Elsevier

LWT

Volume 122, March 2020, 109003
LWT

Effects of pressure-assisted enzymatic hydrolysis on functional and bioactive properties of tilapia (Oreochromis niloticus) by-product protein hydrolysates

https://doi.org/10.1016/j.lwt.2019.109003Get rights and content

Highlights

  • Pressure accelerated the protein hydrolysis and facilitated the release of free amino acids.

  • The solubility and antioxidant activity of fish protein hydrolysates were enhanced.

  • Water and oil holding capacities of fish protein hydrolysates were decreased.

  • Emulsifying properties varied with applied pressure and holding time.

Abstract

Fish by-product protein can be converted into valuable food and nutraceutical ingredients via proteolysis. The existing process suffers from many limitations such as extended reaction time and nonselective hydrolysis. In this study, protein from tilapia fish by-products was transformed into functional peptides using pressure-assisted enzymatic hydrolysis. Proteins were extracted from the tilapia by-products by isoelectric solubilization and precipitation method. The effects of pressure (38–462 MPa) and hydrolysis time (6–35 min) on the properties of the hydrolysates were investigated using a central composite design. Pressure enhanced protein hydrolysis with a maximum trichloroacetic acid-solubility index (TCA-SI) of 23% obtained at 250 MPa for 35 min. Pressure and time were also vital in improving soluble protein content (5.7 mg/mL), reducing power (44 μg AAE/g), and solubility (71%) of the hydrolyzed products. Improved antioxidant activity, indicated by a significant decrease in IC50 values from 653 μg/mL to 304 μg/mL, was recorded. The combined process facilitated the release of low-molecular-weight peptides and essential amino acids. However, water and oil holding capacities were found to be decreased. Pressure-assisted enzymatic hydrolysis could provide an effective approach for recovering bioactive peptides from fish by-products for industrial applications.

Introduction

Tilapia (Oreochromis niloticus) is a major aquaculture species in many countries. The annual tilapia production is about 4.5 million tons (Tveterås, 2014). Fishery industries generally produce frozen fillets as the main commodity whereas by-products such as head, skin, bones, and viscera, accounting for 50–70% of the live fish weight, are considered as waste (FAO, 2016). Around 30% of these are utilized as fertilizer, silage production, and animal feed (Hsu, 2010) while a minor portion is employed as intermediate ingredients in food, nutraceutical, and pharmaceutical sectors (Klompong, Benjakul, Kantachote, & Shahidi, 2007). Larger quantities are rejected and dumped as waste in the absence of effective management systems. Fish by-products can be sources of valuable constituents such as proteins, phospholipids, vitamins, polyunsaturated fatty acids, and bioactive compounds (Shirahigue et al., 2016). Fish protein hydrolysates have been successfully incorporated into foods, such as meat products, cookies, and cereals (Chalamaiah, Rao, Rao, & Jyothirmayi, 2010). Kristinsson and Rasco (2000) reported the applications of FPH as milk replacers in infant formulas. Dekkers, Raghavan, Kristinson, & Marshall (2011) described enzymatic conversion of fish by-products into fish silage and fish sauce. Effective use of these by-products, therefore, will help mitigate environmental pollution and generate additional incomes for fish processors (Chi et al., 2014; FAO, IFAD, & WFP, 2015).

Recently, biologically active peptides derived from fish protein have attracted increasing attention. These peptides are inactive within the sequences of the parent proteins. When released by enzymatic hydrolysis, they exert various bioactivities such as inhibition of the angiotensin-I-converting enzyme (ACE), antioxidant, anti-proliferative, anti-inflammatory, cytomodulatory, antimicrobial, immune-modulatory (Halim, Yusof, & Sarbon, 2016). In addition, the cleavage of protein molecules can modify their functional properties such as emulsification, and gel formation abilities (Queirós, Saraiva, & da Silva, 2018). The existing methods for enzymatic hydrolysis of fish protein suffers from many limitations such as extended reaction time and nonselective hydrolysis. Therefore, innovative hydrolysis technology to convert fish by-products into functional peptides is highly desired. Global market share of peptides is very significant, of about USD 14.4 billion per annum (Uhlig et al., 2014). Market demand is expected to increase due to the increasing number of health-conscious consumers. Therefore, novel bioactive peptides with unique functionalities could have great potential and value in the nutraceutical and food ingredient markets.

