# Heating Quality and Stability of Aqueous Enzymatic Extraction of Fatty Acid-Balanced Oil in Comparison with Other Blended Oils

Authors: Yang Li; Dan Li; Baokun Qi; Sami Rokayya; Wenjun Ma; Jing Liang; Xiaonan Sui; Yan Zhang; Lianzhou Jiang
Journal: Journal of Chemistry
 (2014)

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## Abstract

The heating performance of enzyme-assisted aqueous processing-extracted blended oil (EAEPO), hexane-extracted blended oil (HEBO), and three kinds of blended oils was investigated by varying the heating times. Oil degradation was monitored by analysis of the acid value (AV), peroxide value (PV),p-anisidine value (p-AV), color, and trans-fatty acid composition. The fatty acid ratios of EAEPO, HEBO, and the three kinds of blended oils were very similar (0.27 : 1.03 : 0.96, 0.27 : 1.08 : 1.16, 0.27 : 0.65 : 0.8, 0.27 : 0.6 : 0.84, and 0.27 : 0.61 : 0.79, resp.). The AV and color increased in proportion to the heating time for all the oils. There was a rapid increase in the PV and p-AV of EAEPO and HEBO after heating for only 1 h, whereas the other three blended oils showed a rapid increase after heating for 2 h or 6 h. Despite the highest trans-fatty acid content found for HEBO, this content was relatively low and remained low up to a heating time of 8 h. It was found that after heating, a fatty acid ratio relatively close to its ideal value (0.27 : 0.48 : 0.49) was maintained by EAEPO, which indicates that EAEPO is tolerant to heat treatment and is suitable for maintaining a healthy diet.


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## Body

## 1. Introduction

In recent times, the use of oils with a balanced fatty acid ratio has emerged as one of the most important concerns for maintaining a healthy diet. The blending of several oils with different characteristics is one of the simplest procedures for controlling the characteristics of edible oil [1]. The United States Recommended Dietary Allowances (RDA) and the World Health Organization (WHO) recommend the fatty acid ratio of 1 (SFA, saturated fatty acid(s)) : 1 (MUFA, monounsaturated fatty acid(s)) : 1 (PUFA, polyunsaturated fatty acid(s)) as an ideal ratio for health [2]. In addition, a report from the Chinese Nutrition Society (CNS) regarding dietary reference intakes suggested a fatty acid ratio of 0.27 : 1 : 1 (SFA : MUFA : PUFA) after subtracting the daily consumption of fatty acids derived from animal fat [3]. The use of hexane-extracted blended oil (HEBO) is the main method currently used to achieve this aim. However, the hexane derived from petroleum distillates has environmental and safety issues, and thus, it is necessary to develop alternative methods of hexane extraction. Furthermore, the goal of achieving an acid-balanced oil has been realized by extracting oil from a mixture of soybean, rape seed, purple perilla, fructus cannabis, and scabish using an aqueous enzymatic method (EAEP). The oil extraction yield was maximized by applying the simplex-centroid mixture design method. This balanced fatty acid oil may be one of the major nutritional sources in the diet for maintaining human health by providing the body with unsaturated fatty acids, being particularly rich in omega-3, omega-6, and other nutritional components. However, heat treatment of oils is one of the most important methods for preparing food for human consumption worldwide [4] but may affect the shelf life and quality of the oil directly. Heating may simultaneously induce degradation and oxidation of oil components with the formation of products deleterious to human health. In addition, many reports concluded that the stability of individual vegetable oils against oxidation is unique, being dependent on the fatty acid composition and on the degree of unsaturation in particular [5, 6].The quality characteristics of many oils, including Deglet Nour, Allig, olive, sunflower, and hydrogenated soybean oil, have been investigated [7, 8]. Microwave heating of soybean oil induces severe quality and composition losses, primarily with more than 3 min of microwave heating [9]. Thus, the reactions that occur during heating of oil are an important concern. During heating, lipids may undergo chemical alterations due to hydrolytic and oxidative rancidity [10]. The influence of heating on the antioxidant activity of certain spice essential oils has been discussed in order to control lipid oxidation during food processing [11]. The influence of these reactions is derived from a number of products generated during heating, including volatile compounds, hydrolysis products, cyclic compounds, trans configuration compounds, polymers, and acrylamide. After the first two frying cycles, the free fatty acid content, peroxide value, and total polar materials increase, whereas the radical scavenging activity decreases [5].In this paper, the oxidative stability of oils extracted using various techniques, including solvent and aqueous enzymatic methods, is determined. The major reaction conditions and corresponding physical and chemical alterations during heating are also studied by comparative analysis of three blended oils with fatty acid compositions similar to that of EAEPO and HEBO.


## 2. Materials and Methods

### 2.1. Materials

Dehulled and full-fat soybean flakes were obtained from a company in Shijiazhuang, and rape seed, purple perilla, fructus cannabis, and scabish were obtained from Anhui, Jiangsu, Guangxi, and Muyang, respectively. Three different types of oils, namely, Golden Arowana blend oil (GABO), Fook Lam Moon Grain blend oil (FGBO), and Fook Lam Moon balanced blend oil (FBBO), were obtained from a local market (RT-Mart, Harbin). Alcalase 2.4 L was sourced from Novo-Nordisk A/S (Bagsvaerd, Denmark). The fatty acid methyl ester (FAME) standards were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).


### 2.2. Methods

#### 2.2.1. Extrusion Process

Water was sprayed onto the cracked soybean flakes to achieve the desired moisture content of 14.5% while tumbling the seeds in a mixer (TMV-100, China). The moisture content of 14.5% was chosen on the basis of previous work on oil extraction using EAEP [12]. The extruder (20 mm screw die diameter) used in this work was manufactured by Engineering College, Northeast Agricultural University, Harbin, China. The rotational speed of the extruder was 105 rpm and the barrel temperature was 90°C. The extruded soybeans were immediately used for the EAEP procedure or stored in polyethylene bags at 4°C in a refrigerator prior to use.


#### 2.2.2. Enzyme-Assisted Aqueous Extraction Process (EAEP)

Prior to the extraction procedure, the dehulled forms of rape seed (31.5%), purple perilla (11.8%), fructus cannabis (33.3%), and scabish (21.2%) were individually cracked and then mixed together with the extruded soybean (2.3%). The beaker filled with the seed mixtures and additional water (1 : 6 w/w) was subjected to ultrasonic treatment for 50 min at 55°C at a power of 500 W and then incubated at 55°C in a water bath; the pH of the slurry was adjusted to 9 by the addition of 2 N NaOH [13]. The enzyme dosage of Alcalase 2.4 L was 1.85% (v/w, based on the dry weight of the samples). A continuous stirring device was used to disperse the mixture during the enzymatic hydrolysis process. The reactions were stably maintained at the temperature and pH stated above for 3 h. At the end of the EAEP, the slurry was centrifuged in a 50 mL centrifuge tube at multiples of the force of gravity and expressed at 2,372 g (TGL-16G, China) for 30 min at 20°C. Four distinct layers (oil, cream, skim, and insoluble) were obtained after the centrifugation. The upper oil layer was carefully collected using a Pasteur pipette, and the remaining oil adhering to the cream surface was washed twice with hexane. The hexane was removed by evaporation in a fume hood.


#### 2.2.3. Hexane Extraction (HE) Process

The blending flakes (100 g) (extruded soybean 2.3%, dehulled rape seed 31.5%, dehulled purple perilla 11.8%, dehulled fructus cannabis 33.3%, and dehulled scabish 21.2%) were placed in a Soxhlet extractor equipped with a 0.5 L round-bottom flask and a water condenser. The extraction was carried out on a water bath for 6 h using 3 L ofn-hexane. After the extraction, hexane was removed under reduced pressure using a vacuum rotary evaporator (R205, China) at 45°C. The recovered oil was stored under refrigeration (4°C) prior to use.


#### 2.2.4. Heat Treatment

To simulate conventional times used in cooking, the heating period was varied as 1, 2, 4, 6, and 8 h. For each oil type and time, three subsamples of 80 mL were, respectively, placed in a domestic electronic heater plate with an intelligent magnetic stirrer (ZNCL, China) and the samples were heated at 200°C. Unheated samples of each oil type were used as controls (corresponding to 0 h). After heating, samples were stored in a brown bottle in a refrigerator (4°C) prior to analysis.


