Changes in Soybean (Glycine max L.) Flour Fatty-Acid Content Based on Storage Temperature and Duration

Soybeans are low in saturated fat and a rich source of protein, dietary fiber, and isoflavone; however, their nutritional shelf life is yet to be established. This study evaluated the change in the stability and quality of fatty acids in raw and roasted soybean flour under different storage temperatures and durations. In both types of soybean flour, the fatty-acid content was the highest in the order of linoleic acid (18-carbon chain with two double bonds; C18:2), oleic acid (C18:1), palmitic acid (C16:0), linolenic acid (18:3), and stearic acid (C18:0), which represented 47%, 26%, 12%, 9%, and 4% of the total fatty-acid content, respectively. The major unsaturated fatty acids of raw soybean flour—oleic acid, linoleic acid, and linolenic acid—decreased by 30.0%, 94.4%, and 97.7%, and 38.0%, 94.8%, and 98.0% when stored in polyethylene and polypropylene film, respectively, after 48 weeks of storage under high-temperature conditions. These values were later increased due to hydrolysis. This study presents the changes in composition and content of two soybean flour types and the changes in quality and stability of fatty acids in response to storage temperature and duration. This study shows the influence of storage conditions and temperature on the nutritional quality which is least affected by packing material.


Introduction
Soybean plants are presumed to be native to northeastern China and were not widely distributed, except in China and Japan, until 1890, even after their introduction in Europe. Soybean can easily be mass produced, and the soybean plant is considered to be one of the principal plants that can solve world hunger [1]. Soybean contains low amounts of saturated fat and is a great source of high-quality protein, dietary fiber, and isoflavone, which makes it unique among other legumes [2].
Soybean oil and protein received great commercial interest before the recent attention on soybean protein. According to a United States Department of Agriculture (USDA) report, soybean oil is the second largest vegetable oil produced worldwide after palm oil. Most beans are very low in fat, but soybeans have an exceptionally high fat content [3]. Soybean fat has a uniquely high content of polyunsaturated fatty acids, such as linoleic (18-carbon chain with two double bonds; C18:2) and linolenic (C18:3) acids. They also contain a substantial amount of monounsaturated fatty acids, such as oleic acid (C18:1), and moderate amounts of saturated fatty acids, such as palmitic (C16:0) and stearic storage of soybeans [21,22]. Changes in physical characteristics and nutritional composition were also identified in genotypically different soybean (Glycine max (L.) Merrill) varieties [23].
Though many studies explaining the composition of soybean flour were carried out, very few studies were done to identify the exact storage conditions to enhance the shelf life of soybean flour during storage. Lipid oxidation is a major cause in affecting the nutritional quality of soybean during storage. Lipid oxidation is well known for the development of off-flavors and loss of nutrients leading to the formation of potentially toxic compounds ending in the deterioration of food. Lipids are susceptible to spontaneous oxidation (i.e., autoxidation) and oxidation in the presence of catalytic systems, such as heat, light, and enzymes, leading to complex processes of thermal oxidation, photooxidation, and enzymatic oxidation, respectively [24]. It is generally accepted that lipid autoxidation is a mechanism (a simplified schematic explanation of the mechanism of autoxidation is given in Supplementary Materials Figure S1) that leads to a series of complex chemical changes through free-radical chain mechanisms that undergo three steps: initiation, propagation, and termination.
Thus, identifying the exact shelf life of soybean flour is crucial through identifying the proper storage conditions (i.e., temperature and humidity) and maximum storage duration of soybean flour. In this study, we aimed to evaluate storage stability and changes in soybean flour quality by monitoring changes in fatty-acid content of raw and roasted soybean flour under different storage conditions (i.e., temperature and duration). The effects of packing material and storage condition on the nutritional quality of soybean flour varieties were identified. This study will help in finding suitable conditions in order to retain the nutritional value of soybean flour even after storage.

Lipid Oxidation
Lipid oxidation is a complex process in which the rate and process are affected by various factors, such as the fatty-acid profile, positional distribution of fatty acids, lipid class, minor component, and environmental factors. Lipids are not only susceptible to oxidative change during storage, but also during processing (roasting and deep frying) of the lipid-containing foods. The oxidation sensitivity of lipids depends primarily on the fatty-acid composition, specifically, their degree of unsaturation. Unsaturated fatty acids are the main reactants which undergo significant compositional changes during oxidation and are, therefore, an indirect measure for the extent of oxidation. The oxidation rates of stearic (C18:0), oleic (C18:1), linoleic (C18:2), and linolenic acids (C18:3) were reported to be in the ratio of 1:100:1200:2500 [25]. In this study, we measured the degree of oxidation in soybean flour under different storage conditions by evaluating the fatty-acid composition and content changes as indicators for storage stability.

