Effects of superheated steam processing on common buckwheat grains: Lipase inactivation and its association with lipidomics profile during storage

Background: Buckwheat deteriorates easily during storage, resulting loss of nutrients, rancidity flavor and poor consumer acceptability. Superheated steam (SS) was used to inactivate lipase of common buckwheat grains in this study, in order to retard lipid hydrolytic rancidity and maintain lipid nutrition of common buckwheat. Methods: Buckwheat grains were treated with SS at 110-200°C, for 0-7 min and SS treatment parameters were optimized by moisture content and lipase activity. The changes in free fatty acid (FFA) and lipase activity of SS-treated and untreated buckwheat during 12 weeks storage at 4°C, 25°C and 50°C were determined. Meanwhile, the effects of SS treatment on fatty acid compositions and lipidomics profile of buckwheat before and after storage were also evaluated. Moreover, the associations of hydrolytic rancidity with lipase activity and lipidomics profile were analyzed. Results: SS processing at 170 °C for 5 min was proved to be an effective method for buckwheat stabilization. Better stabilities based on lower FFA accumulation and lipase activity were observed in SS-treated buckwheat samples during storage. Meanwhile, SS could suppress oxidation of unsaturated fatty acids (UFA) in buckwheat, significantly retard the increase of saturated fatty acids (SFA) and the decrease of polyunsaturated fatty acids (PUFA) during storage. Moreover, the lipidomics profile results indicated that SS processing could retard the increased hydrolysis and oxidation of sulfoquinovosyl diacylglycerol (SQDG), phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysophosphatidic acid (LPA) during storage. Conclusion: Thus, SS processing could effectively inactivate lipase, suppress UFA oxidation, change glycerolipids (GLs) and glycerophospholipids (GPs) subclass metabolism, and consequently retard hydrolytic rancidity and lipid nutrition loss of buckwheat during storage. This work was first time to demonstrate the application of SS The scatter plots and linear regression of FFA value vs. lipase activity in untreated and SS-treated

protein, they play a key role in nutritional and functional significance [2]. Lipids are generally categorized into eight classes: fatty acids, glycerolipids (GLs), glycerophospholipids (GPs), sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides [3,4]. GLs and GPs as the structural elements of cell membranes play the unique role in stabilizing cell membranes and protecting membrane lipids from hydrolytic enzymes [5]. Dietary GPs have beneficial effects on a range of human diseases including coronary heart disease, cancer and inflammation [6]. Evidence suggests that a high intake of saturated fatty acids (SFA) from the diet may be associated with elevated cardiovascular disease risk, the change in SFA/unsaturated fatty acid (UFA) ratio may contribute to improve human health [7]. Common buckwheat (Fagopyrum esculentum) as one of the main cultivated buckwheat species in the world contains 8-11% phospholipids (of total lipids in buckwheat), lower SFA amounts and higher essential fatty acid (mainly linoleic acid), which may promote its positive health effects [8].
Although the lipid content in buckwheat ranges from 1.9-3.2%, it mainly contributes to easy deterioration of buckwheat during storage [8]. The content of free fatty acid (FFA) can be increased significantly and exceeded limit within short storage duration, resulting in rancidity flavor and poor consumer acceptability of buckwheat flour. UFA are easily oxidized to produce hydroperoxides and other harmful substances. It has been reported that lipid degradation and oxidation plays an important role in buckwheat quality deterioration during storage [9][10][11]. However, the detailed lipid degradation pathways mechanisms are unclear. Meanwhile, the lipidomics profile of buckwheat is unavailable, which limits the further study and applications of buckwheat.
The lipase activity is an important factor in grain quality deterioration, because it not only affects generation of FFA from lipids hydrolytic rancidity by enzymatic reaction of lipases [12], but also affects subsequent fatty acid oxidation and degradation, involved with the formation of conjugated hydroperoxy fatty acids by lipoxygenase or autoxidation, and then further broken down (nonenzymatic reaction) or oxygenation by peroxidase [13]. Suzuki et al. [9] suggested that lipase activity is relevant to lipid degradation in quality deterioration of buckwheat flour. Moreover, lipase is more thermally stable than lipoxygenase, and lipoxygenase exhibits little activity at the low moisture content of stored grain [14]. Therefore, inactivation of lipase is the common goal for most heat treatment to lengthen the shelf life of cereal grains [14]. However, to our best knowledge, the lipase inactivation methods of buckwheat and their effects on the buckwheat quality is still rarely studied.
To prolong the shelf life and maintain the high quality of cereal grains, various heat treatments designed to inactivate enzymes have been the primary means for cereals processing and storage, such as superheated steam (SS), hot air, infrared heating and microwave [15][16][17][18][19]. SS treatment has became a novel thermal stabilization technology suitable for food processing, because of its several advantages. Firstly, the higher temperature together with higher enthalpy of SS than saturated steam and hot air processing at the same pressure leads to more efficient heating [20]; secondly, high heat penetration of SS can not only increase the temperature of food rapidly, but also result in a reverse moisture transfer induced by the condensation and evaporation of moisture on the food [21,22]; thirdly, an oxygen-free environment can significantly reduce oxidative degradation reactions during SS processing [22]. Recently, SS has been found to be effective for inactivate enzymes: peroxidase and lipolytic enzymes of wheat bran at 170 °C within 7 min [18]; peroxidase of oat groats at 110 °C for 10 and 14 min [23]; lipolytic enzymes of brown rice at a temperature above 125 °C within 2 min [24]. However, there are no detailed studies about the effects of SS processing on buckwheat, especially its effects on lipolytic enzymes inactivation and lipidomics profile.
Given that lipase activity plays an important role in the quality deterioration of buckwheat, we hypothesized that SS treated buckwheat grains prior to storage would inactivate lipase and thus stabilize buckwheat during storage. The aim of this study were to (1) investigate the real effects of SS treatment on lipase inactivation in buckwheat by a quantification of lipase activity under different SStreated conditions; (2) determine the changes in hydrolytic rancidity during different storage conditions of SS-treated buckwheat compared with untreated buckwheat; (3) analyze the effect of SS treatment on the lipidomics profile of buckwheat before and after storage.

