Volatile metabolite profiling of linolenic acid oxidation in the heating process

Abstract

Linolenic acid is an important fatty acid, and volatiles generated by its oxidation are the major components of food flavor. In this study, volatile components generated from linolenic acid during heating were detected and analyzed by using thermal desorption cryo-trapping system coupled with gas chromatography-mass spectrometry. A total of 52 volatile compounds were identified, including aldehydes (18), ketones (12), alcohols (6), furans (4), acids (6) and aromatic compounds (6). The forming temperature of each volatile compound was also determined. It was found that most volatile compounds with shorter carbon chains were mainly generated at lower temperatures, while volatile compounds with longer carbon chains were mainly produced at higher temperatures. Results of principal component analysis show that most of the identified volatiles were considered as the characteristic ones of the high temperature points. Potential thermal oxidation mechanism of linolenic acid was also given at the same time.

Keywords:
linolenic acid; flavor; volatile compounds; heating; GC-MS

1 Introduction

Flavor is one of the most important factors affecting the quality of food, which mainly originates from the oxidation of lipids during the thermal processing of food. Lipids are mainly composed of triacylglycerols (Rivas-Cañedo et al., 2021). It is found that the oxidation of lipids mainly occurs in the fatty acids contained in their molecules, especially the oxidation of unsaturated fatty acids is significantly higher than that of saturated fatty acids, and it is enhanced with the increase of their unsaturation (Oliveira et al., 2020). Vegetable oil is currently the main edible lipid and is the main source of fat intake for humans (Lima et al., 2021; Yuenyong et al., 2021). It contains about 50%-60% linoleic acid (C18:2n-6), 10%-40% linolenic acid (C18:3), 22%-30% oleic acid (C18:1n-9), 7%-10% palmitic acid (C16:0), 5%-9% stearidonic acid (C18:3n-3) and small amounts of n-eicosanoic acid (C20:0) (Zhang et al., 2015). Common vegetable oils such as olive oil, canola oil and soybean oil are widely used in food industry (Chen et al., 2017; Nikzad et al., 2021). During heating of these vegetable oils, the thermal oxidation products of fatty acids are considered to be the main source of flavor in foods (Eckl & Bresgen, 2017), especially the aroma of fried foods. Therefore, exploring the composition and content of volatiles produced from fatty acids is important to investigate the mechanisms of flavor production during heating.

Linolenic acid is an ω-3 fatty acid with three double bonds, and is one of the important fatty acids that constitute fats and oils in the form of glycerides (Babiszewska, 2020; Almas et al., 2022). At the same time, linolenic acid as essential fatty acids, is only through food intake for the normal growth and development of the human body (Rincón-Cervera et al., 2016). Linolenic acid can be used in the body to promote fat metabolism, composition of body cells, synthesis of prostaglandins and related to vision, brain and other aspects of behavioral development (Khan et al., 2020; Bourourou et al., 2016). In our previous study, we analyzed and identified the volatile compounds produced during the heating process of oleic and linoleic acids, and found that these two fatty acids produced a large number of volatile substances during heating, such as volatile aldehydes, alcohols, ketones, acids and furans (Bao et al., 2022). Meanwhile, it is further demonstrated that fatty acids are the main source of food flavor during thermal processing.

Generally, 18-carbon unsaturated fatty acids are the main components of fats and oils, and their oxidation process is one of the important sources of food flavor. In order to further investigate the flavor formation mechanism of lipid oxidation, this study analyzed the volatile components produced by the oxidation of linolenic acid during heating, explored the effect of temperature on the formation of volatiles, and determined the temperature at which each volatile are formed.

The results of this study will serve the food industry in the following aspects: 1. artificial regulation of flavor during thermal processing. 2. theoretical guidance for the development of optimal processes during processing. 3. control of undesirable flavor production while increasing the content of aroma substances by controlling the process technology during thermal processing.

2 Materials and methods

2.1 Chemicals

Standard chemicals: linolenic acid and C4–C20 n-alkanes (analytical grade) were purchased from Sigma-Aldrich (Shanghai, China).

