ABSTRACT
Contents 15.1 Introduction .............................................................................................395 15.2 Development of Large-Scale Peptide Fractionation Method on the
Basis of Diversity of Isoelectric Points of Peptides ....................................397 15.3 Design and Testing of Large-Scale Fractionator .......................................399 15.4 Identication of Biopeptides After Large-Scale Fractionation ..................403 15.5 Industrial Standpoint .............................................................................. 404 15.5 Conclusion .............................................................................................. 406 References ........................................................................................................ 406
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Without doubt, high-performance liquid chromatography (HPLC) has been used most frequently for the laboratory-scale fractionation and isolation of peptides. In particular, reversed-phase (RP)-HPLC is involved in most peptide isolation procedures. Ion-exchange chromatography and size-exclusion (also called gel ltration or gel permeation) chromatography are also preferably used. By optimizing the elution conditions and using a combination of these chromatographic modes, the peptides of interest can be isolated from food protein hydrolysates with high resolution at microgram and milligram levels (see Chapter 2). Coupled with in vitro assay systems, the “active” peptides have been isolated and identied from food protein hydrolysates. However, unlike other functional food ingredients, food-derived peptides may be rapidly and extensively degraded by peptidases during digestion, absorption, and circulation in animal and human bodies. erefore, the peptide with in vitro activity may be degraded and may lose its apparent biological activity during the digestion and absorption processes. On the other hand, some peptides with no in vitro activity might be converted to their active forms by partial digestion in the body. Hence, the activity of a peptide for use as a food ingredient should be evaluated by feeding experiments. For the evaluation of peptide fractions by feeding experiments, relatively high amounts (gram or kilogram orders) of peptides are necessary for even small-scale animal and/or human trials. However, the high initial and production costs of large-scale preparative liquid chromatography systems have hampered the preparation of large amounts of peptide fractions for feeding experiments to identify the real active peptides by ingestion. In addition, some solvents and chemicals frequently used for RP liquid chromatography, such as acetonitorile, methanol, and tri¯uoroacetic acid (TFA), are harmful to animals and humans. It is necessary to completely remove these toxic substances from the nal peptide preparations before the trial. Even nontoxic substances, such as ethanol and salts, should be removed before the feeding experiments, which increases the cost of these experiments. Alternatively, ltration and selective precipitation techniques have been used for both small-and large-scale peptide fractionations. ese techniques are generally used to remove the undigested proteins and proteases from the oligopeptide fractions (see Chapter 2). us, crude protein hydrolysates or peptide fractions prepared by these low selective methods have been used for feeding trials. ese crude preparations are still a mixture of numerous peptides. us, these trials can only conrm the eŸcacy of the peptide preparations by ingestion, but cannot identify the real active peptides. In some cases, peptides with in vitro activity have been chemically synthesized and used in feeding experiments, demonstrating that some single peptides show signicant activity by ingestion (Kagawa et al. 1996; Miguel and Aleixandre 2006). However, more potent peptides may be overlooked by this approach, as the candidate peptide for the feeding trial has been selected by in vitro activity-guided fractionation without considering the bioavailability. To overcome this situation, a large-scale, biocompatible, and low-cost fractionation method for the peptides from the proteolytic digest of a food protein is required, which facilitates the identication of the active peptide by ingestion.