Fatty Acid Composition and Content According to Storage Conditions
Before storage, the total fatty-acid contents of the raw soybean flour and the roasted soybean flour were 137.98 mg·g −1 and 135.56 mg·g −1 , respectively. In both types of soybean flour, the fatty-acid content was the highest in the order of linoleic (C18:2), oleic (C18:1), palmitic (C16:0), linolenic (18:3), and stearic acids (C18:0), which represented 47%, 26%, 12%, 9%, and 4% of the total fatty-acid content, respectively. Our findings are consistent with those of previous studies that showed that linoleic (C18:2), oleic (C18:1), and palmitic acids (C16:0) were the major fatty acids with relative contents of more than 10% in soybean flour. The findings of this study differed slightly from those of previous studies that analyzed the fatty-acid composition of recommended soybean varieties in Korea; however, the differences are expected to be due to the difference in variety, which showed a similar tendency [22,26]. Previous studies showed that the protein content varies slightly, while the fat content varies greatly among soybean varieties [21].
The fatty-acid composition and content changes due to storage conditions are shown in Supplementary Materials Tables S1-S12 and Figures S2-S6.  Except for roasted soybean flour stored at high temperature, the total fatty-acid content of flour stored at refrigeration and room temperature tended to decrease until 12 weeks and then increase for the remaining duration of storage ( Figure 1). This increase is due to hydrolysis reactions that take place during storage, resulting in lipolysis and the liberation of free fatty acids [27].
The total fatty-acid content of the roasted soybean flour stored at high temperature decreased gradually between eight and 24 weeks, regardless of soybean flour type and packing material (Figure 2). At 12 weeks, the total fatty-acid content was lower than that of the initial roasted soybean flour. The decrease in fatty-acid content may be partly due to the heat treatment applied during the roasting process of the soybeans. In the autoxidation process, triplet oxygen reacts after radicals are formed by releasing hydrogen from lipid molecules. Therefore, as the temperature rises, oxidation of the roasted soybean flour, which was preheated, would be promoted, as the release of hydrogen is facilitated by the easy supply of energy to break the hydrogen bond in the lipid molecules [24].
Autoxidation is dependent on the ease with which the alkyl radicals of fatty acids are produced. Linolenic acid (C18:3) has more methylene groups with two double bonds, from which hydrogen Except for roasted soybean flour stored at high temperature, the total fatty-acid content of flour stored at refrigeration and room temperature tended to decrease until 12 weeks and then increase for the remaining duration of storage ( Figure 1). This increase is due to hydrolysis reactions that take place during storage, resulting in lipolysis and the liberation of free fatty acids [27].
The total fatty-acid content of the roasted soybean flour stored at high temperature decreased gradually between eight and 24 weeks, regardless of soybean flour type and packing material (Figure 2). At 12 weeks, the total fatty-acid content was lower than that of the initial roasted soybean flour. The decrease in fatty-acid content may be partly due to the heat treatment applied during the roasting process of the soybeans. In the autoxidation process, triplet oxygen reacts after radicals are formed by releasing hydrogen from lipid molecules. Therefore, as the temperature rises, oxidation of the roasted soybean flour, which was preheated, would be promoted, as the release of hydrogen is facilitated by the easy supply of energy to break the hydrogen bond in the lipid molecules [24].
Autoxidation is dependent on the ease with which the alkyl radicals of fatty acids are produced. Linolenic acid (C18:3) has more methylene groups with two double bonds, from which hydrogen atoms can be cleaved, and a higher oxidation rate than linoleic acid (C18:2) and oleic acid (C18:1) [28]. Compared to roasted soybean flour prior to storage, the major unsaturated fatty acids of the flour, i.e., oleic (C18:1), linoleic (C18:2), and linolenic acids (18:3) decreased by 30.0%, 94.4%, and 97.7%, and 38.0%, 94.8%, and 98.0% when stored in polyethylene and polypropylene film, respectively, for 48 weeks under high temperature. The secondary products of peroxide degradation of these fatty acids-hexanal, nonadienal, and 2,4-heptadienal-are used as indicator compounds for evaluating the oxidation of foods [29]. Regardless of packing material, eicosadienoic acid (C20:2) was not found at 24 weeks in roasted soybean flour stored at high temperature.
