1A comprehensive overview of ‘omic’ analytical methods in production of

2bioactive peptides

4L. Saavedraa, E. M. Heberta*, C. Minahkb, P Ferrantic

6aCentro de Referencia para Lactobacilos (CERELA-CONICET), Chacabuco 145,

74000 San Miguel de Tucumán, Argentina

8bInstituto Superior de Investigaciones Biológicas (CONICET-UNT) e Instituto de

9Química Biológica ‘‘Dr. Bernabe Bloj’’ UNT, Chacabuco 461, 4000,San Miguel de

10Tucumán, Argentina

11cDipartimento di Scienza degli Alimenti, University of Naples Federico II, Parco

12Gussone, Portici I- 80055, Italy.

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14*Corresponding author at:aCentro de Referencia para Lactobacilos (CERELA-

15CONICET), Chacabuco 145, 4000 San Miguel de Tucumán, Argentina Tel.:

16+543814310465; fax: +543814005600.

17E-mail addresses: ehebert@cerela.org.ar

18

19

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20ABSTRACT

21

22Nowadays, there is an increased interest in health-promoting functional foods,

23whereby consumers hold higher expectations of health-promoting benefits beyond

24basic nutrition. Dietary proteins provide a rich source of bioactive peptides, which

25are hidden in a latent state within the native protein, requiring enzymatic

26proteolysis for their release. Bioactive peptides can be produced during in vivo

27gastrointestinal digestion and/or food processing. Lactic acid bacteria are among

28the most widely microorganisms used as starter cultures for the production of

29fermented foods, and through their proteolytic system, they contribute to the

30release of bioactive peptides from dietary proteins. In vitro and in vivo studies

31demonstrated several biological functions attributed to bioactive peptides, such as

32antimicrobial, immunomodulatory, enhancement of mineral absorption,

33antithrombotic, antihypertensive, opioid and antioxidant activities. The great

34complexity and the wide dynamic range of relative peptide abundance in these

35products severely challenge the capabilities of existing analytical methodologies.

36However, functional and comparative genomic studies as well as proteomic

37approaches provide a wealth of knowledge in the way in which these lactic acid

38bacteria can use food proteins releasing bioactive peptides.

39

40

41Keywords: bioactive peptides; lactic acid bacteria; proteomic; genomic; mass

42spectrometry

43

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441. Introduction

45

46 Proteins, carbohydrates and lipids are the three classes of macronutrients

47which provide the necessary energy to maintain body functions as well as the

48structural and functional integrity of the organisms. Nutritionally, the quality of

49proteins depends on the amino acid composition, the absence of co-existing anti-

50nutrients that can limit protein digestibility and absorptive ability. Nowadays, there

51is an increased interest in health-promoting functional foods, whereby consumers

52hold higher expectations of health-promoting benefits beyond basic nutrition.

53Dietary proteins provide a rich source of bioactive peptides, which are hidden in a

54latent state within the native protein, requiring enzymatic proteolysis for their

55release. Biologically active or functional peptides are food derived peptides that

56exert, beyond their nutritional value, a physiological, hormone-like effect in the

57body (Hebert, Saavedra, & Ferranti, 2010). Bioactive peptides derive from a wide

58range of animal and vegetable proteins; such as bovine and human milk, fish, egg,

59meat, soybean, rice, sunflower and cereals (Bouzerzour et al., 2012; Escudero,

60Aristoy, Nishimura, Arihara, & Toldra, 2012; Kussmann & Van Bladeren, 2011;

61Senevirathne & Kim, 2012).

62 Food proteins are selected as sources of bioactive peptides according to

63different criteria (Udenigwe & Aluko, 2012): (i) food industry by-products in order to

64reduce environmental contamination and manufacture value-added products, for

65example whey proteins; (ii) proteins containing specific amino acid sequences for a

66biological activity of interest; iii) a QSAR-based in silico approach was recently

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67proposed for the prediction of food protein than can release bioactive peptides (Gu

68et al., 2012; Udenigwe & Aluko, 2012).

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702. Production of Bioactive Peptides

71 Bioactive peptides are encrypted in the primary structure of animal and plant

72proteins but they can be released by proteolysis in vitro, in vivo or a combination of

73both (Figure 1, Hebert et al. 2010). The in vivo release of bioactive peptides

74involves the gastrointestinal digestion (with digestive enzymes such as pepsin,

75trypsin, chymotrypsin and peptidases from the intestinal brush border membranes)

76as well as enzymes derived from the human microbiota. On the other hand, the in

77vitro production of bioactive peptides includes the enzymatic hydrolysis of the food

78protein by endogenous enzymes present in the food matrix as well as proteolysis

79occurring during food processing or ripening by action of starter cultures or by

80enzymes isolated from proteolytic microorganisms (e.g., Lactobacillus helveticus,

81L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis (Espeche Turbay,

82de Moreno de LeBlanc, Perdigon, Savoy de Giori, & Hebert, 2012; Hebert et al.,

832010). Therefore, certain LAB, mainly the strains belonging to the genera

84Lactobacillus, are currently marketed as health-promoting cultures or probiotics

85(Kleerebezem et al., 2010; Saxelin, Tynkkynen, Mattila-Sandholm, & de Vos,

862005). Microbial fermentation is one of the major processes to generate bioactive

87peptides, mainly in the dairy industry where the lactobacilli cell envelope-

88associates proteinases (Prt) release peptides during milk fermentation (Espeche

89Turbay et al., 2012; Hebert et al., 2008; Hebert et al., 2010).

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913. Main sources and target proteins involved in the production of bioactive

92peptides

93 Cryptic bioactive peptides have been isolated and characterized from a

94variety of sources: soybean, sunflower, corn, wheat, barley, rice, meat, fish, milk

95and its derivatives such as yogurt and cheese (Table1, Bouzerzour et al., 2012;

96Escudero et al., 2012; Kussmann & Van Bladeren, 2011; Senevirathne & Kim,

972012). Depending on their amino acid sequence, bioactive peptides can exhibit

98diverse activities by binding to a specific receptor in the gastrointestinal tract or in

99target organs and tissues after absorption into the bloodstream (Figure 1, Table 1).