### 2.3. Oil Quality Analysis

#### 2.3.1. Determination of Fatty Acid and Trans-Fatty Acid Compositions

The trans-fatty acid composition of the oil types was analyzed using a gas chromatograph connected to a mass spectrometer (GC-MS). The fatty acid methyl esters were prepared in two steps: the oils were first saponified with 0.5 M KOH and subsequently methylated with 40% BF3 in methanol. The separation was performed using an HP-88 capillary column (100 mm × 0.25 mm i.d., 0.2 μm film thickness; Agilent Technologies, USA) connected to a GC/MS (6890/5973, USA). The operating conditions were as follows: carrier gas pressure, 100 kPa; carrier gas, helium; split ratio, 1 : 30; injection temperature, 250°C; scanning scope, 50–550 amu; and ionization voltage, 70 eV. The oven temperature settings were programmed as follows: the temperature was held at 80°C for 5 min, increased to 150°C at 10°C/min and held at 150°C for 2 min, then increased continuously to 230°C at a rate of 5°C/min, and held for 10 min. The individual fatty acids were identified by comparing their retention times with standards [2].


#### 2.3.2. Determination of Acid Value,p-Anisidine Value, and Color

The acid value was determined according to a literature method (AOAC, Cd-63), where 125 mL of the neutralized solvent mixture and 2 mL phenolphthalein indicator solution were placed into an Erlenmeyer flask and neutralized with alkali to a faint but permanent pink color. The oil sample was then weighed (20
±
0.05 g) and added to the solution, and the sample was shaken vigorously while titrating with standard alkali to the first permanent pink color of the same intensity as that of the neutralized solvent before the latter was added to the sample.Thep-anisidine value of the oxidized oil from fried dough after storage was determined according to the AOCS Recommended Practice Ti la-64 (AOCS, 1990).An accurately measured amount of oil (100 mg) was dissolved in 25 mL, and the absorbance was measured at 350 nm using a UV-vis spectrophotometer (1600PC, China). This solution (2.5 mL) was combined with 0.5 mL of 0.5% (w/v)p-anisidine in acetic acid, and the absorbance was read at 350 nm after 10 min.The color was determined using a Lovibond Tintometer (WSL-2, China) in the transmittance mode and expressed as red (R) and yellow (Y) values.


#### 2.3.3. Determination of Peroxide Value

An accurately measured amount of oil (300 mg) was dissolved in 9.9 mL of chloroform/methanol (7 : 3, v/v) after which 50.0μL of 10 mM xylenol orange and 50.0 μL of iron(III) chloride solution were added [14]. The mixture solution was incubated at room temperature for 5 min and then centrifuged at 1000 g for 5 min at 5°C. The supernatant was used for measurement of the absorbance at 560 nm using a UV-vis spectrophotometer (model 1600 PC, China). The iron(III) chloride solution was used to construct a standard curve.


#### 2.3.4. Statistical Analysis

Data from replicate analyses of all samples were subjected to a variance analysis (ANOVA) test using SPSS 18.0 for Windows. The significant difference between the means was determined by Duncan’s New Multiple Range Test (P
<
0.05).


## 2.1. Materials

Dehulled and full-fat soybean flakes were obtained from a company in Shijiazhuang, and rape seed, purple perilla, fructus cannabis, and scabish were obtained from Anhui, Jiangsu, Guangxi, and Muyang, respectively. Three different types of oils, namely, Golden Arowana blend oil (GABO), Fook Lam Moon Grain blend oil (FGBO), and Fook Lam Moon balanced blend oil (FBBO), were obtained from a local market (RT-Mart, Harbin). Alcalase 2.4 L was sourced from Novo-Nordisk A/S (Bagsvaerd, Denmark). The fatty acid methyl ester (FAME) standards were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).


## 2.2. Methods

### 2.2.1. Extrusion Process

Water was sprayed onto the cracked soybean flakes to achieve the desired moisture content of 14.5% while tumbling the seeds in a mixer (TMV-100, China). The moisture content of 14.5% was chosen on the basis of previous work on oil extraction using EAEP [12]. The extruder (20 mm screw die diameter) used in this work was manufactured by Engineering College, Northeast Agricultural University, Harbin, China. The rotational speed of the extruder was 105 rpm and the barrel temperature was 90°C. The extruded soybeans were immediately used for the EAEP procedure or stored in polyethylene bags at 4°C in a refrigerator prior to use.


### 2.2.2. Enzyme-Assisted Aqueous Extraction Process (EAEP)

Prior to the extraction procedure, the dehulled forms of rape seed (31.5%), purple perilla (11.8%), fructus cannabis (33.3%), and scabish (21.2%) were individually cracked and then mixed together with the extruded soybean (2.3%). The beaker filled with the seed mixtures and additional water (1 : 6 w/w) was subjected to ultrasonic treatment for 50 min at 55°C at a power of 500 W and then incubated at 55°C in a water bath; the pH of the slurry was adjusted to 9 by the addition of 2 N NaOH [13]. The enzyme dosage of Alcalase 2.4 L was 1.85% (v/w, based on the dry weight of the samples). A continuous stirring device was used to disperse the mixture during the enzymatic hydrolysis process. The reactions were stably maintained at the temperature and pH stated above for 3 h. At the end of the EAEP, the slurry was centrifuged in a 50 mL centrifuge tube at multiples of the force of gravity and expressed at 2,372 g (TGL-16G, China) for 30 min at 20°C. Four distinct layers (oil, cream, skim, and insoluble) were obtained after the centrifugation. The upper oil layer was carefully collected using a Pasteur pipette, and the remaining oil adhering to the cream surface was washed twice with hexane. The hexane was removed by evaporation in a fume hood.


### 2.2.3. Hexane Extraction (HE) Process

The blending flakes (100 g) (extruded soybean 2.3%, dehulled rape seed 31.5%, dehulled purple perilla 11.8%, dehulled fructus cannabis 33.3%, and dehulled scabish 21.2%) were placed in a Soxhlet extractor equipped with a 0.5 L round-bottom flask and a water condenser. The extraction was carried out on a water bath for 6 h using 3 L ofn-hexane. After the extraction, hexane was removed under reduced pressure using a vacuum rotary evaporator (R205, China) at 45°C. The recovered oil was stored under refrigeration (4°C) prior to use.


### 2.2.4. Heat Treatment

To simulate conventional times used in cooking, the heating period was varied as 1, 2, 4, 6, and 8 h. For each oil type and time, three subsamples of 80 mL were, respectively, placed in a domestic electronic heater plate with an intelligent magnetic stirrer (ZNCL, China) and the samples were heated at 200°C. Unheated samples of each oil type were used as controls (corresponding to 0 h). After heating, samples were stored in a brown bottle in a refrigerator (4°C) prior to analysis.


## 2.2.1. Extrusion Process

Water was sprayed onto the cracked soybean flakes to achieve the desired moisture content of 14.5% while tumbling the seeds in a mixer (TMV-100, China). The moisture content of 14.5% was chosen on the basis of previous work on oil extraction using EAEP [12]. The extruder (20 mm screw die diameter) used in this work was manufactured by Engineering College, Northeast Agricultural University, Harbin, China. The rotational speed of the extruder was 105 rpm and the barrel temperature was 90°C. The extruded soybeans were immediately used for the EAEP procedure or stored in polyethylene bags at 4°C in a refrigerator prior to use.


## 2.2.2. Enzyme-Assisted Aqueous Extraction Process (EAEP)

Prior to the extraction procedure, the dehulled forms of rape seed (31.5%), purple perilla (11.8%), fructus cannabis (33.3%), and scabish (21.2%) were individually cracked and then mixed together with the extruded soybean (2.3%). The beaker filled with the seed mixtures and additional water (1 : 6 w/w) was subjected to ultrasonic treatment for 50 min at 55°C at a power of 500 W and then incubated at 55°C in a water bath; the pH of the slurry was adjusted to 9 by the addition of 2 N NaOH [13]. The enzyme dosage of Alcalase 2.4 L was 1.85% (v/w, based on the dry weight of the samples). A continuous stirring device was used to disperse the mixture during the enzymatic hydrolysis process. The reactions were stably maintained at the temperature and pH stated above for 3 h. At the end of the EAEP, the slurry was centrifuged in a 50 mL centrifuge tube at multiples of the force of gravity and expressed at 2,372 g (TGL-16G, China) for 30 min at 20°C. Four distinct layers (oil, cream, skim, and insoluble) were obtained after the centrifugation. The upper oil layer was carefully collected using a Pasteur pipette, and the remaining oil adhering to the cream surface was washed twice with hexane. The hexane was removed by evaporation in a fume hood.


## 2.2.3. Hexane Extraction (HE) Process

The blending flakes (100 g) (extruded soybean 2.3%, dehulled rape seed 31.5%, dehulled purple perilla 11.8%, dehulled fructus cannabis 33.3%, and dehulled scabish 21.2%) were placed in a Soxhlet extractor equipped with a 0.5 L round-bottom flask and a water condenser. The extraction was carried out on a water bath for 6 h using 3 L ofn-hexane. After the extraction, hexane was removed under reduced pressure using a vacuum rotary evaporator (R205, China) at 45°C. The recovered oil was stored under refrigeration (4°C) prior to use.


