Structure, content, and bioactivity of food-derived peptides in body

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Structure, content, and bioactivity of food-derived peptides in body Kenji Sato J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00390 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Journal of Agricultural and Food Chemistry

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Structure, content, and bioactivity of food-derived peptides in body

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Kenji Sato

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Division of Applied Biosciences, Graduate School of Agriculture,

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Kyoto University, Kitashirakawa Oiwakecho, 606 8502, Kyoto, Japan.

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TEL: +81 75 753 6444

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FAX:+81 75 753 6400

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E-mail: [email protected]

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1 ACS Paragon Plus Environment


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Abstract

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Orally administered peptides are assumed to be degraded into amino acids in the body.

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However, our recent studies revealed some food-derived prolyl and pyroglutamyl

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peptides with 2-3 amino acid residues in the blood of humans and animals, while most

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of the peptides in the endo-proteinase digest of food protein are degraded by

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exo-peptidase. Some food-derived di-peptides in the body display in vitro and in vivo

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biological activities. These facts indicate that the biological activities of food-derived

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peptides in the body, rather than those in food, are crucial to understanding the

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mechanism of the beneficial effects of orally administered peptides.

19 20 21

Keywords: Peptide, food, functional food, bioactive peptide, bioavailability

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Introduction

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Enzymatic hydrolysate of food proteins and fermented foods contain numerous

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peptides with different structures. The ingestion of these preparations has some

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beneficial effects on human health beyond conventional nutritional values such as

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moderation of hypertension, hyperlipidemia, and inflammation.1,2 Some specific

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peptides in the hydrolysate are responsible for the beneficial activity upon ingestion. To

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identify the active peptides, peptides in the food have been isolated using

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high-performance liquid chromatography (HPLC) and other techniques and potential

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biological activities of isolated peptides have been evaluated using in vitro assays

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including cell culture systems and enzyme reaction systems. Many apparent ‘active’

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peptides consisting of 2-10 amino acids have been identified using the in vitro

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activity-guided fractionation and have suggested as being responsible for the biological

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responses by oral administration.1-2 In vitro studies using cell line Caco-2, a widely used

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model for testing the intestinal permeability of compounds, have demonstrated that di-

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and tri-peptides pass through peptide transporter (PepT1) on the intestinal brush border.3

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Furthermore, peptides longer than tri-peptide have been demonstrated to pass by

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paracellular diffusion.4 Recent advances in chromatography separation and tandem



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mass spectrometer (LC-MS/MS) enable determine peptides in complex matrix such as

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blood plasma. However, the contents of the ‘active’ peptides in the body after ingestion

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of the protein hydrolysate are frequently low (nM level), which is far less than the

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concentrations used for in vitro assays.5-7 It has been also demonstrated that some di-

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and tri-peptides are rapidly degraded in enterocyte4 and also blood plasma.8 These

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observations strongly suggest that potential bioactivity of peptides in the food may be

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lost during the digestion and absorption processes. Therefore, the in vitro activity of

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peptides in food is not always associated with the biological response after ingestion of

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the peptide-based food. To understand the mechanism of the beneficial response

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following the ingestion of the hydrolysate, identification of the relevant peptide or its

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metabolite in the target tissue is necessary. To that end, recent knowledge on the

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food-derived peptides in the body and their in vivo and in vitro activities are

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summarized. Furthermore, a prospective approach for identification of food-derived

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peptides in the body is proposed.

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Direct Detection of Food-Derived Peptides


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It has been assumed that most of the orally administered peptides are degraded into

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amino acids during the digestion and absorption processes, since negligible amounts

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(low nM levels) of the active peptide constituent of the food can be detected in the

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body.5-7 However, the demonstration of hydroxylproline (Hyp)-containing peptide in

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human blood at 10-100 µM levels after ingestion of 10-20 g collagen hydrolysate has

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dramatically changed this view.9,10 Hyp is specifically present in collagen, which is the

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main protein in the extracellular matrix (ECM). Increased Hyp-containing peptides in

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the blood after ingestion of collagen hydrolysate can be considered as food-derived

