LUNES Biologically Active Peptides PROCESSES for Their Generation, Purification and Isolation

7232019 LUNES Biologically Active Peptides PROCESSES for Their Generation Purification and Isolation

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Review

Biologically active peptides Processes for their generation puri1047297cationand identi1047297cation and applications as natural additives in the food andpharmaceutical industries

Ruann Janser Soares de Castro Heacutelia Harumi SatoDepartment of Food Science School of Food Engineering University of Campinas 80 Rua Monteiro Lobato Campinas SP Brazil

a b s t r a c ta r t i c l e i n f o

Article historyReceived 19 March 2015Received in revised form 1 May 2015Accepted 8 May 2015Available online 12 May 2015

Keywords

ProteinsEnzymatic hydrolysisFermentationPuri1047297cationBioactive peptides

Recent technological advances have created great interest in the use of biologically active peptides Bioactivepeptides can be de1047297ned as speci1047297c portions of proteins with 2 to 20 amino acids that have desirable biologicalactivities including antioxidant anti-hypertensive antithrombotic anti-adipogenic antimicrobial and anti-in1047298ammatory effects Speci1047297c characteristics including low toxicity and high speci1047297city make these moleculesof particular interest to the food and pharmaceutical industries This review focuses on the production of bioac-tive peptides with special emphasis on fermentation and enzymatic hydrolysis The combination of differenttechnologies and the use of auxiliary processes are also addressed A survey of isolation puri1047297cation and peptidecharacterization methods was conducted to identify the major techniques used to determine the structures of bioactive peptides Finally the antioxidant antimicrobial anti-hypertensive anti-adipogenic activities andprobiotic-bacterial growth-promoting aspects of various peptides are discussed

copy 2015 Elsevier Ltd All rights reserved

Contents

1 Introduction 1852 Major processes for obtaining bioactive peptides 186

21 Fermentation 18622 Enzymatic hydrolysis 187

3 Concentration puri1047297cation and identi1047297cation of bioactive peptides 1874 Biological properties of bioactive peptides 189

41 Peptides with antimicrobial activity 19142 Peptides with antioxidant activity 19143 Peptides with anti-adipogenic activity 19244 Peptides with anti-hypertensive activity 19445 Induction of lactic acid bacteria and probiotic growth 195

5 Conclusion 195References 195

1 Introduction

Proteins are fundamental food components Nutritionally theyare sources of essential amino acids are indispensible for growth andmaintenance and are a source of energy Protein foods are able to affect

physicochemical and sensory properties such as solubility viscositygeli1047297cation and emulsion stability Some proteins in the diet havespeci1047297c biological properties making them potential ingredients forfunctional foods (Korhonen Pihlanto-Leppala Rantamaki amp Tupasela1998) During digestion proteins are hydrolyzed generating a largerange of peptides Someof these peptides have structural characteristicsthat allow them to interact with endogenous peptides Many endoge-nous peptides have important functions acting as neurotransmitters

Food Research International 74 (2015) 185ndash198

Corresponding authorE-mail address ruannjanserhotmailcom (RJS de Castro)

httpdxdoiorg101016jfoodres201505013

0963-9969copy 2015 Elsevier Ltd All rights reserved

Contents lists available at ScienceDirect

Food Research International

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hormones and regulators (Hernaacutendez-Ledesma Garciacutea-NebotFernaacutendez-Tomeacute Amigo amp Recio 2014)

Bioactive peptides can be obtained from animal or plant proteinsPlant sources are generally grains such as wheat rice oats rye andcorn and some legumes such as soy peas and chickpeas Of the plantsources soy is one of the most widely studied as a source of peptidesas it is a signi1047297cant source of dietary protein (Ortiz-Martinez Winkleramp Garciacutea-Lara 2014) Animal protein sources also have great potential

One of the most popular and promising lines of research is the produc-tion of hydrolyzed proteins from meat proteins which in addition tohaving important biological activities and being excellent sources of nu-trients such as minerals and vitamins can be used as 1047298avor enhancersand emulsi1047297ers (Lafarga amp Hayes 2014 Mora Escudero FraserAristoy amp Toldraacute 2014) The biological properties of other animal pro-tein sources such as egg and 1047297sh have also been studied (SakanakaTachibana Ishihara amp Juneja 2004 Theodore Raghavan amp Kristinsson2008)

Recent studies have linked the prevalence of cardiovascular diseaseobesity hypertension diabetes and cancer to nutritional factors In re-sponse to increased awareness of the relationship between food andhealth the market for functional foods has expanded A functionalfood is any food that in addition to basic nutritional functions providesadditional health bene1047297ts regulating one or more functions in the body(Diplock et al 1999 Hernaacutendez-Ledesma Contreras amp Recio 2011)

Processes incorporating protein hydrolysis have been studied to de-termine whether they produce biologically active peptides Mellander(1950) was responsible for the 1047297rst study relating the ingestion of bioactive peptides from hydrolyzed casein protein to increased bonecalci1047297cation in rachitic newborns Since then peptides with countlessbioactivities have been identi1047297ed According to the Biopep and BioPD(Bioactive peptidedatabase) databases morethan 1200 differentbioac-tive peptides have been recorded (Singh Vij amp Hati 2014)

Bioactive peptides are de1047297ned as speci1047297c regions of proteins withamino acid sequences that have biological activity including antioxi-dant anti-hypertensive antithrombotic anti-adipogenic antimicrobialanti-in1047298ammatory and immunomodulatory effects (Ahn Cho amp Je2015 Biziulevicius Kislukhina Kazlauskaite amp Zukaite 2006 Tavares

et al 2011 Tsou Kao Tseng amp Chiang 2010 Zhang Li amp Zhou2010) These peptides have 2ndash20 amino acids and molecular massesof less than 6000 Da Their bioactivity is mainly determined by theircomposition and amino acid sequence (Mora Reig amp Toldraacute 2014Sarmadi amp Ismail 2010 Singh et al 2014 Tsou Kao et al 2010) Thisenormous functional diversity places these peptides and proteins atthe forefront of the biotechnology 1047297elds (Miranda amp Liria 2008) fur-thermore these peptides and proteins have been identi1047297ed by severalauthors as possible substitutes for chemicals used as drugs or foodpreservatives (Hong et al 2008 Uhlig et al 2014)

According to Uhlig et al (2014) there is a very good outlook forusing bioactive peptides in the pharmaceutical 1047297eld Some peptides inthe clinical trial phase have shown very promising results for treatingcardiovascular infectious and metabolic diseases Peptides have an

important competitive advantage over traditional medications for thefollowing reasons 1) They have high speci1047297city for their target tissuesresulting in little or no toxicity and even low concentrations can be ef-fective This characteristic is extremely important for treating chronicdiseases 2) Synthetic chemical compounds that are typically used asdrugs often have a cumulative effect on the organism These syntheticsubstances may represent an environmental problem due to their ex-cretion still in the active form In contrast bioactive peptides undergolittle or no accumulation in the organism and they are easily degradedin the environment (Uhlig et al 2014)

Several methods are usedto obtain bioactive peptides including fer-mentation enzymatic hydrolysis and a combination of the two process-es (Fig 1) In fermentation the addition of lactic acid bacteria withproteolytic activity leads to formation of bioactive peptides especially

during dairy product manufacturing Enzymatic hydrolysis involves

the use of digestive plant or microbial proteolytic enzymes in a partialhydrolysis process leading to a reduction of allergenic factors im-proved digestibility and the formation of biologically active peptides(Korhonen 2009) One strategy used in several scienti1047297c studies dem-onstrated that using lactic acid bacteria together with food-gradeenzymes resulted in1047297nal productswith moreinteresting characteristicsthan either process alone Combining the techniques in addition toincreasing the amount of peptides in the fermented products resulted

in various biological and functional effects (Hafeez et al 2014) ChenTsai and Pan (2007) studied a combined process using enzymatichydrolysis with the microbial protease Prozyme 6 from Aspergillus anda commercial starter culture mixture of 1047297ve lactic acid bacteria as astrategy to enhance the ACE-inhibitory activity of bioactive peptidesfrom milk The results showed that the pre-treatment of the freshmilk with Prozyme 6 presented a positive impact on the ACE inhibitoryactivity of the peptides in which the IC50 value for the combinedprocess was024 mg mL minus1incontrastto064mgmL minus1 for the straightfermentation and 118 mg mL minus1 for fresh milk

In addition to the conventional methods mentioned above combin-ing several technologies has produced effective results for generatingfunctional peptides (Korhonen 2009) Ultra1047297ltration and nano1047297ltrationare examples of technologies that have been used to re1047297ne and isolatebioactive peptides allowing them to be separated by size for use inspeci1047297c applications (Picot et al 2010 Quiroacutes Chichoacuten Recio ampLoacutepez-Fandintildeo 2007)

Understanding the critical parameters for the process is fundamen-tally important for obtaining hydrolyzed proteinswith desirable biolog-icaland functional characteristics Theseparameters include the proteinsource and its characteristics such as chemical composition pH andseasonal variations enzymatic preparation and other aspects relatedto purity substrate speci1047297city speci1047297c activity pH and temperaturefor activity and stability and processing conditions including enzymeand substrate concentrations pH temperature and reaction timePrior knowledge and identi1047297cation of these parameters can be used astools for obtaining products with distinct functions for producing mul-tifunctionalpeptides or even for producing unique peptideswith specif-ic functions (Li-Chan 2015 Samaranayaka amp Li-Chan 2011)

This review describes advances in scienti1047297c research on the process-es of obtaining purifying and identifying the biological activities andpotential applications of bioactive peptides

2 Major processes for obtaining bioactive peptides

21 Fermentation

The use of fermentation processes to make bioactive peptides ismainly relevant to dairy product manufacturing Dairy products natu-rally contain precursor proteins for bioactive molecules (Akalin 2014Schanbacher Talhouk amp Murray 1997) Fermenting milk involves anumber of metabolic pathways responsible for generating metabolites

which signi1047297cantly contribute to the chemical biochemical and nutri-tional properties of the fermented products The proteolytic system of lactic acid bacteria (LAB) is complex andconsists of three major compo-nentsproteases boundto thecell wall that promote the initial hydroly-sis of milk casein into oligopeptides speci1047297c transporters that transferthe oligopeptides to the cytoplasm and intracellular peptidases that1047297n-ish the hydrolysis process to convert oligopeptides intofreeamino acidsandor low molecular weight peptides (Chaves-Loacutepez et al 2014) Theability of these microorganisms to produce proteolytic enzymes makesthem potential producers of bioactive peptides which can be releasedduring fermented product manufacturing Several microorganismshave been extensively reported in the literature as having an effectiveproteolytic system for protein hydrolysis and the release of bioactivepeptides including Lactobacillus helveticus Lactobacillus delbrueckii ssp

bulgaricus Lactococcus lactis ssp diacetylactis Lactococcus lactis ssp

186 RJS de Castro HH Sato Food Research International 74 (2015) 185ndash198



cremoris and Streptococcus salivarius ssp thermophylus (Hernaacutendez-Ledesma et al 2011) In addition to using live microorganisms proteo-lytic enzymes isolated from LAB have also been successfully used inenzymatic hydrolysis processes and for the production of bioactivepeptides (Choi Sabikhi Hassan amp Anand 2012)

Although dairy products have been highlighted in scienti1047297c studiesproducing these peptides by fermentation it has been shown thatfermentation products derived from soy beans rice and wheat arealso biologically active (Hati et al 2014 Inoue et al 2009 Limoacutenet al 2015 Nakahara et al 2010) (Table 1) Species of 1047297lamentousfungi such as Aspergillus oryzae and Aspergillus sojae have a long tradi-tion of safe use in the production of fermented foods in which severalpeptides with biological activities were detected for example antioxi-dant and antihypertensive activities (Giri Osako Okamoto Okazaki amp

Ohshima 2011 Inoue et al 2009 Nakahara et al 2010)

22 Enzymatic hydrolysis

Enzymatic hydrolysis is oneof thefastest safestand most easily con-trolled techniques for producing bioactive peptides and it can be usedto improve the functional and biological properties of the proteinsas well as to add value to byproducts with low commercial value(Luna-Vital Mojica de Mejiacutea Mendoza amp Loarca-Pintildea in press Moraet al 2014 Singh et al 2014 Zarei et al 2014)

Proteases catalyze the hydrolysis of peptide bonds in proteins andmay act on the ester and amide bonds All proteases have a certain de-gree of speci1047297city for the substrate generally based on the sequence of amino acids directly surrounding the bond that is cleaved (Santos amp