100Antimicrobial, immunomodulatory, anticarcinogenic, antitumoral, antithrombotic,

101opioid, and antioxidant activities; enhancement of mineral absorption and/or

102bioavailability; and blood pressure-lowering effect are some of the biological

103activities attributed to food-derived peptides (Hebert et al., 2010). Furthermore,

104some peptides could play a role in prevention and treatment of metabolic

105syndrome via different mechanisms, for instance by decreasing body mass,

106regulation of blood pressure, insulinemia and cholesterolemia levels (Ricci-Cabello,

107Herrera, & Artacho, 2012). An exhaustive description of food bioactive peptides

108currently known is outside the scopes of this review, which is instead aimed to give

109an overview of their main sources, in conjunction with the most representative

110activities and of omic approaches for their characterization.

111

1123.1. Bioactive peptides: main food sources

113 The protein content of soybean is very high (around 37% in average),
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114approximately 80% of total protein corresponds to two storage globulins, 7S

115globulin (β-conglycinin) and 11S globulin (glycinin). A number of bioactive peptides

116were shown to be released from soy protein by means of microbial, gastric and

117pancreatic enzyme digestion of soybean proteins in the last decades (Kwon, Daily,

118Kim, & Park, 2010). For instance, the sequential treatment with pepsin at low pH,

119followed by hydrolysis with pancreatin in mild alkaline conditions resulted in the

120generation of well-defined peptides ranging from approximately 13 kDa to less than

1211 kDa, displaying important activities as antioxidants as well as in improving

122muscle glucose uptake (Roblet et al., 2012). Inoue et al., (2011) digested soy

123protein with endo-type protease derived from Bacillus sp., and the resulted

124peptides were screened for hypotriglyceridemic activity. They were able to

125characterize two dipeptides that lowered triacylglycerols (Lys-Ala, Val-Lys) and one

126promising peptide, Ser-Tyr, that was not only important in decreasing triacylglycerol

127levels but also in reducing apoB secretion in a hepatoma cell line (Inoue et al.,

1282011). Larger soy peptides also displayed hypocholesterolemic activity, although by

129different mechanisms. In this regard, Cho, Juillerat, and Lee (2007) described

130peptides ranging from 200 to 3000 Da that stimulate LDL receptor transcription

131thus inducing a reduction in the levels of circulating LDL particles. In other reports,

132peptides released by hydrolysis from soy proteins were sequenced, characterized

133and based on their sequences, new synthetic peptides were produced with

134enhanced activities. For example, Pak, Koo, Kwon, & Yun (2012) recently identified

135two soy bioactive peptides Leu-Pro-Tyr-Pro and Ile-Ala-Val-Pro-Gly-Asp-Val-Ala,

136which inhibited the enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR)

137that catalyzes the rate-limiting step of cholesterol synthesis. Based on the

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138sequences identified, they were able to design twelve peptides, one of which

139displayed 14,500-fold increase inhibitory activity as compared to the natural

140bioactive peptides (Pak et al., 2012). Other small bioactive peptides from soy were

141characterized as ACE inhibitors and reactive-oxygen species scavengers

142(Farzamirad & Aluko, 2008). Furthermore, an anti-cancer activity has been

143suggested; a nonapeptide obtained by thermolase hydrolysis of de-fatted soy

144protein, X-Met-Leu-Pro-Ser-Tyr-Ser-Pro-Tyr, was shown to arrest mouse monocyte

145macrophage cell line in G2/M phases (Kim et al., 2000). Lunasin, is a promising

146anti-cancer 5.5 kDa peptide from soy that has been shown to bind deacetylated

147histones thus inhibiting their acetylation (Galvez, Chen, Macasieb, & de Lumen,

1482001). However, strictly speaking lunasin is not a bioactive peptide, since it is a

149small subunit of 2S albumin and is not a cryptic peptide released by hydrolysis of a

150larger protein. Lunasin was also found in other sources such as wheat, barley, rye,

151etc (Jeong, Lam, & de Lumen, 2002).

152 Even though much less has been published for other plant sources as

153compared to soy, there is a growing body of evidence showing that promising

154bioactive peptides can be found elsewhere. In this regard, the pentapeptide Glu-

155Gln-Arg-Pro-Arg, isolated from heat stabilized de-fatted rice bran displayed anti-

156cancer property. Actually, its activity was successfully tested on liver, colon, lung

157and breast cancer cells (Kannan, Hettiarachchy, Lay, & Liyanage, 2010). Another

158interesting example is the nonapeptide oryzatensin, which sequence is Gly-Tyr-

159Pro-Met-Tyr-Pro-Leu-Pro-Arg. This peptide was obtained by tryptic hydrolysis of

160rice soluble protein and displayed a dual activity: it was able to enhance the

161antimicrobial activity of leukocytes and it also displayed an ileum-contracting

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162activity (Takahashi, Moriguchi, Yoshikawa, & Sasaki, 1994). In addition, transgenic

163rice turned out to be an excellent tool for delivering bioactive peptides by

164expressing heterologous proteins (Wakasa et al., 2006; Yang, Wakasa, & Takaiwa,

1652008).

166 Corn and wheat are other plant sources of bioactive peptides. In fact, ACE

167inhibitors as well as hypocholesterolemic peptides were isolated from corn (Kongo-

168Dia-Moukala, Nsor-Atindana, & Zhang, 2011; Parris, Moreau, Johnston, Dickey, &

169Aluko, 2008). On the other hand, after proteolysis at low pH, Ile-Ala-Pro tripeptide

170was isolated from wheat gliadin, exhibiting a powerful ACE inhibitory activity (Motoi

171& Kodama, 2003). Interestingly, when some lactic acid bacteria (LAB) belonging to

172the genus Lactobacillus were used for sourdough fermentation of several cereal

173flours, including wheat, anti-oxidant activity was obtained due to the release of

174bioactive peptides ranging from eight to fifty-seven amino acids. Allegedly, these

175peptides were produced from gliadins since the lactobacilli used in this study were

176chosen because of their ability to hydrolyze gliadins (Coda, Rizzello, Pinto, &

177Gobbetti, 2012).

