Abstract: β-D-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23), most commonly known as lactase is widely distributed in nature and has wide scope in food industry, because of its applications in the production of lactose hydrolyzed milk and low lactose dairy products. The present study reports the use of whey as the fermentation medium for the production of β-D-galactosidase using a culture of Kluyveromyces marxianus as the producer organism. The effect of different process parameters such as pH of the medium, temperature, inoculum size, age of inoculum, agitation and incubation time was monitored to enhance the production of β-D-galactosidase. The maximum enzyme activity was observed with pH 5.0, temperature 30°C, inoculum size 6% (v/v) having 20 h age, under shaking conditions 100 rpm after 28 h of incubation.
INTRODUCTION
Whey is the by-product separated from milk during the cheese-making process, which consists of water, lactose, proteins, vitamins and mineral salts. The production of cheese is increasing worldwide, which results in generation of large quantities of whey. Several possibilities of cheese whey exploitation have been investigated; however, approximately half of the world cheese whey production is not treated, but is discarded as effluent. The dumping of whey constitutes a significant loss of potential food and energy. This by-product posses a serious pollution problem, if disposed untreated, as it imposes a high biochemical oxygen demand (Marwaha and Kennedy, 1988; Mawson, 1994). The problem of whey treatment demands simple and economical solutions. A better alternative is subjecting the whey to processes through which the value added products are manufactured which may contribute wholly or partially to the costs. The carbohydrate reservoir of lactose (4-5%) in whey and presence of other essential nutrients makes whey as good natural medium for growth of microorganisms and potential substrate for bio-processing through microbial fermentation (Gandhi, 1989). It can be low priced source of sugars for the fermentation process. Availability of carbohydrate reservoir of lactose in whey and presence of other essential nutrients makes whey as potential substrate for the growth of lactose utilizing microorganisms.
Lactose utilizing yeasts such as Kluyveromyces sp. are important sources for the β-galactosidase production. It is one of the most promising enzymes for biotechnological applications and can be used for the production of lactose hydrolyzed milk and other low-lactose dairy products to meet the dietary requirements of lactose intolerant people (Mahoney, 1997; Panesar et al., 2006). The yeast Kluyveromyces has been reported to be the most important source for the production of β-galactosidase as the enzyme from the yeast has an optimum pH suitable for lactose hydrolysis in milk (Joshi et al., 1989; Stred’ansky et al., 1993; Panesar et al., 2007). Enzymatic lactose hydrolysis in milk can be used to solve the problems of lactose intolerance of the individuals who are deficient in lactase (Gekas and Lopez-Leiva, 1985; Roy and Gupta, 2003; Panesar et al., 2006).
Increasing demand for β-D-galactosidase requires cost-effective production methods to ensure economic feasibility of lactose hydrolysis at commercial scale. The production of β-D-galactosidase through lactose utilizing yeasts could be an alternative processing route for whey lactose utilization. The use of biotechnological means to find the suitability of whey for enzyme production can serve dual purpose while producing valuable product, β-D-galactosidase and addressing to the whey disposal environmental pollution problem. Keeping in view the availability and quantity of the lactose rich dairy by-product whey, the possibility of developing a technology for the production of β-D-galactosidase from whey can be explored to overcome the problems encountered in its disposal.
MATERIALS AND METHODS
Procurement of Micro-Organism
Kluyveromyces marxianus NCIM 3465 was procured from National Collection
of Industrial Micro-organisms, National Chemical Laboratory, Pune (India).
Maintenance of the Culture
The yeast culture was revived on maintenance medium containing (w/v) malt
extract (0.3%), yeast extract (0.3%), peptone (0.5%) and glucose (1.0%). The
culture was incubated under shaking conditions at 30°C for 48 h and maintained
for fortnightly intervals on agar slants at 4°C.
Preparation of Starter Culture
The maintenance medium (50 mL) were taken in Erlenmeyer flask (250 mL),
sterilized by autoclaving at 15 psi for 20 min, cooled and inoculated with a
loopful of culture from slant and incubated under shaking conditions at 30°C
for 24 h.
