3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P41 LIPID DEGRADATION AND ITS APPLICATION
TO LIPID-CONTAINING wASTEwATER
KATEřInA ILLKOVá, JIřInA OMELKOVá and
BOHUMILA VLČKOVá
Institute of Food Chemistry and Biotechnology, Faculty of
Chemistry, Brno University of Technology, Purkynova 118,
612 00 Brno, Czech Republic,
xcillkova@fch.vutbr.cz
Introduction
Food industry and restaurants produce each year about
15 % of the total wastewater and 50 % of the organic pollution.
Character and composition of wastewater depends on
manufacturing technology and current raw material. Wastewater
from food industry and restaurants contain lipids.
These lipids are present in the wastewater. They are difficult
to remove and degrade, because they are difficult to dissolve
in the water 1 .
For facilitation of production unit and treatment wastewater
fats and oils, enzymatic preparations (EP), which are
used, they are accelerated decomposition of organic material
lipids matter. These EP mix of lipases, proteases, amylases
and cellulases, some of them contain suitable combination
of bacteria, and they contain detergents and surfactants, as
well.
In this study was evaluated 5 EP for their ability to degraded
lipids in the wastewater. It was made isolation of lipiddegrading
bacteria and the proof of lipolytic bacteria. They
investigated their lipolytic activities at agar plates by aerobic
and anaerobic conditions. Further ability of lipid degradation
and fatty acids utilization at submerged cultivation and chemical
oxygen demand was followed.
Experimental
Enzymatic preparations are mixtures of enzymes and
bacteriological cultures. These preparations were inoculated
agar plates with tributyrin agar (tributyrin agar base). Plates
were incubed for 7 days at 27 °C for aerobic and anaerobic
conditions. The growth colonies were used for determination
of the lipolytic activity.
Samples for determination of lipid degradation and utilization
fatty acids were taken from submerged cultivation
The medium for submerged cultivation contained per liter
distilled water 1.12 g K 2 HPO 4 ; 0.48 g KH 2 PO 4 ; 5 g naCl;
0.1 g MgSO 4 . 7H2 O; 2 g (nH 4 ) 2 SO 4 and 100 µl EDTA.
Medium was autoclaved at 121 °C for 20 min and then the
medium was supplemented with 1 ml olive oil, as the natural
substrate. In the end, the medium was inoculated by enzymatic
preparations. Medium were incubated under aerobic
conditions at 27 °C and agitated at 160 rev min –1 for 14 days
in Erlenmeyer flasks in a shaker. The % free fatty acids were
determined as an indication of the olive oil degradation by
the tested enzymatic preparations 1 .
s660
S p i r i t B l u e A g a r ( D e t e c t i o n o f
L i p o l y t i c A c t i v i t y )
Microoragnisms was grown on spirit blue agar plates,
to which tributyrin and tween 80 were added as a lipase substrate
in ratio 1 : 1. Plates were incubated at 27 °C. Lipolytic
activity was identified the plates as a transparent halo around
the colonies after 7 days of incubation 2 .
E n z y m e a s s a y
The activity was determined by using culture in 0, 65
ml 0, 05 M phosphate buffer (pH 7.2) a 0.1 ml 0.025M pnPlaurate
in ethanol. The hydrolytic reaction was carried out
at 37 °C for 30 min, after which 0.25 ml 0.1M na 2 CO 3 was
added. The mixture was centrifuged and the activity determined
at 420 nm. One unit of lipase activity is defined as
the amount of enzyme which liberates 1 µg p-nitrophenol
from p-nP-laurate, as a substrate in 30 min under assay condition
3,4 .
D e t e r m i n a t i o n L i p i d D e g r a d a t i o n
a n d f a t t y A c i d s U t i l i z a t i o n
From each Erlenmeyer flasks 20 ml culture medium
was aseptically drawn and transferred to a separating funnel,
where it was mixed with 20 ml of hexane. The mixture was
agitated for 2 min and the upper layer was put to the clean
and weight beaker. The lower layer was re-extracted by a
fresh 20 ml of hexane and the upper layer after the extraction
was collected to the beaker again. The extract in the beaker
was evaporated by heating at 100 °C. Then the dry extracted
lipids were weight and dissolved in 50 ml of alcohol in the
presence of phenolphethalene indicator. The solution was titrated
with 0.1M KOH until the developing of pink color. The
same procedure was repeated within 2, 6, 8, 10 and 14 days.
The free fatty acids % in the sample was indicated lipid
degradation and fatty acids utilization, which was calculated
according to this equation:
%free fatty acids
V . c . M . 100
KOH KOH
= , (1)
1000. m
where V KOH is the volume of 0.1M KOH at the end
point, c KOH actual concentration of the 0.1M KOH, M is the
molecular weight of the oleic acid and m is the weight of the
dry extract 1 .
C h e m i c a l O x y g e n D e m a n d ( C O D )
Chemical oxygen demand (COD) is a measure of the
capacity of the water to consume oxygen during the decomposition
of organic matter and the oxidation of inorganic
chemicals such as ammonia and nitrite. COD measurements
are commonly made with samples of wastewater or natural
water, contaminated by domestic or industrial waste.
COD was determined by the standard potassium dichromate
method, descriebed in the Methods for the Examination
of Waters and Associated Materials (2007).