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Other factors to consider in the formation of chloropropandiol fatty esters in oil processes a
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Muhamad Roddy Ramli , Wai Lin Siew , Nuzul Amri Ibrahim , Ainie Kuntom & Raznim Arni Abd. Razak a
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Protein & Food Technology Unit, Malaysian Palm Oil Board, Kajang, Malaysia
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Analytical & Quality Development Unit, Malaysian Palm Oil Board, Kajang, Malaysia Accepted author version posted online: 23 Mar 2015.Published online: 20 Apr 2015.
Click for updates To cite this article: Muhamad Roddy Ramli, Wai Lin Siew, Nuzul Amri Ibrahim, Ainie Kuntom & Raznim Arni Abd. Razak (2015): Other factors to consider in the formation of chloropropandiol fatty esters in oil processes, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2015.1032368 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1032368
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Food Additives & Contaminants: Part A, 2015 http://dx.doi.org/10.1080/19440049.2015.1032368
Other factors to consider in the formation of chloropropandiol fatty esters in oil processes Muhamad Roddy Ramlia*, Wai Lin Siewa, Nuzul Amri Ibrahima, Ainie Kuntomb and Raznim Arni Abd. Razakb a
Protein & Food Technology Unit, Malaysian Palm Oil Board, Kajang, Malaysia; bAnalytical & Quality Development Unit, Malaysian Palm Oil Board, Kajang, Malaysia
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(Received 6 November 2014; accepted 17 March 2015) This paper examines the processing steps of extracting palm oil from fresh fruit bunches in a way that may impact on the formation of chloropropandiol fatty esters (3-MCPD esters), particularly during refining. Diacylglycerols (DAGs) do not appear to be a critical factor when crude palm oils are extracted from various qualities of fruit bunches. Highly hydrolysed oils, in spite of the high free fatty acid (FFA) contents, did not show exceptionally high DAGs, and the oils did not display a higher formation of 3-MCPD esters upon heat treatment. However, acidity measured in terms of pH appears to have a strong impact on 3-MCPD ester formation in the crude oil when heated at high temperatures. The differences in the extraction process of crude palm oil from current commercial processes and that from a modified experimental process showed clearly the effect of acidity of the oil on the formation of 3-MCPD esters. This paper concludes that the washing or dilution step in palm oil mills removes the acidity of the vegetative materials and that a well-optimised dilution/washing step in the extraction process will play an important role in reducing formation of 3-MCPD esters in crude palm oil upon further heat processing. Keywords: chloropropandiol fatty esters; palm oil; diacylglycerols; acidity
Introduction In recent years, chloropropanediol fatty acid esters (3-MCPD esters – ME) have been detected in oils and fats (Zelinková et al. 2006; Weißhaar 2008, 2011). These contaminants are formed during processing of oils and fats, occurring when lipids such as triacylglycerols (TAGs) or glycerols react with chlorides at high temperatures. Among food products, high levels have been detected in oils and fats or foods containing a high fat content. As the levels are generally higher in refined oils, many researchers (Franke et al. 2009; Hrnčiřík & van Duijn 2011; Nagy et al. 2011; Ramli et al. 2011; Craft et al. 2012; Destaillats et al. 2012) have investigated the factors attributing to the formation, and certainly their studies have contributed to a better understanding of the presence of the esters in the oil. Levels of ME in palm oil have been reported (Abd. Razak et al. 2012; Yamazaki et al. 2013; MacMahon et al. 2013). Refined seed oils in general have lower values. Some researchers (Nagy et al. 2011; Freudenstein et al. 2013) reasoned that possible precursors such as higher diacylglycerol (DAG) contents and chloride levels could be the contributing factors attributing to the higher formation of the esters in refined palm oil. In this study, another possible factor is examined while investigating DAGs in the oil. DAGs are present in all oils and fats, the amount varying from 1% to as much as 8%. Generally, fruit oils such as palm oil (Siew & Ng 1995) and olive oil *Corresponding author. Email: [email protected] © 2015 Malaysian Palm Oil Board. Published by Taylor & Francis.
(Fronimaki et al. 2002) have higher DAG levels than seed oils. Some nut oils such as salfat, shea butter fats and other exotic fats have high DAG contents as well. In recent years, ME and glycidyl esters (GE) have been linked to DAGs present in vegetable oils (Hamlet et al. 2011; Matthäus et al. 2011). Some presentations and publications (Matthäus et al. 2011; Pudel et al. 2012) pointed specifically to DAGs as potential culprits of ME formation, especially observed upon adding DAGs to oils. As all oils and fats have similar components (TAG, DAG, monoacylglycerol (MAG), chlorides), high temperature treatment for physical refining of palm oil has been one of the decisive factors resulting in a greater amount of chloropropandiols. Chemical refining, however, showed less tendency of ME formation as neutralisation and water washing to remove the residual of soap partially eliminates reactive chlorinated precursors from the crude palm oil (CPO). Deodorisation is also conducted at relatively lower temperatures. Chlorides were recently studied (Franke et al. 2009; Nagy et al. 2011) and it would not be a surprise that all oils and fats may have varying quantities of chlorides, being such ubiquitous materials in nature. Franke et al. (2009) reported total chlorine in palm oil and rapeseed oil as 2 and < 2 ppm, respectively, and for chlorides < 1 ppm in both oils. Nagy et al. (2011) discussed the type of chlorides as being both inorganic and organic in nature. While there is a small amount of chlorides present in oils and fats, all oils come
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M.R. Ramli et al.
into contact with chlorides, for example in bleaching clays, water, phosphoric acid, etc. Several analytical measurements of ME and GE have been reported (Crews et al. 2013; Ermacora & Hrnčiřík 2013; Yamazaki et al. 2013; Ermacora & Hrnčiřík 2014; Zhou et al. 2014). The American Oil Chemists' Society has also developed three new methods, namely AOCS Cd13-29a (2013a), AOCS Cd13-29b (2013b) and AOCS Cd13-29c (2013c). These methods made it easier to relate the work of one laboratory with that of another, especially where data on different products were reported. Comprehensive studies have been undertaken to mitigate the formation of these esters. The mitigation procedures can be generally divided into three approaches: removal of precursors in the raw material, modification of the refining process and removal of the esters postrefining. Siew et al. (2012) and Matthäus and Pudel (2013) discussed potential procedures to reduce the formation of ME based on these three approaches. Craft and Nagy (2012) highlighted several ‘suggested practices (SP)’ from upstream (plantation) to downstream (refining) to improve the quality and safety of refined palm oil. A summary of the different mitigation strategies possible to reduce the formation of ME and GE is given in Table 1. The production of palm oil differs very much from that of seed oils, in that seed oils are extracted from dry seeds by solvent or by cold press, while because of the size and nature of the fruit, palm oil is extracted with the whole fruit bunches in the processing steps. Fresh fruit bunches (FFBs) are harvested when ripe, usually 20–22 weeks after flower anthesis. The FFBs are transported in lorries to mills, where they are extracted by a mechanical press. A bunch consists of a bunch stalk and spikelets of fruitlets attached to the stalk. The oil extraction process is described in Figure 1, involving Table 1.
Fresh fruit bunches (FFB)
Sterilization
Steam
Condensate
Sterilized FFB Stripping
Bunch stalks
Fruit Steam
Digestion
Pressing Crude oil
Press cake
Screening
Water
Oil Clarification
Separator
Dirt
Vacuum drying
Crude palm oil (CPO)
Figure 1. oil.
Schematic diagram for the production of crude palm
sterilisation, stripping, digestion, pressing, screening, clarification, centrifugation and drying. In some
Summary of the possible mitigation strategies in reducing the formation of 3-MCPD and glycidyl esters in refined oil. Key finding
Mitigation step Refining aids Glycerol and ethanol Ethanol:water (1:1) Diacetin Carbonates Dual deodorisation process (short term at higher temperature and long term at lower temperature/vice versa) Deodorisation using short-path distillation Neutralisation of degummed oil with Potassium hydroxide Sodium hydroxide Calcium oxide Hot water degumming Washing of crude palm oil before refining Washing of palm fruit pulp before oil extraction
25–35% reduction of MCPD esters Approximately 30% reduction of MCPD esters 50% reduction of MCPD esters and related compounds 66% reduction of MCPD esters and related compounds 63% reduction of MCPD esters
Reference Craft et al. (2012) Matthäus and Pudel (2013)
Matthäus and Pudel (2013)
90% reduction of MCPD esters and related compounds
Matthäus and Pudel (2013)
45% 35% 45% 70% 38% 95%
Matthäus and Pudel (2013)
reduction reduction reduction reduction reduction reduction
of of of of of of
MCPD MCPD MCPD MCPD MCPD MCPD
esters esters esters esters esters esters
Ramli et al. (2011) Ramli et al. (2011) Matthäus and Pudel (2013) Craft et al. (2012)
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Food Additives & Contaminants: Part A variations, decanter systems are used instead of clarification. These steps begin with sterilisation, which kills enzymes such as lipases and softens the fruits for ease of removal from the bunches; cooking the fruits in digesters; pressing, which releases the oil, followed by either addition of water for ease of separation of oil from the vegetative materials or through use of a decanter. Finally the crude oil is separated by centrifugal separators or by decanters, then dried and sent to storage tanks. The difference between oil production from palm and that of other seeds is mainly in the production process, where palm oil comes into contact with vegetative materials of the oil palm (bunch stalk, spikelets, calyx, vegetative fibres), whereas for seed oil production the vegetative materials are removed from the seeds prior to extraction. This difference in production is seen as a possible explanation for the higher occurrence of ME observed upon heat treatment of palm oil. This paper will examine how this is so. This paper also evaluates the different conditions of storage of fruits and their effects on the formation of ME. It also examines the effect of oil acidity on the formation.
measurements. The FFA, DAG and pH of the oils were analysed.