Production of β-D-Galactosidase
Clarified whey was used as the media for the production of enzyme and was
supplemented with peptone (0.5%), yeast extract (0.3%), ammonium sulphate (0.2%)
and potassium dihydrogen orthophosphate (0.1%). The fermentation medium (50
mL in 250 mL flask) was inoculated with 24 h old inoculum (unless otherwise
mentioned), incubated at specified times under shaking conditions. After specific
time intervals, the samples were drawn from flasks and assayed for enzyme activity.
Enzyme Assay
The assay for measurement of enzyme activity was followed as per the method
of Miller (1972). One unit of enzyme activity is defined as one micromole (μM)
of 2-nitrophenol liberated per min under standard assay conditions. The biomass
was washed twice with phosphate buffer (0.1 M, pH 7.0) and resuspended in the
same buffer. The appropriately diluted cell suspension (0.1 mL) was taken in
a test 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-galactopyranoside
was added and incubated for 5 min at the same temperature. The reaction was
stopped by adding 1 mL of sodium carbonate. The liberated colour was measured
at 420 nm using a spectrophotometer. All the enzyme assays were performed in
triplicate and the mean values are reported.
Optimization of Process Parameters
Different process parameters such as pH, temperature, age of inoculum, inoculum
size, agitation and incubation time were optimized by varying the respective
parameters.
RESULTS AND DISCUSSION
The effect of following process parameters was observed to enhance the β-D-galactosidase activity during the course of the present investigation.
Effect of pH
The effect of hydrogen ion concentration on β-D-galactosidase production
was monitored by using fermentation medium having a pH range of 4.0-6.0. The
results of the enzyme activities showed maximum activity (1490 IU g-1
dry weight) at pH 5.0 (Table 1), however, at higher and lower
pH levels, a decrease in the enzyme activity was observed. At pH 4.0, minimum
enzyme activity of 1370 IU g-1 was observed. The pH of a system affects
at least two aspects of microbial cells i.e., functioning of its enzymes and
the transport of nutrients into the cell. The pH values also affect the RNA
and protein synthesis (Klovrychev et al., 1979). When micro-organisms
are grown on either side of their optimum pH range, there may be an increased
lag phase. The pH 5.0 has also been used for β-D-galactosidase production
using different yeast strains by earlier researchers (Inchaurrondo et al.,
1994). However, a pH 5.5 has been used for the enzyme production using K.
marxianus (Artolozaga et al., 1998), whereas, a pH 4.68 has been
found optimum for β-D-galactosidase production using K. lactis (Ramirez-Matheus
and Rivas, 2003).
From these observations, pH 5.0 was considered optimal for maximum β-D-galactosidase activity and was used in the subsequent experiments.
Effect of Temperature
To find out the optimum temperature for β-D-galactosidase production,
whey medium, after inoculation, was incubated at temperatures in the range of
20-40°C (Table 1). The enzyme activity increased with
increase in the temperature up to 30°C, however, a decrease in the activity
was observed at higher temperature with a sharp decrease at temperature range
of 35-40°C. The maximum enzyme activity of 1490 IU g-1 dry weight
was observed at 30°C.
It has been established fact that enzymes are most active at optimum temperature and enzymatic reactions proceed at maximum rate. However, below and above optimal temperature reaction rate is decreased which causes the problems in cell metabolism. The temperature of 28-30°C has been used for β-D-galactosidase production by many workers (Ku and Hang, 1992; Hewitt and GrootWassink, 1984; Artolozaga et al., 1998). Ramirez-Matheus and Rivas (2003) have reported 30.3°C as an optimal temperature for β-D-galactosidase production by K. lactis.
From the above observations, a temperature of 30°C was considered optimal for achieving maximum enzyme activity and it was adopted for further experimentation.