Koblitz 2008) This speci1047297city and the hydrolysis conditions (pH

temperature time) affect the size and the amino acid sequences in thepeptide chains as well as the quantity of free amino acids which can af-fect the biological activity of the hydrolysates (Luna-Vital et al in pressSarmadi amp Ismail 2010 Su Ren Yang Cui amp Zhao 2011 Tsou Kaoet al 2010 Zhou Canning amp Sun 2013) Proteases with speci1047297c activi-ty such as trypsin and chymotrypsin and combinations of differentnon-speci1047297c proteases such as Pronasetrade E from Streptomyces griseusand Flavourzymetrade from A oryzae have been used to produce morestable and effective bioactive peptides by reducing the reaction timesneeded for hydrolysis and by making it possible to obtain different pro-1047297les especially for the composition and molecular mass distribution of thepeptides These processesare especially usefulin the food andphar-maceutical industries which rely on animal plant and microbial prote-ases (de Castro Bagagli amp Sato 2015 Singh et al 2014 Vanderghem

et al 2011)In addition to the commercial enzymes it is important to note that

several studies reported the use of crude microbial enzymes to hydro-lyze proteins suggesting the potential application of novel proteasesources for the production of bioactive peptides

Table 2 summarizes somestudies in which the release of biologicallyactive peptides after protein hydrolysis using commercial and crudeprotease preparations was demonstrated

3 Concentration puri1047297cation and identi1047297cation of bioactive

peptides

Table 3 summarizes different characteristics for some methods of puri1047297cation and identi1047297cation of bioactive peptides including their

principle advantages and limitations Chromatography techniques are

Proteases produced

during fermentation

High pressure

homogenization

Bioactive

Peptides

Protein hydrolysates

Ultrafiltration or nanofiltration

Proteases

vegetable animal or

microbial

Microbial fermentation

Animal or vegetable protein sources

Isolation purification

and identification

Biological activities

in vitro and in vivo methods

Antihypertensive Antimicrobial Anti-adipogenic

Anti-inflamatory Immunomodulatory Antioxidant

Fig 1 Major processes for obtaining bioactive peptides and related bioactivities

187RJS de Castro HH Sato Food Research International 74 (2015) 185ndash198



amongst the most widely used such as high performance liquid chro-matography (HPLC) and ultra high pressure liquid chromatography(UHPLC) (Singh et al 2014) UHPLC has shown great potential in theseparation of small bioactive peptides increasing the throughput of reg-ular HPLC methodsThe main advantages of this method include the in-

crease of throughput resolution and sensitivity(Everley amp Croley 2008Fekete amp Guillarme 2014) Reversed phase HPLC (RP-HPLC) canbe usedto separate peptides by hydrophobicity (Pownall Udenigwe amp Aluko2010) Hydrophilic interaction liquid chromatography (HILIC) hasbeen shown to be a useful method forthe separationof hydrophilic sub-stances This method is based on increases in retention with increasingpolarity of the stationary phase and of the solutes and the decreasingpolarity of the predominantly organic solvent system used for elutionthe opposite principle of that observed in RP-HPLC (Yoshida 2004) LeMaux Nongonierma and FitzGerald (2015) reported HLIC method asa valuable tool to improve theseparation of short peptidesand differen-tiation of peptides with homologous sequences by mass spectrometryGel electrophoresis and ultra1047297ltration techniques have also been usedas auxiliary methods for structural and chemical composition analysis

of peptides (Roblet et al 2012 Singh et al 2014)Mass spectrometry has greatly improved the process of identifying

peptide sequences and studying protein pro1047297les and hydrolysis prod-ucts In particular interfaces have been developed that allow ions tobe generated from analyte molecules that are sensitive to temperatureandor are not very volatile Electrospray ionization and matrix assistedlaser desorptionionization (MALDI-TOF) for example has recently be-come important for the identi1047297cation and characterization of bioactivepeptides and proteins using mass spectrometry Liquid chromatogra-phyndashmass spectrometry is commonly used to identify peptide se-quences (Chiaradia Collins amp Jardim 2008 Contreras Loacutepez-ExpoacutesitoHernaacutendez-Ledesma Ramos amp Recio 2008 Singh et al 2014)

Peptides with anticoagulant activity that were obtained from goby1047297sh ( Awaous guamensis) and a protease from Bacillus licheniformis

were separated by molecular exclusion chromatography and reversed-

phase high-performance liquid chromatography and identi1047297ed bymass spectrometry The hydrolysate solution containing the peptideswas applied to a Sephadex G-25 (52 times 56 cm) gel 1047297ltration columnpre-equilibrated and eluted with distilled water and 45-mL fractionswere collected using a 1047298ow rate of 05 mL minminus1 Absorption at

220 nm was measured to determine the peptide elution pro1047297le Frac-tions with higher anticoagulant activity were recovered and puri1047297edin a reverse-phase Vydac C18 (10 times 250 mm Grace-Vydac) columnand eluted using a linear acetonitrile gradient (0 to 40 vv) and a1047298ow rate of 06 mL minminus1 The molecular mass and amino acidsequence of the peptides were measured using a triple quadrupolemass spectrometer with an electrospray ionization source (AppliedBiosystems API 3000 PE Sciex Toronto Canada) Four peptidesequences had high anticoagulant activity and were identi1047297ed as Leu-Cys-Arg His-Cys-Phe Cys-Leu-Cys-Leu-Arg and Cys-Arg-Arg (Nasriet al 2012)

Tsou Kao Lu Kao and Chiang (2013) puri1047297ed and identi1047297ed bioac-tive peptides from puri1047297ed soy protein and the Flavourzyme proteaseusing sequential fractionation with ultra1047297ltration membranes of various

sizes gel chromatography reversed-phase high-performance liquidchromatography and mass spectrometry The hydrolysates were initial-ly fractionated in ultra1047297ltration membranes of 30 10 and 1 kDa Thefraction retained on the 1-kDa membrane was selected for puri1047297cationdue to its ability to stimulate lipolysis in 3T3-L1 pre-adipocyte cellsThe 1-kDa retained portion was then applied to a Superdextrade peptide10300 GL column (10 times 300 mm GE Healthcare) equilibrated andeluted with 30 acetonitrile and a 1047298ow rate of 05 mL minminus1 One-milliliter fractions were collected and elution curves were constructedbased on absorbance measurements at 214 nm The fractions with thehighest anti-adipogenic activity were collected and puri1047297ed in aDevelosil ODS-HG-5 reverse-phase column (46 times 250 mm NomuraChemical) and eluted using a linear acetonitrile gradient (50 to 750)and a 1047298ow rate of 10 mL minminus1 The fraction with the most anti-

adipogenic activity was puri1047297ed again using a reverse-phase column

Table 1

Obtaining peptides with different biological activities by fermentation using various protein sources

Microorganism Protein source Fermentation conditions Peptides Bioactivity Reference

Streptococcus thermophiles

Lactobacillus bulgaricus

+Protease Flavourzyme

Soy milk Submerged fermentation for 5 hat 43 degC

Tyr-Pro-Tyr-Tyr Antihypertensive Tsai Chen Pan Gongand Chung (2008)

Aspergillus oryzae Rice soy andcasein

Solid-state fermentation for 40 hat 30 degC

Val-Pro-Pro Ile-Pro-Pro Antihypertensive Inoue et al (2009)

Aspergillus oryzaeRhizopus oligosporus Actinomucor elegansBacillus subtilis

Okara Sequential submergedfermentationB subtilis for 48 h at 40 degC A oryzae R oligosporus and A elegans for 60 h at 30 degC

Not identi1047297ed Antioxidant Zhu Cheng WangFan and Li (2008)

Aspergillus sojae Soy and wheat Solid-state fermentation for 192 h at20ndash45 degC and 95 humidity

Gly-Tyr Ala-Phe Val-ProAla-Ile Val-Gly

Antihypertensive Nakahara et al (2010)

Enterococcus faecalis

TH563Lactobacillus delbrueckii subspbulgaricus LA2

Cows milk Submerged fermentation for 24 hat 37 degC (Enterococcus faecalis) or44 degC (Lactobacillus delbrueckii)

Peptides with molecularweights less than 5000 Da

Antihypertensive andimmune-regulatory

Regazzo et al (2010)

L acidophilus ATCC 4356Lc lactis subsp lactis GR5

SodiumCaseinate

Submerged fermentation for 5 h at30 degC (Lactococcus lactis) or 37 degC(L acidophilus) with agitation at140 rpm


Immunomodulatory Stuknyte NoniGuglielmetti Minuzzoand Mora (2011)

Aspergillus oryzae Squid mantles Solid-state fermentation for 365days at 25ndash30 degC


Antioxidant Giri et al (2011)

B subtilis 10160 Rapeseed Solid-state fermentation for 6 daysat 32 degC and 85 plusmn 5 relativehumidity

Peptides with molecularweights between 180 and5500 Da

Antioxidant He et al (2012)

Bi 1047297dobacterium longum KACC91563 Casein Submerged fermentation for 24 h Val-Leu-Pro-Val-Gln Antioxidant Chang et al (2013)Lactobacillus casei spp

pseudoplantarum

Concentratedsoy protein

Submerged fermentation for 36 hat 37 degC

Leu-Ile-Val-Thr-Gln Antihypertensive Vallabha and Tiku (2014)

B subtilis ATCC 6051 Bean Solid-state fermentation for 96 hat 30 degC and 90 relative humidity

Peptides with molecularweights between 62 and2012 kDa

AntioxidantAntihypertensive

Limoacuten et al (2015)




and linear acetonitrile gradients of 10 to 40 Finally the peptides wereidenti1047297ed by liquid-chromatography coupled with mass spectrometryThree peptides with the amino acid sequences Ile-Leu-Leu Leu-Leu-Leu and Val-His-Val-Val were identi1047297ed as being responsible for theanti-adipogenic activity of the protein hydrolysates isolated from soy

Peptideswith anti-hypertensive activity wereisolated andidenti1047297edfrom gelatin hydrolysates extracted from stingray skin (Okamejeikenojei) The hydrolysates1047297rst were subjected to ultra1047297ltration througha 1-kDa membrane and peptides with molecular weights lower thanthis cutoff were collected Puri1047297cation consisted of sequential steps of isolation by fast protein liquid chromatography (FPLC) (AKTAAmersham Bioscience Co Uppsala Sweden) using a HiPrep 1610high 1047298ow ionic exchange column (16 times 100 mm Amersham Biosci-ences Piscataway NJ USA) and a GE Healthcare Superdextrade Peptide10300 GL gel 1047297ltration column (10 times 300 mm) Puri1047297ed peptideswere then identi1047297ed by MALDI-TOF mass spectrometry Two puri1047297edpeptides were found to be very anti-hypertensive and were identi1047297edas Leu-Gly-Pro-Leu-Gly-His-Gln with an estimated molecular weightof 720 Da and Met-Val-Gly-Ser-Ala-Pro-Gly-Val-Leu with a molecular

weight of 829 Da (Ngo et al 2015)Liu et al (2015) developed a UHPLC-Q-TOF MSMS method to iden-

tifypeptides with antioxidant activities derivedfromthe protein hydro-lysate of Mactra veneriformis The hydrolysates were fractionated on aSephadex G-25 gel 1047297ltration column (20 cm times 100 cm GE ChemicalsUppsala Sweden) using distilled water as the eluting solvent at a 1047298owrate of 04 mL minminus1 and separated 1047297ve fractions The two most activefractions were then separated on the basis of their antioxidant activitiesand subjected to an analysis using a Waters ACQUITY UHPLC systemwith a C18 column (100 mm times 21 mm 17 μ m) and a linear gradientof waterndashacetic acid (eluent A) and methanol (eluent B) at a 1047298ow rateof 03 mL minminus1 The UHPLC system was coupled to a Synapt MassQuadrupole Time-of-Flight Mass Spectrometer (Q-TOF MSMS) inwhich the MS spectra were acquired in the mz range of 50ndash2000

This method allowed for the identi1047297cation of 21 peptides and the

most antioxidant peptides were identi1047297ed as Thr-Asp-Tyr Leu-Asp-Tyr Trp-Asp-Asp-Met-Glu-Lys Trp-Gly-Asn-Val-Ser-Gly-Ser-Pro Leu-Tyr-Glu-Gly-Tyr and Met-Glu-Met-Lys

It is important to note that each methodshowed a basic principle forthe separation of the bioactive peptides as shown in Table 3 However

in a complex mixture of peptides common problems are the separationof small and big peptides or peptides with different physicochemicalproperties which makes their subsequent identi1047297cation dif 1047297cultThese problems can be solved by a combination of different separationtechniques before injection into the mass spectrometer A practicalexample is the separation of peptides containing hydrophobic aminoacids and peptides composed of only hydrophilic amino acids In thiscase a combination of RP-HPLC with HILIC can be used for an ef 1047297cientseparation of the peptides with hydrophobic and hydrophilic character-istics respectively (Panchaud Affolter amp Kussmann 2012)

In addition an interesting approach was proposed by Le Maux et al(2015) These authors af 1047297rmed that liquid chromatography coupled tomass spectrometry (LCndashMSMS) providing the necessary data for pep-tide sequencing However although this strategy has been successfully

used for longer peptides the identi1047297cation of short peptides can bemore dif 1047297cult due to the presence of peptides with the same aminoacid composition but a different sequence They showed that themethod HLIC-MSMSandthe parallel determination of the apparenthy-drophilicity of eachpeptidefor the development of a retention timepre-diction model could be used as a valuable tool to improve theseparationof short peptides and the differentiation of peptides with homologoussequences

