Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Evaluation of angiotensin I-converting enzyme inhibitory activity after in vitro digestion of soy protein… Lo, Wendy Man Lee 2005

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2005-0255.pdf [ 5.06MB ]
Metadata
JSON: 831-1.0091962.json
JSON-LD: 831-1.0091962-ld.json
RDF/XML (Pretty): 831-1.0091962-rdf.xml
RDF/JSON: 831-1.0091962-rdf.json
Turtle: 831-1.0091962-turtle.txt
N-Triples: 831-1.0091962-rdf-ntriples.txt
Original Record: 831-1.0091962-source.json
Full Text
831-1.0091962-fulltext.txt
Citation
831-1.0091962.ris

Full Text

EVALUATION OF ANGIOTENSIN I-CONVERTING ENZYME INHIBITORY ACTIVITY AFTER IN VITRO DIGESTION OF SOY PROTEIN ISOLATE  by  WENDY MAN YEE LO  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES  (Food Science)  THE UNIVERSITY OF BRITISH COLUMBIA  April 2005  © Wendy Man Yee Lo, 2005  Abstract The generation of angiotensin I-converting enzyme (ACE) inhibitory activity in soy protein isolate (SPI) was determined after sequential digestion with pepsin and pancreatin using batch or dynamic model digestion systems. During batch digestion, higher A C E inhibitory activity was measured after the first 40 and 60 minutes of pepsin digestion (E:S = 1:25, pH 2, 37°C) than after subsequent digestion with pancreatin (E:S = 1:25, p H 7.5, 37°C, 120 min). At the end of 180 minutes of batch digestion, I C values of 0.28 + 0.04, 0.30 + 0.02, and 0.36 ± 0.01 mg/mL were determined for unheated SPI, blanched (100°C, 10 min)-pasteurized (75°C, 15 s) SPI, and blanched (100°C, 10 min)-sterilized (121°C, 20 min) SPI, respectively. In general, similar trends were observed during dynamic model digestion. However, both the degree of hydrolysis and the A C E inhibitory activity were influenced in the dynamic system by controlling pH and transit time between stomach and duodenum reactors to simulate conditions in the upper gastrointestinal tract. During the first 30 minutes of dynamic model digestion, significantly (p<0.05) higher A C E inhibitory activity was generated from unheated SPI after sequential digestion in both reactors compared to after peptic digestion - only in the stomach reactor. However, after 90 minutes, subsequent digestion by pancreatin of unheated SPI and blanchedsterilized SPI in the duodenum reactor resulted in significantly (p<0.05) lower A C E inhibitory activity compared to the corresponding peptic digests. At that time, IC50 values for unheated SPI, blanched-pasteurized SPI, and blanched-sterilized SPI were 0.38 + 0.01, 0.37 + 0.02, and 0.44 + 0.02 mg/mL, respectively. For both batch and dynamic digestion, heat processing of SPI by blanching and sterilizing decreased the A C E inhibitory activity of resulting soy digests. Chromatographic fractionation of unheated SPI digest resulted in IC50 values of active fractions ranging from 0.13 + 0.03 to 0.93 + 0.08 mg/mL. Although many of the fractions showed A C E inhibition, peptides with lower molecular weights and higher hydrophobicity were most active. This study suggests the potential generation of peptides with A C E inhibitory activity upon physiological digestion of soy protein, including products that have been subjected to heat processing. 50  11  Table of Contents Abstract : ii Table of Contents iii List of Tables v List of Figures vi List of Abbreviations viii Acknowledgement ix Co-Authorship Statement x C H A P T E R I Introduction and Literature Review 1 1.1 Introduction 1 1.2 Renin Angiotensin System (RAS) 2 1.3 Angiotensin I-Converting Enzyme (ACE) : 2 1.3.1 Function 2 1.3.2 Forms and Location 2 1.3.3 Structure : 3 1.3.4 Chloride Activation 3 1.3.5 Substrate Binding 4 1.3.6 Inhibitor Binding 4 1.3.7 In vitro assay for A C E Inhibitory Activity 4 1.3.8 In Vitro A C E Inhibitory Activity and In Vivo Blood Pressure Lowering Effect 5 1.4 A C E Inhibitors and Hypertension 7 1.5 A C E Inhibitory Hydrolysates 7 1.6 Soy Protein Isolate (SPI) 8 1.7 Human Gastrointestinal Digestion 8 1.7.1 Gastric Phase 8 1.7.2 Pancreatic Phase 8 1.7:3 Intestinal Phase 9 1.8 hi Vitro Digestion 10 1.8.1 Batch Digestion 10 1.8.2 Dynamic Model Digestion 11 1.8.3 Degree of Hydrolysis 12 1.9 Chromatographic Techniques for Fractionation 12 1.9.1 Anion Exchange Chromatography 12 1.9.2 Reversed Phase Chromatography 12 1.9.3 Immobilized Metal Affinity Chromatography 13 1.9.4 Gel Filtration Chromatography 13 1.9.5 Chromatographic Fractionation of Soy Protein Hydrolysates 13 1.10 Figures....14 1.11, Tables 22 1.12 Bibliography 26 C H A P T E R II Angiotensin I-Converting Enzyme Inhibitory Peptides from In vitro PepsinPancreatin Digestion of Soy Protein 33 2.1 Introduction 33 2.2 Materials and Methods 34 2.2.1 Materials 34 2.2.2 In vitro Digestion 34 1  2.2.3 Degree of Hydrolysis (DH) 2.2.4 A C E Inhibitory Activity Assay 2.2.5 Anion Exchange Chromatography (AEC).. ; 2.2.6 Ultrafiltration 2.2.7 Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) 2.2.8 Gel Filtration Fast Protein Liquid Chromatography (GF-FPLC) 2.2.9 Immobilized Metal Affinity Chromatography (IMAC) 2.2.10 Amino Acid Composition and Peptide Concentration 2.2.11 Statistical Analysis 2.3 Results and Discussion 2.3.1 Degree of Hydrolysis and A C E Inhibition during In Vitro Digestion 2.3.2 A C E Inhibitory Activity of SPI Peptides 2.3.3 Anion Exchange Chromatography (AEC) 2.3.4 Reversed-phase High Performance Liquid Chromatography (RP-HPLC) 2.3.5 Gel Filtration Fast Protein Liquid Chromatography (GF-FPLC) 2.3.6 Immobilized Metal Affinity Chromatography (IMAC) 2.3.7 Amino Acid Composition... 2.4 Figures 2.5 Tables 2.6 Bibliography .• C H A P T E R III " Angiotensin I-Converting Enzyme Inhibitory Activity of Soy Protein Digests from a Dynamic Model System Simulating the Upper Gastrointestinal Tract 59 3.1 Introduction 3.2 Materials and Methods 3.2.1 Materials 3.2.2 Heat Treatment 3.2.3 Batch Digestion ~. 3.2.4 Dynamic Model Digestion 3.2.5 Degree of Hydrolysis (DH) 3.2.6 A C E Inhibitory Activity Assay 3.2.7 Statistical Analysis 3.3 Results and Discussion 3.3.1 Degree of Hydrolysis during Batch Digestion 3.3.2 Degree of Hydrolysis during Dynamic Model Digestion 3.3.3 Degree of Hydrolysis Comparisons ; 3.3.4 A C E Inhibition during Batch Digestion 3.3.5 A C E Inhibition during Dynamic Model Digestion 3.3.6 Comparison of A C E Inhibition during Batch and Dynamic Model Digestion 3.3.7 IC50 Values from Batch and Dynamic Model Digestions 3.4 Conclusion 3.5 Figures 3.6 Tables 3.7 Bibliography C H A P T E R IV Conclusion 4.1 Bibliography Appendix List of Appendix Figures List of Appendix Tables  List of Tables Table 1.1  Amino acid sequence and composition of A C E inhibitory soy peptides  22  Table 1.2  Other protein sources reported to have A C E inhibitory activity  23  Table 1.3  In vitro digestion methods used to simulate human gastrointestinal digestion.. ..24  Table 1.4  Summary of chromatography methods used to fractionate soy hydrolysates  Table 2.1  IC50  Table 2.2  Molecular weights of fractions eluted from gel-filtration F P L C  Table 2.3  Amino acid composition of SPI digest and fractions from anion exchange  values of SPI, SPI digest and collected fractions. letters are significantly different (p<0.05)  25  Values with different 50 51  chromatography.  52  Table 2.4  Amino acid composition of fractions from reversed-phase H P L C  53  Table 2.5  Amino acid composition of fractions from gel-filtration F P L C  54  Table 2.6  Amino acid composition of fractions from I M A C  55  Table 3.1  Degree of hydrolysis as a function of time course of batch digestion of SPI with  Table 3.2  pepsin (0 - 60 min) and pancreatin (60 - 180 min). Values shown are the mean + SD from digestion experiments performed in triplicates 68 Degree of hydrolysis as a function of time course of dynamic model digestion of SPI with pepsin in stomach reactor and pancreatin in duodenum reactor. Values shown are the mean + SD from digestion experiments performed in triplicates...69  Table 3.3  A C E inhibition as a function of time course of batch digestion of SPI with pepsin (0-60 min) and pancreatin (60-180 min). Aliquots of SPI taken during digestion were adjusted to 0.29 mg/mL for the assay for % A C E inhibition. Values shown are the mean + SD from digestion experiments performed in triplicates 70  Table 3.4  A C E inhibition as a function of time course of dynamic model digestion of SPI with pepsin in stomach reactor and pancreatin in duodenum reactor. Aliquots of SPI taken during digestion were adjusted to 0.29 mg/mL for the assay for % A C E inhibition. Values shown are the mean + SD from digestion experiments performed in triplicates 71  Table 3.5  values of SPI digests after 180 minutes of batch digestion and 90 minutes of model digestion in the duodenum reactor. Values shown are the mean + SD from digestion experiments performed in triplicates 72 IC50  v  List of Figures Figure 1.1  Renin angiotensin system  14  Figure 1.2  Activity of A C E on (A) angiotensin I and (B) hippuryl-L-histidyl-Lleucine 15  Figure 1.3  Structure of captopril  16  Figure 1.4  Typical processing steps of soy protein isolate  17  Figure 1.5  Protein digestion and amino acid absorption  18  Figure 1.6  TNO gastrointestinal model: (a) Gastric compartment; (b) duodenal compartment; (c) jejunal compartment; (d) ileal compartment; (e) basic unit; (f) glass jacket; (g) flexible wall; (h) rotary pump; (i) water bath; (j) peristaltic pump; (1, m) pH electrodes; (n, o) syringe pumps; (p) hollow-fibre device 19  Figure 1.7  Gastrointestinal model: (1) gastric compartment; (2) intestinal compartment; (2) cassette pump; (4) peristaltic pump; (5) pH meters; (6) circulator-heater; (7) stirrers; (8) water bath; (A) simulated gastric fluid; (B) simulated intestinal fluid; (C) N a H C 0 ; (D) 2% bile; (E) 4% bile 20 3  Figure 1.8  Reaction of TNBS with amino groups. X = rest of peptide  21  Figure 2.1  Degree of hydrolysis (—) and A C E inhibition (— ) as a function of time course of digestion of SPI with pepsin (0 - 60 min) and pancreatin (60 - 180 min). Aliquots of SPI taken during digestion were adjusted to 0.29 mg/mL for the assay for % A C E inhibition. Data points with different letters (a, b, c for A C E inhibition, or w, x, y, z for degree of hydrolysis) are significantly different (p<0.05) 42  Figure 2.2  DEAE-anion exchange chromatography profile of SPI digest collected after 180 minutes of sequential pepsin-pancreatin digestion. Fractions were eluted by stepwise gradient of NaCl (0, 0.5, 1.0, 1.5 and 2.0 M). Fractions 1 and 2 were collected and adjusted to 0.66 m M for A C E inhibition assay. Fraction 1 was also subjected to a second anion exchange chromatography (see Figure 2.3) 43  Figure 2.3  Profile from second anion exchange chromatography of fraction 1  Figure 2.4  Reversed-phase FIPLC profile of fraction 1.1 from anion exchange chromatography; fractions were eluted by increasing concentration of Buffer B (acetonitrile containing 0.05% T F A (v/v)) 45  Figure 2.5  GF-FPLC profiles of fractions 1.1.2 (A), 1.1.3 (B), 1.1.4 (C), and 1.1.5 (D) obtained from RP-HPLC of fraction 1. Fractions were eluted by 30% (v/v) acetonitrile containing 0.05% T F A (v/v) 46-7  44  VI  Figure 2.6  A C E inhibitory activity of fractions from GF-FPLC. (Concentration of soy peptides in each fraction was adjusted to 1.2 m M for the assay). Bars with different letters are significantly different (p<0.05) 48  Figure 2.7  I M A C profile of soy peptide digest collected after 180 minutes of sequential pepsin-pancreatin digestion. Fractions were collected after elution with phosphate buffer (0.02 M containing 1.0 M NaCl) at pH 7, 4 and 3 49  Figure 3.1  Set up of the dynamic model digestion system: (1) SPI solution, (2) pump, (3) HC1 and pepsin, (4) stomach reactor, (5) magnetic stirring plate, (6) water bath, (7) computer monitoring pH and temperature, (8) NaOH, (9) pancreatin, (10) oxgall bile, and (11) duodenum reactor 67  vii  List of Abbreviations AA ACE ACE2 sACE tACE AEC DH FPLC GF-FPLC HHL IMAC NMWL RAS RP-HPLC SPI TFA  = = = = = = = = = = = = = = =  amino acid angiotensin I-converting enzyme angiotensin Il-converting enzyme somatic form of A C E germinal form of A C E anion exchange chromatography degree of hydrolysis fast protein liquid chromatography gel filtration fast protein liquid chromatography hippuryl-histidyl-leucine immobilized metal affinity chromatography nominal molecular weight limit renin angiotensin system reversed phase high performance liquid chromatography soy protein isolate trifluoroacetic acid  Acknowledgement I would like to thank and acknowledge the following individuals for providing invaluable advice, guidance, and assistance: Dr. Li-Chan, Dr. Seaman, Dr. Keiver, Pedro, Val, Sherman, Amir, Judy, Bo, Tao, Isabelle, and Dr. Farnworth. I would also like to thank Wilfred, grandma, brother, dad, and mom for their support and encouragements. This research was funded by the Natural Sciences and Engineering Research Council of Canada and a University Graduate Fellowship.  Co-Authorship Statement Chapter II of this thesis has been written with joint authorship of Wendy M . Y . Lo and Eunice C. Y . Li-Chan. Wendy M . Y . Lo is the graduate student who carried out the laboratory experiments, collected the data, and summarized the data as a manuscript for publication. Eunice C. Y . Li-Chan is the professor who supervised Wendy M . Y . Lo, and provided guidance and advice with the planning of laboratory experiments, interpretation of data collected, summary of data as a manuscript and proof-reading of the manuscript.  Wendy M . Y . Lo  Eunice C. Y , Li-Chan  x  CHAPTER I  Introduction and Literature Review  1.1 Introduction Angiotensin I-converting enzyme ( A C E , E C 3.4.15.1) is an enzyme in the renin angiotensin system (RAS) that regulates blood pressure. A C E is responsible for converting the hormone angiotensin I into active hormone angiotensin II (Guyton and Hall 1996; Kovacs and others 2002). Angiotensin II directly increases blood pressure by causing vasoconstriction and aldosterone secretion, which in turn promotes sodium reabsorption and water retention (Guyton and Hall 1996; Kovacs and others 2002). Increases in blood pressure can lead to a serious health condition called hypertension. Hypertension is defined as a systolic blood pressure > 140 mm Hg and a diastolic blood pressure > 90 mm Hg (Kannel and Wilson 2003; Izzo and Black 2003). Hypertension is a major risk factor for coronary heart disease, stroke, peripheral arterial disease, and heart failure (Mark and Davis 2000; Kannel and Wilson 2003). It is termed a silent killer and is the primary or contributing cause of over 200,000 of the 2 million deaths that occur every year worldwide (Li and others 2004). One of the treatments for hypertension is A C E inhibitor drugs, which inhibit A C E activity and prevent the formation of angiotensin II (Sica 2003). Although A C E inhibitor drugs are effective, certain side effects are associated with their use (Sica 2003). Recent research studies have reported discoveries of soy peptides that inhibit A C E in chemical assays as well as rat studies (Okamoto and others 1995; Shin and others 1995; Ahn and others 2000; Shin and others 2001; Wu and Ding 2001; Wu and Ding 2002; Wu and others 2002; Chen and others 2003; Chen and others 2004). These peptides were produced by fermentation or enzyme digestion of soy protein. Enzymes, such as alcalase, papain, trypsin, pancreatin, Bacillus subtilis protease, and pepsin, have been used individually for the hydrolysis of soy (Ahn and others 2000, A n and others 2000, Wu and Ding 2001; Wu and Ding 2002; Wu and others 2002; Chen and others 2003). However, a combination of pepsin and pancreatin has not been used to investigate i f A C E inhibitory peptides will be produced in an in vitro digestion system with enzymes and conditions similar to those in the gastrointestinal tract of humans. Therefore, the objective of this study was to investigate i f A C E inhibitory peptides would be produced from soy protein isolate (SPI) digested by an in vitro batch digestion system and a dynamic model digestion system using enzymes similar to digestive enzymes in humans. In addition to monitoring A C E inhibitory activity in the total soy protein digest, the possibility of generating soy peptide fractions with more potent activity than the unfractionated digest was investigated by measuring activity of fractions obtained after ultrafiltration, anion exchange, reversed-phase, gel filtration and immobilized metal affinity chromatography. In addition, because soy protein is usually heat-treated before consumption, SPI was heat-treated prior to digestion to investigate the effects of heat treatment on A C E inhibitory activity of soy peptides. The hypothesis of this study is that in vitro batch digestion and dynamic model digestion of SPI with pepsin and pancreatin will produce peptides with inhibitory activity against A C E . It is also hypothesized that fractionation of SPI digest will generate a fraction that has higher A C E inhibitory activity than the unfractionated digest, and that heat treatments will enhance digestion and possibly the production of soy peptides with A C E inhibitory activity.  1  1.2 Renin Angiotensin System (RAS) Renin angiotensin system (RAS) is the main regulator of blood pressure homeostasis (Eriksson and others 2002). During times of low arterial blood pressure, the kidneys release the enzyme called renin into the bloodstream (Figure 1.1). Renin converts the plasma protein angiotensinogen to angiotensin I (Guyton and Hall 1996; Skidgel and Erdos 2003). Angiotensin I-converting enzyme (ACE) cleaves angiotensin I (DRVYIHPFHL) at the C-terminal, removing the dipeptide His-Leu, thereby converting angiotensin I to angiotensin II (DRVYIHPF). Angiotensin II causes vasoconstriction, primarily in the arterioles, and results in increased peripheral resistance (Guyton and Hall 1996; Kovacs and others 2002). Angiotensin II also acts on the kidneys to promote sodium reabsorption and decrease water excretion, by stimulating aldosterone secretion from adrenal cortex and by acting on A T I receptor (Guyton and Hall 1996; Kovacs and others 2002). As a result, the extracellular fluid volume is increased (Guyton and Hall 1996; Kovacs and others 2002). Both the increases in peripheral resistance and extracellular fluid volume lead to an increase in blood pressure (Guyton and Hall 1996; Kovacs and others 2002). In addition to A C E , the R A S is also regulated by angiotensin II-converting enzyme (ACE2). A C E 2 hydrolyzes angiotensin I to generate angiotensini_ ) (Tschope and others 2002; Vickers and others 2002). Angiotensin 1.9) can in turn be converted to angiotensin 1.7) by the action of A C E (Eriksson and others 2002). A C E 2 also converts angiotensin II to angiotensin 1.7) (Eriksson and others 2002; Donoghue and others 2000; Turner and others 2002). Angiotensin(i-7) is proposed to be an important regulator of cardiovascular function, to promote vasodilation, apoptosis and growth arrest; however, the significance of this metabolite in humans is controversial (Turner and Hooper 2002; Ren and.others 2002; Lemos and others 2002). Both A C E and A C E 2 may represent fulcrums at which angiotensin II and angiotensin(i_7) are regulated since A C E metabolizes angiotensin^) into angiotensin^) while A C E 2 hydrolyses angiotensin II into angiotensin |_7) (Ferrario and Chappell 2004). Counterregulatory mechanisms occur with the R A S , with actions of opposing peptides angiotensin II and angiotensin 1.7) controlling homeostasis (Ferrario and Iyer 1998; Ferrario 2002a, 2002b, 2003). 9  1.3 Angiotensin I-Converting Enzyme (ACE) 1.3.1 Function A C E has two major activities: acting as dipeptidyl carboxypeptidase to remove carboxyterminal dipeptide from substrates and as endopeptidase on substrates that are amidated at carboxyl terminus (Eriksson and others 2002; Skidgel and Erdos 2003; Sturrock and others 2004). These activities of A C E can affect the blood pressure through the renin angiotensin system (RAS), as previously discussed.  1.3.2 Forms and Location Humans have two distinct A C E isoenzymes: a somatic form and germinal form (Turner and Hooper 2002). The somatic form (sACE) is located on the endothelial surface of lungs and brush-border membranes of kidneys, intestine, placenta and choroids plexus; the germinal form (tACE) is found in the testis (Turner and Hooper 2002). Both s A C E and t A C E are membrane2  bound proteins located at the cell surface, and both function as ectoenzymes that hydrolyze circulating peptides (Eriksson and others 2002). Membrane-bound A C E contributes to most of the physiological function of A C E . The membrane-bound A C E may be cleaved from the cell surface to become a soluble form of the enzymes (Eriksson and others 2002). Soluble A C E is found in the blood, urine, lung edema, amniotic fluid, cerebrospinal fluid, lymph, seminal plasma, prostate and epididymus; however, the biological significance of soluble A C E is unclear (Eriksson and others 2002; Skidgel and Erdos 2003; Sturrock and others 2004).  1.3.3 Structure The human ace gene located on chromosome 17 encodes a 150-180 kDa protein which is A C E (Skeggs and others 1980; Skidgel and Erdos 2003). It is classified as a zinc- and chloridedependent metallopeptidase belonging to the M 2 family of metallopeptidases (Sturrock and others 2004). A C E is a two domain enzyme with an N-terminal domain (612 amino acids) and C-terminal domain (650 amino acids); the N-terminal domain has more than 60% sequence identity to the corresponding segment of the C-terminal domain (Turner and Hooper 2002; Ravi Acharya and others 2003). Each of the two domains contains an active zinc-binding motif, His-Glu-Met-Gly-His, that is characteristic of zinc sites found in endoproteinases (Skeggs and others 1980; Ravi Acharya and others 2003). The zinc sites from both domains have catalytic activity; however, their activity differs and their chloride-activation requirements also differs (Wei and others 1991; Jaspard and others 1993; Turner and Hooper 2002). The N-domain site is 50-fold more active toward haemoregulatory peptide (7V-acetyl-Ser-Asp-Lys-Pro), 1000-fold more sensitive to inhibition by phosphinic peptide RXP407, and 3000-fold less sensitive to inhibition by RXPA380 (Rousseau and others 1995; Geordiadis and others 2003). The activation of the Cdomain site is highly dependent on chloride ions, whereas the activation of the N-domain site is much less dependent on chloride ions (Wei and others 1991, 1992). The A C E structure has been studied using its testis isoform (tACE) and found to be mainly helical with a central cavity (or channel) that extends approximately 30 into the molecule (Sturrock and others 2004). A constriction in the middle of the cavity forms the active site and also divides the cavity into two chambers (or subdomains), which are surrounded by boundaries made of helices a l 3 , al4, a l 5 , a l 7 , and 04 (Sturrock and others 2004). Helix al3 contains the His-Glu-Met-Gly-His zinc binding motif with two zinc coordinating histidines (His 383, 387) (Natesth and others 2003). Highly ordered zinc ion is bound at the active site and two chloride ions are bound in the interior of the A C E structure (Sturrock and others 2004). The two chloride ions are separated by 20.3 ; the first chloride ion ( C l l , 20.7 away from zinc ion) is bound to four ligands (Arg 489, Arg 186, Trp 485 and water) and surrounded by a hydrophobic shell of four tryptophans (Sturrock and others 2004). The second chloride ion (C12, 10.4 away from zinc ion) is bound to three ligands (Arg 522, Tyr 224 and water) (Natesh and others 2003; Sturrock and others 2004).  