Crude palm oil (CPO) from evaporator process In this process shown in Figure 2, the oil/water and vegetative mixture were subjected to a falling film evaporation process, where the water inherent in the fruit was removed. No dilution of water takes place, and removal of vegetative materials takes place by separators. The water obtained from the process is collected for the pH test. The crude oil is also tested for FFA, DAG, ME formation on the heat test, and its acidity in terms of pH. The main difference from the conventional commercial process is the removal of the water step, and no additional water is added to the process, except that which is derived from steam used in the sterilisation of bunches. Fresh fruit bunches (FFB)
Steam
Sterilization
Condensate
Sterilized FFB Stripping
Materials and methods Storage of fresh fruit bunches (FFBs) and oil extraction FFBs were harvested from MPOB oil palm estate in Bangi, Selangor, Malaysia. Spikelets of fruits were cut from the bunches and separated into four portions. The first portion was sterilised fresh for oil extraction. The second portion was bruised with a hammer and kept for 1 day prior to sterilisation and oil extraction. The third portion was incubated at 15°C for 24 h prior to sterilisation; while the last set was incubated at 5°C for 24 h prior to sterilisation. The last two treatments were given cold treatments to increase the speed of hydrolysis, since it was reported that lipase within the fruits are activated at low temperatures (Sambanthamurthi et al. 1991). Sterilisation was carried out in a laboratory autoclave for 30 min at 15 psi. The fruits were then detached from the spikelets and the mesocarps removed using a knife. The mesocarps were pressed in a laboratory-scale hydraulic press and the collected oil was filtered to remove fibres and other impurities. The crude oil was dried by a rotavapor for 30 min. Analysis of ME upon heat test, FFA and DAG levels was carried out. Quadruple experiments were conducted. The mean of four sets of bunches was recorded.
Bunch stalks
Fruit Steam
Digestion
Pressing Crude oil
Press cake
Screening Oil Desanding
Cake
2-Phase separation Oil and water Evaporator Oil and sludge Separator
Vacuum drying
Samples collected from palm oil mills Samples of CPO were collected from commercial palm oil mills. The oil samples were heat treated at 260°C for ME
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Crude palm oil (CPO)
Figure 2.
Modified experimental process.
Sand
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M.R. Ramli et al.
Palm fibre oil (PFO) A sample of PFO is obtained from a local refinery that processes fibre oil from palm fibres by a solvent process. The palm fibres are the vegetative material left over from the extraction of CPO from fruit bunches. These fibres contain 5–8% of oil (Abd. Majid et al. 2012).
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Diacylglycerol (DAG) analysis HPLC was carried out using Gilson (Rue Gambetta, Villers le Bel, France) 303 and 302 pumps and a Waters (Milford, MA, USA) differential refractometer. The two columns were of 25 cm length and 4 mm i.d. with 5 µm Lichrosphere RP18. The mobile phase was acetonitrile: acetone (65:35 v/v) at a flow rate of 1.0 ml min–1. Injection was achieved through a Rheodyne valve fitted with a 20 µl loop. Samples were injected as 10% w/v solution in warm acetone. Identification of TAGs and DAGs was made by comparison with reference standards. The DAG values are given in area per cent. The analysis of DAG was as reported in Yeoh et al. (2014).
1.2 ml min−1. The column temperature was programmed at 60°C (1 min) to 190°C (1 min) at a rate of 6°C min−1, and was then accelerated to 280°C at a rate of 30°C (5 min). The injector was held at 180°C and 1 µl of the sample was injected in a split-less mode. The quantitative analysis was carried out by monitoring characteristic ions at m/z 91, 147 and 196, respectively, for derivatised 3-MCPD; while for 3MCPD-d5, characteristic ions were at m/z 93, 150 and 201. Qualifier ions were m/z 196 towards m/z 201.
Heat test Crude oil samples (2 ml) were heated in a covered vial to 260°C, the vial being held in a sandbath. The oil was maintained at 260°C for 1 h, then cooled and analysed for ME. The measurement may not simulate the different aspects of refining such as degumming and bleaching, but relates to the high temperature as used in deodorisation. It is found to be a reliable indicator of the degree of ME expected for different oil sources.
pH test of oils Free fatty acids (FFAs) FFAs were analysed according to AOCS Official Method Ca 5a-40 (1998).
A total of 100 ml of oil was stirred with a similar amount of water for 1 h at 70°C. The water was removed by separating funnel, and the pH measured with a pH meter.
3-MCPD esters (ME) Analysis of ME was carried out according to Abd. Razak et al. (2012), and recorded in mg kg−1 values. The Federal Institute for Risk Assessment (BfR) Method 008 – Determination of 3-MCPD fatty acid esters in edible oils and solid fats by GC-MS (2009) – was adopted. The internal standard used was deuterated 3-MCPD (3-MCPD-d5). Oil sample of 100 mg was weighed and dissolved in 0.5 ml tert-butyl methyl ether (t-BME) containing the internal standard (0.4 µg). Sulphuric acid solution in methanol (2%, v/v) was added to the sample and the mixture was incubated for 16 h. Sodium hydrogencarbonate solution (0.5 ml) was then added to stop the hydrolysis. To separate the fatty acid methyl ester from the sample, 1.8 ml of an aqueous salt solution were added. Derivatisation was achieved by addition of 250 µl of phenylboronic acid solution to the mixture. The derivative was extracted and analysed. Detection of the analyte was carried out by means of GC-MSD from Agilent Technologies (Santa Clara, CA, USA), equipped with a Series 5975C quadrupole detector and controlled by a programmable GC 7890A. Chromatographic separation was performed on a fused silica capillary column, HP-5MS inert (30 m length, 0.25 mm i.d., 0.25 µm film thickness). Helium (purity 99.999%) was used as a carrier gas at a constant flow of
Statistical analysis Data obtained from the analyses were subjected to oneway analysis of variance (ANOVA) with Fisher’s multiple comparison to determine the significant differences among the samples defined at 95% confidence interval (p < 0.05). All analyses were conducted in duplicate and reported as means ± standard deviation (SD) of independent trials. The statistical analyses were performed using the Minitab software Version 16 statistical package (Minitab Inc., State College, PA, USA).
Results and discussion DAG and ME formation Formation and occurrence of ME in foods have been discussed by Hamlet et al. (2011). Mechanisms of formation are related to lipids, which are possible precursors of formation when conditions are optimum, for example reactions occur in the presence of chloride ions at high reaction temperatures. Thus, it appears easy to find the culprits in the case of palm oil, where DAGs are clearly higher than in other seed oils. Model experiments do show that DAGs, especially those added to the system, are reactive under high temperature conditions (Freudenstein et al. 2013). However, oils from various sources having
Food Additives & Contaminants: Part A Table 2. 3-MCPD esters (ME) formed upon heat treatment of CPO extracted directly from fruit bunches: effect of hydrolysis. Sample
FFA (wt %)
Fresh CPO 0.63 Oil from bruised 1.85 FFB High FFA oil 1 6.75 High FFA oil 2 59.65
DAG (wt %) ME (mg kg−1)
± 0.17a ± 0.71a
3.3 ± 0.3aa 3.8 ± 0.2a
1.55 ± 0.64a 1.45 ± 0.80a
± 2.06a ± 8.02b
5.4 ± 0.9b 4.8 ± 0.5ba
0.74 ± 0.23a 1.01 ± 0.50a
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Notes: Values are the mean ± SD of duplicate analysis. Values within a column with different superscript letters are significantly different (p < 0.05).