Methods Materials
Dehulled common buckwheat grains (Chiqiao 1#, main cultivar of common buckwheat in China) were obtained from the Chifeng Academy of Agriculture and Animal Husbandry Sciences in December, 2018 (Inner Mongolia, China). The initial moisture content of the buckwheat grain was 12.28%. Highperformance liquid chromatography (HPLC)-grade acetonitrile, isopropyl alcohol, and methanol were purchased from Merck (Darmstadt, Germany). Other chemicals used were either analytical or reagent grade.

Optimization of SS treatment
The moisture content of buckwheat grains was adjusted to 20.0% (w.b.) by tempering for further SS treatment according to the reported method of Head et al. [25]. Buckwheat grains with a given mass of distilled water in a sealed bag were preserved at 20-25 °C for 12 h to guarantee the total absorption of water. Buckwheat grains were treated in an SS processing system developed by the Laboratory of Cereal Science at China Agricultural University, Beijing, China, based on our previous method and schematic diagram [21]. The tempered buckwheat grains (about 300 g) were scattered on the metal mesh sample tray in a uniform thin layer (2-3 mm) and conveyed into the processing chamber. The steam velocity of SS was 15.0 m 3 /h, and the temperature was set at 110 °C, 140 °C, 170 °C and 200 °C. The processing time ranged from 1 to 7 min at 2 min intervals. The buckwheat grains were cooled to room temperature by spreading out after SS treatments, and then were used to analyze moisture and lipase activity immediately. The buckwheat grains without SS treatment were considered as the control. All experiments were conducted in triplicate.

Moisture content
Buckwheat grains were ground by a high-speed grinding mill (HY-04A, Beijing Huanyatianyuan Instrument Co., Ltd, Beijing, China) and passed through a 60 mesh sieve. The moisture content of ground buckwheat was determined using a standard method (approved method 44-19.01; AACC International, 2017). All measured response variables were reported on a dry weight basis (d.b.).

Determination of lipase activity
The lipase activity of ground buckwheat was determined according to the previous study of Jha et al. Storage studies After SS treatment at 170 °C for 5 min as a optimized results, the buckwheat grains were sealed in plastic bags and stored at 4 °C, 25 °C, 50 °C in temperature controlled incubators for 12 weeks. After 2, 4, 8 and 12 weeks, samples were withdraw from the storages, ground and sieved as described in section of moisture content. The ground buckwheat flour were re-packaged in plastic bags and stored at -20 °C until further FFA, lipase activity, fatty acid compositions and lipidomics profile analysis. The untreated buckwheat grains were also stored and analyzed as the control.
Free fatty acids value FFA values were determined according to the method of Zhao et al. [27]. The value was expressed as mg KOH which was consumed by neutralizing FFA in 100 g ground buckwheat on a dry weight basis (mg KOH/100 g d.b.). These measurements were carried out in triplicate.

