Profiling of VOCs emitted from soybean seeds during storage
GC-MS/MS profiling of VOCs in soybean seeds which was stored in 25±2°C, 40% relative humidity throughout the storage period results in eight groups. 68 volatile compounds encompass 19 aldehydes, 15 acids, 4 alkanes, 10 alcohols, 8 ketones, 6 esters, 4 alkenes and 2 amides were found. Ethanol, 1-hexadecanol and phenol were found to be the most prevalent alcohol and over the course of storage, their proportions gradually expanded. After four months, the starting strength of ethanol (1.02%) increased dramatically to 12.88%, whereas 1-hexadecanol peaked at 0.86% after six months and then began to decline. After four months, phenol showed a rise to 0.70% and then a significant decline (Fig 1a). Seeds release a wide range of volatile compounds during storage and some of these compounds may affect or result from the aging process
(Lee et al., 2015). As seeds get older, the amount of these compounds released increases with more types and higher amounts being emitted when stored at 25°C compared to 10°C
(Zhang et al., 1993). The volatiles ethanol, 1-hexadecanol, phenol and other alcoholic compounds emission might be due to anaerobic metabolism, lipid peroxidation and glycolysis pathway. Ethanol is released during storage in canola seeds due to anaerobic metabolism
(Buckley and Buckley, 2009). Glycolytic processes caused dry seeds of lettuce, carrot and soybean to release ethanol and other alcoholic compounds when they were stored
(Zhang et al., 1993). During storage, dry sunflower seeds released 1-hexanol and ethanol due to glycolytic processes and lipid peroxidation (
Meenakshi, 2020). These chemicals are also released when the integrity of the mitochondrial membrane is denatured
(Colville et al., 2012).
During the initial five months of storage, acetaldehyde and hexanal were continually released. Acetaldehyde exhibited the highest area percentage among the aldehydes, with its contribution increasing substantially from 0.92% initially to 10.77% before declining at the fourth month. Hexanal followed a similar trend, increasing from 0.61% to 8.57% before decreasing. The most prevalent aldehydes were acetaldehyde and hexanal followed by cyclohexane carboxaldehyde, hexadecenal and tetradecanal (Fig 1b). Lipid peroxidation is responsible for emission of majority of aldehydes
(Grotto et al., 2009). The oxidation of oleic acid, linoleic acid and linolenic acid results in the origination of aliphatic aldehydes
(Solina et al., 2007). Acetaldehyde is released from stored seeds due to the breakdown of linoleic poly unsaturated fatty acids (PUFA) caused by enzymatic oxidation or autooxidation and mitochondrial degradation
(Colville et al., 2012). Acetaldehyde emission in dry seeds may be caused by lipid membrane oxidation during storage
(Zhang et al., 1993). Degradation of linoleic acid causes emission of hexanal, 2, 4-nonadienaland nonanalin stored seeds
(Colville et al., 2012). Hexanal is associated with unsaturated fatty acid oxidation resulting from autoxidation, photo-oxidation, thermal oxidation or isozyme-assisted oxidation (
Gardner, 1996). The oxidative deamination-decarboxylation of amino acids such as leucine, valine and isoleucine
via Strecker degradation is thought to be the primary mechanism for the generation of branched aldehyde (
Ardo, 2006). Lipid peroxidation, which occurs through the action of the enzyme lipoxygenase plays a key role in stored seeds. This process is a major contributor to the release of volatile aldehydes
(Frankel et al., 1981). During seed deterioration, hexanal builds up which serves as a marker for lipid peroxidation
(Frankel, 1983).
The volatile 1,3-Benzenedicarboxylic acid and 9,12-octadecadienoic acid were continuously released over the storage period. The two acids that were most prevalent and their concentrations increases with time were acetic acid and hexanoic acid. In the beginning, acetic acid recorded 0.54% in the second month and hexanoic acid 1.48% in the fifth. But as storage time increases, their proportions exceed noticeably, peaking at 4.12% and 5.23% in the seventh and eighth months, respectively (Fig 1c). The release of esters showed a distinct pattern over time in stored soybean seeds. Bis (2-ethylhexyl) phthalate and palmitic acid vinyl ester were the most abundant, starting at 0.12% and 0.24% at 1st month and 3
th months, respectively, peaking at 4.10% and 2.09% by the 8th month after storage (Fig 1d).
Among alkanes, pentaoxacyclopentadecane were the most abundant, starting at 0.0% at initial month peaking at 2.87% by the 5th month after storage and then gradually decreased (Fig 2a). Octylfuran and furan were the predominant volatiles among alkenes, recording concentrations of 0.72% and 0.44% at the 5
th month, respectively and continued to increase to 1.47% and 1.26% by the 8th month (Fig 2b). The dehydration of carbohydrates through the maillard reaction leads to the formation of furan in seeds
(Monforte et al., 2015). This compound has been utilized as a marker for distinguishing aging in long-duration rice, aiding in its identification and classification
(Wang et al., 2020).
2-pentadecanone and ethanone dominated, with concentrations of 0.66% and 0.14% at the 6th month, respectively, gradually increasing to 1.21% and 1.33% by the 8th month in ketones. Meanwhile, 2-dodecanone exhibited the lowest volatile strength, recording 0.22% (Fig 2c). Only amides, such as decanamide and benzamide were noted in the fifth (0.33%) and sixth month (0.28%), respectively and lasted until the eighth month, at 0.79% and 0.84%. which was dominated by dodecanamide (Fig 2d).
