The quality and antioxidant elucidation of germinated flaxseed treated with acidic electrolyzed water

Abstract The endogenous fortification of antioxidant lipid concomitants in flaxseed was imperative to improve the oxidative stability of α‐linolenic acid (ALA) in flaxseed and flaxseed oil upon processing, storage and gastrointestinal digestion. The comparative effects of acidic electrolyzed water (ACEW) and tap water (TW) on the triglyceride configuration, typical lipid concomitants, and antioxidant properties of flaxseed were conducted during 0–5 days of germination. The results showed that ACEW enhanced the germination rate of flaxseed by 18.25% and simultaneously suppressed the dynamic depletion of ALA by 5.32% when compared with TW (p < .05). The total phenolic acids, lignans, and flavonoids were effectively accumulated in flaxseed following ACEW‐mediated germination with the further increase by 4.82%, 15.48%, and 8.22% in comparison with those induced by TW (p < .05). The total contents of cyclolinopeptides in flaxseed progressively dropped following either ACEW or TW treatment, a slighter decrease by 5.59% for flaxseed treated by ACEW than that by TW. Notably, the maximum accumulation of tocopherols and phytosterols had been early obtained for flaxseed treated with ACEW for 2–3 days due to the de novo synthesis or intermolecular conformational transition (p < .05). Most importantly, ACEW‐mediated germination led to higher increment of the thermal oxidative stability and antioxidant properties of flaxseed and flaxseed oil in comparison to TW. In brief, the initial oxidation temperature increased by 7.09% and 3.06% (p < .05), and the antioxidant activities as evaluated by DPPH, ABTS, and FRAP values raised by 3.86%–28.07% and 4.21%–9.18% (p < .05), respectively. These findings clarify that the germination especailly mediated by ACEW could be an effective method to further optimize the nutritional and functional properties of flaxseed through reconstructing the endogenous antioxidant system.

However, the oxidation of ALA was easily initiated upon the gastrointestinal digestion, which intimately depended on the oxidative degree of flaxseed oil during the processing and storage (Nieva-Echevarria et al., 2017). Fortunately, the simultaneous intake of natural antioxidants, such as phenolic compounds, effectively suppressed the PUFA oxidation (Pilar et al., 2017). These indicated that the endogenous fortification of antioxidant lipid concomitants in flaxseed, especially their migration into oil phase, was profitable and imperative to restrain the oxidative susceptibility of ALA upon processing, storage, and gastrointestinal conditions. Indeed, flaxseed contained bioactive lignans, phenolic acids, flavonoids, tocopherols, phytosterols, cyclolinopeptides (CLs), etc. (Shim et al., 2014). The phenolic compounds and tocopherols were considered as the main components that reflected the antioxidant capacities of flaxseed and flaxseed oil .
Regrettably, the naturally occurring tocopherols in flaxseed mainly existed in form of γ-tocopherol (>90%) but not α-tocopherol, which partly limited its antioxidant potential (Li et al., 2019). Meanwhile, the phenolic acids and flavonoids in native flaxseed were only marginally migrated into the oil phase following cold-pressed extraction, which was still limitedly improved upon the thermal pretreatment of flaxseed due to the relative deficiency and natural hydrophilicity (Suri et al., 2020). The lignan secoisolariciresinol diglucoside (SDG) was considered as a key component to protect ALA-rich oil bodies in flaxseed against environmental stress during seed development and dormancy owing to its oligomeric structure (Nikiforidis, 2019;Toure & Xu, 2010). Although the contents of phytosterols in flaxseed were relatively high, the antioxidant activity of primary β-sitosterol was apparently weaker than that of minor stigmasterol (Singh, 2013).
Flaxseed contained abundant CLs, which possessed relatively weak antioxidant activities, but also contributed to the bitter off-taste of flaxseed oil (Bruhl et al., 2007;Zou et al., 2018). Therefore, a suitable pretreatment method should be explored in order to further fortify the accumulation of antioxidant lipid concomitants in flaxseed based on the de novo synthesis or intermolecular conformational transition without obviously changing the contents of bioactive ALA in flaxseed.
Previous studies had illustrated that the accumulation of lignans, phenolic acids, and flavonoids, as well as the antioxidant activities, subjected to varying degrees of promotion following flaxseed germination treated with tap water (TW) (Li et al., 2019;Wang et al., 2015).
It also had been found that the contents of tocopherols in flaxseed significantly increased after 1-2 days of germination (Li et al., 2019).
Although direct evidence has yet to be sought, Shi et al., (2010) had reported that the levels of total phytosterols in soybeans increased by onefold during 3 days of germination. Regrettably, no definite data had been available on the developmental changes of the CLs, as well as the ALA-rich triglyceride (TAG) during flaxseed germination. The electrolyzed water is produced from the electrolysis of electrolyte solution and divided into acidic and alkaline electrolytic water according to the differences in pH value and available chlorine concentration (ACC) (Liu et al., 2013). Usually, the low-concentration acidic electrolyzed water (ACEW) had been used for fresh-cut lettuce and fresh organic broccoli due to its sanitization effect (Liu et al., 2017;Zhang & Yang, 2017). Notably, the previous study had found that ACEW was more effective than TW on improving the germination of brown rice and buckwheat, which might partly be attributed to its antibacterial activity (Hao et al., 2016;Liu et al., 2013). However, it was still unknown whether the promotive germination induced by ACEW was related to the enhanced accumulation of bioactive phytochemicals in flaxseed. Based on above, the comparative analysis of TAG configuration, bioactive lipid concomitants, and antioxidant properties of flaxseed sprouts initiated by ACEW and TW had been conducted, in order to obtain the flaxseed and flaxseed oil with optimal nutritional and functional properties.