1.3.4 Chloride Activation Monovalent anions, particularly chloride ions, can enhance the activity of A C E by up to 100-fold (Bunning and Riordan 1983; Shapiro and others 1983). The activation of the C-domain of A C E is highly dependent on chloride concentration, but the activation of the N-domain is much less dependent on chloride concentration (Wei and others 1991, 1992). A study by Shapiro and others (1983) found that the degree of chloride activation increases with increasing pH range of 7.5 to 9 for class I substrates (jV-2-furanacryloyl (FA)-blocked tripeptides FA-Phe3  Gly-Gly, -Phe-Ala-Phe, -Phe-Ala-Gly, -Ala-Ala-Ala, -Phe-Leu-Gly, -Leu-Ala-Gly, -Gly-LeuPhe, -Leu-Leu-Gly, -Ala-Leu-Ala, -Phe-His-Leu, and -Gly-Gly-Gly), which require chloride for substrate binding. The amount of chloride required is dependent on the substrate and pH; for example, hydrolysis of angiotensin I is highly chloride dependent, whereas hydrolysis of bradykinin is not chloride dependent (Sturrock and others 2004). The mechanism of chloride activation remains unclear, but chloride ion might restrain Arg 522 from interfering with A C E active site residues or might keep the active site in a conformation that favours substrate binding to A C E (Sturrock and others 2004).  1.3.5 Substrate Binding A C E can bind to a variety of substrates: angiotensin I, gonadotropin-releasing hormone, luteinising hormone-releasing hormone, substance P, P-neoendorphini.g, and neurotensin (Sturrock and others 2004). A study of substrate binding to t A C E found that the substrate's access to tACE's active site is severely limited, with access to the cavity only possible via a small pore in the N-terminal chamber or an occluded slot in the C-terminal chamber (Sturrock and others 2004). Therefore, some flexibility and movements of the domain are likely required for substrate access to A C E , which suggests that A C E might exist in a more open conformation that 'closes' upon substrate binding (Sturrock and others 2004). In addition, three of the N terminal helices that cover the cavity contain several charged residues and restrict the access of large polypeptides to the active site cleft (Sturrock and others 2004). This restrictive nature of the binding clefts could significantly contribute to the specificity of A C E and limit its proteolytic activity to small, disordered peptides, thus accounting for A C E inability to hydrolyze large, folded substrates (Sturrock and others 2004).  1.3.6 Inhibitor Binding The binding of inhibitor, such as lisinopril, to tACE has been studied. Lisinopril binds to tACE in a highly ordered, extended conformation with the phenyl group extended in an N terminal direction and the lysine side chain parallel to the oil3 helix containing the His-Glu-MetGly-His zinc binding motif (Natesh and others 2003). The carboxyalkyl carboxylate of lisinopril is well positioned to bind the active site zinc ion and provides one coordinating ligand (Patchett and others 1980; Patchett and Cordes 1985). The binding does not cause significant rearrangement of the active site residues (Sturrock and others 2004).  1.3.7 In vitro assay for ACE Inhibitory Activity A C E inhibitory activity can be measured by a sensitive, fixed-time spectrophotometric assay (Cushman and Cheung 1971). In this assay, the activity of A C E activity on angiotensin I is estimated by measuring A C E activity on cleaving the synthetic substrate hippuryl-L-histidylL-leucine (HHL), thus resulting in hippuric acid (HA) and His-Leu. The spectrophotometric assay measures the amount of H A produced, which reflects the amount of His-Leu cleaved and the A C E activity. For example, a large amount of H A means a large amount of His-Leu cleaved, which means high A C E activity (or low A C E inhibition). The opposite is true for a small amount of H A measured. The action of A C E on angiotensin I and H H L is shown in Figure 1.2. The Cushman and Cheung's method is most commonly used by pharmaceutical and food industries (Wu and others 2002).  4  The Cushman and Cheung's spectrophotometric method has been widely used by various researchers and some of them made modifications to the A C E concentration and substrate type and concentration. Various A C E concentrations ranging from 20uU to 4 m M (Okamoto and others 1995; Mehanna and Dowling 1999; Hsu and others 2002; Chen and others 2003, 2004) and various H H L concentrations from 5 to 12.5 m M (Wu and Ding 2002; Wu and others 2002; Chen and others 2003) have been used in different studies. Various substrates, such as hippruylGly-Gly, Bz-Gly-His-Leu, and furanacryloyl-Phe-Gly-Gly, have also been used at concentrations of 8.3 to 12.5 m M (Okamoto and others 1995; Hsu and others 2002; Chen and others 2004; Vermeirssen and others 2004). Some methods also included a pre-incubation step of A C E with the substrate lasting for 1 to 5 minutes (Ahn and others 2000; Hsu and others 2002). In addition to the typical spectrophotometric method of Cushman and Cheung, other methods have been used to measure in vitro A C E inhibitory activity. Matsui and others (2002) coupled a colourimetric method to the Cushman and Cheung's method. Instead of H A extraction and quantification, they quantified His-Leu concentration by adding 2, 4, 6 - trinitrobenzene sulfonate to bind specifically to the primary amine (His-Leu), producing a colour that can be detected at wavelength of 420 nm. Chen and others (1999), Mehanna and Dowling (1999), and Wu and Ding (2002) coupled reversed phase - H P L C to the Cushman and Cheung's method. Instead of H A extraction and spectrophotometric quantification, they injected the reaction solution into a reversed phase - H P L C column to separate H A from H H L and also to quantify H A . Another method by Hsu and others (2002) coupled thin-layer chromatography to Cushman and Cheung's method. After the A C E reaction is stopped, the reaction solution is dried, redissolved in methanol, and then spotted onto a silica gel to separate the substrate (furanacryloylPhe-Gly-Gly) and product (furanacryloyl-Phe) by thin-layer chromatography in butanol-acetic acid water, followed by observation under U V light. The variations of in vitro A C E assays used by different studies must be considered when comparing the reported A C E inhibitory activity. For example, the different substrate and enzyme concentration might result in different reaction rates in one study than another study, leading to inaccurate conclusions being made from comparison of the inhibitory effect. The different types of substrates might also have different affinity to A C E ( K ) , leading to comparison of inhibitors against substrates with different levels of affinity for A C E ' s binding site. This could lead to inaccuracy, because the inhibitors would be compared to different levels of competitiveness for A C E ' s binding site. In addition, the various in vitro assays might differ in their level of sensitivity in detection or quantification of the reaction products formed. Although these variations exist, the in vitro A C E assay is still a good screening tool to determine the A C E inhibitory activity; however, one needs to be cautious and consider the possible influence from these variations when comparing the relative potency of the reported A C E inhibitory activity across different studies. m  1.3.8 In Vitro ACE Inhibitory Activity and In Vivo Blood Pressure Lowering Effect Several studies have investigated both in vitro A C E inhibitory activity and in vivo blood pressure lowering effect of peptides from SPI. Chen and others (2003) isolated a fraction of SPI digest that had an IC50 value of 0.24 mg/mL as determined using an in vitro assay. When this fraction of SPI digest was orally fed to spontaneously hypertensive rats (SHR) at a dose level of 2.0 g/kg of body weight, their systolic blood pressure was found to decrease significantly by 10 to 20 mm Hg within six hours after administration. Another study by Shin and others (2001) 5  isolated an A C E inhibitory peptide, His-His-Leu, from fermented soybean paste, and determined an IC50 value of 2.2 ug/mL by an in vitro assay. The His-His-Leu peptide was synthesized and injected into the femoral vein of SHR at a dose level of 5 mg/kg of body weight per injection. Single and triple injections of the synthetic His-His-Leu were found to reduce the rats' systolic blood pressure by 32 and 61 mm Hg, respectively, at 60 minutes after administration. In addition to soy protein, in vitro A C E inhibitory activity and in vivo blood pressure lowering effect were also observed in the digest of another type of protein, spinach leaf protein (Yang and others 2004). Spinach leaf protein digest from pepsin digestion and pepsin-pancreatin digestion had IC50 values of 56 and 120 (ig/mL, respectively, as determine by an in vitro assay. The digests from pepsin digestion and pepsin-pancreatin digestion were orally fed to SHR at dose levels of 0.25 and 0.5 g/kg body weight, respectively. The former digest decreased the rats' systolic blood pressure by 11.1 + 0.75 mm Hg at four hours after administration, while the latter digest decreased the rat's systolic blood pressure by 7.2 + 1.25 mm Hg at two hours after administration. Similarly, a hexapeptide (Val-Leu-Ala-Gln-Tyr-Lys), which was isolated from beef hydrolysates and showed in vitro A C E inhibitory activity, produced beneficial effects in lowering blood pressure, total and L D L cholesterol of SHR when fed daily for 8 wks (Jang and others 2004). Increasing dose of the beef peptide (0.2, 05. or 1.0 g/kg body weight) led to increasing suppression of systolic blood pressure, and was especially noted after 3 weeks of feeding (Jang and others 2004). Significant decrease in blood pressure of SHR was also observed 1-9 hours after oral administration by injection at a lOmg/kg body weight dose, of a purified inhibitor from fermented oyster sauce, with IC50 value of 0.0874 mg/ml in an in vitro assay (Je and others 2004). Although in vitro A C E inhibition assays may serve as a good screening tool for potential inhibitors, the in vivo mechanism of hypotensive action is complex, and may not be directly correlated with in vitro A C E inhibition activity (Fuglsang and others 2003). Furthermore, different values of inhibitory activity may be determined when different substrates, such as angiotensin I-like versus bradykinin-like substrates, are used in the in vitro assay (Fuglsang and others 2003). Suetsuna and others (2004) reported similar blood pressure lowering effect in SHR administered four dipeptides with differing in vitro ACE-inhibitory activity, but noted that the periods for which the effect lasted were different for each dipeptide; it was suggested that variations in the blood pressure-lowering effect result from different absorption and degradation rates of the dipeptides in the GI tract and plasma, respectively. Murakami and others (2004) measured A C E inhibitory activities and antihypertensive activities in spontaneously hypertensive rats of 12 different commercial, food grade peptide products. The tetrapeptide Ala-Leu-Pro-Met, corresponding to residues 142-145 of the whey protein beta-lactoglobulin, did not have particularly potent activity based on its IC50 value in an in vitro assay, but showed strong antihypertensive activity in terms of decreasing blood pressure after 8 hours of oral administration to SHR. Maeno and others (1996) measured the antihypertensive activity (decrease in systolic blood pressure in SHR) as well as IC50 values of ten synthetic peptides corresponding to fragments of milk casein. No direct correlation was observed between in vitro and in vivo results; in fact, the peptide with strongest A C E inhibitory activity (IC50 = 22 uM) showed no significant anti-hypertensive effect for SHR, while another peptide with low A C E inhibitory activity (IC50 = 1000 uM) was found to be antihypertensive. When the latter peptide (Lys-Val-Leu-Pro-Val-Pro-Gln) was incubated with pancreatin, a shorter peptide without the Cterminal Gin residue was produced, which had strong A C E inhibitory activity ((IC50 = 5 uM) as well as strong antihypertensive activity in SHR at a dosage of 1 mg/kg bodyweight. Conversely, the former peptide was partially hydrolyzed by pancreatin to a shorter peptide with low A C E inhibitory activity (IC50 > 1000 uM). These results demonstrated the importance of considering  6  the influence of gastrointestinal digestion of A C E inhibitory peptides on their potential in vivo antihypertensive effects.  1.4 ACE Inhibitors and Hypertension The inhibition of A C E can result in lowering of blood pressure by impaired formation of angiotensin II and reduced degradation of hyoptensive peptides bradykinin and kallidin (Eriksson and others 2002). A C E inhibitors can be categorized according to their characteristics. Competitive inhibitors are able to enter the A C E protein molecule, interact, with the active site and prevent substrate binding to the active site (Wu and Ding 2002). True inhibitors are resistant to cleavage by A C E ; they are often tested and found to withstand cleavage during pre-incubation with A C E (Yang and others 2003). On the contrary, real substrates and pro-drugs are hydrolyzed by A C E (Yang and others 2003). Upon hydrolysis, real substrates would release inactive fragments or fragments with lower A C E inhibitory activity, whereas pro-drugs would release fragments with high A C E inhibitory activity. Hypertension (high blood pressure) is commonly treated with antihypertensive drugs that inhibit A C E . Captopril is a commonly prescribed A C E inhibitor drug ([2S]-N-[3-mercapto-2methylpropionyl]-L-proline, Figure 1.3) at dosages of 75-300 mg per day (Sica 2003). It is a prodrug that is hydrolyzed upon consumption and converts to an active di-acid form in the liver or intestine (Sica 2003). Captopril binds tightly to A C E at its active site and competes with angiotensin I for occupancy (Cushman and others 1987). With captopril bound to its active site, A C E cannot bind to angiotensin I to convert it to. angiotensin II. As a result, captopril decreases the production of angiotensin II and prevents the cascade of events that increases blood pressure (Sica 2003). Many drugs have side effects and so does captopril. Its side effects are coughs, angioedema, hyperkalemia, and generally reversible form of functional renal insufficiency in the presence of renal artery stenosis (Sica 2003).  1.5 ACE Inhibitory Hydrolysates Studies have reported A C E inhibitory activity from protein hydrolysates and also identified their composition. Peptides with A C E inhibitory activity have low molecular weights (Dziuba and others 1999; Wu and Ding 2002) and various types of amino acid. For example, A C E inhibitory soy peptides were found to have nonpolar (Ala, Leu, and Gly), polar (Gin, Asn, and Pro), aromatic (Phe), or negatively charged (Asp and Glu) amino acids at the carboxyl end (Yamauchi and Suetsuna 1996a, 1996b; Shin and others 2001; Wu and Ding 2002; Chen and others 2003). At the amino end, they had nonpolar (He, Val, Gly), aromatic (Tyr and Phe), polar (Gin), positively charged (His), or negatively charged (Asp) amino acids (Yamauchi and Suetsuna 1996a, 1996b; Shin and others 2001; Wu and Ding 2002; Chen and others 2003). Table 1.1 summarizes the amino acid sequence and composition of A C E inhibitory soy peptides reported in the literature. In addition to soy protein, various sources of proteins have been reported to have A C E inhibitory activity and some examples of them are listed in Table 1.2. The fact that so many different protein sources and their hydrolysates exhibit A C E inhibitory activity seem to suggest that A C E inhibitory activity might not be specific to the protein source. The range of potency from these proteins and their hydrolysates suggested that the composition of the peptides in the hydrolysate is the more influential factor on their A C E inhibitory potential. 7  1.6 Soy Protein Isolate (SPI) Soy protein isolate (SPI) is produced from soybeans, which contain 8-10% moisture, 1720% lipids, 38-40%) protein, and 26-29%> carbohydrate (Fukushima 2000). In a typical process, soybeans are first dried, cleaned, cracked and de-hulled to become soy chips (Liu 1997, Figure 1.4). The soy chips are conditioned to 10-11% moisture at 63-74°C and flaked using smooth rolls (Liu 1997). The flakes are defatted by hexane extraction and de-solventized to become soy meal containing approximately 50% protein, 30-35%> carbohydrates, 1%> lipids and 15%> of ash and moisture (Liu 1997). The soy meal is extracted with water (water/flake ratio of 1:1.5) at mild alkaline pH 7-9 and 50-60°C for 20-40 minutes (Liu 1997; Fukushima 2000). The extract is filtered and centrifuged to remove insoluble materials, which are mostly carbohydrates (Liu 1997; Fukushima 2000). The proteins in the extract are precipitated by pH adjustment to 4.5, which is the isoelectric point of soy protein (Liu 1997; Fukushima 2000). The precipitated proteins (or curd) are decanted mechanically, washed with water for several times, and neutralized to pH 6.87 (Liu 1997; Fukushima 2000). The curd and wash water are then spray-dried to produce SPI containing over 90%> protein (Liu 1997; Fukushima 2000). The typical recovery of SPI from defatted soybean flakes is 30-40% (Fukushima 2000). In typical commercial soy milk production, whole or dehulled soybeans are blanched (100°C, 10-20 min) and then ground with hot water (80-100°C) (Golbitz 1995; Liu 1997). After grinding, the soybean slurry formed will be filtered. The filtrate, which is the soy milk, is collected and either pasteurized (75°C, 15 sec) or sterilized (121°C, 20 min).  1.7 Human Gastrointestinal Digestion 1.7.1 Gastric Phase Protein digestion occurs in three phases: gastric phase, pancreatic phase, and intestinal phase (Hunt and Groff 1990). The gastric phase occurs in the stomach, which produces gastric juice containing hydrochloric acid, mucus, water, salts, gelatinase, gastric amylase, gastric lipase and pepsin (Berne and Levy 1996a; Guyton and Hall 2000a). Gastric juice activates pepsin by converting pepsinogen into pepsin, which in turn converts other pepsinogen (Hunt and Groff 1990; Berne and Levy 1996a; Johnson 1998; Guyton and Hall 2000b; Ganong 2001). Pepsin (EC 3.4.23.1) is an endopeptidase that cleaves peptide bonds at the carboxyl end of amino acid residues, such as phenylalanine, tryptophan, tyrosine, leucine, and glutamate (Nelson and Cox 2000; Expasy 2003a). Pepsin functions optimally at pH 1.8 to 3.5 and loses its functionality at pH above 5 (Berne and levy 1996a; Johnson 1998; Guyton and Hall 2000a, 2000b; Ganong 2001). The digestive action of pepsin contributes to 10 to 20%> of total protein digestion in the human body (Berne and Levy 1996a; Berne and Levy 1996b; Guyton and Hall 2000b). After the gastric phase, the end products are products of partial protein digestion, namely proteoses, peptones, and large polypeptides (Guyton and Hall 1996).  1.7.2 Pancreatic Phase The pancreatic phase starts when the protein end products from the gastric phase pass into the small intestine (Hunt and Groff 1990). Immediately upon entering the small intestine, the protein end products are attacked by proteolytic enzymes trypsin, chymotrypsin, 8  carboxypeptidase, and elastase (Guyton and Hall 1996). These enzymes are from the pancreatic juice, which also contains sodium ions, potassium ions, bicarbonate ions, chlorine ions, aamylase, and lipase (Berne and Levy 1996a). The pancreas secretes this juice into the small intestine and the small intestine releases enteropeptidase to activate proenzyme tripsinogen to trypsin (Hunt and Groff 1990; Berne and Levy 1996a, 1996b; Johnson 1998). The trypsin formed in turn converts more trypsinogen as well as activates other proenzymes, such as chymotrypsinogen, procarboxypeptidase, and proelastase to chymotrypsin, carboxypeptidase, and elastase, respectively (Guyton and Hall 2000b). Trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), and elastase (EC 3.4.21.36) are endopeptidases and carboxypeptidase (EC 3.4.17.1 (class A ) , E C 3.4.17.2 (class B)) is an exopeptidase (Hunt and Groff 1990; Johnson 1998; Ganong 2001). A n endopeptidase hydrolyses the interior peptide bonds of a protein sequence, whereas an exopeptidase hydrolyses the peptide bonds at the amino end or carboxyl end of a protein sequence (Hunt and Groff 1990; Johnson 1998; Ganong 2001). Trypsin hydrolyses the peptide bond at the carboxyl end of basic amino acid residues lysine and arginine (Nelson and Cox 2000). Chymotrypsin cleaves the peptide bond at the carboxyl end of aromatic amino acid residues, such as methionine, leucine, phenylalanine, tryptophan, and tyrosine (Nelson and Cox 2000; Expasy 2003b). Trypsin and chymotrypsin split whole and partially digested proteins into peptides of various sizes, but they do not cause release of individual amino acids (Guyton and Hall 2000a). Carboxypeptidase hydrolyses the peptide bonds at the carboxyl ends of polypeptides and produce individual amino acids (Johnson 1998; Guyton and Hall 2000a, 2000b; Ganong 2001). Elastase digests elastin fibers that attach animal meat fibers together and produces peptides with an aliphatic amino acid at the carboxyl end (Johnson 1998; Guyton and Hall 2000b; Ganong 2001). Under the influence of pancreatic proteases, most of the protein digestion occurs in the upper small intestine in the duodenum and jejunum (Guyton and Hall 2000b). Pancreatic proteases are very active in the duodenum and about 50% of ingested proteins are digested and absorbed in the duodenum (Berne and Levy 1996b). Most of the digestion products from the pancreatic phase are dipeptides, tripeptides, and sometimes even larger peptides (Berne and Levy 1996b; Guyton and Hall 2000b; Ganong 2001). Only a small percentage of digestion products from the pancreatic phase are amino acids (Berne and Levy 1996b; Guyton and Hall 2000b; Ganong 2001).  1.7.3 Intestinal Phase The final digestion of protein is in the intestinal lumen achieved by enterocytes lining the villi of the small intestine at the duodenum and jejunum sections (Gutyon and Hall 1996). The digestion products from the pancreatic phase are absorbed through the intestinal cells, enterocytes, in the intestinal phase (Hunt and Gruff 1990; Johnson 1998; Guyton and Hall 2000b; Ganong 2001). The enterocytes line the villi, which are located mainly in the duodenum and jejunum sections of the small intestine (Guyton and Hall 2000b). The enterocytes have a brush border composed of hundreds of microvilli projecting from the surface of each enterocyte (Berne and Levy 1996b; Guyton and Hall 2000b). The microvilli membrane contains multiple peptidases, such as dipeptidases, which hydrolyse the larger polypeptides of four or more amino acids into tripeptides, dipeptides, and sometimes to individual amino acids (Hunt and Gruff 1990; Johnson 1998; Guyton and Hall 2000b; Ganong 2001). The dipeptides, tripeptides, and amino acids are absorbed intact into the enterocytes of the small intestine where they are easily transported through the microvillar membrane to the interior of enterocytes for absorption (Johnson 1998; Guyton and Hall 2000b; Ganong 2001). Any larger peptides are absorbed poorly or not absorbed at all (Johnson 1998). 