2.2 Extraction and collection of volatile compounds from linolenic acid during heating by using a thermal desorption cryo-trapping system

Simulated heating was carried out in a qualified micro-chamber/thermal extractor (M-CTE, M-CTE250 Markes International, UK). M-CTE250 is a quality control tool for screening volatile and semi-volatile organic compounds in organic materials. Before using the M-CTE, it is important to clean it and ensure that it is free of any residues or contaminants to ensure the accuracy of the experiment. First, the M-CTE chamber, o-ring and the sample holders were rinsed with ethanol and cyclohexane. Then, a 20 mL volume of glassware was placed in the sample holder and 20 μL of linolenic acid was added directly into the glassware. Subsequently, the lid of the sample holder was quickly closed. Three sample racks were used simultaneously for parallel experiments (one sample per chamber) and heated at 30, 60, 90, 120, 150, 180 and 210 °C for 30 minutes. Finally, volatile compounds were extracted from the samples by opening the air valve at a flow rate of 100 mL/min for 30 minutes. These compounds were captured in 250 mg Tenax TA absorber tubes (60-80 mesh). As soon as these compounds were trapped the volatile compounds were thermally desorbed from the sorbent tubes using an automated thermal desorber (TD100-xr Markes International) at 280 °C for 5 minutes and cryogenically focused in a cold trap at 10 °C. This was followed by flash heating at 280 °C for 3 minutes to desorb from the cold trap onto a GC-MS column with a flow path temperature of 200 °C.

2.3 GC-MS analysis

Volatile compounds desorbed from the cold trap were analyzed using an Agilent 6890/5975C GC-MS (Santa Clara, CA, USA) system equipped with an HP-5 MS column (30 m × 0.25 mm ID, 0.25 μm film thickness; J&W Scientific). The carrier gas was helium in a non-split mode, delivered at a linear rate of 1 mL/min. The desorption time in the injection port was 5 minutes at a temperature of 250 °C. The temperature was programmed to be held at 35 °C for 3 minutes and increased to 280 °C at a rate of 5 °C/min. The mass selective detector was operated in electron collision ionization mode at 70 eV with a scan range of m/z 40-400. The interface temperature was 230 °C and the retention times for each volatile were converted to Kovats retention indices using n-alkanes (Sigma, Co.) as a reference. Volatile compounds were initially identified by matching mass spectra to spectra of reference compounds from the Wiley Mass Spectrometry (MS) library (version 8) and the NIST/EPA/NIH MS library (version 2014) and validated against mass spectra and Kovats retention indices obtained from the literature compared to those reported in the literature. Finally, identification accuracy was determined by separating relevant standard compounds by GC-MS analysis under the same conditions (Chai et al., 2019).

2.4 Statistical analysis

All experiments were conducted in triplicate. The measured data were analyzed using PCA by the MetaboAnalyst 3.0 (Qin et al., 2020). Significance between related samples was analyzed according to the one-way ANOVA test at the level of 0.05 (p < 0.05) using the SPSS 18.0 (Chicago, IL, USA).

3 Results and discussion

3.1 Composition of volatile compounds from linolenic acid during heating

A total of 52 volatile substances were identified during the heating of linolenic acid, including aldehydes (18), ketones (12), alcohols (6), furans (4), acids (6) and aromatic compounds (6). The volatile compounds detected were dominated by aldehydes, followed by ketones, alcohols, furans, acid esters, aromatic and furans, and the generation of aldehydes was detected throughout the heating process. The results show that aldehydes were the main oxidation products of linolenic acid during heating, and that there were up to 18 species. Most of the aldehydes start to be generated at 90 °C, with individual ones being formed at lower temperatures. Aldehydes are usually considered to be the main product of lipid oxidation (Vandemoortele et al., 2021; Zhang et al., 2022). Low concentrations of volatile aldehydes usually contribute a cereal, cream and caramel aroma, while higher concentrations have a strong solvent odor, which is often referred to as 'rancid' (Xiao et al., 2020). Throughout the heating process, 4 alkyl aldehydes (acetaldehyde, hexanal, nonanal, and decanal) from C2 to C10 were identified, as well as a large variety of alkenals and alkadienals. Acrolein is one of the more representative aldehydes detected in linolenic acid and is inherently toxic to human respiratory and physical health (Kishimoto & Kashiwagi, 2018).