## 2.2.4. Heat Treatment

To simulate conventional times used in cooking, the heating period was varied as 1, 2, 4, 6, and 8 h. For each oil type and time, three subsamples of 80 mL were, respectively, placed in a domestic electronic heater plate with an intelligent magnetic stirrer (ZNCL, China) and the samples were heated at 200°C. Unheated samples of each oil type were used as controls (corresponding to 0 h). After heating, samples were stored in a brown bottle in a refrigerator (4°C) prior to analysis.


## 2.3. Oil Quality Analysis

### 2.3.1. Determination of Fatty Acid and Trans-Fatty Acid Compositions

The trans-fatty acid composition of the oil types was analyzed using a gas chromatograph connected to a mass spectrometer (GC-MS). The fatty acid methyl esters were prepared in two steps: the oils were first saponified with 0.5 M KOH and subsequently methylated with 40% BF3 in methanol. The separation was performed using an HP-88 capillary column (100 mm × 0.25 mm i.d., 0.2 μm film thickness; Agilent Technologies, USA) connected to a GC/MS (6890/5973, USA). The operating conditions were as follows: carrier gas pressure, 100 kPa; carrier gas, helium; split ratio, 1 : 30; injection temperature, 250°C; scanning scope, 50–550 amu; and ionization voltage, 70 eV. The oven temperature settings were programmed as follows: the temperature was held at 80°C for 5 min, increased to 150°C at 10°C/min and held at 150°C for 2 min, then increased continuously to 230°C at a rate of 5°C/min, and held for 10 min. The individual fatty acids were identified by comparing their retention times with standards [2].


### 2.3.2. Determination of Acid Value,p-Anisidine Value, and Color

The acid value was determined according to a literature method (AOAC, Cd-63), where 125 mL of the neutralized solvent mixture and 2 mL phenolphthalein indicator solution were placed into an Erlenmeyer flask and neutralized with alkali to a faint but permanent pink color. The oil sample was then weighed (20
±
0.05 g) and added to the solution, and the sample was shaken vigorously while titrating with standard alkali to the first permanent pink color of the same intensity as that of the neutralized solvent before the latter was added to the sample.Thep-anisidine value of the oxidized oil from fried dough after storage was determined according to the AOCS Recommended Practice Ti la-64 (AOCS, 1990).An accurately measured amount of oil (100 mg) was dissolved in 25 mL, and the absorbance was measured at 350 nm using a UV-vis spectrophotometer (1600PC, China). This solution (2.5 mL) was combined with 0.5 mL of 0.5% (w/v)p-anisidine in acetic acid, and the absorbance was read at 350 nm after 10 min.The color was determined using a Lovibond Tintometer (WSL-2, China) in the transmittance mode and expressed as red (R) and yellow (Y) values.


### 2.3.3. Determination of Peroxide Value

An accurately measured amount of oil (300 mg) was dissolved in 9.9 mL of chloroform/methanol (7 : 3, v/v) after which 50.0μL of 10 mM xylenol orange and 50.0 μL of iron(III) chloride solution were added [14]. The mixture solution was incubated at room temperature for 5 min and then centrifuged at 1000 g for 5 min at 5°C. The supernatant was used for measurement of the absorbance at 560 nm using a UV-vis spectrophotometer (model 1600 PC, China). The iron(III) chloride solution was used to construct a standard curve.


### 2.3.4. Statistical Analysis

Data from replicate analyses of all samples were subjected to a variance analysis (ANOVA) test using SPSS 18.0 for Windows. The significant difference between the means was determined by Duncan’s New Multiple Range Test (P
<
0.05).


## 2.3.1. Determination of Fatty Acid and Trans-Fatty Acid Compositions

The trans-fatty acid composition of the oil types was analyzed using a gas chromatograph connected to a mass spectrometer (GC-MS). The fatty acid methyl esters were prepared in two steps: the oils were first saponified with 0.5 M KOH and subsequently methylated with 40% BF3 in methanol. The separation was performed using an HP-88 capillary column (100 mm × 0.25 mm i.d., 0.2 μm film thickness; Agilent Technologies, USA) connected to a GC/MS (6890/5973, USA). The operating conditions were as follows: carrier gas pressure, 100 kPa; carrier gas, helium; split ratio, 1 : 30; injection temperature, 250°C; scanning scope, 50–550 amu; and ionization voltage, 70 eV. The oven temperature settings were programmed as follows: the temperature was held at 80°C for 5 min, increased to 150°C at 10°C/min and held at 150°C for 2 min, then increased continuously to 230°C at a rate of 5°C/min, and held for 10 min. The individual fatty acids were identified by comparing their retention times with standards [2].


## 2.3.2. Determination of Acid Value,p-Anisidine Value, and Color

The acid value was determined according to a literature method (AOAC, Cd-63), where 125 mL of the neutralized solvent mixture and 2 mL phenolphthalein indicator solution were placed into an Erlenmeyer flask and neutralized with alkali to a faint but permanent pink color. The oil sample was then weighed (20
±
0.05 g) and added to the solution, and the sample was shaken vigorously while titrating with standard alkali to the first permanent pink color of the same intensity as that of the neutralized solvent before the latter was added to the sample.Thep-anisidine value of the oxidized oil from fried dough after storage was determined according to the AOCS Recommended Practice Ti la-64 (AOCS, 1990).An accurately measured amount of oil (100 mg) was dissolved in 25 mL, and the absorbance was measured at 350 nm using a UV-vis spectrophotometer (1600PC, China). This solution (2.5 mL) was combined with 0.5 mL of 0.5% (w/v)p-anisidine in acetic acid, and the absorbance was read at 350 nm after 10 min.The color was determined using a Lovibond Tintometer (WSL-2, China) in the transmittance mode and expressed as red (R) and yellow (Y) values.


## 2.3.3. Determination of Peroxide Value

An accurately measured amount of oil (300 mg) was dissolved in 9.9 mL of chloroform/methanol (7 : 3, v/v) after which 50.0μL of 10 mM xylenol orange and 50.0 μL of iron(III) chloride solution were added [14]. The mixture solution was incubated at room temperature for 5 min and then centrifuged at 1000 g for 5 min at 5°C. The supernatant was used for measurement of the absorbance at 560 nm using a UV-vis spectrophotometer (model 1600 PC, China). The iron(III) chloride solution was used to construct a standard curve.


## 2.3.4. Statistical Analysis

Data from replicate analyses of all samples were subjected to a variance analysis (ANOVA) test using SPSS 18.0 for Windows. The significant difference between the means was determined by Duncan’s New Multiple Range Test (P
<
0.05).


## 3. Results and Discussion

### 3.1. Changes in Acid Value

The acid value is considered in the food industry as an indicator of the quality of the oil and the degree of its degradation during heating. An increase in the acid value leads to the development of unpleasant tastes and odors in oils. Although the initial acid values of the oils varied from 0.08 (EAEPO) to 0.16 (HEBO) as shown in Figure1, at the end of the heating period (8 h), the overall change in the acid value was found to be lowest in the case of EAEPO (0.35), followed by FBBO, FGBO, and GABO. The overall change in the acid value was highest for HEBO (0.48). This variation may be due to differences in the extraction method used. Enzymatic extraction offers the advantages of requiring a lower temperature and not requiring explosive solvents or generating harmful waste relative to solvent extraction. These results are in agreement with the values previously reported for corn oil [15].Figure 1
Changes in acid value.The results also indicated a significant increase in the acid value of the various oil types as heating progressed, which may be attributed to the hydrolysis of TAG and/or cleavage and oxidation of fatty acid double bonds [16]. In addition, the low acid value of the three types of blended oils relative to HEBO might be due to the refining of the former oils. It is recognized that the free fatty acids (FFAs) are removed by saponification; thus, smaller amounts of FFAs are expected for refined oils [17]. However, the lowest acid content of the EAEPO sample might be related to its ideal fatty acid ratio. In a similar study, the stability of soybean and canola oils, with modified fatty acid composition, toward frying was evaluated. The amount of free fatty acids generated during frying was significantly less than that of the corresponding unmodified oils after 5 h of frying [18].


