Minced beef was hydrolysed with 1% w/w Zyactinase using 60qC reaction temperature for 0- 180 minutes. The resulting hydrolysate was reacted with 0.1% TCA to precipitate proteins and the slurry was centrifuged to remove the solid precipitate. The supernatant was further processed. The hydrolysate sample was then diluted with the mobile phase 0.1 % trifloroacetic acid (TFA) (1: 5 dilution). The sample was homogenized using a stomacher for 8 minutes then centrifuged at 10000 x g for 20 minutes. The supernatant was then filtered before the injection into the high performance liquid chromatograph (HPLC) and describe in Section 3.9.1. The extraction was than further separated and analyzed as describe in Section 3.9.2.
The HPLC profile of the hydrolysate processed at 60qC and 1% enzyme concentration sampled at different time intervals up to 180 minutes is shown in Figure 5.2. Similar, chromatograms were also observed at other temperatures and enzyme concentrations. A synthetic peptide standard mixture (Sigma Aldrich, USA) with different molecular weights was injected into the column to find the best chromatographic conditions to obtain an elution profile for the peptides as in Section 3.9.3. The column used was Jupiter 300 C18 columns in which depend on hydrophobicity. It allows one to separate proteins with only slight differences in hydrophobicity.
Figure 5.1: The peptide mixture contained 0.5 mg of five different protein peptides; (1) GLY-TYR, molecular weight of 238.2 g mol-1, (2) Methionine Enkephalin Acetate, MW = 573.7 g mol-1 for free base (TYR-GLY-GLY-PHE-MET), (3) VAL-TYR-VAL, MW = 379.5 g mol-1, (4) Leucine Enkephalin, MW = 555.6 g mol-1 for free base (TYR-GLY-GLY-PHE-LEU) and (5) Angiotensin II Acetate, MW = 1046.2 g mol-1 for free base (ASP-ARG-VAL-TYR-ILE-HIS-PRO-PHE)
The formation of peptides formed during hydrolysis at different time intervals was monitored with HPLC (Figure 5.2). After one minute hydrolysis, a major peak eluted at 4.6 minutes. The peptides began to elute at retention times of 10, 14, 17, 19 and 24 minutes. A comparison of the peak fractions in Figure 5.2 reveals a marked increase in the overall concentration of peptides as the hydrolysis time and enzyme concentration increased. After 90 minutes of hydrolysis, the increases of concentration of peptides containing components were stabilized. After 90 minutes, there was no significant increase in the peptide peak area and no formation of new peptides.
As hydrolysis progressed the HPLC profile changed considerably. A complex series of peaks eluted from 12 - 30 minutes. These reflected the formation of peptides from the protein and show the complexity of the mixture. In addition, the peaks at 3-5 minutes increased in intensity, reflecting a significant increase in these materials. Hydrolysis to peptides (peaks eluting between 12 – 30 minutes) appeared to reach a stable maximum within 90 minutes and did not appear to change significantly after that time. However, the formation of low molecular weight materials (eluting at 3–10 minutes) appeared to increase constantly throughout the entire hydrolysis process.
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min 0 100 200 300 mAU 0.0 2.5 5.0 7.5 10.0 12.5 log(M.W.) PDA Ch1:214nm 1 2 3 4 5
Figure 5.2: HPLC chromatograph of peptides in the hydrolysates processed at 60qqC and 1% enzyme concentration after 1, 30, 60, 90, 120 and 180 minutes hydrolysis.
Fadda et al., (1999) with similar HPLC condition as in 3.9.1 and 3.9.2 divided the peptides based on their retention times. Peptides that emerged between 10 and 15 minutes retention time were considered to be hydrophilic, whereas peptides eluting between 15 and 25 minutes were considered to be hydrophobic in nature. Based on these criteria, this current research shows only two major hydrophilic peptides (eluting at 11 and 14 minutes) were derived from the meat hydrolysates and the others were hydrophobic peptides. The significant increase in the hydrophobic nature of the peptides suggests they will contribute to an undesirable (bitter) hydrolysate taste. According to Ney (1971), bitterness is related to an average hydrophobicity of a peptide (Q value). The Q value was defined as the sum of free energies of transfer of the amino acid side chains from ethanol to water, divided by the number of amino acid residues in the peptide. A peptide was considered bitter if its Q value exceeded 1400 cal/mol and its molecular weight was less than 6000 kDa (Fukui et al., 1983). In addition, Ishibashi et al. (1987a, 1987b), reported that a bitter taste from peptides is more intense when the content of amino acids leucine, phenylalnine and tyrosine are high.The bitter tastes have more instense when hydrophobic acids with l configuration were located at C – terminus of the peptides ( Isibashi et, al.,1987a, Isibashi et, al.,1987b, Shinoda et, al., 1986) Adler–Nissen (1988) proposed that the molar concentration and chain length of most hydrophobic peptides were also important properties responsible for bitterness. The bitterness limits the utilization of enzyme hydrolysates in the food industry. Peptides that contain a hydrophobic residue, especially with a long chain or aromatic amino acid, give bitter tastes (Kanehisa et al,
1984). Bitterness in meat hydrolysate is asscociated with low molecular weight peptides such as 5 kDa of hydrophobic peptides derives from bovine haemoglobin hydrolysates (Aubes –Dufau et al..,1995)
Food protein hydrolysates have known to have various application in food, cosmetic and pharmaceutical company. The use and characteristic of protein hydrolysates is based on their molecular size (Gauthier et al. 1986). Digestion of food protein released peptide that may exhibit a diverse range of bioactivities (Rutherfurd- Makwick and Moughan, 2005, Kaur et al, 2010a). Large molecular weight peptides (more than 20 amino acid residues) have improvement in the functional properties wheras low molecular weight peptides (di- and tripeptides, and amino acid) have high nutritional and therapeutic values such as preventing disease of hypertension (Bautista et al.