Please cite this article as: Ortiz-Martinez MProt (2014), http://dx.doi.org/10.1016/j.jpro
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RECTED Epidemiological studies have shown that regular consumption of food based on whole-grain
cereals and their products is associated with reduced risks of various types of degenerativechronic diseases. Food proteins are considered an important source of nutraceutical peptidesand amino acids that can exert biological functions to promote health and prevent disease,including cancer. There have been several reports on peptides with anti-tumour activity inrecent years. Plant-derived peptides, such as rapeseed, amaranth and soybean lunasin havereceivedmain attention. In this review,we extend this vision to analyse the evidence of currentadvances in peptides in cereals such as wheat, maize, rice, barley, rye and pseudocerealscompared with soybean. We also show evidence of several mechanisms through whichbioactive peptide exerts anti-tumour activity. Finally, we report the current status of majorstrategies for the fractionation, isolation and characterisation of bioactive peptides in cereals.
Biological significanceIn recent reports, it has been shown that peptides are an interesting alternative in thesearch for new treatments for cancer. One of the most studied sources of these peptides isfood proteins; however, a review that includes more recent findings for cereals as apotential source of bioactive peptides in the treatment of cancer, the techniques for theirisolation and characterisation and the assays used to prove their bioactivity is not available.This review can be used as a tool in the search for new sources of anti-cancer peptides. Theauthors have no conflicts of interest, financial or otherwise.This article is part of a Special Issue entitled: Proteomics, mass spectrometry andpeptidomics, Cancun 2013.
Epidemiological studies have shown that regular consump-tion of certain foods is associated with reduced risks ofvarious types of chronic diseases, such as cardiovasculardisease, type II diabetes, some types of cancer and majorcauses of mortality. One of the components of the dietreported to have the potential to prevent these diseases arewhole grains and whole grain products. There are reportsindicating that diets rich in whole grains or whole seed areassociated with lower cancer mortality rates, particularlycolon, breast and prostate cancers. This has led to moredetailed studies on their disease-prevention activity and thepotential therapeutic use of isolated components of food [1,2].
One of the most relevant groups of food derivatives withbiological activity areproteins andpeptidederivatives.Numerousstudies have shown that foodproteins are an important source ofbioactive peptides. Those peptides are encrypted in the proteinsequence, and once they are released, bioactive peptides exhibitseveral bio-functionalities and may have diverse therapeuticroles in human body systems. Abundant food-derived peptidesexhibiting activities such as opiate, antithrombotic, anticancer,antihypertensive, immunomodulation, mineral-binding, antimi-crobial or antioxidant properties have been reported [3,4].Biologically active peptides are either naturally occurring orproduced by enzymatic digestion or fermentation. Bowman–Birk-type and Kunitz-type trypsin inhibitors are examples ofnaturally occurring proteinswith knownnutraceutical functions,while products of protein enzymatic digestionorhydrolysates arethe main sources of biologically active peptides in food-basedresearch [5,6].
Sources of bioactive peptides are from animal origin andplant origin. Plant sources usually include cereals, suchaswheat,corn, rice, barley, rye and pseudocereals, such as buckwheat andamaranth (Table 1). Other plant sources are legumes (soy, peaand chickpea), brassica species (mustard, rapeseed) and others
Please cite this article as: Ortiz-Martinez M, et al, Preventive and tProt (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044
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(sunflower). Among plant sources, soybean is the most studiedsource of bioactive proteins and peptides. This can be explainedby the fact that soybean is an important protein source, on theaverage, soybean contains about 40% protein [7,8].
Therefore, food proteins can be considered a source ofnutraceutical peptides and amino acids which can exertbiological functions to promote health and prevent disease,including cancer [5,6]. Proteins and peptides show potentialresults in preventing the different stages of cancer, includinginitiation, promotion and progression.
Although there have been many reports on peptides withanti-tumour activity in recent years, these peptides are mainlyderived from animals or microorganisms. Plant-derived pep-tides, such as rapeseed peptide, amaranth peptides and soybeanlunasin have received great attention [9].
There are several mechanisms through which bioactivepeptide exert anti-tumour activity, including:
I. Induction of apoptosis; the process of apoptosis is carefullycontrolled, involving an energy-dependent cascade ofmolecular events led by cysteinyl aspartate-specificproteases called caspase. Strategies to overcome tumourresistance to either extrinsic or intrinsic apoptotic path-ways includes activation of the extrinsic pathway throughproapoptotic receptors, restoration of p53 activity, inhibi-tion of the Bcl-2 family of proteins, BH3-only mimicproteins, caspase modulation, IAP inhibition and protea-some inhibition [10,11].