9  During absorption, dipeptides or tripeptides require an active transport system using N a co-transport or a secondary transport system using H co-transport, while amino acids require a passive transport system or an active transport system using N a co-transport (Guyton and Hall 2000b; Ganong 2001). For example, in the active transport system, the sodium ion moves down the electrochemical gradient to the interior of the enterocyte and transports the dipeptide, tripeptide, or amino acid along with it (Guyton and Hall 2000b). The active transport system has high affinity for dipeptides and tripeptides, but has low affinity for peptides of four or more amino acids (Berne and Levy 1996b). On the other hand, the secondary active transport system of dipeptides and tripeptides is powered by the electrochemical potential difference of H ions across the membrane of the enterocyte (Berne and Levy 1996b; Ganong 2001). In a passive transport system, the amino acids are absorbed through facilitated diffusion (Johnson 1998; Guyton and Hall 2000b; Ganong 2001). The rate of transport of dipeptides or tripeptides usually exceeds the rate of transport of individual amino acids. The dipeptides and tripeptides are more efficiently absorbed than amino acids into the enterocyte (Berne and Levy 1996b; Johnson 1998). Two thirds of the total amount of protein absorbed is in the form of peptides containing up to six amino acids, and the remainder is absorbed in the form of free amino acids (Hunt and Groff 1990). Inside the cytosol of the enterocyte are multiple other peptidases specific for the remaining types of linkages between amino acids (Hunt and Groff 1990; Guyton and Hall 2000b; Ganong 2001). The cytosolic peptidases are active against dipeptides and tripeptides (Berne and Levy 1996b). Within minutes, all dipeptides and tripeptides are digested to the final stage of single amino acids (Berne and Levy 1996b; Johnson 1998; Guyton and Hall 2000b; Ganong 2001). The amino acids leave the enterocytes, exit the basolateral membrane via carriers, and enter into the portal blood (Johnson 1998; Guyton and Hall 2000b Ganong 2001). Most of the final protein digestion products absorbed into the blood are individual amino acids, but a small percentage of peptides may be absorbed into the blood. The peptides would have entered into the portal blood via the paracellular route passing through the tight junctions of enterocytes (Hunt and Groff 1990; Berne and Levy 1996b; Johnson 1998; Guyton and Hall 2000b). Therefore, the digestive products flowing into the portal vein are mainly free amino acids and dipeptides and tripeptides (Hunt and Groff 1990). Some proteins (2 to 5%) escape digestion and enter the colon; the colonic bacteria digest and ferment the proteins to produce short-chain fatty acids, dicarboxylic acids, phenolic compounds, and ammonia (Ganong 2001). Figure 1.5 provides a summary of protein digestion and amino acid absorption. +  +  +  1.8 In Vitro Digestion 1.8.1 Batch Digestion Table 1.3 is a summary of some in vitro batch digestion methods used in various studies in order to mimic human gastrointestinal digestion. Most in vitro batch digestion methods focus on simulating the gastric phase and the pancreatic phase of human gastrointestinal digestion. To simulate these phases, porcine bile extract and porcine enzymes, such as pepsin, pancreatin, and lipase, are used to mimic human bile, pepsin, trypsin, chymotrypsin, and lipase released during gastrointestinal digestion. In addition, the pH and temperature during in vitro digestion are carefully adjusted to mimic the acidic environment of the gastric phase, the neutral environment of the pancreatic phase, and the body temperature of 37°C during human gastrointestinal  10  digestion. In the method of Shen and others (1994), dialysis was used to remove the products formed during digestion.  1.8.2 Dynamic Model Digestion In addition to the in vitro digestion models exemplified in Table 1.3, more sophisticated and advanced dynamic models have been developed to mimic conditions in the gastrointestinal tract. Three models have been reported in the literature: the T N O gastrointestinal model described by Minekus and others (1995), the dynamic model described by Mainville and others (2004), and the gastrointestinal model described by Koo and others (2001). The TNO gastrointestinal tract model (Minekus and others 1995) contains 4 successive compartments that simulate the stomach, duodenum, jejunum, and ileum (Figure 1.6). Each compartment is a glass jacketed beaker with tube-like flexible walls. The 4 compartments are connected by peristaltic pumps consisting of three connected T-tubes. Water is pumped from a water bath into the glass jackets and around the flexible walls to control the temperature inside the beaker and to exert pressure on the flexible walls. Changes in water pressure cause alternate compression and relaxation of the flexible walls, which allow for mixing of the chyme. The flexible walls also facilitate the passage of chyme into the tubes that connects the compartments. A computer controls the frequency of peristaltic cycles of the pumps and thus controls the flow rate of the chyme being transferred from one compartment to the next. The computer also controls the pumping of a predetermined quantity of meal into the stomach compartment within a pre-set period of time. Level sensors and pH electrodes are equipped within each compartment and connected to the computer to monitor volume and pH. Computer-controlled syringe pumps regulate the secretion of water, HC1, NaHC03, gastric electrolytes, enzymes, bile and pancreatic juices into the stomach and duodenal compartments. The jejunal and ileal compartments are connected with hollow-fiber devices to absorb digestion products and water from the chyme and to modify the electrolyte and bile salt concentrations in the chyme. Various studies have adapted this model to simulate the gastrointestinal digestion (Arkbage and others 2003; Krul and others 2000; Larsson and others 1997; Marteau and others 1997; Verwei and others 2003, 2004; Mainville and others 2004). The dynamic model (Mainville and others 2004) contains 2 successive compartments to simulate the stomach and duodenum. Each compartment is a glass jacketed beaker connected to a 37°C water bath to control the temperature inside the beaker. The two beakers are also connected to each other via a peristaltic pump to facilitate gastric emptying of chyme from the stomach compartment to the duodenum compartment. The temperature and pH in each compartment are monitored and the contents are mixed by magnetic stirrers. Peristaltic pumps are used to regulate the amount of meal added to the stomach compartment at the start of digestion. Throughout digestion, peristaltic pumps are used to regulate the adding of HC1 and pepsin to the stomach compartment and also the adding of NaOH, pancreatin, and bile to the duodenum compartment. The gastrointestinal model (Koo and others 2001) contains two beakers that simulate the gastric compartment and intestinal compartment (Figure 1.7). The p H in each beaker is monitored and the chyme is mixed by magnetic stirrers. The beakers are placed in a water bath (37°C) and connected by a peristaltic pump that facilitates gastric emptying from the gastric compartment to the intestine compartment. The meal is manually poured into the gastric compartment at the start of the digestion. The intestinal compartment continuously collects chyme emptied from the gastric compartment. Simulated gastric fluid (containing pepsin, mucin, NaCl, and distilled water adjusted to pH 2), simulated intestinal fluid (containing trypsin, pancreatin, and distilled water), and bile solution are pumped into the gastric or intestine 11  compartments via cassette pumps. The pH of the gastric and intestine compartments are monitored and adjusted by manually adding 1 N HC1 into the gastric compartment or by adding 0.1 or 0.3 M NaHCCh via cassette pumps into the intestine compartment.  1.8.3 Degree of Hydrolysis The degree of hydrolysis (DH) is one of the key parameters used to monitor protein hydrolysis (Nielsen and others 2001). It was used in this experiment to measure the extent of hydrolysis after in vitro digestion of the soy protein isolate. D H is defined as the percentage of peptide bonds cleaved during protein hydrolysis (Adler-Nissen 1979, 1986; Clegg and others 1982; Nielsen and others 2001). DH-h/htotX 100% h (meqv/g) = number of peptide bonds cleaved during hydrolysis process h (meqv/g) = total number of peptide bonds in given protein (Adler-Nissen 1979, 1986; Nielsen and others 2001) tot  Several procedures for determining the degree of hydrolysis have been developed, including methods based on trinitrobenzenesulfonic acid, fluorescamine, o-phthaldialdehyde, pH-stat, osmometry, and soluble nitrogen content (Adler-Nissen 1979; Kwan and others 1983; Nielsen and others 2001). The trinitrobenzenesulfonic acid (TNBS) method was introduced by Satake and others (1960) and modified by Adler-Nissen (1979). The TNBS method determines the number of peptide bonds cleaved (h) by measuring the concentration of primary amino groups through their reaction with TNBS to form chromophores that are measured spectrophotometrically (Adler-Nissen 1979, 1986, Figure 1.8). The reaction between the primary amino group and TNBS takes place under slightly alkaline condition and is terminated by lowering of the pH.  1.9 Chromatographic Techniques for Fractionation 1.9.1 Anion Exchange Chromatography In ion-exchange chromatography, the sample compounds are separated based on differences in their ionic charges (Niessen 1999). Anion exchange chromatography is a type of ion exchange chromatography where the positively charged counter-ions (i.e. cations) on the stationary phase bind to negatively charged sample molecules in the mobile phase (Rounds and Gregory III 1998; Niessen 1999). Any positively charged or neutral sample molecules will elute out of the column (Rounds and Gregory III 1998; Niessen 1999). To elute the bound sample molecules, the pH or ionic strength of the mobile phase is typically adjusted (Rounds and Gregory III 1998; Niessen 1999).  1.9.2 Reversed Phase Chromatography Reversed phase chromatography separates sample molecules on the basis of their polarities (Rounds and Gregory III 1998; Niessen 1999). The stationary phase of the column is typically silica chemically modified with alkyl chains, such as octyl (C$), octadecyl (Cis), phenyl, n-propylamine, and alkyldiol (Rounds and Gregory III 1998; Niessen 1999). Since the 12  stationary phase is nonpolar, the nonpolar sample molecules will adhere to it (Rounds and Gregory III 1998; Niessen 1999). Polar sample molecules that do not adhere to the stationary phase will elute out of the column with the mobile phase and thus be separated from the nonpolar sample molecules (Rounds and Gregory III 1998; Niessen 1999). The nonpolar sample molecules can be released and eluted from the column by decreasing the polarity of the mobile phase (Rounds and Gregory III 1998; Niessen 1999).  1.9.3 Immobilized Metal Affinity Chromatography In immobilized metal affinity chromatography, separation is based on interactions between sample molecules and the immobilized metals on the stationary phase (Li Chan 1996). Metal ions are chelated onto the spacer arm of the stationary phase by passing a solution of metal ions through the column. Amino acid residues, particularly histidine that are on the surface of protein molecules form complexes with the chelated metal ion, thus binding to the stationary phase. Sample molecules without available histidine do not bind to the stationary phase and are instead eluted out of the column with the mobile phase. Therefore, sample molecules with and without available histidine are separated. The bound sample molecules can be released and eluted from the column by modifying the pH of the mobile phase (Li Chan 1996).  1.9.4 Gel Filtration Chromatography In gel filtration chromatography, sample molecules are separated on the basis of size (Rounds and Gregory III 1998; Niessen 1999). The difference in molecular size affects the sample molecule's ability to diffuse in and out of the pores in the stationary phase (Rounds and Gregory III 1998; Niessen 1999). The porosity of the stationary phase results from the column packing material, such as wide pore silicagel, polysaccharides, and synthetic polymers (Rounds and Gregory III 1998; Niessen 1999). Due to the porous beads in the stationary phase, small sample molecules will enter the beads and subsequently require a longer elution time (Rounds and Gregory III 1998; Niessen 1999). The elution time is much shorter for larger sample molecules, because they are unable to enter the beads and are eluted out quickly with the mobile phase (Rounds and Gregory III 1998; Niessen 1999).  1.9.5 Chromatographic Fractionation of Soy Protein Hydrolysates Table 1.4 is a summary of chromatographic methods, such as cation exchange, gel filtration, and reversed phase chromatography that were used in studies to separate soy protein hydrolysates to collect fractions for testing on their A C E inhibitory activity (Shin and others 1995; Ahn and others 2000; Shin and others 2001; Wu and Ding 2001; Wu and Ding 2002; Chen and others 2003).  13  1.10 Figures Angiotensinogen Renin ACE2  Angiotensin I ACE  JJ, A C E ACE2  Angiotensin II  Vasoconstriction  V.  Angiotensin i-9)  Renal retention of salt & water  Angiotensin(i-7)  ACE  Vasodilation  Bradykinin & Kallidin ft  ft  Increase blood pressure  Angiotensin^)  Decrease blood pressure  ACE  Inactive products  Figure 1.1 Renin angiotensin system. (Guyton and Hall 1996; Eriksson and others 2002; Ferrario and Chappell 2004).  14  Asp - Arg - Val - Tyr - He - His - Pro - Phe - His - Leu Angiotensin I ACE Asp - Arg - Val - Tyr - He - His - Pro - Phe Angiotensin II B  o -C-Gly-His-Leu  Hippuryl - l - Histidyl - L - Leucine  ACE  „ / /  +  \\  Hippuric acid  His - Leu Dipeptide o "C - Gly +  His - Leu  Dipeptide  Figure 1.2 Activity of A C E on (A) angiotensin I and (B) hippuryl-L-histidyl-L-leucine (Cushman and Cheung 1971).  15  V  HSCH ^ 2  ^ CH C-  H  Q  II  3  Figure 1.3 Structure of captopril.  Soybeans  y  Drying Cleaning Cracking De-hulling  Soy Chips ^  Conditioning Flaking  Full Fat Flakes Solvent Fat Extraction Solvent Removal  Defatted soy meal, ueous Extraction  Extract Filtration Centrifugation Protein Precipitation  Protein Curd jj^ Spray Drying  Soy Protein Isolate  Figure 1.4 Typical processing steps of soy protein isolate (Jenks and others 1997; L i u 1997; Fukushima 2000).  17  Protein  Polypeptides & oligopeptides  > Stomach  Polypeptides r Lumen of small intestine Tripeptides  Dipeptides Microvilli Amino acids  Dipeptides V Absorptive cell of small intestine ^Arnino acids  Blood capillary  Figure 1.5 Protein digestion and amino acid absorption. (Wardlaw and Kessel 2002).  18  Figure 1.6 TNO gastrointestinal model: (a) Gastric compartment; (b) duodenal compartment; (c) jejunal compartment; (d) ileal compartment; (e) basic unit; (f) glass jacket; (g) flexible wall; (h) rotary pump; (i) water bath; (j) peristaltic pump; (1, m) pH electrodes; (n, o) syringe pumps; (p) hollow-fibre device (Source: Minekus and others 1995 with permission).  19  Figure 1.7 Gastrointestinal model: (1) gastric compartment; (2) intestinal compartment; (2) cassette pump; (4) peristaltic pump; (5) pH meters; (6) circulator-heater; (7) stirrers; (8) water bath; (A) simulated gastric fluid; (B) simulated intestinal fluid; (C) N a H C 0 ; (D) 2% bile; (E) 4% bile (Source: Koo and others 2001 with permission). 3  20  °K o 2N  S(V  •  o ,N —<^ (T  J)  )-NH-X  +S0 "+H 2  +  3  [0,  Figure 1.8 Reaction of TNBS with amino groups. X = rest of peptide (Adler-Nissen 1979)  21  1.11 Tables Table 1.1 Amino acid sequence and composition of A C E inhibitory soy peptides. Treatment Pepsin hydrolysis  A C E Inhibitory Soy Peptides Ile-Ala Tyr-Leu-Ala-Gly-Asn-Gln Phe-Phe-Leu Ile-Tyr-Leu-Leu Val-Met-Asp-Lys-Pro-Gly-Gly  Alcalase hydrolysis  Asp-Gly Asp-Leu-Pro  Pepsin hydrolysis  Gln-Val-Val-Phe Ile-Thr-Pro-Leu Val-Val-Phe-Asp Gly-Asp-Ala-Pro-Asn Ile-Val-Phe-Asp-Ala Val-Gln-Val-Val-Phe Gly-Glu-Leu-Phe-Glu Val-Thr-Val-Pro-Gln  Fermented paste  His-His-Leu  Fermented paste  Gly(3 .58 Lys (2 .28 Pro (1..90 A l a ( l .65 V a l ( l .64 T h r ( l .24 Phe(l .00  %) %) %) %) %) %) %)  IC 153 14 37 42 39 50  uM uM uM uM uM  4.8 u M 12.3 u M 46 52 39 28 41 34 23 63  uM uM uM uM uM uM uM uM  2.2 p.g/mL 26.52 ug/mL  Reference Chen and others 2003  Wu and Ding 2002  Yamauchi and Suetsuna 1996a, 1996b  Shin and others 2001 Ahn and others 2000  22  Table 1.2 Examples of some of the other protein sources reported to have A C E inhibitory activity. Reference IC 50 6.2 u M Miguel and 4.7 u M others 2004  Source Egg White (pepsin, trypsin and chymotrypsin hydrolysis) -  Hydrolysate/Peptide Arg-Ala-Asp-His-Pro-Phe-Leu Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-IleLeu  Chickpea legumin (alcalase hydrolysis)  Hydrolysate  Wakame seaweed (Undaria pinnatifida) (Protease S 'Amano' hydrolysis)  Val-Tyr Ile-Tyr Ala-Trp Phe-Tyr Val-Trp Ile-Trp Leu-Trp  Skimmed milk (Proteinase B hydrolysis)  Hydrolysate  0.42 mg/mL Kumar and others 2000  Fermented Oyster Sauce  Fermented sauce Purified fraction  2.45 mg/mL Je and 0.087 mg/mL others 2004  Chicken Muscle (Thermolysin hydrolysis)  Hydrolysate  Ovalbumin (Thermolysin, pepsin, trypsin, and chymotrypsin hydrolysis)  Hydrolysate Hydrolysate Hydrolysate Hydrolysate  Dried Bonito fish (Thermolysin hydrolysis)  Leu-Lys-Pro Ile-Trp-His  Dioscorin (yam)  Extract  Pea Whey (pepsin, trypsin and chymotrypsin hydrolysis)  Hydrolysate Hydrolysate  0.18 mg/mL Yust and others 2003 35.2 6.1 18.8 42.3 3.3 1.5 23.6  uM uM uM uM uM uM uM  Sato and others 2002  45 (ig/mL Fujita and others 2000  (thermolysin) (pepsin) (trypsin) (chymotrypsin)  83.0 ug/mL Fujita and 45.3 ug/mL others 2000 >1000 [ig/mL >1000 ug/mL  0.32 u M Fujita and 3.5 u M Yoshikawa 1999 6.404 u M Hsu and others 2002 0.076 mg/mL 0.048 mg/mL  Vermeirsseri and others 2004  23  Table 1.3 In vitro digestion methods used to simulate human gastrointestinal digestion. Methodology Steps Protocol 1 1) Adjust (w/ HC1) pH to T ' ' 2) Add p e p s i n ' ' ^ 3) Incubate at 37°C for l ' ' or 2 hours 4) Adjust (w/ N a H C 0 ) pH to 5.3 ' or 5 5) Add pancreatin & bile extract * ' ' 6) Add lipase 7) Adjust (w/ NaOH) pH to 7 ' ' or 7.5 8) Incubate at 37°C for 2 ' ' hours b c d  3  Sample Milk-based & soy-based infant formulas  Reference Shen and others 1994  carrots, spinach, meat & tomato paste puree  b  a  a  15  a  b  d  b  d  3  3  a  c  c  C  Garrett and others 1999  b c d  d  a >b  b  C  d  c  Mineral water  b  d  Spinach puree  Ekmekcioglu and others 1999  c  Ferruzzi and others 2001  d  Protocol 2 1) Add simulated gastric fluid (papsin, NaCl, HC1, p H 1.2) 2) Incubate at 37°C for 1.5 hours 3) Add simulated intestinal fluid (pancreatin, K H P 0 , NaOH, pH 7.5) 4) Incubate at 37°C for 4 hours  Purified soy & corn protein  Okunuki and others 2001  Protocol 3 1) Add artificial saliva (amylase, lysozyme, p H 6.7) 2) Incubate at 37°C for 5 minutes 3) Add simulated gastric juice (pepsin, p H 2) 4) Adjust (w/ HC1) pH to 2.5 5) Incubate 37°C for 2 hours  Soy sauce, beer & soybean paste  K i m and others 2002  Protocol 4 1) Add HC1 and pepsin 2) Incubate at 37°C for 1 hour 3) Neutralized pH (w/ N a H C 0 ) 4) Add ileal digesta 5) Incubate at 37°C for 3 hours  Ground maize seeds & texturized soya protein  Martin-Orue and others 2002  2  4  3  24  Table 1.4 Summary of chromatography methods used to fractionate soy hydrolysates. Chromatography steps Protocol 1 1) cation exchange 2) gel filtration 3) gel filtration  Protocol 2 1) cation exchange (strong acid) 2) gel filtration 3) gel filtration 4) reversed-phase (HPLC) 5) reversed-phase (HPLC) Protocol 3 1) reversed-phase (HPLC) 2) cation exchange (HPLC)  Protocol 4 1) gel filtration 2) gel filtration 3) reversed-phase 4) reversed-phase (HPLC) Protocol 5 1) reversed-phase (HPLC) 2) cation exchange (HPLC)  Column  Elution buffer  Refere -nee  1) Dowex 50W 2) Sephadex G-25 3) Sephadex G-25  1) 5% ammonia 2) 3% NaCl (linear gradient) 3) 3% NaCl (linear gradient)  Chen and others 2003  1) 732 cation resin  1) not described  2) Sephadex G-15 3) Sephadex G-25 4) Hi-Pore CI8  2) 1 M acetic acid 3) 0.5 M ammonium formate 4) 35% acetonitrile with 0.1% trifluoroacetic acid 5) acetonitrile with 0.1 % trifluoroacetic acid  Wu& Ding 2002  5) Supelcosil LC-18-DB  2) Shodex asahipak ES2502N-7C  1) 0.05% trifluoroacetic acid / acetonitrile (95:5) 2) 20 m M tris[hydroxymethyl]aminometh ane-HCl buffer with 125 m M NaCl  Shin and others 2001  1) Sephadex G-25 2) Sephadex LH-20 3) Octadecyltrichlorosilane 4) Supelcosil PLC-18  1) distilled water 2) 20% methanol 3) 15, 30, 60, 90% methanol 4) water / acetonitrile (85:15)  Ahn and others 2000  1) JAIGEL-ODS-A-343-10  1) water / acetonitrile (98:2,96:4,35:65) 2) 10 m M sodium succinate with 20% acetonitrile  Shin and others 1995  1) JAIGEL-ODS-A-343-10  2) JAIGEL-ES-502 CP  25  1.12 Bibliography Adler-Nissen J. 1979. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry 27:1256-62. Adler-Nissen J. 1986. The degree of hydrolysis. In: Enzymic Hydrolysis of Food Proteins. London: Elsevier Applied Science Publishers, p 12-5. Ahn SW, K i m K M , Y u K W , Noh DO, Suh HJ. 2000. Isolation of angiotensin I converting enzyme inhibitory peptide from soybean hydrolysate. Food Science and Biotechnology 9:378-81. A n C W , Lee H B , Nam HS, K i m JH, inventors. Nong Shim Co., Ltd., assignee. 2000 Mar 15. Manufacturing method of soy protein enzyme hydrolyzate containing hypotensive factor. Korean patent K R 2000014663. Arkbage K , Verwei M , Havenaar R, Witthoft C. 2003. Bioaccessibility of folic acid and (6S)-5methyltetrahydrofolate decreases after the addition of folate-binding protein to yogurt as in a dynamic in vitro gastrointestinal model. Journal of Nutrition 133:3678-83. Ariyoshi Y . 1993. Angiotensin-converting enzyme inhibitors derived from food proteins. Trends in Food Science & Technology 4:139-44. Berne R M , Levy M N . 1996a. Gastrointestinal secretions. In: Principles of Physiology. 2nd ed. St. Louis: Mosby-Year Book Inc. p 459-80. Berne R M , Levy M N . 1996b. Digestion and Absorption. In: Principles of Physiology. 2nd ed. St. Louis: Mosby-Year Book Inc. p 481-99. Bunning P, Riordan JF. 1983. Activation of angiotensin converting enzyme by monovalent anions. Biochemistry 22:110-6. Chen GR, Liu ST, Shi B H , Zhang RZ, L i JC, Chen R M , L i L , Gao W H , Chen TB, Zhen Y Q , Rao PF. 1999. H P L C determination of angiotensin-converting enzyme activity on Toyopearl HW-40S column. In: Whitaker JR, editor. Food for Health in the Pacific Rim: International Conference of Food Science and Technology. 3rd ed. Davis: Food & Nutrition Press, p 363-70. Chen JR, Okada T, Muramoto K , Suetsuna K , Yang SC. 2003. Identification of angiotensin I converting enzyme inhibitory peptides derived from the peptic digest of soybean protein. Journal of Food Biochemistry 26:543-54. Chen JR, Yang SC, Suetsuna K , Chao JCJ. 2004. Soybean protein-derived hydrolysate affects blood pressure in spontaneously hypertensive rats. Journal of Food Biochemistry 28:6173. Clegg K M , Lee Y K , McGilligan JF. 1982. Technical note: trinitrobenzenesulphonic acid and ninhydrin reagents for the assessment of protein degradation in cheese samples. Journal of Food Science & Technology 17:517-20. Cushman DW, Cheung HS. 1971. Spectrophotometric assay and properties of the angiotensinconverting enzyme of rabbit lung. Biochemical Pharmacology 20:1649-60. Cushman DW, Ondetti M A , Gordon E M , Natarajan S, Karanewsky DS, Krapcho J, Petrillo E W Jr. 1987. Rational design and biochemical utility of specific inhibitors of angiotensinconverting enzyme. Journal of Cardiovascular Pharmacology 10:S17-30. Donoghue M , Hsieh F, Baronas E, Godbout K , Gosselin M , Stagliano N , Donovan M , Woolf B, Robison K , Jeyaseelan R. 2000. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circulation Research 87:El-9. Dziuba J, Minkiewicz P, Nalecz D. 1999. Biologically active peptides from plant and animal proteins. Polish Journal of Food And Nutrition Sciences 8:3-16. 