Effect of Inoculum Age
To find the effect of inoculum age on β-D-galactosidase production,
whey fermentation medium was inoculated with 12-28 h old cultures. The results
displayed progressive increase in the enzyme activity between 12-20 h inoculum
age and the maximum enzyme activity (1530 IU g-1 dry weight) was
observed with 20 h old yeast culture (Table 2).
| Table 1: | Effect of pH and temperature on β-D-galactosidase production by K. marxianus |
| Table 2: | Effect of inoculum age and size on β-D-galactosidase production by K. marxianus |
| Table 3: | Effect of agitation on β-D-galactosidase production by K. marxianus |
| Process conditions: Inoculum age: 20 h; pH: 5.0; Inoculum size: 6%, (v/v) Temperature: 30°C; Incubation period: 32 h | |
However, suppression in enzyme activity was observed with 24 and 28 h old inoculum. The low enzyme activity with inoculum age of 12 h may be attributed to the fact that yeast culture may have not yet entered the log phase of growth. The maximum enzyme activity observed with inoculum of 20 h, which may be due to the exponential phase of the yeast culture used as an inoculum. Similarly, 20 h old culture of K. fragilis for β-D-galactosidase production has been used (Chen et al., 1992). However, Ku and Hang (1992) have used 24 h old culture of K. marxianus strains for β-D-galactosidase production.
Since, 20 h yeast culture displayed maximum β-D-galactosidase activity, it was selected for further studies.
Effect of Inoculum Size
Different inoculum levels (4-12%, v/v) were added to the whey medium to
study the influence of inoculum size on the β-D-galactosidase activity
(Table 2). The enzyme activity increased with the increase
in inoculum size up to 6% (v/v), thereafter no improvement in enzyme activity
was observed. The maximum enzyme activity of 1540 IU mg-1 dry weight
was observed with 6% (v/v) inoculum of yeast culture. The low enzyme activity
at 4% (v/v) inoculum level may be attributed to the low density of starter culture.
Therefore, an inoculum of 6% (v/v) can be considered optimal for achieving maximum
enzyme activity using 20 h old yeast culture.
Effect of Agitation
To study the effect of agitation on the β-D-galactosidase production
by the yeast culture, the cultivation was carried under stationary conditions
in an incubator and shaking condition (60-120 rpm) on a rotary shaker (Table
3). The agitation mode of cultivation supported an increase in enzyme activity
as compared to the stationary conditions. During the experimentation, a progressive
increase in the enzyme activity with increase in the agitation rate up to 100
rpm was observed. However, no improvement in the enzyme activity was observed
at higher agitation rate. The increase in enzyme activity with agitation mode
may be attributed to the uniform distribution of the yeast culture in the medium
resulting in better nutrient availability and oxygen transfer rate. The earlier
studies have also supported the agitation mode for β-D-galactosidase production
(Pedrique and Castillo, 1982; Champluvier et al.,1988; Chen et al.,
1992).
Since, maximum β-D-galactosidase activity was observed with agitation rate of 100 rpm, it was selected for further investigations.
| Fig. 1: | Effect of incubation period on β-D-galactosidase production by K. marxianus. Bars indicate the standard deviation from triplicate determinations |
Effect of Incubation Period
To find out the optimal incubation time for the maximal β-D-galactosidase
activity, the whey medium inoculated with yeast cultures was incubated for 36
h under the above optimized conditions (Fig. 1). The results
displayed an increase in enzyme activity up to 24 h and thereafter no improvement
in this function was observed. However, a decrease in the enzyme activity was
observed with further increase in the incubation time (28-36 h), which may be
attributed to the growth of the culture having reached the stationary phase
and as a consequence of metabolism. A maximum enzyme activity of 1580 IU g-1
dry weight was observed after 24 h of incubation. The earlier workers also reported
that maximum enzyme yield is obtained at the beginning of the stationary phase
of growth, after which the yield of the enzyme decreases (Pinheiro et al.,
2003). Ku and Hang (1992) have reported 24 h as an optimal incubation period,
however, a fermentation time of 18.5 h has also been reported optimal using
K. lactis (Ramirez-Matheus and Rivas, 2003). Keeping the above observations
in view, the incubation time of 24 h was considered optimal for maximum β-D-galactosidase
activity under the above optimized conditions.
CONCLUSIONS
From the foregoing account and the observations made during the present investigation, it can be concluded that whey is suitable medium for the production of β-D-galactosidase. The maximum enzyme activity can be obtained with the process conditions of pH 5.0, temperature 30°C and inoculum size 6% (v/v) of 20 h old yeast culture under shaking conditions (100 rpm) after an incubation period of 24 h. The different optimal conditions reported by various researchers for maximum β-D-galactosidase activity could be explained by the differences in the nature of the strains and medium composition used in their studies.