Development of a Large-Scale (50 L) Apparatus for Ampholyte-free Isoelectric Focusing (Autofocusing) of Peptides in Enzymatic Hydrolysates of Food Proteins

Abstract

It has been demonstrated that peptides in enzymatic hydrolysates of proteins can be fractionated on the basis of the amphoteric nature of the sample peptides, by a laboratory-scale isoelectric focusing apparatus, without adding a chemically synthesized carrier ampholyte. This approach is referred to as autofocusing. In the present study, a large-scale (up to 50 L) autofocusing apparatus was developed and tested. A tank (125 cm x 25 cm x 20 cm) was divided into 12 compartments by 11 plates, each with a window covered in a thin agarose gel layer supported by a nylon screen (100 mesh). The compartments at both ends were filled with 0.1 N phosphoric acid (anode) and 0.1 N NaOH (cathode), respectively, functioning as electrode compartments. The remaining compartments were used for sample compartments. Autofocusing was carried out at constant voltage according to two different methods. In method 1, all sample compartments were filled with a 1% water solution of casein or milk whey protein hydrolysates. In method 2, two compartments located in the center of the tank were filled with 5% sample solution and the others were filled with deionized water. Compositional and sequence analyses of the autofocusing fractions revealed that peptides in the two hydrolysates can be fractionated within 24 h by the present apparatus. Better fractionation was obtained by method 2, whereas enrichment of some peptides occurred by using method 1.

Novel antibacterial lactoferrin peptides generated by rennet digestion and autofocusing technique

“…In addition, basic autofocusing fractions exhibited higher antibacterial activity than the crude digest. Autofocusing does not use harmful chemicals and solvents (Hashimoto, Sato, Nakamura, & Ohtsuki, 2005), therefore, it would be useful to scale-up the potent antibacterial fractions in rennin LFH, even in an industrial setting. 5.…”

In vitroandin vivoanti-inflammatory activity of digested peptides derived from salmon myofibrillar protein conjugated with a small quantity of alginate oligosaccharide

“…An aliquot of the Mf peptides was hydrolyzed by vapor HCl at 150°C, derivatized with phenyl isothiocyanate, and then analyzed using a reverse phase HPLC system. Peptide content of each fraction was represented as a summation of whole amino acids content (mmole) according to the method of Hashimoto et al 34) The peptide fractions were diluted with water and adjusted the pH to 7.0 by HCl or NaOH, and then lyophilized, and stored at −30°C until the assessment of anti-inflammatory activity.…”

Utilization of Aspergillus niger Phytase Preparation for Hydrolysis of Phytate in Foods

“…For fractionation of peptides in the crude digests of food proteins, a preparative ampholyte-free isoelectric focusing has been developed (Hashimoto et al, 2005), which has a potential for purification of enzymes. This technique depends on amphoteric nature of the sample compounds.…”

“…Then these compounds can act as ampholine for isoelectric focusing. This phenomenon is referred to autofocusing (Yata et al, 1996;Akahosi et al, 2000;Hashimoto et al, 2005). This technique does not require harmful reagents and solvents just requires water and agarose gel in sample compartments and diluted phosphoric acid and sodium hydroxide in the electrode compartments.…”

“…By loading direct electric current into the electrode compartments at constant voltage mode at 500 -1000 V, the compounds in sample start to migrate to their own isoelectric points. This technique has been successfully used for peptide fractionation (Hashimoto et al, 2005;Higaki-Sato et al, 2006;Park et al, 2008;Murota et al, 2010;Elbarbary et al, 2010).…”