Phytate is one of the major organic storage forms of phosphorous of phosphorus in
seeds, cereal, oil and legume; in nature about 75%-80% of total phosphorus is available in
this form. Phosphorus is stored in roots and in seeds and cereals as phytate. Phytases are
responsible for breakdown of phytic acid (phytate) into inorganic monophosphates and
free myo- inositol. Phytases are a class of phophatases which hydrolyze phytic acid into
inorganic phosphate and myo inositol or less phosphorylated phosphates. Monogasteric
animal like poultry, human and fish lack phytase due to which they cannot derive
phosphate from phytate and phosphorus is unavailable to them.
So, present study is designed as a first step in a multi-step project to develop
indigenous phytase producing probiotic lactobacilli from different sources, the
optimization of phytase production and effect of physical & chemical factors on the
phytase stability and activity. Lactobacillus isolated from poultry was checked for phytase
production on Phytase screening media (PSM). Enzyme from the isolates showing activity
were quantified by ammonium molybadate method, the enzyme production were
optimized at different physical and chemical parameters such as temperature (30, 35 &
42°C), pH (4,5,6,7 & 8), osmotic pressure (1%,2% and 4%), aerobic/anaerobic conditions,
carbon (glucose, lactose, sucrose), nitrogen sources (peptone, tryptone & urea) and bile
salts (0.3%,1% and 2). Enzyme was partially purified and characterized by SDS-PAGE.
In present study 20 samples of poultry droppings (SP01-SP10) and fermented food (SY01-
SY10) were processed for isolation of lactobacilli. A total of 90 isolates (PDP01-PDP45;
FYP01-FYP45) were selected from MRS plates. Isolates were preliminary confirmed as
Gram positive rods with Catalase negative. All isolates were further purified and stored in
MRS broth supplemented with 15% glycerol at -20oC. Purified lactobacilli isolates were
screened for phytase production on phytase screening medium and zone o f hydrolysis was
Summary
77
measured in mm. Out of total of 90 isolates 62 isolates showed phytate hydrolysis. Out of
62 isolates, 16 were selected on the basis of retention of hydrolysis zone after cobalt
chloride staining. Out of 16 selected isolates, eight isolates PDP05, PDP09, PDP10,
PDP16, PDP23,PDP24, PDP30 and PDP35 were of poultry origin and eight FYP12,
FYP15, FYP17, FYP21, FYP26, FYP31, FYP38 and FYP42 were of fermented foods.
Selected isolates and retention of their zone of hydrolysis after cobalt chloride staining are
given in table 4.4. Phytase activity of selected lactobacilli isolates was checked in
modified MRS broth containing 0.2% sodium phytate at 37°C after 24 hrs. Cell free
supernatant was used as crude source of enzyme. Enzyme activity was determined by
using ammonium molybadate method. Amplification and sequencing of 16Sr DNA
(≈1500bp) was done by using universal primers which revealed PDP10, PDP24 and
FYP38 had >99% similarity with Lactobacillus gallinarum, Lactobacillus reutri and
Lactobacillus fermentum respectively with GenBank accession no. MF980924,
MF980925 and MF980923 respectively.
Phytase production by lactobacilli was optimized at different parameters e.g.
temperature (30, 35 and 42°C), pH (4, 5, 6, 7 and 8), osmotic pressure 1%, 2% and 4%.
The effect of oxygen was determined by growing lactobacilli isolates in aerobic and
anaerobic conditions followed by measuring enzyme activity. PDP10, PDP24 and FYP38
showed the best activity at 35°C (6.86 ± 0.15 IU/ml, 5.12 ± 0.12 IU/ml and 5.65 ± 0.13
IU/ml respectively) at pH 5 (6.86 ± 0.15 IU/ml, 5.12 ± 0.12IU/ml and 5.50 ± 0.13 IU/ml
respectively). Maximum phytase activity was recorded at 1% NaCl 4.78 ± 0.14, 4.18 ±
0.13 and 5.58 ± 0.12 IU/ml respectively) whereas anaerobic conditions were favourable for
the production of phytase by selected isolates.
Effect of carbon, nitrogen sources and bile salts was determined by growing
isolates MRS broth (0.2% sodium phytate) modified with different carbon (glucose,
Summary
78
lactose, sucrose) and nitrogen sources (peptone, tryptone and urea) and bile salts (0.3% ,
1% and 2%) followed by measuring enzyme activity. In this study isolates PDP10, PDP24
and FYP38 exhibited maximum phytase activity in the presence of 2% glucose as
compared to other carbon sources lactose and sucrose (4.36 ± 0.11, 4.38 ± 0.18 and 5.01 ±
0.15 IU/ml respectively). Present study revealed 0.1 % peptone as an optimal source of
nitrogen for PDP10 and PDP24 (4.54 ± 0.13 and 4.23 ± 0.19 IU/ml respectively) while
FYP38 (4.56 ± 0.14 IU/ml) showed best result in presence of 0.1% tryptone. All the
isolates showed maximum phytase activity at 0.3% bile salt concentration as compared to
1% and 2% concentration. Enzyme activity of phytase obtained from PDP10 was not
varied while treating it at different pH (4, 5, 6, 7 and 8) at different intervals of time.
Enzyme activity of phytase obtained from PDP24 was lower at pH 8 for 15, 30, 45 and
60min (3.41± 0.10, 3.40 ± 0.09, 3.42 ± 0.08 and 3.41 ± 0.11IU/ml respectively). Enzyme
activity of phytase obtained from FYP38 was lower down from pH 7 to 8 for 15, 30, 45
and 60min (4.41 ± 0.09, 4.42 ± 0.11, 4.43 ± 0.10, 4.41 ± 0.12 IU/ml respectively) & (4.40
± 0.09, 4.31 ± 0.11, 4.33 ±0.10 and 4.34 ± 0.12 respectively). Enzyme activity was
inhibited at 1mM and 5mM concentrations of Ca2+ while metal ions like Mg2+ and Na2+
addition stimulated the phytase activity. Approximate molecular weight of extracellular
protein precipitated from the cell free supernatant of PDP10, PDP24 and FYP38 was
~50kDa.