells
The permeabilized yeast cells were centrifuged (5000 rpm × 5 min at 4 °C) and
washed twice with phosphate buffer (0.1M, pH 7.0). The immobilization of
permeabilized yeast cells was carried out by entrapment under aseptic conditions. The
different matrices namely sodium alginate, chitosan, k-carrageenan, agarose, pectin and
agar-agar were used for the immobilization of permeabilization of yeast cells.
3.11.1.1 Alginate
The procedure of Marwaha and Kennedy (1984) was used for the entrapment of
yeast cells in sodium alginate. The permeabilized yeast cells were mixed thoroughly
with different concentration of sodium alginate (1.5-3.0%, w/v) and the solution was
sterilized at 121 °C for 15 min. The resultant slurry was extruded as drops through a
sterilized glass syringe, into calcium chloride (0.075M) solution. The beads were left
suspended in calcium chloride solution for 5 h to allow complete gelation. The beads
were washed with sterilized distilled water prior to their use to remove excess of
calcium ions and unentrapped cells.
3.11.1.2 Chitosan
The immobilization of yeast cells in chitosan matrix was carried out using the
procedure of Liang et al. (2005) with slight modifications. Chitosan solution (1.0-3.0%,
w/v) was prepared by dissolving chitosan in acetic acid (1.0%, w/v) at room
temperature. The yeast pellet was dissolved into the above prepared chitosan solution
60
and the resultant slurry was extruded as drops through a sterilized glass syringe into
2.0% (w/v) sodium tripolyphosphate solution. The solution was stirred for 1 h followed
by filtration and rinsing with water.
3.11.1.3 k-Carrageenan
The procedure of Foster et al. (1983) was employed for entrapping the yeast
cells in k-carrageenan. The permeabilized yeast cells were mixed thoroughly with k-
carrageenan (2.0-3.0%, w/v) and the resultant slurry was then dripped through a
sterilized glass syringe into potassium chloride (1.0%, w/v) solution.
3.11.1.4 Agarose
The immobilization of yeast cells in agrarose matrix was carried out using the
procedure of Madrib et al. (1989) with slight modifications. The permeabilized yeast
cells were mixed thoroughly with agarose (1.0-3.0%, w/v) and the resultant slurry was
poured into sterilized petriplate, allowed to solidify and cubes were made by cutting of
solidified gel.
3.11.1.5 Pectin
The cell entrapment into calcium pectate gel bead was accomplished following
the method of Kurillova et al. (1992). The commercial citrus pectin (3.0-6.0%, w/v,
unless otherwise specified) was neutralized with with concentrated ammonia solution to
pH 7.0. The yeast biomass mixed thoroughly with the pectate solution and resultant
slurry was extruded as drops through a sterilized syringe into calcium chloride (0.2M)
solution. The resultant beads were washed with sterile distilled water to remove excess
of calcium ions and unentrapped cells. The beads obtained were kept overnight at 4 °C
and then washed with aluminum nitrate (0.1 M) and sterile water.
61
3.11.1.6 Agar-Agar
The cells were immobilized by entrapping into agar-agar as described by Toda
and Shoda (1975). This agar solution was prepared by dissolving agar (2.0-3.0%, w/v)
in distilled water, holding it in a boiling water bath, and then cooling it to 45 °C. The
permeabilized cells were mixed thoroughly and the resultant slurry was then dripped
through a sterilized glass syringe into an ice cold, toluene-chloroform (3: 1) mixture.
The resulted beads were washed with phosphate buffer, immediately air dried.
3.11.1.7 Alginate-Pectin
The immobilization of yeast cells in alginate-xanthan was carried out using the
procedure of Satar et al. (2008) with slight modifications. Sodium alginate (1.5%, w/v)
and pectin (0.5%, w/v) was dissolved and the solution was sterilized at 121 °C for 15
min. The pH 7 of the slurry maintain by using KOH. The mixture solution was extruded
dropwise through a syringe with a thin needle into 0.075M CaCl2 solution. The drops
were shaken gently for 1 h and left in calcium chloride solution for 5 h to allow
complete gelation. The beads were washed several times with sterilized phosphate
buffer (pH 7.0) to remove excess of calcium ions and unentrapped cells.