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peptides. This finding initiated studies on isolation and identification of food-derived

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collagen peptides in the blood. An oligo peptide fraction containing Hyp-peptide was

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prepared from blood plasma by size-exclusion chromatography (SEC) (Figure 1,

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lower).9 Food-derived collagen peptides in the oligo peptide fraction were isolated by

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reversed phase HPLC. Using this technique, some collagen-derived peptides in human

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blood have been identified (Table 1).9-12 All are di- and tri-peptides. These peptides

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reached maximum levels 30-60 min after the ingestion depending sequence. The

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leading food-derived collagen peptides in human blood are Pro-Hyp (approximately



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>50% of total food-derived collagen peptides in blood) in all cases9-12 with Hyp-Gly

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accounting for approximately 15%.12 These peptides resist the degradative action of

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peptidases in plasma.9,11 On the other hand, the collagen hydrolysate prepared by

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bacterial protease used for ingestion was shown to consist of peptides with an average

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molecular weight of 5,000 daltons, and lacking di- and tri-peptides (Figure 1, upper).

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The bacterial protease has strong endo-proteinase activity but low exo-peptidase

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activity.13 Thus, the collagen hydrolysate consisted of relatively large peptides. These

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observations clearly demonstrate that peptides in food are further degraded by

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exo-peptidases during digestion and that the indigestible di- and tri-peptide are

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absorbed.

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The same approach has been used to identify food-derived peptides after ingestion of

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enzymatic hydrolysates of non-ECM proteins, such as soy, wheat, corn, and fish meat.

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However, it has been difficult to directly detect food-derived peptides in blood, possibly

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due to the lower concentration of these protein-derived peptides in the blood compared

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to the concentrations of collagen- and elastin-derived peptides. There are repeated

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motifs in collagen (Pro-Hyp-Gly) and elastin (Val-Gly-Val-Ala-Pro-Gly). Thus,


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Pro-Hyp and Pro-Gly could be released in multiple regions of collagen and elastin,

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which explains the high concentration of these peptides in human blood. In order to

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detect food-derived peptides after ingestion of enzyme hydrolysate of food protein that

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lacks these repeated motifs, a new approach is necessary.

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Pre-Identification of Peptidase-Resistant Peptide.

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In most cases, a food-grade enzymatic hydrolysate of protein is prepared using

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bacterial protease from Bacillus subtilis, B. thermoproteolyticus, B. licheniformis, and

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other bacteria.13 As mentioned above, these have strong endo-proteinase activity but low

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exo-peptidase activity. The peptides in the hydrolysate could be further degraded by

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exo-peptidase

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Tyr-Leu-Asp-Asn-Tyr, isolated from a thermolysin (B. thermoproteolyticus) digest of

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shark cartilage and which attenuated oxonate-induced hyperuricemia in rat upon

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ingestion, was degraded into Asp-Asn-Tyr and other peptide fragments by incubation

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with plasma for 60 min.14 This observation clearly indicates that peptides in the

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endo-proteinase digest could be further degraded by exo-peptidases in plasma.14 The

in

the

digestive

tract

and

blood.


In

fact,

a

pentapeptide,


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same peptides can be released by in vitro digestion using carboxypeptidase A and

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leucine aminopeptidase (data not shown). Peptides in the in vitro digest of

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exo-peptidases including above peptidases or crude extracts of intestine and blood

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plasma, rich in exo-peptidase, could be candidate for the food-derived peptides in the

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body. They would be more easily isolated and identified compared to peptides in blood

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and organ extracts, which is a more complicated matrix. Our preliminary results have

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revealed that most of the exo-peptidase-resistant peptides are prolyl peptides, which

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include Pro-Hyp, Pro-Gly, Pro-Ala, and Pro-Gln and pyroglutamyl peptides including

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pyroGlu-Leu, pyroGlu-Gln, and pyroGlu-Pro. The amide in the side chain of glutamine

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is easily released from the carbonyl group and the latter is coupled to an amino group of

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glutamine to forms a lactam (Figure 2 upper). This cyclic glutamic acid derivative is

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referred to as pyroglutamic acid. The pyroglutamyl peptide has a pyroglutamyl residue

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at the amino terminus (Figure 2 lower). Short chain pyroglutamyl peptides resist

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exo-peptidase digestion.15,16 These exo-peptidase-resistant peptides likely survive in the

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digestive tract and blood and are present in the body. Pro-Hyp, Pro-Gly, and

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pyroGlu-Leu have been detected in the blood of humans9-12,17,18 and animals19 bloods.