26  Ekmekcioglu C, Pomazal K , Steffan I, Schweiger B , Marktl W. 1999. Calcium transport from mineral waters across Caco-2 cells. Journal of Agricultural and Food Chemistry 47:25949. Eriksson U , Danilczyk U , Penninger J M . 2002. Just the beginning: novel functions for angiotensin-converting enzymes. Current Biology 12:R745-52. Expasy. 2003a. NiceZyme View of Enzyme: E C 3.4.23.1. Enzyme nomenclature database. <http://ca.expasy.org/enzyme/> (accessed October 12, 2004) Expasy. 2003b. NiceZyme View of Enzyme: E C 3.4.21.1. Enzyme nomenclature database. <http://ca.expasy.org/enzyme/> (accessed October 12, 2004) Ferrario C M . 2002a. Angiotensin I, angiotensin II and their biologically active peptides. J. Hypertension 20:805-7. Ferrario C M . 2002b. Does angiotensin^ 1-7) contribute to cardiac adaptation and preservation of endothelial function in heart failure? Circulation 105:1523-5. Ferrario C M . 2003. Commentary on Tikellis et al.: there is more to discover about angiotensin converting enzyme. Hypertension 41:390-1. Ferrario C M , Chappell M C . 2004. Novel angiotensin peptides. Cellular and Molecular Life Sciences 61:2720-7. Ferrario C M , Iyer SN. 1998. Angiotensin^ 1-7): a bioactive fragment of the renin-angiotensin system. Regulatory Peptides 78:13-8. Ferruzzi M G , Failla M L , Schwartz SJ. 2001. Assessment of degradation and intestinal cell uptake of carotenoids and chlorophyll derivatives from spinach puree using an in vitro digestion and caco-2 human cell model. Journal of Agricultural and Food Chemistry 49:2082-9. Fuglsang A , Nilsson D, Nyborg N C B . 2003. Characterization of new milk-derived inhibitors of angiotensin converting enzyme in vitro and in vivo. Journal of Enzyme Inhibition and Medicinal Chemistry 18:407-12. Fujita H , Yoshikawa M . 1999. L K P N M : a prodruy-type ACE-inhibitory peptide derived from fish protein. Immunopharmacology 44:123-7. Fujita H , Yokoyama K , Yoshikawa M . 2000. Classification and antihypertensive activityof angiotensin I-converting enzyme inhibitory peptides derived from food proteins. Journal of Food Science 65:564-9. Fukushima D. 2000. Soybean processing. In: Nakai S, Modler W, editors. Food Proteins:Processing Applications. New York: Wiley-VCH Inc. p 309-42. Ganong WF. 2001. Digestion and absorption. In: Review of Medical Physiology. 20th ed. NewYork: The McGraw-Hill Companies Inc. p 453-63. Garrett D A , Failla M L , Sarama RJ. 1999. Development of an in vitro digestion method to assesscarotenoid bioavailability from meals. Journal of Agricultural and Food Chemistry 47:4301-9. Georgiadis D, Beau F, Czarny B , Cotton J, Yiotakis A , Dive V . 2003. Roles of the two activesites of somatic angiotensin-converting enzyme in the cleavage of angiotensin I and bradykinin: insights from selective inhibitors. Circulation Research 93:148-54. Gobbetti M , Ferranti P, Smacchi E, Goffredi F. 2000. Production of angiotensin-I convertingenzyme inhibitory peptides in fermented milks started by Lactobacillus delbruekii subsp bulgaricus SSI and Lactococcus lactis subsp cremoris FT5. Applied & Environmental Microbiology 66:3898-904.Golbitz P. 1995. Traditional soyfoods: processing and products. Journal of Nutrition 125:570S-2S. Guyton A C , Hall JE. 1996. Dominant role of the kidneys in long-term regulation of arterial pressure and in hypertension. In: Textbook of Medical Physiology. 9th ed. Philadephia: W.B. Sanders Company, p 221-37. 27  Guyton A C , Hall JE. 2000a. Secretory functions of the alimentary tract. In: Textbook of Medical Physiology. 10th ed. Philadephia: W.B. Sanders Company, p 738-53. Guyton A C , Hall JE. 2000b. Digestion and absorption. In: Textbook of Medical Physiology. 10th ed. Philadephia: W.B. Sanders Company, p 754-63. Hsu FL, Lin Y H , Lee M H , Lin C L , Hou WC. 2002. Both Dioscorin, the tuber storage protein of yam (Dioscorea alt acv. Tainong No. 1), and its peptide hydrolysates exhibited angiotensin converting enzyme inhibitory activities. Journal of Agricultural and Food Chemistry 50:6109-13. Hunt S M , Groff JL. 1990. Proteins. In: Hunt S M , Groff JL, editors. Advanced Nutrition and Human Metabolism. New York: West Publishing Company, p 134-9. Hyun C K , Shin H K . 2000. Utilization of bovine blood plasma proteins for the production of angiotensin-I converting enzyme inhibitory peptides. Process Biochemistry 36:65-71. Izzo JL, Black HR. 2003. Approach to the management of hypertension. In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel LJ, Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 378-381. Jang A , Cho Y J , Lee JL Shin JH, K i m IS, Lee M . 2004. The effect of beef peptide on blood pressure and serum lipid concentration of spontaneously hypertensive rat (SHR). Journal of Animal Science and Technology 46:107-114. Jaspard E, Wei L , Alhenc-Gelas F. 1993. Differences in the properties and enzymatic specificities of the two active sites of angiotensin I-converting enzyme (kininase II). Studies with bradykinin and other natural peptides. Journal of Biological Chemistry 268:9496-503. Je JY, Park JY, Jung W K , Park PJ, K i m SK. 2005. Isolation of angiotensin I converting enzyme (ACE) inhibitor from fermented oyster sauce, Crassostrea gigas. Food Chemistry 90:809-14. Jenks B H , Waggle D H , Henley EC. 1997. Isolated soy protein technology: Potential for new developments. In: Lachance P A , editor. Nutraceuticals: Designer Foods III Garlic, Soy, and Licorice. Trumbull: Food & Nutrition Press Inc. p 203-17. Johnson L R . 1998. Digestion and absorption. In: Johnson L R , editor. Essential Medical Physiology. 2nd ed. Philadelphia: Lippincott-Raven Publishers, p 474-88. Kannel WB, Wilson PWF. 2003. Cardiovascular risk factors and hypertension. In: Izzo JL, Black HR, Goodfriend T L , Sowers JR, Weder A B , Appel L J , Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 235-8. K i m K R , Lee SJ, Shin JH, Seo JK, Shon M Y , Sung NJ. 2002. The formation of N-nitrosamine in soy sauce, soybean paste, and beer under simulated gastric digestion. Journal of the Korean Scoiety of Food Science and Nutrition 31:378-83. Kohama Y , Oka H , Kayamori Y , Tsujikawa K , Mimura T, Nagase Y , Satske M . 1991. Potent synthetic analogues of angiotensin-converting enzyme inhibitors derived from tuna muscle. Agriculture and Biological Chemistry 55:2169-217. Koo J, Marshall D L , DePaola A . 2001. Antacid increases survival of Vibrio vulnificus and Vibrio vulnifucus phage in a gastrointestinal model. Applied and Environmental Microbiology 67:2895-902. Kovacs G, Peti-Peterdi J, Rosivall L , Bell PD. 2002. Angiotensin II directly stimulates macula densa Na-2C1-K co-transport via apical AT(1) receptors. American Journal of Physiology: Renal Physiology 282:F301-6. Krul C, Luiten-Schuite A , Baan R, Verhagen H , Mohn G , Feron V , Havenaar R. 2000. Application of a dynamic in vitro gastrointestinal tract model to study the availability of food mutagens using heterocyclic aromatic amines s model compounds. Food and Chemical Toxicology 38:783-2. 28  Kumar R, Watanabe Y , Tamai Y . 2000. Yeast protease B-digested skimmed milk inhibits angiotensin-I-converting-enzyme activity. Biotechnology and Applied Biochemistry 31:95-100. Kwan K K H , Nakai S, Skura BJ. 1983. Comparison of four methods for determining protease activity in milk. Journal of Food Science 48:1418-21. Larsson M , Minekus M , Havenaar R. 1997. Estimation of the bioavailability of iron and phosphorus in cereals using a dynamic in vitro gastrointestinal model. Journal of the Science of Food and Agriculture 74:99-106. Lemos V S , Cortes SF, Silva D M , Campagnole-Santos M J , Santos R A . 2002. Angiotensin 1-7 is involved in the endothelium dependent modulation of phenylephrine-induced contraction in the aorta of mRen-2 transgenic rats. British Journal of Pharmacology 135:1743-8. Li-Chan E. 1996. Separation and purification. In: Nakai S, Modler HW, editors. Food Proteins: Properties and Characterization. New York: V C H . p 429-503. L i G H , Le GW, Shi Y H , Shrestha S. 2004. Angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrition Research 24(7):469-86. Liu K . 1997. Chemistry and nutritional value of soybean components. In: Soybeans: Chemistry, Technology, and Utilization. New York: International Thomson Publishing, p 25-113. Maeno M , Yamamoto N , Takano T. 1996. Identification of an antihypertensive peptide from casein hydrolysate produced by a proteinase from lactobacillus helveticus CP790. Journal of Dairy Science 79:1316-21. Mainville I, Arcand Y , Farnworth ER. 2005. A dynamic model that simulates the human upper gastrointestinal tract for the study of probiotics. International Journal of Food Microbiology 9:287-96. Mark K S , Davis TP. 2000. Stroke: development, prevention and treatment with peptidase inhibitors. Peptides 21:1965-73. Marteau P, Minekus M , Havenaar R, Huis in't Veld JHJ. 1997. Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine : validation and the effects of bile. Journal of Dairy Science 80:1031-7. Martin-Orue S M , O'Donnell A G , Arino J, Netherwood T, Gilbert HJ, Mathers JC. 2002. Degradation of transgenic D N A from genetically modified soya and maize in human intestinal simulations. British Journal of Nutrition 87:533-42. Matsui T, Matsufuji H , Osajima Y . 1992. Colorimetric measurement of angiotensin I-converting enzyme inhibitory activity with trinitrobenzene sulfonate. Bioscience, Biotechnology & Biochemistry 56:517-8. Matsui T, Matsufuji H , Seki E, Osajima K , Nahashima M , Osajima Y . 1993. Inhibition of angiotensin I converting enzyme by Bacillus licheniformis alkaline protease hydrolyzates derived from sardine muscle. Bioscience, Biotechnology & Biochemistry 57:922-5. Mehanna A , Dowling M . 1999. Liquid chromatographic determination of hippuric acid for the evaluation of ethacrynic acid as angiotenisn converting enzyme inhibitor. Journal of Pharmaceutical and Biomedical Analysis 19:967-73. Miguel M , Recio I, Gomez-Ruiz JA, Ramos M , Lopez-Fandino R. 2004. Angiotensin I converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis. Journal of Food Protection 67:1914-20. Minekus M , Marteau P, Havenaar R, Huis in't Veld JHJ. 1995. A Multicompartmental dynamic computer-controlled model simulating the stomach and small intestine. Alternatives to Laboratory Animals 23:197-209.  29  Murakami M , Tonouchi H , Takashashi R, Kitazawa H , Kawai Y , Negishi H , Saito T. 2004. Structural analysis of a new anti-hypertensive peptide (P-Lactosin B) isolated from a commercial whey product. Journal of Dairy Science 87:1967-74. Nakamura Y , Yamamoto N , Sakai K , Okubo A , Yamazaki S, Takano T. 1995a. Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. Journal of Dairy Science 78:777-83. Nakamura Y , Yamamoto N , Sakai K , Takano T. 1995b. Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme. Journal of Dairy Science 78:1253-7. Natesh R, Schwager SL, Sturrock ED, Ravi Acharya K R . 2003. Crystal structure of the human angiotensin-converting enzyme-lisinopril complex. Nature 421:551-4. Nelson D L , Cox M M . 2000. Amino acids, peptides, and proteins. In: Lehninger Principles of Biochemistry. 3rd ed. New York: Worth Publishers, p 115-158. Nielsen P M , Petersen D, Dambmann C. 2001. Improved method for determining food protein degree of hydrolysis. Journal of Food Science 66:642-6. Niessen W M A . 1999. Introduction to Liquid Chromatography. In Liquid Chromatography Mass Spectrometry. 2nd ed. New York: Marcel Dekker, Inc. p 3-29. Okamoto A , Hanagata H , Kawamura Y , Yanagida F. 1995. Anti-hypertensive substances in fermented soybean, natto. Plant Foods for Human Nutrition 47:39-47. Okunuki H , Teshima R, Shigeta T, Sakushima J, Akiyama H , Goda Y , Toyoda M , Sawada J. 2001. Increased digestibility of two products in genetically modified food (CP4-EPSPS and Cryl Ab) after preheating. Journal of the Food Hygiene Society of Japan 43:68-73. Patchett A A , Harris E, Tristram EW, Wyvratt M J , Wu M T , Taub D. 1980. A new class of angiotensin-converting enzyme inhibitors. Nature 288:280-3. Patchett A A , Cordes EH. 1985. The design and properties of N-carboxyalkyldipeptide inhibitors of angiotensin-converting enzyme. Adv. Enzymol. Relat. Areas Mol. Biol. 57:1-84. Pihlanto-Leppala A . 1999. Isolation and characterization of milk-derived bioactive peptides.[Academic dissertation]. Trku: University of Turku. 349 p. Available from: booklet, T E M A - T E A M 6898-99, ISBN 951-29-1547-2. Ravi Acharya K , Sturrock ED, Riordan JF, Ehlers M R W . 2003. A C E revisited: a new target for structure-based drug design. Nature Reviews Drug Discovery 2:891-902. Ren Y , Garvin JL, Carraetero OA. 2002. Vasodilator action of angiotensin^ 1-7) on isolated rabbit afferent arterioles. Hypertension 39:799-802. Rounds M A , Gregory III JF. 1998. High Performance Liquid Chromatography. In: Nielsen SS, editor. Food Analysis. 2nd ed. Gaithersburg: Aspen Publishers Inc. p 511-25. Rousseau A , Michaud A , Chauvet M T , Lenfant M , Corvol P. 1995. The hemoregulatory peptide N acetyl-Ser-Asp-Lys-Pro is a natural and specific substrate of the N-terminal active site of human angiotensin-converting enzyme. Journal of Biological Chemistry 270:3656-661. Satake K , Okuyama T, Ohashi M , Shinoda T. 1960. The spectrophotometric determination of amine, amino acid and peptide with 2, 4, 6 - trinitrobenzene 1 - sulfonic acid . Journal of Biochemistry 47:654-60. Sato M , Hosokawa T, Yamaguchi T, Nakano T, Muramoto K , Kahara T, Funayama K , Kobayashi A , Nakano T. 2002. Angiotensin I-converting enzyme inhibitory peptides derived from wakame (Undaria pinnatifida) and their antihypertensive effect in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry 50:6245-52. Seki E, Osajima K . 1996. Quantitative analysis of digestion resistant A C E inhibitory peptides by small intestinal mucosa. Nippon Shokuhin Kagaku Kogaku Kaishi 43:967-9. Shapiro R, Holmquist B , Riordan JF. 1983. Anion activation of angiotensin converting enzyme: dependence on nature of substrate. Biochemistry 22:3850-7. 30  Shen L H , Luten J, Robberecht H , Bindels J, Deelstra H . 1994. Modification of an in-vitro method for estimating the bioavailability of zinc and calcium from foods. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung (European Journal of Food Research and Technology) 199:442-5. Shin ZI, Ahn C W , Nam HS, Lee HJ, Lee HJ, Moon T H . 1995. Fractionation of angiotensin converting enzyme inhibitory peptide from soybean paste. Korean Journal of Food Science and Technology 27:230-4. Shin ZI, Y u R, Park SA, Chung D K , Ahn CW, Nam HS, K i m K S , Lee HJ. 2001. His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste exerts antihypertensive activity in vivo. Journal of Agricultural and Food Chemistry 49:3004-9. Sica D A . Angiotensin-converting enzyme inhibitors. 2003. In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel L J , Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 426-9. Skeggs L T , Dorer FE, Levine M , Lentz K E , Kahn JR. 1980. The biochemistry of the rennin angiotensin system. Advances in Experimental Medicine and Biology 130:1-27. Skidgel R A , Erdos E G . 2003. Angiotensin I-converting enzyme and neprilysin (neutral endopeptidase). In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel LJ, Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 17-9. Sturrock E D , Natesh R, van Rooyen J M , Acharya K R . 2004. Structure of angiotensin I converting enzyme. Cellular and Molecular Life Sciences 61:2677-86. Suetsuna K , Maekawa K , Chen JR. 2004. Antihypertensive effects of Undaria Pinnatifida (wakame) peptide on blood pressure in spontaneously hypertensive rats. Journal of Nutritional Biochemistry 15:267-72. Tschope C, Schultheiss HP, Walther T. 2002. Multiple interactions between the rennin angiotenisin and the kallikrein-kinin systems: role of A C E inhibition and A T I receptor blockage. Journal of Cardiovascular Pharmacology 39:478-87. Turner AJ, Hooper N M . 2002. The angiotensin-converting enzyme gene family: genomics and . pharmacology. Trends in Pharmacological Sciences 23:177-83. Turner AJ, Tipnis SR, Guy JL, Rice G, Hooper N M . 2002. A C E H / A C E 2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to A C E inhibitors. Canadian Journal of Physiology and Pharmacology 80:346-53. Vermeirssen V , van der Bent A , Van Camp J, van Amerongen A , Verstraete W. 2004. A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests. Biochimie 86:231 -9. Verwei M , Arkbage K , Havenaar R, Berg H van den, Witthoft C, Schaafsma G. 2003. Folic acid and 5-methyltetrahydrofolate in fortified milk are bioaccessible as determined in a dynamic in vitro gastrointestinal model. Journal of Nutrition 133:2377-83. Verwei M , Arkbage K , Mocking H, Havenaar R, Groten J. 2004. The binding of folic acid and 5 methyltetrahydrofolate to folate-binding proteins during gastric passage differs in a dynamic in vitro gastrointestinal model. Journal of Nutrition 134:31-7. Vickers C, Hales P, Kaushik V , Dick L , Gavin J, Tang J, Godbout K , Parsons T, Baronas E, Hsieh F. 2002. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. Journal of Biological Chemistry 277:14838-43. Wardlaw G M , Kessel M . 2002. Proteins. In: Wardlaw G M , Kessel M , editors. Perspectives in Nutrition. 5th ed. Boston: McGraw-Hill, p 267-70.  31  Wei L , Alhenc-Gelas F, Corvol P, Clauser E. 1991. The two homologous domains of human angiotensin I-converting enzyme are both catalytically active. Journal of Biological Chemistry 266:9002-8. Wei L , Clauser E, Alhenc-Gelas F, Corvol P. 1992. The two homologous domains of human angiotensin I-converting enzyme interact differently with competitive inhibitors. Journal of Biological Chemistry 267:13398-405. Wu J, Ding X . 2001. Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry 49:501-6. Wu J, Ding X . 2002. Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides. Food Research International 35: 367-75. Wu J, Aluko R E , Muir A D . 2002. Improved method for directed high-performance liquid chromatography assay of angiotensin-converting enzyme-catalyzed reactions. Journal of Chromatography A 950:125-30. Yamauchi F, Suetsuna K , inventors; Japan Kokai Tokkyo Koho, assignee. 1996a Sep 3. New tetrapeptides and pentapeptides of hydrolyzate of soy protein and their chemical synthesis as angiotensin converting enzyme inhibitors. Japan patent JP 08225593. Yamauchi F, Suetsuna K , inventors; Japan Kokai Tokkyo Koho, assignee. 1996b Oct 15. Tetrapeptides and pentapeptides from soy protein hydrolyzate as antihypertensives. Japan patent JP 08269087. Yang Y , Marczak E D , Yokoo M , Usui H , Yoshikawa M . 2003. Isolation and antihypertensive effect of ACE-inhibitory peptides from spinach rubisco. Journal of Agricultural and Food Chemistry 51:4897-902. Yang Y , Marczak ED, Usui H , Kawamura Y , Yoshikawa M . 2004. Antihypertensive properties of spinach leaf protein digest. Journal of Agricultural and Food Chemistry 52:2223-5. Yano S, Suzuki K , Funatsu G. 1996. Isolation from a-zein hydrolysate of thermolysin peptides with angiotensin converting inhibitory activity. Bioscience Biotechnology & Biochemistry 60:661-3. Yokoyama K , Chiba H , Yoshikawa M . 1992. Peptide inhibitors for angiotensin I converting enzyme from thermolysin digest of dried bonito. Bioscience Biotechnology & Biochemistry 56:1541-5. Yust M M , Pedroche J, Calle-Giron J, Alaiz M , Millan F, Vioque J. 2003. Production of A C E inhibitory peptides by digestion of chickpea legumin with alcalase. Food Chemistry 81:363-9.  32  CHAPTER II Angiotensin I-Converting Enzyme Inhibitory Peptides from In vitro Pepsin-Pancreatin Digestion of Soy Protein 1  2.1 Introduction Soybean is a traditional food in Asia and has been a part of the Asian diet for many years. In 1999, the United States Food and Drug Administration approved a health claim stating 'diets low in saturated fat and cholesterol that include 25 grams of soy protein per day may reduce the risk of heart disease' (US F D A 1999). Heart disease is a major health concern in Canada and the United States, because it is the number one leading cause of death (US F D A 1999; Health Canada 2002). Heart diseases, such as arteriosclerosis, coronary heart disease, stroke, peripheral arterial disease and heart failure may be caused by hypertension or high blood pressure greater than 140 mm Hg systolic and/or 90 mm Hg diastolic pressure (Kannel and others 2003; Izzo and others 2003). Hypertension is commonly treated with antihypertensive or blood pressure lowering drugs, such as captopril, benazepril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril and trandolapril (Sica 2003). These drugs are angiotensin I converting enzyme (ACE) inhibitors; they inhibit A C E and subsequently prevent conversion of inactive hormone angiotensin I (DRVYIHPFHL) to active hormone angiotensin II (DRVYIHPF) (Guyton and Hall 1996; Sica 2003; Skidgel and Erdos 2003). Angiotensin II raises blood pressure by vasoconstriction as well as stimulation of synthesis and release of aldosterone, a hormone that promotes sodium and water retention in the kidneys, and thus increases blood volume in blood vessels. Furthermore, inhibition of A C E prevents degradation of bradykinin, a vasodilator. In addition to A C E inhibitors, other antihypertensive drugs include diuretics, beta-blockers, calcium channel blockers, and angiotensin receptor blockers (Basile 2004). However, A C E inhibitors are the most commonly prescribed drugs, because they cause fewer adverse side effects than other antihypertensive drugs (Fotherby and Panayiotou 1999). Recent research studies have reported discoveries of soy peptides that inhibit A C E in chemical assays as well as rat studies (Okamoto and others 1995; Shin and others 1995; Ahn and others 2000; A n and others 2000; Shin and others 2001; Wu and Ding 2001, 2002; Rhyu and others 2002; Wu and others 2002; Chen and others 2003, 2004). These peptides were produced by fermentation or enzyme digestion of soy protein. Enzymes, such as alcalase, papain, trypsin, pancreatin, Bacillus subtilis protease, and pepsin, have been used individually for the hydrolysis of soy (Ahn and others 2000; A n and others 2000; Wu and Ding 2001, 2002; Wu and others 2002; Chen and others 2003). However, a combination of pepsin and pancreatin has not been used to investigate if A C E inhibitory peptides will be produced in an in vitro digestion model system with enzymes similar to those in the gastrointestinal digestive system of humans.  'A version of this chapter has been accepted for publication. Lo WMY, Li-Chan ECY. 2005. Angiotensin Iconverting enzyme inhibitory peptides from in vitro pepsin-pancreatin digestion of soy protein. Journal of Agricultural and Food Chemistry 53. Available online March 30, 2005.  33  Therefore, the objective of this study was to investigate i f A C E inhibitory soy peptides would be produced in an in vitro digestion model system using enzymes similar to digestive enzymes in humans. In addition to monitoring A C E inhibitory activity in the total soy protein digest, the possibility of generating soy peptide fractions with more potent activity than the unfractionated digest was investigated by measuring activity of fractions obtained after ultrafiltration, anion exchange, reversed-phase, gel filtration and immobilized metal affinity chromatography.  2.2 Materials and Methods 2.2.1 Materials Soy protein isolate (SPI, A D M Protein Specialities Division, Decatur, IL) was donated by Yves Veggie Cuisine Inc., a division of Hain Celestial Canada (Delta, B C , Canada). Pepsin (2500-3500 units/mg protein, catalogue no. P-7012, E C 3.4.23.1), pancreatin (8xUSP, catalogue no. P-7545, E C 2324689, contains many enzymes, including amylase, trypsin, lipase, ribonuclease and protease), hippuryl-histidyl-leucine (HHL, catalogue no. H-1635), A C E (from rabbit lung, 3.1 units/mg protein, catalogue no. A-6778, E C 3.4.15.1), A C E inhibitor (pGlu-TrpPro-Arg-Pro-Gln-Ile-Pro-Pro, catalogue no. A-0773), captopril (catalogue no. C-8856) and L leucine (catalogue no. L-8912) were purchased from Sigma-Aldrich (St. Louis, M O , USA). 