3.11.1.8 Alginate-Carrageenan
The preparation of alginate-carrageenan beads was performed by using the
method of Mohamadnia et al. (2007) with slight modifications. Sodium alginate (1.5%,
w/v) and k-carrageenan (0.5%, w/v) was dissolved and the solution was sterilized at 121
°C for 15 min. The mixture solution was extruded dropwise through a syringe with a
thin needle into a stirring salt solution containing 0.075M CaCl2 and 0.135M KCl. The
drops were shaken gently for 1 h and left in CaCl2-KCl solution for 5 h to allow
complete gelation. The beads were washed several times with sterilized phosphate
buffer (pH 7.0) to remove excess of calcium ions and unentrapped cells.
62
3.11.1.9 Alginate-Gelatin
The preparation of alginate-gelatin beads was performed by using the method of
Roy et al. (2007) with slight modifications. Sodium alginate (1.5%, w/v) and gelain
(0.5%, w/v) was dissolved and the solution was sterilized at 121 °C for 15 min. The
mixture solution was extruded dropwise through a syringe with a thin needle into
0.075M CaCl2 solution. The drops were shaken gently for 1 h and left in calcium
chloride solution for 5 h to allow complete gelation. The beads were washed several
times with sterilized phosphate buffer (pH 7.0) to remove excess of calcium ions and
unentrapped cells.
3.11.1.10 Alginate-Xanthan
The immobilization of yeast cells in alginate-xanthan was carried out using the
procedure of Pongjanyakul and Puttipipatkhachorn (2007) with slight modifications.
Sodium alginate (1.5%, w/v) and xanthan gum (0.5%, w/v) was dissolved and the
solution was sterilized at 121 °C for 15 min. The mixture solution was extruded
dropwise through a syringe with a thin needle into 0.075M CaCl2 solution. The drops
were shaken gently for 1 h and left in calcium chloride solution for 5 h to allow
complete gelation. The beads were washed several times with sterilized phosphate
buffer (pH 7.0) to remove excess of calcium ions and unentrapped cells.
3.11.1.11 Alginate-Agarose
For the preparation of alginate-agarose beads, sodium alginate (1.5%, w/v) and
(0.5%, w/v) agarose was dissolved and the solution was sterilized at 121 °C for 15 min.
The mixture solution was extruded dropwise through a syringe with a thin needle into
0.075M CaCl2 solution. The drops were shaken gently for 1 h and left in calcium
chloride solution for 5 h to allow complete gelation. The beads were washed several
63
times with sterilized phosphate buffer (pH 7.0) to remove excess of calcium ions and
unentrapped cells.
3.11.1.12 Alginate-Agar
The cells were immobilized by entrapping into agar-agar using the procedure of
Rao et al. (1986) with slight modifications. Sodium alginate (1.5%, w/v) and (0.5%,
w/v) agar-agar was dissolved and the solution was sterilized at 121 °C for 15 min. The
mixture solution was extruded dropwise through a syringe with a thin needle into
0.075M CaCl2 solution. The drops were shaken gently for 1 h and left in calcium
chloride solution for 5 h to allow complete gelation. The beads were washed several
times with sterilized phosphate buffer (pH 7.0) to remove excess of calcium ions and
unentrapped cells.
3.11.1.13 Alginate-Chitosan
The immobilization of yeast cells in alginate-chitosan matrix was carried out
using the procedure of Sezer and Akbuga (1999) with slight modifications. Alginate-
chitosan beads were prepared by dripping 1.5% (w/v) sodium alginate solution into a
cross-linking solution composed of 0.075M CaCl2, 0.5% (w/v) chitosan and 0.5% (v/v)
acetic acid. The drops were shaken gently for 1 h and left in calcium chloride solution
for 5 h to allow complete gelation. The beads were washed several times with sterilized
phosphate buffer (pH 7.0) to remove excess of calcium ions and unentrapped cells.
3.11.2 Characterization of Alginate and Hybrid Beads (Alginate-Carrageenan and
Alginate-Xanthan Beads)
Characterization of alginate and hybrid beads were carried out by studying the
various parameters like morphology, functional group characterization, flow properties,
hardness of beads, swelling property, and thermal stability.
64
3.11.2.1 Morphology
The morphological characterizations of alginate and hybrid beads were carried
out using scanning electron microscope (SEM) to check the cell entrapment. The
sample for SEM analysis has been carried out by following the procedure of Kwon et al.