II. Blockage of intermediate tumour generation because itsbinding to cellular components related to cell proliferationand survival or biosynthetic pathways may modulate thegrowth rate of a tumour or even decrease its size [12,13].
III. Regulation of immune system may stimulateimmunosurveillance by acting on cancer cells in severalways, for example by increasing the expression orpresentation of tumour-associated antigens on the
herapeutic potential of peptides from cereals against cancer, J
HPLC (comparison with lunasin standard)Western blot (also for quantification)
Bioavailability of lunasin from tissues of rats fed LEB (lunasin-enrichedbarley): four-wk-old male Sprague–Dawley rats fed LEB; the liver, kidneyand blood were collected, and lunasin was isolated by ion-exchangecolumn chromatography and purified by HPLC; lunasin was quantified byWestern blotInhibition assay of HAT activity: HAT Activity Colorimetric Assay kit(BioVision)Internalisation of barley lunasin: visualised by fluorescence microscopyafter stain with antibodies labelled with fluorescent dye (NIH 3T3 cells)Inhibitory effect of lunasin on the cell cycle: determined the expressionlevel of p21, p15INK4b, cyclin D1 and CDK4; NIH 3T3 cells byimmunofluorescence stain
t1:8 Nakurte et al. (2013) Oats HPLC reverse phase coupled to an electrospray ionisation tandem mass spectrometer Radical scavenging assay: DPPH radical scavenging assayCell culturing: Human embryonic kidney HEK 293 (ATCC, catalogue noCRL-1573)Cell proliferation assay: MTT viability assay (HEK 293 cells)
t1:9 Hyung Jin Jeong et al.(2009)
Rye HPLC reverse phase HPLC (comparison with lunasin standard)Western blot
Bioavailability of lunasin from tissues of rats fed LER (lunasin-enrichedrye): four-wk-old male Sprague–Dawley rats fed LER; the liver, kidney andblood were collected, and lunasin was isolated by ion-exchange columnchromatography and purified by HPLC; lunasin was quantified by WesternblotInhibition assay of HAT activity: HAT Activity Colorimetric Assay kit(BioVision)Internalisation experiment: Immunostaining of 95% lunasin purified fromrye and tissue lunasin internalised into the mouse fibroblast cell line NIH3T3
t1:10 Nakurte et al. (2012) Triticale HPLC reverse phase coupled to an electrospray ionisation tandem mass spectrometer Bioactivity assays are not reportedt1:11t1:12 Pseudocerealst1:13 Silva-Sánchez
et al. (2008)Amaranth Immunoprecipitation prior to
identification assaysELISA Western MALDI-TOF peptide massmapping
Apoptosis and cell cycle distribution: The fraction containing thelunasin-like peptides was proved for their potential induction of apoptosisin HeLa cells; apoptosis assay was performed (Tunel Labeling Kits,RnDSystems) and cell cycle distribution using a FACS(fluorescence-activated cell sorting) apparatus; primary culture offibroblasts was used as the control of normal cells
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surface of cancer cells (antigenicity), by causing tumourcells to emit danger signals that stimulate innate orcognate immune responses by operating as adjuvants(immunogenicity) or by augmenting the propensity oftumour cells to be recognised and killed by immuneeffectors (susceptibility) [9,14].
In 2010, 50 peptidedrugswere approved formarketing,mostlysynthetic and recombinant hormone analogues, with annualglobal sales of around US$ 1 billion associated with the followingpeptide drugs: cyclosporine (e.g. Neoral®, Novartis), goserelinacetate (Zoladex®,AstraZeneca), glatiramer acetate (Copaxone®,Teva Pharmaceuticals), leuprolide acetate (e.g. Lupron®, AbbottLaboratories) and octreotide acetate (Sandostatin®, Novartis).The increasing interest by the pharmaceutical industry indeveloping peptides as drugs is at least partially a conse-quence of the now widespread acceptance of protein thera-peutics by physicians and patients and the development ofsolutions to problems such as a short half-life and moleculedelivery [15,16].
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2. Peptide-based cancer therapies
Cancer remains a major source of mortality and morbidityaround the world, despite numerous recent advances in treat-ment alternatives. Conventional cytotoxic therapies, such asradiation and chemotherapy, are the methods of choice forcancer management [17]. Chemotherapy is still the choicetreatment for advanced and metastatic disease. However,both therapies have low therapeutic indices and are oftenhighly toxic, with a broad spectrum of severe side effects. Thedevelopment of a new class of anticancer that lack toxicity tohealthy cells and are unaffected by common mechanisms ofresistance would be a major advance in cancer chemotherapy[17,18].