Molecules 2018, 23, x 5 of 12 atoms can be cleaved, and a higher oxidation rate than linoleic acid (C18:2) and oleic acid (C18:1) [28]. Compared to roasted soybean flour prior to storage, the major unsaturated fatty acids of the flour, i.e., oleic (C18:1), linoleic (C18:2), and linolenic acids (18:3) decreased by 30.0%, 94.4%, and 97.7%, and 38.0%, 94.8%, and 98.0% when stored in polyethylene and polypropylene film, respectively, for 48 weeks under high temperature. The secondary products of peroxide degradation of these fatty acids-hexanal, nonadienal, and 2,4-heptadienal-are used as indicator compounds for evaluating the oxidation of foods [29]. Regardless of packing material, eicosadienoic acid (C20:2) was not found at 24 weeks in roasted soybean flour stored at high temperature. Previous studies concluded that the stability of vegetable oil depends on the type of plastic film and oxygen permeability, the level of natural antioxidants in the oil, the type and initial physicochemical properties of oil, and the storage duration and temperature. In particular, the stability was shown to differ greatly in response to different storage durations and temperatures in all types of packing materials [30]. The increase in the fatty-acid value of soybean flour of sprouted soybeans was affected by storage temperature more than humidity [31]. The relative contributions of factors studied for the retention of olive oil quality were temperature ≈ light > container headspace > packing material oxygen transmission rate [32]. Previous studies concluded that the stability of vegetable oil depends on the type of plastic film and oxygen permeability, the level of natural antioxidants in the oil, the type and initial physicochemical properties of oil, and the storage duration and temperature. In particular, the stability was shown to differ greatly in response to different storage durations and temperatures in all types of packing materials [30]. The increase in the fatty-acid value of soybean flour of sprouted soybeans was affected by storage temperature more than humidity [31]. The relative contributions of factors studied for the retention of olive oil quality were temperature ≈ light > container headspace > packing material oxygen transmission rate [32].
Tables 1 and 2 shows the effects of packing material, storage temperature, and storage duration on the fatty-acid composition and content of the raw and roasted soybean flour, respectively.    In this study, we showed that the most impactful of the three main factors of storage conditions was storage duration, followed by storage temperature and then packing material, regardless of the type of soybean flour. The fatty-acid composition and content of raw and roasted soybean flour, detected as C16:0, C16:1, C17:0, C18:0, C20:0, C20:3n6&21:0, and C22:0, showed considerable changes in both flour types. The fatty acids tend to change more when stored until 48 weeks than at initial storage. More changes were observed in storage duration, followed by storage temperature and packing material, as the latter two variables showed a lesser effect on fatty acids compared to storage duration. Differences were also observed in the fatty-acid composition and content sorted at different temperature based on individual components. Thus, variations between C16:0, C16:1, C17:0, C18:0, C20:0, C20:3n6&21:0, and C22:0 composition and content stored in polyethylene and polypropylene showed the effect of packing materials on fatty acids.
The findings represent the influence of storage duration and temperature on fatty acids. Drastic variations were observed between the initial and 48-week storage of C16:0, C17:0, C18:1c&t, and C18:2c&t in raw soybean flour, whereas, in roasted soybean flour, C14:0, C16:0, C17:0, C18:1c&t, C18:2c&t, and C18:3n3 were affected. Other fatty-acid content also produced changes during storage, albeit by a lesser amount. These results showcase the impact created on each fatty-acid content of raw and roasted soybean flour which, in turn, emphasizes the importance of storage conditions. Thus, controlled storage conditions are essential for the preservation of soybean quality [33]. By controlling the soybean flour type and storage temperature conditions, and by analyzing changes in fatty-acid composition and content during long-term storage, the findings of this study further the understanding of how fatty acids can be used as indicators of quality change and storage stability in soybean flour. Furthermore, the findings of the present study provide basic data that can be used to establish the shelf life and suitable storage conditions for raw and roasted soybean flour.

