Factor Influence Degradation of Cell Wall to Enhance Palm Oil Extraction-A Preliminary Results
Nurul Hasimah Kasmin1, Azwani Shah Mat Lazim1, Saiful Irwan Zubairi2, Roila Awang3*
1 Department of Chemical Sciences, Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
2Department of Food Sciences, Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
3Malaysian Palm Oil Board, No. 6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia
*Corresponding Author: [email protected] Accepted: 15 April 2023 | Published: 30 April 2023
DOI:https://doi.org/10.55057/ajfas.2023.4.1.6
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Abstract: The cell walls of palm mesocarp can be degraded by heating oil palm fruits or by the application of enzymes to increase the release of oil from the oil globules thus higher oil yield can be obtained. In this study, the effect of dry heating and the application of enzyme as a medium to degrade the cell wall of palm fruits were studied. The cell wall degradation by dry heating was found to be slightly different from wet heating treatment of oil palm fruits when observed using transmission electron microscope (TEM). It was found that the cell wall by dry heating had not fully decomposed as compared to wet heating. However, the combination of dry heating followed by application of enzyme achieved a maximum oil yield with 6% enzyme Celluclast 1.5L concentration, 200 rpm speed and 4 hours extraction time at 50˚C. Moreover, pineapple juice at the corresponding condition was also studied in an attempt to replace the commercial enzyme to extract the oil from palm mesocarp.
Keywords: enzyme, cell wall, mesocarp, degradation, heating
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1. Introduction
The main feature of oilseed cells is the existence of discrete cellular organelle called lipid and protein bodies which are embedded in the grain. The wall which surrounds the cell are primarily composed of cellulose, hemicellulose and lignin in addition to pectin (Rosenthal et al. 1996). The complex arrangement of polysaccharide in the cell wall can be broken into smaller molecules by mechanical action, i.e. (grinding the grain to flakes) causing the cell wall to rupture and thus exposing the oil located inside the cell. Another method is by breaking the bonds of polysaccharide molecules found in the cell wall and this process is known as hydrolysis.
When a polysaccharide bond is broken, the molecule will form monomers and exit the material.
Hydrolysis can occur by using heat or enzymes to break the structure of cotyledon cell walls,
fruits by heating the fruits at higher temperature and longer time, besides other purposes such as stripping of fruits from the bunches, facilitating the separation of the mesocarp from the kernel and deactivation of lipase activity (Vincent et al. 2014).
Enzymes are also involved in breaking bonds of material which serves the same purpose of exposing and releasing the oil more easily from the material. The role of most hydrolytic enzymes such as cellulase, hemicellulase and pectinase in this process is to break the structure of cotyledon cell walls by hydrolysis. Considering the specificity of each carbohydrase, a rational choice of the enzyme can be made after gaining in depth understanding of the complex arrangement of polysaccharide in the cell wall.
However, the use of commercial enzymes to obtain the highest possible oil yields is expensive.
Besides, commercial enzyme preparations must contain a mixture of cellulase, hemicellulase, pectinase and even protease. Thus, the main operations and the potential use of enzymes from other sources that are much cheaper should be considered to replace the existing use of commercial enzymes. In this study, the impact of heat treatments by drying of the palm fruit to replace conventional sterilization was studied. Our study includes further treatment which involves utilization of enzymes to facilitate the degradation of cell walls. Therefore, a study on the combination of both types of processes to yield a product with high oil yield and avoid waste water production during the conventional sterilization process may be required.
2. Materials and Method
2.1 Material
Oil palm fruits (Elaeis guineensis of tenera) were obtained from a local palm oil mill situated in Labu, Negeri Sembilan. Palm fruits were cleaned to remove any dirt on the surface using tap water.
The fruits were peeled and the nuts were removed from the fruit mesocarp. The peeled mesocarp was later mashed using a blender. For heat treatment analysis, the palm fruits were sterilized at 90°C for 90 min for both types of heat treatments which were wet (SW) and dry (SD) heating.