### 3.2. Changes in Peroxide Value (PV)

Figure2 shows the changes in the PV of the oils with heating from 0 h to 8 h. Prior to the heating process, EAEPO presented the lowest peroxide value (3.18 mEq O2/kg), followed by HEBO (3.51 mEq O2/kg), FGBO (3.65 mEq O2/kg), and FBBO (3.77 mEq O2/kg). The highest PV was detected for GABO (3.88 mEq O2/kg). The PV of all the samples was within the acceptable limit of below 10 mEq O2/kg for vegetable oils [18]. In addition, the lowest PV of EAEPO is plausibly due to tocopherol release during the EAEP, which is related to hydrolysis by the enzymes [19].Figure 2
Changes in peroxide value.The peroxide levels of all of the oil types remained similar to the original values during heating with slight variations from the initial amounts (2–4 mEq O2/kg) due to the volatile nature of peroxides [17]. However, the concentration of peroxides in the various oil types differed significantly.The PV of the EAEPO and HEBO samples increased during the first 1 h, followed by a decline to the initial levels (3.63 mEq O2/kg and 2.76 mEq O2/kg, resp.). In the case of FGBO and FBBO, the PV continued to increase up to 6 h with an eventual return to the initial levels of ca. 4.02 mEq O2/kg and 3.96 mEq O2/kg, respectively. The peroxide value of GABO increased to 3.81 mEq O2/kg after 2 h. The rapid increase in the PV of the EAEPO and HEBO samples indicates that these oils were unstable to oxidative degradation. The EAEPO and HEBO samples had the highest percentage of polyunsaturated fatty acids and demonstrated less stability to oxidation, which is in agreement with the data previously reported for olive oil [17]. The PV decreased after reaching a maximum for all of the oil types, which is attributed to the fact that primary oxidation reactions cause an increase in the concentration of peroxides to a maximum value, beyond which the peroxide concentration decreases due to thermal decomposition thereof into secondary products [20].


### 3.3. Changes inp-Anisidine Value (p-AV)

Thep-AV is a measure of the content of aldehydes (principally 2-alkenals and 2, 4-alkadienals), which are relatively stable, and is thus a useful tool for monitoring the oxidative state of oil during heating. Therefore, for an accurate estimation of the oxidation status of the oils, both the PV andp-AV should be interpreted simultaneously.Figure3 shows the changes in thep-AV of the oils with heating from 0 h to 8 h. Thep-AV of all oil types increased with time. Furthermore, thep-AV of all the oil types exhibited significant changes at the same heating time. This may be attributed to further decomposition of the less-stable primary oxidative products (hydroperoxides) to form aldehydic compounds, as previously reported [17, 21].Figure 3
Changes inp-anisidine value.It was also noted that the trend in the variation of thep-AV with heating for FGBO was similar to that for FBBO with a maximum rate of change at 6 h of treatment compared to GABO for which the maximum rate of change was achieved at 2 h. However, in the case of EAEPO and HEBO, rapid hydroperoxide formation was detected after 1 h of heat treatment, probably due to the high amounts of PUFA in the two oils. The slightly higherp-AV of HEBO may be due to the ability of the solvent to extract other lipid associated substances such as sterols, fat soluble compounds, and hydrocarbons. This increasedp-AV level was consistent with the PV data presented in Figure 2, which confirms that EAEPO and HEBO present minor resistance to oxidation under heating conditions relative to the other three blended oils.


### 3.4. Changes in Color

Color is an important food evaluation indicator for rapid monitoring of the quality of heated oil. For the unheated oils, HEBO had the highest R parameter of 34.97 compared to the other blended oils due to the extraction of pigments by the organic solvent. The yellow color of EAEPO was more prominent than that of the other blended oils (55.01), indicative of the presence of more yellow pigments, such as carotenoids, in the EAEPO sample (Tables1 and 2). However, the lighter color of the other three oils is related to decolorization during oil refining.Table 1
Variation of yellow color of blended oils.
Y
0 h
1 h
2 h
4 h
6 h
8 h
FGBO
15.08 ± 0.14aB
15.53 ± 0.09bB
15.94 ± 0.05cB
16.09 ± 0.04cB
16.37 ± 0.03215dB
16.65 ± 0.08eB
FBBO
19.70 ± 0.02aC
20.17 ± 0.03bC
20.25 ± 0.03bC
20.39 ± 0.08cC
20.60 ± 0.02082dC
20.87 ± 0.04eC
GABO
29.29 ± 0.05aD
29.49 ± 0.06bD
29.87 ± 0.08cD
30.09 ± 0.01dD
30.26 ± 0.06110dD
30.57 ± 0.09eD
EAEPO
55.01 ± 0.09aE
55.32 ± 0.03bE
55.37 ± 0.07bE
55.80 ± 0.09cE
55.85 ± 0.07000cdE
56.05 ± 0.07eE
HEBO
2.13 ± 0.09aA
2.74 ± 0.03bA
3.07 ± 0.03cA
3.07 ± 0.06dA
3.52 ± 0.02517dA
3.78 ± 0.02eA
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.Table 2
Variation of red color of blended oils.
R
0 h
1 h
2 h
4 h
6 h
8 h
FGBO
1.21 ± 0.02aA
1.57 ± 0.11bA
2.00 ± 0.06cA
2.56 ± 0.06dB
2.79 ± 0.04eC
3.50 ± 0.09fB
FBBO
1.49 ± 0.02aC
1.87 ± 0.04bC
2.00 ± 0.05bcA
2.07 ± 0.11cA
2.63 ± 0.07cB
2.76 ± 0.04dA
GABO
1.37 ± 0.04aB
1.77 ± 0.04bB
1.97 ± 0.04cA
2.04 ± 0.04cdA
2.15 ± 0.04dA
4.11 ± 0.109eC
EAEPO
2.57 ± 0.05aD
3.60 ± 0.03bD
3.64 ± 0.02bB
4.55 ± 0.05cC
4.67 ± 0.02cD
5.86 ± 0.04dD
HEBO
34.97 ± 0.03aE
38.03 ± 0.09bE
38.27 ± 0.07bC
38.90 ± 0.11cD
39.37 ± 0.05dE
39.72 ± 0.10eE
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.Heat treatment induced a considerable increase in the R and Y parameters. In general, the reddish and yellowish coloration intensifies when the pigments developed during oxidation and thermal decomposition of fatty acids diffuse into the oil during heating. In addition, these changes may also be due to traces of carotenoids [22]. The difference in the R parameter of the nontreated and treated oils was slightly higher (34.97 versus 39.72) in the case of HEBO, which is more sensitive to oxidation due to its higher relative percentage of PUFA. This also holds for EAEPO (2.57 versus 5.86).