Cancer cells provide their own growth signals to ignoregrowth inhibitory signals, avoid cell death, replicate withoutlimit, sustain angiogenesis and invade tissues through base-ment membranes and capillary walls. In addition, the immunesystem fails to eliminate cancer cells due to the immunosup-pressive effects mediated by tumour-infiltrating host cells.Cancer cells have an elevated apoptotic threshold, and theinduction of apoptosis in cancer cells is increasingly seen as atherapeutic desirable goal [18,19].
Food proteins are considered not only nutrients for theproper maintenance of body functions but also as a source ofimportant peptides with known biological activities. Foodproteins can be considered a source of nutraceutical peptidesthat can exert biological functions to promote health andprevent disease, including cancer. Bioactive peptides havebeen known to be a part of the human diet for several years.With the appearance of chromatographic methods, thenumber of studies on bioactive peptides from animal andplant sources has increased. As the findings of these studieshave shown, peptides exert regulatory functions besides theirnutritional roles. Several studies have shown the anti-cancerpotential of dietary proteins, peptides and amino acids,whether naturally occurring or the product of fermentation,enzymatic hydrolysis or gastrointestinal digestion, in the
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mediation of apoptosis and angiogenesis, important steps tocontrol tumour metastasis [5,20].
Peptides have attracted attention as drug candidates owingto their possession of certain key advantages over alternativechemotherapy molecules. In contrast to most small-moleculedrugs, peptides have high affinity, strong specificity and lowtoxicity, and in contrast to chemotherapeutic antibodies, theyhave good tissue penetration because of their small size. Thisprovides impetus to the study of bioactive peptides as possibletherapeutic agents, although the therapeutic use of peptideshas remained limited due to their high instability in biologicalenvironments, rapid depuration from the blood, poor mem-brane transportability and effective digestion in the gastroin-testinal tract. In vivo experiments and clinical trials are neededto demonstrate the physiological effect of peptides, but in vitrostudies remain important prospective tools because peptidefunctionality is based on biological mechanisms. They cannot,however, replace in vivo and clinical studies because it is verydifficult to establish a direct relationship between invitro and invivo biological activity. Peptide bioavailability after oral admin-istration is one of themain reasons for this incomparability andone of the primary aspects to study before bioactive peptidescan be incorporated into food or drug systems [18,21].
The implementation of a peptide-based therapy dependslargely on its ability to remain intact until it reaches the targetorgan. Bioactive peptides must remain active and intact duringgastrointestinal digestion and absorption to reach the cardiovas-cular system and potentially exercise their physiological effects,although once in the organism, all peptides must pass through aseries of barriers that can inactivate themand consequently theirbiological action. This performance of anticancer peptides can beenhanced byusing different delivery systems to improve stabilityand longevity, as well as to generate enhanced permeability andretention in the body (Table 2) [18,21].
3. Plants as sources of bioactive peptides
Initially, the search for bioactivepeptideswasmainly focussedonanimal products such as milk. In the last years, the studies inplants were intensified, motivated by their huge diversity. Alsonutritional studies with epidemiological basis have associatedthe consumptionof certain foodswith benefits for humanhealth.
Legumes are the plant source for which the most peptideswith anticancer are reported. In the first instance this may bedue to the high content of high quality proteins of legumes,but this first impulse has been fuelled by the discovery ofproteins and peptides with interesting bioactivity, such ashemagglutinin, defensins and protease inhibitors.
3.1. Soybean
Legumes play an important role in a diet strategy for reducingcancer risk. Soybean (Glycine max) has undoubtedly receivedthe most research attention, because it contains a variety ofphytochemicals with demonstrated anti-cancer activity. Themost widely studied bioactive substances are the isoflavones,the Bowman–Birk protease inhibitor (BBI) and the less purifiedBBI concentrate (BBIC). Soybeans also contains other proteinsand peptides with biological activity, which may contribute to
herapeutic potential of peptides from cereals against cancer, J
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the prevention and treatment of cancer [12,22,23]. Thesoybean Bowman–Birk protease inhibitor (BBI) is a 8 kDapolypeptide consisting of 71 amino acids and exhibiting twoprotease inhibitor sites, one for trypsin and one for chymo-trypsin [24]. BBI works by inhibiting proteases involved in theinitiation and promotion of carcinogenesis. Its capacity forpreventing or suppressing carcinogenic processes has beendemonstrated in different cell lines in vitro and in vivo [12,24].The first reports of this proteases inhibitor are from the 1970s[25], but it is remarkable that the soybean was not consideredas a potential source for other types of bioactive proteins orpeptides until the 1980s, when a group reported the isolationof a polypeptide with an unusually high concentration ofaspartic acid [26], later named lunasin.