178 In meat, short peptides commonly increase their concentrations during post-

179mortem storage even at low temperatures (Nishimura, Rhue, Okitani, & Kato,

1801988). However, there is scarce information on the formation of beneficial bioactive

181peptides in such conditions. On the other hand, muscle hydrolysis by enzymatic

182digestion in controlled conditions may release important peptides. As opposed to

183milk, LAB do not exert an important effect in bioactive peptide generation in meat

184(Arihara, 2006). Nevertheless, they do modulate digestion by modifying pH during

185fermentation, which may result in increased proteolysis by endogenous meat

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186proteases (Kato et al., 1994). Among beneficial properties of meat bioactive

187peptides, ACE-inhibition is the most common activity found. For instance, two

188pentapeptides released from the heavy chain of porcine myosin by thermolysin

189were sequenced and characterized as anti-hypertensive compounds. Interestingly,

190these peptides were also tested in vivo showing that they were very effective in

191lowering blood pressure in animal model of spontaneously hypertensive rats

192(Arihara, Nakashima, Mukai, Ishikawa, & Itoh, 2001). On the other hand, pepsin

193also generated important ACE-inhibitors from the light chain of porcine myosin,

194such as the octapeptide Val-Lys-Lys-Val-Leu-Gly-Asn-Pro, which was also effective

195in the same animal model of spontaneously hypertensive rats (Katayama et al.,

1962007). Myosins are not the only sources of bioactive peptides in muscle, troponin

197and collagen are also important contributors to the peptide production (Katayama

198et al., 2008; Saiga et al., 2008). Even titin, the largest protein described so far, was

199recently shown to contribute with bioactive peptides. In fact, the ACE-inhibitor

200pentapeptide Lys-Ala-Pro-Val-Ala was released from this protein after treatment of

201porcine muscle with a sequential digestion of pepsin and pancreatic proteases

202(Escudero, Sentandreu, Arihara, & Toldra, 2010). In addition to ACE inhibition,

203antioxidant peptides derived from meat were reported in the literature (Li, Chen,

204Wang, Ji, & Wu, 2007). Moreover, there is also a mention of four meat-derived

205peptides formerly ascribed as ACE-inhibitors that turned out to be antimicrobial and

206anti-cancer agents (Jang, Jo, Kang, & Lee, 2008). A comprehensive analysis of

207bioactive peptides from meat can be found elsewhere (Ryan, Ross, Bolton,

208Fitzgerald, & Stanton, 2011).

209 Fish and other aquatic organisms are also sources of bioactive peptides,
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210including antioxidants such as the anionic nonapeptide Leu-Gly-Leu-Asn-Gly-Asp-

211Asp-Val-Asn from conger eel (Ranathunga, Rajapakse, & Kim, 2006) and the two

212decapeptides from rotifer (Byun, Lee, Park, Jeon, & Kim, 2009). Antioxidant

213peptides were also reported from fishes like tuna (Je, Qian, Byun, & Kim, 2007).

214Tuna was also shown to be the source of a quite interesting peptide with anti-

215hypertensive activity. In fact, pepsin digestion of tuna frame protein released a 21-

216mer cationic peptide that acted as ACE-inhibitor (Lee, Qian, & Kim, 2010). Other

217aquatic organisms also seem to be very promising as sources of powerful ACE-

218inhibitors, recently reviewed in Ngo, Vo, Ngo, Wijesekara, and Kim (2012). Anti-

219coagulant (Jung & Kim, 2009), anti-obesity (Cudennec, Ravallec-Plé, Courois, &

220Fouchereau-Peron, 2008), anticancer (Naqash & Nazeer, 2012), an even anti-HIV

221activities were also reported. In the latter case, among several peptides active

222against HIV, two almost identical hexapeptides from oyster with the sequence Leu-

223Leu-Glu-Tyr-Ser-Leu/Ile proved to be highly effective at the nanomolar range.

224Actually, these peptides blocked HIV protease by competitive inhibition (Lee &

225Maruyama, 1998).

226 Bioactive peptides can also be released by hydrolysis with digestive

227enzymes from milk and dairy products. These peptides can be released from α, β,

228and -caseins and whey proteins such as α-lactalbumin, β-lactoglobulin, lactoferrin

229and immunoglobulins (Belem, Gibbs, & Lee, 1999; Fitzgerald & Murray, 2006).

230Caseins are hydrolyzed not only in the stomach but also in the small intestine. On

231the other hand, even though pepsin may digest whey protein to some extent, the

232fact that whey proteins are less retained in the stomach as compared to caseins

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233(which precipitate because of the low pH and hence stay there for longer period of

234times) results in quicker exposure of whey proteins to pancreatic enzymes and

235therefore rapid absorption as well (Jakubowicz & Froy, 2012). For instance,

236lactoferricin, a 25 amino acid peptide derived from the digestion of the lactoferrin

237by pepsin, displayed an outstanding antimicrobial activity, even higher than the

238activity of its precursor protein (Oo, Cole, Garthwaite, Willcox, & Zhu, 2010). Potent

239antimicrobial peptides can be generated by trypsin digestion of whey proteins;

240these peptides are active against both Gram-positive and Gram-negative bacteria

241(Pellegrini, 2003). In addition, pepsin, trypsin or chymotrypsin digestion of α-

242lactalbumin results in the production of peptides with both immunomodulatory as

243well as antimicrobial activities against not only bacteria but also viruses and even

244fungi (Pellegrini, Thomas, Bramaz, Hunziker, & von Fellenberg, 1999). Beyond

245antimicrobial properties described for some whey-derived peptides, other activities

246have been reported to be generated from whey proteins by using enzymatic

247digestion like the emulsifying peptides released from β- lactoglobulin. Another

248interesting whey-derived bioactive compound is the tripeptide Ile-Pro-Ala that is

249formed from β-lactoglobulin hydrolysis and may act as inhibitor of dipeptidyl

250peptidase-4, reducing glucose levels and stimulate insulin (Tulipano, Sibilia, Caroli,

251& Cocchi, 2011). In the same trend, other reports point out the possible role of

252whey peptides in reducing type II diabetes as well as obesity (Jakubowicz & Froy,

2532012).

254 Caseins are also targeted by digestive proteases and release promising

255bioactive peptides. Actually, β-casein treated with pepsin released 125 peptides,

256three of them were phosphopeptides. These peptides ranged from 4 to around 100

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257amino acids, many of them may display some activity (Schmelzer et al., 2007). In

258fact, the digestion of β-casein with pepsin or trypsin and chymotrypsin led to the

259release of neocasomorphin-6, a hexapeptide with opioid properties. On the other

260hand, α-casein and β-casein are also sources of bioactive peptides released by

261digestive enzymes. For instance, two peptides derived from α s1-casein, released by

262enzymatic treatment, were found to be involved in the regulation of the cholesterol

2637α-hydroxylase expression i.e. the key enzyme of bile synthesis. They were

264identified and sequenced (Nass et al., 2008). -casein in turn was shown to

265release at least four anti-hypertensive peptides from 3 to 11 amino acid length;

266these peptides were very potent ACE-inhibitors (Gomez-Ruiz, Ramos, & Recio,

2672007).