varying FFA and DAG contents point to the contrary of the expectations of the above (Lacoste et al. 2010). When a FFB is harvested, FFA increases in the oil, especially if there is considerable bruising, usually caused by the ripe fruit dropping to the ground from a height of several feet. Inherent and extraneous lipases come into contact with the oil released from ruptured cell walls as a result of bruising. A rise in FFA is slower if fruit bruising is minimised. Table 2 shows results of ME formed in heat-treated CPO extracted from palm fruits of varying state of hydrolysis. In the fruit experiments, spikelets from a bunch were separately treated as (1) freshly extracted, (2) bruised fruits and (3 and 4) hydrolysed fruits. The oils extracted were heat treated for their potential to form ME. In these experiments, the degree of hydrolysis of palm oil was affected to show the different levels of FFA and DAG. In fresh oil, the FFA of the CPO was only 0.63%, while oil from slightly bruised fruits resulted in an FFA of 1.85%. Chilling the fruits can enhance lipolytic hydrolysis by lipase present in the oil palm fruit, where it was observed that palm fruits subjected to 5°C chilling resulted in FFA levels of up to 70% (Sambanthamurthi et al. 1991, 1995; Cadena et al. 2013). Although FFA levels were high, the DAG levels were unexpectedly low. In our experiments, hydrolysed fruits caused by chilling at different temperatures (15 and 5°C) resulted in high FFA oils as indicated by values of 6.75% and 59.65%, respectively. Structural changes in mesocarp tissue as a result of chilling resulted in high enzyme substrate interaction. Even at FFA levels as high as 59.65%, DAG is still relatively low, being only slightly higher than the low FFA oils. In fact, 8% is the highest limit observed for DAG in most CPOs. The observations appeared contradictory to expectations of high DAG to be found in such highly hydrolysed oils. It is hypothesised that whatever DAG and MAG formed, they are being quickly hydrolysed to FFA. Sambanthamurthi et al. (1995) discussed evidence of an active endogenous lipase in the oil palm mesocarp which is located in the oil body. While in vitro experiments found the lipase maximum activity at 18°C, an in vivo assay showed maximum activity at 5°C. Further evidence of low-temperature activation
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of lipase of oil palm fruits was also shown by Cadena et al. (2013). In commercial oils, the FFA limit for traded CPO is 5% and any oil of values of > 10% would have been traded as high FFA oils. Keeping other variables such as the temperature of treatment for the oil constant, the experiment shows that the ME of the oils with higher FFA and DAG levels derived from same bunch of fruits did not differ significantly (p ≥ 0.05) from each other. In experiments involving added DAGs from commercial sources, Freudenstein et al. (2013) reported an increase in ME when 1,3-dipalmitin is added to palm oil. However, the process by which such 1,3-dipalmitin is produced is not known. Any impurities or residue (if acidic) left from the production process may cause an effect of increasing the ME formation. The experiments from fruits using DAG formed within the fruit illustrated conclusively that DAG alone would not be very reactive unless other preconditions are present. Other researchers who have also shown that DAG is not necessarily the critical factor are Hrnčiřík and Ermacora (2011) and Lacoste et al. (2010). Destaillats et al. (2012) showed that TAG is the main acylglycerol class involved in MCPD diester formation when oils are heated. DAG was found to form ME as well, but it has relatively lower reactivity than TAG. Crude oil from evaporation process In an experiment where CPO is processed in a different manner from that of commercial mill operation, it was found that the way palm oil is extracted from FFBs may play a role in determining the formation of ME. Figure 1 shows CPO being extracted in conventional mills. The process takes the fruit bunches through sterilisation, stripping of spikelets from the bunch, digestion of the fruits, pressing and dilution with water, followed by clarification. The clarification process allows settling of the oil and water phase and the oily phase is passed to separators for removal of dirt, and the crude oil is finally vacuumed dried. In Figure 2 oil extraction is carried out from a modified process in a small pilot plant. In the initial few steps, processing is generally similar as the conventional process. After digestion and pressing, the oil together with the fruit liquor is passed to a desander and a two-phase separation, removing most of the vegetative material. The oil and fruit liquor is retained in an evaporator, where the fruit liquor and water from steam sterilisation of bunches are removed via a falling film evaporator. The oil obtained is centrifuged and dried as before. The difference between this process and that of the current commercial mill process is in the use of the evaporator to remove any water present either inherently in the fruits or introduced through sterilisation. On the other hand, in current commercial process, a large quantity of water is added to dilute the oil to allow better separation of the oil from other
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M.R. Ramli et al. Table 3.
pH of crude palm oil and other properties: effect on the formation of 3-MCPD esters (ME).
Sample
FFA (wt %)
Commercial sample number 1 Commercial sample number 2 Commercial sample number 3 CPO, from evaporation process
2.66 2.66 3.66 3.04
± ± ± ±
0.03a 0.04a 0.06b 0.02c
DAG (wt %) 5.5 5.3 5.4 5.6
± ± ± ±
0.2a 0.1a 0.1a 0.2a
pH
ME (mg kg−1)
6.2 5.5 7.1 4.0
0.84 ± 0.05 0.99 ± 0.03 < LOD 12.96 ± 0.76
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Notes: Values are the mean ± SD of duplicate analysis. Values within a column with different superscript letters are significantly different (p < 0.05). LOD = 0.25 m g kg−1.
vegetative material. The evaporator water from this experimental process is found to have a very high acidity with a pH 3. Interestingly, the CPO obtained from the process has also high acidity with pH 4, while in comparison samples from commercial processes showed a pH of about 5–7 (Table 3). It has been generally observed that palm oil mill effluent (POME), which is the effluent waters removed during the oil extraction process, is acidic in nature (Igwe & Onyegbado 2007; Irenosen et al. 2014). The sample of CPO with pH 7 was found to have a lower ME upon heat treatment. The results pointed to an acidic nature in CPO, which is generally mainly reduced during its extraction process when there is much water used in the clarification step. The acidity is due to its contact with vegetative materials of the palm fruit bunch, which are generally acidic in nature. In conventional mills, the acidity in the fruits is further diluted by water just before the clarification process, and therefore is washed out. Any further washing carried out by some mills will have removed the acidity completely and also remove chlorides to a minimum level. Oil obtained from the experimental process (Figure 2), when heated to 260°C for 1 h, showed higher contents of ME than any commercial CPO as shown in Table 3. The high ME being formed would be caused by a higher reactivity of the lipids, resulting from the acidity present. Reactivity is enhanced when acidic conditions are present (Hamlet et al. 2011) as indicated in mechanisms of formation. Also, work by Ramli et al. (2011) has indicated the effect of acidity from the degumming agent and bleaching clays, providing further evidence of the effect of acidity on the formation of ME. The screening and clarification steps in palm oil mills may need to be optimised for better reduction of acidity level or, alternatively, an additional water wash step could be introduced into the process. In areas where soils are acidic, harvested fruit bunches that fall to the ground could also pick up acidic soil. It would be a good idea to wash the fruit bunches on conveyor belts to remove the additional acidity. Effect of oils from palm fibre Palm fibres are the by-products of extraction of CPO. These are the vegetative materials from the mesocarp and empty
bunch. Some mills have installed additional solvent plants to extract further the oil retained in the fibres. PFOs are sold for industrial uses, animal feeds or for biodiesel production. They contain higher quantities of carotenes (1400–1600 ppm) and tocols (1700–2600 ppm) (Subramaniam et al. 2013) and are potentially good raw materials for extraction of these components. However, the possibility of such oils being added to normal palm oil is considered as it enhances the oil extraction rate (OER) of palm oil production at the particular mill. With a residual oil recovery system (RORS) installed in a mill, oil recovery from the mesocarp ranging from 0.15% to 0.45% per tonne of FFB has been reported (Subramaniam et al. 2013). The effects of blending a small proportion of PFO to normal oils were examined. In this experiment, the PFO was added in small proportions to CPO. Results in Figure 3 show that PFO had a significant (p < 0.05) effect on the formation of ME. DAG level was considered high (7.9%) compared with normal CPO. A blend of 5% PFO into CPO would not contribute much to the DAG level. On the other hand, the pH of the PFO is found to be a low value of 4.6. Blending this oil to CPO would be detrimental to ME formation. The results confirmed that acidity is an important factor contributing to the ease of formation of ME when the oil is heated to a high temperature.
Figure 3. Effect of PFO in the formation of 3-MCPD esters (ME) in crude palm oil. Mean of duplicate analysis ± SD. Points with different letters are significantly different (p < 0.05).
Food Additives & Contaminants: Part A Conclusions
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While DAG may appear to be a culpable precursor of ME, their role in several situations as given in fruit experiments give some doubts as to their impact on formation. This paper presents some evidence for the case against making DAG content of palm oil the inherent critical factor for the formation of ME. It is noted that the acidity of CPO is the more important factor to consider; it should be removed during processing steps as a way towards reducing the formation of these esters. The chlorides present can also be removed in the process. The addition of water during the process has already been included in most mills and only needs further optimisation to enhance the better reduction of acidity. The possible inclusion of other types of extracted oils via solvents or additional pressings should be thoroughly discouraged. Disclosure statement No potential conflict of interest was reported by the authors.
Funding The authors thank the Director-General for financially supporting the study and for subsequent permission to publish this research effort.