High-pressure processing (HPP) is considered as one of the most important innovations in the last 30 years. The unique features of HPP offer food industry means to produce novel foods, textures, and tastes. It was reported that high pressure treatment has unique effects on proteolysis. Pressure-assisted protein unfolding reduces hydrolysis time (Graham, Penac, Frias, Gomez, & Martinez-Villaluenga, 2015), enhances proteolysis via increased exposure of susceptible peptide bonds to enzymatic cleavage (Girgih, Chao, He, Jung, & Aluko, 2015). High pressure stabilizes and increases the activity of some enzymes during the hydrolysis of proteins (Maresca & Ferrari, 2017). Pressure treatment also increases protein digestibility (Quirós, Chichón, Recio, & López-Fandiño, 2007), facilitates the enzymatic release of antioxidant peptides (Girgih et al., 2015), and enhances the formation of antioxidant peptides in the hydrolysates (Zhang, Jiang, Miao, Mu, & Li, 2012). Moreover, some antimicrobial peptides are only active under high pressure (Masschalck, Houdt, Haver, & Michiels, 2001). These results suggest the feasibility of producing unique, bioactive peptides via pressure-assisted enzymatic proteolysis (Chao, He, Jung, & Aluko, 2013; Girgih et al., 2015). Nevertheless, the application of HPP for hydrolyzing the protein from fish by-products has not been fully explored. The main objective of this study was to investigate the effects of pressure-assisted enzymatic hydrolysis on physicochemical, functional, and bioactive properties of fish protein hydrolysate (FPH) based on tilapia by-products. The role of process parameters (pressure and holding time) in modulating key properties of the hydrolysates were subsequently evaluated.

Section snippets

Chemicals and sample preparation

Alcalase enzyme from Bacillus licheniformis was supplied by EMD Millipore Corp. (Burlington, MA, USA). Bovine serum albumin (BSA) was purchased from Sigma Aldrich (St. Louis, MO, USA). Coomassie brilliant blue dye, 2,2-diphenyl-1-picrylhydrazyl (DPPH), phosphate buffer saline (PBS), sodium azide, potassium ferricyanide, sodium thiosulfate, trichloroacetic acid (TCA), Tris hydrochloride and all other chemicals were of analytical grade and provided by Brenntag Ingredients Public Company Limited

Soluble protein content

The effects of pressure-assisted hydrolysis on soluble protein content of FPH are presented in Fig. 4a. Both pressure and holding time had significant influence on soluble protein content of HPP-FPH (p < 0.05) (Table 1). Soluble protein content of HPP-FPH generally increased with increasing pressure and holding time. The highest concentration (5.7 mg/mL) was obtained at 250 MPa and 35 min, which was significantly higher than that of the original crude protein sample (1.3 mg/mL). Pressure

Conclusions

This study provided information on the pressure-assisted enzymatic hydrolysis of tilapia by-product protein as a new substrate. Pressure and holding time were found to have significant impacts on the physicochemical, functional, and bioactive properties of hydrolyzed products. The HP process accelerated the hydrolysis and facilitated the release of free amino acids. The treatment also considerably improved solubility, and emulsifying properties as well as antioxidant activity of FPH. However,

Authors’ contributions

  • Ashutosh Kumar Hemker: Prepared experimental plans, logistics; conducted the experiments and data analysis; drafted the manuscript.

  • Dr. Loc Thai Nguyen: Initiated the research ideas; collaborated the research activities and preparation of the manuscript.

  • Dr. Deepti Salvi: Jointly developed the research ideas, collaborated the experimental activities using high pressure processing unit, involved in the preparation of the manuscript.

  • Prof. Mukund Karwe: Involved in development of the research ideas

Declaration of competing interest

None.

Acknowledgements

This project was supported by the Global Advancement and International Affairs (GAIA) International Collaborative Research Grants award of Rutgers, The State University of New Jersey, USA. The authors are also thankful to Washington State University, USA for facilitating the high-pressure processing of protein samples. The author would also like to acknowledge help from Sawali Naware in high-pressure processing of protein samples.

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