Soybean Material
Raw and oven-roasted (110-130 • C for 30 min) soybean flour (Glycine max L.) was provided by the Rural Development Administration. The soybean cultivar used was "SaeDanbaek", which has a high protein content and was developed in 2010 to improve its suitability for processing tofu.

Storage Condition
Raw and roasted soybean flour samples were subdivided into polypropylene film (PE; 10 × 15 cm, width × length; thickness 0.05 mm) and polyethylene film (PP; 15 × 22 cm, width × length; thickness 0.04 mm) packets and immediately sealed with vinyl adhesive. The subdivided soybean flour was stored in the dark at three different temperatures: refrigeration (4 • C), room temperature (20 • C), and high temperature (45 • C, as the accelerated condition). Three packets containing each type of soybean flour, stored under each condition, were opened and used for fatty-acid analysis after 1,2,4,8,12,24,36, and 48 weeks of storage [34].

Chemicals
All solvents for fatty-acid extraction were high-performance liquid chromatography grade.

Sample Preparation for Fatty-Acid Measurement
Transesterification was conducted to convert the fatty acids in the soybean flour to FAMEs prior to gas chromatography/flame ionization detector (GC-FID) analysis. Soybean flour samples (50 mg) were placed in amber vials and combined with pentadecanoic acid (0.2 mg) as the internal standard. For lipid extraction and transesterification, 400 µL of n-heptane and 640 µL of methylation solvents (MeOH, benzene, DMP, and H 2 SO 4 at 39:20:5:2, v/v/v/v) were added to the sample vials, which were capped with teflon-lined caps. The vials were shaken gently for 2 h at 60 rpm in a water bath maintained at 80 • C. The vials were then cooled to room temperature (20 • C) and centrifuged at 4000 rpm, for 1 min. The supernatant (containing FAMEs) was separated and transferred to a new container insert in the amber vial [35,36].

Analysis by GC-FID
The fatty-acid composition was determined using a GC-FID (Agilent 7890B, Agilent Co. Ltd., Santa Clara, CA, USA) system. A capillary column (HP-INNOWAX 19091N, 30 m × 0.25 mm, 0.25 µm, Agilent Co. Ltd., Santa Clara, CA, USA) was used to separate the 37 FAMEs. The injection volume was 1 µL with 1:50 split mode. The carrier gas was helium set at a flow rate of 10 mL·min −1 . The flame gas consisted of H 2 at 35 mL·min −1 and mixed gas at 300 mL·min −1 . The initial oven temperature was 100 • C for 2 min, later increased at the rate of 5 • C·min −1 from 150 • C to 240 • C, and then held isothermally. The inlet temperature was 230 • C and the FID temperature was 250 • C. The acquisition was done from 1 to 55 min for a total analysis time of 65 min [34].

Quantification of Fatty Acids
The stock solution was produced by dissolving 1 mL of the 37-component FAME standard mixture in 9 mL of dichloromethane (CH 2 Cl 2 ). Individual fatty acids in the sample were identified by comparing their retention times with those of the 37-component FAME standard mixture. The fatty-acid content of each sample was calculated using the analytical method specified in the Food Code issued by the Korean Food Drug Administration, as follows: Fatty acid (mg.g −1 ) = ((P ti × F i × F IS × Wt IS )/ Pt IS × R i × W spl ) × 100 (1) R i = Ps i /Ps IS × W IS /W i (2) where Pt i is the peak area of fatty acid i, Pt IS is the peak area of the internal standard, F i is the conversion factor of fatty acid i, F IS is the conversion factor of the internal standard, Wt IS is the amount of internal standard (mg), W spl is the amount of sample (g), R i is the response factor of fatty acid i, Ps i is the peak area of fatty acid i in the standard, Ps IS is the peak area of the internal standard in the standard, W IS is the amount of internal standard in the standard (mg), and W i is the amount of fatty acid i in the standard (mg).

Statistical Analyses
Statistical analyses were performed using the general linear model of the statistical analysis program SAS (version 9.4; SAS Institute Inc., Cary, NC, USA). The experimental design and collection of samples were completely randomized, and samples were analyzed in triplicate. The significant differences of the sample means were determined using a least significant difference test at the 0.05 probability level.

Conclusions
This study determined the changes in fatty-acid content in raw and roasted soybean flour stored in different packaging materials at different temperatures for 48 weeks. Packaging material was found to have the least influence, and storage duration and temperature were found to have the greatest influence on the composition and content of fatty acids in both raw and roasted soybean flour. The present study shows the importance of selecting suitable storage temperature and duration to retain the nutritional value of soybean flour for human consumption.
Supplementary Materials: Steps of lipid autoxidation; Chromatograms of the 37 FAMEs; Chromatograms of the raw and roasted soybean flour prior to storage; and Chromatograms and tables of raw and roasted soybean flour packed in polyethylene and polypropylene film stored at refrigeration (4 • C), room temperature (20 • C), and high temperature (45 • C, as the accelerated condition) are available online.