References Abd. Majid R, Mohammad AW, Choo YM. 2012. Properties of residual palm pressed fibre oil. J Oil Palm Res. 24:1310– 1317. Abd. Razak RA, Kuntom A, Siew WL, Ibrahim NA, Ramli MR, Hussein R, Nesaretnam K. 2012. Detection and monitoring of 3-monochloropropane-1, 2-diol (3-MCPD) esters in cooking oils. Food Control. 25:355–360. American Oil Chemists’ Society. 2013a. Official methods Cd 29a13, Sampling and analysis of commercial fats and oils, 2- and 3-MCPD fatty acid esters and glycidyl fatty acid esters in edible oils and fats by acid transesterification. Urbana, IL: AOCS Press. American Oil Chemists’ Society. 2013b. Official methods Cd 29b-13, Sampling and analysis of commercial fats and oils, Determination of bound monochloropropanediol- (MCPD) and bound 2, 3-epoxy-1-propanol (glycidol-) by gas chromatography/mass spectrometry (GC/MS). Urbana, IL: AOCS Press. American Oil Chemists’ Society. 2013c. Official methods Cd 29c-13, Sampling and analysis of commercial fats and oils, Fatty-acid-bound 3-chloropropane-1,2-diol (3-MCPD) and 2,3- epoxi-propane-1-ol (glycidol), determination in oils and fats by GC/MS (Differential measurement). Urbana, IL: AOCS Press. American Oil Chemists’ Society (AOCS). 1998. Official methods and recommended practices of the American Oil Chemists’ Society. 5th ed. Urbana, IL: AOCS Press. Cadena T, Prada F, Perea A, Romero HM. 2013. Lipase activity, mesocarp oil content and iodine value in oil palm fruits of Elaeis Guineensis (E.G.), Elaeis Oleifera (E.O.), and the
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interspecific hybrid (E.O. x E.G.). J Sci Food Agric. 93:674–680. Craft BD, Nagy K. 2012. Mitigation of MCPD-ester and glycidyl ester levels during the production of refined palm oil. Lipid Technol. 24:155–157. Craft BD, Nagy K, Sandoz L, Destaillats F. 2012. Factors impacting the formation of monochloropropanediol (MCPD) fatty acid diesters during palm (Elaeis guineensis) oil production. Food Addit Contam: Part A. 29:354–361. Crews C, Chiodini A, Granvogl M, Hamlet C, Hrnčiřík K, Kuhlmann J, Lampen A, Scholz G, Weisshaar R, Wenzl T, Jasti PR, Seefelder W. 2013. Analytical approaches for MCPD esters and glycidyl esters in food and biological samples: a review and future perspectives. Food Addit Contam: Part A. 30:11–45. Destaillats F, Craft BD, Sandoz L, Nagy K. 2012. Formation mechanisms of Monochloropropanediol (MCPD) fatty acid diesters in refined palm (Elaeis guineensis) oil and related fractions. Food Addit Contam: Part A. 29:29–37. Ermacora A, Hrnčiřík K. 2013. A novel method for simultaneous monitoring of 2-MCPD, 3-MCPD and glycidyl esters in oils and fats. J Am Oil Chem Soc. 90:1–8. Ermacora A, Hrnčiřík K. 2014. Development of an analytical method for the simultaneous analysis of MCPD esters and glycidyl esters in oil-based foodstuffs. Food Addit Contam: Part A. 31:985–994. Federal Institute for Risk Assessment. 2009. BfR Method_82_FC-008-01: Determination of 3-MCPD fatty acid esters in edible oils and solid fats by GC-MS – An indirect determination by detection of free 3-MCPD released from 3-MCPD-esters by acid hydrolysis and by derivatization with phenylboronic acid. Dahlem: BfR Press. Franke K, Strijowski U, Fleck G, Pudel F. 2009. Influence of chemical refining process and oil type on bound 3-chloro-1, 2-propanediol contents in palm oil and rapeseed oil. Food Sci Tech. 42:1751–1754. Freudenstein A, Weking J, Matthäus B. 2013. Influence of precursors on the formation of 3-MCPD and glycidyl esters in a model oil under simulated deodorization conditions. Eur J Lipid Sci Tech. 115:286–294. Fronimaki P, Spyros A, Christophoridou S, Dais P. 2002. Determination of the diglyceride content in Greek virgin olive oils and some commercial olive oils by employing 31 P NMR Spectroscopy. J Agric Food Chem. 50:2207–2213. Hamlet CG, Asuncion L, Velíšek J, Doležal M, Zelinková Z, Crews C. 2011. Formation and occurrence of esters of 3-chloropropane-1,2 diol (3-CPD) in foods: what we know and what we assume. Eur J Lipid Sci Tech. 113:279–303. Hrnčiřík K, Ermacora A. 2011. Formation of 3-MCPD esters in palm oil: Effect of partial acylglycerols. Informative Meeting of 3-MCPD and glycidyl esters in Foodstuffs, organized by OVID and BLL, Jan 18. Berlin: Association of Oilseed Processing. Hrnčiřík K, van Duijn G. 2011. An initial study on the formation of 3-MCPD esters during oil refining. Eur J Lipid Sci Tech. 113:374–379. Igwe JC, Onyegbado CC. 2007. A review of palm oil mill effluent (POME) water treatment. Global J Environ Res. 1:54–62. Irenosen OG, Oluyemi AS, Korede AO, Samuel AS. 2014. Integration of physical, chemical and biological methods for the treatment of palm oil mill effluent. Sci J Anal Chem. 2:7–10.
Downloaded by [University of Victoria] at 16:05 25 April 2015
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Lacoste F, Joffre F, Soulet B, Griffon H. 2010. 3-MCPD esters in edible oils: analytical aspects, 101st AOCS Annual Meeting and Expo; May 16-19; Urbana, IL: AOCS Press. MacMahon S, Begley TH, Diachenko GW. 2013. Occurrence of 3-MCPD and glycidyl esters in edible oils in the United States. Food Addit Contam: Part A. 30:2081–2092. Matthäus B, Pudel F. 2013. Mitigation of 3-MCPD and glycidyl esters within the production chain of vegetable oils especially palm oil. Lipid Technol. 25:151–155. Matthäus B, Pudel F, Fehling P, Vosmann K, Freudenstein A. 2011. Strategies for the reduction of 3-MCPD esters and related compounds in vegetable oils. Eur J Lipid Sci Tech. 113:380–386. Nagy K, Sandoz L, Craft B, Destaillats F. 2011. Mass-defect filtering of isotope signatures to reveal the source of chlorinated palm oil contaminants. Food Addit Contam: Part A. 28:1492–1500. Pudel F, Matthäus B, Freudenstein A, Rudolph T. 2012. Mitigation of 3-MCPD and glycidyl esters in refined palm oils, 103rd AOCS Annual Meeting and Expo, April 29-May 2. Urbana, IL: AOCS Press. Ramli MR, Siew WL, Ibrahim NA, Hussein R, Kuntom A, Abd Razak RA, Nesaretnam K. 2011. Effects of degumming and bleaching on 3-MCPD esters formation during physical refining. J Am Oil Chem Soc. 88:1839–1844. Sambanthamurthi R, Chong CL, Oo KC, Yeo KH, Rajan P. 1991. Chilling-induced lipid hydrolysis in the oil palm (Elaeis guineensis) mesocarp. J Exp Bot. 42:1199–1205. Sambanthamurthi R, Oo KC, Parman SH. 1995. Factors affecting lipase activity in the oil palm (Elaeis Guineensis) mesocarp.
In: Kader JC, Mazliak P, editor. Plant Lipid Metabolism. Dordrecht: Kluwer Academic Publications. Siew WL, Kuntom A, Ibrahim NA, Ramli MR, Abd. Razak RA. 2012. The possible mitigation procedures for the reduction of chloropropanol esters and related compounds. Palm Oil Develop. 57:21–27. Siew WL, Ng WL. 1995. Diglyceride content and composition as indicators of palm oil quality. J Sci Food Agric. 69:73–79. Subramaniam V, Menon NR, Sin H, Choo YM. 2013. The development of a residual oil recovery system to increase the revenue of a palm oil mill. J Oil Palm Res. 25:116–122. Weißhaar R. 2008. 3-MCPD-esters in edible fats and oils – a new and worldwide problem. Eur J Lipid Sci Tech. 110:671–672. Weißhaar R. 2011. Fatty acid esters of 3-MCPD: Overview of occurrence and exposure estimates. Eur J Lipid Sci Tech. 113:304–308. Yamazaki K, Ogiso M, Isagawa S, Urushiyama T, Ukena T, Kibune N. 2013. A new, direct analytical method using LS-MS/MS for fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD esters) in edible oils. Food Addit Contam: Part A. 30:52–68. Yeoh CM, Phuah ET, Tang TK, Siew WL, Abdullah LC, Thomas Choong SY. 2014. Molecular distillation and characterization of diacylglycerol-enriched palm olein. Eur J Lipid Sci Tech. 116:1654–1663. Zelinková Z, Svejkovská B, Velíšek J, Doležal M. 2006. Fatty acid esters of 3-chloropropane-1,2-diol in edible oils. Food Addit Contam: Part A. 23:1290–1298. Zhou H, Jin Q, Wang X, Xu X. 2014. Direct measurement of 3chloropropane-1,2-diol fatty acid esters in oils and fats by HPLC method. Food Control. 36:111–118.