2, 4, 6 - Trinitrobenzenesulfonic acid (TNBS, product no. 8746) was purchased from Eastman Kodak Co. (Rochester, N Y , USA). Purified bovine lactoferricin (3196 Da) was purchased from the Centre for Food Technology (Hamilton, Australia).  2.2.2 In vitro Digestion In vitro digestion was carried out in triplicate according to the method of Garrett and others (1999). Soy protein isolate (SPI) solution (5% w/v, in distilled, deionized water containing 0.02% sodium azide) was adjusted to pH 2.0 with 1 N HC1 and pepsin (4% w/w, protein basis) was added. The solution was incubated at 37°C for 1 hour before pH was adjusted to 5.3 with 0.9 M NaHC03. Pancreatin (4%> w/w, protein basis) was added, and pH was adjusted to 7.5 with 1 N NaOH. The solution was incubated at 37°C for 2 hours and then submerged in a boiling water bath for 10 minutes to terminate the digestion. The SPI peptide digest was centrifuged at 16 000 g for 10 minutes and the supernatant containing soy peptides (58%> yield) was collected and stored at -25°C until used. For the monitoring of % A C E inhibition at intervals during digestion, aliquots'of SPI digest were removed at 0, 20, 40, 60, 90, 120, and 180 minutes during in vitro digestion. The aliquots were submerged in a boiling water bath for three minutes to inactivate pepsin and pancreatin. The aliquots were cooled and stored at -25°C until used to carry out A C E inhibitory assay.  2.2.3 Degree of Hydrolysis (DH) Degree of hydrolysis was analyzed in triplicate according to the method of Adler-Nissen (1979) and Kwan and others (1983) with modifications by Liceaga-Gesualdo and Li-Chan (1999). Aliquots (1.0 mL) of SPI peptide digest were removed after 30, 60, 120 and 180 minutes of in vitro digestion. The aliquot was mixed with trichloroacetic acid (1.0 mL, 24%>) and 34  centrifuged at 12 350 g for 5 minutes. The supernatant (0.2 mL) was added to sodium borate buffer (2.0 mL, 0.05 M , pH 9.2) and 2,4,6-trinitrobenzenesulfonic acid (1.0 mL, 4.0 mM) and incubated at room temperature for 30 minutes in the dark. A n aliquot of NaH^PO^ (1.0 mL, 2.0 M) containing Na S03 (18 mM) was added and the absorbance was measured at 420 nm using a spectrophotometer. Degree of hydrolysis was calculated as % D H = (h / h t) x 100, where D H = percent ratio of the number of peptide bonds broken (h) to total number of bonds per unit weight (h ) and h = 7.75 mequiv/g of soy protein (22). L-leucine was used as a standard in the D H assays. 2  to  tot  tot  2.2.4 ACE Inhibitory Activity Assay A C E inhibitory activity assay was carried out in triplicate according to the method of Cushman and Cheung (1971) with modifications by Wu and others (2002). SPI or digest sample (30 uL), H H L (150 uL, 6.5 mM) and A C E (25 uL, 2.5 mU) were incubated at 37°C for 1 h. HC1 (250 uL, 1 N) and ethyl acetate (1.5 mL) were added and the mixture was mixed by vortexing and centrifuged at 2000 g for 5 minutes. After centrifugation, 1.0 mL of the top layer (containing hippuric acid extracted into ethyl acetate) was taken and ethyl acetate was evaporated off. The residual hippuric acid was re-dissolved with distilled, deionized water (1 mL) prior to measurement of the absorbance at 228 nm. IC50 value was defined as the amount of peptide required to inhibit A C E activity by 50%. The %> A C E inhibition was defined as the percentage of A C E activity inhibited by a specific amount of peptide. Results were reported as mean + SD. A C E inhibitory activity of SPI digest during digestion and after 3 hours of sequential digestion was based on triplicate in vitro digestion experiments, each.assayed in triplicate. Triplicate A C E inhibitory assays were carried out on a single SPI digest that was subjected to various chromatographic fractionation steps. Captopril and A C E inhibitor (pGluTrp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) were used as standards.  2.2.5 Anion Exchange Chromatography (AEC) The supernatant (45 mL) from in vitro digestion was loaded onto a column (2.5 x 20 cm, i.d., Bio-Rad Laboratories Inc., Hercules, C A , U S A ) packed with D E A E Sephacel anion exchange resin (Amersham Bioscience Piscataway, N J , U S A ) and equilibrated in phosphate buffer (0.05 M , pH 7) (Amersham Bioscience Piscataway, N J , USA). Fractions were eluted using the equilibration buffer containing NaCl (0.2, 0.5, 1, 1.5 and 2 M ) , at a flow rate of 2.5 mL/min, and the elution was monitored at 280 nm. . SPI peptide fractions (1, 1.1, 1.2, 1.3, 2, 3) were collected. Corresponding fractions from two replicate chromatography runs were pooled. The pooled fractions were lyophilized and stored at -25°C until used.  2.2.6 Ultrafiltration Each of the lyophilized soy peptide fractions from A E C was ultrafiltered sequentially using an ultrafiltration unit (Amicon®, Millipore Corporation, model 8050, Beverly, M A , USA) through membranes (Amicon®) of 10 000 and 3000 nominal molecular weight limit (NMWL) under the conditions of 40-psi nitrogen gas and 4°C. The filtrate was collected, lyophilized, and stored at -25°C until used.  35  2.2.7 Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) Lyophilized SPI peptide fraction (1.1) collected from A E C was re-dissolved into 6 mL distilled, deionized water and 50 uL of the fraction was loaded onto a Jupiter300™ C-18 reversed-phase column (4.6 x 250 nm, i.d., Phenomenex, U S A ) that was equilibrated in 2% acetonitrile containing T F A (0.05%, v/v) and connected to a high performance liquid chromatography (HPLC) system (Hewlett Packard Series 1050, Waldbrunn, Germany). SPI peptide fractions were eluted using a gradient of acetonitrile (2 - 16.5%, v/v, over 60 minutes, followed by 16.5 - 100%, v/v, over 20 minutes) containing T F A (0.05%, v/v) at a flow rate of 0.5 mL/min and elution was monitored at 214 nm. SPI peptide fractions (1.1.1, 1.1/2, 1.1.3, 1.1.4, and 1.1.5) were collected and corresponding fractions from 36 replicate chromatography runs were pooled. The pooled fractions were lyophilized and stored at -25°C until used.  2.2.8 Gel Filtration Fast Protein Liquid Chromatography (GF-FPLC) Lyophilized SPI peptide fractions (1.1.2, 1.1.3, 1.1.4, and 1.1.5) collected from H P L C were re-dissolved in distilled, deionized water, and 30 to 250 uL of each fraction was loaded onto a Superdex Peptide 10/300 G L gel filtration column (10 x 300-310 nm, i.d., Amersham Bioscience, Piscataway, N J , USA) that was equilibrated in 30% acetonitrile containing T F A (0.05%, v/v) and connected to a fast protein liquid chromatography (FPLC) system (Amersham Bioscience, Piscataway, N J , USA). SPI peptide fractions were eluted using acetonitrile (30%, v/v) containing T F A (0.05%, v/v) at a flow rate of 0.2 mL/min and elution was monitored at 214 nm. SPI peptide fractions were collected and corresponding fractions from four replicate chromatography runs were pooled. The pooled fractions were lyophilized and stored at -25°C until used. Phenylalanine (FW 165), lactoferricin (FW 3196 and 3124), and A C E inhibitor (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, F W 1101.3) were used as molecular weight standards.  2.2.9 Immobilized Metal Affinity Chromatography (IMAC) Chelating Sepharose Fast Flow resin (Amersham Bioscience) was packed into a column (1.5 x 10 cm, i.d., Bio-Rad Laboratories Inc., Hercules, C A ) and charged with 0.05 M Q 1 O 2 solution. SPI peptide supernatant (17.5 to 35 mL) from in vitro digestion was loaded onto the column that was equilibrated using 0.02 M phosphate buffer (at pH 7) containing 1.0 M NaCl. SPI peptide fractions were eluted using the equilibration buffer at pH 4 and 3. Elution was monitored at 280 nm. SPI peptide fractions were collected and corresponding fractions from four replicate chromatography runs were pooled. The pooled fractions were lyophilized and stored at -25°C until used.  2.2.10 Amino Acid Composition and Peptide Concentration Lyophilized SPI peptide fractions were sent to The Advanced Protein Technology Centre at The Hospital for Sick Children (Toronto) for the analysis of amino acid (AA) content after pre-column derivatization based on Waters Pico-Tag H P L C System. Peptide concentration was calculated based on A A analysis results.  2.2.11 Statistical Analysis Using Minitab™ Statistical Software (version 13.30), analysis of variance (ANOVA) using general linear model and pairwise comparisons with Tukey's method were carried out to compare the IC50 values of SPI samples. 36  2.3 Results and Discussion 2.3.1 Degree of Hydrolysis and ACE Inhibition during In Vitro Digestion Degree of hydrolysis increased most rapidly from 60 to 120 minutes of digestion with pancreatin (Figure 2.1). The hydrolysis tended to slow down during 120 to 180 minutes of digestion with pancreatin, as shown by the levelling off effect in degree of hydrolysis. In contrast to the time course of hydrolysis, the inhibitory activity against A C E increased most rapidly from 0 to 20 minutes of digestion with pepsin and remained at a plateau during the next 20 to 60 minutes of digestion with pepsin (Figure 2.1). Inhibitory activity against A C E decreased during 60 to 90 minutes of digestion with pancreatin and remained at that level upon further incubation until the termination of digestion at 180 minutes. These results indicate that the soy peptides generated during pepsin digestion had greater A C E inhibitory activity than soy peptides after subsequent digestion with pancreatin. The study by Ahn and others (2000) observed similar findings with respect to the time course of digestion of defatted soybean peptides with Bacillus subtilis protease (40°C, 6 hours). The A C E inhibitory activity of defatted soybean peptides increased most rapidly at the start of digestion from 0 to 1 hour, followed by levelling off to a plateau during the remaining 1 to 6 hours. Besides soy protein, in vitro digestion of other food proteins also revealed a similar pattern, in which peptides produced during the initial stages of digestion had greater A C E inhibitory activity than peptides produced during the later stages of digestion. Moreover, peptides produced during pepsin digestion had greater A C E inhibitory activity than peptides produced during pancreatin digestion (Megias and others 2004; Hernandez-Ledesma and others 2004; Yang and others 2004). In the study by Megias and others (2004), sunflower protein was digested sequentially with pepsin (37°C, 3 hours) and pancreatin (37°C, 2.5 hours). The sunflower peptides produced after 10 minutes of digestion with pepsin had greater A C E inhibitory activity than peptides produced during the remaining pepsin digestion and pancreatin digestion. In another study, commercial fermented bovine milk was digested with pepsin (37°C, 1.5 hours) and then with corolase (porcine pancreatic enzymes containing trypsin, chymotrypsin, aminopeptidase, and carboxylpeptidase) for 2.5 hours (Hernandez-Ledesma and others 2004). The milk peptides produced after 1.5 hours of digestion with pepsin had greater A C E inhibitory activity than the peptides produced during digestion with corolase. Furthermore, Hernandez-Ledesma and others (2004) found that increasing digestion time with corolase led to production of peptides with decreasing A C E inhibitory activity. Another study by Yang and others (2004) digested spinach leaf protein at 37°C using three different methods: digestion with pepsin for 5 hours, digestion with pancreatin for 5 hours, and digestion with pepsin for 5 hours followed by subsequent digestion with pancreatin for 5 hours. Yang and others (2004) found that spinach leaf peptides produced after pepsin digestion had greater A C E inhibitory activity than spinach leaf peptides produced after pancreatin digestion or spinach leaf peptides produced after pepsin and pancreatin digestion. Several studies provide possible reasons for the stronger A C E inhibitory peptides after pepsin digestion. The C-terminal residues of A C E inhibitory peptides play a predominant role in competitive binding to the active site of A C E (Cheung and others 1980). Peptides with hydrophobic and aromatic amino acids at the C-terminal are among the most favourable for strong competitive binding to A C E (Cheung and others 1980; Clare and Swaisgood 2000; Je and others 2004). Since pepsin cleaves at the carboxyl end of hydrophobic and aromatic amino acids (Phe, Tyr, Tip, Leu), it results in peptides with hydrophobic and aromatic amino acids at the C37  terminal (Nelson and Cox 2000). These peptides bind tightly to A C E at its active site and compete with angiotensin I for occupancy; therefore, A C E cannot bind to angiotensin I to convert it to angiotensin II (Cushman and others 1987). Another study by Gibbs and others (2004) found that protease (pepsin) of lower specificity produced more oligopeptides and a higher percentage of bioactive peptides from whey than protease (trypsin) of higher specificity.  2.3.2 ACE Inhibitory Activity of SPI Peptides SPI digest collected after 180 minutes of digestion had an IC50 value of 0.28 + 0.04 mg/mL (2.5 + 0.4 mM), whereas 0.73 mg/mL of undigested SPI (collected at 0 minutes) demonstrated no inhibition against A C E (Table 2.1). The IC50 value of SPI digest found in the present study after 180 minutes of sequential pepsin-pancreatin digestion is comparable to the IC50 values of soy digest reported in other studies using other enzymes. For example, IC50 values of 0.34, 0.1964 and 0.73 mg/mL were reported for soy digest collected from alcalase digestion (50°C, 12 hours), Bacillus subtilis protease digestion (40°C, 1 hour), and pepsin digestion (39°C, 12 hours), respectively (Ahn and others 2000; Wu and Ding 2002; Chen and others 2004), while Shin and others (2001) reported that soy peptides in a commercial fermented soybean paste had IC50 value of 0.2763 mg/mL. A C E inhibitory activity of captopril and A C E inhibitor (pGlu-Trp-Pro-Arg-Pro-Gln-IlePro-Pro) were determined and compared to that of soy peptides. Captopril and A C E inhibitor had stronger A C E inhibitory activity than that of soy peptides. The IC50 values of captopril and A C E inhibitor were found to be 7.2 ng/mL and 455 ng/mL, respectively (Table 2.1), which compares closely to the reported IC50 values of 1.3 to 8.9 ng/mL for captopril (Mullally 1997; Fujita and Yoshikawa 1999; Hsu and others 2002; Vermeirssen and others 2002) and 100 to 300 ng/mL for A C E inhibitor (Ondetti and others 1971).  2.3.3 Anion Exchange Chromatography (AEC) After 180 minutes of in vitro digestion, SPI digest was collected and the supernatant containing digested SPI (soy peptides) was collected. The supernatant was loaded onto an anion exchange column to separate soy peptides on the basis of charge. Soy peptides eluted from the A E C column as three fractions: fraction 1 eluted at 0 M NaCl, fraction 2 eluted at 0.5 M NaCl, and fraction 3 eluted at 1 M NaCl (Figure 2.2). Each fraction was filtered sequentially through two ultrafiltration membranes with molecular weight cut-offs of 10 000 and 3000 N M W L . Each of the fractions passed through the 10 000 N M W L membrane and the filtrate collected also passed through the 3000 N M W L membrane. Subsequently, the filtrate that passed through the 3000 N M W L membrane was collected and adjusted to 0.66 m M and the % A C E inhibitory activity of the filtrate was determined. Fraction 1 filtrate exhibited 30% inhibition against A C E , whereas fraction 2 filtrate showed 0% inhibition against A C E . The IC50 value of fraction 1 filtrate was 0.26 + 0.02 mg/mL (2.3 ± 0.2 mM) (Table 2.1). Due to the negligible % A C E inhibitory activity of fraction 2 filtrate, its IC50 value was not investigated. Fraction 1 filtrate was loaded to the A E C column for a second run through the column, because a large amount of soy peptides eluted in fraction 1. The second run was carried out to confirm that the A E C column capacity had not been exceeded during the first run. As shown in Figure 3, the majority of soy peptides in the fraction 1 filtrate eluted at 0 M NaCl, which indicated the A E C column was not unreasonably overloaded during the first run. From the second run, fraction 1 filtrate eluted as three fractions: fractions 1.1 (eluted at 0 M NaCl), 1.2 (eluted at 0 M NaCl), and 1.3 (eluted at 0.2 M NaCl) (Figure 2.3). Fractions 1.1 and 1.2 had I C values of 0.21 + 0.02 mg/mL (1.9 + 0.2 mM) and 0.93 ± 0.08 mg/mL (8.3 ± 0.7 mM), 50  38  respectively (Table 1). Fraction 1.3 at 1.9 m M had no inhibition against A C E ; therefore, the IC50 value of fraction 1.3 was not investigated. The IC50 values of fractions 1 filtrate, 1.1, and 1.2 were comparable to the IC50 values reported in the study by Chen and others (2003). In that study, soy peptides from pepsin digestion (37°C, 24 hours) and subsequently eluted from A E C column had IC50 values of 0.24 mg/mL (fraction SP-I) and 1.2 mg/mL (fraction SP-II). It is' interesting to note that the IC50 values of fraction 1 filtrate and fraction 1.1 are similar to the IC50 value of the unfractionated SPI digest, while fraction 2 filtrate and fractions 1.2 and 1.3 had no or lower A C E activity than the digest. This indicates that A E C of SPI digest did not yield soy peptide fractions with greater A C E inhibitory activity (lower IC50 value) than the digest.  2.3.4 Reversed-phase High Performance Liquid Chromatography (RP-HPLC) Fraction 1.1 from the second A E C was loaded onto a RP-HPLC column to be further separated on the basis of nonpolarity. Fraction 1.1 eluted from RP-HPLC column as five fractions. Fractions 1.1.1, 1.1.2, 1.1.3, 1.1.4 and 1.1.5, eluting at 3.5%, 6%, 9%, 14% and 74% (v/v) acetonitrile, respectively (Figure 2.4), had IC50 values of 0.60 + 0.02 mg/mL (4.5 + 0.2 mM), 0.24 + 0.03 mg/mL (1.8 ± 0.2 mM), 0.13 ± 0.03 mg/mL (1.1 ± 0.3 mM), 0.31 ± 0.02 mg/mL (2.5 + 0.2 mM), and 0.14 + 0.01 mg/mL (1.2 + 0.1 mM), respectively, compared to 0.21 ± 0.02 mg/mL (1.9 ± 0.2 mM) for the starting fraction 1.1 (Table 2.1). The results of this study are contrary to findings reported in other studies, in which fractions with much greater inhibitory activity were obtained after RP-HPLC. Although fractions 1.1.3 and 1.1.5 had A C E inhibitory activity that was significantly (p<0.05) greater than that of SPI digest, the activity of these fractions was not significantly greater than the starting fraction 1.1 prior to RP-HPLC, while fraction 1.1.1 had significantly lower A C E inhibitory activity than SPI digest as well as the starting fraction 1.1. In comparison, Ahn and others (2000) fractionated soy peptides using RP-HPLC and isolated soy peptides (IC50 = 26.52 ug/mL) with greater A C E inhibitory activity than soy peptides (IC50 = 58.82 |ag/mL) from the preceding hydrophobic interaction chromatography. Shin and others (2001) fractionated soy peptides using RP-HPLC and isolated peptides (IC50 = 6.8 ng/mL) with greater A C E inhibitory activity than soy peptides (IC50 = 41.8 ug/mL) from the preceding ultrafiltered fraction. Chen and others (2003) fractionated a soy peptide fraction using RP-HPLC and isolated 4 peptides (IC50 = 39 to 153 uM) with greater A C E inhibitory activity than the preceding fraction (IC50 = 0.24 mg/mL)fromA E C .  2.3.5 Gel Filtration Fast Protein Liquid Chromatography (GF-FPLC) Each of the fractions from RP-HPLC, except fraction 1.1.1, was loaded to GF-FPLC column and separated on the basis of size (Figure 2.5). Fraction 1.1.1 was not selected, because it had lower A C E inhibitory activity than SPI digest. The eluted fractions were collected; their molecular weights and probable peptide lengths were estimated (Table 2.2). It is interesting to note that peptide fractions from GF-FPLC had low molecular weights, which is consistent with previous studies that also noted peptides with A C E inhibitory activity have low molecular weight (Wu and Ding 2002; Dziuba and others 1999). The fractions collected from GF-FPLC were adjusted to 1.2 m M and tested for % inhibition against A C E (Figure 2.6). Fractions 1.1.2.2, 1.1.4.3, 1.1.5.3 were not tested, because of insufficient peptide concentration; despite the high absorbance at 214 nm of these fractions, their actual peptide content as determined by amino acid analysis was too low for the A C E inhibition assay. The A C E inhibitory activity of fractions 1.1.4 and 1.1.5, which were collected from RP-HPLC, increased after further separation using GF-FPLC. Fraction 1.1.4.2 had greater 39  A C E inhibition than fraction 1.1.4; the former (at 1.2 m M , collected from GF-FPLC) had 43 + 5% inhibition against A C E , whereas the latter required two-folds the amount (2.5 + 0.2 m M , collected from RP-HPLC) for 50% inhibition against A C E . Fraction 1.1.5.2 also had greater A C E inhibition than fraction 1.1.5; the former (at 1.2 m M , collected from GF-FPLC) inhibited 73 + 5 %> of A C E activity, whereas the latter (at 1.2 m M , collected from RP-HPLC) inhibited 50% of A C E activity. The A C E inhibitory activity of GF-FPLC fractions from fraction 1.1.2 and 1.1.3 remained about the same as before GF-FPLC. Overall out of all the GF-FPLC fractions, fraction 1.1.5.2 had significantly (p<0.05) strongest inhibition against A C E and also had greater A C E inhibitory activity than the unfractionated SPI digest. On comparison, fraction 1.1.5.2 had lower molecular weight than fraction 1.1.5.1 and was more hydrophobic than fractions 1.1.1, 1.1.2, 1.1.3 and 1.1.4. Therefore, the results suggest that peptides with low molecular weights that are hydrophobic may have stronger inhibition against A C E .  2.3.6 Immobilized Metal Affinity Chromatography (IMAC) The study by Shin and others (2001) reported a soy peptide, His-His-Leu, with strong A C E inhibitory activity of IC50 = 2.2 ug/mL. Therefore, SPI digest from in vitro digestion was loaded onto an I M A C column to isolate histidine-containing soy peptides. SPI digest was collected after 180 minutes of in vitro digestion and centrifuged to remove undigested SPI. The supernatant containing soy peptides was loaded to the I M A C column to separate the peptides on the basis of accessible histidine. Soy peptides with available histidine residues will bind to the I M A C column, whereas other soy peptides will pass through in the unbound fraction Soy peptides eluted from the I M A C column as three fractions at pHs 7, 4, and 3 (Figure 2.7). A C E inhibitory assay was carried out on the fractions that eluted at pH 7 and 4 and their IC50 values were 0.24 + 0.03 m M and 0.53 ± 0.04 m M , respectively. The fraction at pH 7 includes soy peptides that did not bind to the I M A C column. The significantly (p<0.05) lower IC50 value of the fraction at pH 7 indicated that soy peptides without acessible histidine had significantly (p<0.05) greater A C E inhibitory activity than histidine-rich soy peptides (fraction eluted at pH 4). The findings from RP-HPLC and GF-FPLC also showed a similar trend in that the fractions (1.1.3, 1.1.5, and 1.1.5.2) with high A C E inhibitory activity had very low amounts of His, as shown by subsequent amino acid analysis results in Tables 2.3 and 2.4. These findings suggested that peptides with accessible histidine may not necessarily have high A C E inhibitory activity. Other factors, such as amino acid sequence, may also contribute to the A C E inhibitory activity. For example, the sequence similarity of His-His-Leu to the dipeptide (His-Leu) at the C-terminal of angiotensin I may have aided the binding of His-His-Leu to A C E and thus inhibition against A C E . A C E inhibitory assay was not carried out for the fraction that eluted at pH 3, because the peak was small and had insufficient amount of SPI peptides.  