Formation of polymerization compounds during thermal oxidation of cottonseed oil , partially hydrogenated cottonseed oil and their blends

Muestras de aceite de algodón, aceite de algodón parcialmente hidrogenado y sus mezclas, con índices de yodo de 60 a 110, tratadas o no con óxido de aluminio, fueron sometidas a termoxidación, a 180 °C, durante 10 horas. Se retiraron muestras en los tiempos 0, 2, 5, 8 y 10 horas, para determinación de dímeros y polímeros (compuestos de degradación) y de tocoferoles. Se verificó la influencia del grado de hidrogenación sobre la formación de dímeros y polímeros, y también el papel de los tocoferoles originalmente presentes en el aceite y en las grasas, en la protección contra la degradación térmica. Las curvas de degradación de los tocoferoles mostraron una destrucción bastante rápida de los tocoferoles presentes en el aceite y en las grasas de algodón (α y γ-tocoferoles), con niveles residuales próximos a cero después de 10 horas de termoxidación. Aún así, muestras con sus tocoferoles naturales tuvieron una degradación térmica más lenta. El grado de insaturación parece ser más importante en la protección contra la degradación térmica que el contenido de tocoferoles.


INTRODUCTION
Frying has become one of the most extensively used methods for the preparation of foods in developed countries, making frying fats absorbed by fried products an important component of contemporary diets.The popularity of frying continues to grow and this is demonstrated by the remarkable increase in new fried and pre-fried foods found on the market (Velasco et al., 2004).Oil is the best heat transfer medium for the rapid preparation of foods, adequate for modern lifestyles and food-service systems; also giving the fried product desirable organoleptic characteristics (crispness, flavor, etc.) (Pérez-Camino et al., 1988).
During frying, fats and oils decompose forming volatile and non-volatile products that modify their functional, organoleptic and nutritional properties.The main degradation products absorbed by the food are non-volatile, including triacylglycerol polymers, triacylglycerol dimers, oxidized triacylglycerol monomers, cyclic substances and hydrolytic decomposition products (diacylglycerols, monoacylglycerols and free fatty acids) (Warner, 1997).
There are means to retard degradation, such as: the process of hydrogenation, which reduces the number of double bonds in the fat or oil, reducing reactive sites, concomitantly reducing iodine value; the presence or addition of antioxidants, such as naturally occurring tocopherols (Kamal-Eldin and Appelqvist, 1996) or synthetic tert-butylhydroquinone (TBHQ) (Allam and Mohamed, 2002;Gordon and Kourimska, 1995); and the addition of anti-foaming agents, such as dimethylpolysiloxane (DMPS), which reduce the contact with oxygen (Márquez- Ruiz et al., 2004).
Frying oils must be resistant to elevated temperatures, have adequate consistency to ease handling, have high stability in the finished product and low absorption by the fried product.In Brazil, high performance vegetable frying fats are produced using hydrogenation as an intermediate step, followed by blending with other bases and/or liquid oils (Block, 1997).The two main oils used for producing frying shortenings are soybean oil and cottonseed oil.
Cottonseed fats and oils are often considered superior to other fats and oils in frying applications, due to their higher stability (lower levels of linolenic acid) and better organoleptic characteristics (Warner et al, 1997;NPCA, 2001).
Cottonseed fats used for frying in Brazil have iodine values that vary from 70 to 105, melting points from 24 to 38 °C and may contain tertbutyl-hydroquinone (TBHQ), citric acid and dimethylpolysiloxane to prevent oxidative and thermal degradation.
Naturally occurring tocopherols in cottonseed fats and oils are also a means to retard degradation of the oil.Antioxidant activity of tocopherols is mainly due to their capacity for donating their phenolic hydrogens to lipidic free radicals.But many other factors can affect the in vitro activity, such as concentration, light and temperature, type of substrate and solvent, and other chemical species that act as pro-oxidants or synergists in the system (Kamal-Eldin and Appelqvist, 1996).
The objective of this research was to evaluate the formation of polymerization compounds when refined cottonseed oil, partially hydrogenated cottonseed oil and three blends of both were submitted to thermal oxidation conditions.Towards this aim, two series of oils (with and without natural tocopherols) were heated under standard conditions, thus allowing the analysis of the influence of two factors: the degree of fat unsaturation and the level of tocopherols.

