Effect of Additives on Subcritical Water Hydrolysis of Whey Protein Isolate Ashley D. Espinoza and Rube ́ n O. Morawicki* Department of Food Science, University of Arkansas, 2650 North Young Avenue, Fayetteville, Arkansas 72704, United States ABSTRACT: The objective was to examine the effect of the additives acetic acid, lactic acid, sodium bicarbonate, sodium chloride, and sodium hydroxide on the hydrolysis of whey protein isolate with subcritical water. A screening experimental design was used to study the effect of temperature, time, and additives. The most influential additive, sodium bicarbonate, along with temperature and time was used in a second experimental design to predict the treatment conditions to maximize the degree of hydrolysis and production of free amino acids. The maximum degree of hydrolysis achieved was 50% at a concentration of 1.24 M sodium bicarbonate, 291 °C, and 28 min. The highest concentration of total amino acids was 83.0 mg/g of whey protein isolate with 0.83 M sodium bicarbonate at 264 °C for 29 min. Compared to water alone, sodium bicarbonate increased the degree of hydrolysis 4-fold and the production of amino acids by 44% and decreased peptides’ molecular weight. KEYWORDS: additives, amino acids, degree of hydrolysis, subcritical water, whey protein ■ INTRODUCTION Subcritical water hydrolysis (SWH) is an alternative to traditional acid/base hydrolysis and to enzymatic methods when the cleavage points are not very important. During SWH, water is maintained in the subcritical state, between its boiling (100 °C and 0.10 MPa) and critical point (374 °C and 22 MPa), where it remains as a liquid due to the high pressure. 1 As the temperature and pressure approach the critical point, the ionic product of water increases and, therefore, its potential to act as an acid- or base-like catalyst increases. In previous research, the highest degree of hydrolysis (DH) attained when whey protein isolate (WPI) was hydrolyzed with subcritical water was 12% for a temperature of 298 °C and a duration of 17 min; and the highest production of total amino acids (AAs) was 57.4 mg/g of WPI at 300 °C for 40 min. 2 Consequently, if the objective is to obtain a higher DH, or a higher production of AA, the conditions of the reaction need modification, for instance, by using additives. Alternatively, the incorporation of modifiers could facilitate these reactions by decreasing the temperature of the reaction for a fixed DH, increasing the DH for a fixed temperature, enhancing the production of AAs, or altering the molecular weight (MW) distributions of peptides. Other researchers have used modifiers in SWH. The inclusion of nitrogen and carbon dioxide as modifiers has successfully increased the production of AAs during in the case of fish waste and biomass hydrolysis. 3,4 Carbon dioxide alone has also increased hydrolysis rates by promoting acid-catalyzed hydrolysis. 5-8 Degradation products of AAs, such as ammonia, formic acid, acetic acid, and fatty acids, were studied as potential additives because they have been shown to increase AA yields during SWH. 9 Other additives have been used to accelerate the SWH of silk fibroin including sodium hydroxide, strong acids (sulfuric acid and hydrochloric acid), and sodium chloride. 10 To the extent of our knowledge, the use of modifiers during the hydrolysis of WPI using subcritical water has not been studied. Whey is an abundant byproduct of cheese manufactur- ing produced at a rate of approximately 145 million metric tons per year in the world, and half ends up in surface water. 11 Because whey is composed of approximately 13% proteins (dry basis), 12 it has intrinsic nutritional and functional value. 13 Furthermore, hydrolyzed whey protein has numerous potential applications because hydrolysis improves heat stability, reduces allergenicity, enhances digestibility, and liberates bioactive peptides. 14-17 This research has the scientific objective of studying the effect of several additives (acetic acid, lactic acid, sodium bicarbonate, sodium chloride, and sodium hydroxide) on the hydrolysis of whey protein with subcritical water using WPI as material. The goals are to determine the influence of temperature, time, and additives on the DH, production of AA, and MW of peptides. ■ MATERIALS AND METHODS Materials. WPI (with 90% protein content) was obtained from Davisco Foods, Inc. (Eden Prairie, MN, USA). Acetonitrile, hydro- chloric acid, and methanol were purchased from VWR (Radnor, PA, USA). AA standard kits, MW standard kits, o-phthaldialdehyde (OPA) (97% purity), acetic acid (97.7%), disodium phosphate (99%), lactic acid (90%), sodium bicarbonate (100%), sodium chloride (99.5%), and sodium hydroxide (5.4 N) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All solutions were prepared with deionized water, and the mobile phases were prepared with HPLC grade water. The OPA derivatizing solution was prepared fresh daily according to the method of Nielsen et al. 18 and used for spectrophotometric and HPLC analyses. Methods. Experimental Designs. Two experimental designs were used in this research. The first one, a custom 2 3 factorial screening design, was used to determine whether the factors additive, temperature, and reaction time influenced the DH of Received: February 9, 2012 Revised: April 18, 2012 Accepted: April 19, 2012 Published: April 19, 2012 Article pubs.acs.org/JAFC © 2012 American Chemical Society 5250 dx.doi.org/10.1021/jf300581r | J. Agric. Food Chem. 2012, 60, 5250-5256