3.1.1. Lunasin propertiesLunasin is a 43-amino acid soy peptide that has been shown tobe chemopreventive against oncogenes and chemical carcino-gens in mammalian cells and in a skin cancer mouse model(Fig. 1). Soybean varieties display varying amounts of lunasin,which correlate with the extent of inhibition of core histoneacetylation. Both, soy lunasin and synthetic lunasin inhibit corehistone acetylation in a dose-dependent manner. Syntheticlunasin is heat-stable, resisting temperatures of up to 100 °C for10 min. Animal studies indicate, that lunasin resists digestionand enters the target tissues after absorption [22,27].
During seed development, the lunasin peptide appears fiveweeks after flowering and persists in the mature seed.Western blot analysis of different soybean varieties andcommercially available soy proteins shows the presence ofthe peptide in varying amounts. These results demonstratethe feasibility of producing large quantities of natural lunasinfrom soybean for animal and human studies. The high cost ofsynthesising lunasin makes it impractical to use syntheticlunasin for animal experiments and human studies. There-fore, there is a need to isolate, characterise and demonstratethe biological activity of lunasin [2].
Bioavailability studies carried out with animals have con-firmed the preliminary results obtained by in vitro analysis. Afterthe ingestion of lunasin-enriched soy and lunasin-enrichedwheat by rats, lunasin was found as an intact and active peptidein the blood and liver of these animals. One of the properties ofan ideal cancer-preventive agent is that it can be taken orally.Thismeans being able to survive degradation by gastrointestinaland serum proteinases and peptidases and to reach the targetorgan or tissue in an active form. Simulation of the gastrointes-tinal digestion of lunasinhas demonstrated that,while syntheticpure lunasin is easily hydrolysed by pepsin and pancreatin,lunasin in soy protein is resistant to the action of these enzymes.These results suggest that the combined protection provided byBBI and other naturally protease inhibitors, such as Kunitztrypsin inhibitor, against digestion plays a major role in makinglunasin available in soy and wheat protein [12].
There have been several attempts to express the lunasingene in E. coli. Sequence modifications should produce fusionpeptideswith desired characteristics [28,29]. Extensive searchesof transcriptome and DNA sequence databases for wheat andother cereals have failed to identify sequences encoding eitherthe lunasin peptide or a precursor protein, which leads tospeculations about its real origin [30].
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3.2. Common bean
Beans exert many effects beneficial to health, including thelowering blood glucose levels, blood lipids and incidence ofcancer. Additionally, beans represent an inexpensive, but richsource of dietary proteins [31], whereas. Common bean (Phaseolusvulgaris L) accounts for 50% of legumes used for humanconsumption. Intensive investigations on various legume seedsrevealed anti-tumour, anti-viral and antifungal activities [31–33].Wang and Ng investigated a 7.3 kDa peptide from P. vulgaris cv.“Spotted bean” and which has considerable homology withdefensins from other sources. The peptide displays potentantiproliferative activity for leukaemia cell line L1210 andlymphoma cell line MBL2 [34]. 2011, the isolation of a dimerichemagglutinin was reported, which suppressed the proliferationof breast cancer MCF-7 cells (IC50 of 0.2 μM). Thehemagglutinin-treatedMCF-7 cells showed anumber of changes,including cell cycle arrest in G2/M phase, phosphatidylserineexternalisation and mitochondrial membrane depolarisation.The hemagglutinin induced apoptosis by activating the deathreceptor-mediated pathway, involving Fas ligands, caspase-8activation, BID truncation, p53 release, caspase-9 activation andLamin A/C truncation [31].
ED4. Cereals source of bioactive peptides
Cereals can be defined as a grain or edible seed of the grassfamily, Gramineae (see Fig. 2). They are grown for their highlynutritious edible seeds, which are often referred to as grains.The grains consist of an embryo (or germ), the endosperm,which is packed with starch grains, and bran (fibre). If thecereal grain germinates, the seedling uses the nutrientsprovided by the endosperm until the development of a newplant occurs. Cereals are the most important sources of food,and cereal-based foods are a major source of energy fromcarbohydrates, protein, B vitamins and minerals for the worldpopulation [35]. Wheat, rice and corn are the major importantgrains in the human diet. The minor grains include oats,barley, rye, triticale, sorghum, millet and buckwheat. Thecereals are protein-rich sources and therefore are a potentialsource of bioactive protein and peptides, already documentedas imparting several physiological functions, including anti-oxidant, immunomodulatory, chemopreventive and anti-cancer functions [1,36]. (See Fig. 3.)
The storage protein fractions of the cereal grains arecategorised into four classes depending on their solubility:water-soluble albumins, globulins soluble in salt solution,prolamins soluble in alcohol solution and glutenins insolubleinneutral aqueousor saline solution andethanol. Theprolaminsare monomeric polypeptide chains with molecular weightsbetween 30 and 80 kDa. They are rich in proline and glutamine(20–55%). Prolamins in wheat are known as gliadins, in barley ashordeins, in rye as secalins and in oats as avenins [37].