Aluminium oxide treatment
A portion of each sample was treated to remove originally present tocopherols.This stripping procedure was carried out as described by Yoshida et al. (1992), by passing 50 -100 g of oil through a column with approximately double this amount of activated aluminium oxide (Al 2 O 3 : aluminium oxide 90, standardised for column chromatographic adsorption analyses, acc. to Brockmann, Merck, item n o 1.01097.1000).Aluminium oxide was activated in an oven at 200 °C for at least 4 hours.After this treatment, each sample had its stripped or treated counterpart.

Thermal oxidation
8 ± 0.01 g of each sample were weighed in Rancimat tubes and inserted into the heating block previously heated to 180 ± 1 °C.Two samples of approximately 50 mg were collected after 2, 5, 8 and 10 hours heating and kept at -30 °C until analysis.
Rancimat instructions were carefully observed for glassware cleaning.During heating, the tubes were left open and no bubbling of air was applied.This procedure was described in detail, including reproducibility data, in a previous publication (Barrera-Arellano et al., 1997).

Analysis of thermally oxidized samples
1. Dimers and polymers were quantitated directly in the oil samples, using high performance sizeexclusion chromatography (HPSEC), according to the IUPAC Standard Method 2.508 (IUPAC, 1992).Conditions applied for HPSEC were as follows: a Waters 510 HPLC pump and a Rheodyne 7725i injector with a 10 μm sample loop (Waters Associates, Milford, MA, USA), a Merck L-7490 refractive index detector (Merck, Darmstadt, Germany) and a Hewlett-Packard HP 3390A integrator (Hewlett-Packard, Pittsburgh, PA, USA) were used.Two Ultrastyragel columns (Waters Associates, Milford, MA, USA), 100 and 500 Å, connected in series, were used.The columns were 25 cm long x 0.77 cm inner diameter, packed with a porous, highly cross-linked styrenedivinylbenzene copolymer (< 10 μm).High performance liquid chromatography grade tetrahydrofuran was used as mobile phase, with a flow rate of 1 mL/min.Sample solutions of about 50 mg of oil/mL tetrahydrofuran were prepared for analyses.
FORMATION OF POLYMERIZATION COMPOUNDS DURING THERMAL OXIDATION OF COTTONSEED OIL, PARTIALLY… 2. Tocopherols were determined using high performance liquid chromatography (HPLC), according to the IUPAC Standard Method 2.432 (IUPAC, 1992).Conditions applied for HPLC were as follows: a Waters 600 HPLC pump with a 20 μL sample loop or a Waters Model M45 Solvent Delivery System (Waters Associates, Milford, MA, USA) were used.A LiChrosorb SI 60 (250 x 4 mm) column, packed with silica (average particle size of approximately 5 μm) (Merck, Darmstadt, Germany), was used.A Hewlett-Packard 1046A programmable fluorescence detector (290 -330 nm) and a Hewlett-Packard HP 3390A integrator (Hewlett-Packard, Pittsburgh, PA, USA) were used.Oil samples (approximately 50 mg) were dissolved in 1 mL n-hexane.Mobile phase was n-hexane:isopropanol (99:1), with a flow rate of 1 mL/min.