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Other factors to consider in the formation of chloropropandiol fatty esters in oil processes a
a
a
b
Muhamad Roddy Ramli , Wai Lin Siew , Nuzul Amri Ibrahim , Ainie Kuntom & Raznim Arni Abd. Razak a
b
Protein & Food Technology Unit, Malaysian Palm Oil Board, Kajang, Malaysia
b
Analytical & Quality Development Unit, Malaysian Palm Oil Board, Kajang, Malaysia Accepted author version posted online: 23 Mar 2015.Published online: 20 Apr 2015.
Click for updates To cite this article: Muhamad Roddy Ramli, Wai Lin Siew, Nuzul Amri Ibrahim, Ainie Kuntom & Raznim Arni Abd. Razak (2015): Other factors to consider in the formation of chloropropandiol fatty esters in oil processes, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2015.1032368 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1032368
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Food Additives & Contaminants: Part A, 2015 http://dx.doi.org/10.1080/19440049.2015.1032368
Other factors to consider in the formation of chloropropandiol fatty esters in oil processes Muhamad Roddy Ramlia*, Wai Lin Siewa, Nuzul Amri Ibrahima, Ainie Kuntomb and Raznim Arni Abd. Razakb a
Protein & Food Technology Unit, Malaysian Palm Oil Board, Kajang, Malaysia; bAnalytical & Quality Development Unit, Malaysian Palm Oil Board, Kajang, Malaysia
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(Received 6 November 2014; accepted 17 March 2015) This paper examines the processing steps of extracting palm oil from fresh fruit bunches in a way that may impact on the formation of chloropropandiol fatty esters (3-MCPD esters), particularly during refining. Diacylglycerols (DAGs) do not appear to be a critical factor when crude palm oils are extracted from various qualities of fruit bunches. Highly hydrolysed oils, in spite of the high free fatty acid (FFA) contents, did not show exceptionally high DAGs, and the oils did not display a higher formation of 3-MCPD esters upon heat treatment. However, acidity measured in terms of pH appears to have a strong impact on 3-MCPD ester formation in the crude oil when heated at high temperatures. The differences in the extraction process of crude palm oil from current commercial processes and that from a modified experimental process showed clearly the effect of acidity of the oil on the formation of 3-MCPD esters. This paper concludes that the washing or dilution step in palm oil mills removes the acidity of the vegetative materials and that a well-optimised dilution/washing step in the extraction process will play an important role in reducing formation of 3-MCPD esters in crude palm oil upon further heat processing. Keywords: chloropropandiol fatty esters; palm oil; diacylglycerols; acidity
Introduction In recent years, chloropropanediol fatty acid esters (3-MCPD esters – ME) have been detected in oils and fats (Zelinková et al. 2006; Weißhaar 2008, 2011). These contaminants are formed during processing of oils and fats, occurring when lipids such as triacylglycerols (TAGs) or glycerols react with chlorides at high temperatures. Among food products, high levels have been detected in oils and fats or foods containing a high fat content. As the levels are generally higher in refined oils, many researchers (Franke et al. 2009; Hrnčiřík & van Duijn 2011; Nagy et al. 2011; Ramli et al. 2011; Craft et al. 2012; Destaillats et al. 2012) have investigated the factors attributing to the formation, and certainly their studies have contributed to a better understanding of the presence of the esters in the oil. Levels of ME in palm oil have been reported (Abd. Razak et al. 2012; Yamazaki et al. 2013; MacMahon et al. 2013). Refined seed oils in general have lower values. Some researchers (Nagy et al. 2011; Freudenstein et al. 2013) reasoned that possible precursors such as higher diacylglycerol (DAG) contents and chloride levels could be the contributing factors attributing to the higher formation of the esters in refined palm oil. In this study, another possible factor is examined while investigating DAGs in the oil. DAGs are present in all oils and fats, the amount varying from 1% to as much as 8%. Generally, fruit oils such as palm oil (Siew & Ng 1995) and olive oil *Corresponding author. Email: [email protected] © 2015 Malaysian Palm Oil Board. Published by Taylor & Francis.
(Fronimaki et al. 2002) have higher DAG levels than seed oils. Some nut oils such as salfat, shea butter fats and other exotic fats have high DAG contents as well. In recent years, ME and glycidyl esters (GE) have been linked to DAGs present in vegetable oils (Hamlet et al. 2011; Matthäus et al. 2011). Some presentations and publications (Matthäus et al. 2011; Pudel et al. 2012) pointed specifically to DAGs as potential culprits of ME formation, especially observed upon adding DAGs to oils. As all oils and fats have similar components (TAG, DAG, monoacylglycerol (MAG), chlorides), high temperature treatment for physical refining of palm oil has been one of the decisive factors resulting in a greater amount of chloropropandiols. Chemical refining, however, showed less tendency of ME formation as neutralisation and water washing to remove the residual of soap partially eliminates reactive chlorinated precursors from the crude palm oil (CPO). Deodorisation is also conducted at relatively lower temperatures. Chlorides were recently studied (Franke et al. 2009; Nagy et al. 2011) and it would not be a surprise that all oils and fats may have varying quantities of chlorides, being such ubiquitous materials in nature. Franke et al. (2009) reported total chlorine in palm oil and rapeseed oil as 2 and < 2 ppm, respectively, and for chlorides < 1 ppm in both oils. Nagy et al. (2011) discussed the type of chlorides as being both inorganic and organic in nature. While there is a small amount of chlorides present in oils and fats, all oils come
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into contact with chlorides, for example in bleaching clays, water, phosphoric acid, etc. Several analytical measurements of ME and GE have been reported (Crews et al. 2013; Ermacora & Hrnčiřík 2013; Yamazaki et al. 2013; Ermacora & Hrnčiřík 2014; Zhou et al. 2014). The American Oil Chemists' Society has also developed three new methods, namely AOCS Cd13-29a (2013a), AOCS Cd13-29b (2013b) and AOCS Cd13-29c (2013c). These methods made it easier to relate the work of one laboratory with that of another, especially where data on different products were reported. Comprehensive studies have been undertaken to mitigate the formation of these esters. The mitigation procedures can be generally divided into three approaches: removal of precursors in the raw material, modification of the refining process and removal of the esters postrefining. Siew et al. (2012) and Matthäus and Pudel (2013) discussed potential procedures to reduce the formation of ME based on these three approaches. Craft and Nagy (2012) highlighted several ‘suggested practices (SP)’ from upstream (plantation) to downstream (refining) to improve the quality and safety of refined palm oil. A summary of the different mitigation strategies possible to reduce the formation of ME and GE is given in Table 1. The production of palm oil differs very much from that of seed oils, in that seed oils are extracted from dry seeds by solvent or by cold press, while because of the size and nature of the fruit, palm oil is extracted with the whole fruit bunches in the processing steps. Fresh fruit bunches (FFBs) are harvested when ripe, usually 20–22 weeks after flower anthesis. The FFBs are transported in lorries to mills, where they are extracted by a mechanical press. A bunch consists of a bunch stalk and spikelets of fruitlets attached to the stalk. The oil extraction process is described in Figure 1, involving Table 1.
Fresh fruit bunches (FFB)
Sterilization
Steam
Condensate
Sterilized FFB Stripping
Bunch stalks
Fruit Steam
Digestion
Pressing Crude oil
Press cake
Screening
Water
Oil Clarification
Separator
Dirt
Vacuum drying
Crude palm oil (CPO)
Figure 1. oil.
Schematic diagram for the production of crude palm
sterilisation, stripping, digestion, pressing, screening, clarification, centrifugation and drying. In some
Summary of the possible mitigation strategies in reducing the formation of 3-MCPD and glycidyl esters in refined oil. Key finding
Mitigation step Refining aids Glycerol and ethanol Ethanol:water (1:1) Diacetin Carbonates Dual deodorisation process (short term at higher temperature and long term at lower temperature/vice versa) Deodorisation using short-path distillation Neutralisation of degummed oil with Potassium hydroxide Sodium hydroxide Calcium oxide Hot water degumming Washing of crude palm oil before refining Washing of palm fruit pulp before oil extraction
25–35% reduction of MCPD esters Approximately 30% reduction of MCPD esters 50% reduction of MCPD esters and related compounds 66% reduction of MCPD esters and related compounds 63% reduction of MCPD esters
Reference Craft et al. (2012) Matthäus and Pudel (2013)
Matthäus and Pudel (2013)
90% reduction of MCPD esters and related compounds
Matthäus and Pudel (2013)
45% 35% 45% 70% 38% 95%
Matthäus and Pudel (2013)
reduction reduction reduction reduction reduction reduction
of of of of of of
MCPD MCPD MCPD MCPD MCPD MCPD
esters esters esters esters esters esters
Ramli et al. (2011) Ramli et al. (2011) Matthäus and Pudel (2013) Craft et al. (2012)
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Food Additives & Contaminants: Part A variations, decanter systems are used instead of clarification. These steps begin with sterilisation, which kills enzymes such as lipases and softens the fruits for ease of removal from the bunches; cooking the fruits in digesters; pressing, which releases the oil, followed by either addition of water for ease of separation of oil from the vegetative materials or through use of a decanter. Finally the crude oil is separated by centrifugal separators or by decanters, then dried and sent to storage tanks. The difference between oil production from palm and that of other seeds is mainly in the production process, where palm oil comes into contact with vegetative materials of the oil palm (bunch stalk, spikelets, calyx, vegetative fibres), whereas for seed oil production the vegetative materials are removed from the seeds prior to extraction. This difference in production is seen as a possible explanation for the higher occurrence of ME observed upon heat treatment of palm oil. This paper will examine how this is so. This paper also evaluates the different conditions of storage of fruits and their effects on the formation of ME. It also examines the effect of oil acidity on the formation.
measurements. The FFA, DAG and pH of the oils were analysed.