(2009). The cross-section of the bead was photographed using a SEM (Carl Zeiss
EVO40, Germany). After being treated in 30, 50, 70 and 90% (v/v) of ethanol each for 5
min, the beads were placed in absolute ethanol for 15 min for removing water. The
dehydrated beads and as well as their cross-section were mounted on aluminium stubs
and placed in the desiccator to dry overnight or until needed. The samples on coated
with gold and examined under a scanning electron microscope.
3.11.2.2 Fourier Transform Infrared Spectroscopy
FTIR spectra of alginate and hybrid beads were carried out to identify the
functional groups. FTIR spectra of the beads were obtained by using a FTIR
spectrophotometer (RX-FTIR, PerkinElmer, USA). The dry samples (alginate, xanthan,
carrageenan, alginate-carrageenan and alginate-xanthan) were mixed with dry
potassium bromide and pressed into plate for measurement and recording the FTIR
spectrum (Sankalia et al., 2005).
3.11.2.3 Flow Properties
The flow properties of cell loaded beads were investigated by measuring the
angle of repose using fixed-base cone method (Sevukarajan et al., 2011). For this,
funnel was fixed at 1cm above the horizontal flat surface and beads (alginate, alginate-
carraginan and alginate-xanthan) were allowed to fall freely through the funnel until the
apex of conical pile just touched the tip of the funnel. The angle of repose (ø) was
determined by formula:
ø = tan-1
(h/r),
65
Where, h =Cone height of microspheres;
r = Radius of circular base formed by the beads on the ground.
3.11.2.4 Texture Analysis of Beads
The hardness of alginate and hybrid beads was measured by using a Texture-
analyzer (TA-XT2i, Texture analyzer, Stable Micro System, UK). The apparatus was
equipped with a 5mm cylinder probe (P/5). The hardness of the beads was expressed as
the load (g force) that the beads could withstand for 1 mm compression (Dey et al.,
2003).
3.11.2.5 Swelling Characterization
The evaluation of pH sensitive behavior of alginate and hybrid beads was
determined by the percentage swelling ratio. The removal of excess surface-adhered
liquid was carried out by blotting paper and the samples transferred into separate tubes
of phosphate buffer solution (pH 5-9) for at least 24 h at room temperature. During this
process, the beads were removed from the buffer solution and frequently weighed by an
electronic microbalance after pressing between two filter papers to remove excess
surface water. Then, beads were dried in a vacuum oven at 60 °C to constant weight.
Percentage swelling ratio was calculated by using the formula of Wan Ngah et al.
(2002).
Where, Ws = weight of beads in swollen state,
Wd = weight of beads in dry state,
Ws = initial weight of beads
66
3.11.2.6 Differential Scanning Calorimetry Analysis
Thermograms of alginate and alginate-carrageenan beads were obtained using a
Differential Scanning Calorimetry (DSC 4000, PerkinElmer, USA) by following the
method of Sankalia et al. (2005) with slight modifications. The powdered sample of
beads was sealed in an aluminium pan and heated at a constant rate of 10 ºC/min, over a
temperature range of 20-445 °C. Inert atmosphere was maintained by purging nitrogen
at the flow rate of 10 mL/min.
3.11.2.7 Cell Entrapment Efficiency
The counting of cells has been carried out by using hemocytometer (Covarrubias
et al., 2012). 50 mgDW of yeast biomass was mixed properly with 10 mL of matrix
solution. Cells entrapment efficiency of bead was measured by the counting of yeast
cells using hemocytometer. For this, 10 beads (alginate and composite gel beads) were
dissolved in 1 mL of sodium citrate (1.0%, w/v) and cells were counted by taking the
average of 5 chambers of hemocytometer.
3.11.2.8 Stability of Beads as a Function of β-Galactosidase Activity
To check the stability of beads, recycling of yeast cells entrapped alginate and
hybrid beads (alginate-carrageenan and alginate-xanthan) was carried out for the
biotransformation of lactose by β-galactosidase at phosphate buffer pH 7.0 and
temperature of 50 oC and measuring the β-galactosidase activity after each cycle. The
residual enzyme activity was calculated by taking the enzyme activity of the first cycle
as 100%.