4.1. Barley
Barley (Hordeum vulgare L.) is the fourthmost widely cultivatedcereal in the world after wheat (Triticum aestivum L.), rice
herapeutic potential of peptides from cereals against cancer, J
Table 2t2:1 –Mechanism of action of anticancer peptides from legumes and cereals.t2:2t2:3t2:4 Reference Source Treatment Cell line or animal
modelMechanism Evidence
t2:5 Legumes Lam and Ng (2011) Common bean(Phaseolus vulgariscultivar “Legumisecchi”)
Isolateddimerichemagglutininwith a relativemolecularmass of 62000
MCF-7(human breastadenocarcinomacell line)
Apoptosis through deathreceptor-mediated pathway
Using a flow cytometer and differential stainingthey observe:• G2/M phase arrest• Phosphatidylserine externalisation• Mitochondrial membrane depolarisation
DNA fragmentation was indiscernibleby agarose electrophoresisActivation of death receptor-mediatedpathway was detected by Westernblot: Fas ligands aumented, caspase-8activation, BID truncation, p53 release,caspase-9 activation and Lamin A/Ctruncation
Hydrolysatefrom soybeangerminated for6 days fraction>10 kDa
HeLa (human cervicaladenocarcinomacells) and HaCaT,non-canceroushuman keratinocytescell line
Apoptosis Apoptotic cells were identified throughfluorescence microscopic observation ofsamples using Hoechst, staining revealed thetypical changes, such as nuclear shrinkage,chromatin condensation and fragmentation.Images of phase contrast microscopy of HeLacells treated with the peptide fraction showthe membrane blebbing and cell shrinkagetypical of apoptosis.The internucleosomal DNA fragmentationwas determined by the Apoptotic DNA LadderKit that is based on Real-time quantitativePCR, the treated cells showed thecharacteristic DNA ladder pattern of apoptosisThe caspase activity was evaluated using afluorescence microscope after stain with afluorescent kit for caspase 8 and 9respectively, both caspase activity was foundin the treated cellsThe PTTGl and TOP2A mRNA expression wasdetermined by real-time quantitative PCR, theexpression of both genes was markedlydecreased by treatment.
t2:7 Dia and Mejia (2010) Soybean Lunasin HT-29 coloncancer cells
Analysis of cell cycle distribution was performedusing flow cytometry after staining withpropidium iodide shows that lunasin caused aG2/M cell cycle arrest on HT-29 colon cancer cells.
Reference Source Treatment Cell line or animalmodel
Mechanism Evidence
The apoptotic status of the HT-29 coloncancer cells was confirmed by determiningthe presence of phosphatidylserine on the cellmembrane using an Annexin V-FITCapoptosis detection kit by flow cytometry.Caspase-3 activity in HT-29 colon cancer cellswas analysed using a fluorescence assay kit,the results were a statistically significantincrease in caspase-3 activityThe Bax protein expression increases upontreatment of lunasin. On the other hand, theexpression of the anti-apoptotic Bcl-2 proteinwas dose-dependently reduced by lunasintreatment. Lunasin caused a dose-depen-dent increase in the expression of p21.This determination was done by Western blot
t2:8 de Mejia, Wang,and Dia (2010)
Soybean Hydrolysatesrich on lunasin
Leukaemia cellline L1210
Apoptosis through a caspasedependent pathway
Analysis of the cell cycle was performed byflow cytometry, treatment of L1210 leukaemiacells with LES for 24 h led to an increase inthe amount of cells in the sub-G1 fraction in adose-dependent manner.The apoptotic inducing effect was confirmedby microscopic analysis of the cells treatedand stained with Hoechst reagent.Using a kit based on fluorescence theexpressions of caspases 3, 8 and 9 weredetermined, treatment increased theexpression of caspases 8 and 9 inconcentration-dependent manner but mostlyincreased the expression of caspase-3Analysis of p21 and p27 expression wasperformed by western blot, treatment showedno effect on the expression of p21 and p27.
t2:9 Cereals Chen, Chen, Wu, Yu,and Liao (2010)
Rice (O. sativaL. Japonica)
Prolaminfraction
Human mono-blastoid leukaemiccell line U937
Potentiating of immuneresponses
Medium supplemented with prolaminpromoted monocyte differentiation of U937cells, cell morphology was evaluated bycytocentrifugation onto a microscope slidestained with Wright's stain and observedunder an inverted microscope for determiningmonocyte differential counts.The amount of TNF-α secreted significantlyincreased with prolamin treatment measuredby enzyme- linked immunoassay (EIA).