4 Biological properties of bioactive peptides

Bioactive peptides from dietary proteins have been extensivelystudied over the last decade to determine their potential uses andtheir effects on themajor systems of thehumanbody such as thediges-

tive cardiovascular nervous and immune systems Several bioactive

Table 2

Using proteases to generate biologically active peptides from various protein sources

Protease Hydrolysisconditions

Proteinsource

Bioactivity of the peptides

Peptides Identi1047297cation methods Reference

Alcalasetrade pH 80 50 degC 3 hES = 120[S] = 50

Soy Antiadipogenesis Peptides with molecularweights between 754 and3897 Da

Liquid chromatography massspectrometry

Mejia et al (2010)

Flavourzymetrade pH 70 50 degC 2 hES = 1100

[S] = 25

Puri1047297ed soyprotein

Antiadipogenesis Peptides with molecularweights less than 1300 Da

High-performance molecularexclusion chromatography

Tsou Kao et al 2010

Neutrasetrade pH 60 45 degC 4 hES = 1100[S] = 25


Antiadipogenesis Peptides with molecularweights between 1300and 2200 Da


Tsou Lin et al 2010

Pepsintrade pH 55 23 degC[S] = 10

Bovinehemoglobin

AntimicrobialAntihypertensive


Electrospray ionization massspectrometry(ESIMS)

Adje et al (2011)

Alcalasetrade pH 80 50 degC 3 h[E] = 02 mgmL [S] = 80

Bean AntioxidantAnti-in1047298ammatory

Peptides with molecularmasses between 445 and2148 Da

Matrix-assisted laserdesorptionionization massspectrometry(MALDI-TOF)

Oseguera-ToledoMejia Dia andAmaya-Llano (2011)

Crude protease fromBacillus licheniformis

pH 100 50 degC 55 h[S] = 100

Goby muscle Anticoagulant Leu-Cys-ArgHis-Cys-PheCys-Leu-Cys-ArgLeu-Cys-Arg-Arg

Liquid chromatographyElectrospray ionization massspectrometry(ESIMS)

Nasri et al (2012)

AlcalasetradeFlavourzymetrade

Protamextrade

NeutrasetradePepsinTrypsin

pH 70 50 degC 8 hpH 70 50 degC 8 h

pH 70 50 degC 8 hpH 70 50 degC 8 hpH 20 37 degC 8 hpH 80 37 degC 8 h

Salmon AntioxidantAnti-in1047298ammatory

Peptides with molecularmasses between 1000

and 2000 Da


Ahn Je and Cho (2012)

Crude protease fromBacillus mojavensis

pH 100 50 degC[S] = 50

Cuttle1047297sh(Sepia

of 1047297cinalis)muscle

Antihypertensive Peptides with molecularmasses between 163 and1047 Da

Liquid chromatographyElectrospray ionization massspectrometry (ESIMS)Tandem mass spectrometry(ESI-MSMS)

Balti et al (2015)




peptides have biological activities that are bene1047297cial for human healthincluding antimicrobial (Adje Balti Kouach Guillochon amp Nedjar-

Arroume 2011) anti-hypertensive (Alemaacuten et al 2011) antioxidant

(Zhang et al 2009) anticancer (Alemaacuten et al 2011) anti-adipogenic(Tsou Kao et al 2010) immunomodulatory (Huang Chen Chen

Hong amp Chen 2010) and anti-in1047298ammatory effects (Ahn et al 2015)

Table 3

The main characteristics of the different analytical methods for the puri1047297cation and identi1047297cation of bioactive peptides

Method of puri1047297cationidenti1047297cation

Mechanism Advantage Limitation Reference

Reversed phase high pressure liquidchromatography (RP-HPLC)

Based on the hydrophobicity of proteins or peptides that can interactdifferently to the reversed-phase

material of the chromatographycolumn

Useful method for theisolation of complexpeptide mixtures

Lack of retention of polarmolecules Slow intrapore diffusiontimes The presence of unresolved

structural microheterogeneity andconformational isomers Secondaryinteractions with the stationaryphase

Everley and Croley (2008)Le Maux et al (2015)Yang Boysen Chowdhury

Alam and Hearn (2015)

Af 1047297nity chromatography Based on the af 1047297nity of bioactivepeptides to interact speci1047297cally andreversibly with a complementarymolecule bound to a solid supportimmobilized on a column

The 1047298exibility of using alarge number of bindingagents allows for theseparation of differenttypes of peptides

Tone must know thephysicochemical properties of theligands which limits its use for acomplex mixture of unknownpeptides

Hage et al (2012)Ortiz-Martinez et al (2014)

Ion-exchange chromatography (IEC) Based on the ability of chargedbioactive peptides to interact with asolid support bearing the oppositecharge

Appropriate method forthe separation of highlycationic or anionicpeptides

Low selectivity and requirescomplementary steps for theseparation of the fractions

Bouhallab Henry andBoschetti (1996)Ortiz-Martinez et al (2014)

Isoelectric focusing (IEF) Based on the separation of proteinpeptide solutions according totheir isoelectric points (pI) A focusingcell containing a mixture of proteinspeptides and a carrier

ampholyte is subjected to an electricpotential causing the migration of theproteinspeptides to a position in anestablished pH gradient equivalent totheir respective pI

The method allows one tofractionate a complexmixture of peptidesaccording to their pI

Loss of highly hydrophobicproteins in the samplepreparation and precipitation of neutral proteins at their pI whichcan result in overlapping between

different fractions

Issaq Conrads Janini andVeenstra (2002)Guijarro-Diacuteez Garciacutea Cregoand Marina (2014)

Size exclusion chromatography(SEC)

Based on the fractionation of bioactivepeptides according to the retentiontime of the molecules in the stationaryphases particles with a carefullycontrolled pore size in which themolecules are separated from eachother according to their molecular size

The elution conditions areconsidered mild allowingthe characterization of theprotein with minimalimpact on theconformational structureand the local environment

Long columns are required forcomplex peptide mixtures whichcan be obtained by joiningmultiple columns in a series Thisstrategy is necessary to improvethe separation resolution

Mora et al (2014)Fekete Beck Veuthey andGuillarme (2014)

Ultra high pressure liquidchromatography (UHPLC)

Based on separation of the moleculesusing experimental columns packedwith very small particles of anon-porous material carrying out theanalyses at very high pressures

Increased throughputresolution and sensitivityin separation of complexprotein mixtures

The heat dissipated from the useof small particles at ultra-highpressures may increasechromatographic bandbroadening and compromise

ef 1047297ciency of the column

Everley and Croley (2008)UliyanchenkoSchoenmakers and van derWal (2011)Fekete and Guillarme (2014)

Hydrophilic interaction liquidchromatography (HILIC)

Based on the polarity andhydrophilicity of bioactive peptidesseparated using polar chromatographicsurfaces (stationary phase) and ahighly organic mobile phase(N70 solvent) also containing a smallpercentage of aqueous solventbuffer orother polar solvent

The method shows greatpotential for the separationof short peptide sequences(b5 amino acids) andimproves the identi1047297cationusing mass spectrometry

Compared to RP-HPLC themethod shows limited 1047298exibilityand applicability problems withsample solubility and theretention mechanisms are poorlyunderstood

Gray et al (2013)Le Maux et al (2015)

Electrospray ionization massspectrometry (ESIMS)

Based on the transformation of anaqueous solution with uniformelectrical density to gas-phase ions bypassing a high voltage through a thincapillary The gas-phase is transferredinto a mass analyzer and separatedaccording to the mass-to-charge(mz) ratio

Production of singly andmultiply charged ionsallowing for an accuratemeasurement of themolecular weight of thepeptides

The ef 1047297ciency of identi1047297cation isdirectly related to thechromatographic method used forthe prior separation of thebioactive peptides beforeinjection into the massspectrometer Therefore acombination of different

separation techniques isnecessary for accurateidenti1047297cation

Mano and Goto (2003)Contreras et al (2008)Panchaud et al (2012)

Matrix-assisted laserdesorptionionization-time-of- 1047298ightmass spectrometry(MALDI-TOFMS)

Based on co-crystallization of theanalytes when they are mixed with amatrix solution on a target plate Theco-crystal is subjected to the action of pulsed laser causing the accumulationof high-density energy which results invaporization of the analyte and matrixmolecule MALDI is usually connectedto TOF mass spectrometer whichmeasures the 1047298ight time of ions to theion detector and provides the mz ratiomapping results

The method has notheoretical upper limit tothe mz ratio allowing forthe analysis of complexsamples with a wide rangeof molecular weights




Therefore they can potentially be incorporated into functional foodsnutraceuticals and medications where this bioactivity can aid inpreventing and controlling diseases (Agyei amp Danquah 2012)

This review describes efforts to identify and characterize peptideswith antimicrobial antioxidant anti-adipogenic and anti-hypertensiveactivity and it discusses their use in the growth of lactic acid bacteriaand other probiotic bacteria

41 Peptides with antimicrobial activity

Over the lastfew decadesa growing number of pathogenic microor-ganisms have developed resistance to conventional antibiotics causingserious problems treating infections especially in immunocompro-mised individuals In addition the development of new antibiotics hasslowed over this same period Two major causes underlie the increasein antibiotic resistance in microorganisms the indiscriminateuse of an-tibiotics for in small doses or with ineffective treatment times and thegenetic mutation capacity of the microorganisms which increases thedif 1047297culty of developing drugs based on speci1047297c mechanisms of action(Harrison Abdel-Rahman Miller amp Strong 2014) Thus using naturalsources of antimicrobial compounds has enormous potential becausethey have characteristics such as low toxicity and high speci1047297city Themechanisms of these natural antimicrobial compounds can be betterunderstood if we compare their modes of action against bacterial(unicellular) and animal (multicellular) cells Bacterial cells have alayer rich in negatively charged phospholipids pointing toward the ex-ternal environment facilitating their interactions with peptides mostof which are positively charged In contrast animal cells are mainlycomposedof uncharged lipids in the outermost layer andthe negativelycharged regions are pointed toward the cell interior (cytoplasm)(Matsuzaki 1999)

Antimicrobial peptides are widely distributed in nature and are es-sential to the immune system They are the organisms 1047297rst line of de-fense against colonization by exogenous microorganisms and theyplay a fundamental role in regulating bacterial populations on the mu-cosa and other epithelial surfaces (Bevins amp Zasloff 1990 Boman amp

Hultmark 1987 Zasloff 2002) More than 800 antimicrobial peptideshave been described in plants and animals (Boman 2003) Despitegreat diversity in their primary structures most antimicrobial peptidesare similar in that they are short amino acid chains composed primarilyof cationic and hydrophobic amino acids (Dashper Liu amp Reynolds2007Zasloff 2002) The lowmolecular weightsof the peptide fractionsthe resulting higher exposure of the amino acids and their charges andthe formation of small channels in the lipid bilayer are related to theirantimicrobial activity These features promote interactions betweenthe peptide and the membrane (Gobbetti Minervini amp Rizzello 2004Goacutemez-Guilleacuten et al 2010 Patrzykat amp Douglas 2005)

The exact mechanisms of action for many antimicrobial peptideshave not been well established Dueto the large number of known pep-tides it is likely that there are additional mechanisms of action yet to be

discovered (Dashper et al 2007)In addition to the peptides that are naturally present in the defense

systems of plants and animals peptides with antimicrobial activityhave been identi1047297ed in several protein hydrolysates

Hydrolysates of casein from cows milk obtained by enzymatic hy-drolysis using chymosin were analyzed for their antimicrobial powerFive different antibacterial peptides were isolated from the carboxylicend of αs2-casein Peptide fractions f (181ndash207) f (175ndash207) and f (164ndash207) had a wide spectrum of activity andwere able to inhibit sev-eral Gram+ and Gramminus bacteria the minimum inhibitory concentra-tion (MIC) of each fraction ranged from 210 to 1680 mg mL minus1 107to1712mgmL minus1 and48to762mgmL minus1 respectively The inhibitorypower of these peptides against Gram+ bacteria was as strong as theknown antimicrobial peptides nisin and lactoferricin B (Mccann et al

2005)

Peptides with antimicrobial activity were prepared from gelatin hy-drolysate with Alcalasetrade24 L (Sigma-Aldrich United States) Fractionsobtained from ultra1047297ltration through 1- and 10-kDa membranes wereused for antimicrobialtests against 18 bacteria Themostsensitivebacte-ria in the presence of the tested fractions were Lactobacillus acidophilusBi 1047297dobacterium lactis Shewanella putrafaciens and Photobacterium

phosphoreum (Goacutemez-Guilleacuten et al2010) Hydrolysates of bovinehemo-globin treated with pepsin were puri1047297ed by HPLC and tested for their

antimicrobial power against two Gramminus

(Escherichia coli Salmonellaenteritidis) and three Gram+ strains (Kocuria luteus A270 Staphylococcusaureus and Listeria innocua) The results showed that the puri1047297ed peptidefractionshad a wide spectrum of action affecting4 of the5 testedbacteria(Kocuria luteus A270 L innocua E coli and S aureus) with a MIC between352 and 1871 μ M (Adje et al 2011)