Total VOCs strength emitted from soybean seeds during storage were 15.07% alcohol, 37.84% aldehyde, 26.03% acid, 8.14% ester, 5.84% alkane, 3.32% alkene, 2.52% ketone and 1.23% amide (Fig 3). The Strecker degradation of Maillard reaction, non-enzymatic degradation of macromolecules, glycolysis and lipid bi-layer cell membrane oxidation produce acid, alkene, alkane, ketones, esters and ethers
(Mira et al., 2016). Linoleic auto-oxidation in seeds results in the emission of esters (methyl formate,
etc.), alcohols (butanol, propanol, pentanol, etc.), ketones (2-heptanone,
etc.), aldehydes (propanal, pentanal, hexanal, butanal,
etc.) and three to six carbon alkanes (propane, pentane, butane,
etc.)
(Knutson et al., 2000). Lipid peroxidation in stored pea seeds produces alcohols, ketones, esters and alkanes (
Bhattacharjee, 2019). Volatile aldehydes emission from heated soybean might be due to the thermal breakdown of lipid hydroperoxides (
Hailstone and Smith, 1989). Weathering-related deterioration of soybean seeds also results in the emission of volatile aldehydes (
Tyagi, 1992).
Understanding the relationship: VOC emission and physiological seed quality
The current study found a clear association between the amounts of volatile organic compounds (VOC) emitted and seed germination. Germination started at 76% but dropped to 59% after the eighth month of storage. During the first three months, there was small decline in germination, which coincided with a VOC emission strength of 29.12%. However, there was a substantial decrease in germination during the fourth month, when VOC emission strength exceeded by 47.60%. The sharp decline in germination during the fourth month coincided with a peak in VOC emissions, particularly from aldehydes. By the end of the 8
th month, germination had reached 59%, resulting in a VOC emission strength of 60.36%. Root length reduced from 17.6 cm to 12.5 cm after eight months of storage, while shoot length decreased from 15.7 cm to 11.8 cm with in the same time period (Table 1).
Seedling dry weight decreased from 1.189 to 0.755 g per 10 seedlings and the vigour index considerably reduced from 2531 to 1434 during the first and eighth month of storage (Table 1). In the first month of storage, total volatiles accounted for only 2.43%, but over the course of eight months, this figure surged to 60.36%. This increase coincided with a significant increase in volatile groupings’ individual strengths. In the initial month, alcohol, aldehyde and acids had values of 0.42%, 1.68% and 0.33%, respectively, but by the eighth month, those numbers had risen to 14.35%, 10.52% and 13.96%, respectively. Esters, alkanes, alkenes, ketones and amide grew from 0.0% in the initial month to 9.49%, 2.68%, 3.51%, 4.22% and 1.63% by 8
th month respectively (Fig 4). This substantial rise demonstrates the dynamic nature of volatile compound generated from soybean seeds over the storage period.
Volatile compounds produced through fermentation and lipid oxidation of the lipid bilayer membrane are likely responsible for the decline in physiological parameters, such as reduced mitochondrial activity, which leads to decreased seed germination and seedling vigour. Studies have reported a negative correlation between seed quality and volatile emissions during storage
(Mira et al., 2010; Colville et al., 2012). Aldehydes, alcohols and ketones have been shown to adversely impact the germination and vigour of sunflower seeds
(Balesevic et al., 2005). Similarly, volatile aldehydes released from stored dry seeds have been associated with reduced germination and vigour in pea and soybean seeds
(Harman et al., 1982). A decline in the viability of
Pyrus communis and
Sorbus aucuparia seeds has been linked to fermentation-related volatiles such as ethanol, acetaldehyde, methyl acetate, acetic acid and ethyl acetate
(Michalak et al., 2021). Additionally, soybean seed germination has been negatively affected by ethanol and acetaldehyde emissions during storage
(Zhang et al., 1994). Studies indicate that the seed quality of a variety of crop species is decreased by volatile organic compounds (VOCs) especially ethanol and methanol
(Rutzke et al., 2008). Seed viability is greatly reduced by volatile aldehydes particularly malondialdehyde generated as a result of lipid peroxidation as observed in
Ammopiptanthus mongolica and hazelnuts
(Pastorelli et al., 2006; Yi et al., 2010).
Understanding the relationship: VOC emission and biochemical seed quality
The increased VOCs emissions positively correlated with increased electrical conductivity of seed leachate, lipoxygenase activity and lipid peroxidation and negatively correlated with catalase, peroxidase and dehydrogenase enzyme activity (Table 2).The biochemical parameters of seeds were closely associated with elevated levels of VOCs, likely due to the damaging effects of free radicals and the catabolic processes affecting the cell membrane. Volatiles emitted from stored seeds have been shown to reduce biochemical quality attributes in cabbage
Bicanic et al., (2003) and pine
Tammela et al., (2003). Volatile compounds such as aldehydes, alkanes, carboxylic acids, ketones and other polymerization products can readily diffuse through and penetrate biological membranes, affecting both cellular and extracellular matrix components which leads to a decline in the biochemical quality of seeds (
Bhattacharjee, 2019).