| Preparation of acidic electrolyzed water
ACEW was prepared using XYS-C-12 Electric Sterilizing Water Generator (Baoji xinyuguang electromechanical Co., Ltd., Baoji, China). Briefly, the sodium chloride solution (0.5‰, m/v) was electrolyzed for 8 min under the operating current of 6.1 A and voltage of 8 V, respectively. The pH value of ACEW was measured using a pH meter (Model PHS-3E, Shanghai, China). The ACC concentration was measured by iodometry method. The pH and ACC values of ACEW in this study were 3.50 ± 0.05 and 35.30 ± 3.25 mg/L, respectively.

| Germination of flaxseed
The germination of flaxseed (Jinya 7#) was performed according to the method reported by Wang et al., (2015) with slight modification.

| Morphology observation of flaxseed
The morphological changes of flaxseed spouts were observed during 5 days of germination, and the pictures were taken using a Canon EOS 90D digital camera.

| Extraction of phenolic compounds
Briefly, 0.3 g of freeze-dried and crushed flaxseed sprouts was extracted with 5 ml of ethanol-water (80:20, v/v) using vortex mixing for 10 min coupled with ultrasonic bath for 15 min at 25℃ and then centrifuged at 6800 g for 10 min. The resulting supernatant was combined and stored at 4℃ in the dark until used.

| Total phenolic acids and flavonoids
The contents of total phenolic acids and flavonoids in flaxseed sprouts were determined by using the Folin-Ciocalteu colorimetric method and aluminum nitrate method, respectively (Yu et al., 2020).
The results were expressed as milligram gallic acid equivalents (GAE) and rutin/100 g of flaxseed (dry basis), respectively. The free phenolic acids were further analyzed using an Agilent 1290 ultraperformance liquid chromatograph (UPLC) (Agilent, California, USA) equipped with a photodiode array(PDA) detector (Cong et al., 2020
The detector wavelength was set at 200-400 nm, and the injection volume was 2.0 μl. The SDG and p-CouAG were quantified using an external standard method (for SDG, y = 1433x+249.1; for p-CouAG, y = 15086x+16258). The FeAG content was expressed as the ferulic acid equivalent following the molecular weight conversion between ferulic acid and FeA.

| Cyclolinopeptides
The contents of CLs in flaxseed sprouts were conducted as described previously with a slight modification (Lao et al., 2014). Briefly, the extracts were subjected to HPLC system with a Phenomenex Kinetex Pheny-Hexyl analytical column (2.6 μm, 150 mm×4.6 mm) main-
The contents of phytosterols were analyzed by Agilent 6,890 GC with a flame ionization detector and capillary DB-5HT column (30.0 m × 220 μm × 0.10 μm) following derivation with 160 μl of Tri-Sil at 105℃ for 15 min and redissolved in 1.0 ml of n-hexane. The phytosterols were identified in comparison with authentic standards and quantified relative to the 5α-Cholestane. were maintained at unit resolution. Quantification was conducted using the area under the mass spectral peak for individual SRM ion channels for each TAG molecules.