3.11.3 Process Optimization for Biotransformation of Lactose to Lactulose Using
Immobilized Cell System
To optimize the immobilized technique for the efficient lactulose production,
the following parameters were investigated.
67
3.11.3.1 Effect of Bead Size
To investigate the effect of different bead size on the lactulose production, the
beads of different size range (2.25-3.55 mm) were used. These beads were made using
syringe needles of variable sizes.
3.11.3.2 Effect of Biomass Load
To find the effect of yeast biomass concentration on the production of lactulose,
various biomass loads (2-5 gDW/L) of permeabilized cells were used.
3.11.3.3 Effect of pH
The effect of hydrogen ion (pH) concentration on lactulose production was
monitored by using different pH (5.5-7.5) of the reaction mixture.
3.11.3.4 Effect of Reaction Temperature
The flasks containing reaction mixture were incubated at different temperature
(40-70 °C) under shaking conditions, to find out the optimal temperature for lactulose
production.
3.11.3.5 Effect of Reaction Time
The influence of reaction period on lactulose synthesis was analyzed for
different time intervals (3-5 h).
3.11.4 Recycling of Alginate-Carrageenan Entrapped Yeast Cells
To investigate the effect of immobilization on reusability of yeast cells, the
immobilized yeast cells after each cycle of 4 h reaction time at 50 °C was washed with
phosphate buffer and then suspended again in a fresh reaction mixture to measure
lactulose production, enzyme activity, cells entrapment efficiency, hardness and
percentage swelling ratio.
68
3.11.4.1 Lactulose Production
To test the suitability and stability of beads for repeated use in lactulose
production from alginate-carrageenan beads containing yeast cells were recycled. After
every cycle of 4 h, the beads were removed and replaced by fresh reaction medium after
washing the beads with phosphate buffer (pH 7.0).
3.11.4.2 β-Galactosidase Activity
The β-galactosidase assay of beads was carried out by the method of Miller
(1972). A decrease in enzyme activity was observed at each cycle. The residual activity
was calculated by taking the enzyme activity of the first cycle as 100%.
3.11.4.3 Hardness of Beads
The hardness of beads of each cycle was calculated by following the method of
Dey et al. (2003).
3.11.4.4 Water Uptake of Beads
The swelling ratio of beads for each cycle was calculated by followed the
method of Wan Ngah et al. (2002).
Where Ws, Wi and Wd are the weights of the swollen beads, initial beads and dry
beads, respectively.
3.11.4.5 Morphology of Beads
The morphological characterizations of beads (initial and after 10th cycle) were
carried out using scanning electron microscope (SEM) to check the cell entrapment as
well as cell leakage after the 10th cycle.
69
3.12 Biotransformation of Whey Lactose to Lactulose Using
Immobilized Cell System
For the production of lactulose, the physico-chemical characterization of whey,
processing of whey and optimization of process parameters for lactulose production
using yeast cells entrapped in alginate-carrageenan beads have been carried out.
3.12.1 Physico-Chemical Characterization of Cheese Whey
The physico-chemical characterization such as pH, lactose concentration,
mineral content, citrate content, chloride content as well as total protein, fat and solid
content of cheese whey were examine by using different techniques.
3.12.2 Processing of Whey
During the course of lactulose production, clarification of whey was carried
through protein precipitation induced by heating the whey at 90 °C for 20 min.
Precipitated proteins were removed by centrifugation at 4,000 rpm for 15 min.
Furthermore, whey was concentrated by using vacuum evaporator, so that the final
concentration of lactose in whey reaches 40% (w/v).
3.12.3 Optimization of Process Parameters
Various process parameters (biomass load, pH, reaction temperature and
reaction time) were optimized to enhance the production of lactulose from whey using
immobilized cells. For this, concentrated whey (containing 40%, w/v of lactose) was
supplemented with fructose (20%, w/v) adjusted to 7.0 for the production of lactulose.
3.12.3.1 Effect of Biomass Load
To find the effect of yeast biomass concentration on the production of lactulose,
various biomass loads (2-5 gDW/L) of permeabilized cells were used.
70
3.12.3.2 Effect of pH
The effect of hydrogen ion (pH) concentration on lactulose production was
monitored by using different pH (6.0-7.5) of the reaction mixture.
