Elsevier

Food Chemistry

Volume 315, 15 June 2020, 126237
Food Chemistry

Effect of soaking conditions on the formation of lipid derived free radicals in soymilk

https://doi.org/10.1016/j.foodchem.2020.126237Get rights and content

Highlights

  • Soybean soaked at lower temperature retain more antioxidants.

  • Soybean soaked at 50 °C lead to a higher amount of free radicals in the soymilk.

  • Soybean exudates were positively related to lipid derived free radicals.

  • The free radicals in soymilks will have profound effects on flavors of soymilks.

Abstract

Lipid derived free radical in soymilks were studied by combining 5,5-dimethyl-pyrroline-l-oxide (DMPO) spin trap, chloroform-methanol extraction and electron spin resonance (ESR) spectroscopy. Five lipid derived free radical adducts: DMPO-Xradical dot, DMPO-Lradical dot, DMPO-Rradical dot, DMPO-LOOradical dot, and DMPO-ROradical dot were presented in soymilks. The total amounts of spins increased as the soaking temperature increased from 4 °C to 50 °C and the soaking pH increased from 3 to 9 and in paralleled with the diffusion of soybean exudates to soaking water. Prolonged soaking of soybean at 50 °C resulted in a higher signal intensity of DMPO-Rradical dot than that of DMPO-LOOradical dot. Soybean lipoxygenases (LOXs) were responsible for the formation of lipid derived free radicals in soymilks. Soybean exudates affected the total amounts of lipid radicals in linoleic acid (LA) – LOX model system. The relative signal intensities of DMPO-Rradical dot and DMPO-LOOradical dot were depended on the contents of soybean exudates in the system.

Introduction

Soymilk itself is a vegetable protein beverage made from soybeans and is getting accepted worldwide due to its health benefits in preventing heart disease, cancers, osteoporosis, syndromes, and aging (Ramdath et al., 2017, Singh et al., 2007). At the same time, soymilk is the starting materials of many other soy foods, such as tofu, sofu, yuba and soy yogurt. In most cases, soaking is the first step of soymilk making. After draining off soaking water, the soaked soybean is ground with added water followed by removing solid residues (okara) in a separation procedure.

A radical is an atom or group of atoms with one or more unpaired electrons. In chemical terms, free radicals are molecules with an uneven number of electrons (Koppenol, 2000). More precisely, free radicals are molecules that contain one or more unpaired electrons and thus are paramagnetic (Ganten & Ruckpaul, 2006). Lipid peroxidation is such a process which produces many different lipid-derived radicals (Andersen and Skibsted, 2002, Chamulitrat and Mason, 1989). Most free radicals are very reactive with other components. In food systems, free radicals will attack other food components, such as proteins (Huang et al., 2006, Wang et al., 2014), vitamins (Tsai, Chang, & Chang, 2007), tryptophan (Ganhao et al., 2010, Yi et al., 2015) and phenolic compounds (Çelik, Gökmen, & Skibsted, 2015), causing a series of unfavorable effects. The free radicals produced from lipid peroxidation would destroy proteins, leading to changing the functional properties. Huang reported that free radical transfer from oxidizing linoleic acid to soybean proteins resulted in the formation of large amounts of protein radicals which further led to the formation of protein aggregates (Huang et al., 2006).

ESR spin trapping has been used to detect a wide variety of free radicals. A spin trap is a diamagnetic compound that reacts with a radical to give a more stable ESR observable spin adduct. 5,5-Dimethyl-pyrroline-1-oxide (DMPO) is a commonly used spin trap, which has provided information on carbon-centered, oxygen-centered, nitrogen-centered, sulfur-centered, and a variety of other radicals. However, DMPO spin adducts can also react with other radicals to form non-radical products. By solvent extraction, the lifetimes of DMPO lipid-derived radical adducts can be increased from about 20 min to more than 10 h (Qian, Wang, Schafer, & Buettner, 2000).