### 3.5. Changes in Fatty Acids Composition

The fatty acid composition is the most important oil constituent parameter, and the nature of these fatty acids, particularly the degree of unsaturation, determines the oxidative stability of the oils. The unheated oils comprised four major fatty acids: palmitic, stearic, oleic, and linoleic acids (Table3, Figure 4). The oleic acid content (40.49–47.35%) was found to be the highest, followed by linoleic acid, (35.17–44.63%), palmitic acid (6.61–12.12%), and stearic acid (3.14–9.15%). EAEPO and HEBO, respectively, had the highest linoleic acid content (40.46%, 44.63%) and the lowest palmitic acid content (7.60%, 6.61%). After heating for 4 h, the palmitic acid and stearic acid contents of EAEPO increased significantly, whereas the content of oleic and linoleic acids decreased (Figure 5). Both linoleic acid and palmitic acid are considered as good indicators of the extent of fat deterioration given that linoleic acid is more susceptible to oxidation, whereas palmitic acid is more stable toward oxidation [23].Table 3
Main fatty acids (%) profile.
FAs
0 h
1 h
2 h
4 h
6 h
8 h
C
16
:
0
FGBO
9.87 ± 0.03aD
17.25 ± 0.03bB
17.70 ± 0.01bcB
17.48 ± 0.01cB
17.48 ± 0.35cB
18.05 ± 0.02dB
FBBO
9.15 ± 0.02aC
12.41 ± 0.03bC
13.07 ± 0.02cE
13.14 ± 0.03dD
13.71 ± 0.03eD
16.42 ± 0.04fD
GABO
12.12 ± 0.02aE
13.12 ± 0.02bD
17.11 ± 0.04cD
17.76 ± 0.04dE
17.76 ± 0.01dE
17.74 ± 0.02dC
EAEPO
7.60 ± 0.02aB
15.50 ± 0.04bE
16.32 ± 0.01cC
16.71 ± 0.02dA
16.78 ± 0.02eA
16.89 ± 0.02fA
HEBO
6.61 ± 0.04aA
15.92 ± 0.01bA
16.03 ± 0.02cA
17.22 ± 0.02dC
17.31 ± 0.02eC
18.15 ± 0.01fE
C
18
:
0
FGBO
4.10 ± 0.010aC
6.37 ± 0.02bD
7.14 ± 0.03cD
7.60 ± 0.02dD
7.53 ± 0.13dC
9.07 ± 0.02eC
FBBO
9.15 ± 0.011aE
12.41 ± 0.03bE
13.05 ± 0.02cE
13.13 ± 0.03dE
13.75 ± 0.01eD
16.44 ± 0.00fD
GABO
4.54 ± 0.047cD
4.15 ± 0.04bC
4.21 ± 0.00bC
4.05 ± 0.01aC
4.07 ± 0.02aB
4.56 ± 0.04cB
EAEPO
3.60 ± 0.030cB
3.46 ± 0.03aB
3.12 ± 0.00dB
3.48 ± 0.01aB
3.54 ± 0.01bA
3.59 ± 0.00cA
HEBO
3.14 ± 0.036aA
3.23 ± 0.020bA
3.12 ± 0.01aA
3.36 ± 0.01cA
3.44 ± 0.02dA
3.61 ± 0.02eA
C
18
:
1
FGBO
46.74 ± 0.036fD
40.90 ± 0.02dD
40.54 ± 0.04cD
40.09 ± 0.02bC
41.21 ± 0.03eD
40.02 ± 0.02aC
FBBO
47.35 ± 0.025fE
46.53 ± 0.04eE
45.75 ± 0.01cE
46.13 ± 0.03dE
44.87 ± 0.02bE
42.62 ± 0.04aE
GABO
45.81 ± 0.032eC
39.79 ± 0.02aC
39.99 ± 0.12bC
40.21 ± 0.03cD
40.55 ± 0.04dC
40.30 ± 0.03cD
EAEPO
44.49 ± 0.036fB
37.91 ± 0.03eB
35.12 ± 0.02dA
34.67 ± 0.06cA
34.37 ± 0.04bA
34.28 ± 0.02aA
HEBO
40.49 ± 0.025fA
35.79 ± 0.03eA
35.45 ± 0.01dB
35.23 ± 0.02cB
34.94 ± 0.02bB
34.84 ± 0.03aB
C
18
:
2
FGBO
36.34 ± 0.025fB
30.79 ± 0.03eA
30.51 ± 0.04dA
30.18 ± 0.03cA
29.97 ± 0.03bA
27.79 ± 0.03aA
FBBO
35.17 ± 0.015dA
33.58 ± 0.03cB
32.40 ± 0.02aB
32.37 ± 0.08aB
33.24 ± 0.03bC
32.35 ± 0.02aA
GABO
35.57 ± 0.05cA
36.30 ± 0.53dC
32.22 ± 0.037abB
32.60 ± 0.07bC
32.40 ± 0.02abB
32.05 ± 0.03aA
EAEPO
40.46 ± 0.55bC
39.51 ± 0.03aD
39.63 ± 0.03aC
39.72 ± 0.06aE
39.74 ± 0.02aE
39.82 ± 0.04aB
HEBO
44.63 ± 0.01eD
39.96 ± 0.01cE
40.20 ± 0.01dD
38.97 ± 0.02bD
38.94 ± 0.02bD
38.68 ± 0.04aB
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.Figure 4
Typical chromatogram of fatty acid methyl ester prepared from EAEPO before heating. Peaks: 1: hexadecanoic acid; 2: octadecanoic acid; 3: 9-octadecenoic acid; 4: 9-octadecenoic acid; 5: 9,12-octadecadienoic acid; 6: 12,15-octadecadienoic acid.Figure 5
Typical chromatogram of fatty acid methyl ester prepared from EAEPO after heating. Peaks: 1: hexadecanoic acid; 2: octadecanoic acid; 3: 9-octadecenoic acid; 4: 9-octadecenoic acid; 5: 9,12-octadecadienoic acid; 6: 12,15-octadecadienoic acid.Table4 shows the variation of the content of SFA, MUFA, and PUFA of the oils from the initial to the final heating time. Heat treatment induced a decrease in the relative percentages of the unsaturated fatty acids and an increase of the relative percentages of the saturated fatty acids, as has previously been demonstrated in various studies [17]. Progressive heating may induce modification of fatty acids that contain two or three double bonds. Both oxidation and oxidative polymerization occur with progressive heating. Furthermore, the relative oxidation rate of linolenic acid is much higher than that of stearic acid [24].Table 4
Changes in saturated, monounsaturated, and polyunsaturated fatty acids.
SFA, MUFA, and PUFA
0 h
1 h
2 h
4 h
6 h
8 h
SFA
FGBO
15.28 ± 0.04 aC
26.83 ± 0.04bE
27.75 ± 0.01cE
28.94 ± 0.03dD
27.87 ± 0.04eE
31.45 ± 0.01fE
FBBO
15.51 ± 0.04aD
18.34 ± 0.02bA
19.92 ± 0.02cA
19.85 ± 0.04dA
20.22 ± 0.02eA
25.32 ± 0.01fC
GABO
17.93 ± 0.06aE
19.78 ± 0.03bB
24.23 ± 0.01cD
24.74 ± 0.02dC
24.69 ± 0.07dD
25.35 ± 0.01eD
EAEPO
11.93 ± 0.03aB
20.13 ± 0.02bD
21.11 ± 0.02cC
21.30 ± 0.02dB
21.44 ± 0.00eB
21.65 ± 0.01fA
HEBO
10.57 ± 0.02aA
20.01 ± 0.02bC
20.03 ± 0.02bB
21.36 ± 0.02cB
21.57 ± 0.02dC
22.54 ± 0.01eB
MUFA
FGBO
47.12 ± 0.02fCD
41.64 ± 0.02dC
41.11 ± 0.01cC
40.70 ± 0.02cB
41.78 ± 0.04eB
40.54 ± 0.02aC
FBBO
48.15 ± 0.04eD
47.43 ± 0.02dE
46.93 ± 0.05cE
47.65 ± 0.49cD
45.85 ± 0.02bC
41.65 ± 0.03aD
GABO
45.97 ± 0.01eBC
45.13 ± 0.03dD
41.53 ± 0.01aD
41.55 ± 0.04cC
41.86 ± 0.08cB
41.77 ± 0.01bE
EAEPO
44.48 ± 0.04cB
40.12 ± 0.02bB
39.28 ± 0.04abA
38.87 ± 0.03abA
38.53 ± 0.02abA
38.46 ± 0.02aA
HEBO
42.22 ± 0.02dA
39.98 ± 0.02cA
40.18 ± 0.04bB
39.30 ± 0.58cA
38.53 ± 0.65aA
38.74 ± 0.02abB
PUFA
FGBO
37.11 ± 0.01fC
31.46 ± 0.02eA
31.16 ± 0.02cA
30.63 ± 0.02cA
30.31 ± 0.01bA
27.96 ± 0.02aA
FBBO
36.29 ± 0.03eA
34.24 ± 0.02dB
33.12 ± 0.02bB
33.14 ± 0.03bB
33.91 ± 0.03cC
32.95 ± 0.04aB
GABO
36.44 ± 0.02fB
35.65 ± 0.01eC
34.23 ± 0.03cC
33.73 ± 0.03cC
33.41 ± 0.02bB
32.95 ± 0.01aB
EAEPO
42.13 ± 0.03dD
39.54 ± 0.02aD
39.58 ± 0.03aD
39.73 ± 0.05bE
39.74 ± 0.03bE
39.84 ± 0.03cD
HEBO
45.64 ± 0.02fE
39.97 ± 0.01dE
40.18 ± 0.04dE
38.96 ± 0.02cD
38.89 ± 0.02bD
38.74 ± 0.02aC
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.The fatty acid ratio of EAEPO and HEBO (0.27 : 1.03 : 0.96 and 0.27 : 1.08 : 1.16) approached the ideal fatty acid ratio of 0.27 : 1 : 1 (SFA : MUFA : PUFA), as shown in Table5; the other three kinds of blended oils also approached this ratio prior to heating (0.27 : 0.65 : 0.8, 0.27 : 0.6 : 0.84, and 0.27 : 0.61 : 0.79 for FGBO, FBBO, and GABO, resp.). At the end of the heating time, the ratio was 0.27 : 0.48 : 0.49 for EAEPO, which indicates that a relatively close to ideal fatty acid ratio for a healthy diet could be maintained for the EAEPO sample as heating progressed.Table 5
Variation of SFA : MUFA : PUFA ratio of blended oils.
SFA, MUFA, and PUFA
0 h
1 h
2 h
4 h
6 h
8 h
FGBO
0.27 : 0.65 : 0.8
0.27 : 0.42 : 0.32
0.27 : 0.40 : 0.30
0.27 : 0.38 : 0.29
0.27 : 0.41 : 0.29
0.27 : 0.35 : 0.24
FBBO
0.27 : 0.6 : 0.84
0.27 : 0.70 : 0.50
0.27 : 0.64 : 0.45
0.27 : 0.64 : 0.45
0.27 : 0.61 : 0.45
0.27 : 0.44 : 0.35
GABO
0.27 : 0.61 : 0.79
0.27 : 0.62 : 0.49
0.27 : 0.46 : 0.38
0.27 : 0.45 : 0.37
0.27 : 0.45 : 0.35
0.27 : 0.45 : 0.35
EAEPO
0.27 : 1.03 : 0.96
0.27 : 0.54 : 0.53
0.27 : 0.49 : 0.50
0.27 : 0.48 : 0.50
0.27 : 0.48 : 0.50
0.27 : 0.48 : 0.49
HEBO
0.27 : 1.08 : 1.16
0.27 : 0.51 : 0.54
0.27 : 0.50 : 0.54
0.27 : 0.47 : 0.49
0.27 : 0.46 : 0.49
0.27 : 0.43 : 0.46