268

2693.2. Release of bioactive peptides by lactic acid bacteria

270 In milk, LAB undoubtedly have the greatest impact in releasing bioactive

271peptides. Protein digestion, in fermented milk products, is attributed to the typical

272LAB protease system composed by an extracellular serine proteinase and a

273number of intracellular peptidases alongside the specific transport systems for

274oligopeptides. In general, peptides from four to forty amino acid residues can be

275released from milk-related matrices (Kunji, Mierau, Hagting, Poolman, & Konings,

2761996). Among LAB, the most common species involved in bioactive peptides

277production are Lactococcus (Lc.) lactis subsp. cremoris, L. delbrueckii subsp.

278bulgaricus and L. helveticus. (Gobbetti, Ferranti, Smacchi, Goffredi, & Addeo,

2792000). For instance, proteases from L. helveticus were able to hydrolyze milk-

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280derived caseinates from different sources generating bioactive peptides that were

281sequenced and characterized as either antimicrobial agents or ACE inhibitors

282(Minervini et al., 2003). Interestingly, bovine milk-derived peptides were

283hydrophobic and rich in proline, for example the 19-mer peptide Leu-Val-Tyr-Pro-

284Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro while sheep-derived

285peptides were shorter and cationic such as the hexapeptide Arg-Pro-Lys-His-Pro-

286Ile (Minervini et al., 2003). On the other hand, L. delbrueckii subsp. bulgaricus was

287also able to generate ACE inhibitors from casein by milk fermentation. It can be

288mentioned the nonapeptide Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu and the

289octapeptide Asn-Val-Pro-Gly-Glu-Ile-Val-Glu (Gobbetti et al., 2000). It is important

290to note that these peptides are anionic with no similarity with the ACE-inhibitors

291produced by L. helveticus. Another important activity reported in the literature that

292can be ascribed to bioactive peptides produced by LAB from milk and dairy

293products is the immunomodulatory activity. For instance, L. acidophilus and Lc.

294lactis were shown to hydrolyze bovine milk generating small peptides that induced

295an important reduction in the activation of the nuclear factor NF-κB in an intestinal

296cell line (Stuknyte, De Noni, Guglielmetti, Minuzzo, & Mora, 2011). NF-κB is known

297to be a critical transcription factor that enhances the transcription of inflammatory

298cytokines e.g. tumour necrosis factor, IL-1, IL-6, and IL-8, hence it regulates the

299immune responses, inflammation and cell death (Stuknyte et al., 2011). A related

300activity was recently reported by Espeche Turbay, de Moreno de LeBlanc,

301Perdigon, Savoy de Giori, & Hebert, (2012). In this case, β-casein was digested by

302Lactobacillus delbrueckii ssp. lactis CRL 581 proteinase and the hydrolysate

303induced a strong anti-inflammatory action in a Crohn's disease murine model. This

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304effect was mediated by, at least in part, an increase in interleukin 10 levels and a

305decrease in IFN-γ (Espeche Turbay et al., 2012).

306 As stated above, bioactive peptides can be released not only from caseins

307but also from whey fraction mainly composed by α-lactalbumin and β-lactoglobulin,

308which represent roughly the 20% total protein of milk (Krissansen, 2007). In this

309regard, Yamamoto et al. (1999) reported a dipeptide released from whey proteins

310by the action of L. helveticus CPN4. This peptide, Tyr-Pro, acted as an ACE-

311inhibitor (Yamamoto, Maeno, & Takano, 1999). Even an osteoblast proliferation

312activity has been suggested. In this regard, the tripeptides Val-Pro-Pro and Ile-Pro-

313Pro released by L. helveticus fermentation were shown to be very effective in doing

314so (Narva, Halleen, Vaananen, & Korpela, 2004). More interestingly, Val-Pro-Pro

315was later tested in an animal model of ovariectomized rats, confirming that it was

316very important in preventing bone loss. However, because of its poor solubility in

317water, the tripeptide was ineffective in a pure form in solution and needed to be

318generated by L. helveticus and stay as part of the fermented milk in order to be

319active (Narva et al., 2007).

320 Several aspects are important for determining the biological activity of

321peptides including the enzymes used for hydrolysis, food processing conditions,

322and the size of the resulting peptides, which influences their absorption across the

323enterocytes and bioavailability in target tissues (Udenigwe & Aluko, 2012).

324 Theoretically, any food protein can be used as a potential source of

325bioactive peptides. Several bioactive peptides are produced in vitro by enzymatic

326proteolysis or fermentation. A combination of enzymatic hydrolysis by

327gastrointestinal digestion and fermentation of food proteins with proteolytic starter

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328cultures has been demonstrated to be effective in producing short functional

329peptides (Hebert, Saavedra, & Ferranti, 2010; Korhonen & Pihlanto, 2007). The

330choice of proteolytic enzymes or microorganisms used for food processing has a

331crucial impact on the composition of the released peptides (Panchaud, Affolter, &

332Kussmann, 2012). In this sense, the most important application of lactic acid

333bacteria (LAB) is their use as starter cultures in the manufacturing processes of

334various fermented food, mainly because they contribute to raw-material

335preservation due to acidification, but also because of their contribution to the

336development of flavor and texture of fermented products (Canchaya, Claesson,

337Fitzgerald, van Sinderen, & O'Toole, 2006; Kleerebezem et al., 2010). The natural

338habitat of lactobacilli includes dairy, meat and vegetal material fermentations to the

339gastrointestinal and the genital and tracts of humans and animals (Kleerebezem et

340al., 2010; Satokari et al., 2003; Vaughan et al., 2002). Therefore, any existing food

341proteins could be used as possible substrate for generating bioactive peptides.