t2:12 Reference Source Treatment Cell line or animalmodel
Mechanism Evidence
t2:10 Li et al. (2013) Corn Gluten meal(Zea mays)
Hydrolysatemolecularweight < 5 kDa
HepG2 (Humanhepatoma cellline HepG2)
Apoptosis Microscopical observations revealed thatthe treatment affects the cell morphologyand nuclear condensation andfragmentation appear in a dose-dependentmanner.Cell-cycle phase distribution was analysedby flow cytometry with PI staining, the Sphase cells increased, meanwhile the G0/G1phase cells were markedly decreased.The expressions of several critical apoptosisrelated protein were checked by westernblot analysis. Anti-apoptotic Bcl-2 expressionwas significantly inhibited in adose-dependent manner, whereas that in Baxwas relatively constant. The level of p53 wassignificantly increased. Simultaneously, theexpression of Cleaved-caspase-3 wasincreased.
The treatment could stimulate the growthand development of the thymus gland andspleen in H22-bearing miceEffect of CPs on IL-2 and TNF-a level inmurine serum were determined by ELISA,the levels of IL-2, IFN-c and TNF-a wererestored and enhanced in a dose- dependentmanner
Fig. 1 – Taxonomy of the Gramineae Family.(Modified of McKevith, 2004 and Cavazos & Mejia, 2013).
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(Oryza sativa L.) and corn (Zea mays L.). The low lysine contentin the barley storage proteins limits their wide application as anutritional ingredient in foods, so they are normally soldcheaply as animal feed [38].
Hordein, a barley prolamin, is the major protein in barleyby-products, and it is the main storage barley protein. It isenrichedwith Glu, Pro, Leu, Val, Phe and Tyr,most of which havebeen reported to be related to antioxidant activity in their freeforms or as residues in proteins and peptides. Hordein iscomposed of three sub-fractions, B hordein (sulphur-rich), Chordein (sulphur-poor), andDhordein (highmolecular weight). Chordeinhas been reported as the fractionwithhigher antioxidantpotency. Although limited recent data indicates that the antiox-idant activity of barley hordein can be enhanced after enzymatichydrolysis, there is little information regarding the effects of thetype of protease and the hydrolysis process on the peptidestructures and their antioxidant activity [38,39].
U
Fig. 2 – Lunasin
Please cite this article as: Ortiz-Martinez M, et al, Preventive and tProt (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044
In 2002, a peptide similar to lunasin was reported in barley,a cereal seed. Partially purified lunasin showed in vitro and invivo bioactivity. This discovery motivated the search forlunasin in other cereal crops [40]. Currently, lunasin wasfound in seed crops such as soybean, amaranth, solanumfamily, wheat and rye. The presence of other bioactivecomponents aside from peptides similar to lunasin is currentlybeing investigated in these crops [41]. Nevertheless, sometimesthe correct identification of lunasin is questioned, due to theuse of methodology with low selectivity. [30,42].
4.2. Corn
Originating in the highlands of Mexico between 5000 and10,000 years ago, maize (Zea mays L.) has become the mostextensively cultivated cereal crop, followed by wheat and rice.Corn is an important source of protein. Globally, it contributes
sequence.
herapeutic potential of peptides from cereals against cancer, J
Fig. 3 – Diagram with the most common strategies to produce and analyse bioactive peptides.
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UNCOapproximately 42 million tons of protein a year, which corre-
sponds to approximately 15% of the world's annual productionof food-crop protein [43,44]. Maize has a wide range of food andnon-food applications, and even when they are predominantlycomposed of carbohydrates, they contain considerable amountsof protein. The chemical composition of the grain is variable indifferent parts of the grain,withhigher concentrations of proteinin the endosperm (74%) and the germ (26%). The proteins ofmaize can be grouped according to their solubility as follows:albumins (water-soluble), globulins (saline-soluble), prolamins(soluble in strong alcohol solution) and glutelin (soluble inalkaline medium). The prolamin fraction (α-zein) representsthehighest concentration inmaize, representing 50 to 60%of thetotal protein [45].
