Two-stage selective enzymatic hydrolysis generates protein hydrolysates rich in Asn-Pro and Ala-His for enhancing taste attributes of soy sauce
Introduction
Soy sauce is a traditional liquid condiment with a high popularity among consumers especially those in Asia, owing to its unique and appetizing flavor. Soy sauce has various taste-active substances including free amino acids, small peptides, nucleotides, organic acids, sugars and salts (Kong et al., 2018). However, how these substances contribute to the complex flavor of soy sauce and the precise mechanisms underlying such actions remain unclear, even though a great deal of research has been conducted (Onuma et al., 2018, Yang et al., 2018).
The savory umami taste is one of the attributes of soy sauce wins over consumers. Therefore, efforts are made to improve the flavor of soy sauce and enhance its competitiveness, including the investigations on the effects of raw materials (Chou & Ling, 1998), microorganisms (Sugiyama, 1984), exogenous enzymes (Kijima & Suzuki, 2007), manufacturing processes (Lee, Seo, & Kim, 2006). Studies have shown that free amino acids, peptides, nucleotides (e.g. inosinic acid (IMP: I) and guanylic acid (GMP: G)), organic acids and salts in soy sauce could be the main factors affecting the umami taste of soy sauce (Keast and Breslin, 2003, Lioe et al., 2010, Zhuang et al., 2016), although there were controversies. Free umami amino acids such as Glu and Asp which were of high content in soy sauce and their synergistic effects with sodium salt were reported to affect the umami taste of soy sauce (Lioe et al., 2004). Some small molecular peptides in soy sauce were also found to play important roles in umami taste perception of soy sauce, including the umami peptides like pGlu-Gly, pGlu-Gly, pGlu-Glu, pGlu-Pro, pGlu-Ile, pGlu-Leu, pGlu-Phe and pGlu-Gln (Kaneko, Kumazawa, & Nishimura, 2011), and some are tasteless themselves but can impart enhancement of the umami taste (Lioe et al., 2010). Certain peptides and their derivatives generated during fermentation, irrespective of low contents, can elicit intense umami taste perception and fleshy and nicely balanced mouthfeel on the palate (Zhao, Schieber, & Gänzle, 2016), such as the oligopeptides smaller than 500 Da that can exert a strong umami taste or a great impact on the umami taste perception of soy sauce (Apriyantono, Setyaningsih, & Purwiyatno Hariyadi, 2004).
This study aims to identify umami-tasting and umami-enhancing substances that can elicit a pleasant umami taste in soy sauce, and establish an industrially viable approach to generate these substances through two-stage hydrolysis, coming up with a novel and efficient access to intensify flavor quality of commercial soy sauce product by adding resultant hydrolysate from non-soy sauce protein preparations. Firstly, comparison was made on the taste profiles between the original soy sauce and its reconstituted product with the same mass concentration of free amino acids to identify the possible peptide contributors to the umami taste. Secondly, synthesized peptides (Asn-Pro, Ala-His, Gly-Pro, Gly-Leu, Leu-Pro, Glu-Leu, Thr-Pro, Val-Pro, Glu-Phe and Asp-Gly-Tyr; the same peptides in the original soy sauce that exhibit an umami taste) were commercially produced by the solid-phase synthesis technique in Shanghai Jier Biochemical Co., Ltd., and then subjected to the evaluation on the taste profiles and umami-enhancing capacity in monosodium glutamate (MSG) or soy sauce through sensory evaluation and electronic tongue analysis. Finally selective enzymatic hydrolysates (SEHs) containing high level (p < 0.05) of relative contents of the objective peptides compared to the original soy sauce were prepared from the protein preparations rich in umami peptides (e.g. soy protein isolate (SPI) (Rhyu & Kim, 2011), rice protein (Yamana et al., 2020), wheat protein (Schlichtherle-Cerny & Amadò, 2002), peanut protein (Su et al., 2012), pea protein (Yamana et al., 2020)), through selective enzymatic hydrolysis with an endoprotease (neutral protease, alkaline protease, trypsin, papain, pepsin or bromelain), or an exoprotease (aminopeptidase or carboxypeptidase) for different enzymatic hydrolysis times. Then the effects of the obtained SEHs on soy sauce were evaluated.
Section snippets
Materials and chemicals
Soy sauce was provided by Zhu Jiang Qiao Biotechnology Co., Ltd (Zhongshan, China) and considered as the original soy sauce. Citric acid, sucrose, caffeine, sodium salt, monosodium glutamate (MSG) (containing 20% sodium chloride), glucose, and amino acids were of food grade and provided by Guangdong Tianqi Biotechnology Co., Ltd (Foshan, China). Soy protein isolate (SPI) (protein content: 86.69% dry weight) and peanut protein (protein content: 50.01% dry weight) were purchased from Henan
Taste profiles of the original and reconstituted soy sauce samples
As shown in Fig. 1, the scores of the five taste qualities of RS were all different from those of the original soy sauce, with their saltiness more closely resembling. Compared with the original soy sauce, RS had a substantially higher sourness, significantly higher bitterness, marginally lower saltiness, moderately lower sweetness, and substantially lower umaminess (scored 3.10 only). RS was created through mixing the same amounts of the amino acids occurring in the original soy sauce (Table 1
Conclusions
This study demonstrated the important contribution of umami-active peptides to the desired and distinct taste of soy sauce, and the feasibility to produce a protein hydrolysate rich in in umami-tasting and umami-enhancing peptides from non-soy sauce protein preparations for enhancing the desirable taste of soy sauce. For the first time, an industrially viable two-stage hydrolysis (a 12-h hydrolysis with the neutral protease at 55 °C, followed by a 12-h hydrolysis at 55 °C with the
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.
Acknowledgements
The authors gratefully acknowledge Special Support Project of Guangdong Province for Science and Technology Innovative Young Talents (Guowan Su, 2016TQ03N728), and Special Support Project of Foshan for Program for Innovative Research Team (Guangdong Tianqi Biotechnology Co., Ltd., AA17204075).
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