Tellez Corredig Turner Morales and Grif 1047297ths (2011)demonstratedthe ef 1047297ciency of a peptide fraction isolated from milk fermented withL helveticus against an experimental infection of S enteritidis in ratsThe survival rate of the group fed with the peptide fraction (002 μ gper day) was higher than the group fed with half of the dose (001 μ gper day) and higher than the control group

The antimicrobial powers of protein isolated from whey hydrolyzedwith various gastrointestinal enzymes were demonstrated by TheacuteolierHammami Labelle Fliss and Jean (2013) These authors showed thathydrolyzed proteins from trypsin and chymotrypsin digests did nothave antibacterial activity against Listeria ivanovii HPB28 and E coliMC4100 but they found that hydrolysates of pepsin had signi1047297cantactivity Hydrolysates were fractionated by reverse-phase high-performance liquid chromatography resulting in 1047297ve fractions withhigh antibacterial activity and MIC values between 200 and350 μ g mL minus1 A peptide fraction obtained from wastewater fromcooking anchovies (Engraulis japonicus) was digested by the Protamexenzyme and had high antimicrobial activity against S aureus The iden-ti1047297ed fraction had the peptide sequence Gly-Leu-Ser-Arg-Leu-Phe-Thr-Ala-Leu-Lys andan estimated molecular weight of 11kDa (Tang ZhangWangQian amp Qi 2015) Dueto their hydrophobicity bioactivepeptidescontaining sequences rich in the amino acids Gly andLeu were reportedas potent antimicrobial molecules The presence of the Arg residue in

the peptide sequence also plays an important role in antimicrobialactivity increasing interactions with bacterial cell walls due its cationiccharacteristic (Amadou Le Amza Sun amp Shi 2013 Sousa et al 2009Tang et al 2015)

42 Peptides with antioxidant activity

The creation of free radicals such as superoxide (O2minus) and hydroxyl

(OH) is one of the inevitable consequences of respiration in aerobicorganisms These radicals are very unstable and react quickly withother groups or substances in the organism causing cellular and tissuedamage (Zhang et al 2009) An excessive amount of these radicals inthe organism has been linked to the development of several diseasessuch as atherosclerosis arthritis diabetes and cancer (Gu et al 2015)

Because they are highly reactive species free radicals can damageproteins mutate DNA oxidize membrane phospholipids and modifylow-density lipoproteins (LDL) (Pihlanto 2006) In food oxidationalso directly affects quality negatively affecting characteristics such astaste aromaand color Thussubstances that inhibit oxidation reactionsare useful for maintaining food quality

An antioxidants ability to remove free radicals is determined by var-ious factors including chemical reactivity the rate of removal of thecompound the fate of the product of the antioxidantndashradical reactioninteractions with other antioxidants concentration and mobility in theenvironment and the compounds absorption distribution retentionand metabolism (Niki 2010)

Antioxidants are thought to be important nutraceuticals with vari-ous health bene1047297ts They are de1047297ned as substances that signi1047297cantly

slow or inhibit the oxidation of a substrate (Bougatef et al 2009)






fractions and found that subunits from the β-conglicinin fraction had alarger number of peptides responsible for the inhibition of lipid accu-mulation in 3T3-L1 cells than the glycinin subunits

Tsou Kao et al 2010 studied the use of commercial preparationsof Flavourzymetrade proteases on protein hydrolysates isolated from soyby analyzing the anti-adipogenic capacity of the hydrolysate fractions

obtained by ultra1047297ltration The results revealed thatthe partial hydroly-sis of proteins isolated from soy provided hydrolysates with stronganti-adipogenic capacity and fractions obtained by ultra1047297ltrationmore ef 1047297ciently inhibited GPDH activity Speci1047297cally the 1-kDa mem-brane fraction was the most effective (590 inhibition) The anti-adipogenicactivityof the hydrolysates from soy protein after enzymatic

Table 4

Major methods for measuring antioxidant activities of peptides in vitro and their respective mechanisms

Method Mechanism Reaction Measurement

DPPH DPPH capture DPPH radical (22-diphenyl-1-picryl-hydrazyl) reacts withhydrogen-donating antioxidants changing the color fromviolet to yellow

Reduction in theabsorbance at 517 nm

Sharma and Bhat (2009)

ORAC Peroxyl radical capture The peroxyl radical generated from the breakdown of AAPH [22prime-Azobis(2-amidinopropane) dihydrochloride]in the presence of atmospheric oxygen reacts with a

1047298uorescent indicator to produce a non-1047298uorescentproduct In the presence of antioxidants the 1047298uorescenceis maintained

Reduction in 1047298uorescence(excitation at 485 nm andemission at 520 nm)

Daacutevalos Goacutemez-Cordoveacutes andBartolomeacute (2004)

FRAP Iron reducing power In the presence of electron-donating antioxidants theFe3+-TPTZ [246-Tripyridyl-S-Triazine] complex isreduced to Fe2+-TPTZ changing the color from light blueto dark blue

Increase in the absorbanceat 593 nm

Ou Huang Hampsch-WoodillFlanagan and Deemer (2002)

ABTS ABTS capture The radical ABTS(22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))is stabilized in the presence of hydrogen-donating freeradicals changing the color from dark green to light green


Goacutemez-Guilleacuten et al (2010)

Ability to chelatetransition metals(Cu2+)

Chelation of Cu2+ Complexation reaction of Cu2+ with pyrocatechol violet togenerate a colored product The presence of antioxidantsdecreases the formation of the Cu2+-pyrocatecholcomplex reducing the intensity of the color


Theodore et al (2008)

Ability to chelatetransition metals

(Fe

2+

)

Chelation of Fe2+ Complexation reaction of Fe2+ with ferrozine generatinga colored product The presence of antioxidants decreases

the formation if the Fe

2+

-ferrozine complex reducingcolor intensity


Nazeer and Kulandai (2012)

TBARS Quanti1047297cation of lipidperoxidation products

Reaction of thiobarbituric acid with hydroperoxidedecomposition products Malonaldehyde is the maincompoundquanti1047297ed Absorbance and antioxidant activityare inversely proportional


Raghavan and Kristinsson(2008)

Table 5

Major methods for measuring antioxidant activities of peptides in vivo and their respective mechanisms

Method Sample analyzed Animals Tissueorgananalyzed

Mechanism of action and principleunderlying measurement

Reference

Superoxide dismutase (SOD) Peptide isolate fromhydrolyzed pig plasma

Male adultWistar rats

Liver SOD is an enzyme that catalyzes thedismutation of superoxide radicals intohydrogen and oxygen thus playing animportant role in protecting cells againstreactive oxygen species

Liu Kong Li Liu andXia (2011)

Catalase (CAT) Peptide isolated fromhydrolyzed 1047297sh protein

Albino maleadult Wistar rats

Erythrocytelysate (blood)

CAT is an enzyme that converts hydrogenperoxide into water and oxygen thushaving one of the major mechanisms forremoving free radicals in the organism

Nazeer Kumar andGanesh (2012)

Level of reduced glutathione

(GSH)

Protein isolates from

seeds from Syrian rue(Peganum harmala)

Albino male rats Liver and blood

plasma

GSH is an intracellular reducer that plays

an important role for protecting cellsfrom free radicals peroxides and othertoxic compounds

Soliman Abu-El-Zahab

and Alswiai (2013)

Glutathione-S-transferase(GST)

Peptide isolated fromhydrolyzed musselprotein

Male adult rats Liver GST is an enzymatic complex in thecytosol that catalyzes the binding of reactive electrophilic molecules withglutathione facilitating the metabolismand excretion of toxins and consequentlyreducing cell damage and DNA damage

Kim et al (2013)

Glutathione peroxidase(GPx)

Hydrolyzed corn gluten Male and femaleKunming mice

Liver and bloodplasma

GPx is an enzyme that catalyzes thereaction between hydroperoxide andreduced glutathione leading to theformation of glutathione disul1047297te and theproduct of hydroperoxide reduction

Liu et al (in press)

Measurement of the level of malonaldehydes

Hydrolyzed 1047297sh protein(Salaria basilisca)



Malanodialdehyde is an intermediateproduct for lipid peroxidation and thuscan be used as an indicator for thepresence of free radicals

Ktari et al (2014)




treatment with Neutrase and the effect of fractionation by ultra1047297ltrationon activity were studied byTsou Lin et al 2010 Similar to thepreviousstudy the resultsshowed that low molecular weight peptides (between1300 and 2200 Da) most effectively inhibited GPDH activity

MejiaMartinez-VillaluengaRomanand Bringe(2010)analyzed theeffects of soy protein hydrolysates enriched with β-conglycinin (aprotein naturally found in soy) on FAS activity and adipogenesis inhuman adipocytes in vitro The resultsshowedthat genotypic alterations

in the subunits of the soy protein (enriched with β-conglycinin) pro-duced peptide pro1047297les that inhibited FAS and decreased lipid accumula-tion in vitro The quantity of soy protein hydrolysates necessary toinhibit 50 of FAS activity (IC50) ranged from 50ndash175 μ M A peptidewith anti-adipogenic abilities was isolated by ultra1047297ltration gel 1047297ltra-tion and reversed-phase HPLC from soy protein hydrolysates and itsanti-adipogenic ability was con1047297rmed by the inhibition of 3T3-L1 pre-adipocyte celldifferentiationThe inhibitor wasidenti1047297ed as a tripeptide(Ile-Gln-Asn) with an IC50 value of 0014 mg mL minus1 (Kim et al 2007)

44 Peptides with anti-hypertensive activity

Arterialhypertension affects approximately 25 of the adult popula-tion worldwide andis predicted to reach 29 of thepopulation by 2025representing a total of 156 billion people (Ngo et al 2015) Although itis a controllable disease hypertension is associated with several cardio-vascular diseases such as atherosclerosis myocardial infarction andstroke (Sheih Fang amp Wu 2009) Angiotensin converting enzyme(ACE) plays an important role in the regulation of arterial pressure be-cause it catalyzes the conversion of angiotensin-I (the inactive form)to angiotensin-II (a vasoconstrictor)and inactivates bradykinin (a vaso-dilator) Consequently synthetic ACE inhibitors such as captopril andenalapril are often used to treat hypertension and other related heartdiseases However synthetic inhibitors can cause various side effectssuch as coughalteredtaste rash and angioedema (Alemaacuten et al 2011)

It is well known that dietary proteins have primary sequences of peptides able to modulate speci1047297c physiological functions (Hong et al2008) Many types of bioactive peptides that inhibit ACE were isolatedfrom protein hydrolysates and fermented products The dipeptide

Ala-Pro and the tripeptide Phe-Ala-Pro for example have structuresanalogous to the drugs captopril and enalapril respectively (Fig 2)

The presence of aromatic and aliphatic amino acids such as Pro Pheor Tyr at the C-terminal and of Val and Ile at the N-terminal position of the bioactive peptides has been associated with ACE-inhibitory activity(Kapel Rahhou Lecouturier Guillochon amp Dhulster 2006 WijesekaraQian Ryu Ngo amp Kim 2011) Cian Vioque and Drago (2015) studiedthe ACE-inhibitory activity of peptides from wheat gluten hydrolysate

and obtained an interesting result in which no relationship was foundbetween hydrophobicitysize and the IC50 values of the ACE-inhibitoryactivity indicating that it was the sequence of peptides from thewheat gluten hydrolysate that was mainly responsible for the ACE-inhibitory activity although several studies have shown that the sizeand hydrophobic character of the peptides exert a strong in1047298uence onthis bioactivity (Aluko et al in press Li Le Shi amp Shrestha 2004Wijesekara et al 2011) According to Li et al (2004) the hydrophilicndash

hydrophobic partitioning in the peptide sequence was a critical factorin ACE-inhibitory activity because hydrophilic amino acid residuescould disrupt the access of the peptide to the active site of ACE Thisconcept was reinforced by Yuan Wu and Aluko (2007) and Alukoet al (in press) who showed an important role of branched-chainamino acids such as ValLeu andIlein enhancing thehydrophobic char-acter of peptides which is an important structural feature that enablesstrong peptide interactions with non-polar amino acid residues withinthe enzyme active site

Peptide fractions from soy protein hydrolyzed with pepsin wereseparated by ion exchange chromatography gel 1047297ltration and HPLCand they were found to have ACE-inhibiting activity Four aminoacid sequences were identi1047297ed as potential ACE inhibitors Ile-Ala(IC50 153 μ M) Tyr-Leu-Ala-Gly-Asn-Gln (IC50 14 μ M) Phe-Phe-Leu(IC50 37 μ M) and Ile-Tir-Leu-Leu (IC50 42 μ M) When administered ata dose of 20 gkg body weight in hypertensive rats over 15 weeks thepeptide fractions considerably reduced arterial pressure (Chen OkadaMuramoto Suetsuna amp Yang 2003)