2.3.7 Amino Acid Composition SPI digest and soy peptides from A E C , RP-HPLC, and GF-FPLC were analyzed for amino acid composition. The amino acid composition of the fractions reflected the type of chromatography used to obtain the fractions (Tables 2.3 to 2.6). As shown in Table 2.3, much higher amounts of negatively charged amino acids (Asp and Glu) were found in the bound fractions (2, 1.2 and 1.3) than the unbound fractions (1 and 1.1) from A E C . Table 2.4 showed much higher amounts of hydrophilic amino acids (Arg and Lys) in the fraction (1.1.1) that eluted early than the fractions (1.1.2, 1.1.3, 1.1.4 and 1.1.5) that eluted later during RP-HPLC. Fraction 1.1.1 also had much lower amounts of certain hydrophobic amino acids (lie and Leu) than 40  fractions 1.1.2, 1.1.3, 1.1.4 and 1.1.5. Moreover, as expected, the bound fraction (pH 4) had much higher amounts of His than the unbound fraction (pH 7) from I M A C (Table 2.6). A n interesting finding from Table 2.5 showed that although fraction 1.1.3.3 had extremely high amounts of Phe (95%), it had similar A C E inhibitory activity as the other fractions that had much lower amounts of Phe, such as fractions 1.1.2.1 (0.51% Phe), 1.1.3.1 (0.31% Phe), 1.1.3.2 (4.89% Phe), 1.1.4.1 (0.96% Phe), 1.1.4.2 (7.06% Phe) and 1.1.5.1 (4.01% Phe). In general, the results in Tables 2.3 to 2.6 show a diverse amino acid composition among fractions with A C E inhibitory activity, which reflects the findings in the published literature. Other studies that have reported A C E inhibitory peptides have also identified the amino acid compositions of the peptides as being composed of a variety of amino acids (Shin and others 2001; Wu and Ding 2002; Chen and others 2003; Dziuba and others 1999; Yamauchi and Suetsuna 1996b;Kim and others 1999). However, the C-terminal residues of A C E inhibitory peptides played a predominant role in competitive binding to the active site of A C E (Cheung and others 1980). Peptides with hydrophobic and aromatic amino acids at the C-terminal are among the most favourable for strong competitive binding to A C E (Cheung and other 1980; Clare and Swaisgood 2000; Je and others 2004). Overall, in reviewing the literature, soy peptides with A C E inhibitory activity have been reported to contain Gly, nonpolar amino acid (Ala or Leu), aromatic amino acid (Phe), polar amino acid (Gin, Asn or Pro), or negatively charged amino acid (Asp or Glu) at the carboxyl terminal, and, Gly, nonpolar amino acid (He or Val), aromatic amino acid (Tyr or Phe), polar amino acid (Gin), positively charged amino acid (His) or negatively charged amino acid (Asp) at the amino terminal. Val was the most frequently observed amino acid in A C E inhibitory soy peptides, followed by these amino acids in decreasing frequency: Leu, Phe, Asp, Pro, Gin, Gly, Ala, He, Asn, Glu, His, Thr, Arg, Met, and Lys. The results from this study showed that in vitro digestion of SPI with pepsin and pancreatin produced soy peptides with A C E inhibitory activity. This finding suggests the potential production of A C E inhibitory peptides upon consumption and digestion of SPI, since the pepsin and pancreatin enzymes used in this study are similar to digestive enzymes in a gastrointestinal digestive system. Inhibitory activity was observed within the first 20 minutes of pepsin digestion; subsequent digestion with pancreatin resulted in lower activity. The soy peptides produced after 3 hours of in vitro sequential digestion with pepsin and pancreatin were all smaller than 3000 Daltons. Fractionation of SPI digest based on reversed-phase, gel filtration and immobilized metal chelate affinity chromatography did result in soy peptide fractions with significantly greater A C E inhibitory activity than the unfractionated digest. Despite their diversity in amino acid composition, many of the fractions exhibited A C E inhibitory activity, and no single fraction was isolated with extraordinarily high activity. Nonetheless, peptides with low molecular weights that were hydrophobic had higher inhibition against A C E . In conclusion, these findings suggest that the A C E inhibitory activity after 3 hours of in vitro sequential digestion of SPI with pepsin and pancreatin may be attributed to the generation of numerous peptides contributing to the overall A C E inhibitory activity of the digest.  41  2.4 Figures •  inhibition (%)  Digestion Time (min)  Figure 2.1 Degree of hydrolysis (—) and A C E inhibition (— ) as a function of time course of digestion of SPI with pepsin (0 - 60 min) and pancreatin (60 - 180 min). Aliquots of SPI taken during digestion were adjusted to 0.29 mg/mL for the assay for % A C E inhibition. Data points with different letters (a, b, c for A C E inhibition, or w, x, y, z for degree of hydrolysis) are significantly different (p<0.05).  42  Figure 2.2 DEAE-anion exchange chromatography profile of SPI digest collected after 180 minutes of sequential pepsin-pancreatin digestion. Fractions were eluted by stepwise gradient of NaCl (0, 0.5, 1.0, 1.5 and 2.0 M). Fractions 1 and 2 were collected and adjusted to 0.66 m M for A C E inhibition assay. Fraction 1 was also subjected to a second anion exchange chromatography (see Figure 2.3).  43  Volume (mL)  Figure 2.3 Profile from second anion exchange chromatography of fraction 1.  Time (min)  Figure 2.4 Reversed-phase H P L C profile of fraction 1.1 from anion exchange chromatography; fractions were eluted by increasing concentration of Buffer B (acetonitrile containing 0.05% T F A (v/v)).  45  20  40  60  80  100  120  140  160  -1.0 Time (min)  Figure 2.5 GF-FPLC profiles of fractions 1.1.2 (A), 1.1.3 (B), 1.1.4 (C), and 1.1.5 (D) obtained from RP-HPLC of fraction 1. Fractions were eluted by 30% (v/v) acetonitrile containing 0.05% T F A (v/v).  47  80  d  c o  5 uu o <  ab  ab  ab  40  i  20]  1.1.2.1  1.1.3.1  1.1.3.2  1.1.3.3  1.1.4.1  1.1.4.2  1.1.5.1  1.1.5.2  Fractions  Figure 2.6 A C E inhibitory activity of fractions from GF-FPLC. (Concentration of soy peptides in each fraction was adjusted to 1.2 m M for the assay). Bars with different letters are significantly different (p<0.05).  48  Figure 2.7 I M A C profile of soy peptide digest collected after 180 minutes of sequential pepsinpancreatin digestion. Fractions were collected after elution with phosphate buffer (0.02 M containing 1.0 M NaCl) at pH 7, 4 and 3.  49  2.5 Tables Table 2.1 IC50 values of SPI, SPI digest and collected fractions. Values with different letters are significantly different (p<0.05). Fraction  IC " 50  SPI SPI digest  (mg/mL) ND' 0.28 ± 0.04  Anion Exchange 1 filtrate 2 filtrate  0.26 ± 0.02 ND  1.1 1.2 1.3  0.21 +0.02 0.93 ± 0.08 ND  RP-HPLC 1.1.1 1.1.2 1.1.3 1.1.4 1.1.5  0.60±0.02 0.24 ± 0.03 0.13±0.03 0.31 ± 0 . 0 2 0.14±0.01  Captopril A C E inhibitor  7.2 ng/mL 455 ng/mL  (mM) (ND ) (2.5 ± 0.4 )  Peptide Content (mg)  J  Peptide Yield (%)  1  b  b  1  2896  100  (2.3 + 0.2 ) (ND )  2030 738  70 25  (1.9 + 0.2 ) (8.3 + 0.7 ) (ND )  1569 230 107  54 8.0 3.7  (4.5 ± 0.2 ) (1.8 + 0.2 ) (1.1 +0.3 ) (2.5 ± 0.2 ) (1.2 ± 0.1 )  764 220 67 69 368  26 7.6 2.3 2.4 13  b  b  1  ab  d  1  ab d  1  c  ab a b a  C  ab  a  b a  33 n M 413 n M  N D = not determined, 0% A C E inhibition at 0.73 mg/mL of SPI, 0.66 m M of fraction 2 filtrate and 1.9 m M of fraction 1.3 IC5o value was calculated from total amino acid content (picomoles or nanograms) from amino acid analysis. Peptide content was calculated from amino acid analysis and volume of the digest and collected fractions !  2  3  50  Table 2.2 Molecular weights of fractions eluted from gel-filtration FPLC. Fraction 1.1.2.1 1.1.2.2 1.1.3.1 1.1.3.2 1.1.3.3 1.1.4.1 1.1.4.2 1.1.4.3 1.1.5.1 1.1.5.2 1.1.5.3  Molecular Weight (Daltons) 350±27 < 165 519 + 0 321 + 14 < 165 653±27 371 +0 < 165 894 + 53 . 405 ± 4 8 < 165  Probable Peptide Length (expressed as number of amino acids) 2-3 1 4-5 2-3 1 4-5 2-3 1 7-8 3 1  51  Table 2.3 Amino acid composition of SPI digest and fractions from anion exchange chromatography.  Asx (Asp + Asn) Glx (Glu + Gin) Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys lie Leu Phe Lys Trp  % mole Anion Exchange Chromatography Fractions 1.3 1 2 1.1 1.2  SPI Digest 11.80  11.30  20.90  10.10  18.20  12.90  17.90  16.00  42.10  12.90  29.40  31.60  6.95 8.43 2.08 6.04 4.14 6.21 6.50 2.20 5.51 1.23 ND 4.93 7.76 4.11 4.14 ND  7.09 8.27 2.10 6.63 4.45 6.44 6.62 2.33 5.67 1.20 ND 4.96 7.96 4.22 4.69 ND  5.94 7.46 1.66 2.80 1.55 1.64 6.53 1.09 1.98 0.57 ND 0.49 2.02 0.78 2.53 ND  7.51 8.60 2.48 7.44 4.72 7.01 6.45 2.44 6.16 1.31 ND 5.30 8.75 4.18 4.69 ND  4.46 8.16 1.61 2.03 4.28 5.19 7.14 1.79 5.03 0.97 ND 4.27 4.77 1.80 0.86 ND  5.11 8.39 2.58 3.43 3.96 4.43 8.71 1.54 4.14 0.74 ND 3.98 4.62 2.08 1.79 ND  1  1  1  1  1  1  1  1  1  1  1  1  ' N D = not determined  52  Table 2.4 Amino acid composition of fractions from reversed-phase H P L C .  Asx (Asp + Asn) Glx (Glu + Gin) Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys He Leu Phe Lys Trp 1  1.1.1 8.10  % mole Reversed-Phase H P L C Fractions 1.1.3 1.1.2 1.1.4 3.83 12.10 8.38  1.1.5 9.53  18.50  8.40  16.00  11.10  10.10  4.46 5.38 1.35 2.52 2.88 3.92 3.15 8.40 3.48 0.79 ND 11.50 37.18. 0.10 2.64 ND  7.67 9.59 2.45 2.53 6.43 7.71 4.43 1.57 6.39 0.70 ND 8.26 10.20 0.30 3.63 ND  6.84 9.52 2.38 2.82 5.37 5.88 11.70 1.59 10.70 1.19 ND 7.40 6.92 3.99 4.12 ND  6.06 10.30 1.61 2.44 4.84 5.39 13.90 2.02 7.19 0.96 ND 9.61 8.85 4.78 2.41 ND  7.78 6.62 3.41 19.50 3.98 . 10.60 1.85 0.08 6.74 1.00 ND 0.27 0.30 0.06 11.20 ND 1  1  1  1  1  1  1  1  1  1  N D = not determined  53  Table 2.5 Amino acid composition of fractions from gel-filtration FPLC.  Asx (Asp + Asn) Glx (Glu + Gin) Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys He Leu Phe Lys Trp  % mole Gel Filtration F P L C Fractions 1.1.3.2 1.1.3.3 1.1.4.1 1.1.4.2 7.32 9.80 0.08 11.00  1.1.2.1 3.42  1.1.3.1 11.20  8.45  18.60  9.24  0.08  11.60  5.20 5.99 1.60 2.55 3.13 3.74 3.18 0.87 3.92 1.00 ND 13.40 38.80 0.51 4.24 ND  5.31 8.94 3.03 3.09 5.09 8.85 7.75 0.43 7.69 0.81 ND 3.46 6.29 0.31 9.23 ND  6.93 7.68 1.54 1.88 5.09 4.98 1.91 2.13 3.84 0.84 ND 13.70 22.90 4.89 2.69 ND  0.14 0.14 0.00 0.02 0.09 0.06 0.03 3.22 0.00 0.00 ND 0.43 0.74 95.00 0.00 ND  6.90 9.82 1.86 2.92 5.68 7.42 12.80 0.78 7.71 1.01 ND 6.76 4.98 0.96 7.86 ND  1  1  1  1  1  1  1  1  1  1  1.1.5.1 9.28  1.1.5.2 11.10  10.10  10.40  6.77  5.05 7.26 2.51 2.75 4.84 5.10 10.00 0.10 14.40 1.87 ND 8.72 9.98 7.06 2.95 ND  5.80 10.20 1.84 2.49 5.08 5.41 15.50 1.83 6.59 1.43 ND 8.72 7.76 4.01 3.67 ND  5.44 8.44 0.94 1.64 3.20 4.82 9.35 2.41 6.90 1.30 ND 13.10 12.40 10.50 1.73 ND  1  1  1  1  1  1  ' N D = not determined  54  Table 2.6 Amino acid composition of fractions from I M A C . % mole I M A C Fractions Asx (Asp + Asn) Glx (Glu + Gin) Ser Gly His Arg TinAla Pro Tyr Val Met Cys He Leu Phe Lys Trp  pH 7 11.10 15.80 7.48 8.93 2.19 6.43 4.50 6.54 6.87 2.55 5.80 1.19 ND 5.09 8.06 3.91 3.65 ND 1  1  pH 4 4.58 8.08 3.85 6.59 16.90 13.20 3.16 3.45 9.35 6.11 3.75 1.25 ND • 3.08 7.22 6.84 2.68 ND 1  1  ' N D = not determined  55  2.6 Bibliography Adler-Nissen J. 1979. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry 27:1256-62. Ahn SW, K i m K M , Y u K W , Noh DO, Suh HJ. 2000. Isolation of angiotensin I converting enzyme inhibitory peptide from soybean hydrolysate. Food Science and Biotechnology 9:378-81. A n C W , Lee H B , Nam HS, K i m JH, inventors. Nong Shim Co., Ltd., assignee. 2000 Mar 15. Manufacturing method of soy protein enzyme hydrolyzate containing hypotensive factor. Korean patent K R 2000014663. Basile J. 2004. A n evidence-based approach to selecting initial antihypertensive therapy. In: Egan B M , Basile JN, Lackland DT, editors. Hypertension. Philadelphia: Hanley & Belfus, Inc. p 231-5. Chen JR, Okada T, Muramoto K , Suetsuna K , Yang SC. 2003. Identification of angiotensin I converting enzyme inhibitory peptides derived from the peptic digest of soybean protein. Journal of Food Biochemistry 26:543-54. Chen JR, Yang SC, Suetsuna K , Chao JCJ. 2004. Soybean protein-derived hydrolysate affects bipod pressure in spontaneously hypertensive rats. Journal of Food Biochemistry 28:6173. Cheung HS, Wang F L , Ondetti M A , Sabo EF, Cushman D W . 1980. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOHterminal dipeptide sequence. Journal of Biological Chemistry 255:401-7. Clare D A , Swaisgood HE. 2000. Bioactive milk peptides: a prospectus. Journal of Dairy Science 83:1187-95. Cushman D W , Cheung HS. 1971. Spectrophotometric assay and properties of the angiotensin converting enzyme of rabbit lung. Biochemical Pharmacology 20:1637-48. Cushman DW, Ondetti M A , Gordon E M , Natarajan S, Karanewsky DS, Krapcho J, Petrillo E W Jr. 1987. Rational design and biochemical utilityof specific inhibitors of angiotensinconverting enzyme. Journal of Cardiovascular Pharmacology 10:S17-30. Dziuba J, Minkiewicz P, Nalecz D. 1999. Biologically active peptides from plant and animal proteins. Polish Journal of Food and Nutrition Sciences 8:3-16. Fotherby M D , Panayiotou B . 1999. Antihypertensive therapy in the prevention of stroke: what, - when, and for whom? Drugs 58:663-74. Fujita H , Yoshikawa M . 1999. L K P N M : a prodrug-type ACE-inhibitory peptide derived from fish protein. Immunopharmacology 44:123-7. Garrett D A , Failla M L , Sarama RJ. 1999. Development of an in vitro digestion method to assess carotenoid bioavailability from meals. Journal of Agricultural and Food Chemistry 47:4301-9. Gibbs B F , Alexandre Z, Masse R, Mulligan C. 2004. Production and characterization of bioactive peptides from soy hydrolysate and soy fermented food. Food Research International 37:123-31. Guyton A C , Hall JE. 1996. Dominant role of the kidneys in long-term regulation of arterial pressure and in hypertension. In: Textbook of Medical Physiology. 9 ed. Philadephia: W.B. Sanders Company, p 221-37. Health Canada. 2002. Cardiovascular disease surveillance on-line, <http://dsol-smed.hcsc.gc.ca/dsol-smed/cvd/c_ind_e.html#top_list, 2002> (accessed August 15, 2004) th  56  Hernandez-Ledesma B , Amigo L , Ramos M , Recio I. 2004. Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion. Journal of Agricultural and Food Chemistry 52:1504-10. Hsu FL, Lin Y H , Lee M H , Lin C L , Hou WC. 2002. Both dioscorin, the tuber storage protein of yam (dioscorea alata cv. Tainong no. 1), and its peptide hydrolysates exhibited angiotensin converting enzyme inhibitory activities. Journal of Agricultural and Food Chemistry 50:6109-13. Izzo JL, Black HR. 2003. Approach to the management of hypertension. In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel LJ, Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 378-81. Je J Y , Park PJ, Kwon JY, K i m SK. 2004. A novel angiotensin I converting enzyme inhibitory peptide from Alaska Pollack (Theragra chalcogramma) frame protein hydrolysate. Journal of Agricultural and Food Chemistry 52:7842-5. Kannel W B , Wilson PWF. 2003. Cardiovascular risk factors and hypertension. In: Izzo JL, Black HR, Goodfriend T L , Sowers JR, Weder A B , Appel L J , Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. P 235-8. K i m SH, Lee Y J , Kwon D Y . 1999. Isolation of angiotensin converting enzyme inhibitor from Doenjang. Korean Journal of Food Science and Technology 31:848-54. Kwan K K H , Nakai S, Skura BJ. 1983. Comparison of four methods for determining protease activity in milk. Journal of Food Science 48:1418-21. Liceaga-Gesualdo A M , Li-Chan E C Y . 1999. Hydrolysate from herring (Clupea harengus). Journal of Food Science 64:1000-4. Megias C, Del Mar Yust M , Pedroche J, Lquari H , Giron-Calle J, Alaiz M , Millan F, Vioque J. 2004. Purification of an A C E inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates. Journal of Agricultural and Food Chemistry 52:1928-32. Mullally M M , Meisel H , FitzGerald RJ. 1997. Identification of a novel angiotensin-I converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine p-lactoglobulin. FEBS Letters 402:99-101. Nelson D L , Cox M M . 2000. Amino acids, peptides and proteins. In: Lehninger Principles of Biochemistry. New York: Worth Publishers, p 115-58. Okamoto A , Hanagata H , Kawamura Y , Yanagida F. 1995. Antihypertensive substances in fermented soybean, natto. Plant Foods for Human Nutrition 47:39-47. Ondetti M A , Williams N J , Sabo EF, Pluscec J, Weaver ER, Kocy O. 1971. Angiotensin converting enzyme inhibitors from the venom of Bothrops jararaca. Isolation, elucidation of structure and synthesis. Biochemistry 19:4033-9. Rhyu M R , K i m E Y , Han JS. 2002. Antihypertensive effect of the soybean paste fermented with the fungus Monascus. International Journal of Food Science and Technology 37:585-8. Shin ZI, Ahn C W , Nam HS, Lee HJ, Lee HJ, Moon T H . 1995. Fractionation of angiotensin converting enzyme (ACE) inhibitory peptides from soybean paste. Korean Journal of Food Science and Technology 27:230-4. Shin ZI, Y u R, Park SA, Ghung D K , A n CW, Nam HS, K i m K S , Lee HJ. 2001. His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo. Journal of Agricultural and Food Chemistry 49:3004-9. Sica D A . 2003. Angiotensin-converting enzyme inhibitors. In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel L J , Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 426-9. 57  Skidgel R A , Erdos E G . 2003. Angiotensin I-converting enzyme and neprilysin (neutral endopeptidase). In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel LJ, Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 17-9. US F D A (Food and Drug Administration). 1999. F D A Talk Paper, <http://www.fda.gov/bbs/ topics/ANSWERS/ANS00980.html> (accessed August 15, 2004) Vermeirssen V , Camp JV, Verstraete W. 2002. Optimisation and validation of an angiotensinconverting enzyme inhibitory assay for the screening of bioactive peptides. Journal of Biochemistry & Biophysical Methods 51:75-87. Vermeirssen V , van der Bent A , Van Camp J, van Aart A , Verstraete W. 2004. A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests. Biochimie 86:231-9. Wu J, Ding X . 2001. Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry 49:501-6. Wu J, Ding X . 2002. Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides. Food Research International 35:367-75. Wu J, Aluko R E , Muir A D . 2002. Improved method for direct high-performance liquid chromatography assay of angiotensin-converting enzyme-catalyzed reactions. Journal of Chromatography A 950:125-30. Yamauchi F, Suetsuna K , inventors; Japan Kokai Tokkyo Koho, assignee. 1996a Sep 3. New tetrapeptides and pentapeptides of hydrolyzate of soy protein and their chemical synthesis as angiotensin converting enzyme inhibitors. Japan patent JP 08225593. Yamauchi F, Suetsuna K , inventors; Japan Kokai Tokkyo Koho, assignee. 1996b Oct 15. Tetrapeptides and pentapeptides from soy protein hydrolyzate as antihypertensives. Japan patent JP 08269087. Yang Y , Marczak ED, Usui H , Kawamura Y , Yoshikawa M . 2004. Antihypertensive properties of spinach leaf protein digests. Journal of Agricultural and Food Chemistry 52:2223-5.  58  CHAPTER III Angiotensin I-Converting Enzyme Inhibitory Activity of Soy Protein Digests from a Dynamic Model System Simulating the Upper Gastrointestinal Tract 3.1 Introduction Hypertension is a condition of high blood pressure defined as a systolic blood pressure > 140 mm Hg and/or a diastolic blood pressure > 90 mm Hg (Kannel and Wilson 2003; Izzo and Black 2003). It is a major risk factor for coronary heart disease, stroke, peripheral arterial disease, and heart failure (Mark and Davis 2000; Kannel and Wilson 2003). Angiotensin Iconverting enzyme (ACE) is an enzyme in the renin angiotensin system, a system that regulates blood pressure. The activity of A C E can cause an increase in blood pressure. A C E converts inactive hormone angiotensin I (DRVYIHPFHL) to active hormone angiotensin II (DRVYIHPF) (Sica 2003; Guyton and Hall 1996; Skidgel and Erdos 2003). Angiotensin II raises blood pressure by vasoconstriction as well as stimulation of the synthesis and release of aldosterone, a hormone that promotes sodium and water retention in the kidneys, thus increasing blood volume in the vessels. Furthermore, A C E degrades the vasodilator bradykinin. Hypertension is commonly treated with A C E inhibitor drugs, such as captopril, benazepril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril and trandolapril (Sica 2003). A C E inhibitor drugs are effective, but they can cause undesired side effects (Sica 2003). In a previous study (Lo and Li-Chan 2005), peptides with inhibitory activity against A C E were produced after sequential pepsin-pancreatin digestion of soy protein isolate (SPI) using an in vitro batch digestion system, suggesting potential production of A C E inhibitory peptides upon consumption and in vivo digestion of SPI. Therefore, the objective of this study was to investigate i f A C E inhibitory peptides would be produced after digestion of SPI using an in vitro dynamic model system that more closely simulates gastrointestinal digestion conditions than the batch digestion system. In addition, in order to investigate the effects of heating on the production of peptides with A C E inhibitory activity, SPI was also blanched and either pasteurized or sterilized prior to digestion using the batch or dynamic model digestion systems.  3.2 Materials and Methods 3.2.1 Materials Soy protein isolate (SPI, A D M Protein Specialities Division, Decatur, IL) was donated by Yves Veggie Cuisine Inc., a division of Hain Celestial Canada (Delta, B C , Canada). Pepsin (2331 units/mg solid, catalogue no. P-7012, E C 3.4.23.1), pancreatin (8xUSP, catalogue no. P7545, E C 2324689, contains many enzymes, including amylase, trypsin, lipase, ribonuclease and protease), hippuryl-histidyl-leucine (HHL, catalogue no. H-1635), A C E (from rabbit lung, 3.1 units/mg protein, catalogue no. A-6778, E C 3.4.15.1), A C E inhibitor (pGlu-Trp-Pro-Arg-ProGln-Ile-Pro-Pro, catalogue no. A-0773), captopril (catalogue no. C-8856) and L-leucine (catalogue no. L-8912) were purchased from Sigma-Aldrich (St. Louis, M O , USA). 2, 4, 6 59  Trinitrobenzenesulfonic acid (TNBS, product no. 8746) was purchased from Eastman Kodak Co. (Rochester, N Y , USA).  3.2.2 Heat Treatment Heat treatment was carried out in triplicate at the temperature and time typically used for blanching, pasteurization and sterilization during processing of commercial soy milk (Golbitz, 1995; Liu, 1997). SPI solution (5% w/v, in distilled, deionized water containing 0.02% sodium azide) was blanched (held at 100°C for 10 minutes) by submersion into a 100°C water bath. The SPI solution was cooled immediately in an ice-water bath. Once cooled to approximately 75°C, the SPI solution was pasteurized (held at 75°C for 15 seconds) by submersion into a 75°C water bath or sterilized at 121°C for 20 minutes in an autoclave chamber, to yield the "blanchedpasteurized" and "blanched-sterilized" SPI solutions. The solutions were cooled immediately in an ice-water bath and stored at -25°C until used.  3.2.3 Batch Digestion Batch digestion was carried out in triplicate according to the method of Garrett and others (1999) as modified by Lo and Li-Chan (2005). SPI solution (5g SPI in 100 mL distilled, deionized water containing 0.02% sodium azide) was adjusted to pH 2.0 with 1 N HC1 and pepsin (200 mg) was added. The solution was incubated at 37°C for 1 hour before pH was adjusted to 5.3 with 0.9 M NaHC03. Pancreatin (200 mg) was added, and pH was adjusted to 7.5 with 1 N NaOH. The solution was incubated at 37°C for 2 hours and then submerged in a boiling water bath for 10 minutes to terminate the digestion. The SPI peptide digest was collected and stored at -25°C until used. For the monitoring of degree of hydrolysis (DH) and A C E inhibition at intervals during digestion, aliquots of SPI digest were removed at 0, 20, 40, 60, 90, 120, and 180 minutes during batch digestion. The aliquots were submerged in a boiling water bath for three minutes to inactivate pepsin and pancreatin. The aliquots were cooled and stored at -25°C until used to carry out D H and A C E inhibitory assays.  