Fatty acid composition
Buckwheat flour (0.5 g) were mixed with 1 mL toluene, 2 mL sulfuric acid in methanol (v/v = 1:99), then left overnight in a water bath at 50 °C. After cooling to room temperature, the mixture was further mixed with sodium chloride (5 mL, 5% w/v) and extracted with hexane twice. The hexane layer was collected and washed with 4 mL of 2% (w/v) KHCO 3 -water solution. The upper layer of hexane containing the fatty acid methyl esters was evaporated with N 2 , and dissolved in 1 mL hexane for the determination of fatty acids composition using an Agilent 7890A Gas Chromatography/Agilent 5975C Mass Spectroscopic (GC-MS) system (Agilent Technologies Inc., Beijing, China) equipped with an Agilent HP-5-MS capillary column (30 m × 0.25 mm × 0.25 µm). The oven temperature was programmed using the method of Liang et al. [28]. Fatty acids were identified by comparison of the retention times to those of a standard fatty acid methyl esters mixture, and the results were expressed as percentage of total fatty acids. These measurements were carried out in triplicate.

Lipidomics profiling
The lipid extraction was performed according to Folch and Bligh methods [29,30] with minor modification. Ground buckwheat (60 mg) and chloroform-methanol-water (v/v/v = 2:1:1, 600 µL) were sonicated for 10 min and kept in freezer (-20 °C) for 20 min, followed by frozen centrifugation at 14,000 g for 10 min. The lipid-containing chloroform layer was collected and the residue was extracted twice more. The chloroform layer was combined and evaporated with N 2 . The dried lipid residue was redissolved in 200 µL of methanol-isopropanol (v/v = 1:1) and filtered through 0.22 µm organic filter before analysis.

Results
Effect of superheated steam on moisture content  Table 1. Before storage, the predominant fatty acids in untreated buckwheat were palmitic acid (18.04 ± 0.35%), oleic acid (34.31 ± 0.43%) and linoleic acid (35.00 ± 0.58%), which together constituted 87.35% of the total fatty acids. The percentages of stearic acid, behenic acid and docosenoic acid were significantly (P < 0.05) increased after SS treatment. There was no significant difference (P > 0.05) in percentage distribution of total SFA, total monounsaturated fatty acids (MUFA), and total polyunsaturated fatty acids (PUFA) between untreated and SS-treated buckwheat.
After 12 weeks storage, the percentage of total SFA in untreated buckwheat increased significantly and that of total PUFA decreased significantly. The biggest reduction of PUFA was found in linoleic acid. However, SS treatment significantly retard the increase of SFA (mainly palmitic acid, stearic acid, arachidic acid and behenic acid) and the reduction of PUFA (mainly linoleic acid) during storage.
The fatty acid compositions of stored SS-treated buckwheat were similar to those of fresh untreated and SS-treated buckwheat. Lipidomics profile differences of buckwheat samples  Table 2. The sum of lipid molecular species of the buckwheat were: stored SS-treated buckwheat > fresh SS-treated buckwheat > stored untreated buckwheat > fresh untreated buckwheat. Before storage, the total species of GPs decreased and that of GLs increased after SS processing. Storage significantly increased the GLs and GPs species in untreated buckwheat, and SS treatment slow down this change in GPs species during storage. The lipids categories were analyzed by summing the relative abundance of individual lipids in the same class (Fig. 3).  Association of hydrolytic rancidity with lipase activity and lipidomics profile The scatter plots and linear regression of FFA value vs. lipase activity in untreated and SS-treated buckwheat are presented in Fig. 4a