| Analysis of antioxidant and oxidation stability of flaxseed and flaxseed oil
The 2

| Statistical analysis
The results were presented as mean ±standard deviation (n = 3).
Data were statistically analyzed using the SPSS statistical software (version 21.0, IBM, Chicago, IL, USA). Statistical analysis was conducted using one-way analysis of variance (ANOVAs) and Duncan's multiple-range test. A value of p <.05 was considered significant.

| Changes in morphology of flaxseed during germination
As depicted in Figure 1, the flaxseed began to germinate after 2 days of ACEW and TW treatments. The germination percentage of flaxseed treated with ACEW reached 97.45%, whicha increased by 18.25% when compared with that of TW (p <.05) following 5 days of treatment (seen in Figure S1), revealing a promotive effect on seed germination. The mean length of hypocotyls and radicles of flaxseed sprouts exposed to TW were 6.1 cm and 3.2 cm upon 5 days of germination, respectively. By contrast, the flaxseed sprouts increased by 35.56% in the length of radicles (p <.05), but not the hypocotyls when exposed to ACEW. Our findings were consistent with the previous study conducted by Liu et al., (2013), who found that ACEW F I G U R E 1 Changes in the morphology of flaxseed during germination. Abbreviations: ACEW, acidic electrolyzed water; TW, tap water was more favorable to the germination of brown rice due to its potential antibacterial activity.

| Total phenolic and flavonoids
As shown in Table 1, the content of total phenolic acids in native flaxseed reached 97.89 mg/100 g, which was consistent with the result reported by Deng et al., (2018), but lower than the data found by Wang et al., (2015) due to the different flaxseed varieties. During 5 days of germination, the contents of total phenolic acids in flaxseed significantly and linearly increased, reaching the maximum value of 279.07 mg/100 g (p <.05). Although it showed a relatively small increment, the changing trend was in agreement with the previous findings obtained by Wang et al., (2015). The contents of total flavonoids in native flaxseed were 21.67 mg/100 g and increased by 2.73-fold during 5 days of germination (p <.05), which was less than those reported by Wang et al., (2015) due to the different germinating conditions. Notably, ACEW further heightened the accumulation of total phenolic acids and flavonoids in flaxseed by 4.82% and 8.22% in comparison with TW after 5 days of germination (p <.05).
The accumulation of phenolic acids could be explained by the activation of key enzymes or cofactors responsible for seed germination . As depicted in Figure 2 As shown in Table 1 The inconsistent abundance and profiles of free phenolic acids in flaxseed were reported by Deng et al., (2018), which might be explained by the different planting area, harvest year, and seed maturity. The amounts of free phenolic acids in flaxseed increased by 2.68-fold following 5 days of germination treated with TW (p <.05).
Surprisingly, the protocatechuic acid was synthesized after 2 days of flaxseed germination with the value of 14.77 μg/g and then increased by 2.14-fold after 5 days of germination (p <.05). A further ascension of free phenolic acids by 27.83% had been displayed for flaxseed treated with ACEW when compared to those of TW (p <.05). Notably, the ferulic acid and p-coumaric acid were specifically accumulated in flaxseed with the increment of 41.35% and 33.32%, respectively (p <.05).
As previously reported, the gallic acid could be directly formed after the dehydration of shikimic acid, and the 4-hydroxybenzoic acid could be synthesized by chorismate under the catalysis of isochorismate and pyruvate lyase (Vogt, 2010). The p-coumaric acid was generated under the action of PAL and cinnamate 4-hydroxylase (C4H) and was further formed caffeic acid and p-coumaroyl-CoA, which could further converted into lignan secoisolariciresinol diglucoside, flavonoids, and ferulic acid (Vogt, 2010). Moreover, the syringic acid and vanillin content in flaxseed displayed no obvious change, which might be the biosynthesis and conversion depletion of syringic acid and vanillin were in dynamic equilibrium. The relatively lagging synthesis of protocatechuic acid might be due to the activation of 3-dehydroshikimate dehydratase via the shikimate synthesis pathway (Li et al., 1999). However, the specific enrichments of ferulic acid and p-coumaric acid in flaxseed were thanks to the further activation of the PAL and C4H enzyme activities by ACEW.