3.2.4 Dynamic Model Digestion Digestion of SPI was carried out in triplicate according to the method of Mainville and others (2005), which was modified to incorporate pepsin and pancreatin in the stomach and duodenum reactors, respectively. The dynamic model system at the Food Research and Development Centre, Agriculture and Agri-Food Canada in Saint Hyacinthe, Quebec, (Figure 3.1) consisted of two lL-jacketed glass beakers (Kontes, Vineland, N J , USA) to represent the stomach and duodenum. For each beaker, a cover was designed to accommodate a Radiometer Copenhagen "Red Rod" pH electrode (London Scientific, London, ON, Canada), a temperature probe, and entry ports for SPI, HC1 and pepsin delivery into the stomach reactor and stomach digesta, NaOH, Oxgall bile and pancreatin into the duodenum reactor. A magnetic stir bar was placed inside each vessel and agitation was controlled via a magnetic stirrer plate. The temperature inside the reactor was controlled by circulating water at 37°C through the jacketed beakers. Peristaltic pumps (Minipuls 3, Gilson Inc., Middleton, WI, USA) were used to control the delivery of the products to be added, as well as the emptying rate of the stomach reactor (6.4 mL/min) into the duodenum reactor. The emptying rate of the stomach reactor was controlled to approximate the conditions in the stomach following yoghurt consumption in humans (Berrada 60  and others 1991). The quantity of SPI solution remaining in the stomach reactor was calculated by considering the rate of addition of each solution into, and the rate of emptying from, the stomach vessel. HC1 addition in the stomach vessel was controlled to reproduce the p H curve found in humans during and after milk consumption (Minekus and others 1995). The p H of the duodenum vessel was maintained at pH 6. Biogenie ID system (Ste-Foy, QC, Canada) was used to record pH and temperature values during the 90 minute digestion period. The 90 minute study period began when the SPI solution (20 g SPI in 400 mL distilled deionized water containing 0.02% sodium azide) started to enter the stomach reactor, followed by addition of distilled deionized water (50 mL). The SPI solution and water were added at a rate of 100 mL/min, simulating the in vivo consumption rate of a liquid product. At the beginning of the SPI solution delivery, 17.5 mL of 150 m M HC1 with 0.429 mg/mL of pepsin was added to the stomach reactor to simulate the cephalic phase of acid secretion (Guyton and Hall 2000; Fruton 1970). The rate of pepsin/HCl delivery during digestion was 3.5 mL/min until the stomach reactor reached a pH of 3.0, at which time it was lowered to 0.9 mL/min, to simulate gastrin inhibition. The total amount of pepsin added during the entire 90-minute digestion was 83.5 to 94.7 mg. NaOH (1 M solution) was added to the duodenum reactor (at 0.08 mL/min from 0 to 46 minutes and 0.35 mL/min from 47 to 90 minutes) to maintain the p H at 6 during the experiment. At time 0, the duodenum reactor contained 12 mL of a 4% Oxgall bile solution. Pancreatin (72.5 mg/mL water) was pumped into the duodenum reactor at 0.5 mL/min for the entire 90 minutes and the total amount of pancreatin added was 3.3 g (Layer and others 1986). 4%> Oxgall solution was pumped at 0.5 mL/min into the duodenum reactor for the first 30 min, and then 2% solution was used for the remainder of the experiment in order to have Oxgall in the duodenum at close to 0.3% throughout the experiment. For the monitoring of D H and A C E inhibition at intervals during digestion, approximately 5 mL aliquots of SPI digest were removed from the stomach reactor at 0, 30, 60 and 90 min and the duodenum reactor at 30, 60 and 90 min during dynamic model digestion. The aliquots were submerged in a boiling water bath for three minutes to inactivate pepsin and pancreatin. The aliquots were cooled and stored at -25°C until used to carry out D H and A C E inhibitory assays.  3.2.5 Degree of Hydrolysis (DH) Degree of hydrolysis was analyzed in triplicate according to the method of Adler-Nissen (1979) and Kwan and others (1983) with modifications by Liceaga-Gesualdo and Li-Chan (1999) as described by Lo and Li-Chan (2005). Aliquots (1.0 mL) of SPI peptide digest (from batch digestion and model digestion) were mixed with trichloroacetic acid (1.0 mL, 24%) and centrifuged at 12 350 g for 5 minutes. The supernatant (0.2 mL) was added to sodium borate buffer (2.0 mL, 0.05 M , pH 9.2) and 2,4,6-trinitrobenzenesulfonic acid (1.0 mL, 4.0 mM) and incubated at room temperature for 30 minutes in the dark. A n aliquot of NaH2P04 (1.0 mL, 2.0 M) containing Na2S03 (18 mM) was added and the absorbance was measured at 420 nm using a spectrophotometer. Degree of hydrolysis was calculated as % D H = (h / h t) x 100, where D H = percent ratio of the number of peptide bonds broken (h) to total number of bonds per unit weight (h ) and h t = 7.75 mequiv/g of soy protein (Adler-Nissen 1979). L-leucine was used as a standard in the D H assays. to  tot  to  3.2.6 ACE Inhibitory Activity Assay A C E inhibitory activity assay was carried out in triplicate according to the method of Cushman and Cheung (1971) with modifications by Wu and others (2002) as described by Lo 61  and Li-Chan (2005). SPI digest sample (30 uL), H H L (150 uL, 6.5 mM) and A C E (25 uL, 2.5 mU) were incubated at 37°C for 1 h. HC1 (250 uL, 1 N) and ethyl acetate (1.5 mL) were added and the mixture was mixed by vortexing and centrifuged at 2000 g for 5 minutes. After centrifugation, 1.0 mL of the top layer (containing hippuric acid extracted into ethyl acetate) was taken and ethyl acetate was evaporated off. The residual hippuric acid was re-dissolved with distilled, deionized water (1 mL) prior to measurement of the absorbance at 228 nm. IC50 value was defined as the amount of peptide required to inhibit A C E activity by 50%. %> A C E inhibition was defined as the percentage of A C E activity inhibited by a specific amount of peptide. Results were reported as mean + SD. A C E inhibitory activity of SPI digest during digestion and after 90 minutes of digestion was based on triplicate in vitro digestion experiments, each assayed in triplicate. Captopril and A C E inhibitor (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) were used as standards.  3.2.7 Statistical Analysis Using Statistica™ Statistical Software (version 6.0), analysis of variance (ANOVA) of repeated measures using general linear model and pairwise comparisons with Tukey's method were carried out.  3.3 Results and Discussion 3.3.1 Degree of Hydrolysis during Batch Digestion The degree of hydrolysis (DH) of all three SPI solutions (unheated, blanched-pasteurized and blanched-sterilized) increased slowly during batch digestion with pepsin from 0 to 60 minutes, then increased rapidly during batch digestion with pancreatin at 90 to 180 minutes. As shown in Table 3.1, a similar pattern was observed for D H of all three SPI solutions; however, the D H was significantly (p<0.05) greater for blanched-pasteurized and blanched-sterilized SPI solutions throughout most of the digestion, as observed at 40, 90, 120, and 180 minutes. At 60 minutes of batch digestion, the D H of blanched-sterilized SPI solution was significantly (p<0.05) greater than the D H of blanched-pasteurized and unheated SPI solutions.  3.3.2 Degree of Hydrolysis during Dynamic Model Digestion The D H of all three SPI solutions did not differ significantly (p>0.05) between 30, 60 and 90 minutes of digestion in the stomach reactor; however, the D H of all three SPI solutions increased significantly (p<0.05) overtime during the 90 minutes of digestion in both the stomach and duodenum reactors (Table 3.2). The highest D H was obtained during 60 to 90 minutes after digestion in both the stomach and duodenum reactors. Overall, the D H of all three solutions was significantly higher (p<0.05) after digestion in both the stomach and duodenum reactors than after digestion in only the stomach reactor. The D H of the three types of SPI solutions did not differ significantly (p<0.05) with each other at each given time interval; however the results showed a trend that blanched-sterilized SPI had lower D H than unheated SPI and blanchedpasteurized SPI. This trend suggested that blanching and sterilizing might have caused conformation changes to the SPI that affected its digestibility by pepsin and pancreatin under the pH and enzymatic conditions used in the dynamic model digestion, thus leading to lower D H . Since the % D H of blanched-pasteurized SPI was similar to that of unheated SPI, it suggested that 62  the milder heat treatment from blanching and pasteurizing might not have caused the same conformational changes from blanching and sterilizng that had decreased the digestibility of SPI.  3.3.3 Degree of Hydrolysis Comparisons Overall, the results showed that the D H of unheated SPI, blanched-pasteurized SPI, and blanched-sterilized SPI was higher during batch digestion than during dynamic model digestion (Tables 3.1 and 3.2). This suggested that the different conditions during the two digestion systems affected the D H of the samples. On comparison, higher amounts of pepsin but lower amounts of pancreatin were used during batch digestion than during dynamic model digestion. However, the pH conditions for both pepsin and pancreatin were more optimal during batch digestion than during dynamic model digestion (Appendix Tables 1 and 2, Appendix Figures 1 to 3). The SPI concentration in the samples was also higher during batch digestion than during dynamic model digestion (Appendix Tables 3 and 4, Appendix Figures 4 to 6). The cumulative effects from these conditions might have consequently led to greater enzymatic activity, resulting in more hydrolysis and higher D H , during batch digestion than during dynamic model digestion. In terms of heat treatment effects, blanching-pasteurizing and blanching-sterilizing significantly (p<0.05) increased the D H of SPI during batch digestion, suggesting that heating enhanced the digestibility of SPI, probably by causing conformational changes on SPI that exposed more cleavage sites for enzyme hydrolysis. However, an opposite effect was observed during dynamic model digestion in which heating by blanching and sterilizing decreased the digestibility of SPI, suggesting that the digestion condition might also have affected hydrolysis. Since the pH conditions of the dynamic model system is different than that of the batch system, it might be possible that the conditions in the dynamic model system affected the conformation of blanched-sterilized SPI and led to fewer accessible cleavage sites for enzyme hydrolysis.  3.3.4 ACE Inhibition during Batch Digestion Unheated SPI had strong inhibitory activity against A C E throughout 20 to 60 minutes of digestion with pepsin, while blanched-pasteurized SPI and blanched-sterilized SPI had highest inhibitory activity against A C E at 40 to 60 minutes of digestion with pepsin (Table 3.3). For all three SPI solutions, the A C E inhibitory activity of the digest was significantly (p<0.05) higher during parts of the initial digestion with pepsin than subsequent digestion with pancreatin. Similar findings were also observed in a previous study on unheated SPI (Lo and Li-Chan 2005). Pepsin cleaves at the carboxyl end of hydrophobic and aromatic amino acids (Phe, Tyr, Tip, Leu), resulting in peptides with hydrophobic and aromatic amino acids at the C-terminal (Nelson and Cox 2000; Expasy 2003). These peptides bind tightly to A C E at its active site and compete with angiotensin I for occupancy; therefore, A C E cannot bind to angiotensin I to convert it to angiotensin II (Cushman and others 1987). A study by Pripp and others (2004) also reported correlation between the A C E inhibitory activity of the milk-derived peptides and the amino acid residues located at the C-terminal end. A C E inhibitory potential of the peptides were found to increase with increased side chain hydrophobicity of the amino acid residues and the absence of positive charge at the C-terminal position (Pripp and others 2004). At 20 and 180 minutes during batch digestion, A C E inhibitory activity of soy peptides from blanched-sterilized SPI was found to be significantly lower (p<0.05) than those from unheated SPI and both unheated SPI and blanched-pasteurized SPI, respectively. The A C E inhibitory activity of soy peptides from blanched-pasteurized SPI was found to be similar to soy peptides from unheated SPI throughout batch digestion. 63  3.3.5 ACE Inhibition during Dynamic Model Digestion For all three SPI solutions, the A C E inhibitory activity of the soy peptides increased over time throughout the 90 minutes of pepsin digestion in the stomach reactor (Table 3.4). This finding reflected the finding from batch digestion. As shown in Table 3.2, the D H of all three SPI solutions did not increase overtime throughout the 90 minutes of pepsin digestion in the stomach reactor. This finding combined with the A C E inhibition results suggested that D H did not seem to affect the A C E inhibitory activity of the soy peptides, because the % A C E inhibition increased despite no changes in % D H . For blanched-pasteurized SPI that was digested by pepsin in the stomach reactor and then subsequently digested by pancreatin in the duodenum reactor, the A C E inhibitory activity of the soy peptides increased overtime throughtout the 90 minutes of digestion (Table 3.4). For unheated SPI and blanched-sterilized SPI that were digested by pepsin in the stomach reactor and subsequently digested by pancreatin in the duodenum reactor, the A C E inhibitory activity of their soy peptides did not change overtime throughout the 90 minutes of digestion. The results after digestion by pancreatin in the duodenum reactor, following digestion by pepsin in the stomach reactor, were the most important because peptides are absorbed while in the duodenum during in vivo gastrointestinal digestion. For unheated SPI at 30 minutes, subsequent digestion in the duodenum reactor following digetsion in stomach reactor produced soy peptides with significantly higher (p>0.05) A C E inhibitory activity than peptides produced after digestion only in the stomach reactor. However, at 90 minutes, soy peptides from subsequent digestion in the duodenum reactor following digestion in the stomach reactor had significantly (p<0.05) lower A C E inhibitory activity thn peptides produced after digesiton in the stomach reactor only. A similar trend was observed for blanched-sterilized SPI in which soy peptides from subsequent digestion in the duodenum reactor at both 30 and 60 minutes had similar A C E inhibitory activity as peptides produced after digestion in the stomach reactor at both 30 and 60 minutes; however, soy peptides from subsequent digesiton in the duodenum reactor at 90 minutes had significantly (p<0.05) lower A C E inhibitory activity than peptides produced after digestion in the stomach reactor at 90 minutes. For blanched-pasteurized SPI, soy peptides produced from subsequent digestion in duodenum reactor at 30, 60 and 90 minutes had similar A C E inhibitory activity as soy peptides produced from digestion in the stomach reactor at respective times. Subsequent digestion with pancreatin in the duodenum reactor was able to produce soy peptides with high A C E inhibitory activity at the beginning of digestion when there was less hydrolysis of the SPI in the stomach reactor. However, as digestion time increased and pepsin had hydrolyzed more SPI and its peptides in the stomach reactor, subsequent digestion with pancreatin in the duodenum reactor produced soy peptides with low A C E inhibitory activity. This finding suggested that the longer time might have allowed pancreatin to hydrolyze the peptides from pepsin digestion that had strong A C E inhibitory activity, turning them into peptides with low A C E inhibitory actiivty. This finding also reflect the finding from batch digestion in which soy peptides produced after subsequent digestion with pancreatin, following 60 minutes of prior digestion with pepsin, had significantly (p<0.05) lower A C E inhibitory activity than soy peptides produced after digestion with pepsin only. Hence, the A C E inhibitory activity of soy peptides produced during gastrointestinal digestion of SPI might vary depending on at which stage of digestion these peptides were absorbed from the gastrointestinal tract. Peptides that are absorbed early from the duodenum might have higher A C E inhibitory activity than peptides that are absorbed later from the duodenum.  64  3.3.6 Comparison of ACE Inhibition during Batch and Dynamic Model Digestion During both batch digestion and dynamic model digestion, the A C E inhibitory activity of the soy peptides produced during pepsin digestion increased significantly (p<0.05) overtime. The A C E inhibitory activity of the soy peptides produced after 60 minutes of pepsin digestion had decreased significantly (p<0.05) after subsequent digestion of those peptides by pancreatin. For the effects that heat treatment had on % A C E inhibition, blanching followed by sterilizing significantly (p<0.05) decreased the A C E inhibitory activity of SPI after both batch digestion (at 20 min and 180 min) and dynamic model digestion (at all except 30 min in stomach reactor). Although D H by batch digestion was higher after blanching and sterilizing, the % A C E inhibition was not, suggesting that other factors besides D H affected the % A C E inhibition. It might have been possible that blanching and sterilizing caused coagulation of the protein or conformational changes that caused different cleavage sites to be exposed and available for enzyme hydrolysis. For example, there might have been more hydrophobic interaction within the protein, thus leaving fewer hydrophobic sites available for cleavage. As a result, peptides with lower A C E inhibitory activity were produced from blanched-sterilized SPI.  3.3.7 I C Values from Batch and Dynamic Model Digestions 50  The IC50 values were determined for soy peptides produced at the end of 180 minutes of batch digestion and 90 minutes of dynamic model digestion in the stomach and duodenum reactors (Table 3.5). Similar to the % A C E inhibition results, heat treatment by blanching and sterilizing of SPI, prior to digestion, decreased the A C E inhibitory activity of the resulting soy peptides. Since the dynamic model digestion was more representative of in vivo digestion than batch digestion was, IC50 values from the former might be closer to those that would be obtained from in vivo gastrointestinal digestion of SPI. The IC50 values from this study are comparable to those reported in the literature for other soy treatments. Shin and others (2001) reported an IC50 value of 0.2763 mg/mL for a commercial fermented soybean paste, while Ahn and others (2000), Wu and Ding (2002), and Chen and others (2004) reported IC50 values of 0.34, 0.1964, and 0.73 mg/mL for soy digests collected from alcalase digestion (50°C, 12 hours), Bacillus subtilis protease digestion (40°C, 1 hour), and pepsin digestion (39°C, 12 hours), respectively.  3.4 Conclusion Digestion of SPI by dynamic model digestion produced peptides with A C E inhibitory activity. Pepsin digestion of SPI in the stomach reactor produced soy peptides with A C E inhibitory activity that increased in inhibition potential with increased digestion time. Pancreatin digestion of SPI in the duodenum reactor, following prior digestion of the SPI in the stomach reactor, produced soy peptides with A C E inhibitory activity that decreased in inhibition potential with increased digestion time. However, the duodenum reactor of the dynamic model was not a true simulation of the duodenum in the gastrointestinal tract, because the peptides were collected in the duodenum reactor, whereas peptides are absorbed in the duodenum of the gastrointestinal tract. Therefore, some of the peptides in the duodenum reactor might have received more extensive hydrolysis than they would have received i f they were in the duodenum of the gastrointestinal tract. This prolonged digestion in the duodenum reactor might have contributed to the production of peptides with low A C E inhibitory activity and might not be reflective of all 65  the peptides that are absorbed in the duodenum of the gastrointestinal tract. A modification of the dynamic model duodenum reactor to empty out peptides in order to mimic absorption would have provided a closer simulation of the duodenum in the gastrointestinal tract. For the effects of heat treatments, blanching and pasteurizing of SPI did not affect the A C E inhibitory activity of soy peptides; however heating by blanching and sterilizing decreased the A C E inhibitory activity of soy peptides. Overall, the results from this study suggested the potential production of peptides with A C E inhibitory activity upon physiological digestion of soy protein, including products that have been subjected to heat processing. Clinical trials would be required to provide final evidence of efficacy of the soy peptides.  66  3.5 Figures  Figure 3.1 Set up of the dynamic model digestion system: (1) SPI solution, (2) pump, (3) HC1 and pepsin, (4) stomach reactor, (5) magnetic stirring plate, (6) water bath, (7) computer monitoring pH and temperature, (8) NaOH, (9) pancreatin, (10) oxgall bile, and (11) duodenum reactor.  67  3.6 Tables Table 3.1 Degree of hydrolysis as a function of time course of batch digestion of SPI with pepsin (0 - 60 min) and pancreatin (60 - 180 min). Values shown are the mean + SD from digestion experiments performed in triplicates. DH(%) Blanched-Pasteurized SPI 0+0 6.6±0.3 ' 8.9 + 0 . 3 ' 12 + 2 32 + 2 ' 45±3 47 + 2 a  Time (min) 0 20 40 60 90 120 180  Unheated SPI 0+0 4.9 + 0.3 6.6 + 0.3 ' 11 + 2 ' 26+ l ' 35 ± 3 ' 42 + 2 '  a,v  a  a  w  a  x  a  y  a  z  v  a  v  b  a , x  b  y  b , z  b , z  w  Blanched-Sterilized SPI 0.1+0 6.9 + 0.3 9.4 + 0 . 3 18 + 2 34 + 2 ' 46±2 ' 47 + 2 a,v  b,w  b , x  b  b  y  z  b , z  The sample treatments (unheated, blanched-pasteurized and blanched-sterilized) were compared at each given time interval and data points with different letters (a, b) are significantly different (p<0.05). The time intervals (20, 40, 60, 90, 120 and 180 min) were compared within each sample treatment and data points with different letters (v, w, x, y, z) are significantly different (p<0.05). a  68  Table 3.2 Degree of hydrolysis as a function of time course of dynamic model digestion of SPI with pepsin in stomach reactor and pancreatin in duodenum reactor. Values shown are the mean + SD from digestion experiments performed in triplicates.  Stomach  Time (min) 0  Unheated SPI 0±0  D H (%) Blanched-Pasteurized SPI 0±0  Stomach Duodenum  30 30  3.4±0.4 25±3  4.1 +0.4 24±l  Stomach Duodenum  60 60  5.9 + 0.5 29 ± 3  Stomach Duodenum  90 90  7.5 + 1.8" 33±4  !  