### 3.6. Changes in Trans-Fatty Acids

Epidemiological evidence has suggested that the level of trans-fatty acid (TFA) intake is connected to the risk of cardiovascular disease [25]; thus, there have been several studies on TFAs contained in food materials. The amount of total TFAs in the evaluated oil types was in the range of 0.01-0.02 mg/mL, which clearly approximates the requirement for a “zero trans” content. However, a substantial increase in the TFAs of the various oils was observed when the oils were heated for 6 h to 8 h (Table 6). At the final heating time, EAEPO and HEBO presented the highest total TFA content. Several factors, such as the FA composition of each oil and coexisting antioxidants, may contribute to the variation in the thermal TFA accumulation profiles [26].Table 6
Changes in trans-fatty acid profile (mg/mL).
TFAs
0 h
1 h
2 h
4 h
6 h
8 h
FGBO
0.01 ± 0.01aA
4.94 ± 0.01bA
17.18 ± 0.02cA
59.68 ± 0.03dA
157.83 ± 0.08eA
280.16 ± 0.18fA
FBBO
0.01 ± 0.04aC
5.78 ± 0.03bC
21.32 ± 0.01cC
68.79 ± 0.04dC
188.48 ± 0.03eC
257.14 ± 0.08fC
GABO
0.01 ± 0.01aB
5.07 ± 0.06bB
18.54 ± 0.05cB
64.61 ± 0.08dB
162.39 ± 0.03eB
333.44 ± 0.09fB
EAEPO
0.02 ± 0.01aD
9.55 ± 0.03bD
29.88 ± 0.05cD
88.64 ± 0.02dD
232.52 ± 0.07eD
542.39 ± 0.08fD
HEBO
0.02 ± 0.01aC
12.43 ± 0.02bE
30.51 ± 0.03cE
93.09 ± 0.037dE
256.70 ± 0.06eE
558.60 ± 0.14fE
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.


## 3.1. Changes in Acid Value

The acid value is considered in the food industry as an indicator of the quality of the oil and the degree of its degradation during heating. An increase in the acid value leads to the development of unpleasant tastes and odors in oils. Although the initial acid values of the oils varied from 0.08 (EAEPO) to 0.16 (HEBO) as shown in Figure1, at the end of the heating period (8 h), the overall change in the acid value was found to be lowest in the case of EAEPO (0.35), followed by FBBO, FGBO, and GABO. The overall change in the acid value was highest for HEBO (0.48). This variation may be due to differences in the extraction method used. Enzymatic extraction offers the advantages of requiring a lower temperature and not requiring explosive solvents or generating harmful waste relative to solvent extraction. These results are in agreement with the values previously reported for corn oil [15].Figure 1
Changes in acid value.The results also indicated a significant increase in the acid value of the various oil types as heating progressed, which may be attributed to the hydrolysis of TAG and/or cleavage and oxidation of fatty acid double bonds [16]. In addition, the low acid value of the three types of blended oils relative to HEBO might be due to the refining of the former oils. It is recognized that the free fatty acids (FFAs) are removed by saponification; thus, smaller amounts of FFAs are expected for refined oils [17]. However, the lowest acid content of the EAEPO sample might be related to its ideal fatty acid ratio. In a similar study, the stability of soybean and canola oils, with modified fatty acid composition, toward frying was evaluated. The amount of free fatty acids generated during frying was significantly less than that of the corresponding unmodified oils after 5 h of frying [18].


## 3.2. Changes in Peroxide Value (PV)

Figure2 shows the changes in the PV of the oils with heating from 0 h to 8 h. Prior to the heating process, EAEPO presented the lowest peroxide value (3.18 mEq O2/kg), followed by HEBO (3.51 mEq O2/kg), FGBO (3.65 mEq O2/kg), and FBBO (3.77 mEq O2/kg). The highest PV was detected for GABO (3.88 mEq O2/kg). The PV of all the samples was within the acceptable limit of below 10 mEq O2/kg for vegetable oils [18]. In addition, the lowest PV of EAEPO is plausibly due to tocopherol release during the EAEP, which is related to hydrolysis by the enzymes [19].Figure 2
Changes in peroxide value.The peroxide levels of all of the oil types remained similar to the original values during heating with slight variations from the initial amounts (2–4 mEq O2/kg) due to the volatile nature of peroxides [17]. However, the concentration of peroxides in the various oil types differed significantly.The PV of the EAEPO and HEBO samples increased during the first 1 h, followed by a decline to the initial levels (3.63 mEq O2/kg and 2.76 mEq O2/kg, resp.). In the case of FGBO and FBBO, the PV continued to increase up to 6 h with an eventual return to the initial levels of ca. 4.02 mEq O2/kg and 3.96 mEq O2/kg, respectively. The peroxide value of GABO increased to 3.81 mEq O2/kg after 2 h. The rapid increase in the PV of the EAEPO and HEBO samples indicates that these oils were unstable to oxidative degradation. The EAEPO and HEBO samples had the highest percentage of polyunsaturated fatty acids and demonstrated less stability to oxidation, which is in agreement with the data previously reported for olive oil [17]. The PV decreased after reaching a maximum for all of the oil types, which is attributed to the fact that primary oxidation reactions cause an increase in the concentration of peroxides to a maximum value, beyond which the peroxide concentration decreases due to thermal decomposition thereof into secondary products [20].


## 3.3. Changes inp-Anisidine Value (p-AV)

Thep-AV is a measure of the content of aldehydes (principally 2-alkenals and 2, 4-alkadienals), which are relatively stable, and is thus a useful tool for monitoring the oxidative state of oil during heating. Therefore, for an accurate estimation of the oxidation status of the oils, both the PV andp-AV should be interpreted simultaneously.Figure3 shows the changes in thep-AV of the oils with heating from 0 h to 8 h. Thep-AV of all oil types increased with time. Furthermore, thep-AV of all the oil types exhibited significant changes at the same heating time. This may be attributed to further decomposition of the less-stable primary oxidative products (hydroperoxides) to form aldehydic compounds, as previously reported [17, 21].Figure 3
Changes inp-anisidine value.It was also noted that the trend in the variation of thep-AV with heating for FGBO was similar to that for FBBO with a maximum rate of change at 6 h of treatment compared to GABO for which the maximum rate of change was achieved at 2 h. However, in the case of EAEPO and HEBO, rapid hydroperoxide formation was detected after 1 h of heat treatment, probably due to the high amounts of PUFA in the two oils. The slightly higherp-AV of HEBO may be due to the ability of the solvent to extract other lipid associated substances such as sterols, fat soluble compounds, and hydrocarbons. This increasedp-AV level was consistent with the PV data presented in Figure 2, which confirms that EAEPO and HEBO present minor resistance to oxidation under heating conditions relative to the other three blended oils.