Discussion
The aim of this study were to investigate the real effects of SS treatment on lipase inactivation, hydrolytic rancidity and lipidomics profile of buckwheat, and to verify the hypothesis that SS treated buckwheat grains prior to storage would inactivate lipase and thus stabilize buckwheat during storage. The study firstly optimize the best stabilization condition by SS treatment as 170 °C-5 min, based on the retaining moisture and relative high lipase inactivation in buckwheat grains, for the following storage experiments. Tempering prior to SS treatment was observed as an efficient way to eliminate enzymes in buckwheat grains, because it increased the moisture content and protected buckwheat from drying to very low final moisture content after SS processing. SS processing at 170 °C-5 min was better for retaining moisture of buckwheat grains at 12.09%. A moisture content of 16% is considered stable for avoiding buckwheat deterioration during storage [31], accordingly, buckwheat grains treated with 170 °C-5 min SS could be stored directly without drying or other additional moisture adjustment. Meanwhile, higher temperature and longer treatment of SS could improve the efficiency of lipase inactivation, and more than 50% lipase activity could be inactivated at 170 °C for 5 min. Although the efficiency of 200 °C SS was more significant than 170 °C, when the treatment time was extended to 5 min or more, the color of buckwheat grains changed greatly due to a mass loss of water within short processing time and thus partial scorch, which seriously affected the quality and acceptability. Moreover, lipase could not be completely inactivated in the present conditions, which might because the lipase in buckwheat was relatively stable compared with other grains, and the lipase activity in buckwheat was too low to reach a lower level. The lipase activity of oat (30 mg/g) was inactivated 78% by 160 °C-2 min SS treatment and inactivated completely at 170 °C-5 min [32]. The lipase activity of brown rice (5.25-12 mg/g) became constant at about 5% when the SS treatment time was extended to 2 min, regardless of its temperature [24]. The present lipase results of buckwheat were in accordance to the previous study in highland barley that the lipase content in barley was too low to inactivated totally, and 160 °C-8 min SS treatment could inactivated only 50% of lipase [33]. Whether SS could stabilize the quality of buckwheat by inactivating more than 50% lipase was determined in subsequent storage experiments.
Lipase hydrolysis results in the release of FFA and glycerol. Thus, an increase of the amount of FFA released from buckwheat grains during storage provides necessary information on potential development of hydrolytic rancidity. In this study, the lipidic acidity rates of untreated buckwheat significantly increased with an increase in storage period. The time to reach maximum level of FFA in the lipid hydrolysis process was related to enzyme activity and storage condition. Lipid oxidation and the inhibition of mould growth at extremely high storage temperature were key for the FFA inflection point of buckwheat at 50 °C storage [34,35]. Similar results were reported by Li et al. [36] study that FFA value of brown rice initially increased at the beginning of storage and reached maximum levels after 225 d of storage, then followed by a slight decline. According to the GB/T35028-2018 [37], the FFA value of buckwheat flour with fresh and good quality must be below 120 mg KOH/100 g d.b..
The FFA value in untreated buckwheat exceeded this prescribed limit seriously after stored at 50 °C for 8 weeks, accompanied with rancidity flavor. However, the SS-treated buckwheat could maintain good quality after 12 weeks of storage. SS treatment could retard FFA accumulation and inhibit lipid hydrolytic rancidity in buckwheat during storage, even SS processing was more effective than low temperature (4 °C) storage.
The inhibitory effect of SS treatment on lipid hydrolytic rancidity was also observed based on the lipase activity change during storage. The significantly lower lipase activity in SS-treated samples suggested that SS treatment could inactivate lipase and slow down the conversion rate of lipid, thus reduce the hydrolytic rancidity of lipid during storage. Meanwhile, the lower level of lipase variation in SS-treated buckwheat during storage, further indicated that inactivation of more than 50% lipase by SS could effectively stabilize the quality of buckwheat. SS could change the structures or conformations of lipase [38], so as to change the active site of the enzyme and the interaction with the substrate, and finally passivate the lipase activity and stabilize the quality of buckwheat.
The fatty acid composition was in accordance with FFA and lipase activity, supporting that SS treatment could suppress lipid hydrolysis and oxidation during storage. UFA accounted for a large proportion of fatty acids (75.28%) in buckwheat, which was in accordance with the studies of lightly milled rice [15] and wheat bran [18].
Compared with SFA, UFA are important for functional properties of buckwheat and considered more beneficial to human health. However, UFA are highly susceptible to thermo-oxidation during heating due to the presence of π bonds with high reactivity [15]. SS treatment was an oxygen-free heating medium, which protected UFA from oxidation. This is the reason why SS treatment significantly retard the increase of SFA (palmitic acid, stearic acid, arachidic acid, behenic acid) and the reduction of PUFA (linoleic acid) induced by storage. Maximum reduction in linoleic acid induced by storage indicated it might be the preferential substrate for oxidative rancidity [15]. These findings supported that SS processing was effective in suppressing lipid hydrolytic and oxidative rancidity while maintaining/improving buckwheat nutritional attributes and membrane stability during storage. Even though, SS treatment still caused slight lipid hydrolysis and changed lipid composition to some extent, according to the changes in fatty acid composition in fresh SS-treated buckwheat. It might be because heating treatment could result in disintegration of membranous structures or inactivation of heat labile antioxidants in cereals [39]. This was subsequently confirmed by the lipidomics profile results.
SS processing led to the decrease of total GPs species and the increase of total GLs species in buckwheat before storage, which was consistent with the previous results about changes in lipid molecule species by other thermal processing [40]. GPs and galactolipids are major structural constituents of cellular membranes and they play a key role in maintaining cellular homeostasis [41]. During the storage, the significant hydrolysis and oxidation of GPs and GLs occurred, based on the results of increased GLs and GPs species in stored untreated buckwheat, which caused the changes in components and structure of the phospholipid membrane, and further led to the release of TG [2]. However, SS treatment could slow down the change of GPs (mainly PE, PS and LPA) during storage, which might be because of the phospholipase inactivation. The decrease in TG, DG and MG of stored SStreated buckwheat indicated that GLs partially hydrolyzed, probably due to the destruction of phospholipid membrane by SS treatment prior to storage. The destruction of phospholipid membrane resulted in the TG leak which could contact with lipases in the aleurone and germ tissues [2]. Although SS-treated samples contained sufficient lipase substrates (e.g. TG), the degree of lipid hydrolysis and rancidity was much lower than that of untreated samples. Therefore, the enzyme activity, action sites and environment were particularly important for the stabilization of SS-treated buckwheat during storage. It was necessary to balance the inactivation of lipase and the destruction of phospholipid membrane by heat treatment to achieve better storage effect. This was also supported by our above results of FFA value and lipase activity that SS treatment altered the lipase site where the interaction with the lipase substrate were changed so that reducing lipase activity to low enough to prevent excessive accumulation of fatty acids.
Maintaining membrane integrity of cereals by reducing the saturation of fatty acids and the synthesis of galactolipids (e.g. DGDG, MGDG, SQDG) plays a key role in their growth adaptation [41,42]. Liner regression analysis of FFA value in response to lipase activity showed that SS suppressed the rate of hydrolytic rancidity as compared to untreated buckwheat. With each unit decrease of lipase activity, a lower unit of FFA response were found for these SS-treated buckwheat. Correlation analysis showed that the increase of FFA mainly comes from the hydrolysis of GL, while the increase of SQDG, PE and PIP2 could be used as a marker of lipid hydrolytic rancidity. These results suggested that the inactivation of lipase by SS could effectively reduce the lipid hydrolytic rancidity of buckwheat during storage, and the change in lipidomics profile was effective to characterize the change of buckwheat quality.