Peptides with anti-hypertensive activity were isolated from proteinhydrolysates of milk after fermentation with lactic bacteria and enzy-matic hydrolysis withthe commercial proteaseProzyme 6 The peptideswere identi1047297ed as Gly-Thr-Trp and Gly-Val-Trp and had ACE inhibitoractivity with IC50 values of 4644 and 2400 μ M respectively (Chenet al 2007) Hernaacutendez-Ledesma Quiroacutes Amigo and Recio (2007)hydrolyzed human milk proteins with pepsin and pancreatin to studythe anti-hypertensive properties of the peptides and showed thathydrolysates derived from β-casein were strong inhibitors of ACEwith an IC50 of 21 μ M

Chaves-Loacutepez et al (2014)studied the effects of combinedmicrobial

cultures previously identi1047297ed as proteolytic and their ability to releaseACE-inhibiting peptides during the production of fermented milk Theyeast strains Torulaspora delbruekii KL66A Galactomyces geotrichumKL20B Pichia kudriavzevii KL84A and Kluyveromyces marxianus KL26Aand the lactic acid bacteria Lactobacillus plantarum LAT03 Lb plantarumKLAT01 and Enterococcus faecalis KE06 (non-virulent) were used Theresults indicated that the combination of different cultures can signi1047297-cantly increase the levels of anti-hypertensive peptides The most

H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro

Fig 2 Structures of ACE inhibitor drugs and their analogous peptides ( Matsui amp Matsumoto 2006)




effective combination for producing these peptides was a mixture of P kudriavzevii KL84A Lb plantarum LAT3 and E faecalis KL06 whichhad an IC50 for ACE inhibition of 3063 μ g mL minus1

The anti-hypertensive effect of a bovine casein peptide previouslyidenti1047297ed as Met-Lys-Pro was analyzed in vitro and in vivo The in vitroanalyses were based on the ability to inhibit ACE and thein vivo studieswere conducted using groups of naturally hypertensive rats Animalswere treated with peptide solutions (10 mgkg) two times per day

and a single daily dose of enalapril (10 mgkg) for 28 consecutivedays The in vitro assay showed that the peptide had ACE inhibition ac-tivity with an IC50 of 043 μ MFor the in vivo assays the arterialpressureof the animals was 1717 1633 and 1397 for the control peptide solu-tion and enalapril groups respectively indicating signi1047297cant differencesand reductions in arterial pressure between the control group and thepeptide ( p b 005) and enalapril ( p b 001) treatment groups (Yamadaet al 2015)

45 Induction of lactic acid bacteria and probiotic growth

Lactic acid bacteria are not able to synthesize all of the amino acidsnecessary for their growth Thusthese microorganisms must hydrolyzeproteins while fermenting dairy productsto obtain free amino acids andsmall peptides as nutritional sources The proteolytic system of lacticacid bacteria has three basic mechanisms 1) one or more proteolyticenzymes located in the cell wall also known as cell-envelope proteaseswhich can hydrolyze milk proteins into peptides containing 4 to 30amino acid residues 2) a peptide transport system composed of bind-ing proteins two permeases to create the transport channels and twoATPases to provide energy to the system and 3) a group of intracellularpeptidasesthatcatalyzes the hydrolysis of the peptides transportedintothe cell interior into amino acids (Hafeez et al 2014)

Despite the existence of this proteolytic apparatus several studieshave shown that supplementing milk with sources of hydrolyzed pro-teins positively affects the growth of lactic acid and probiotic bacteriaThese studies are mainly driven by two main characteristics of thisgroup of microorganisms 1) lactic acid and probiotic bacteria are re-quired nutritionally especially for amino acid uptake and 2) the free

amino acids and peptides in the milk are not suf 1047297cient to ensure idealbacterial growth on this substrate possibly impeding fermentation bythese microorganisms (Zhang et al 2011) Thus various proteinsources have been analyzed as supplements to be used in the culturemedia to study the induction of lactic acid andprobiotic bacteriagrowth(Mccomas amp Gilliland2003Prasanna Grandison amp Charalampopoulos2012 Zhang et al 2011)

Mccomas and Gilliland (2003) studied the growth of lactic acid andprobiotic bacteriain cows milk samplessupplementedwith hydrolyzedwhey protein The results showed that the hydrolysates did not affectL delbrueckii ssp bulgaricus and Streptococcus thermophiles growth butsigni1047297cant increases inBi 1047297dobacterium longumand L acidophilus growthwere observed

Prasanna et al (2012) studied the supplementation of skim milk

with various hydrolyzed proteins and their effects of probiotic bacteriagrowth and found that the type or fraction of the protein used directlyaffected microorganism growth The 1047297nal concentration of B longumsubsp infantis CCUG 52486 and B infantis NCIMB 702205 cells washigher when thesestrainswere culturedin milk supplementedwith hy-drolyzed casein than with the other hydrolyzed protein fractions fromlacto-albumin concentrated whey protein or puri1047297ed whey protein

5 Conclusion

Biologically active peptides can be de1047297nedas speci1047297c amino-acid se-quences that promote bene1047297cial physiological effects Technologies forobtaining bioactive peptides include protein hydrolysis by exogenousmicrobial plant or animal enzymes and fermentation using fungi or

bacteria The wide biochemical diversity of the proteins and the

existence of protein sources with various amino-acid compositionsmake it possible to obtain peptides with distinct biological functionsandor multiple functions Important techniques for puri1047297cation andidenti1047297cation include chromatography and mass spectrometry Studieson production processes studies on multifunctionality and analysesusing in vitro and in vivo methods all contribute to our understandingof bioactive peptides as powerful natural biological agents that can beused together with or in place of synthetic substances in food preserva-

tion functional foods and pharmaceutical production

References

Adje EY Balti R Kouach M Guillochon D amp Nedjar-Arroume N (2011) α 67ndash106 of bovine hemoglobin A new family of antimicrobial angiotensin I-converting enzymeinhibitory peptides European Food Research and Technology 232 637ndash646

Agyei Damp Danquah MK(2012) Rethinking food-derivedbioactive peptidesfor antimi-crobial and immunomodulatory activities Trends in Food Science amp Technology 2362ndash69

Ahn C -B Cho Y -S amp Je J -Y (2015) Puri1047297cation and anti-in1047298ammatory action of tripeptide from salmon pectoral 1047297n byproduct protein hydrolysate Food Chemistry168 151ndash156

Ahn C -B Je J -Y amp Cho Y -S (2012) Antioxidant and anti-in1047298ammatory peptide frac-tion from salmon byproduct protein hydrolysates by peptic hydrolysis Food ResearchInternational 49 92ndash98

Akalin AS (2014) Dairy-derived antimicrobial peptides Action mechanisms pharma-

ceutical uses and production proposals Trends in Food Science amp Technology 36 79ndash95Alam MN Bristi NJ amp Ra1047297quzzaman M (2013) Review on in vivo and in vitro

methods evaluation of antioxidant activity Saudi Pharmaceutical Journal 21143ndash152

Alemaacuten A Peacuterez-Santiacuten E Bordenave-Juchereau S Arnaudin I Goacutemez-GuilleacutenMC ampMontero P (2011)Squid gelatin hydrolysates with antihypertensive anticancer andantioxidant activity Food Research International 44 1044ndash1051

Aluko RE GirgihAT He R Malomo S Li H Offengenden M et al (2015) Structuraland functional characterization of yellow 1047297eld pea seed (Pisum sativum L) protein-derived antihypertensive peptides Food Research International httpdxdoiorg101016jfoodres201503029 (in press)

Amadou I Le G -W Amza T Sun J amp Shi Y -H (2013) Puri1047297cation and characteriza-tion of foxtail millet-derived peptides with antioxidant and antimicrobial activitiesFood Research International 51 422ndash428

Aoyama T Fukui K Takamatsu K Hashimoto Y amp Yamamoto T (2000) Soy proteinisolate and its hydrolysate reduce body fat of dietary obese rats and geneticallyobese mice (yellow KK) Nutrition 16 349ndash354

Balti R Bougatef A Sila A Guillochon D Dhulster P amp Nedjar-Arroume N (2015)

Nine novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttle-1047297sh (Sepia of 1047297cinalis) muscle protein hydrolysates and antihypertensive effect of the potent active peptide in spontaneously hypertensive rats Food Chemistry 170519ndash525

Bevins CL amp Zasloff M (1990) Peptides from frog skin Annual Review of Biochemistry59 395ndash414

Biziulevicius GA Kislukhina OV Kazlauskaite J amp Zukaite V (2006) Food-protein en-zymatic hydrolysates possess both antimicrobial and immunostimulatory activitiesA lsquocause and effectrsquo theory of bifunctionality FEMS Immunology and MedicalMicrobiology 46 131ndash138

Boman HG (2003) Antibacterial peptides Basic facts and emerging concepts Journal of Internal Medicine 254 197ndash215

Boman HG amp Hultmark D (1987) Cell-free immunity in insect Annual Review of Microbiology 41 103ndash126

Bougatef A Hajji M Balti R Lassoued I Triki-Ellouz Y amp Nasril M (2009) Antioxi-dant and free radical-scavenging activities of smooth hound (Mustelus mustelus)muscle protein hydrolysates obtained by gastrointestinal proteases Food Chemistry114 1198ndash1205

Bouhallab S Henry G amp Boschetti E (1996) Separation of small cationic bioactive pep-tides by strong ion-exchange chromatography Journal of Chromatography A 724(1)137ndash145

Carrasco-Castilla J Hernaacutendez-Aacutelvarez AJ Jimeacutenez-Martiacutenez C Jacinto-Hernaacutendez CAlaiz M Giroacuten-Calle J et al (2012) Antioxidant and metal chelating activities of Phaseolus vulgaris L var Jamapa protein isolates phaseolin and lectin hydrolysatesFood Chemistry 131 1157ndash1164

Chang OK Seol KH Jeong SG Oh MH Park BY Perrin C et al (2013) Casein hy-drolysis by Bi 1047297dobacterium longum KACC91563 and antioxidant activities of peptidesderived therefrom Journal of Dairy Science 96(9) 5544ndash5555

Chaves-Loacutepez C Serio A Paparella A Martuscelli M Corsetti A Tofalo R et al(2014) Impact of microbial cultures on proteolysis and release of bioactive peptidesin fermented milk Food Microbiology 42 117ndash121

Chen HM MuramotoK Yamauchi F Fujimoto K amp NokiharaK (1998)Antioxidativeproperties of histidine-containing peptides designed from peptide fragments foundin the digests of a soybean protein Journal of Agricultural and Food Chemistry 4649ndash53

Chen J Okada T Muramoto K Suetsuna K amp Yang S (2003) Identi1047297cation of angio-tensin I-converting enzyme inhibitory peptides derived from the peptic digest of

soybean protein Journal of Food Biochemistry 26 543ndash

554




Chen G Tsai J amp Pan B (2007) Puri1047297cation of angiotensin I-converting enzyme inhib-itory peptides and antihypertensive effect of milk produced by protease-facilitatedlactic fermentation International Dairy Journal 17 641ndash647

Chi C -F Hu F -Y Wang B Li T amp Ding G -F (2015a) Antioxidant and anticancerpeptides from the protein hydrolysate of blood clam (Tegillarca granosa) muscle Journal of Functional Foods 15 301ndash313

Chi C -F Wang B Wang Y -M Zhang B amp Deng S -G (2015b) Isolation and charac-terization of three antioxidant peptides from protein hydrolysate of blue1047297nleatherjacket (Navodon septentrionalis) heads Journal of Functional Foods 12 1ndash10

Chiaradia MC Collins CH amp Jardim ICSF (2008) O estado da arte da cromatogra1047297aassociada agrave espectrometria de massas acoplada agrave espectrometria de massas na

anaacutelise de compostos toacutexicos em alimentos Quimica Nova 31(3) 623ndash

636Choi J Sabikhi L Hassan A amp Anand S (2012) Bioactive peptides in dairy productsInternational Journal of Dairy Technology 65(1) 1ndash12

Cian R E Vioque J amp Drago S R (2015) Structurendashmechanism relationship of antiox-idant and ACE I inhibitory peptides from wheat gluten hydrolysate fractionated bypH Food Research International 69 216ndash223

Contreras MM Loacutepez-Expoacutesito I Hernaacutendez-Ledesma B Ramos M amp Recio I (2008)Application of mass spectrometry to the characterization and quanti1047297cation of food-derived bioactive peptides Journal of AOAC International 91(4) 981ndash994

Dashper SG Liu SW amp Reynolds EC (2007) Antimicrobial peptides and theirpotential as oral therapeutic agents International Journal of Peptide Research andTherapeutics 13(4) 505ndash516

Daacutevalos A Goacutemez-Cordoveacutes C amp Bartolomeacute B (2004) Extending applicability of theoxygen radical absorbance capacity (ORAC-1047298uorescein) assay Journal of Agriculturaland Food Chemistry 52 48ndash54

de Castro RJS Bagagli MP amp Sato HH (2015) Improving the functional properties of milk proteins Focus on the speci1047297cities of proteolytic enzymes Current Opinion inFood Science 1 64ndash69

Diplock AT Aggett PJ Ashwell M Bornet F Fern EB amp Roberfroid MB (1999) Sci-

enti1047297c concepts of functional foods in Europe Consensus document British Journal of Nutrition 81 1ndash27