2.2. Methods Solvent extraction
Solvent extraction was carried out to compare with the performance of an aqueous enzymatic oil extraction. The mesocarp was dried using an oven at 90°C for 90 min. Then the mesocarp was immersed in hexane for 4h. The oil was then extracted from the mesocarp using a hydraulic press.
The solvent was then removed from the oil by using a rotary evaporator. The same procedure was repeated for unsterilized mesocarp. For soxhlet extraction, 20g of peeled mesocarp was subjected to hexane extraction for 6h by using a soxhlet extractor, at fruit to solvent ratio of 1:4 (w/v). All procedures as described above were also performed for each extraction technique. The total oil yield was calculated according to the following equation (eq. 1).
Oil yield = Weight of oil obtained, g
Weight of mesocarp, g X 100% eq. 1
Aqueous enzymatic extraction
The mesocarp was dried using an oven at 90°C for 90 min. Then, the mesocarp and 6% of Celluclast 1.5L enzyme was suspended in distilled water. The mixture was incubated in a water bath shaker at 50°C and 200 rpm for 4h. The oil obtained was diluted with hexane and was centrifuged at 5000 rpm for 5 min to yield a clarified oil suspension. The solvent was then removed from the oil by using a rotary evaporator. The same procedure was repeated for unsterilized mesocarp. A mixture without Celluclast 1.5L was used as a control.
To determine the effect of treatment conditions on oil yield, 10g of mashed mesocarp and Celluclast 1.5L enzyme was suspended in distilled water. The mixture was incubated in a water bath shaker. The oil obtained was diluted with hexane and was centrifuged at 5000 rpm for 5 min to yield a clarified oil suspension. The oil was weighed and the percent of oil was calculated.
Enzyme concentrations of 2, 6, 10, 20 and 30% were used. Extraction times ranged from 15-240 min. Shaking speeds of 50, 100, 150, 200, 250 rpm and extraction temperatures of 40, 45, 50, 55, 60°C are used.
For the treatment of palm mesocarp using pineapple enzyme, the pineapple waste (skin and middle part of the fruit) was ground using a grinder (Waring, Germany). Distilled water was added at a ratio of distilled water to fruit of 1:1 and the mixture was then filtered using a siever. The residue of pineapple waste was removed by centrifuge at 5000 rpm for 5 min. Distilled water was then added to the pineapple juice until pH 4. Then, 10g of mashed mesocarp was mixed with 10 ml pineapple juice. The dilution ratio of distilled water to fruit was at 1:1(v:w). The mixture was incubated in a water bath shaker at 50°C and 200 rpm for 4h. The oil obtained was then diluted with 10 ml hexane and was centrifuged at 5000 rpm for 5 min to yield a clarified oil suspension.
The solvent was then removed from the oil by using a rotary evaporator. The oil was weighed and the oil yield was calculated.
2.3. Transmission Electron Microscopy (TEM)
Samples were prepared by a standard method and analyzed using transmission electron microscope Model CM 12, Philips 12-KV at several magnifications. The aim was to investigating the influence of heat treatment on the cell wall degradation.
2.4. Quality Analysis
The oils extracted were analysed for FFA, DOBI and carotene content according to MPOB test method p2.5:2004, p2.9:2004 and p2.6:2004, respectively.
3. Results and Discussion
3.1 Morphology of the mesocarp of oil palm tissue after heat treatment
Sterilization processes currently employed to obtain oil from mesocarp fruit require heat treatment such as steam sterilization at 140˚C for about 75 to 90 min. Previous studies indicated that sterilization by dry heat treatment (SD) of palm fruit mesocarp is possible at low temperatures although the yield obtained was lower than those obtained using wet heat treatment (SW) (Hasimah et al. 2015). Thus, in the present study, the morphology of cell wall for both SW and SD
Hemavathi and Jamaliah (2015) reported that the cell structure of mesocarp before sterilization is very rigid and organized. The cell wall that protects the cell can be clearly seen. Thus, the oil globules and cell walls present on the tissue samples determine whether the sterilization process performed is adequate for the hydrolysis reaction to take place. Besides, a thinning or decomposing cell wall causes a loss of shape on the cell wall, indicating a hydrolytic reaction has taken place and causes the release of oil globules from the cell.