3.12.3.3 Effect of Reaction Temperature
The flasks were incubated at different temperatures (40-70 °C) under shaking
conditions, to find out the optimal temperature for lactulose production.
3.12.3.4 Effect of Reaction Time
The influence of reaction period on lactulose synthesis was analyzed for
different time intervals (2-5 h).
3.12.4 Effect of Reusability of Beads on Lactulose production
To test the suitability and stability of beads for repeated use in lactulose
production from whey using alginate-carrageenan beads containing yeast cells were
recycled. After every cycle of 4 h, the beads were removed and replaced by fresh
reaction medium after washing the beads with phosphate buffer (pH 7.0).
3.13 Analytical Techniques
The following analytical techniques were used during the course of the present
investigation.
3.13.1 Measurement of β-Galactosidase Activity
The enzyme assay was carried out by the method of Miller (1972).
Reagents
(i) O-Nitrophenyl-β-D-galactopyanoside: 4.0 mg/mL in 0.1M phosphate buffer
(pH 7.0)
71
(ii) Z-buffer: Z-buffer was prepered by dissolved (g/L) Na2HPO4.7H2O: 16.1;
NaH2PO4. H2O: 5.5; KCl: 0.75; MgSO4. H2O: 0.246 and β-mercaptoethanol:
2.7 mL
(iii) Sodium dodecyl sulphate: 0.1% (w/v)
(iv) Sodium carbonate: 0.5M
Procedure
The yeast cells from the broth were centrifuged (5000 rpm × 10 min at 4 °C).
The biomass was washed twice with phosphate buffer (0.1M, pH 7.0), resuspended in
the same buffer and diluted. The appropriately diluted cell suspension (0.1 mL) was
taken in a tube and to this 0.9 mL of Z- buffer was added. The cells were lysed by
adding chloroform (50 μl) and sodium dodecyl sulphate (20 μl). The reaction mixture
was incubated at 30 °C for 10 min. Then, 0.2 mL of O-Nitrophenyl-β-D-
galactopyanoside (ONPG) was added and incubated for 5 min at the same temperature.
The reaction was stopped by added 1 mL of Na2CO3 (0.5 M). The liberated colour was
measured at 420 nm using a UV-Vis spectrophotometer (DR 5000, HACH, Germany).
One unit of enzyme activity is defined as one micromole of o-nitrophenol liberated per
min under standard assay condition. The standard curved was prepared using
o-nitrophenol as standard.
3.13.2 Dry Weight Determination
A known amount of the yeast culture was centrifuged (5000 rpm × 10 min at
4 °C) and washed twice with distilled water. The cell suspension was filtered through
the pre weight Whatman filter paper. The biomass was dried in oven at 80 °C, till a
constant weight was obtained.
72
3.13.3 Lactulose estimation
The estimation of lactulose in the sample was carried out following the method
of Montilla et al. (2005a) with slight modifications.
Reagents
(i) Pyridine
(ii) N-trimethylsilylimidazole (TMSI)
Procedure
For the analysis, the freeze dried sample was resuspended in
N-trimethylsilylimidazole and pyridine (1: 4). Approximate, 2: 1 molar ratio of TMSI
and pyridine to active hydrogen was used to silylate the carbohydrates; the reaction was
completed in 1 h at 65 °C. Volumes in the range of 1µl of were injected into the
stainless steel column Rtx®-5MS. The separation was performed at 100 °C for 1 min,
followed by an increase up to 250 °C at rate of 15 °C min and finally temperature rise
up to 300 °C for 8 min. Injections were carried out in split mode 1: 10. The lactulose
was identified by comparison with the retention time of the standards. Furthermore,
Gas-chromatography (GC 2010, Shimadzu, Japan) was performed again with the
addition of internal lactulose into the reaction mixture to confirm the lactulose peak.
3.13.4 Whey Lactose Estimation
The lactose estimation in whey was carried following the procedure of
Nickerson et al. (1976).
Reagents
(i) Zinc acetate-phosphotungstic (ZAPT) reagent-Zinc acetate (25.0 g) and
phosphotungstic acid (12.5 g) were dissolved in distilled water. Then 20 mL glacial acid
and the final volume were made 100 mL with distilled water
73
(ii) Glycine-NaOH buffer- 150 mL of glycine solution containing 2.48 g glycine and
1.93 g NaCl was added to 850 mL of 0.385N NaOH and pH was adjusted to 12.8
(iii) Methylene solution- 5.0% (w/v) methylamine-HCl in distilled water
(iv) Sodium sulfite solution- 1.0% (w/v) Na2SO3 in distilled water
Procedure
0.1 mL ZAPT reagent was added into 0.8 mL of whey and incubated for 10 min.