RESULTS AND DISCUSSION
Physicochemical characteristics of cottonseed oil and partially hydrogenated cottonseed oil (PHCO) and their blends are shown in Table 1.It can be observed that oxidative stability was iodine value (IV) dependent.Comparing the oxidative stability of the cottonseed samples of this study with those of soybean samples evaluated in previous work (Steel et al., 2005), values close to half can be observed in the present study for the cottonseed samples with similar iodine values.For example, a soybean sample with IV = 60 had a Rancimat oxidative stability, at 110 °C, of 192.1 h, while the stability of PHCO (IV = 60) was of 89.2 h.One of the reasons for this difference could be the difference in the quantities and composition of tocopherols.Levels of 1245 mg/kg and 905 mg/kg total tocopherols were found in the soybean sample and in the PHCO sample, respectively.Apart from this, the soybean sample had a greater quantity of γ− and δ−tocopherols, to which a higher antioxidant activity in vitro is attributed (Kamal-Eldin & Appelqvist, 1996).All other quality related analytical indexes were within expected ranges for refined fats and oils.
Table 2 shows the fatty acid composition of cottonseed oil and partially hydrogenated cottonseed oil (PHCO) and their blends.The results clearly show the effect of blending (oil into PHCO) on the simultaneous increase of iodine value and of the more unsaturated fatty acids, mainly linoleic acid (C18:2), with a concomitant reduction of the monounsaturated and saturated C18 fatty acids (oleic acid, C18:1, and stearic acid, C18:0).
Table 3 presents the tocopherol composition of cottonseed oil, PHCO and their blends.In this work, an abnormally low quantity of tocopherols was observed in cottonseed oil.This could indicate that the oil was intensely deodorized or that it was stored for a very long period.However, the low peroxide value indicates that the oil was recently refined.It can also be observed, in Table 1, that in parallel to the low antioxidant protection, the oxidative stability of the cottonseed oil studied was very low (2.27 h, Rancimat 110 °C).
The levels of tocopherols found in the PHCO sample are close to those reported for crude cottonseed oil (402 mg/kg α−tocopherol and 572 mg/kg γ−tocopherol) (Gunstone et al., 1994) and its oxidative stability is very high when compared to the sample of refined cottonseed oil.Both factors, iodine value and tocopherol content, favored the oxidative stability of the more saturated samples.
Polar compounds (quantitation and distribution) in the samples before and after aluminium oxide treatment can be seen in Table 4. Differences in the levels of polar compounds among the cottonseed samples were observed.Before aluminium oxide treatment, total polar compounds, which represent the degradation compounds in an oil or fat, were present in levels of 10.2% in cottonseed oil (IV = 110) and 6.1% in PHCO (IV = 60).The blends with intermediate iodine values presented intermediate values for polar compounds and correspond to those expected considering the proportions of the two initial fats.Lumley (1988) determined total polar compounds in fifty oil samples without use in frying and found a typical range of 0.4 -6.4%.Four of the oils analyzed had polar compounds above 7.5%, but these values were considered exceptionally high.In this work, all the cottonseed samples, except the most saturated (PHCO, IV = 60), presented initial polar compound levels above 7.5%.
In the distribution of polar compounds, it can be seen that in PHCO (IV = 60), the sample with the 286 GRASAS Y ACEITES, 57 (3), JULIO-SEPTIEMBRE, 284-291, 2006,  The absence of tocopherols in aluminium oxide treated samples demonstrates the efficacy of the procedure in their removal, while fatty acid composition was not affected by the treatment (data not included).The percentages of total polar compounds in the treated samples varied from 2.4% in the blend with IV = 70 to 4.9% in cottonseed oil (IV = 110).As can be observed in Table 4, the lower concentration of polar compounds in the treated samples was mainly due to the adsorption on the aluminium oxide of two groups of compounds: oxidized triglycerides and diglycerides.
As expected, a drastic reduction in the oxidative stability of tocopherol-stripped samples was observed (Table 5).Higher oxidative stability was observed for the more saturated samples, but the removal of tocopherols caused a drastic reduction in oxidative stability, demonstrating the enormous influence of natural antioxidants on the stability against oxidation.Also, differences in stability of the series (OR or TR) increased as the iodine value decreased.
GRASAS Y ACEITES, 57 (3), JULIO-SEPTIEMBRE, [284][285][286][287][288][289][290][291]2006  lowest quantity of total polar compounds, the predominant species were oxidized triglycerides (oxidized monomers) and diglycerides.As the unsaturation of the samples increased (and also their total polar compound level, in this case), the level of oxidized triglycerides increased, from 2.7% in PHCO (IV = 60) to 4.0% in cottonseed oil (IV = 110), and also the levels of triglyceride polymers, from 0.8% in PHCO (IV = 60) to 4.0% in cottonseed oil (IV = 110).In general, the results found for the blends are those expected from the proportions of the cottonseed oil and PHCO.Also, as commented above, the presence of a high amount of polymerization compounds in the cottonseed oil, would justify its low tocopherol content, as these compounds are mainly nonpolar dimers formed during the deodorization step of the refining process.The higher the amount of these compounds, the more severe the deodorization conditions and the more probable the loss of minor compounds like tocopherols, due to their volatility (Ruiz-Méndez et al., 1997).