In our study, it was found that the oil cell had ruptured after heat treatment processes (Figure 1).
The morphological differences of SW and SD palm mesocarp tissues are shown in Figure 1 (a–f).
Micrographs of the mesocarp after SW treatment showed that the cell wall underwent decomposition and the oil had completely melted and flowed out of the ruptured cell wall (Figure 1c). These findings are similar to the study by Owolarafe and Faborode (2008), which found that there were many oil globules that had melted and lost shape on the cell wall after heat treatment.
The cell wall of SD treatment was also found to undergo loss of shape similar to SW treatment (Fig. 1d). There was a fine layer of cellulose that had not decomposed completely as with the SW tissue (Figure 1f). This caused the percentage of oil extraction for SD treatment to be lower than SW treatment. This observation was due to the absence of water which breaks the cellulose bonds that make up the cell wall. Abdul Aziz (2018) stated that hemicellulose functions to bind each cell while cellulose is the component that forms the cell wall. In this study, it was found that hemicellulose bonds were disrupted based on the loss of cell wall shape.
Thus, further treatment using enzyme that is capable of attacking cell walls such as cellulase was required to hydrolyze this component. The limitation of the conventional process such as waste water production can be avoided by a combination of this two-step procedure and can produce better or similar oil yield.
Figure 1: TEM micrograph for SW at a magnification of (a) 2600x (b) 5300x (c) 5300x and SD at (d) 2600x (e) 5300x (f) 17500x. CW: cell wall; O: oil globule. Dotted lines represent degradation of cell wall (a)
(b)
(c)
(d)
(e)
(f) CW
O
CW
O CW
CW
O
CW
O
O
CW O
3.2 Optimization process of Celluclast 1.5L
As shown in Figure 2, an optimum condition has to be derived to achieve a higher degree of hydrolysis. Extraction time, speed, temperature and enzyme concentration highly influence the activity of Celluclast 1.5L enzyme and thus contribute to higher oil yield. From the results, an increase in extraction time and speed increases oil yield (Figure 2 & 3). Oil yield also increases as heating time increases. However, an extraction time of more than 4h is not advisable since this does not increase yield significantly and in certain cases, it even decreases oil yield. Speeds above 200 rpm are however not to be used as these speeds do not increase oil yield significantly. An increase in heating temperature up to 50oC increases oil yield (Figure 4). The % oil yield drastically reduced when the heating temperature was increased to 55oC and 60oC. This is probably because beyond a critical temperature, the enzyme may have been deactivated. Lastly, an increase in enzyme concentration increases yield by up to 10%. Above 10% enzyme concentration, the oil yield do not increase significantly (Figure 5).
Figure 2: Effect of extraction time on oil yield. The mesocarps (10g) were dispersed in 10 ml distilled water.
The suspension was then subjected to extraction for 15min - 4h after adding 6% Celluclast 1.5L and incubated at 50°C with constant shaking at 100 rpm.
Figure 3: Effect of shaking speed on oil yield. The mesocarps (10g) were dispersed in 10 ml distilled water.
The suspension was then subjected to extraction for 4h after adding 6% Celluclast 1.5L and incubated at 50°C with constant shaking at 50, 100, 150, 200, 250 rpm.
Figure 4: Effect of extraction temperature on oil yield. The mesocarps (10g) were dispersed in 10 ml distilled water. The suspension was then subjected to extraction for 4h after adding 6% Celluclast 1.5L and incubated at 45, 50, 55, 60°C with constant shaking at 200 rpm.
Figure 5: Effect of enzyme concentration on oil yield
3.3 Effect of various extraction methods
Most of the previous studies were focused on the effect of the enzyme used to break up the chemical backbone of cell walls of related fragments during extraction process. Therefore, in this study we examined the effect of the extraction process before and after sterilization on the percentage of the oil yield. Celluclast 1.5L and pineapple extract were chosen as a hydrolytic enzyme of palm mesocarp. In this study, the oil yield obtained from the mesocarp fruit showed different trends depending on the type of extraction process used (Table 1).
The sterilized palm fruits with low moisture content normally resulted in higher oil yield as compared to unsterilised fruits as obtained by hexane, soxhlet and aqueous extraction processes.