After that, sample mixture was centrifuged at 5000 rpm for 10 min. To, 0.5 mL of
NaOH (1N) was added and the final volume was made 10 mL. After recentrifugation, 5
mL of prepared whey sample was taken in a test tube. To this, 5 mL of glycine-NaOH
buffer was added. Then 0.5 mL each of methylamine solution (5.0%, w/v) and sodium
sulfite solution (1.0%, w/v) was added. After thoroughly mixing the sample mixture
was kept at 65 °C in a water bath for 25 min. Then, the sample mixture was cooled
immediately in an ice-water bath for 2 min to stop the reaction. The absorbance of the
sample was taken at 540 nm on a spectrophotometer. The standard curve was prepared
using lactose as standard.
3.13.5 Chloride Content
Chloride content in whey was determined by the Mohr test, in which silver
nitrate is use for titration and potassium chromate as an indicator.
Reagents
(i) Potassium chromate-5% (w/v)
(ii) 0.1 M AgNO3
Procedure
Transfer 50 mL of sample into 250 mL of Erlenmeyer flasks. Add 1 mL of
potassium chromate indicator. Titrate sample with standerlized 0.1M AgNO3 to the pale
red-brown colour that persists for 30 s. Record the volume of titrate used.
74
Calculation
3.13.6 Estimation of Protein Content
Estimation of protein content is done as per procedure given by A.O.A.C.
(1984).
Reagents
(i) Mixed indicator: Prepared 0.1% bromocresol green and 0.1% methyl red indicator in
95% alcohol separately. Mix 10 mL of the bromocresol green with 2 mL of the methyl
red solution in a bottle provided with dropper
(ii) 2% Boric acid
(iii) 0.1N Hydrochloric acid
(iv) 40% Sodium hydroxide
(v) Catalyst for digestion: Mix 2.5 g of powdered selenium oxide, 100 g of potassium
sulphate, and 20 g of copper sulphate
Apparatus
(i) Kjeldhal digestion flask
(ii) Distillation flask
Procedure
5.0 ±0.1 mL of sample was taken in to digestion tube and 25 mL of concentrated
sulfuric acid was added. Digestion was carried out at 440 °C heating temperature and
20-25 mL of 40% NaOH was injected into the digestion tube. The distillate was
collected in 25 mL of saturated boric acid solution already consisting of 4 drops of
indicator. The distillate (Ammonium bromated) was then titrated against 0.1N HCl until
75
the color was changed from bluish green to colorless. The volume of 0.1 N HCl used
was recorded. A blank estimation was also carried out with distill water instead of
sample in the similar conditions.
Calculation
Protein (%) = Nitrogen (%) × 6.25
3.13.7 Estimation of Crude Fat
Estimation of crude fat is done as per procedure given by A.O.A.C. (1984).
Procedure
Fat is extracted from an oven dried sample using a Soxhlet extraction apparatus.
Transferred the dried sample remaining after moisture determination to a thimble and
plug the top of the thimble with a wad of fat-free cotton. Dropped the thimble into the
fat extraction tube of a Soxhlet apparatus and attached the bottom of the extraction tube
to a Soxhlet flask. Poured approximately 75 mL or more of anhydrous ether in the flask
through the fat extraction tube and attach top of the fat extraction tube with condenser.
Extract the sample for 16 h or longer on a heater. When the ether has reached a small
volume, pour it into a small, dry (previously weighed) beaker through a small funnel
containing a plug of cotton. Rinse the flask and filter thoroughly, using several small
portions of ether. Evaporate the ether on a steam bath at low heat, preferably under a
current of air. Dry it 100 °C for 1 h, cool and weigh. The difference in the weights gives
the ether-soluble material present in the sample.
76
Calculation
3.13.8. Total Solid Content
Whey sample was taken into petri plate and dry in oven (force draft or
circulating) at constant temperature of 105 °C. After that, dried whey sample put in
desiccator (gypsum or silica gel) to remove the remaining moisture.
