Development of an Integrative Process for the Production of Bioactive Peptides from Whey by Proteolytic Commercial Mixtures

Abstract

The aim of this work is to develop an integrative process based on the use of an ion exchange resin for the enzymatic hydrolysis of b-lactoglobulin to produce peptides with angiotensin converting enzyme (ACE) inhibitory activity using whey as a feed stock. Unlike the conventional methods, the main advantage of this approach is that by integrating the selective separation of b-lactoglobulin from whey and its hydrolysis less complex mixtures of peptides are produced. Furthermore, peptides of similar charge as b-lactoglobulin remain adsorbed achieving further purification. In this work, the enzyme protease N Amano at an enzyme to substrate ratio of 1/100 (wt/wt) was added directly to the adsorbed proteins in a thermostatically controlled membrane reactor operated in batch mode. Separation of the smaller peptides from the enzyme and larger peptides was achieved with a 1 kDa molecular weight cut-off ultrafiltration membrane. Also, this step enables the recycling of nonhydrolyzed substrates, large peptides, and enzyme. The adsorbed protein was re-solubilised in a 10 mM potassium phosphate buffer (pH 7 and 45 C). The different fractions were assayed for their bioactivity in terms of angiotensin converting enzyme inhibition percentage (ACEi%) and IC 50 which is the concentration of peptides that can inhibit the ACE activity by 50%. Results show that permeates of 2 and 6 hrs hydrolysis have the highest bioactivity with IC 50 ¼ 67 and 98 lg=ml respectively.

Production of acid whey hydrolysates applying an integrative process: Effect of calcium on process performance

“…Its application using sweet whey produced hydrolysates with potent 99 ACE-inhibitory activity with peptides derived from β-lactoglobulin and CMP. Furthermore, 100 Welderufael and Jauregi [31] demonstrated that the process require no purification or enrichment 101 step since hydrolysates produced were of high potency. The different composition of acid whey 102 as compared to sweet whey such as, high concentration of calcium and the absence of CMP in 103 acid whey could lead to hydrolysates of different composition and/or potency.…”

“…In our group, we have developed an integrative process combining ion exchange and 98 microfiltration [31,32]. Its application using sweet whey produced hydrolysates with potent 99 ACE-inhibitory activity with peptides derived from β-lactoglobulin and CMP.…”

“…The integrative process developed for the production of hydrolysates was shown in Fig. 1 136 following the work from Welderufael and Jauregi [31] with several modification. DEAE 137 Sepharose ® resin was prepared by equilibrating it using 10 mM potassium phosphate buffer at 138 pH 6.5.…”

“…Enzymatic production of bioactive peptides 91 either using digestive enzymes such as pepsin, trypsin, chymotrypsin or microbial enzymes such 92 as proteinase K, protease N and thermolysin have proven to produce specific peptide sequences 93 with high bioactivity [27][28][29]. High cost of operation, membrane fouling and loss of enzyme 94 activity during hydrolysis, prove to be economical disadvantage for industrial scale production 95 [30,31]. Interestingly, there is a plausible solution for cost reduction by simplifying the 96 production through process integration.…”

“…Acid whey at pH 4.6 and 6.5 were compared for total protein, major whey protein and ionic 279 calcium as shown in Table 1 and Table 2. Neutralisation of acid whey (pH 6.5) was carried out to 280 perform the integrative process using an anion exchanger resin [31]. In general, acid whey (pH 281 4.6 and 6.5) tends to have slightly lower protein as compared to sweet whey [2].…”