Several types of corn peptides have been reported to havebiological activity. In the search for peptides with inhibitoryeffects for the angiotensin-converting enzyme (ACE), the meth-od of choice according to the reports is enzymatic hydrolysis,linked to separation using ultrafiltration membranes. Corn
Please cite this article as: Ortiz-Martinez M, et al, Preventive and tProt (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044
gluten meal (CGM), a major by-product of corn wet milling,contains 67–71% protein (w/w). At present, CGM is mainly usedas forage. However, it may be considered a good source for thepreparation ofACE inhibitor and antioxidant peptides because ofits high proportion of hydrophobic amino acid and proline[46–49]. Corn gluten meal (CGM), a by-product of the starchindustrywith abundant protein, ismainly comprised of zein andglutelin. Corn protein is isolated from CGM, and in turn, cornpeptides (CPs) are obtained from hydrolysis of the corn protein.Previous studies have found that CPs exhibited anti-breastcancer activity [50]; however, they ignored the effects of CPs oncancer cells and their underlying mechanisms. Some factors,such as molecular weight, hydrolysate concentration, degree ofhydrolysis (DH) and amino acid composition, affects theiractivity [9,46–49].
More recently, a study was aimed at evaluating the anti-tumour mechanism of corn peptides (CPs). In vitro, the resultsshowed that CPs significantly inhibited cell viability in both adose- and a time-dependent manner. CP treatment induced S
herapeutic potential of peptides from cereals against cancer, J
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cell-cycle arrest and caused apoptotic death in HepG2 cells. Itwas observed that CPs caused an increase in the Bax/Bcl-2 ratio(measuring the protein expression after treatment with CPsusing Western blot) and triggered the activation of caspase-3,andp53 inHepG2 cells. In vivo, the results showed thatCPs couldnot only inhibit the growth of the tumour but also enhance thespleen index [calculated by dividing the spleen weight (mg) bythe total body weight (g)]; the importance of this index it isthat the size of the spleen has a direct correlation with theprogression of tumours in vivo, as well as the level of the serumcytokines of interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumournecrosis factor-α (TNF-α). Moreover, CPs could prolong survivaltime in H22-bearingmice. This study demonstrated that CPs arean apoptosis inducer in HepG2 cells, could effectively inhibithepatocellular carcinoma in vivo via enhancement of the hostimmune system function and may be a safe and effectiveanticancer, bioactive agent or functional food [9].
4.3. Oats
Oat (Avena sativa L) is distinct among the cereals due to itsmultifunctional characteristics and its nutritional profile. Oatand oat by-products are used as complementary treatments forpatientswithdiabetes and cardiovascular diseases. Recently, theingestion of oat bran in a meal has been shown to affect genesets associated with insulin secretion and b-cell development,protein synthesis and genes related to cancer diseases [51,52].Oats also contain peptides similar to lunasin.Monitoring lunasinlevels in different oat genotypes showed genotype-relatedvariations over time. The results of antioxidant assays indicatedthat this oat lunasin-like peptide is bioactive [51].
4.4. Rice
Rice is not only an important cereal as a staple food worldwidebut is also nutritional for human health, with fewer allergenicproperties and easier digestion. Several ingredients isolated andderived from rice possess pharmacological and biologicalactivities. Rice seeds contain about 8–9% protein. Four impor-tant fractions of rice proteins are identifiable by their differen-tial solvent solubility. Of those, rice seeds contain 5–10%alcohol-soluble proteins (prolamin), 4–10% salt-soluble proteins(globulin and albumin) and 80–90% alkali soluble proteins(glutelin). The portion prolamin has proven to have a beneficialeffect on activating anti-leukaemia immunity [53].
Rice protein isolate (RPI) has been reported to reduce theincidence of 7, 12-dimethylbenz[a]-anthracene-induced mam-mary tumours in rats. The potential role of phytochemicalsassociated with the RPI has been studied, but not the activity ofthe proteins and peptides, which are themain components [54].
Rice bran is a cheap co-product of rough rice milling, and itcontains nutrients including B vitamins, minerals and fibre,including oil, which has health benefits. It is used as a low-costanimal feed. Defatting the bran, and directly hydrolysing thehigh-quality protein using endoprotease can sustainably re-lease peptides in a consistentmanner. The proteins in rice branare complexed within carbohydrates and lipids and henceprovide difficulties for protein extraction. Therefore, the directhydrolysis of heat-stabilised defatted rice bran (HDRB) wasperformed to obtain high-quality and high-yield peptides for
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determining anti-cancer activities. This approach not only wasunique but also can prove to be an economicalway of producinganti-cancer peptides from rice bran on a large scale. Similarstudieswere able to obtain bioactive peptides fromHDRB and toprove that the products of their hydrolysis with digestiveenzymes retain and even improve their anti-cancer activity,but this information must also be tested in vivo [55].
In 2010, findings were published showing that the <5 kDapeptide fraction from HDRB was the most active; it was selectedfor further characterisation to obtain single pure peptide(s)with enhanced anti-cancer properties. The pure peptide at a600–700 μg/mL dose caused 84% inhibition against the growth ofcolon cancer cells (Caco-2, HCT-116), 80% against breast cancercells (MCF-7, MDA-MB-231) and 84% against liver cancer cells(HepG-2). Mass spectrometry analysis and de novo sequencingrevealed the sequence of Glu-Gln-Arg-Pro-Arg for the peptidewith a molecular mass of 685.378 Da. A novel pentapeptide wasisolated from rice bran possessing cancer growth inhibitoryproperties on colon, breast, lung and liver cancer cells [56].