During heating, 12 ketones were identified from linolenic acid. They were the second largest proportion of volatile substances. Volatile ketones are important flavor components in fried foods (Du et al., 2020). High concentrations of volatile ketones provide a strong solvent flavor, while low concentrations of ketones usually contribute a nut flavor (Jackson & Penumetcha, 2018). Ketones can be obtained directly from the oxidation of the linolenic acid carbon chain or from the oxidation of the corresponding alcohols. Current research indicates that the majority of volatile ketones are 2-ketones, such as 3-penten-2-one, 2-hexanone, 3-hexen-2-one, 6-octen-2-one, (E,E)-3,5-octadien-2-one and 6-methyl-3,5-hepten-2-one (Table 1).

Table 1
Volatile compounds from linolenic acid oxidation in the heating process.

No.	Compounds	RI	Identification method	Peak area (×106)
				30 °C	60 °C	90 °C	120 °C	150 °C	180 °C	210 °C
Aldehydes
1	Acetaldehyde	382	RI, MS, STD	nd	nd	13.2 ± 2.07b	15.41 ± 2.38a	nd	nd	nd
2	2- Acrolein	456	RI, MS	nd	79.51 ± 3.77b	34.04 ± 5.07d	34.85 ± 2.31d	59.11 ± 5.06c	104.26 ± 8.04a	52.71 ± 9.25c
3	2-Butenal	606	RI, MS, STD	39.05 ± 4.2d	148.36 ± 22.55c	nd	203.81 ± 28.45b	361.98 ± 47.44a	376.83 ± 19.06a	334.95 ± 42.39a
4	(E)-2-Butenal	629	RI, MS	nd	nd	104.74 ± 5.64a	nd	nd	nd	nd
5	2-Methyl-2-butenal	632	RI, MS, STD	nd	nd	nd	180.7 ± 26.96a	nd	nd	nd
6	(E)-2-Methyl-2-butenal	745	RI, MS	nd	nd	nd	nd	263.5 ± 50.6a	5.86 ± 5.86b	2.1 ± 0.73b
7	(E)-2-Pentenal	754	RI, MS, STD	3.43 ± 1.57e	28.26 ± 1.29d	101.41 ± 3.86c	8.14 ± 2.43e	160.8 ± 17.65b	282.39 ± 11.91a	324.25 ± 64.47a
8	2-Methyl-4-pentenal	793	RI, MS	nd	13.06 ± 0.43b	nd	99.57 ± 12.63a	nd	1.53 ± 0.17c	1.41 ± 0.46c
9	Hexanal	800	RI, MS	nd	nd	nd	nd	75.49 ± 12.72b	67.55 ± 23.94b	106.37 ± 23.08a
10	Furfural	833	RI, MS, STD	nd	nd	nd	nd	5.21 ± 2.03b	42.86 ± 11.04a	44.14 ± 3.63a
11	(E)-2-Hexenal	851	RI, MS, STD	nd	nd	nd	nd	nd	59.74 ± 5.72a	nd
12	(Z)-4-heptenal	900	RI, MS, STD	nd	nd	nd	8.92 ± 2.67b	36.75 ± 11.75b	21.28 ± 11.82c	74.34 ± 7.27a
13	(E,E)-2,4-Hexadienal	911	RI, MS, STD	nd	7.85 ± 1.79d	26.04 ± 5.23c	125.85 ± 7.55a	108.24 ± 20.1b	nd	nd
14	(Z)-2-heptenal	958	RI, MS, STD	nd	nd	nd	16.56 ± 2.14b	14.19 ± 4.18b	15.15 ± 2.54b	23.96 ± 4.25a
15	(E,E)-2,4-heptadienal	1012	RI, MS, STD	7.2 ± 1.7c	330.76 ± 34.78b	4.45 ± 0.04c	435.11 ± 60.74b	nd	1999.72 ± 152.31a	nd
16	(Z,Z)-3,6-nonadienal	1100	RI, MS, STD	nd	nd	7.34 ± 1.94d	23.68 ± 4.17c	33.08 ± 6.68b	19.7 ± 9.24c	78.84 ± 9.34a
17	Nonanal	1104	RI, MS, STD	nd	nd	nd	38.12 ± 5.81b	25.26 ± 2.37c	9.7 ± 0.65d	73.1 ± 8.31a
18	Decanal	1211	RI, MS, STD	nd	nd	nd	88.38 ± 18.33a	nd	nd	nd
Alcohols
19	Ethanol	427	RI, MS, STD	nd	nd	11.08 ± 1.33b	15.57 ± 2.45a	17.32 ± 1.34a	nd	8.45 ± 1.58c
20	1-penten-3-ol	684	RI, MS, STD	11.71 ± 3.6c	32.23 ± 2.86b	31.18 ± 3.33b	49.04 ± 6.46b	206.47 ± 42.18a	163.65 ± 76.39a	49.3 ± 18.92b