## 3.4. Changes in Color

Color is an important food evaluation indicator for rapid monitoring of the quality of heated oil. For the unheated oils, HEBO had the highest R parameter of 34.97 compared to the other blended oils due to the extraction of pigments by the organic solvent. The yellow color of EAEPO was more prominent than that of the other blended oils (55.01), indicative of the presence of more yellow pigments, such as carotenoids, in the EAEPO sample (Tables1 and 2). However, the lighter color of the other three oils is related to decolorization during oil refining.Table 1
Variation of yellow color of blended oils.
Y
0 h
1 h
2 h
4 h
6 h
8 h
FGBO
15.08 ± 0.14aB
15.53 ± 0.09bB
15.94 ± 0.05cB
16.09 ± 0.04cB
16.37 ± 0.03215dB
16.65 ± 0.08eB
FBBO
19.70 ± 0.02aC
20.17 ± 0.03bC
20.25 ± 0.03bC
20.39 ± 0.08cC
20.60 ± 0.02082dC
20.87 ± 0.04eC
GABO
29.29 ± 0.05aD
29.49 ± 0.06bD
29.87 ± 0.08cD
30.09 ± 0.01dD
30.26 ± 0.06110dD
30.57 ± 0.09eD
EAEPO
55.01 ± 0.09aE
55.32 ± 0.03bE
55.37 ± 0.07bE
55.80 ± 0.09cE
55.85 ± 0.07000cdE
56.05 ± 0.07eE
HEBO
2.13 ± 0.09aA
2.74 ± 0.03bA
3.07 ± 0.03cA
3.07 ± 0.06dA
3.52 ± 0.02517dA
3.78 ± 0.02eA
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.Table 2
Variation of red color of blended oils.
R
0 h
1 h
2 h
4 h
6 h
8 h
FGBO
1.21 ± 0.02aA
1.57 ± 0.11bA
2.00 ± 0.06cA
2.56 ± 0.06dB
2.79 ± 0.04eC
3.50 ± 0.09fB
FBBO
1.49 ± 0.02aC
1.87 ± 0.04bC
2.00 ± 0.05bcA
2.07 ± 0.11cA
2.63 ± 0.07cB
2.76 ± 0.04dA
GABO
1.37 ± 0.04aB
1.77 ± 0.04bB
1.97 ± 0.04cA
2.04 ± 0.04cdA
2.15 ± 0.04dA
4.11 ± 0.109eC
EAEPO
2.57 ± 0.05aD
3.60 ± 0.03bD
3.64 ± 0.02bB
4.55 ± 0.05cC
4.67 ± 0.02cD
5.86 ± 0.04dD
HEBO
34.97 ± 0.03aE
38.03 ± 0.09bE
38.27 ± 0.07bC
38.90 ± 0.11cD
39.37 ± 0.05dE
39.72 ± 0.10eE
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.Heat treatment induced a considerable increase in the R and Y parameters. In general, the reddish and yellowish coloration intensifies when the pigments developed during oxidation and thermal decomposition of fatty acids diffuse into the oil during heating. In addition, these changes may also be due to traces of carotenoids [22]. The difference in the R parameter of the nontreated and treated oils was slightly higher (34.97 versus 39.72) in the case of HEBO, which is more sensitive to oxidation due to its higher relative percentage of PUFA. This also holds for EAEPO (2.57 versus 5.86).


## 3.5. Changes in Fatty Acids Composition

The fatty acid composition is the most important oil constituent parameter, and the nature of these fatty acids, particularly the degree of unsaturation, determines the oxidative stability of the oils. The unheated oils comprised four major fatty acids: palmitic, stearic, oleic, and linoleic acids (Table3, Figure 4). The oleic acid content (40.49–47.35%) was found to be the highest, followed by linoleic acid, (35.17–44.63%), palmitic acid (6.61–12.12%), and stearic acid (3.14–9.15%). EAEPO and HEBO, respectively, had the highest linoleic acid content (40.46%, 44.63%) and the lowest palmitic acid content (7.60%, 6.61%). After heating for 4 h, the palmitic acid and stearic acid contents of EAEPO increased significantly, whereas the content of oleic and linoleic acids decreased (Figure 5). Both linoleic acid and palmitic acid are considered as good indicators of the extent of fat deterioration given that linoleic acid is more susceptible to oxidation, whereas palmitic acid is more stable toward oxidation [23].Table 3
Main fatty acids (%) profile.
FAs
0 h
1 h
2 h
4 h
6 h
8 h
C
16
:
0
FGBO
9.87 ± 0.03aD
17.25 ± 0.03bB
17.70 ± 0.01bcB
17.48 ± 0.01cB
17.48 ± 0.35cB
18.05 ± 0.02dB
FBBO
9.15 ± 0.02aC
12.41 ± 0.03bC
13.07 ± 0.02cE
13.14 ± 0.03dD
13.71 ± 0.03eD
16.42 ± 0.04fD
GABO
12.12 ± 0.02aE
13.12 ± 0.02bD
17.11 ± 0.04cD
17.76 ± 0.04dE
17.76 ± 0.01dE
17.74 ± 0.02dC
EAEPO
7.60 ± 0.02aB
15.50 ± 0.04bE
16.32 ± 0.01cC
16.71 ± 0.02dA
16.78 ± 0.02eA
16.89 ± 0.02fA
HEBO
6.61 ± 0.04aA
15.92 ± 0.01bA
16.03 ± 0.02cA
17.22 ± 0.02dC
17.31 ± 0.02eC
18.15 ± 0.01fE
C
18
:
0
FGBO
4.10 ± 0.010aC
6.37 ± 0.02bD
7.14 ± 0.03cD
7.60 ± 0.02dD
7.53 ± 0.13dC
9.07 ± 0.02eC
FBBO
9.15 ± 0.011aE
12.41 ± 0.03bE
13.05 ± 0.02cE
13.13 ± 0.03dE
13.75 ± 0.01eD
16.44 ± 0.00fD
GABO
4.54 ± 0.047cD
4.15 ± 0.04bC
4.21 ± 0.00bC
4.05 ± 0.01aC
4.07 ± 0.02aB
4.56 ± 0.04cB
EAEPO
3.60 ± 0.030cB
3.46 ± 0.03aB
3.12 ± 0.00dB
3.48 ± 0.01aB
3.54 ± 0.01bA
3.59 ± 0.00cA
HEBO
3.14 ± 0.036aA
3.23 ± 0.020bA
3.12 ± 0.01aA
3.36 ± 0.01cA
3.44 ± 0.02dA
3.61 ± 0.02eA
C
18
:
1
FGBO
46.74 ± 0.036fD
40.90 ± 0.02dD
40.54 ± 0.04cD
40.09 ± 0.02bC
41.21 ± 0.03eD
40.02 ± 0.02aC
FBBO
47.35 ± 0.025fE
46.53 ± 0.04eE
45.75 ± 0.01cE
46.13 ± 0.03dE
44.87 ± 0.02bE
42.62 ± 0.04aE
GABO
45.81 ± 0.032eC
39.79 ± 0.02aC
39.99 ± 0.12bC
40.21 ± 0.03cD
40.55 ± 0.04dC
40.30 ± 0.03cD
EAEPO
44.49 ± 0.036fB
37.91 ± 0.03eB
35.12 ± 0.02dA
34.67 ± 0.06cA
34.37 ± 0.04bA
34.28 ± 0.02aA
HEBO
40.49 ± 0.025fA
35.79 ± 0.03eA
35.45 ± 0.01dB
35.23 ± 0.02cB
34.94 ± 0.02bB
34.84 ± 0.03aB
C
18
:
2
FGBO
36.34 ± 0.025fB
30.79 ± 0.03eA
30.51 ± 0.04dA
30.18 ± 0.03cA
29.97 ± 0.03bA
27.79 ± 0.03aA
FBBO
35.17 ± 0.015dA
33.58 ± 0.03cB
32.40 ± 0.02aB
32.37 ± 0.08aB
33.24 ± 0.03bC
32.35 ± 0.02aA
GABO
35.57 ± 0.05cA
36.30 ± 0.53dC
32.22 ± 0.037abB
32.60 ± 0.07bC
32.40 ± 0.02abB
32.05 ± 0.03aA
EAEPO
40.46 ± 0.55bC
39.51 ± 0.03aD
39.63 ± 0.03aC
39.72 ± 0.06aE
39.74 ± 0.02aE
39.82 ± 0.04aB
HEBO
44.63 ± 0.01eD
39.96 ± 0.01cE
40.20 ± 0.01dD
38.97 ± 0.02bD
38.94 ± 0.02bD
38.68 ± 0.04aB
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.Figure 4
Typical chromatogram of fatty acid methyl ester prepared from EAEPO before heating. Peaks: 1: hexadecanoic acid; 2: octadecanoic acid; 3: 9-octadecenoic acid; 4: 9-octadecenoic acid; 5: 9,12-octadecadienoic acid; 6: 12,15-octadecadienoic acid.Figure 5
Typical chromatogram of fatty acid methyl ester prepared from EAEPO after heating. Peaks: 1: hexadecanoic acid; 2: octadecanoic acid; 3: 9-octadecenoic acid; 4: 9-octadecenoic acid; 5: 9,12-octadecadienoic acid; 6: 12,15-octadecadienoic acid.Table4 shows the variation of the content of SFA, MUFA, and PUFA of the oils from the initial to the final heating time. Heat treatment induced a decrease in the relative percentages of the unsaturated fatty acids and an increase of the relative percentages of the saturated fatty acids, as has previously been demonstrated in various studies [17]. Progressive heating may induce modification of fatty acids that contain two or three double bonds. Both oxidation and oxidative polymerization occur with progressive heating. Furthermore, the relative oxidation rate of linolenic acid is much higher than that of stearic acid [24].Table 4
Changes in saturated, monounsaturated, and polyunsaturated fatty acids.
SFA, MUFA, and PUFA
0 h
1 h
2 h
4 h
6 h
8 h
SFA
FGBO
15.28 ± 0.04 aC
26.83 ± 0.04bE
27.75 ± 0.01cE
28.94 ± 0.03dD
27.87 ± 0.04eE
31.45 ± 0.01fE
FBBO
15.51 ± 0.04aD
18.34 ± 0.02bA
19.92 ± 0.02cA
19.85 ± 0.04dA
20.22 ± 0.02eA
25.32 ± 0.01fC
GABO
17.93 ± 0.06aE
19.78 ± 0.03bB
24.23 ± 0.01cD
24.74 ± 0.02dC
24.69 ± 0.07dD
25.35 ± 0.01eD
EAEPO
11.93 ± 0.03aB
20.13 ± 0.02bD
21.11 ± 0.02cC
21.30 ± 0.02dB
21.44 ± 0.00eB
21.65 ± 0.01fA
HEBO
10.57 ± 0.02aA
20.01 ± 0.02bC
20.03 ± 0.02bB
21.36 ± 0.02cB
21.57 ± 0.02dC
22.54 ± 0.01eB
MUFA
FGBO
47.12 ± 0.02fCD
41.64 ± 0.02dC
41.11 ± 0.01cC
40.70 ± 0.02cB
41.78 ± 0.04eB
40.54 ± 0.02aC
FBBO
48.15 ± 0.04eD
47.43 ± 0.02dE
46.93 ± 0.05cE
47.65 ± 0.49cD
45.85 ± 0.02bC
41.65 ± 0.03aD
GABO
45.97 ± 0.01eBC
45.13 ± 0.03dD
41.53 ± 0.01aD
41.55 ± 0.04cC
41.86 ± 0.08cB
41.77 ± 0.01bE
EAEPO
44.48 ± 0.04cB
40.12 ± 0.02bB
39.28 ± 0.04abA
38.87 ± 0.03abA
38.53 ± 0.02abA
38.46 ± 0.02aA
HEBO
42.22 ± 0.02dA
39.98 ± 0.02cA
40.18 ± 0.04bB
39.30 ± 0.58cA
38.53 ± 0.65aA
38.74 ± 0.02abB
PUFA
FGBO
37.11 ± 0.01fC
31.46 ± 0.02eA
31.16 ± 0.02cA
30.63 ± 0.02cA
30.31 ± 0.01bA
27.96 ± 0.02aA
FBBO
36.29 ± 0.03eA
34.24 ± 0.02dB
33.12 ± 0.02bB
33.14 ± 0.03bB
33.91 ± 0.03cC
32.95 ± 0.04aB
GABO
36.44 ± 0.02fB
35.65 ± 0.01eC
34.23 ± 0.03cC
33.73 ± 0.03cC
33.41 ± 0.02bB
32.95 ± 0.01aB
EAEPO
42.13 ± 0.03dD
39.54 ± 0.02aD
39.58 ± 0.03aD
39.73 ± 0.05bE
39.74 ± 0.03bE
39.84 ± 0.03cD
HEBO
45.64 ± 0.02fE
39.97 ± 0.01dE
40.18 ± 0.04dE
38.96 ± 0.02cD
38.89 ± 0.02bD
38.74 ± 0.02aC
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.The fatty acid ratio of EAEPO and HEBO (0.27 : 1.03 : 0.96 and 0.27 : 1.08 : 1.16) approached the ideal fatty acid ratio of 0.27 : 1 : 1 (SFA : MUFA : PUFA), as shown in Table5; the other three kinds of blended oils also approached this ratio prior to heating (0.27 : 0.65 : 0.8, 0.27 : 0.6 : 0.84, and 0.27 : 0.61 : 0.79 for FGBO, FBBO, and GABO, resp.). At the end of the heating time, the ratio was 0.27 : 0.48 : 0.49 for EAEPO, which indicates that a relatively close to ideal fatty acid ratio for a healthy diet could be maintained for the EAEPO sample as heating progressed.Table 5
Variation of SFA : MUFA : PUFA ratio of blended oils.
SFA, MUFA, and PUFA
0 h
1 h
2 h
4 h
6 h
8 h
FGBO
0.27 : 0.65 : 0.8
0.27 : 0.42 : 0.32
0.27 : 0.40 : 0.30
0.27 : 0.38 : 0.29
0.27 : 0.41 : 0.29
0.27 : 0.35 : 0.24
FBBO
0.27 : 0.6 : 0.84
0.27 : 0.70 : 0.50
0.27 : 0.64 : 0.45
0.27 : 0.64 : 0.45
0.27 : 0.61 : 0.45
0.27 : 0.44 : 0.35
GABO
0.27 : 0.61 : 0.79
0.27 : 0.62 : 0.49
0.27 : 0.46 : 0.38
0.27 : 0.45 : 0.37
0.27 : 0.45 : 0.35
0.27 : 0.45 : 0.35
EAEPO
0.27 : 1.03 : 0.96
0.27 : 0.54 : 0.53
0.27 : 0.49 : 0.50
0.27 : 0.48 : 0.50
0.27 : 0.48 : 0.50
0.27 : 0.48 : 0.49
HEBO
0.27 : 1.08 : 1.16
0.27 : 0.51 : 0.54
0.27 : 0.50 : 0.54
0.27 : 0.47 : 0.49
0.27 : 0.46 : 0.49
0.27 : 0.43 : 0.46


