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.