21	(E)-2-Penten-1-ol	769	RI, MS, STD	nd	nd	8.54 ± 1.72c	13.24 ± 2.38c	95.21 ± 23.6a	51.01 ± 22.98b	45.55 ± 5.11b
22	2-Methyl-1-penten-3-ol	845	RI, MS	nd	nd	26.79 ± 7.26a	nd	nd	nd	nd
23	2-Methyl-1,3-pentanediol	1005	RI, MS	nd	nd	nd	16.21 ± 3.15a	nd	nd	nd
24	2-Butyl-1-octanol	1393	RI, MS, STD	nd	8.4 ± 0.96a	nd	nd	nd	nd	nd
Ketones
25	Acetone	475	RI, MS, STD	102.67 ± 14.37a	nd	nd	nd	nd	nd	nd
26	3-Penten-2-one	657	RI, MS, STD	nd	nd	nd	26.72 ± 3.83a	nd	nd	nd
27	1-penten-3-one	681	RI, MS, STD	nd	98.37 ± 8.32bc	66.32 ± 7.68c	133.38 ± 18.38bc	382.53 ± 50.79a	80.36 ± 80.36bc	140.53 ± 15.88b
28	1-Hydroxy-2-butanone	739	RI, MS	nd	nd	8.25 ± 1.62a	nd	nd	nd	nd
29	2-Hexanone	761	RI, MS, STD	4.07 ± 0.6a	1.5 ± 0.17b	nd	nd	nd	nd	nd
30	5-Methyl-4-hexen-3-one	792	RI, MS	nd	nd	20.2 ± 0.96b	125.59 ± 12.99a	nd	nd	nd
31	3-Hexen-2-one	845	RI, MS, STD	nd	nd	3.16 ± 1.89d	21.41 ± 1.72c	79.42 ± 14.27a	43.8 ± 2.38b	30.93 ± 5.96c
32	3-Heptanone	887	RI, MS, STD	nd	nd	nd	nd	12.47 ± 3.95b	9.33 ± 4.84b	34.16 ± 6.41a
33	6-octen-2-one	985	RI, MS, STD	nd	nd	nd	35.07 ± 6.47a	24.42 ± 12ab	22.49 ± 8.06b	26.26 ± 4.65b
34	(E,E)-3,5-octadien-2-one	1073	RI, MS, STD	nd	45.09 ± 5.59b	33.78 ± 4.83b	158.51 ± 22.47a	28.96 ± 5.14bc	17.01 ± 4.51c	35.25 ± 2.27b
35	6-Methyl-3,5-hepten-2-one	1107	RI, MS	nd	nd	nd	nd	23.12 ± 10.09a	24.68 ± 15.49a	10.46 ± 2.81b
36	Dihydro-3-methylene-5-methyl-2-furanone	2048	RI, MS	nd	54.7 ± 5.3b	30.4 ± 1c	503.0 ± 35.2a	nd	nd	nd
Furans
37	2-Ethylfuran	672	RI, MS, STD	nd	nd	8.44 ± 0.07d	139 ± 11.98c	118.88 ± 3.9c	228.47 ± 3.78a	187.94 ± 29.39b
38	cis-2-(2-Pentenyl)-furan	767	RI, MS	nd	nd	nd	nd	1740.61 ± 424.37a	1135.91 ± 257.67b	1293.18 ± 149.04b
39	2-pentylfuran	977	RI, MS, STD	nd	nd	nd	17.26 ± 3.18c	32.14 ± 12.05b	38.04 ± 2.31ab	43.56 ± 7.14a
40	5-Methyl-2-ethyl-furan	990	RI, MS	nd	nd	nd	nd	9.4 ± 3.24c	28.96 ± 7.97b	40.24 ± 6.86a
Acids and esters
41	Formic acid	526	RI, MS, STD	nd	nd	nd	228.94 ± 18.58a	69.01 ± 23.41c	158.91 ± 47.01b	190.8 ± 13.58a
42	Acetic acid	610	RI, MS, STD	nd	nd	nd	547 ± 83.32a	99.9 ± 33.06d	209.31 ± 43.23c	305.34 ± 72.55b
43	Propionic acid	702	RI, MS, STD	nd	nd	4.26 ± 0.39b	9.81 ± 0.01a	nd	nd	nd
44	3-Methylbutyric acid	863	RI, MS	nd	nd	nd	nd	2.01 ± 0.67b	4.17 ± 0.57a	4.11 ± 0.69a
45	Heptanoic acid	1078	RI, MS, STD	nd	nd	nd	nd	89.55 ± 8.53a	19.92 ± 10.87b	12.58 ± 2.01b
46	Octanoic acid	1180	RI, MS, STD	nd	nd	36.06 ± 6.4a	36.72 ± 4.64a	27.61 ± 2.12a	18.92 ± 1.92b	33.32 ± 5.25a
Aromatic compounds
47	Toluene	760	RI, MS, STD	25.69 ± 1.14a	nd	nd	nd	nd	nd	nd
48	Ethylbenzene	855	RI, MS, STD	20.12 ± 0.32c	nd	nd	nd	8.68 ± 2.87d	30.27 ± 0.98b	59.44 ± 9.58a
49	p-Xylene	865	RI, MS, STD	nd	60.62 ± 3.17a	6.6 ± 0.05b	nd	nd	nd	nd
50	Propylbenzene	992	RI, MS	32.2 ± 0.11a	nd	nd	nd	nd	nd	nd
51	Benzyl alcohol	1036	RI, MS	nd	nd	nd	nd	26.35 ± 6.07a	20.54 ± 1.02b	15.41 ± 3.01c
52	3-Ethylbenzaldehyde	1168	RI, MS	nd	nd	nd	nd	68.87 ± 13.8b	27.65 ± 6.79c	344.13 ± 57.27a