However, the extraction of oil from unsterilized palm fruits treated with Celluclast 1.5L enzyme exhibited opposite characteristics and more yield was obtained than sterilized fruits. This might be due to the solubility of non-polar triacylglycerols (TAG) in the solvent used for the extraction.
A hydrolysis reaction does not only occur in polysaccharides but also in lipids. Hydrolysis of lipids occur to an ester group of TAG that causes the separation of oil molecules and the addition of water molecules to produce glycerol and free fatty acids (FFA). During the sterilization process, the quantity of FFA is reduced by deactivated lipase activity. The lipase activity in unsterilised palm fruits hydrolyse TAG to FFA which is easily soluble in a solvent system and results in higher oil yield as compared to sterilized palm fruits. This proves that a higher degree of hydrolysis of cell wall occurred during the addition of Celluclast 1.5L enzyme to facilitate the release of FFA as shown in Table 2. However, other quality characteristics remained unchanged.
From Table 1, it can be seen that enzymatic treatment facilitates the rupture of cell wall to release oil globules even in the absence of heat treatment compared to without the addition of enzyme.
However, the quality of oil produced was low due to the high level of FFA. As mentioned earlier, sterilization of palm fruit not only breaks the cell wall in the mesocarp tissue to produce high oil yield but also deactivates the lipase activity.
In this study, pineapple juice with adjusted pH was used other than Celluclast 1.5L. The main advantage of using pineapple juice is the operation cost might be cheaper due to the abundance of this waste compared to Celluclast 1.5L enzyme. Also, pineapple juice produced high oil yield when unsterilized fruit were used in this study, proving the higher degree of hydrolysis due to degradation of cell wall, although it had not completely ruptured in a similar manner when using Celluclast 1.5L. Thus, further studies should be carried out to investigate the composition of structural carbohydrate and other components in pineapple juice. Besides, the optimum condition for pineapple juice extraction has to be determined as each enzyme has their own specific conditions.
Table 1: Effect of Enzyme Treatment on Quality of Oil Extracted
Extraction process Oil yield (%)
Sterilized Unsterilized
Hexane 51.31±5.10 34.59±4.88
Soxhlet 83.16±3.42 73.95±5.18
Aqueous enzyme treatment (Celluclast 1.5L)* 57.56±2.47 66.66±2.12
Aqueous treatment (no enzyme) 31.80±2.45 16.82±3.08
Pineapple - 50.82±0.96
* Extraction condition: enzyme concentration: 6%, temperature: 50˚C, time: 4hrs, speed: 200 rpm
Table 2: Effect of Enzyme Treatment on Quality of Oil Extracted
Extraction process Sterilized Unsterilized
FFA
(%) DOBI
Carotene content
(ppm)
FFA
(%) DOBI
Carotene content
(ppm)
Soxhlet 2.56
±0.03
2.67
±0.08
786.70
±3.12
33.21
±0.11
1.97
±0.09
746.23
±12.38 Aqueous enzyme
treatment (Celluclast 1.5L)*
2.01
±0.14
2.91
±0.04
964.60
±2.41
25.47
±0.06
1.32
±0.06
554.98
±12.28 Aqueous treatment
(no enzyme)
2.35
±0.01
3.46
±0.03
1091.93
±2.71
42.45
±0.11
1.88
±0.08
681.37
±7.47
* Extraction condition: enzyme concentration: 6%, temperature: 50˚C, time: 4hrs, speed: 200 rpm
4. Conclusion
The extraction methods and parameters involved in increasing oil yield have been investigated. A combination of dry heating followed by aqueous enzymatic extraction was capable of degrading the cell wall of mesocarp in an attempt to halt the release of effluent to the environment. In-depth studies on pineapple juice as an enzyme replacement to Celluclast 1.5L should be done since both gave almost equal oil yield.
Acknowledgement
The authors would like to thanks MPOB for the financial support, Universiti Kebangsaan Malaysia (UKM), Cik Suhaida and En. Syakir from UKM EM and UKM CRIM for microscopy technical advises and services provided.
References
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