Crude palm oil (CPO) from evaporator process In this process shown in Figure 2, the oil/water and vegetative mixture were subjected to a falling film evaporation process, where the water inherent in the fruit was removed. No dilution of water takes place, and removal of vegetative materials takes place by separators. The water obtained from the process is collected for the pH test. The crude oil is also tested for FFA, DAG, ME formation on the heat test, and its acidity in terms of pH. The main difference from the conventional commercial process is the removal of the water step, and no additional water is added to the process, except that which is derived from steam used in the sterilisation of bunches. Fresh fruit bunches (FFB)
Steam
Sterilization
Condensate
Sterilized FFB Stripping
Materials and methods Storage of fresh fruit bunches (FFBs) and oil extraction FFBs were harvested from MPOB oil palm estate in Bangi, Selangor, Malaysia. Spikelets of fruits were cut from the bunches and separated into four portions. The first portion was sterilised fresh for oil extraction. The second portion was bruised with a hammer and kept for 1 day prior to sterilisation and oil extraction. The third portion was incubated at 15°C for 24 h prior to sterilisation; while the last set was incubated at 5°C for 24 h prior to sterilisation. The last two treatments were given cold treatments to increase the speed of hydrolysis, since it was reported that lipase within the fruits are activated at low temperatures (Sambanthamurthi et al. 1991). Sterilisation was carried out in a laboratory autoclave for 30 min at 15 psi. The fruits were then detached from the spikelets and the mesocarps removed using a knife. The mesocarps were pressed in a laboratory-scale hydraulic press and the collected oil was filtered to remove fibres and other impurities. The crude oil was dried by a rotavapor for 30 min. Analysis of ME upon heat test, FFA and DAG levels was carried out. Quadruple experiments were conducted. The mean of four sets of bunches was recorded.
Bunch stalks
Fruit Steam
Digestion
Pressing Crude oil
Press cake
Screening Oil Desanding
Cake
2-Phase separation Oil and water Evaporator Oil and sludge Separator
Vacuum drying
Samples collected from palm oil mills Samples of CPO were collected from commercial palm oil mills. The oil samples were heat treated at 260°C for ME
3
Crude palm oil (CPO)
Figure 2.
Modified experimental process.
Sand
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Palm fibre oil (PFO) A sample of PFO is obtained from a local refinery that processes fibre oil from palm fibres by a solvent process. The palm fibres are the vegetative material left over from the extraction of CPO from fruit bunches. These fibres contain 5–8% of oil (Abd. Majid et al. 2012).
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Diacylglycerol (DAG) analysis HPLC was carried out using Gilson (Rue Gambetta, Villers le Bel, France) 303 and 302 pumps and a Waters (Milford, MA, USA) differential refractometer. The two columns were of 25 cm length and 4 mm i.d. with 5 µm Lichrosphere RP18. The mobile phase was acetonitrile: acetone (65:35 v/v) at a flow rate of 1.0 ml min–1. Injection was achieved through a Rheodyne valve fitted with a 20 µl loop. Samples were injected as 10% w/v solution in warm acetone. Identification of TAGs and DAGs was made by comparison with reference standards. The DAG values are given in area per cent. The analysis of DAG was as reported in Yeoh et al. (2014).
1.2 ml min−1. The column temperature was programmed at 60°C (1 min) to 190°C (1 min) at a rate of 6°C min−1, and was then accelerated to 280°C at a rate of 30°C (5 min). The injector was held at 180°C and 1 µl of the sample was injected in a split-less mode. The quantitative analysis was carried out by monitoring characteristic ions at m/z 91, 147 and 196, respectively, for derivatised 3-MCPD; while for 3MCPD-d5, characteristic ions were at m/z 93, 150 and 201. Qualifier ions were m/z 196 towards m/z 201.
Heat test Crude oil samples (2 ml) were heated in a covered vial to 260°C, the vial being held in a sandbath. The oil was maintained at 260°C for 1 h, then cooled and analysed for ME. The measurement may not simulate the different aspects of refining such as degumming and bleaching, but relates to the high temperature as used in deodorisation. It is found to be a reliable indicator of the degree of ME expected for different oil sources.
pH test of oils Free fatty acids (FFAs) FFAs were analysed according to AOCS Official Method Ca 5a-40 (1998).
A total of 100 ml of oil was stirred with a similar amount of water for 1 h at 70°C. The water was removed by separating funnel, and the pH measured with a pH meter.
3-MCPD esters (ME) Analysis of ME was carried out according to Abd. Razak et al. (2012), and recorded in mg kg−1 values. The Federal Institute for Risk Assessment (BfR) Method 008 – Determination of 3-MCPD fatty acid esters in edible oils and solid fats by GC-MS (2009) – was adopted. The internal standard used was deuterated 3-MCPD (3-MCPD-d5). Oil sample of 100 mg was weighed and dissolved in 0.5 ml tert-butyl methyl ether (t-BME) containing the internal standard (0.4 µg). Sulphuric acid solution in methanol (2%, v/v) was added to the sample and the mixture was incubated for 16 h. Sodium hydrogencarbonate solution (0.5 ml) was then added to stop the hydrolysis. To separate the fatty acid methyl ester from the sample, 1.8 ml of an aqueous salt solution were added. Derivatisation was achieved by addition of 250 µl of phenylboronic acid solution to the mixture. The derivative was extracted and analysed. Detection of the analyte was carried out by means of GC-MSD from Agilent Technologies (Santa Clara, CA, USA), equipped with a Series 5975C quadrupole detector and controlled by a programmable GC 7890A. Chromatographic separation was performed on a fused silica capillary column, HP-5MS inert (30 m length, 0.25 mm i.d., 0.25 µm film thickness). Helium (purity 99.999%) was used as a carrier gas at a constant flow of
Statistical analysis Data obtained from the analyses were subjected to oneway analysis of variance (ANOVA) with Fisher’s multiple comparison to determine the significant differences among the samples defined at 95% confidence interval (p < 0.05). All analyses were conducted in duplicate and reported as means ± standard deviation (SD) of independent trials. The statistical analyses were performed using the Minitab software Version 16 statistical package (Minitab Inc., State College, PA, USA).
Results and discussion DAG and ME formation Formation and occurrence of ME in foods have been discussed by Hamlet et al. (2011). Mechanisms of formation are related to lipids, which are possible precursors of formation when conditions are optimum, for example reactions occur in the presence of chloride ions at high reaction temperatures. Thus, it appears easy to find the culprits in the case of palm oil, where DAGs are clearly higher than in other seed oils. Model experiments do show that DAGs, especially those added to the system, are reactive under high temperature conditions (Freudenstein et al. 2013). However, oils from various sources having
Food Additives & Contaminants: Part A Table 2. 3-MCPD esters (ME) formed upon heat treatment of CPO extracted directly from fruit bunches: effect of hydrolysis. Sample
FFA (wt %)
Fresh CPO 0.63 Oil from bruised 1.85 FFB High FFA oil 1 6.75 High FFA oil 2 59.65
DAG (wt %) ME (mg kg−1)
± 0.17a ± 0.71a
3.3 ± 0.3aa 3.8 ± 0.2a
1.55 ± 0.64a 1.45 ± 0.80a
± 2.06a ± 8.02b
5.4 ± 0.9b 4.8 ± 0.5ba
0.74 ± 0.23a 1.01 ± 0.50a
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Notes: Values are the mean ± SD of duplicate analysis. Values within a column with different superscript letters are significantly different (p < 0.05).