342

3434. Influence of whole genome sequencing in the generation of bioactives

344peptides from LAB

345 Beginning with the genome of Lactobacillus plantarum WCFS1 in 2003

346(Kleerebezem et al., 2003), currently public databases contain 42 complete

347Lactobacillus genomes, while 105 more Lactobacillus genome sequencing projects

348containing scaffolds or contigs are available on-line

349(http://www.ncbi.nlm.nih.gov/genome/browse/). The availability of microbial

350genomes and extensive comparative analyses of the Lactobacillus (and other LAB)

351genomes have allowed to reveal the genomic features that contribute to the

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352ecological adaptability and examine the evolution of the species and their

353phenotypic diversities (Broadbent et al., 2012; Cai, Thompson, Budinich,

354Broadbent, & Steele, 2009; Canchaya et al., 2006; Kleerebezem et al., 2010; Liu,

355Bayjanov, Renckens, Nauta, & Siezen, 2010; Makarova et al., 2006). Comparative

356analysis of several LAB genomes showed that a combination of gene gain and

357gene loss occurred during the evolution of LAB with different ecological habitats

358(Broadbent et al., 2012; Cai et al., 2009; Canchaya et al., 2006; Kleerebezem et

359al., 2010; Liu et al., 2010; Makarova et al., 2006). Niche-specific genomic

360adaptations are reflected within the genomes (Cai et al., 2009; Kleerebezem et al.,

3612010). Adaptation to the dairy niche has been associated with a trend toward

362metabolic simplification. Thus, representative dairy LAB, Lactobacillus delbrueckii

363subsp. bulgaricus and Lactobacillus helveticus, contain many pseudogenes related

364to the utilization of several carbohydrates, reflecting their dedication to growth on

365lactose (Cai et al., 2009). In addition, their genome lost amino acid biosynthetic

366and cofactor biosynthetic genes while an increased in genes for peptide transport

367and proteolysis was observed, suggesting their adaptation to the protein rich dairy

368niche (Cai et al., 2009; Callanan et al., 2008; van de Guchte et al., 2006).

369Conversely, the lactobacilli commonly associated with the gastrointestinal tract

370display a great array for transport of a diverse group of carbohydrates and specific

371extracellular enzyme complexes that could be involved in complex carbohydrate

372degradation (Azcarate-Peril et al., 2008; Cai et al., 2009; Kleerebezem et al.,

3732010). These gastrointestinal lactobacilli encode other functions related with the

374gastric survival (e.g. genes encoding bile salt hydrolase) and with the intestinal

375mucosa interactions (e.g. mucus binding proteins).

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376 Comparative genome analysis of lactobacilli revealed that amino acid

377biosynthetic pathways are deficient in different degrees in LAB, being related with

378the adaptation to a specific-niche. Thus, dairy LAB L. delbrueckii subsp. bulgaricus

379and L. helveticus have lost the majority of their amino acid biosynthetic genes (Cai

380et al., 2009; Callanan et al., 2008; van de Guchte et al., 2006). Lactobacillus

381acidophilus and Lactobacillus gasseri, typical LAB found in the human

382gastrointestinal tract are auxotrophic for 14 and 17 amino acids, respectively

383(Azcarate-Peril et al., 2008; Cai et al., 2009). L. plantarum and Lactobacillus casei,

384ubiquitous microorganisms, possesses enzyme for biosynthesis of all amino acids

385except the branched-chain amino acids leucine, isoleucine, and valine (Cai et al.,

3862009).

387 Compensating for the inability of dairy-lactobacilli to synthesize most amino

388acids, these genomes contain several genes related to the proteolytic system that

389allows them to acquire amino acids from proteins present in their environment. This

390proteolytic enzyme system is vital to obtain essential amino acids and likely

391provides these lactobacilli with a selective advantage in protein-rich environments

392due to it is energetically favorable to obtain amino acids from environmental

393proteins than de novo synthesis (Cai et al., 2009; Hebert et al., 2008). The

394proteolytic system of LAB consists of a cell envelope-associated proteinase (Prt),

395transport systems to allow uptake of the resulting peptides, and several

396intracellular peptidases, which degrade peptides to amino acids (Hebert et al.,

3972008; Savijoki, Ingmer, & Varmanen, 2006). The Prt is the key enzyme of this

398system as it is involved in the first step of casein degradation (Savijoki et al., 2006),

399this enzyme also contributes to the development of flavor and texture of fermented

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400products (Savijoki et al., 2006). Additionally, certain Prts can release bioactive

401health-beneficial peptides during food fermentation (Hebert et al., 2008; Hebert et

402al., 2010; Savijoki et al., 2006).

403 Liu et al. (2010) performed a genome comparison of the proteolytic system

404of 22 LAB, including Prt, peptide transporters and peptidases. The 22 LAB

405genomes analyzed included L. acidophilus NCFM, L. johnsonii NCC 533, L.

406gasseri ATCC 33323, L. delbrueckii subsp. bulgaricus ATCC 11842, L. delbrueckii

407subsp. bulgaricus ATCC BAA365, L. plantarum WCFS1, L. brevis ATCC 367, L.

408sakei 23 K, L. salivarius UCC118, Oenococcus oeni PSU1, Pediococcus

409pentosaceus ATCC 25745, Leuconostoc mesenteroides ATCC 8293, L. casei

410ATCC 334, Lactococcus (Lc.) lactis subsp. lactis IL1403, Lc. lactis subsp. cremoris

411MG1363, Lc. lactis subsp. cremoris SK11, Streptococcus thermophilus CNRZ1066,

412St. thermophilus LMG18311, St. thermophilus LMD9, L. reuteri F275, L. helveticus

413DPC 4571, and L. rhamnosus GG (Liu et al., 2010). The LAB genomes of L.

414acidophilus, L. johnsonii, L. gasseri, L. delbrueckii subsp. bulgaricus and L.

415helveticus, encode a relatively higher number and variety of proteolytic system

416components (Liu et al., 2010). On the other hand, the Prt enzyme, responsible for

417the hydrolysis of food proteins, was only found in a few LAB strains, such as on the

418chromosome of L. acidophilus, L. johnsonii, L. delbrueckii subsp. bulgaricus, L.

419casei, L. rhamnosus and S. thermophilus strain LMD9, as well as on the plasmid of

420Lc. lactis subsp. cremoris SK11. This information on the distribution of the

421proteolytic system genes can be used to predict the proteolytic potential of the LAB

422strains. For example, L. delbrueckii subsp. bulgaricus and L. helveticus have a very

423broad set of proteolytic enzymes, which agrees with the role of these bacteria in

35 18
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424milk, L. delbrueckii subsp. bulgaricus serves as the proteolytic microorganism in

425yoghurt and L. helveticus is a cheese starter culture that has been used to degrade

426bitter peptides in cheese.

