1 Introduction
Palm fatty acid distillate is a by-product which is obtained from the
deodorization step in the refinery of crude palm oil for human
consumption. The deodorization stage is designed to handle the
elimination of unwanted substances present in the crude vegetable oil as
it operates by stripping of volatile compounds, components, and elements
that causes the vegetable oil to deteriorate to a state of instability.
Furthermore, this refinery stage removes compounds that confer odour,
and make better the flavour of the product by thermal destruction of
pigments (de Greyt, 2012). In the deodorization stage, steam
distillation which hinders both oxidation of the vegetable oil by
contact with atmospheric oxygen and oil hydrolysis, is the main unit
operation that take place. The operating range of temperatures in this
unit generally lies between 200 and 250, and column pressure is
under vacuum as low as 10mmHg (Cibelem et al., 2014). Conventionally,
distillation columns are designed to yield two product streams – top
and bottom streams. The top product stream in this context is the
deodorizer distillate while the bottom product stream contains the
refined palm oil. Palm deodorized distillate contains mainly sterols,
stanols, hydrocarbons, lipids and tocopherols. These substances have
their respective uses and applications in science.
In clinical studies sterols and stanols have been discovered to have an
effect in reducing plasma cholesterol concentration by inhibiting the
absorption of cholesterol from small intestine (Jones et al., 1999;
Sierksma et al., 1999).
Lipids are regarded as one of the most elemental nutrients by man
because it produces several bioactive molecules that are fundamental
mediators of multiple signalling pathways, and they are also
indispensable compounds of the cell membranes of living organisms (Jana
et al., 2015). The major constituent of lipids is fatty acids, which are
grouped according to the presence of the type of bond each possesses in
nature. Fatty acid is a hydrocarbon chain, saturated or not, with methyl
group at one end and a carboxylic functional group at the other end.
Saturated fatty acids have no double bonds, monounsaturated fatty acids
have just one single bond, and polyunsaturated fatty acids have more
than one bond, but up to six double bonds can be present in their
chemical structure. Humans cannot synthesize polyunsaturated fatty acids
with the first double bond on C3 and C6 from the methyl-end because of
the absence of a proper enzymes for metabolism.
PFAD also contains another valuable product called tocopherols.
Tocopherols are class of compounds with vitamin E activity. Until early
1940s, the main commercial sources of vitamin E were crude vegetable
oils. Soybean and wheat-germ oil were considered to be the best sources
of vitamin E but containing only minute amounts of the vitamin (Siew et
al., 2017). Lately, other than crude vegetable oil sources, vitamin E
can be extracted from a much cheaper, richer and non-competitive source
- the fatty acid distillate (FAD), a residue from refining vegetable
oils. This discovery was made by Hickman K.C.D. (Hickman, 1944). The
vitamin E levels in FAD are many times greater than that of the original
crude vegetable oils from which they are derived.
Free fatty acids and tocopherols can be obtained from palm fatty acid
distillate by enzymatic hydrolysis - an extraction method that provides
high yields of the product using an appropriate lipase to catalyse the
reaction. According to Camilla et al. (2008), hydrolytic
lipase-catalysed reaction provides the needed advantage of extracting
these substances at lower energy cost and high efficiency with maximum
yield. In addition, the application lipase-catalysed systems further
concentrate tocopherols and fatty acids from PFAD, and this area have
not received much study. In enzymatic operations, water-soluble
substrates like proteins enzyme concentration, substrate concentration,
temperature of the system, pH and metallic ions present are major
factors that determine the rate of enzyme hydrolysis. However, in
heterogeneous reactions such as the hydrolysis of FAD, water ratio,
agitation speed, orientation and shape of the reactor and the presence
of surfactants have large effects on the rate reaction and formation of
stable suspension fatty acids and tocopherols in water (Tanaka et al.,
1992). In this work, enzymatic hydrolysis was done before applying a
neutralization method to pre-concentrate tocopherols by separating it
from free fatty acid. Since process variables play a large role for an
effective reaction to occur, there is need to optimize the variables in
order to have optimum yield of the desired product.
Response surface methodology (RSM) is an acceptable statistical
technique that applies quantitative data from appropriate experimental
designs to determine and simultaneously solve multivariate equations in
order to optimize processes or, increase or decrease the yield of
products (Camilla et al. 2008) as the case may be. In practise, the RSM
is a design constructed in a way that correlations between the chosen
variables are either minimised or totally eliminated thereby allowing
the independent estimation of variable effects and their potential
interactions. According to Mikko (2017), a response surface model is
based on approximating the true behaviour of a response:
\begin{equation}
\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ y=f\ \left(\beta_{1},\beta_{2},\ldots,\beta_{k}\right)+\ \delta\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (1)\nonumber \\
\end{equation}where y is referred the measured response as a function of\(\left(\beta_{1},\beta_{2},\ldots,\beta_{k}\right)\) variables and\(\delta\) represents other sources of variabilities. The variables are
coded to compare their effects within the design range:
\begin{equation}
\text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ θ}_{i}=\ \frac{\left(\beta_{i}-\ \beta_{\min}\right)}{\frac{\beta}{2}}-1\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (2)\nonumber \\
\end{equation}where \(\theta_{i}\) denotes a coded value and\(\beta_{i},\ \beta_{\min}\) and \(\beta\) the respective variable value
and variable range in their original units. A quadratic regression
equation is mainly used to approximate the response y :
\begin{equation}
\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ y=\ \varphi_{0}+\ \sum_{i=1}^{k}{\varphi_{i}\ x_{i}}+\ \sum_{i=1}^{k-1}{\sum_{j=i+1}^{k}{\varphi_{\text{ij}}\ x_{i}\ x_{j}}+\ \sum_{i=1}^{k}{\varphi_{\text{ii}}\ x_{i}^{2}+\ \varepsilon}}\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (3)\nonumber \\
\end{equation}where \(\varphi_{0}\) describe the mean value of y,\(\varphi_{i}\), \(\varphi_{\text{ij}}\) and\(\varphi_{\text{ii}}\) the first order, interaction and quadratic
coefficients, respectively, \(x_{i}\) the coded factors and\(\varepsilon\) the model residual. Most programs operate the matrix
notation, and this is expressed as:
y = Xb + e (4)
where y is an n\(\times\)1 vector response value, X an
n\(\times\)p matrix of coded values, b is a p\(\times\)1 vector
coefficients and e is an n\(\times\)1 vector of model residuals.
The design matrix X concludes columns for determining the
interaction and quadratic coefficients included in Eq. (3).
In terms of the application of RSM, a mathematical prediction of the
model for the yields of tocopherol has been developed with regards to
the selected experimental parameters. The effect and description of
these parameters on the yield of tocopherols have been investigated. In
studying oil and fat applications, RSM is well adapted for the
determination of the coefficients of a second order model that describes
the system parameters and is used as a valuable tool for design
optimisation of the extraction of tocopherols and improve its yield.
In this study, RSM was applied to evaluate usefulness in optimizing the
effects of several variables pertaining enzymatic hydrolysis of PFAD.
Furthermore, the investigation of the relationship among selected
process parameters – water weight, lipase weight and reaction time –
affecting the yield of tocopherols as the response, and the
determination of the optimal conditions for enzymatic hydrolysis with
Aspergillus niger lipase for maximum yield of tocopherols are the
objectives of this study. The Aspergillus niger is among the most
well-known lipase produced and its enzymatic activity is suitable for
many industrial applications. In addition, the effect of process
parameters (reaction time, water content, lipase concentration, and
temperature for the release of FFA and tocopherol during hydrolysis
using a lipase were studied as these areas have received very little
attention in the course of research.