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The comprehensive identification of exo-peptidase-resistant peptides in food protein

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hydrolysates and their detection in blood and organs are in progress.

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Biological Activity of Food-Derived Peptides in the Body

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Food-derived peptides present in the body are almost always di- or tri-peptides at

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concentrations of up to 100 µM. An important issue to consider is whether these small

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peptides have significant biological activity at these concentrations and are responsible

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for the biological response by ingestion of the hydrolysate, or whether they are just

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non-active metabolites. It has been demonstrated that some food-derived peptides in

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human blood possess in vitro and in vivo activities. The Pro-Hyp and Hyp-Gly,

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collagen-derived peptides, trigger the growth of primary cultured fibroblasts attached to

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collagen gel.11,20 In addition, Pro-Hyp increases the production of hyaluronic acid by

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fibroblasts.21 Pro-Gly, elastin-derived peptide, increases the production of elastin from

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cultured fibroblast22 and protects aortic endothelial cells against hypertensive

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endothelial injury in a spontaneous hypertensive rat (SHR) model.23 The oral ingestion

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of pyroGlu-Leu, which was first identified in wheat gluten hydrolysate and which has



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been detected in the blood of rat after ingestion of the hydrolysate, attenuates both

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D-glucosamine-induced acute hepatitis (20 mg/kg body weight)19 and experimental

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colitis-induced dysbiosis in mice (0.1 mg/kg body weight).24 These observations

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indicate that small food-derived peptides can produce substantial in vitro and in vivo

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biological activities.

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Conclusion

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While most of the peptides in food are degraded into amino acids by exo-peptidases,

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some exo-peptidase-resistant peptides can reach target organs and can circulate in the

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blood. These small food-derived peptides in the body can produce significant biological

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activities. The collective evidence leads me to propose a new approach for identification

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of active peptide, in which the peptides are first identified in the target organ followed

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by an examination of their in vitro and in vivo activities. For the identification of the

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food-derived peptides in the body, pre-identification of peptides present in the in vitro

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exo-peptidase digests of food peptide is helpful, as these peptides are frequently present

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in the body. This approach has an advantage over the conventional in vitro


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activity-guided fractionation approach, as the conventional approach does not consider

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bio-availability of peptides and must assay many peptides in food. Recently, in silico

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digestion has been used to predict the production of bioactive peptides from protein. If

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the software does not consider exo-peptidase digestion, it can predict the release of

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peptides with in vitro activity in food but cannot predict the real bioactive peptide in the

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body.

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ACKNOWLEDGEMENT

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The present study was partially supported by the Grant-in-Aid for Scientific Research

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(C) 16K07735.

162 163

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Table 1. Structures of food-derived collagen peptides in human blood

256 257 258 259 260 261 262

Sequence

Reference

Pro-Hyp

9, 10, 11,12, 17,18

Ala-Hyp

9, 10, 12, 17,18

Hyp-Gly

11, 12

Glu-Hyp

12

Ile-Hyp

9, 10, 12, 17, 18

Leu-Hyp

9, 10, 12, 17, 18

Phe-Hyp

9, 10, 17

Ala-Hyp-Gly

9, 10, 17

Pro-Hyp-Gly

9, 10, 12, 17

Ser-Hyp-Gly

10, 12, 17

263 264 265 266 267 268 269 270 271


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Legends for Figures

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Figure 1. Distribution of collagen peptides in food for ingestion and those in

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human blood 1 hour after ingestion.

Adapted from Iwai et al.6

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Figure 2.

Formation of pyroglutamic acid and pyroglutamyl peptide.

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281 282 283

Figure 1

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Figure 2

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TOC

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Structure, content, and bioactivity of food-derived peptides in body

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