Duan X Ocen D Wu F Li M Yang N Xu J et al (2014) Puri1047297cation and character-ization of a natural antioxidant peptide from fertilized eggs Food ResearchInternational 56 18ndash24

EverleyRA amp Croley TR (2008) Ultra-performance liquid chromatographymass spec-trometry of intact proteins Journal of Chromatography A 1192 239ndash247

Fekete S Beck A Veuthey J -L amp Guillarme D (2014) Theory and practice of sizeexclusion chromatography for the analysis of protein aggregates Journal of Pharmaceutical and Biomedical Analysis 101 161ndash173

Fekete S amp Guillarme D (2014) Ultra-high-performance liquid chromatography for thecharacterization of therapeutic proteins Trends in Analytical Chemistry 63 76ndash84

Giri A Osako KOkamotoA Okazaki Eamp Ohshima T (2011) Antioxidative propertiesof aqueous and aroma extracts of squid miso prepared with Aspergillus oryzae-inoculated koji Food Research International 44(1) 317ndash325

Gobbetti M Minervini F amp Rizzello CG (2004) Angiotensin I-converting enzyme-inhibitory and antimicrobial bioactive peptides International Journal of DairyTechnology 57 173ndash188

Goacutemez-Guilleacuten MC Loacutepez-Caballero ME Loacutepez De Lacey A Alemaacuten A Gimeacutenez B ampMontero P (2010) Antioxidant and antimicrobial peptide fractions from squid andtuna skin gelatin In EL Bihan amp N Koueta (Eds) Sea by-products as a real materialNew ways of application (pp 89ndash115)Kerala TransworldResearch Network Signpost

Gray N Heaton J Musenga A Cowan DA Plumb RS amp Smith NW (2013) Compar-ison of reversed-phase and hydrophilic interaction liquid chromatography for thequanti1047297cation of ephedrines using medium-resolution accurate mass spectrometry Journal of Chromatography A 1289 37ndash46

Gu MChenH -P Zhao M -M Wang X Yang B Ren J -Y et al (2015)Identi1047297cationof antioxidant peptides released from defatted walnut ( Juglans Sigillata Dode) mealproteins with pancreatin LWT mdash Food Science and Technology 60 213ndash220

Guijarro-Diacuteez M Garciacutea MC CregoAL amp MarinaML (2014) Off-line two dimension-al isoelectrofocusing-liquidchromatographymass spectrometry (time of 1047298ight) forthe determination of the bioactive peptide lunasin Journal of Chromatography A 1371 117ndash124

Hafeez Z Cakir-Kiefer C Roux E Perrin C Miclo L amp Dary-Mourot A (2014) Strate-gies of producing bioactive peptides from milk proteins to functionalize fermentedmilk products Food Research International 63 71ndash80

Hage DS Anguizola JA Bi C Li R Matsuda R Papastavros E et al (2012) Pharma-

ceutical and biomedical applications of af 1047297nity chromatography Recent trends anddevelopments Journal of Pharmaceutical and Biomedical Analysis 69 93ndash105Harding JW Pyeritz EA Copeland ES amp White HB (1975) Role of glycerol

3-phosphate dehydrogenase in glyceride metabolism Effect of diet on enzymeactivities in chicken liver Biochemistry Journal 146 223ndash229

Harrison PL Abdel-Rahman MA Miller K amp Strong PN (2014) Antimicrobialpeptides from scorpion venoms Toxicon 88 115ndash137

Hati S Vij S Mandal S Malik RK Kumari V amp Khetra Y (2014) α-Galactosidaseactivity and oligosaccharides utilization by lactobacilli during fermentation of soymilk Journal of Food Processing and Preservation 38 1065ndash1071

He R Ju X Yuan J Wang L Girgih AT amp Aluko RE (2012) Antioxidant activities of rapeseed peptides produced by solid state fermentation Food Research International49 432ndash438

Hernaacutendez-Ledesma B Contreras MM amp Recio I (2011) Antihypertensive peptidesProduction bioavailability and incorporation into foods Advances in Colloid andInterface Science 165 23ndash35

Hernaacutendez-Ledesma B Garciacutea-Nebot MJ Fernaacutendez-Tomeacute S Amigo L amp Recio I(2014) Dairy protein hydrolysates Peptides for health bene1047297ts International Dairy Journal 38 82ndash100

Hernaacutendez-Ledesma B Quiroacutes A Amigo L amp Recio I(2007) Identi1047297cation of bioactivepeptides after digestion of human milk and infant formula with pepsin and pancrea-tin International Dairy Journal 17 42ndash49

Hirai S Yamanaka M Kawachi H Matsui T amp Yano H (2005) Activin A inhibits dif-ferentiation of 3T3-L1 preadipocyte Molecular and CellularEndocrinology 232 21ndash26

Hong F Ming L Yi S Zhanxia L Yongquan W amp Chi L (2008) The antihypertensiveeffect of peptides A novel alternative to drugs Peptides 29 1062ndash1071

Huang S Chen K -N Chen Y -P Hong W -S amp Chen M -J (2010) Immunomodula-tory properties of the milk whey products obtained by enzymatic and microbialhydrolysis International Journal of Food Science and Technology 45 1061ndash1067

Inoue K Gotou T Kitajima Hirokuni Mizuno S Nakazawa T amp Yamamoto N (2009)

Releaseof antihypertensive peptides in miso paste during its fermentation by thead-dition of casein Journal of Bioscience and Bioengineering 108(2) 111ndash115Issaq HJ Conrads TP Janini GM amp Veenstra TD (2002) Methods for fractionation

separation and pro1047297ling of proteins and peptides Electrophoresis 23 3048ndash3061Kapel R Rahhou E Lecouturier D Guillochon D amp Dhulster P (2006) Characteriza-

tion of an antihypertensive peptide from an alfalfa white protein hydrolysateproduced by continuous enzymatic membrane reactor Process Biochemistry 41 1961ndash1966

Kim HJ Bae IY Ahn C Lee S amp Lee HG (2007) Puri1047297cation and identi1047297cation of ad-ipogenesis inhibitory peptide from black soybean protein hydrolysate Peptides 282098ndash2103

Kim E -K Oh H -J Kim Y -S Hwang J -W Ahn C -B Lee JS et al (2013) Puri1047297ca-tion of a novel peptide derived from Mytilus coruscus and in vitroin vivo evaluation of its bioactive properties Fish amp Shell 1047297sh Immunology 34 1078ndash1084

Korhonen H (2009) Milk-derived bioactive peptides From science to applications Journal of Functional Foods 1 177ndash187

Korhonen H Pihlanto-Leppala A Rantamaki P amp Tupasela T (1998) Impact of processing on bioactive proteins and peptides Trends in Food Science amp Technology9 307ndash319

Ktari N Nasri R Mnafgui K Hamden K Belguith O Boudaouara T et al (2014)Antioxidative and ACE inhibitory activities of protein hydrolysates from zebrablenny (Salaria basilisca) in alloxan-induced diabetic rats Process Biochemistry49 890ndash897

Lafarga T amp Hayes M (2014) Bioactive peptides from meat muscle and by-productsGeneration functionality and application as functional ingredients Meat Science 98227ndash239

Le Maux S Nongonierma AB amp FitzGerald RJ (2015) Improved short peptide identi-1047297cation using HILICndashMSMS Retentiontime predictionmodel based on theimpactof amino acid position in the peptide sequence Food Chemistry 173 847ndash854

Li GH Le GW Shi YH amp Shrestha S (2004) Angiotensin I-converting enzyme inhib-itory peptides derived from food proteins and their physiological and pharmacologi-cal effects Nutrition Research 24 469ndash486

Li X Luo Y Shen H amp You J (2012) Antioxidant activities and functional properties of grass carp (Ctenopharyngodon idellus) protein hydrolysates Journal of the Science of Food and Agriculture 92 292ndash298

Li-Chan EC (2015) Bioactive peptides and protein hydrolysates Research trends andchallenges for application as nutraceuticals and functional food ingredients Current Opinion in Food Science 1 28ndash37

Limoacuten RI Pentildeas E Torino MI Martiacutenez-Villaluenga C Duentildeas M amp Frias J (2015)Fermentation enhances the content of bioactive compounds in kidney bean extractsFood Chemistry 172 343ndash352

Liu Q Kong B LiG Liu Namp XiaX (2011) Hepatoprotective and antioxidanteffects of porcine plasma protein hydrolysates on carbon tetrachloride-induced liver damagein rats Food and Chemical Toxicology 49 1316ndash1321

Liu R Zheng W Li J Wang L Wu H Wang X et al (2015) Rapid identi1047297cation of bioactive peptides with antioxidant activity from the enzymatic hydrolysate of Mactra veneriformis by UHPLCndashQ-TOF mass spectrometry Food Chemistry 167 484ndash489

Liu X Zheng X Song Z Liu X Kopparapu NK Wang X et al (2014) Preparation of enzymatic pretreated corn gluten meal hydrolysate and in vivo evaluation of its an-tioxidant activity Journal of Functional Foods httpdxdoiorg101016jjff201410013 (in press)

Luna-Vital DA MojicaL de Mejiacutea EG Mendoza S amp Loarca-PintildeaG (2014) Biologicalpotential of protein hydrolysates and peptides from common bean ( Phaseolusvulgaris L) A review Food Research International httpdxdoiorg101016jfoodres201411024 (in press)

Maier T Leibundgut M amp Ban N (2008) The crystal structure of mammalian fatty acidsynthase Science 321 1315ndash1322Mano N amp Goto J (2003) Biomedical and biological mass spectrometry Analytical

Sciences 19 3ndash14Martinez-Villaluenga C Bringe NA Berhow MA amp Mejia EG (2008) β-Conglycinin

embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytesin vitro Journal of Agricultural and Food Chemistry 56 10533ndash10543

Matsui T amp Matsumoto K (2006) Antihypertensive peptides from natural resources InMTH Khan amp A Ather (Eds) Lead molecules from natural products (pp 255ndash271)Amsterdam Elsevier

Matsuzaki K (1999) Why and how are peptide-lipid interactions utilized for self-defense Magainins and tachyplesins as archetypes Biochimica et Biophysica Acta1462 1ndash10

Mccann KB Shiell BJ Michalski WP Lee A Wan J Roginski H et al (2005) Isola-tion and characterisation of antibacterial peptides derived from the f (164ndash207)region of bovine αS2-casein International Dairy Journal 15 133ndash143

Mccomas KA Jr amp Gilliland SE (2003) Growth of probiotic and traditional yogurt cul-tures in milk supplemented with whey protein hydrolysate Journal of Food Science68 2090ndash2095




Mejia EG Martinez-Villaluenga C RomanM amp Bringe NA (2010)Fatty acid synthaseandin vitro adipogenic response of human adipocytes inhibited byα andαprime subunitsof soybean β-conglycinin hydrolysates Food Chemistry 119 1571ndash1577

Mellander O (1950) The physiological importance of the casein phosphopeptides calci-um salts II Per oral calcium dosage of infants Acta Society Medicine Uppsala 55247ndash255

Miranda MTM amp Liria CW (2008) Teacutecnicas de anaacutelise e caracterizaccedilatildeo de peptiacutedeos eproteiacutenas In A PessoaJr amp BV Kilikian (Eds) Puri 1047297caccedilatildeo de Produtos Biotecnoloacutegicos(pp 411ndash427) Barueri Manole

Mora L Escudero E Fraser PD Aristoy M -C amp Toldraacute F (2014) Proteomic identi1047297-cation of antioxidant peptides from 400 to 2500 Da generated in Spanish dry-cured

ham contained in a size-exclusion chromatography fraction Food ResearchInternational 56 68ndash76Mora L Reig Mamp Toldraacute F (2014) Bioactive peptidesgenerated from meat industry by

products Food Research International 65(Part C) 334ndash349Naja1047297an L amp Babji AS (2015) Isolation puri1047297cation and identi1047297cation of three novel

antioxidative peptides from patin (Pangasius sutchi) myo1047297brillar protein hydroly-sates LWT mdash Food Science and Technology 60 452ndash461

Nakahara T Sano A Yamaguchi H Sugimoto K Chikata H Kinoshita E et al (2010)Antihypertensive effect of peptide-enriched soy sauce-like seasoning and identi1047297ca-tion of its angiotensin I-converting enzyme inhibitory substances Journal of Agricultural and Food Chemistry 58 821ndash827

Nasri R Amor IB Bougatef A Nedjar-Arroume N Dhulster P Gargouri J et al(2012) Anticoagulant activities of goby muscle protein hydrolysates FoodChemistry 133 835ndash841

NazeerRA amp Kulandai KA(2012) Evaluation of antioxidant activity of muscle andskinprotein hydrolysates from giant king1047297sh Caranx ignobilis (Forsskal 1775)International Journal of Food Science and Technology 47 274ndash281

Nazeer RA Kumar NSS amp Ganesh RJ (2012) In vitro and in vivo studies on the anti-oxidant activity of 1047297sh peptide isolated from thecroaker (Otolithes ruber ) muscle pro-

tein hydrolysate Peptides 35 261ndash268Ngo D -H Kang K -H Ryu B Vo T -S Jung W -K Byun H -G et al (2015)