Several lipid derived radicals, such as alkyl radicals (Lradical dot), epoxyallylic radicals (OLradical dot), dihydroxyallylic radicals (radical dotL[OH]2), and a variety of Rradical dot or radical dotRCOOH were identified in the soybean LOX–polyunsaturated fatty acid (PUFA) system (Qian et al., 2003, Zhuang et al., 1991). When PUFA was mixed with LOX in air-saturated borate buffer (pH 9), lipid peroxyl radicals without extraction were readily detected with a g-value of 2.014, whereby the activated enzyme (E-Fe3+) was reduced to the ferrous state with the formation the PUFA peroxyl radicals (Chamulitrat and Mason, 1989, Groot, 1975, Mozuraityt et al., 2008). While alkoxyl radicals can be produced during the oxidation of ferrous enzyme by hydroperoxides. Chamulitrat et al. identified the DMPO adducts of peroxyl, acyl, carbon-centered, and hydroxyl radicals in incubations (pH 7.8) containing linoleic acid and lipoxygenase (type V) through means of a rapid sampling device combined with EPR, while only the signals of carbon-centered radical adducts could be detected when the experiment were carried out under anaerobic conditions (Chamulitrat, Hughes, Eling, & Mason, 1991).

Soybean contains various biologically active phytochemicals that provide health benefits including protection against oxidative stress (Balwinder, 2017, Yasmin et al., 2008). Significant higher free radical-scavenging activity in the black soybean could be contributed by the higher anthocyanins content in the soybean hull was reported (Zhang, 2011). Soaking of soybean is a process of water imbibing by soybean seeds as well as the diffusion of soluble solids from seeds to water. It has been reported that the losses of nutritional compounds of white beans during soaking accelerated at high soaking temperature (over 50 °C) and obtained maximum at 90 °C (Samuel, 1979). Hydrolysis of isoflavone glucosides to isoflavone aglycones during soybean soaking was also reported (Egounlety & Aworh, 2003). Our previous studies showed that volatiles components in soymilk were affected by soaking conditions. The objectives of this study were to carry out a comprehensive examination on the effect of soaking conditions on the lipid derived free radicals in soymilks, including soaking time, temperature, and pH, and also to find the relationship between soybean exudates and the formation of lipid radicals. The present study is aimed to get a better understanding of the mechanism of the off-flavor formation in soymilk as affected by the soaking of soybean. In addition, the study will also provide useful information for the quality control in the processing plant of soymilk and soy foods.

Section snippets

Materials

Soybeans (Glycine max) with different origins were used in this study. Two varieties of soybeans were used: ordinary soybean Jilin 20 and LOX-deficient 05J-27 (harvested in 2018) were provided by the Northeast Agricultural University (Harbin, China). The soybeans were stored in a cool and dry air-conditioned room until used. The spin trap DMPO, was purchased from J&K Chemical technology (Shanghai, China) and further purified with activated carbon/benzene and stored at −20 °C before use. Soybean

Proximate analysis and fatty acid of soybeans

The chemical components of ordinary soybeans and LOX-deficient soybeans are given in Table 2A. The two soybeans showed significant difference (p < 0.05) in contents of protein, fat and ash. The fat and protein contents in LOX-deficient soybeans were 2.5% and 1.27% higher than those in ordinary soybeans, respectively.

There were remarkable differences in the species and contents of fatty acids (FA) in the two soybean varieties. As shown in Table 2B, the total contents of FA in ordinary and

Conclusion

In summary, five free radical adducts: DMPO-Xradical dot, DMPO-Lradical dot, DMPO-Rradical dot, DMPO-LOOradical dot, DMPO-ROradical dot were identified in raw soymilks prepared from ordinary soybeans while no free radical signals were observed in soymilk from LOX-deficient soybeans. The result indicated the correlation between LOX and lipid free radicals. The total amounts of spins of free radicals increased as the soaking temperature increased from 4 °C to 50 °C and the soaking pH increased from 3 to 9, and is positively correlated with water

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 31801590), the Nature Science Foundation of Jiangsu Province (Grants No. BK20180609).

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