Table 3 Tocopherol Table 6 shows the evolution of dimers and polymers in cottonseed fat samples, during 10 hours under thermal oxidation conditions.In the first place, aluminium oxide treated samples, without their natural tocopherols, all showed greater degradation than their original counterparts after 2 h, demonstrating the importance of tocopherols in retarding thermal degradation.At time 0 h, dimers and polymers were lower for the treated samples as most of the polar compounds present in the original samples were retained in the aluminium oxide column.Secondly, the effect of iodine value was also observed; the higher the iodine value, the greater the thermal degradation (more oligomeric compounds formed).An exception was the blend with IV = 70, which will be commented below.However, the differences were lower than expected, considering the enormous differences in oxidative stabilities shown in Table 5. Results were in agreement with those reported for used frying fats and oils where differences in unsaturation degree were translated into low differences in degradation (Márquez-Ruiz et al., 1995;Warner et al., 1997).
The blend with IV = 70 showed an unexpected behavior: having a higher IV than PHCO (IV = 60), thus a higher unsaturation degree, it degraded less than PHCO under the thermal oxidation conditions applied in this study.The quantity of D+P in the original blend with IV = 70, after 10 h, 4.6%, was notably lower than the quantity (8.2%) found in original PHCO.
The relationship between the amount of polymers and time fitted a pseudo-zero order kinetic in all the experiments.Table 7 shows the parameters of the lines (slopes and intercepts) as well as the correlation coefficients found.As can be observed, not only correlation coefficients higher than 0.99 were found, but also the intercept increases with iodine value, parallel to the experimental data found for the initial samples.Also, the slopes corresponding to the data of thermoxidation assays without original tocopherols were, as expected, higher than their counterparts with original tocopherols.Finally, with the exception of the sample with IV = 70, the slopes increased with iodine values.
For those experiments carried out with original oils, Figure 1 shows the formation of polymers (the amounts in Table 6 minus the amount at the initial time) and the loss of natural tocopherols expressed as residual percentages.With the exception of the sample with IV = 70 commented above, the loss of tocopherols was very rapid in all the samples and they were exhausted at the end of the heating   period (10 hours).On the other hand, polymer formation was higher when the unsaturation degree increased.As high polymerization involves high oil degradation, these results suggest that fats of low unsaturation degree would become unprotected at low degradation levels.Consequently, fat degradation should be controlled in saturated fats used for the preparation of fried foods to be stored, as they have to maintain a significant part of antioxidants in order to delay oxidation before consumption (Márquez-Ruiz et al., 1999).A possible explanation of the results obtained for the samples with IV = 60 and 70 could be related to the complex relationship between the formation of new compounds and the loss of natural antioxidants, considering that tocopherols are lost very rapidly in highly saturated fats at frying temperatures (Yuki and Ishikawa, 1976).As can be observed in Figure 1, the total loss of tocopherols was very rapid, around 5 hours, for the most saturated sample (IV = 60).After 5 hours, the increase in polymers was exponential and justifies the fact that the lowest correlation coefficient among those shown in Table 7 was found for the linear fitting of this sample.The existence of an uncontrolled variable, for example, a higher content of pro-oxidants in the sample with IV = 60 could accelerate the rapid loss of tocopherols and the subsequent rapid decomposition of the main substrate.Moreover, when tocopherols were still present in both samples (2 and 5 hours), it can be observed that there were no differences in practice between the samples with IV = 60 and 70.
In Figure 2, the degradation of individual tocopherols in cottonseed samples, submitted to thermal oxidation conditions, can be observed.Alpha-tocopherol was destroyed faster than γ− tocopherol.This can be seen at time 5 h, where GRASAS Y ACEITES, 57 (3), JULIO-SEPTIEMBRE, 284-291, 2006, ISSN:    The blend with IV = 70 was the less degraded sample considering the formation of polymerization compounds (D+P).It also kept its tocopherols longer (had higher percentages of residual tocopherols): approx.50% α−tocopherol and 60% γ−tocopherol after 10 h under thermal oxidation conditions.
Overall, these results suggest the complexity of degradation at high temperatures.In contrast to what occurs at low or medium temperatures, substrates of low iodine values, more stable at high temperatures, may be unprotected from natural antioxidants very rapidly and at low levels of polymerization.

Table 2 Fatty acid composition of cottonseed oil, partially hydrogenated cottonseed oil and their blends
PHCO: partially hydrogenated cottonseed oil; IV: iodine value.

Table 6 Evolution of dimers and polymers (%) in cottonseed oil, partially hydrogenated cottonseed oil and their blends, with and without aluminium oxide treatment, submitted to thermal oxidation conditions
OR: original; TR: treated with aluminium oxide to remove tocopherols; PHCO: partially hydrogenated cottonseed oil; IV: iodine value.

Table 7 Parameters of the equation (Polymers (%) = a t (h) + b) and correlation coefficients
OR: original; TR: treated with aluminium oxide to remove tocopherols.