Reactor  X  3.4±0.6 23 ± l  X  6.0+ 1.4 29 ± 5  4.7±1.4 28 + 2  X  6.1 +0.3 31 ± 5  4.9±0.5 32±4  X  X  y  y  x  yz  Z  Blanched-Sterilized SPI 0±0  y  X  yz  Z  X  yz  Z  The sample treatments (unheated, blanched-pasteurized and blanched-sterilized) were compared at each given time interval and data points were not significantly different (p>0.05). The time intervals (stomach 30, 60, 90 and duodenum 30, 60, 90 min) were compared for each sample treatment and data points with different letters (x, y, z) are significantly different (p<0.05). a  69  Table 3.3 A C E inhibition as a function of time course of batch digestion of SPI with pepsin (060 min) and pancreatin (60-180 min). Aliquots of SPI taken during digestion were adjusted to 0.29 mg/mL for the assay for % A C E inhibition. Values shown are the mean + SD from digestion experiments performed in triplicates. A C E Inhibition (%) Blanched-Pasteurized SPI 0+0 a  Time (min) 0 20 40 60 90 120 180  Unheated SPI 0±0 60 + 2 63 + 2 64 + 2 47 + 2 ' 48 + l ' 50 + 4 ' b , z a , z a , z  a  y  a  y  b  y  5  5  +  2  a b , y  58 + 2 ' 65±0 46 + 2 46 + 3 49 + 4 a  y z  a , z a , x  a , x  b , x y  Blanched-Sterilized SPI 0+0 48 + l 56±2 ' 58 + 2 48 + 2 47±2 ' a , y  a  z  a , z  a , y  a  3  7  ±  2  y  a , ! x  The sample treatments (unheated, blanched-pasteurized and blanched-sterilized) were compared at each given time interval and data points with different letters (a, b) are significantly different (p<0.05). The time intervals (20, 40, 60, 90, 120 and 180 min) were compared within each sample treatment and data points with different letters (x, y, z) are significantly different (p<0.05).  a  70  Table 3.4 A C E inhibition as a function of time course of dynamic model digestion of SPI with pepsin in stomach reactor and pancreatin in duodenum reactor. Aliquots of SPI taken during digestion were adjusted to 0.29 mg/mL for the assay for % A C E inhibition. Values shown are the mean + SD from digestion experiments performed in triplicates. A C E Inhibition (%) Blanched-Pasteurized SPI 0±0  Blanched-Sterilized SPI 0+0  24 + 3 ' 27±5 '  16±5 ' 13±5 '  ;  Reactor  Unheated SPI 0±0  Stomach  Time (min) 0  Stomach Duodenum  30 30  16 + 4 32±7  Stomach Duodenum  60 60  45 + 5 ' 42 + 3 '  Stomach Duodenum  90 90  56 + 5 ' 35±4 '  a , x  a  b  b , y  b  yz  b  b  b  y  y  z  3  0  34  +  ± 4  8  a  x  x  a  ab,xy  ab  b  y z  x  25 + 5 * 29±5 a  ab,xy  46+10 ' 39±3 '  x y  z  y  a , y  42 + 6 24±4 ' a  a,z x y  The sample treatments (unheated, blanched-pasteurized and blanched-sterilized) were compared at each given time interval and data points with different letters (a, b) are significantly different (p<0.05). The time intervals (stomach 30, 60, 90 and duodenum 30, 60, 90 min) were compared within each sample treatment and data points with different letters (x, y, z) are significantly different (p<0.05). a  71  Table 3.5 IC50 values of SPI digests at the end of digestion after 180 minutes of batch digestion or 90 minutes of model digestion in the stomach and duodenum reactors. Values shown are the mean + SD from digestion experiments performed in triplicates. Samples  IC  50  value (mg/mL)'  Batch Digestion Unheated SPI Blanched-Pasteurized SPI Blanched-Sterilized SPI  0.28 ± 0.04' 0.30 ± 0.02 0.36 + 0.01  Model Digestion Unheated SPI Blanched-Pasteurized SPI Blanched-Sterilized SPI  0.38±0.01 0.37 ± 0.02 0.44 ± 0.02  Captopril A C E inhibitor  a  b  ca cd d  7.2 ng/mL 455 ng/mL  The sample treatments (unheated, blanched-pasteurized and blanched-sterilized) were compared across the two types of digestion and data points with different letters (a, b, c, d) are significantly different (p<0.05). a  72  3.7 Bibliography Adler-Nissen J. 1979. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry 27:1256-62. Ahn SW, K i m K M , Y u K W , Noh DO, Suh HJ. 2000. Isolation of angiotensin I converting enzyme inhibitory peptide from soybean hydrolysate. Food Science and Biotechnology 9:378-81. Berrada N , Lemeland JF, Laroche G, Thouvenot P, Piaia M . 1991. Bifidobacterium from fermented milks: survival during the gastric transit. Journal of Dairy Science 74:403-13. Chen JR, Yang SC, Suetsuna K , Chao JCJ. 2004. Soybean protein-derived hydrolysate affects blood pressure in spontaneously hypertensive rats. Journal of Food Biochemistry 28:6173. Cushman D W , Cheung HS. 1971. Spectrophotometric assay and properties of the angiotensin converting enzyme of rabbit lung. Biochemical Pharmacology 20:1637-48. Cushman DW, Ondetti M A , Gordon E M , Natarajan S, Karanewsky DS, Krapcho J, Petrillo E W Jr. 1987. Rational design and biochemical utility of specific inhibitors of angiotensinconverting enzyme. Journal of Cardiovascular Pharmacology 10:S17-30. Expasy. 2003. NiceZyme View of Enzyme: Enzyme nomenclature database. <http://ca.expasy.org/enzyme/> (accessed October 12, 2004) Fruton JS. 1970. In: Boyer PD, editor. The Enzymes. New York: Academic Press, p 119-64. Garrett D A , Failla M L , Sarama RJ. 1999. Development of an in vitro digestion method to assess carotenoid bioavailability from meals. Journal of Agricultural and Food Chemistry 47:4301-9. Golbitz, P. 1995. Traditional soyfoods: processing and products. Journal of Nutrition 125:570S2S. Guyton A C , Hall JE. 1996. Dominant role of the kidneys in long-term regulation of arterial pressure and in hypertension. In: Textbook of Medical Physiology. 9th ed. Philadephia: W.B. Sanders Company, p 221-37. Guyton A C , Hall JE. 2000. Secretory Functions of the Alimentary Tract. In: Textbook of Medical Physiology. 10th ed. Philadephia: W.B. Sanders Company, p 738-53. Izzo JL, Black HR. 2003. Approach to the management of hypertension. In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel LJ, Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 378-81. Kannel WB, Wilson PWF. 2003. Cardiovascular risk factors and hypertension. In: Izzo JL, Black HR, Goodfriend T L , Sowers JR, Weder A B , Appel L J , Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 235-8. Kwan K K H , Nakai S, Skura BJ. 1983. Comparison of four methods for determining protease activity in milk. Journal of Food Science 48:1418-21. Layer P, Go V L , DiMango EP. 1986. Fate of pancreatin enzymes during small intestinal aboral transit in humans. American Journal of Physiology 251 :G475-80. Liceaga-Gesualdo A M , Li-Chan E C Y . 1999. Hydrolysate from herring (Clupea harengus). Journal of Food Science 64:1000-4. Liu K . 1997. Nonfermented oriental soyfoods. In: Soybeans: Chemistry, Technology & Utilization. New York: International Thomson Publishing, p 137-217. Lo W M Y , Li-Chan E C Y . 2005. Angiotensin I-converting enzyme inhibitory peptides from in vitro pepsin-pancreatin digestion of soy protein. Journal of Agricultural and Food Chemistry. In Press. (Article accepted February 24, 2005) 73  Mainville I, Arcand Y , Farnworth ER. 2005. A dynamic model that simulates the human upper gastrointestinal tract for the study of probiotics. International Journal of Food Microbiology 99:287-96. Mark K S , Davis TP. 2000. Stroke: development, prevention and treatment with peptidase inhibitors. Peptides 21:1965-73. Minekus M , Marteau P, Havenaar R, Huis in't Veld JHJ. 1995. A multicompartimental dynamic computer-controlled model simulating the stomach and the small intestine. Alternatives to Laboratory Animals 23:197-209. Nelson D L , Cox M M . 2000. Amino Acids, Peptides, and Proteins. In: Lehninger Principles of Biochemistry. 3rd ed. New York: Worth Publishers, p 115-58. Pripp A H , Isaksson T, Stepaniak L . 2004. Quantitative structure-activity relationship modeling of ACE-inhibitory peptides derived from milk proteins. European Food Research and Technology 219:579-83. Shin ZI, Y u R, Park SA, Ghung D K , A n CW, Nam HS, K i m K S , Lee HJ. 2001. His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo. Journal of Agricultural and Food Chemistry 49:3004-9. Sica, D. A . 2003. Angiotensin-converting enzyme inhibitors. In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel L J , Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 426-9. Skidgel, R. A . ; Erdos, E. G. 2003. Angiotensin I-converting enzyme and neprilysin (neutral endopeptidase). In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel LJ, Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 17-9. Wu J, Ding X . 2002. Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides. Food Research International 35:367-75. Wu J, Aluko R E , Muir A D . 2002. Improved method for direct high-performance liquidChromatography assay of angiotensin-converting enzyme-catalyzed reactions. Journal of Chromatography A 950:125-30.  74  CHAPTER IV  Conclusion  In conclusion, the results from this thesis demonstrated that A C E inhibitory soy peptides can be produced from SPI that has been digested by pepsin and pancreatin in a batch digestion system or in a dynamic model digestion system. Multiple chromatographic fractionation of SPI digest from batch digestion system generated fractions with higher A C E inhibitory activity than the unfractionated digest. Heat treatment of 5% SPI solution by sequential blanching (100°C, 10 min) and pasteurization (75°C, 15 s) prior to digestion had no effect on the A C E inhibitory activity of soy peptides, whereas, blanching (100°C, 10 min) followed by sterilization (121°C, 20 min) decreased the A C E inhibitory activity of soy peptides. The results from both batch and dynamic model in vitro digestion of SPI showed that peptides resulting from pepsin digestion had higher A C E inhibitory activity than those from subsequent sequential pancreatin digestion. The batch digestion system is a simple simulation of gastrointestinal digestion, mainly simulating the pH and type of enzymes in a gastrointestinal system, whereas the dynamic model digestion system more closely simulates in vivo gastrointestinal digestion. In addition to mimicking the pH and types of enzymes, the dynamic model digestion system also mimics the gradual introduction of food into the stomach, the gradual emptying of food from the stomach into the duodenum, and the introduction of bile into the duodenum. Therefore, the finding of A C E inhibitory peptides from dynamic model in vitro digestion of SPI suggests potential production of A C E inhibitory peptides from in vivo gastrointestinal digestion of SPI. If in vivo gastrointestinal digestion of SPI were to produce A C E inhibitory peptides that are similar to the ones produced during dynamic model in vitro digestion and i f all of the peptides were intact and remained functional after absorption into the blood, then a calculated dose of approximately 4 to 18 kg of SPI per day might yield similar levels of A C E inhibitory effect as captopril (refer to Appendix Table 5 for calculation). Captopril is an antihypertensive drug typically prescribed at a dosage of 75 to 300 mg per day (Sica 2003). The A C E inhibitory activity of captopril, expressed as an IC50 value, is 1.3 to 8.9 ng/mL (Mullally and others 1997; Fujita and Yoshikawa 1999; Hsu and others 2002; Vermeirssen and others 2002). The amount of 4 to 18 kg SPI is approximately several hundred fold higher than the intake associated with the US Food and Drug Administration health claim, which states that "diets low in saturated fat and cholesterol that includes 25 grams of soy protein per day may reduce the risk of heart disease" (US F D A 1999). Such a large amount of SPI in kilogram quantities would be impossible to consume on a daily basis, suggesting SPI might not be suitable as a direct replacement for captopril in treating high blood pressure. However, as shown in some rat studies, the A C E inhibitory activity of some protein hydrolysates might have increased or decreased inhibitory potential upon hydrolysis during in vivo digestion or further processing in the enterocytes. Therefore, it might be possible that in vivo digestion of SPI could yield soy peptides with higher A C E inhibitory activity than those produced after in vitro digestion in the thesis study. To confirm the production and absorption of A C E inhibitory soy peptides and to determine their in vivo A C E inhibitory activity, clinical studies involving human volunteers would be necessary to determine i f A C E inhibitory peptides would be produced after gastrointestinal digestion of SPI and whether or not these peptides would be absorbed into the blood to exert their activity. SPI can be provided to human volunteers to consume in their diets for a period of time. The human volunteers can be examined throughout the study for reductions in blood pressure, which would indicate A C E inhibitory activity. Chyme samples can be collected from the gastrointestinal tract of these volunteers to obtain peptides and to study their properties, such as molecular size, amino acid composition or sequence, and A C E inhibitory 75  activity. In addition, blood samples can be collected from the human volunteers to investigate i f there are any A C E inhibitory peptides absorbed from the gastrointestinal tract into the blood stream. These suggestions for the clinical study are based on a study by Chabance and others (1998) in which milk peptides were determined from the duodenum and blood of human volunteers after they have ingested a milk or yogurt sample. ' Another limitation of this thesis study was the fact that it investigated digestion of SPI alone. Digestion of SPI along with other components of a meal, such as lipids, carbohydrates, and other proteins, was not investigated. These components might have interactions with SPI and might reduce its digestibility, by possibly causing steric hinderance and blocking the digestive enzymes' access to its peptide bonds. Possible interactions of SPI peptides with these components might also decrease the accessibility of the peptides to bind to A C E ' s active site, thus reducing their A C E inhibitory potential. In addition, this thesis study did not investigate digestion at various gastric pHs. Gastric pH is sensitive to increasing age, pathological conditions, and drug-induced changes (Charman and other 1997). Although gastric pH is typically around 2, it might be different for some people, such as the elderly who have diminished (hypochlorhydria) or no gastric acid secretion (achlorhydria) might have higher gastric p H level than that tested in this thesis. High gastric p H might reduce the specificity of pepsin, resulting in less cleavage at the aromatic or hydrophobic amino acid residues Phe, Tyr, Tip, and Leu (Expasy 2003). Thus, fewer peptides with hydrophobic or aromatic amino acids at the C-terminal would be produced. Consequently, the production of A C E inhibitory peptides might be reduced, because peptides with hydrophobic or aromatic C-terminal amino acids residues tend to have greater inhibitory activity against A C E . Therefore, further research is needed to investigate the possible effects that other food components and varying gastric pH might have on the production of A C E inhibitory peptides from SPI. Furthermore, this thesis did not investigate the single amino acids that are produced after protein digestion to determine i f they contribute to the A C E inhibitory activity determined from the SPI digests. There has been no research in the literature on the in vitro or in vivo A C E inhibitory activity of single amino acids that are produced after in vitro or in vivo digestion of protein. Therefore, further research is needed to investigate i f single amino acids would contribute A C E inhibitory activity.  76  4.1 Bibliography Aldridge  S.  2004. Cultured milk drink reduces blood pressure. Health and Age. <http://vv ww.healthandage.com/Home/gidl=6228> (accessed Feb 27, 05) Chabance B , Marteau P, Rambaud JC, Migliore-Samour D, Boynard m, Perrotin P, Guillet R, Jolles P, Fiat A M . 1998. Casein peptide release and passage to the blood in humans during digestion of milk or yogurt. Biochimie 80:155-65. Charman W N , Porter CJH, Mithani S, Dressman JB. 1997. Physicochemical and physiological mechanisms for the effects of food on drug absorption: the role of lipids and pH. Journal of Pharmaceutical Sciences 86:269-82. Expasy. 2003. NiceZyme View of Enzyme: E C 3.4.23.1. Enzyme nomenclature database. <http://ca.expasy.org/enzyme/> (accessed October 12, 2004) Fujita H , Yoshikawa M . 1999. L K P N M : a prodrug-type ACE-inhibitory peptide derived from fish protein. Immunopharmacology 44:123-7. Hsu FL, L i n Y H , Lee M H , L i n CL, Hou WC. 2002. Both dioscorin, the tuber storage protein of yam (Dioscorea alata cv. Tainong no. 1), and its peptide hydrolysates exhibited angiotensin converting enzyme inhibitory activities. Journal of Agricultural and Food Chemistry 50:6109-113. Mullally M M , Meisel H , FitzGerald RJ. 1997. Identification of a novel angiotensin-I converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine p-lactoglobulin. FEBS Letters 402:99-101. Nakashima Y , Arihara K , Mio H , Itoh M . 2002. Antihypertensive activities of peptides derived from porcine skeletal muscle myosin in spontaneously hypertensive rats. Journal of Food Science 67:434-7. Sica D A . 2003. Angiotensin-converting enzyme inhibitors. In: Izzo JL, Black HR, Goodfriend TL, Sowers JR, Weder A B , Appel L J , Sheps SG, Sica D A , Vidt D G , editors. Hypertension Primer. 3rd ed. Baltimore: Lippincott Williams & Wilkins. p 426-9. US FDA (Food and Drug Administration). 1999. FDA Talk Paper. <http://www.fda.gov/bbs/topics/ ANSWERS/ANS00980.html> (Accessed Sep 20, 04) Vermeirssen V , Camp JV, Verstraete W. 2002. Optimisation and validation of an angiotenisn converting enzyme inhibitory assay for the screening of bioactive peptides. Journal of Biochemistry & Biophysical Methods 51:75-87. r  77  Appendix  78  List of Appendix Figures Appendix Figure 1. The pH profile of unheated SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions 81 Appendix Figure 2. The pH profile of blanched-pasteurized SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions 82 Appendix Figure 3. The pH profile of blanched-sterilized SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions 83 Appendix Figure 4. The concentration profile of unheated SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions 84 Appendix Figure 5. The concentration profile of blanched-pasteurized SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions 85 Appendix Figure 6. The concentration profile of blanched-sterilized SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions 86  79  List of Appendix Tables Appendix Table 1.  The pH of digest as a function of time course of batch digestion of SPI with pepsin (0 - 60 min) and pancreatin (60 - 180 min). Values shown are the mean + SD from digestion experiments performed in triplicates 87  Appendix Table 2.  The pH of digest as a function of time course of dynamic model digestion of SPI with pepsin in stomach reactor and pancreatin in duodenum reactor. Values shown are the mean + SD from digestion experiments performed in triplicates 88  Appendix Table 3.  The concentration of digest as a function of time course of batch digestion of SPI with pepsin (0 - 60 min) and pancreatin (60 - 180 min). Values shown are the mean + SD from digestion experiments performed in triplicates 89  Appendix Table 4.  The concentration of digest as a function of time course of dynamic model digestion of SPI with pepsin in stomach reactor and pancreatin in duodenum reactor. Values shown are the mean + SD from digestion experiments performed in triplicates 90  Appendix Table 5.  Calculation of SPI Required to Exert Similar A C E Inhibitory Activity as Captopril 91  80  stomach reactor - unheated SPI •a— duodenum reactor - unheated SPI  1  o -I 0  , 10  , 20  , 30  1 40  , 50  , 60  , 70  , 80  1 90  Time (min)  Appendix Figure 1. The pH profile of unheated SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions.  81  "0— stomach reactor - blanched-pasteurized SPI •a— duodenum reactor - blanched-pasteurized SPI  0  10  20  30  40  50  60  70  80  90  Time (min)  Appendix Figure 2. The pH profile of blanched-pasteurized SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions.  82  Appendix Figure 3. The pH profile of blanched-sterilized SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions.  83  —s— stomach reactor - unheated SPI - h — duodenum reactor - unheated SPI 50  T  0  10  20  30  40  50  60  70  80  90  Time (min)  Appendix Figure 4. The concentration profile of unheated SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions.  84  Appendix Figure 5. The concentration profile of blanched-pasteurized SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions.  85  — s t o m a c h reactor - blanched-sterilized SPI —a—duodenum reactor - blanched-sterilized SPI 50  T  0  10  20  30  40  50  60  70  80  90  Time (min)  Appendix Figure 6. The concentration profile of blanched-sterilized SPI during digestion in the stomach and duodenum reactors. Bars represent standard deviation values from three replicate digestions.  86  Appendix Table 1. The pH of digest as a function of time course of batch digestion of SPI with pepsin (0 - 60 min) and pancreatin (60 - 180 min). Values shown are the mean ± SD from digestion experiments performed in triplicates.  Time (min) 0 1 60 61 180  Unheated SPI 7.1 + 0.1 2.0 ± 0 . 0 2.6 + 0.1 7.5 ± 0 . 0 7.1 ± 0 . 1  pH of Digest Blanched-Pasteurized SPI 7.1 ± 0 . 1 2.0 ± 0 . 0 2.2 ± 0 . 1 7.5 ± 0 . 0 7.3 ± 0 . 1  Blanched-Sterilized SPI 7.1 ± 0 . 1 2.0 ± 0 . 0 2.0 ± 0 . 1 7.5 ± 0 . 0 7.3 ± 0 . 1  87  Appendix Table 2. The pH of digest as a function of time course of dynamic model digestion of SPI with pepsin in stomach reactor and pancreatin in duodenum reactor. Values shown are the mean ± SD from digestion experiments performed in triplicates.  Stomach  Time (min) 0  Unheated SPI 7.1 ± 0 . 1  pH of Digest Blanched-Pasteurized SPI 7.1 ± 0 . 1  Blanched-Sterilized SPI 7.1 ± 0 . 1  Stomach Duodenum  30 30  3.9 ± 0 . 1 6.2 ± 0 . 2  3.8 ± 0 . 1 5.6 ± 0 . 0  3.7 ± 0 . 1 5.5 ± 0 . 1  Stomach Duodenum  60 60  2.7 ± 0 . 2 5.9 ± 0 . 3  2.5 ± 0 . 1 5.5 ± 0 . 2  2.6 ± 0 . 0 5.8 ± 0 . 1  Stomach Duodenum  90 90  2.2 + 0.1 6.0 ± 0 . 3  2.1 ± 0 . 0 5.7 ± 0 . 3  1.8 ± 0 . 1 5.8 ± 0 . 1  Reactor  88  Appendix Table 3. The concentration of digest as a function of time course of batch digestion of SPI with pepsin (0 - 60 min) and pancreatin (60 - 180 min). Values shown are the mean ± SD from digestion experiments performed in triplicates.  Time (min) 0 20 40 60 90 120 180  Unheated SPI 50.0 + 0.0 50.0 + 0.0 50.0 + 0.0 50.0 ± 0 . 0 44.1+0.1 44.1+0.1 44.1+0.1  Concentration of Digest (mg/mL) Blanched-Sterilized Blanched-Pasteurized SPI SPI 44.7 ± 0 . 1 45.0 + 0.1 45.0 ± 0 . 1 44.7 ± 0 . 1 44.7 ± 0 . 1 45.0 ± 0 . 1 44.7 ± 0 . 1 45.0 ± 0 . 1 38.9 ± 0 . 1 38.8 ± 0 . 1 38.9 ± 0 . 1 38.8 ± 0 . 1 38.8 ± 0 . 1 38.9 ± 0 . 1  89  Appendix Table 4. The concentration of digest as a function of time course of dynamic model digestion of SPI with pepsin in stomach reactor and pancreatin in duodenum reactor. Values shown are the mean ± SD from digestion experiments performed in triplicates.  Reactor  Concentration of Digest (mg/mL) B lanched- Sterilized Blanched-Pasteurized SPI SPI 50.0 ± 0 . 0 50.0 ± 0 . 0  Stomach  Time (min) 0  Unheated SPI 50.0 + 0.0  Stomach Duodenum  30 30  33.4 ± 0 . 0 31.7 ± 0 . 0  3.3.4 ± 0 . 0 31.7 ± 0 . 0  33.4 ± 0 . 0 31.7 ± 0 . 0  Stomach Duodenum  60 60  27.9 ± 0 . 6 28.3 ± 0 . 1  28.3 ± 0 . 6 28.4 ± 0 . 1  29.1 ± 0 . 1 28.6 ± 0 . 0  Stomach Duodenum  90 90  23.5 ± 0 . 3 26.0 ± 0 . 2  23.7 ± 0 . 3 26.2 ± 0 . 2  24.0 + 0.0 26.4 ± 0.0  90  Appendix Table 5. Calculation of SPI Required to Exert Similar A C E Inhibitory Activity as Captopril. Sample Captopril Unheated SPI Blanched-Pasteurized SPI Blanched-Sterilized SPI  IC  50  7.2 ng/mL 0.38 + 0.01 mg/mL 0.37 ± 0.02 mg/mL 0.44 + 0.02 mg/mL  Approximate Difference with Captopril (# of folds higher IC50 value) 1 50 000 50 000 60 000  Recommended dose for Captopril: 7 5 - 3 0 0 mg For unheated SPI and blanched-pasteurized SPI: (50 000)*(75 mg) = 3.75 kg and (50 000)*(300 mg) - 15 kg For blanched-sterilized SPI (60 000)*(75 mg) = 4.5 kg and (60 000)*(300 mg) = 18 kg Therefore, calculated amount of SPI required is 4 - 18 kg.  91  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0091962/manifest

Comment

Related Items