• nd = Compound not detected in the sample. MS = Identification by MS spectra, RI = Kovat’s retention indexes, STD = Comparison with a standard compound; Peak area is the mean ± standard error of three replicates; “a,b, c, d” indicates a significant difference (p < 0.05).

Six alcohols from C2 to C12 were identified from linolenic acid. These alcohols include both saturated and unsaturated alcohols. Among them, ethanol, and 2-butyl-1-octanol are saturated alcohols; 1-penten-3-ol, (E)-2-penten-1-ol, 2-methyl-1,3-pentanediol and 2-methyl-1-penten-3-ol are unsaturated alcohols. The alcohols are mainly derived from the reduction of carbonyl compounds, and they usually contribute a soft or vegetal odor (Jackson & Penumetcha, 2018).

Furans usually impart a certain sweetness. During heating, 4 furans, including 2-pentylfuran, were identified from linolenic acid oxidation. This result was consistent with the results reported by Becalski and Seaman that furans are oxidation products of polyunsaturated fatty acids (Becalski et al., 2005). In addition, amino acids and α, β-unsaturated aldehydes can be catalyzed into alkyl furans by the Maillard reaction (Adams et al., 2011). During heating, six acids were identified from linolenic acid and all of them were saturated. The saturated acids usually producing sweetness and fruit aromas (Wahman et al., 2020). They are also considered to be oxidation products of the corresponding aldehydes (Wu et al., 2021). Six aromatic compounds were identified, and aromatic compounds are usually considered as solvent residues. However, the analysis of aromatic compounds represented by ethylbenzene showed that the substance appeared at 30 °C and could not be detected at 60 °C, 90 °C and 120 °C.This substance was detected again after 150 °C and showed a clear trend of increasing content with the temperature increased. This phenomenon suggested that the aromatic compounds as solvent residues were almost completely volatilized at 30 °C, while the aromatic analogues detected at 150 °C were obtained from the oxidation of linolenic acid. However, the forming mechanism of these aromatic compounds was still unknown.