427 LAB species of plant origin such as L. plantarum, O. oeni, and Leuc.

428mesenteroides encode less proteolytic enzymes in their genomes, which is

429consistent with their habitat that is fiber-rich but contains less proteins (Liu et al.,

4302010). In addition, this comparative genomic approach allowed exploring the

431diversity of proteolytic system genes in various strains of Lc. lactis, confirming the

432proteolytic diversity between the two Lc. lactis subspecies, i.e. subsp. lactis and

433subsp. cremoris. These genomic approaches may allow the prediction of

434proteolytic and flavor-forming potential of bacterial strains. In addition, this

435knowledge could be used to improve the functional properties of dairy and other

436fermented food products by supporting the strain selection process.

437

4385. Strategies for the discovery of bioactive peptides

439 There are in silico and in vitro approaches aimed at discovering and

440identifying bioactive peptides in food matrix (Kussmann & Van Bladeren, 2011).

441The in vitro method consists of the following steps (1) selection of an appropriate

442food protein source; (2) enzymatic hydrolysis by selected enzymes, fermentation

443and/or gastrointestinal digestion; (iii) in vitro screening for the potential bioactivity

444properties; (iv) fractionation of the peptide mixture; (v) analysis of the peptide

445structure; and (vi) design of synthetic structural analogues or peptide mimetic to

446validate bioactivity in vitro and in vivo (Figure 2).

37 19
38
447 After enzymatic digestion or microbial-driven hydrolysis e.g. LAB

448fermentation, different activities should be analyzed and bioactive peptides

449identified following several strategies. When large volumes are being handled, a

450concentration step should be used. An interesting strategy is the enzymatic

451membrane reactor for continuous production of peptides. In fact, ultrafiltration

452membrane reactors have been applied to the production and concentration of milk-

453related peptides since more than twenty years ago with great success (Bordenave,

454Sannier, Ricart, & Piot, 1999). Alternatively, stepwise ultrafiltration with membranes

455of low molecular mass cut-off represents a useful tool for isolating bioactive

456peptides that are being produced (Pihlanto-Leppala, Koskinen, Piilola, Tupasela, &

457Korhonen, 2000). Regardless crude peptide fraction was filtered or not, several

458chromatographic steps need to be taken in order to purify the peptides. On one

459hand, either hydrophobic or hydrophillic adsorbent can be used as an initial step

460(Inoue et al., 2011) followed by an alternative fractionation with an ion-exchange

461chromatography (Contreras, Lopez-Exposito, Hernandez-Ledesma, Ramos, &

462Recio, 2008). Finally a HPLC-FPLC step has to be performed in order to separate

463peptides. This chromatographic step may involve either a size-exclusion column

464such as the Superdex Peptide HR10/30 column (Inoue et al., 2011; Seppo,

465Jauhiainen, Poussa, & Korpela, 2003), a reverse phase column (RP-HPLC) such

466as C8, C18 (Chen, Yang, Sun, Niu, & Liu, 2012; Haileselassie, Lee, & Gibbs, 1999),

467or a combination of both (Ghassem, Arihara, & Babji, 2012). Usually, peaks are re-

468chromatographed in RP-HPLC prior to the identification by mass spectrometry

469(Gobbetti et al., 2000). However, when hydrophilic peptides need to be analyzed

470there is a major problem with reverse phase chromatography. These peptides may

39 20
40
471elute in the first part of the chromatogram, but this part is usually discarded just to

472avoid troubles with salts in the mass spectrometer. Therefore, a different approach

473needs to be used. For instance, capillary electrophoresis coupled to MS/MS proved

474to be an excellent alternative (Gomez-Ruiz et al., 2007).

475 Even though soft ionization MS techniques such as field desorption (Mullally,

476Meisel, & FitzGerald, 1997; Singh, Fox, & Healy, 1997) and fast atom

477bombardment (Ferranti et al., 1997; Suetsuna & Nakano, 2000) were successfully

478used in the past for studying bioactive peptides, newer advanced MS

479methodologies are used nowadays, which constitute the core itself of the so-called

480peptidomic approach to analysis of food peptide matrices. For characterization and

481sequencing purposes, two different ionization techniques are generally applied:

482electrospray ionization (ESI (Herrero, Simo, Garcia-Canas, Ibanez, & Cifuentes,

4832012) and matrix-assisted laser desorption ionization (MALDI). In ESI, sample

484solutions are sprayed from a capillary tube under the action of an electric field.

485Droplets are formed and solvent evaporation under vacuum causes the charge to

486increase on the droplets (Crotti, Seraglia, & Traldi, 2011). This procedure generates

487multi-charged species, hence caution has to be taken when analyzing results

488(Tamvakopoulos, 2007). For its own nature, ESI-MS is easily on-line coupled with

489liquid chromatography (LC/ESI-MS). This allows to obtain de novo peptide

490sequencing by analysis of the fragmentation spectra generate by tandem MS

491(MS/MS). The availability of new generation high resolution instruments makes

492nowadays possible analysis of the complex peptide mixtures such as those

493normally found in food matrices or those generated by hydrolysis of food proteins

41 21
42
494to screen for bioactive species.

495 On the other hand, when MALDI is used, an off-line procedure is normally

496followed: peptides are first purified and mixed with a matrix, generally α-cyano-4-

497hydroxycinnamic acid. This ionization technique is more tolerant to impurities than

498ESI, and allows partially impure peptide mixtures to be analyzed. Furthermore,

499MALDI lets optimize the conditions of sample separation and since less than 5% of

500the sample is consumed for one analysis, different MS and MS/MS determinations

501can be done with a single chromatographic separation. However, hydrophobic

502peptide identification is not favored (Cech & Enke, 2000). In addition, chemical

503noise may become a problem in this technique (Krutchinsky & Chait, 2002). Both

504methodologies i.e. ESI and MALDI were extensively used for identifying bioactive

505peptides (Contreras et al., 2008; Leonil, Gagnaire, Molle, Pezennec, & Bouhallab,

5062000) and even though the final result should be independent from the ionizing

507technique used, there are some reports in the literature stressing that quite

508different results can be obtained when ESI and MALDI were used for comparison

509(Stapels, Cho, Giovannoni, & Barofsky, 2004). Therefore, it is convenient to use

510both approaches not only for comparing results but for combining them in order to

511get the complete information. In this regard, the study of bovine α-casein

512performed by (Schmelzer et al., 2007) represents a really nice example of this and

513the right way to tackle such a problem. The combination of these approaches is

514especially important when a large number of peptides are being analyzed because

515complementary information is crucial for getting the identity of all of them. An

516interesting example is the full characterization of bioactive peptides released from

517αs1- and β-Casein by the cell envelope proteinase from L. delbrueckii subsp. lactis

43 22
44
518CRL 581. In fact, more than sixty peptides were identified (Hebert et al., 2008).