varying FFA and DAG contents point to the contrary of the expectations of the above (Lacoste et al. 2010). When a FFB is harvested, FFA increases in the oil, especially if there is considerable bruising, usually caused by the ripe fruit dropping to the ground from a height of several feet. Inherent and extraneous lipases come into contact with the oil released from ruptured cell walls as a result of bruising. A rise in FFA is slower if fruit bruising is minimised. Table 2 shows results of ME formed in heat-treated CPO extracted from palm fruits of varying state of hydrolysis. In the fruit experiments, spikelets from a bunch were separately treated as (1) freshly extracted, (2) bruised fruits and (3 and 4) hydrolysed fruits. The oils extracted were heat treated for their potential to form ME. In these experiments, the degree of hydrolysis of palm oil was affected to show the different levels of FFA and DAG. In fresh oil, the FFA of the CPO was only 0.63%, while oil from slightly bruised fruits resulted in an FFA of 1.85%. Chilling the fruits can enhance lipolytic hydrolysis by lipase present in the oil palm fruit, where it was observed that palm fruits subjected to 5°C chilling resulted in FFA levels of up to 70% (Sambanthamurthi et al. 1991, 1995; Cadena et al. 2013). Although FFA levels were high, the DAG levels were unexpectedly low. In our experiments, hydrolysed fruits caused by chilling at different temperatures (15 and 5°C) resulted in high FFA oils as indicated by values of 6.75% and 59.65%, respectively. Structural changes in mesocarp tissue as a result of chilling resulted in high enzyme substrate interaction. Even at FFA levels as high as 59.65%, DAG is still relatively low, being only slightly higher than the low FFA oils. In fact, 8% is the highest limit observed for DAG in most CPOs. The observations appeared contradictory to expectations of high DAG to be found in such highly hydrolysed oils. It is hypothesised that whatever DAG and MAG formed, they are being quickly hydrolysed to FFA. Sambanthamurthi et al. (1995) discussed evidence of an active endogenous lipase in the oil palm mesocarp which is located in the oil body. While in vitro experiments found the lipase maximum activity at 18°C, an in vivo assay showed maximum activity at 5°C. Further evidence of low-temperature activation
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of lipase of oil palm fruits was also shown by Cadena et al. (2013). In commercial oils, the FFA limit for traded CPO is 5% and any oil of values of > 10% would have been traded as high FFA oils. Keeping other variables such as the temperature of treatment for the oil constant, the experiment shows that the ME of the oils with higher FFA and DAG levels derived from same bunch of fruits did not differ significantly (p ≥ 0.05) from each other. In experiments involving added DAGs from commercial sources, Freudenstein et al. (2013) reported an increase in ME when 1,3-dipalmitin is added to palm oil. However, the process by which such 1,3-dipalmitin is produced is not known. Any impurities or residue (if acidic) left from the production process may cause an effect of increasing the ME formation. The experiments from fruits using DAG formed within the fruit illustrated conclusively that DAG alone would not be very reactive unless other preconditions are present. Other researchers who have also shown that DAG is not necessarily the critical factor are Hrnčiřík and Ermacora (2011) and Lacoste et al. (2010). Destaillats et al. (2012) showed that TAG is the main acylglycerol class involved in MCPD diester formation when oils are heated. DAG was found to form ME as well, but it has relatively lower reactivity than TAG. Crude oil from evaporation process In an experiment where CPO is processed in a different manner from that of commercial mill operation, it was found that the way palm oil is extracted from FFBs may play a role in determining the formation of ME. Figure 1 shows CPO being extracted in conventional mills. The process takes the fruit bunches through sterilisation, stripping of spikelets from the bunch, digestion of the fruits, pressing and dilution with water, followed by clarification. The clarification process allows settling of the oil and water phase and the oily phase is passed to separators for removal of dirt, and the crude oil is finally vacuumed dried. In Figure 2 oil extraction is carried out from a modified process in a small pilot plant. In the initial few steps, processing is generally similar as the conventional process. After digestion and pressing, the oil together with the fruit liquor is passed to a desander and a two-phase separation, removing most of the vegetative material. The oil and fruit liquor is retained in an evaporator, where the fruit liquor and water from steam sterilisation of bunches are removed via a falling film evaporator. The oil obtained is centrifuged and dried as before. The difference between this process and that of the current commercial mill process is in the use of the evaporator to remove any water present either inherently in the fruits or introduced through sterilisation. On the other hand, in current commercial process, a large quantity of water is added to dilute the oil to allow better separation of the oil from other
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M.R. Ramli et al. Table 3.
pH of crude palm oil and other properties: effect on the formation of 3-MCPD esters (ME).
Sample
FFA (wt %)
Commercial sample number 1 Commercial sample number 2 Commercial sample number 3 CPO, from evaporation process
2.66 2.66 3.66 3.04
± ± ± ±
0.03a 0.04a 0.06b 0.02c
DAG (wt %) 5.5 5.3 5.4 5.6
± ± ± ±
0.2a 0.1a 0.1a 0.2a
pH
ME (mg kg−1)
6.2 5.5 7.1 4.0
0.84 ± 0.05 0.99 ± 0.03 < LOD 12.96 ± 0.76
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Notes: Values are the mean ± SD of duplicate analysis. Values within a column with different superscript letters are significantly different (p < 0.05). LOD = 0.25 m g kg−1.
vegetative material. The evaporator water from this experimental process is found to have a very high acidity with a pH 3. Interestingly, the CPO obtained from the process has also high acidity with pH 4, while in comparison samples from commercial processes showed a pH of about 5–7 (Table 3). It has been generally observed that palm oil mill effluent (POME), which is the effluent waters removed during the oil extraction process, is acidic in nature (Igwe & Onyegbado 2007; Irenosen et al. 2014). The sample of CPO with pH 7 was found to have a lower ME upon heat treatment. The results pointed to an acidic nature in CPO, which is generally mainly reduced during its extraction process when there is much water used in the clarification step. The acidity is due to its contact with vegetative materials of the palm fruit bunch, which are generally acidic in nature. In conventional mills, the acidity in the fruits is further diluted by water just before the clarification process, and therefore is washed out. Any further washing carried out by some mills will have removed the acidity completely and also remove chlorides to a minimum level. Oil obtained from the experimental process (Figure 2), when heated to 260°C for 1 h, showed higher contents of ME than any commercial CPO as shown in Table 3. The high ME being formed would be caused by a higher reactivity of the lipids, resulting from the acidity present. Reactivity is enhanced when acidic conditions are present (Hamlet et al. 2011) as indicated in mechanisms of formation. Also, work by Ramli et al. (2011) has indicated the effect of acidity from the degumming agent and bleaching clays, providing further evidence of the effect of acidity on the formation of ME. The screening and clarification steps in palm oil mills may need to be optimised for better reduction of acidity level or, alternatively, an additional water wash step could be introduced into the process. In areas where soils are acidic, harvested fruit bunches that fall to the ground could also pick up acidic soil. It would be a good idea to wash the fruit bunches on conveyor belts to remove the additional acidity. Effect of oils from palm fibre Palm fibres are the by-products of extraction of CPO. These are the vegetative materials from the mesocarp and empty
bunch. Some mills have installed additional solvent plants to extract further the oil retained in the fibres. PFOs are sold for industrial uses, animal feeds or for biodiesel production. They contain higher quantities of carotenes (1400–1600 ppm) and tocols (1700–2600 ppm) (Subramaniam et al. 2013) and are potentially good raw materials for extraction of these components. However, the possibility of such oils being added to normal palm oil is considered as it enhances the oil extraction rate (OER) of palm oil production at the particular mill. With a residual oil recovery system (RORS) installed in a mill, oil recovery from the mesocarp ranging from 0.15% to 0.45% per tonne of FFB has been reported (Subramaniam et al. 2013). The effects of blending a small proportion of PFO to normal oils were examined. In this experiment, the PFO was added in small proportions to CPO. Results in Figure 3 show that PFO had a significant (p < 0.05) effect on the formation of ME. DAG level was considered high (7.9%) compared with normal CPO. A blend of 5% PFO into CPO would not contribute much to the DAG level. On the other hand, the pH of the PFO is found to be a low value of 4.6. Blending this oil to CPO would be detrimental to ME formation. The results confirmed that acidity is an important factor contributing to the ease of formation of ME when the oil is heated to a high temperature.
Figure 3. Effect of PFO in the formation of 3-MCPD esters (ME) in crude palm oil. Mean of duplicate analysis ± SD. Points with different letters are significantly different (p < 0.05).
Food Additives & Contaminants: Part A Conclusions
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While DAG may appear to be a culpable precursor of ME, their role in several situations as given in fruit experiments give some doubts as to their impact on formation. This paper presents some evidence for the case against making DAG content of palm oil the inherent critical factor for the formation of ME. It is noted that the acidity of CPO is the more important factor to consider; it should be removed during processing steps as a way towards reducing the formation of these esters. The chlorides present can also be removed in the process. The addition of water during the process has already been included in most mills and only needs further optimisation to enhance the better reduction of acidity. The possible inclusion of other types of extracted oils via solvents or additional pressings should be thoroughly discouraged. Disclosure statement No potential conflict of interest was reported by the authors.
Funding The authors thank the Director-General for financially supporting the study and for subsequent permission to publish this research effort.