Angiotensin-I converting enzyme inhibitory peptides from antihypertensive skate(Okamejei kenojei) skin gelatin hydrolysate in spontaneously hypertensive ratsFood Chemistry 174 37ndash43

Niki E (2010) Assessment of antioxidantcapacity in vitro and in vivo Free Radical Biologyamp Medicine 49 503ndash515

Ortiz-Martinez M Winkler R amp Garciacutea-Lara S (2014) Preventive and therapeutic po-tential of peptides from cereals against cancer Journal of Proteomics 111 165ndash183

Oseguera-Toledo ME Mejia EG Dia VP amp Amaya-Llano SL (2011) Common bean(Phaseolus vulgaris L) hydrolysates inhibit in1047298ammation in LPS-induced macro-phages through suppression of NF-κB pathways Food Chemistry 127 1175ndash1185

Ou B Huang D Hampsch-Woodill M Flanagan JA amp Deemer EK (2002) Analysis of antioxidant activities of common vegetables employing oxygen radical absorbancecapacity(ORAC) andferric reducing antioxidant power (FRAP)assaysA comparativestudy Journal of Agricultural and Food Chemistry 50 3122ndash3128

Panchaud A Affolter M amp Kussmann M (2012) Mass spectrometry for nutritionalpeptidomics How to analyze food bioactives and their health effects Journal of Proteomics 75 3546ndash3559

Patrzykat A amp Douglas SE (2005) Antimicrobial peptides Cooperative approaches toprotection Protein and Peptide Letters 12 19ndash25

Picot L Ravallec R Fouchereau-Peacuteron M Vandanjon L Jaouen P Chaplain-Derouiniot M et al (2010) Impact of ultra1047297ltration and nano1047297ltration of an indus-trial 1047297sh protein hydrolysate on its bioactive properties Journal of the Science of Food and Agriculture 90 1819ndash1826

Pihlanto A (2006) Antioxidative peptidesderived from milk proteins International Dairy Journal 16 1306ndash1314

Pownall TL Udenigwe CC amp Aluko RE (2010) Amino acid composition and antioxi-dant properties of pea seed (Pisum sativum L) enzymatic protein hydrolysatefractions Journal of Agricultural and Food Chemistry 58 4712ndash4718

Prasanna PHP Grandison AS amp Charalampopoulos D (2012) Effect of dairy-basedprotein sources and temperature on growth acidi1047297cation and exopolysaccharideproduction of Bi 1047297dobacterium strains in skim milk Food Research International 47 6ndash12

Quiroacutes A Chichoacuten R Recio I amp Loacutepez-Fandintildeo R (2007) The use of high hydrostaticpressure to promote the proteolysis and release of bioactive peptides from ovalbu-min Food Chemistry 104 1734ndash1739

Raghavan S amp Kristinsson HG (2008) Antioxidative ef 1047297cacy of alkali-treated tilapiaprotein hydrolysates A comparative study of 1047297ve enzymes Journal of Agriculturaland Food Chemistry 56 1434ndash1441

Rahman MA Kumar SG Kim SW Hwang HJ Baek YM Lee SH et al (2008) Pro-teomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3-L1 adipocytedifferentiation Proteomics 8 569ndash581

Rajapakse N Mendis E Jung WJe J amp KimS (2005) Puri1047297cationof a radical scaveng-ing peptide from fermented mussel sauce and its antioxidant properties FoodResearch International 38 175ndash182

Rao S Sun J Liu Y Zeng H Su Y amp Yang Y (2012) ACE inhibitory peptides andantioxidant peptides derived from in vitro digestion hydrolysate of hen egg whitelysozyme Food Chemistry 135(3) 1245ndash1252

Regazzo D Da Dalt L Lombardi A Andrighetto C Negro A amp Gabai G (2010)Fermented milks from Enterococcus faecalis TH563 and Lactobacillus delbrueckiisubsp bulgaricus LA2 manifest different degrees of ACE-inhibitory and immunomod-ulatory activities Dairy Science amp Technology 90 469ndash476

Roblet C Amiot J Lavigne C Marette A Lessard M Jean J et al (2012) Screening of in vitro bioactivities of a soy protein hydrolysate separatedby hollow1047297ber andspiral-wound ultra1047297ltration membranes Food Research International 46 237ndash249

Saidi S Deratani A Belleville M -P amp Amar RB (2014) Antioxidant properties of peptide fractions from tuna dark muscle protein by-product hydrolysate producedby membrane fractionation process Food Research International 65 329ndash336

Sakanaka S Tachibana Y Ishihara N amp Juneja LR (2004) Antioxidant activity of egg-yolk protein hydrolysates in linoleic acid oxidation system Food Chemistry86(1) 99ndash103

Samaranayaka AGP amp Li-Chan ECY (2011) Food-derived peptidic antioxidants A re-view of their production assessment and potential applications Journal of FunctionalFoods 3 229ndash254

Santos LF amp Koblitz MGB (2008) Proteases In MGB Koblitz (Ed) Bioquiacutemica de Alimentos (pp 78ndash103) Rio de Janeiro Guanabara Koogan

Sarmadi BH amp Ismail A (2010) Antioxidative peptides from food proteins A reviewPeptides 31 1949ndash1956Schanbacher FL Talhouk RS amp Murray FA(1997)Biologyand originof bioactive pep-

tides in milk Livestock Production Science 50 105ndash123Senphan T amp Benjakul S (2014) Antioxidative activities of hydrolysates from seabass

skin prepared using protease from hepatopancreas of Paci1047297c white shrimp Journalof Functional Foods 6 147ndash156

Sharma OP amp Bhat TK (2009) DPPH antioxidant assay revisited Food Chemistry 1131202ndash1205

Sheih IC Fang TJ amp Wu TK (2009)Isolation and characterisation of a novel angioten-sin I-converting enzyme (ACE) inhibitory peptide from the algae protein waste FoodChemistry 115 279ndash284

Singh BP Vij S amp Hati S (2014) Functional signi1047297cance of bioactive peptides derivedfrom soybean Peptides 54 171ndash179

Soliman AM Abu-El-Zahab HS amp Alswiai GA (2013) Ef 1047297cacy evaluation of theprotein isolated from Peganum harmala seeds as an antioxidant in liver of rats Asian Paci 1047297c Journal of Tropical Medicine 6(4) 285ndash295

Sousa JC Berto RF Gois EA Fontenele-Cardi NC Honoacuterio-Juacutenior JER Konno Ket al (2009) Leptoglycin A new glycineleucine-rich antimicrobial peptide isolated

from the skin secretion of the South American frog Leptodactylus pentadactylus(Leptodactylidae) Toxicon 54 23ndash32

Stuknyte M Noni ID Guglielmetti S Minuzzo M amp Mora D (2011) Potential immu-nomodulatory activity of bovine casein hydrolysates produced after digestion withproteinases of lactic acid bacteria International Dairy Journal 21 763ndash769

Su G Ren J Yang B Cui C amp Zhao M (2011) Comparison of hydrolysis characteristicson defatted peanut meal proteins between a protease extract from Aspergillus oryzaeand commercial proteases Food Chemistry 126 1306ndash1311

Tang W ZhangH Wang LQianH amp Qi X(2015) Targeted separation of antibacterialpeptide from protein hydrolysate of anchovy cooking wastewater by equilibriumdialysis Food Chemistry 168 115ndash123

Tavares TG Contreras MM Amorim M Martiacuten-Aacutelvarez PJ Pintado ME Recio Iet al (2011) Optimization by response surface methodology of degree of hydrolysisand antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtainedwith cardoon extract International Dairy Journal 21 926ndash933

Tellez A Corredig M Turner PV Morales R amp Grif 1047297ths M (2011) A peptidic fractionfrom milk fermented with Lactobacillus helveticus protects mice against Salmonellainfection International Dairy Journal 21 607ndash614

Theodore AE Raghavan S amp Kristinsson HG (2008) Antioxidative activity of proteinhydrolysates prepared from alkaline-aided channel cat1047297sh protein isolates Journalof Agricultural and Food Chemistry 56 7459ndash7466

Theacuteolier J Hammami R Labelle P Fliss I amp Jean J (2013) Isolation and identi1047297cationof antimicrobial peptides derived by peptic cleavage of whey protein isolate Journalof Functional Foods 5 706ndash714

Tsai J -S Chen T -J Pan BS Gong S -D amp Chung M -Y (2008) Antihypertensiveeffect of bioactive peptides produced by protease-facilitated lactic acid fermentationof milk Food Chemistry 106 552ndash558

TsouM -J KaoF -J Lu H -C KaoH -C amp ChiangW -D (2013) Puri1047297cationand iden-ti1047297cation of lipolysis-stimulating peptides derived from enzymatic hydrolysis of soyprotein Food Chemistry 138 1454ndash1460

Tsou M -J Kao FJ Tseng CK amp Chiang WD (2010a) Enhancing the anti-adipogenicactivity of soy protein by limited hydrolysis with 1047298avourzyme and ultra1047297ltrationFood Chemistry 122 243ndash248

Tsou M -J Lin W Lu H Tsui Y amp Chiang W (2010b) The effect of limited hydrolysiswith neutrase and ultra1047297ltration on the anti-adipogenic activity of soy proteinProcess Biochemistry 45 217ndash222

UhligT Kyprianou TMartinelli FG Oppici CA HeiligersD Hills Det al(2014) The

emergence of peptides in the pharmaceutical business From exploration toexploitation EuPA Open Proteomics 4 58ndash69Uliyanchenko E Schoenmakers PJ amp van der Wal S (2011) Fast and ef 1047297cient size-

based separations of polymers using ultra-high-pressure liquid chromatography Journal of Chromatography A 1218 1509ndash1518

Vallabha VS amp Tiku PK (2014) Antihypertensive peptides derived from soyprotein by fermentation International Journal of Peptide Research and Therapeutics 20 161ndash168

Vanderghem C Francis F Danthine S Deroanne C Paquot M Pauw ED et al(2011) Study on the susceptibility of the bovinemilk fat globule membrane proteinsto enzymatic hydrolysis and organization of some of the proteins International Dairy Journal 21 312ndash318

Wang WY amp De Mejia EG (2005) A new frontier in soy bioactive peptides that mayprevent age-related chronic diseases Comprehensive Reviews in Food Science andFood Safety 4 63ndash78

Wang B Gong YD Li ZR Yu D Chi CF amp Ma JY (2014) Isolation and characterisa-tion of 1047297ve novel antioxidant peptides from ethanol-soluble proteins hydrolysate of spotless smoothhound (Mustelus griseus) muscle Journal of Functional Foods 6 176ndash185




Wijesekara I Qian Z Ryu B Ngo D amp Kim S (2011) Puri1047297cation and identi1047297cationof antihypertensive peptides from seaweed pipe1047297sh (Syngnathus schlegeli) muscleprotein hydrolysate Food Research International 44 703ndash707

Yamada A Sakurai T Ochi D Mitsuyama E Yamauchi K amp Abe F (2015) Antihyper-tensive effect of the bovine casein-derived peptide Met-Lys-Pro Food Chemistry 172441ndash446

Yang Y Boysen RI Chowdhury J Alam A amp Hearn MTW (2015) Analysis of pep-tides and protein digests by reversed phase high performance liquid chromatogra-phyndashelectrospray ionisation mass spectrometry using neutral pH elution conditions Analytica Chimica Acta 872 84ndash94

Yasufumi K Shigeki M Keiji I amp Tomoyuki O (2001) Antioxidative peptides from

milk fermented with Lactobacillus delbrueckii subsp bulgaricus IFO 13953 Journal of the Japanese Society for Food Science and Technology 48 44ndash50Yoshida T (2004) Peptide separation by hydrophilic-interaction chromatography A

review Journal of Biochemical and Biophysical Methods 60 265ndash280Yuan L Wu J amp Aluko RE (2007) Size of the aliphatic chain of sodium houttuyfonate

analogs determines their af 1047297nity for renin and angiotensin I converting enzymeInternational Journal of Biological Macromolecules 41 274ndash280

Zarei M Ebrahimpour A Abdul-Hamid A Anwar F Bakar FA Philip R et al (2014)Identi1047297cation and characterization of papain-generated antioxidant peptides frompalm kernel cake proteins Food Research International 62 726ndash734

Zasloff M (2002) Antimicrobial peptides of multicellular organisms Nature 145389ndash395

Zhang L Li J amp Zhou K (2010) Chelating and radical scavenging activities of soy pro-tein hydrolysates prepared from microbial proteases and their effect on meat lipidperoxidation Bioresource Technology 101 2084ndash2089

Zhang Q Ren J Zhao H Zhao M Xu J amp Zhao Q (2011) In1047298uence of casein hydroly-sates on the growth and lactic acid production of Lactobacillus delbrueckii subspbulgaricus and Streptococcus thermophilus International Journal of Food Science andTechnology 46 1014ndash1020