519 In peptidomic MS analysis, ionized peptides are directed to the mass

520spectrometer analyzer, which may have different configurations, i.e. time of flight

521(TOF), TOF-TOF, triple quadrupole spectrometers, quadrupole-TOF (QTOF), ion

522trap and Fourier-transform ion cyclotron resonance (FT-ICR) (Aebersold & Mann,

5232003), which provides the accurate measurement for peptide identification (Lubec

524& Afjehi-Sadat, 2007). However, even ion trap (IT) based instruments, with a

525relatively poor resolving power, have been successfully used for peptide

526identification and quantification. In fact, ESI-IT-MS was successfully used for the

527identification of a number of ACE-inhibitors as well as antibacterial and antioxidant

528peptides (Hayes et al., 2007; Hernandez-Ledesma, Davalos, Bartolome, & Amigo,

5292005; Losito et al., 2006). On the other hand, triple quadrupole was effective in

530identifying ACE-inhibitors such as those from marine shrimp Acetes chinensis with

531Lactobacillus fermentum SM 605 (Wang et al., 2008) and antibacterial peptides

532(Recio & Visser, 1999) among others. Liquid chromatography-ESI MS/MS systems

533like QTOF were also utilized. In this regard, four antimicrobial peptides from royal

534jelly designed as jelleines were identified by RP-HPLC associated to ESI-QTOF

535(Fontana et al., 2004). Furthermore, two new ACE-inhibitors from caseins were

536reported using this powerful configuration i.e. MAP (β-casein f102-104) and ITP (α-

537s2-casein f119-121) (van Platerink, Janssen, & Haverkamp, 2007). Finally, for citing

538just a few examples, antioxidant peptides from bovine liver sarcoplasmic proteins

539that were released after digestion with thermolysin were recently identified as well

540by ESI-Q-TOF analysis (Di Bernardini et al., 2011).

541 On the other hand, MALDI-associated mass spectrometric analyses usually

45 23
46
542involve TOF or TOF-TOF equipments. For instance, the production of the well-

543known antimicrobial peptide lactoferricin and its purification could be studied by

544MALDI-TOF analysis (Chan & Li-Chan, 2007). Because this is a highly sensitive

545technique, the authors were able to found new cationic peptides, besides

546lactoferricin, released by peptic digestion of lactoferrin and found the proper

547conditions for lactoferricin production with just two-step purification protocol (Chan

548& Li-Chan, 2007). In addition to MALDI-TOF, MALDI-TOF-TOF tandem mass

549spectrometry proved to be a valuable procedure in bioactive peptide analysis.

550Indeed, three peptides with important ACE-inhibitor activity were identified by this

551technique from alcalase-hydrolyzed Mung bean protein (Li, Wan, Le, & Shi, 2006).

552Another example of the usefulness of MALDI-TOF-TOF is represented by the

553characterization of the highly water-soluble pentapeptide Glu-Gln-Arg-Pro-Arg,

554which displays anti-cancer activity against colon, breast and liver cancer cells. Heat

555stabilized de-fatted rice bran was hydrolyzed with digestive enzymes and <5 kDa

556fraction was further purified and characterized by MALDI-TOF-TOF (Kannan et al.,

5572010). In addition to the time of flight tandem measurements, MALDI can be

558adapted to Q-TOF as well. This technique is not only sensitive but also has a high

559mass resolution that allows studying posttranslational-modified peptides. For

560instance, it was successfully used in the localization of phosphorylated amino acids

561in phosphopeptides from β-casein and egg ovalbumin (Bennett et al., 2002).

562Moreover, high-quality data from the MALDI-QTOF result in protein identification,

563even determining protein modifications, where regular MALDI-TOF fails (Zhu,

564Miller, Barder, & Lubman, 2004). However, this approach was not fully utilized for

565cryptic bioactive peptides so far.

47 24
48
566 Besides identification, mass spectrometry is certainly an interesting strategy

567for quantifying already known peptides (Saz & Marina, 2008; Tamvakopoulos,

5682007). It should be noted that MALDI-based analyses are not as suitable for

569peptide quantification as LC-ESI-MS because peptides in the MALDI matrix are

570non-uniformly distributed (Lubec & Afjehi-Sadat, 2007). Nevertheless, attention has

571to be paid to biological matrices because they can interfere with ionization, thus

572misleading calculations (John, 2005). Actually, several peptides have been already

573quantified by these methods. For example, three ACE-inhibitor peptides from goat

574milk hydrolysate were quantified by mass spectrometry using synthetic peptides for

575calibration (Geerlings et al., 2006). There are a number of peptides that can be

576reliably quantified nowadays with either LC-MS or LC-MS/MS (Contreras et al.,

5772008). Finally, it should be mention that besides food, peptides can also be

578quantified from biological fluids. For instance, blood levels of several ACE-inhibitor

579peptides were quantified in volunteers that have orally taken up these peptides. For

580this purpose, a LC-ESI-triple quadrupole was used, which proved to be extremely

581sensitive and reliable (van Platerink, Janssen, Horsten, & Haverkamp, 2006).

