Record Details

Characterization of lipase from lactic acid bacteria isolated from fish processing waste.

IR@CSIR-CFTRI

View Archive Info
 
 
Field Value
 
Relation http://ir.cftri.com/11769/
 
Title Characterization of lipase from lactic
acid bacteria isolated from fish
processing waste.
 
Creator Vrinda, R.
 
Subject 05 Enzymes
07 Waste utilization
Fish
 
Description Lactic acid bacteria (LAB) are capable of producing a variety of metabolites
which are of utmost importance to the food industry. In the present study, 78 LAB
isolates were screened for lipolytic activity from fish processing waste (FPW) of
which 30 most potent lipase producing cultures were selected for further
characterization. FPW was chosen as the source for isolation of these lipolytic LAB,
as it is rich in lipids and there is a greater possibility of occurrence of lipase producers
enabling convenient screening and isolation of potent lipolytic LAB isolates.
Phylogenetic analysis based on 16S rRNA gene sequences revealed that most of these
cultures belonged to the genus Enterococcus whereas a few belonged to the genus
Pediococcus. Genetic diversity of LAB isolates was analyzed by RAPD and Simpson
index was calculated in order to quantitatively determine the degree of occurrence of
different genotypes in a dataset. Consequently, two most potent isolates with respect
to lipolytic and antibacterial activity; namely- Enterococcus faecium MTCC5695
(MTCC5695; also deposited as E. faecium NCIM5363) and Enterococcus durans
NCIM5427 (ED-27) were selected for further studies. The lipases produced by these
isolates were found to be intracellular in nature.
Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) are inducible enzymes
whose production depends upon several process variables, such as pH, temperature,
incubation period, substrate concentration, inoculum level and inducer concentration.
Therefore, the conditions for lipase production by MTCC5695 and ED-27 were
optimized by Response Surface Methodology (RSM). The optimized conditions were
found to be 5% v/v FWO, 0.15mg/ml FWPH at 24 h of fermentation time for
MTCC5695, and 5% v/v FWO, 0.2 mg/ml FWPH and 48h of fermentation time
for ED-27, which were further validated. MTCC5695 showed a 3.15 fold increase
(543.63 to 1715U/ml) whereas ED-27 showed a fold increase of 3.0 (207.6 U/ml to
612.53 U/ml) in lipase production under optimized conditions in comparison to the
unoptimized conditions. Additionally, unstructured mathematical models were used to
describe the fermentation kinetics of growth and lipase production of MTCC5695 and
ED-27. Logistic model for cell growth and Luedeking-Piret model for lipase
production predicted the fermentation profile accurately and the kinetic model
parameters α and β evidently implied that the lipase produced by MTCC5695 and ED-
27 to be growth- associated.
Intracellular lipases obtained from the bacterial cultures MTCC5695 and ED-
27 upon cultivation at optimized conditions was subjected to partial purification and
concentration using aqueous two-phase systems (ATPS). The optimum conditions for
MTCC5695 lipase were found to be Disodium Hydrogen phosphate-Polyethylene
glycol (Na2HPO4 /PEG8000) at 41.24% tie line length and 0.25 phase volume ratio
whereas for ED-27 lipase it was (NH4)2SO4 /PEG 6000 at 41.24% tie line length and
0.5 phase volume ratio. The integration of ultra filtration with ATPE facilitated the
selective removal of PEG from the partially purified lipase. The molecular weight of
the lipases was found to be around 19.2 kDa and 21.6 kDa for MTCC5695 and ED-
27, respectively. MTCC5695 lipase showed optimal activity at pH 10.8 and
temperature 40°C indicating that it was of alkaline nature whereas ED-27 produced an
acidic lipase that showed maximal activity at pH 4.6 and 30°C.
Characterization of lipases enabled the selective application of these cultures
for two different purposes suited to its specific property. MTCC5695 was used as a
starter culture for the preparation of curd owing to its lipolytic, antibacterial and
probiotic properties. The conditions for curdling were optimized by RSM and were
found to be 26.48 h and 2.17%v/v inoculum level for which the second order model
was validated. Further co-cultivation studies were performed which indicated that
MTCC5695 was effective against a wide spectrum of Gram-negative bacteria and
thereby possesses the ability to prevent the spoilage caused by food borne pathogens
which in turn increases the shelf-life of the product. This finding holds significance in
the fact that MTCC5695 lipase besides its organoleptic properties was also active
against Gram-negative bacteria and works synergistically with the enterocin resulting
in their complete destruction. This is an added advantage for the food industry as
Gram-negative bacteria pose serious threat as they are the major food borne pathogens
associated with food spoilage leading to diarrhoea and chronic gastroenteritis on their
ingestion. Usually, most of the bacteriocins from LAB show activity only towards
Gram-positive bacteria and not Gram-negative pathogens due to the presence of the
LPS layer on their cell wall. In this study, it was proven that the synergistic effect of
enterocin with lipase had a lethal effect on Gram-negative bacteria.
On the contrary, ED-27 exhibited poor curdling properties and the lipase
extracted from it was subjected to the degradation of lipids present in the slaughter
house effluent in view of its ability to produce an acidic lipase. The rate of enzymatic
hydrolysis was optimized for the parameters namely; concentration of lipase used,
rate of agitation and time of reaction. The optimal enzyme concentration was found to
be 600 U/ml; at agitation rate of 25 rpm for 48 h of reaction time. These results were
found to be significant as they help to overcome the problems related to free radical
generation when mineral acids are being used. In addition, chemicals like
polyelectrolytes are often expensive and their utilization is not feasible both in terms
of costs and environmental balance. Thus the enzymatic hydrolysis method
demonstrated in this study proved to be noteworthy in terms of pollution control and
effective waste management.
 
Contributor Prakash, M. Halami
 
Date 2013
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
Rights —
 
Identifier http://ir.cftri.com/11769/1/Vrinda%20Ramakrishnan%20Corrected%20final.pdf
Vrinda, R. (2013) Characterization of lipase from lactic acid bacteria isolated from fish processing waste. PhD thesis, University of Mysore.