Zhang J Zhang HWang L Guo X Wang X amp Yao H (2009) Antioxidant activities of the rice endosperm protein hydrolysate Identi1047297cation of the active peptideEuropean Food Research and Technology 229 709ndash719

Zhou K Canning C amp Sun S (2013) Effects of rice protein hydrolysates prepared by

microbial proteases and ultra1047297ltration on free radicals and meat lipid oxidationLWT mdash Food Science and Technology 50 331ndash335Zhu YP ChengYQ Wang LJ Fan JF amp Li LT (2008) Enhanced antioxidative activity

of chinese traditionally fermented Okara (Meitauza) prepared with various microor-ganism International Journal of Food Properties 11 519ndash529

Zhuang Y amp Sun L (2011) Preparation of reactive oxygen scavenging peptides fromtilapia (Oreochromis niloticus) skin gelatin Optimization using response surfacemethodology Journal of F ood Science 76(3) 483ndash489




hormones and regulators (Hernaacutendez-Ledesma Garciacutea-NebotFernaacutendez-Tomeacute Amigo amp Recio 2014)

Bioactive peptides can be obtained from animal or plant proteinsPlant sources are generally grains such as wheat rice oats rye andcorn and some legumes such as soy peas and chickpeas Of the plantsources soy is one of the most widely studied as a source of peptidesas it is a signi1047297cant source of dietary protein (Ortiz-Martinez Winkleramp Garciacutea-Lara 2014) Animal protein sources also have great potential

One of the most popular and promising lines of research is the produc-tion of hydrolyzed proteins from meat proteins which in addition tohaving important biological activities and being excellent sources of nu-trients such as minerals and vitamins can be used as 1047298avor enhancersand emulsi1047297ers (Lafarga amp Hayes 2014 Mora Escudero FraserAristoy amp Toldraacute 2014) The biological properties of other animal pro-tein sources such as egg and 1047297sh have also been studied (SakanakaTachibana Ishihara amp Juneja 2004 Theodore Raghavan amp Kristinsson2008)

Recent studies have linked the prevalence of cardiovascular diseaseobesity hypertension diabetes and cancer to nutritional factors In re-sponse to increased awareness of the relationship between food andhealth the market for functional foods has expanded A functionalfood is any food that in addition to basic nutritional functions providesadditional health bene1047297ts regulating one or more functions in the body(Diplock et al 1999 Hernaacutendez-Ledesma Contreras amp Recio 2011)

Processes incorporating protein hydrolysis have been studied to de-termine whether they produce biologically active peptides Mellander(1950) was responsible for the 1047297rst study relating the ingestion of bioactive peptides from hydrolyzed casein protein to increased bonecalci1047297cation in rachitic newborns Since then peptides with countlessbioactivities have been identi1047297ed According to the Biopep and BioPD(Bioactive peptidedatabase) databases morethan 1200 differentbioac-tive peptides have been recorded (Singh Vij amp Hati 2014)

Bioactive peptides are de1047297ned as speci1047297c regions of proteins withamino acid sequences that have biological activity including antioxi-dant anti-hypertensive antithrombotic anti-adipogenic antimicrobialanti-in1047298ammatory and immunomodulatory effects (Ahn Cho amp Je2015 Biziulevicius Kislukhina Kazlauskaite amp Zukaite 2006 Tavares

et al 2011 Tsou Kao Tseng amp Chiang 2010 Zhang Li amp Zhou2010) These peptides have 2ndash20 amino acids and molecular massesof less than 6000 Da Their bioactivity is mainly determined by theircomposition and amino acid sequence (Mora Reig amp Toldraacute 2014Sarmadi amp Ismail 2010 Singh et al 2014 Tsou Kao et al 2010) Thisenormous functional diversity places these peptides and proteins atthe forefront of the biotechnology 1047297elds (Miranda amp Liria 2008) fur-thermore these peptides and proteins have been identi1047297ed by severalauthors as possible substitutes for chemicals used as drugs or foodpreservatives (Hong et al 2008 Uhlig et al 2014)

According to Uhlig et al (2014) there is a very good outlook forusing bioactive peptides in the pharmaceutical 1047297eld Some peptides inthe clinical trial phase have shown very promising results for treatingcardiovascular infectious and metabolic diseases Peptides have an

important competitive advantage over traditional medications for thefollowing reasons 1) They have high speci1047297city for their target tissuesresulting in little or no toxicity and even low concentrations can be ef-fective This characteristic is extremely important for treating chronicdiseases 2) Synthetic chemical compounds that are typically used asdrugs often have a cumulative effect on the organism These syntheticsubstances may represent an environmental problem due to their ex-cretion still in the active form In contrast bioactive peptides undergolittle or no accumulation in the organism and they are easily degradedin the environment (Uhlig et al 2014)

Several methods are usedto obtain bioactive peptides including fer-mentation enzymatic hydrolysis and a combination of the two process-es (Fig 1) In fermentation the addition of lactic acid bacteria withproteolytic activity leads to formation of bioactive peptides especially

during dairy product manufacturing Enzymatic hydrolysis involves

the use of digestive plant or microbial proteolytic enzymes in a partialhydrolysis process leading to a reduction of allergenic factors im-proved digestibility and the formation of biologically active peptides(Korhonen 2009) One strategy used in several scienti1047297c studies dem-onstrated that using lactic acid bacteria together with food-gradeenzymes resulted in1047297nal productswith moreinteresting characteristicsthan either process alone Combining the techniques in addition toincreasing the amount of peptides in the fermented products resulted

in various biological and functional effects (Hafeez et al 2014) ChenTsai and Pan (2007) studied a combined process using enzymatichydrolysis with the microbial protease Prozyme 6 from Aspergillus anda commercial starter culture mixture of 1047297ve lactic acid bacteria as astrategy to enhance the ACE-inhibitory activity of bioactive peptidesfrom milk The results showed that the pre-treatment of the freshmilk with Prozyme 6 presented a positive impact on the ACE inhibitoryactivity of the peptides in which the IC50 value for the combinedprocess was024 mg mL minus1incontrastto064mgmL minus1 for the straightfermentation and 118 mg mL minus1 for fresh milk

In addition to the conventional methods mentioned above combin-ing several technologies has produced effective results for generatingfunctional peptides (Korhonen 2009) Ultra1047297ltration and nano1047297ltrationare examples of technologies that have been used to re1047297ne and isolatebioactive peptides allowing them to be separated by size for use inspeci1047297c applications (Picot et al 2010 Quiroacutes Chichoacuten Recio ampLoacutepez-Fandintildeo 2007)

Understanding the critical parameters for the process is fundamen-tally important for obtaining hydrolyzed proteinswith desirable biolog-icaland functional characteristics Theseparameters include the proteinsource and its characteristics such as chemical composition pH andseasonal variations enzymatic preparation and other aspects relatedto purity substrate speci1047297city speci1047297c activity pH and temperaturefor activity and stability and processing conditions including enzymeand substrate concentrations pH temperature and reaction timePrior knowledge and identi1047297cation of these parameters can be used astools for obtaining products with distinct functions for producing mul-tifunctionalpeptides or even for producing unique peptideswith specif-ic functions (Li-Chan 2015 Samaranayaka amp Li-Chan 2011)

This review describes advances in scienti1047297c research on the process-es of obtaining purifying and identifying the biological activities andpotential applications of bioactive peptides

2 Major processes for obtaining bioactive peptides

21 Fermentation

The use of fermentation processes to make bioactive peptides ismainly relevant to dairy product manufacturing Dairy products natu-rally contain precursor proteins for bioactive molecules (Akalin 2014Schanbacher Talhouk amp Murray 1997) Fermenting milk involves anumber of metabolic pathways responsible for generating metabolites

which signi1047297cantly contribute to the chemical biochemical and nutri-tional properties of the fermented products The proteolytic system of lactic acid bacteria (LAB) is complex andconsists of three major compo-nentsproteases boundto thecell wall that promote the initial hydroly-sis of milk casein into oligopeptides speci1047297c transporters that transferthe oligopeptides to the cytoplasm and intracellular peptidases that1047297n-ish the hydrolysis process to convert oligopeptides intofreeamino acidsandor low molecular weight peptides (Chaves-Loacutepez et al 2014) Theability of these microorganisms to produce proteolytic enzymes makesthem potential producers of bioactive peptides which can be releasedduring fermented product manufacturing Several microorganismshave been extensively reported in the literature as having an effectiveproteolytic system for protein hydrolysis and the release of bioactivepeptides including Lactobacillus helveticus Lactobacillus delbrueckii ssp

bulgaricus Lactococcus lactis ssp diacetylactis Lactococcus lactis ssp
















peptides



Proteases produced

during fermentation

High pressure

homogenization

Bioactive

Peptides



Proteases

vegetable animal or

microbial




and identification




















Table 1
























SodiumCaseinate











pseudoplantarum
























Table 2



Proteinsource






Mejia et al (2010)


[S] = 25




Tsou Kao et al 2010





Tsou Lin et al 2010


Bovinehemoglobin




Adje et al (2011)







pH 100 50 degC 55 h[S] = 100



Nasri et al (2012)


Protamextrade






and 2000 Da




pH 100 50 degC[S] = 50





Balti et al (2015)








Table 3
























different fractions








































2005)























Table 4





















(Fe

2+

)



2+








Table 5




Reference











(GSH)




plasma




and Alswiai (2013)




Kim et al (2013)











Ktari et al (2014)


















H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro















5 Conclusion





References


























554




































































































































































peptides



Proteases produced

during fermentation

High pressure

homogenization

Bioactive

Peptides



Proteases

vegetable animal or

microbial




and identification




















Table 1
























SodiumCaseinate











pseudoplantarum
























Table 2



Proteinsource






Mejia et al (2010)


[S] = 25




Tsou Kao et al 2010





Tsou Lin et al 2010


Bovinehemoglobin




Adje et al (2011)







pH 100 50 degC 55 h[S] = 100



Nasri et al (2012)


Protamextrade






and 2000 Da




pH 100 50 degC[S] = 50





Balti et al (2015)








Table 3
























different fractions








































2005)























Table 4





















(Fe

2+

)



2+








Table 5




Reference











(GSH)




plasma




and Alswiai (2013)




Kim et al (2013)











Ktari et al (2014)


















H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro















5 Conclusion





References


























554



































































































































































Table 1
























SodiumCaseinate











pseudoplantarum
























Table 2



Proteinsource






Mejia et al (2010)


[S] = 25




Tsou Kao et al 2010





Tsou Lin et al 2010


Bovinehemoglobin




Adje et al (2011)







pH 100 50 degC 55 h[S] = 100



Nasri et al (2012)


Protamextrade






and 2000 Da




pH 100 50 degC[S] = 50





Balti et al (2015)








Table 3
























different fractions








































2005)























Table 4





















(Fe

2+

)



2+








Table 5




Reference











(GSH)




plasma




and Alswiai (2013)




Kim et al (2013)











Ktari et al (2014)


















H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro















5 Conclusion





References


























554





































































































































































Table 2



Proteinsource






Mejia et al (2010)


[S] = 25




Tsou Kao et al 2010





Tsou Lin et al 2010


Bovinehemoglobin




Adje et al (2011)







pH 100 50 degC 55 h[S] = 100



Nasri et al (2012)


Protamextrade






and 2000 Da




pH 100 50 degC[S] = 50





Balti et al (2015)








Table 3
























different fractions








































2005)























Table 4





















(Fe

2+

)



2+








Table 5




Reference











(GSH)




plasma




and Alswiai (2013)




Kim et al (2013)











Ktari et al (2014)


















H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro















5 Conclusion





References


























554




























































































































































Table 3
























different fractions








































2005)























Table 4





















(Fe

2+

)



2+








Table 5




Reference











(GSH)




plasma




and Alswiai (2013)




Kim et al (2013)











Ktari et al (2014)


















H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro















5 Conclusion





References


























554


































































































































































2005)























Table 4





















(Fe

2+

)



2+








Table 5




Reference











(GSH)




plasma




and Alswiai (2013)




Kim et al (2013)











Ktari et al (2014)


















H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro















5 Conclusion





References


























554





























































































































































Table 4





















(Fe

2+

)



2+








Table 5




Reference











(GSH)




plasma




and Alswiai (2013)




Kim et al (2013)











Ktari et al (2014)


















H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro















5 Conclusion





References


























554



























































































































































Table 4





















(Fe

2+

)



2+








Table 5




Reference











(GSH)




plasma




and Alswiai (2013)




Kim et al (2013)











Ktari et al (2014)


















H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro















5 Conclusion





References


























554






































































































































































H

H C

2

N C

H

CH3

C

O

NCOOH

CO

C2

CH2

H

H

2CH

2C

COOHN

O

C

3CH

H

CNC

H

H N

O

HS C

H

H

C

H

CH3

C

O

NCOOH

COOHN

O

C

3CH

H

CH N2

Captopril

Ala-Pro

Enalapril

Phe-Ala-Pro















5 Conclusion





References


























554


































































































































































5 Conclusion





References


























554
















































































































































































































































































































































































































Documents

LUNES Biologically Active Peptides PROCESSES for Their Generation, Purification and Isolation