References Abd. Majid R, Mohammad AW, Choo YM. 2012. Properties of residual palm pressed fibre oil. J Oil Palm Res. 24:1310– 1317. Abd. Razak RA, Kuntom A, Siew WL, Ibrahim NA, Ramli MR, Hussein R, Nesaretnam K. 2012. Detection and monitoring of 3-monochloropropane-1, 2-diol (3-MCPD) esters in cooking oils. Food Control. 25:355–360. American Oil Chemists’ Society. 2013a. Official methods Cd 29a13, Sampling and analysis of commercial fats and oils, 2- and 3-MCPD fatty acid esters and glycidyl fatty acid esters in edible oils and fats by acid transesterification. Urbana, IL: AOCS Press. American Oil Chemists’ Society. 2013b. Official methods Cd 29b-13, Sampling and analysis of commercial fats and oils, Determination of bound monochloropropanediol- (MCPD) and bound 2, 3-epoxy-1-propanol (glycidol-) by gas chromatography/mass spectrometry (GC/MS). Urbana, IL: AOCS Press. American Oil Chemists’ Society. 2013c. Official methods Cd 29c-13, Sampling and analysis of commercial fats and oils, Fatty-acid-bound 3-chloropropane-1,2-diol (3-MCPD) and 2,3- epoxi-propane-1-ol (glycidol), determination in oils and fats by GC/MS (Differential measurement). Urbana, IL: AOCS Press. American Oil Chemists’ Society (AOCS). 1998. Official methods and recommended practices of the American Oil Chemists’ Society. 5th ed. Urbana, IL: AOCS Press. Cadena T, Prada F, Perea A, Romero HM. 2013. Lipase activity, mesocarp oil content and iodine value in oil palm fruits of Elaeis Guineensis (E.G.), Elaeis Oleifera (E.O.), and the
7
interspecific hybrid (E.O. x E.G.). J Sci Food Agric. 93:674–680. Craft BD, Nagy K. 2012. Mitigation of MCPD-ester and glycidyl ester levels during the production of refined palm oil. Lipid Technol. 24:155–157. Craft BD, Nagy K, Sandoz L, Destaillats F. 2012. Factors impacting the formation of monochloropropanediol (MCPD) fatty acid diesters during palm (Elaeis guineensis) oil production. Food Addit Contam: Part A. 29:354–361. Crews C, Chiodini A, Granvogl M, Hamlet C, Hrnčiřík K, Kuhlmann J, Lampen A, Scholz G, Weisshaar R, Wenzl T, Jasti PR, Seefelder W. 2013. Analytical approaches for MCPD esters and glycidyl esters in food and biological samples: a review and future perspectives. Food Addit Contam: Part A. 30:11–45. Destaillats F, Craft BD, Sandoz L, Nagy K. 2012. Formation mechanisms of Monochloropropanediol (MCPD) fatty acid diesters in refined palm (Elaeis guineensis) oil and related fractions. Food Addit Contam: Part A. 29:29–37. Ermacora A, Hrnčiřík K. 2013. A novel method for simultaneous monitoring of 2-MCPD, 3-MCPD and glycidyl esters in oils and fats. J Am Oil Chem Soc. 90:1–8. Ermacora A, Hrnčiřík K. 2014. Development of an analytical method for the simultaneous analysis of MCPD esters and glycidyl esters in oil-based foodstuffs. Food Addit Contam: Part A. 31:985–994. Federal Institute for Risk Assessment. 2009. BfR Method_82_FC-008-01: Determination of 3-MCPD fatty acid esters in edible oils and solid fats by GC-MS – An indirect determination by detection of free 3-MCPD released from 3-MCPD-esters by acid hydrolysis and by derivatization with phenylboronic acid. Dahlem: BfR Press. Franke K, Strijowski U, Fleck G, Pudel F. 2009. Influence of chemical refining process and oil type on bound 3-chloro-1, 2-propanediol contents in palm oil and rapeseed oil. Food Sci Tech. 42:1751–1754. Freudenstein A, Weking J, Matthäus B. 2013. Influence of precursors on the formation of 3-MCPD and glycidyl esters in a model oil under simulated deodorization conditions. Eur J Lipid Sci Tech. 115:286–294. Fronimaki P, Spyros A, Christophoridou S, Dais P. 2002. Determination of the diglyceride content in Greek virgin olive oils and some commercial olive oils by employing 31 P NMR Spectroscopy. J Agric Food Chem. 50:2207–2213. Hamlet CG, Asuncion L, Velíšek J, Doležal M, Zelinková Z, Crews C. 2011. Formation and occurrence of esters of 3-chloropropane-1,2 diol (3-CPD) in foods: what we know and what we assume. Eur J Lipid Sci Tech. 113:279–303. Hrnčiřík K, Ermacora A. 2011. Formation of 3-MCPD esters in palm oil: Effect of partial acylglycerols. Informative Meeting of 3-MCPD and glycidyl esters in Foodstuffs, organized by OVID and BLL, Jan 18. Berlin: Association of Oilseed Processing. Hrnčiřík K, van Duijn G. 2011. An initial study on the formation of 3-MCPD esters during oil refining. Eur J Lipid Sci Tech. 113:374–379. Igwe JC, Onyegbado CC. 2007. A review of palm oil mill effluent (POME) water treatment. Global J Environ Res. 1:54–62. Irenosen OG, Oluyemi AS, Korede AO, Samuel AS. 2014. Integration of physical, chemical and biological methods for the treatment of palm oil mill effluent. Sci J Anal Chem. 2:7–10.
Downloaded by [University of Victoria] at 16:05 25 April 2015
8
M.R. Ramli et al.
Lacoste F, Joffre F, Soulet B, Griffon H. 2010. 3-MCPD esters in edible oils: analytical aspects, 101st AOCS Annual Meeting and Expo; May 16-19; Urbana, IL: AOCS Press. MacMahon S, Begley TH, Diachenko GW. 2013. Occurrence of 3-MCPD and glycidyl esters in edible oils in the United States. Food Addit Contam: Part A. 30:2081–2092. Matthäus B, Pudel F. 2013. Mitigation of 3-MCPD and glycidyl esters within the production chain of vegetable oils especially palm oil. Lipid Technol. 25:151–155. Matthäus B, Pudel F, Fehling P, Vosmann K, Freudenstein A. 2011. Strategies for the reduction of 3-MCPD esters and related compounds in vegetable oils. Eur J Lipid Sci Tech. 113:380–386. Nagy K, Sandoz L, Craft B, Destaillats F. 2011. Mass-defect filtering of isotope signatures to reveal the source of chlorinated palm oil contaminants. Food Addit Contam: Part A. 28:1492–1500. Pudel F, Matthäus B, Freudenstein A, Rudolph T. 2012. Mitigation of 3-MCPD and glycidyl esters in refined palm oils, 103rd AOCS Annual Meeting and Expo, April 29-May 2. Urbana, IL: AOCS Press. Ramli MR, Siew WL, Ibrahim NA, Hussein R, Kuntom A, Abd Razak RA, Nesaretnam K. 2011. Effects of degumming and bleaching on 3-MCPD esters formation during physical refining. J Am Oil Chem Soc. 88:1839–1844. Sambanthamurthi R, Chong CL, Oo KC, Yeo KH, Rajan P. 1991. Chilling-induced lipid hydrolysis in the oil palm (Elaeis guineensis) mesocarp. J Exp Bot. 42:1199–1205. Sambanthamurthi R, Oo KC, Parman SH. 1995. Factors affecting lipase activity in the oil palm (Elaeis Guineensis) mesocarp.
In: Kader JC, Mazliak P, editor. Plant Lipid Metabolism. Dordrecht: Kluwer Academic Publications. Siew WL, Kuntom A, Ibrahim NA, Ramli MR, Abd. Razak RA. 2012. The possible mitigation procedures for the reduction of chloropropanol esters and related compounds. Palm Oil Develop. 57:21–27. Siew WL, Ng WL. 1995. Diglyceride content and composition as indicators of palm oil quality. J Sci Food Agric. 69:73–79. Subramaniam V, Menon NR, Sin H, Choo YM. 2013. The development of a residual oil recovery system to increase the revenue of a palm oil mill. J Oil Palm Res. 25:116–122. Weißhaar R. 2008. 3-MCPD-esters in edible fats and oils – a new and worldwide problem. Eur J Lipid Sci Tech. 110:671–672. Weißhaar R. 2011. Fatty acid esters of 3-MCPD: Overview of occurrence and exposure estimates. Eur J Lipid Sci Tech. 113:304–308. Yamazaki K, Ogiso M, Isagawa S, Urushiyama T, Ukena T, Kibune N. 2013. A new, direct analytical method using LS-MS/MS for fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD esters) in edible oils. Food Addit Contam: Part A. 30:52–68. Yeoh CM, Phuah ET, Tang TK, Siew WL, Abdullah LC, Thomas Choong SY. 2014. Molecular distillation and characterization of diacylglycerol-enriched palm olein. Eur J Lipid Sci Tech. 116:1654–1663. Zelinková Z, Svejkovská B, Velíšek J, Doležal M. 2006. Fatty acid esters of 3-chloropropane-1,2-diol in edible oils. Food Addit Contam: Part A. 23:1290–1298. Zhou H, Jin Q, Wang X, Xu X. 2014. Direct measurement of 3chloropropane-1,2-diol fatty acid esters in oils and fats by HPLC method. Food Control. 36:111–118.