| Flaxseed lignans
As seen in Table 1, the contents of SDG, FeAG, and p-CouAG in native flaxseed were 5.25 mg/g, 55.06 μg/g, and 80.93 μg/g, respectively, which were relatively lower than that reported by Deng

| Cyclolinopeptides
The CLs are hydrophobic cyclic peptides mainly existed in cotyledons of flaxseed. As shown in Figure 3 and Table S1, eight CLs were identified in native flaxseed with 77.14 mg/100 g of total CLs, in-

| Phytosterols
Phytosterols are one of the key substances of plant cell membrane as scaffolds (Moreau et al., 2018). As shown in Figure 5, the content of total phytosterols in native flaxseed achieved 381.74 mg/100 g oil, which was in agreement with the results from Herchi et al., (2009). Similar to Deng et al., (2018), the β-sitosterol, cycloartenol, campesterol, Δ5-oatosterol, and stigmasterol were identified, accounting for 36.50%, 24.85%, 18.49%, 15.67%, and 4.49% of total phytosterols, respectively. The contents of total phytosterols increased by 13.89% after 3 days of germination (p <.05) and then declined by 14.59% when the germination time further extended to 5 days (p <.05). The above results were similar to those observed in germinated soybeans (Shi et al., 2010). In which, the contents of β-sitosterol, campesterol, F I G U R E 3 Changes in the contents of total CLs (a) and CL-B, E, C, L (b) in flaxseed during germination. Abbreviations: ACEW, acidic electrolyzed water; CL, cyclolinopeptide; TW, tap water. The means with different letters are significantly different at p <.05 level.

| Changes in lipid content, fatty acid profiles, and triglyceride configuration of flaxseed sprouts during germination
The lipids exist in form of oil bodies in oilseed, which provides energy for seed germination as a high-energy carbon reserve, and protected the lipid oxidation against various environmental stress (Nikiforidis, 2019). As shown in Table 2, the total lipid content of native flaxseed was 39.64%, which belonged to the ranges reported by Deng et al., (2018). As expected, no obvious changes in total lipid content had been observed for flaxseed treated with TW during 2 days of germination and then declined by 45.51% upon 5 days of  Herchi et al., (2015). As previously reported (Wanasundara et al., 1999), the lipase activity in flaxseed might be initially upregulated and then reached a plateau after 2 days of germination. By contrast, the total fat content of flaxseed treated with ACEW started to decrease after 3 day of germination and then dropped by 41.32% with the lowest value of 23.26% (p <.05). The ACEW might reduce the number of microorganisms, which shared the energy required for the growth of flaxseed sprouts (Liu et al., 2013). In particular, ACEW led to the preferential depletion of carbohydrates as demonstrated by the lower total sugar contents in flaxseed (Seen in Table S2). As presented in Table 2 and 0.55% for LA and SA during 5 days of germination.
The initial oxidation temperature (IOT) is an important indicator for evaluating the thermal oxidation stability of flaxseed and flaxseed oil. A superior thermal oxidation stability was accompanied by a higher IOT value. As exhibited in Figure 6(d), the IOT values of native flaxseed and flaxseed oil extracted by Soxhlet were 178.42℃ and 162.27℃, respectively, which ascended by 5.94% and 7.45% after 5 days of germination when treated with TW (p <.05).
Comparatively, higher values of IOT had been observed for flaxseed and flaxseed oil during 5 days of germination by ACEW, reaching 200.68℃ and 180.13℃, respectively (p <.05).
As depicted in Figure 7

| CON CLUS ION
In conclusion, the ACEW promotes the growth of flaxseed sprouts and simultaneously suppresses the preferential degradation of ALA that of TW. Moreover, the accumulation of bioactive lipid concomitants in flaxseed sprouts, including lignans, phenolic acids, flavonoids, tocopherols, and phytosterols, are further favorably induced by ACEW following appropriate germination in comparison with those by TW, which is parallel with the optimized thermal oxidation stability and antioxidant capacities of flaxseed and flaxseed oil. Therefore, the application of germination for flaxseed especially induced by ACEW can be favorable for further improving the nutritional and functional properties of flaxseed.

ACK N OWLED G M ENT
This work was partly supported by the National Natural Science Foundation of China (31801502, 31771938).

CO N FLI C T S O F I NTE R E S T
The authors declare no competing financial interest.