3.2 Characterization of volatile compounds forming from linolenic acid oxidation during heating

The heating process of linolenic acid is a complex thermal oxidation reaction, with many reactions occurred. One of the important parameters in this complex process is the temperature, which directly affects the degree of oxidation and final product composition (Semenov et al., 2011). In this study, a simulated heating process was carried out in which seven temperature points (30 °C, 60 °C, 90 °C, 120 °C, 150 °C, 180 °C and 210 °C) were selected to investigate the effect of temperature on the volatile compounds generated from linolenic acid. Total ion current chromatogram based on GC/MS of linolenic acid at different temperature was shown in Figure 1.

Figure 1
Total ion current chromatogram based on GC/MS at different temperature during linolenic acid heating.

Figure 2 visualizes the formation temperatures of the different volatiles from linolenic acid oxidation during heating and the composition of volatiles in each temperature points. The general trends indicate that most volatiles with short carbon chains are generated at low temperatures, and the forming temperatures of volatiles in descending order are volatile ketones, alcohols, aldehydes, acids, and furans. As shown in Figure 2, 9 volatile compounds (aldehydes (3), alcohols (1), ketones (2), and aromatic compounds (3)) were generated at 30 °C, including 2-butenal, (E)-2-pentenal, (E, E)-2,4-heptadienal, 1-penten-3-ol, acetone, 2-hexanone, toluene, ethylbenzene and propyl benzene. Eight volatile compounds (aldehydes (3), alcohols (1), ketones (3), aromatic compounds (1)) were produced at 60 °C, including 2-acrolein, (E,E)-2,4-hexadienal, 2-methyl-4-pentenal, 2-butyl-1-octanol, 1-penten-3-one, (E,E)-3,5-octadien-2-one, dihydro-3-methylene-5-methyl-2-furanone and p-xylene. This phenomenon indicated that linolenic acid is very unstable and could be oxidated under lower temperature conditions during heating.

Figure 2
Formation temperatures of different volatile compounds from linolenic acid oxidation during heating.

In order to see the variation tendency of compounds more intuitively at different temperatures, a heatmap is drawn to assess the changing trend of the volatile compounds at different temperatures. The heatmap shows the amount of volatile compounds during heating by the warm and cold color, where the cold colors represent low levels and the warm colors represent high levels. As shown in Figure 3, the color of most volatile compounds gradually changes from cold to warm as the temperature increased.

Figure 3
Heatmaps of volatile compounds from linolenic acid oxidation during heating.

With the heating temperature increased, 12, 11 and 11 volatile compounds were detected at 90 °C, 120 °C and 150 °C, respectively. It is obviously that a large number of new volatile compounds were generated at 90 °C and 150 °C, accounting for nearly 65% or more of the total volatile compounds, and that the volatiles are mainly composed of volatile aldehydes and ketones, which are usually key flavor substances in foods (Wang et al., 2021; Cao et al., 2021). However, few new volatile compounds were generated at 180 °C and 210 °C, and the content of most volatiles dramatically increased at these two temperature points (Figure 3), such as 2-butenal, (E)-2-pentenal, (Z, Z)-3,6-nonadiene, 3-ethylbenzaldehyde, 3-hexen-2-one 2-ethylfuran, 2-pentylfuran and 6-octen-2-one, because the linolenic acid oxidation becomes more intense at higher temperature conditions. By comparing with our previous experimental data on heating oleic and linoleic acids, we found that the amount of volatiles generated by thermal oxidation of linolenic acid was the highest (Bao et al., 2022). Meanwhile, the number of products containing carbon-carbon unsaturated double bonds is also the highest for linolenic acid, especially for the two unsaturated degrees. Moreover, linoleic acid has the highest number of lower temperatures products, followed by linolenic acid, and oleic acid has the least. This phenomenon indicates that linoleic and linolenic acid residues are more susceptible to oxidation compared to oleic acid residues during the thermal oxidation of lipids.