582 Recently, Bouzerzour et al. (2012) exhaustively studied in vivo, the kinetics

583of hydrolysis of a complex matrix like infant formula in the different compartments

584of the gut (proximal jejunum, median jejunum and ileum) at different postprandial

585times (after 30, 90 and 210 min of the meal) using the piglet as a model; the

586residual proteins were quantified in each compartment by using an inhibition ELISA

587while the peptides were identified by nano-liquid chromatography–MS/MS

588(Bouzerzour et al., 2012). Some peptides which can promote bioactivity, such as

49 25
50
589peptide β-casein (f60–66) and peptide β-casein (f80–90) that carry

590immunomodulatory activity in vitro and anti-hypertensive activity in spontaneously

591hypertensive rats, respectively (Abubakar, Saito, Kitazawa, Kawai, & Itoh, 1998;

592Kayser & Meisel, 1996), were present in the jejunum and ileum of piglets

593(Bouzerzour et al., 2012). On the other hand, Picariello et al. (2012) analyzed the

594human milk proteome by using a gel-free shotgun proteomic analysis to overcome

595the limitations of the classical electrophoresis-based approach. Conventional 2DE

596for descriptive proteomics shows some limitations that are particularly critical for

597characterizing complex mixtures such as very low-abundance proteins, as well as

598those with extreme isoelectric points or molecular weight values which can escape

599detection on the gels. Briefly, the shotgun proteomic strategy consisted of the

600reduction of protein disulphide bridges and alkylation of cysteine residues,

601following by the trypsin digestion and dephosphorylation, this late step to prevent

602the missed detection of peptides due to phosphorylation. To enlarge the proteome

603coverage, the peptides are subjected to N-deglycosylation by PNGase F and

604analyzed separately. Finally, the complex mixture is analyzed by nanoflow-high

605performance liquid chromatography (HPLC)/Fourier Transform-Ion Cyclotron

606Resonance (FT-ICR) mass spectrometry (MS) (Picariello et al., 2012).

607 These studies aimed to elucidate the structure-activity of peptides, which

608provides essential information for the design of novel therapeutics or functional

609food ingredients. Thus, in order to guarantee address questions of bioavailability

610and bioefficacy, both systemically (i.e., in blood or target tissue) and locally (e.g., in

611the gastrointestinal tract), bioactive peptides should be identified and quantified all

612across from the food to the target tissues (Kussmann & Van Bladeren, 2011).

51 26
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613 In addition to this classical peptide discovery strategy, an in silico prediction

614and discovery of bioactive peptides approaches have been developed (Grigorov &

615van Bladeren, 2007; Panchaud et al., 2012). These in silico methods, named

616“reverse genome engineering” allow the prediction of bioactive peptides and their

617enzymatic release from a target protein diminishing the number of bioactivity tests

618developed (Grigorov & van Bladeren, 2007; Panchaud et al., 2012). Bioinformatics

619approaches together with the available protein and peptide databases offer the

620possibility to simulate the hydrolysis of a target animal of plant protein and predict

621the release of bioactive peptides. Several plant and animal genomes are available

622such as rice, maize, soy and bovine. On the other hand, some industrial and

623probiotic LAB are sequenced, being possible to predict the enzymatic hydrolysis of

624food proteins based on the specific enzyme activity of a selected LAB. Thus,

625nowadays, it is possible the in silico prediction of bioactive peptide produced by

626gastrointestinal digestion and/or food processing, providing a rapid and cost-

627effective strategy for novel bioactive peptide discovery.

628

6296. Conclusions

630 The importance attributed to food proteins and bioactive peptides in nutrition

631and health is undeniable. Scientific evidence demonstrated that peptides derived

632from enzymatic food protein hydrolysates possess several activities such as

633antimicrobial, immunomodulatory, enhancement of mineral absorption,

634antithrombotic, antihypertensive, opioid and antioxidant activities, which have a

635positive impact in human health. These bioactive peptides can be produced during

636in vivo gastrointestinal digestion and/or food processing. Lactic acid bacteria are

53 27
54
637among the most widely microorganisms used as starter cultures for the production

638of fermented foods, and through their proteolytic system, they contribute to the

639release of bioactive peptides from dietary proteins. Therefore, microbial

640fermentation especially by LAB, acquires a major role, since the production of key

641bioactive peptides can be triggered as foods are being produced. In this sense, the

642genomic and proteomic analysis of new LAB strains capable of releasing milk-

643derived bioactive peptides constitute the basis for the development of novel and

644improved functional foods. Mass spectromic analysis will help to improve our

645knowledge on food-derived peptides identifying those bioactive peptides that have

646a beneficial effect on health.

647

648Acknowledgements

649This work was supported by grants from CONICET, ANPCyT and CIUNT.

650

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1058

91 46
92
1059Table 1. Bioactive properties of food protein-derived peptides significant to human

1060health

Food sources Activities displayed by bioactive References

peptides derived from different food
sources
Soy bean Antioxidant Roblet et al., 2012
  Hypotriglyceridemic Inoue et al., 2011
  Hypocholesterolemic Cho et al., 2007
  HMG-CoA reductase inhibitor Pak et al., 2012
  ACE inhibitor Farzamirad & Aluko
2008
  Reactive oxigen species scanvengers Farzamirad & Aluko
2008
  Anticancer Kim et al., 2000, Galvez
et al., 2001
Rice Anticancer Kannan et al., 2010
  Antimicrobial and ileum-contracting activity Takahashi et al., 1994
Corn ACE inhibitor Parris et al., 2008
  Hypocholesterolemic Kongo-Dia-Moukala et
al., 2011
Wheat ACE inhibitor Motoi & Kodama 2003
Meat Anti-hypertensive Arihara et al., 2001
  ACE-inhibitor Katayama et al., 2007
    Escudero et al., 2010
  Antimicrobial and anti-cancer Jang et al., 2008
    Ryan et al., 2011
Fish and other Antioxidants Ranathunga et al., 2006
aquatic organisms
    Byun et al., 2009
    Je et al., 2007
  ACE-inhibitor Lee et al., 2010
    Ngo et al., 2012
  Antiviral (anti-HIV) Lee & Maruyama, 1998
Milk and dairy Antimicrobial Oo et al., 2010
products
  Immunomodulatory Pellegrini et al., 1999,
Espeche Turbay et al.,
2012, Stuknyte et al.,
2011
  Inhibitor of dipeptidyl peptidase-4 Tulipano et al., 2011
  Phosphopeptides Schmelzer et al., 2007
  Regulation of the key enzyme of bile Nass et al., 2008
synthesis

93 47
94
   ACE-inhibitors Gomez-Ruiz et al., 2007,
Yamamoto et al., 1999,
Minervini et al., 2003,
Gobbetti et al., 2000
  Osteoblast proliferation Narva et al., 2004
1061

1062

95 48
96
1063Figure Legends

1064

1065Figure 1. Hydrolysis of food proteins by the proteolytic system of the lactic acid

1066bacteria or by several digestive enzymes during the digestion process, which

1067results in the release of bioactive peptides

1068

1069Figure 2. Food bioactive peptide analysis workflow showing steps of production

1070and purification integrated strategies for the structural and functional

1071characterization.

1072

97 49
98