Conclusion
The present study focused on the effects of SS processing on lipase activity, lipid hydrolytic rancidity, and lipidomics profile of buckwheat. SS processing at 170 °C for 5 min was proved to be an effective method for buckwheat stabilization. Inactivation of more than 50% lipase by SS could effectively inhibit lipid hydrolytic rancidity of buckwheat during storage. SS processing considerably suppressed FFA accumulation, lipase activity variation, UFA oxidation, and GPs hydrolysis (mainly PE, PS and LPA) during storage. SS processing could improve membrane integrity and fluidity of buckwheat during storage by regulating the UFA/SFA ratio and the content of galactolipids (mainly SQDG, MGMG, MGDG, DGMG and DGDG). Moreover, the correlation analysis indicated that the lipase inactivation had an important effect on lipid metabolism. Therefore, SS processing should be a new efficient technology that could inactivate lipase of buckwheat, change its GLs and GPs subclass metabolism during storage while maintain its lipid nutrition at the same time, so as to improve the storage quality of buckwheat. In addition, it is also demonstrated that lipidomics analysis is a workable approach to monitor the dynamic changes in lipid characteristics of buckwheat during storage, which provides scientific support for buckwheat quality control.
approved the final manuscript.  Pearson correlation coefficients of hydrolytic rancidity and different lipid species (c). Data with single asterisk (*) and double asterisk (**) are statistically significant at P < 0.05 and P < 0.01, respectively. Pearson correlation coefficients of hydrolytic rancidity and different lipid species (c). Data with single asterisk (*) and double asterisk (**) are statistically significant at P < 0.05 and P < 0.01, respectively.

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