In order to better understand the composition and changing characteristics of the volatile compounds of linolenic acid during heating, a principal component analysis was performed by normalizing the peak area of each compound. The scoring scatter plots and loading scatter plots are shown in Figure 4A and Figure 4B, respectively. In the Figure 4A, it can be seen that the cumulative contribution of PC1 (48.2%) and PC2 (20.6%) is 68.8%. Furthermore, the scoring points at different temperatures can be clearly separated. This result indicates that volatile components and changing characteristics of linolenic acid at each heating temperature points are different from each other. It is obviously that lower temperature scoring points (30 °C, 60 °C and 90 °C) were distributed in the 3^rd quadrant of scoring plot, while the high temperature scoring points (150 °C, 180 °C and 210 °C) were distributed in the 4^th quadrant of scoring plot, indicating that the volatiles produced at lower temperature points or higher temperature points were relatively similar. Corresponding to the loading plot (Figure 4B), it is easy to see that compounds such as p-xylene and 2-hexanone contribute more for the lower temperatures. The score points at 120 °C located in the 2^nd quadrant of scoring plot, separately. And compounds such as 2-methyl-1,3-pentanediol, decanal, 3-penten-2-one, and 5-methyl-4-hexen-3-one contribute more to this temperature point (Figure 4B). As can be seen from Figure 4A, the scoring points at 150, 180 and 210 °C are located in the positive loading region of PC1, which is significantly different from the distribution of scoring points at low temperature conditions. At the same time, the distribution of scoring points at these three temperatures is relatively concentrated, indicating that the composition of volatiles at 150 °C, 180 °C and 210 °C is very similar. As shown in Figure 4B, most of volatile compounds have a big contribution to these higher temperature points, such as (E)-2-pentenal, (E)-2-hexenal and ethylbenzene.

Figure 4
PCA plots of volatile compounds from linolenic acid oxidation during heating. (A) Scoring scatter plot; (B) loading scatter plot.

3.3 Mechanism of linolenic acid oxidation in the heating process

As shown in Figure 5, during heating, due to the electron absorption of the carbon-carbon unsaturated double bond in the linolenic acid molecule, the electron density of the hydrogen atoms on both sides of the α-carbon reduced, which makes it very reactive and susceptible to oxidation by other electron-rich groups, such as hydrogen atoms on the 8,11,14 and 17 carbon atoms (Jackson & Penumetcha, 2018). During the heating process, the hydrocarbon bonds at these carbons firstly absorb energy and break, forming the corresponding olefin radicals and hydrogen radicals. The olefin radicals and the double bonds on either side of them form a conjugated π-bond system, which results in the rearrangement of the double bonds of the carbon chain. Specifically, the carbon at position 8 forms the C8-C9-C10 π-bond system, the carbon at position 11 forms the C9-C10-C11-C12-C13 π-bond system, the carbon at position 14 forms the C12-C13-C14-C15-C16 π-bond system, and the carbon at position 17 forms the C15-C16-C17 π-bond system. The positions of the double bonds on the carbon chain after rearrangement are shown in Figure 5. The olefin radical is then attacked by oxygen to form a peroxide radical. Finally, the peroxide radical continues to attack the carbon atoms on both sides leading to the breakage of the carbon chain, and form the volatile aldehydes.

Figure 5
Potential oxidative mechanism of linolenic acid.

4 Conclusion

Thermal desorption coupled with gas chromatography-mass spectrometry is an effective method for studying the forming mechanism of volatile compounds from linolenic acid oxidation during heating. Temperature is the most important parameters in this complex thermal oxidation system and plays an essential role. It is possible for us to regulate the production of food flavor during heating by controlling the heating temperature. Meanwhile, during the thermal oxidation of lipids, linoleic and linolenic acid residues are more susceptible to oxidation than oleic acid residues.

• Practical Application: By controlling the heating temperature, the types and contents of volatile flavor substances generated by the oxidation of linolenic acid residues in lipids were regulated during heating.

• Funding

  We thank the support of National Natural Science Foundation of China (No. 31871760).

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Publication Dates

• Publication in this collection
  27 Mar 2023
• Date of issue
  2023

History

• Received
  30 Oct 2022
• Accepted
  22 Dec 2022