## 3.6. Changes in Trans-Fatty Acids

Epidemiological evidence has suggested that the level of trans-fatty acid (TFA) intake is connected to the risk of cardiovascular disease [25]; thus, there have been several studies on TFAs contained in food materials. The amount of total TFAs in the evaluated oil types was in the range of 0.01-0.02 mg/mL, which clearly approximates the requirement for a “zero trans” content. However, a substantial increase in the TFAs of the various oils was observed when the oils were heated for 6 h to 8 h (Table 6). At the final heating time, EAEPO and HEBO presented the highest total TFA content. Several factors, such as the FA composition of each oil and coexisting antioxidants, may contribute to the variation in the thermal TFA accumulation profiles [26].Table 6
Changes in trans-fatty acid profile (mg/mL).
TFAs
0 h
1 h
2 h
4 h
6 h
8 h
FGBO
0.01 ± 0.01aA
4.94 ± 0.01bA
17.18 ± 0.02cA
59.68 ± 0.03dA
157.83 ± 0.08eA
280.16 ± 0.18fA
FBBO
0.01 ± 0.04aC
5.78 ± 0.03bC
21.32 ± 0.01cC
68.79 ± 0.04dC
188.48 ± 0.03eC
257.14 ± 0.08fC
GABO
0.01 ± 0.01aB
5.07 ± 0.06bB
18.54 ± 0.05cB
64.61 ± 0.08dB
162.39 ± 0.03eB
333.44 ± 0.09fB
EAEPO
0.02 ± 0.01aD
9.55 ± 0.03bD
29.88 ± 0.05cD
88.64 ± 0.02dD
232.52 ± 0.07eD
542.39 ± 0.08fD
HEBO
0.02 ± 0.01aC
12.43 ± 0.02bE
30.51 ± 0.03cE
93.09 ± 0.037dE
256.70 ± 0.06eE
558.60 ± 0.14fE
Mean values within each row followed by different letters (a, b, c, etc.) are significantly (P
<
0.05) different. Mean values within each column followed by different letters (A, B, C, etc.) are significantly (P
<
0.05) different.


## 4. Conclusions

The results of this study demonstrate that the quality of enzyme-assisted aqueous processing extracted blended oil (EAEPO) is superior to that of hexane-extracted blended oil (HEBO) from the initial to the final heating time, which is possibly due to the extraction method of the former that utilizes water as an extraction and separation medium thereby maintaining a higher antioxidant content. However, compared to the other three refined oil samples, the EAEPO and HEBO samples were less stable towards oxidative degradation. Nevertheless, EAEPO was found to have the lowest acid value and offers the additional advantage of containing an ideal fatty acid ratio after heat treatment of the oils (0.27 : 0.48 : 0.49), which has been linked to reduced risk of high cholesterol and heart disease. The advantageous characteristics may be attributed to the superior initial fatty acid composition of EAEPO.



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*Source: 530787-2014-05-28.xml*