4.5. Rye
Rye (Secale cereale L.) is, in addition to wheat, the major breadgrain in Europe. Rye ismainly produced and consumed as breadin northern Europe; it is an important source of dietary fibre.Rye bread is oftenmade using sourdough, a process influencingboth the nutritional quality and the taste of rye bread. There isconvincing evidence that the consumption of whole grain foodsis associated with reduced incidence of chronic diseases, e.g.diabetes, cardiovascular disease and certain cancers. In addi-tion to dietary fibre, various phytochemicals, vitamins andminerals havebeen suggested to contribute to thehealth effectsof whole grain foods; however, little attention has been given toits proteins and peptides [57]. Peptides similar to lunasin werereported also for rye. The peptides are found in relevantconcentrations, and the researchers also reported their bio-availability and bioactivity [58].
4.6. Triticale
Nowadays, triticale is very rarely studied as a healthy food.Triticale (X Triticosecale Wittmack) is a synthetic cereal grainspecies resulting from a plant breeder's cross between wheat(Triticum) and rye (Secale). Historically, triticale, incorporatingthe functionality and high yield of wheat and the durability ofrye, has mostly been used as animal food. In 2012, a reportwas published on the first discovery of lunasin in triticale,finding that triticale was the most lunasin-rich cereal. Thehighest lunasin content was 6.46 mg/g [59]. This data can betaken as an incentive to explore the potential of triticale in thehuman diet and as a source of bioactive peptides.
In contrast, a recent paper states the absence of lunasin inwheat, which is congruent with studies of the groups of Dinelli[42] and Mitchell [30]. According to this reports, extensivesearches of transcriptome and DNA sequence databases forwheat and other cereals have failed to identify sequencesencoding either the lunasin peptide or a precursor protein,which feeds the controversy about the presence or lunasin ingroups of plants with high genetic distance (legumes andcereals). The authors recommend further detailed studies to
herapeutic potential of peptides from cereals against cancer, J
Table 3t3:1 – Summary of the reported techniques for the generation, isolation, characterisation and bioactivity assays of anti-cancer peptides from cereals corn and rice.t3:2t3:3t3:4 Reference Source Techniques Product obtained Bioactivity assays
After tumourinductionusing DMBA, thetumour size wasmeasured
Female rats(35 daysof age) of theSprague–Dawleystrain
t3:7 Li et al. (2013) Corn glutenmeal
Corn protein solutionwas hydrolysed byAlcalase
5 kDaultra-filtrationmembrane
– Mixture of peptides of5 kDa or less obtainedby hydrolysis
Cell viability assay byan MTT-based assayAnalysis of apoptoticcells by flowcytometerCell morphology byfluorescencemicroscopyPro-apoptotic factordetermination byWestern blot analysis
Human hepatomacell line HepG2cells(HepG2),
t3:8The volume of thesolid tumour wasmeasured; inhibitionrate of thymus andspleen indices weredeterminedThe serum of mice ineach group wascollected for thedetection of IL-2,IFN-c and TNF-a levelusing a commercialELISA kitDetection of lifeprolongation rate.
Human colon(Caco-2), breast(MCF-7), liver(HepG-2), and lung(A549) cancer celllines
t3:11 Chen et al. (2010) Rice extractsfrom rice bran,endospermand total riceseeds
Rice extracts were treatedwith protease K
– Two-dimensionalelectrophoresisMass spectrometry(MALDI-QUAD-TOF)analysisWestern blot SDS-PAGE
Crude rice extracthydrolisate andprolamin
Growth inhibitionassessed using thetrypan blue dyeexclusion testCell morphologyevaluated bycytocentrifugationstained with Wright'sstain and observedunder an invertedmicroscope
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resolve the origin of the lunasin in samples of wheat and othercereals [30,42].
4.7. Pseudocereals (Amaranth)
Amaranth seeds are rich in protein with a high nutritionalvalue, but little is known about their bioactive compoundsthat could benefit health. In 2008, a paper reported thepresence, characterisation and anti-carcinogenic propertiesof the peptide lunasin in amaranth seeds. Western blotanalysis revealed a band at 18.5 kDa, and MALDI-TOF analysisshowed that this peptide matched more than 60% of thesoybean lunasin peptide sequence. Glutelin extracts digestedwith trypsin showed the induction of apoptosis against HeLacells. Predictions of other bioactive peptides in globulins andglutelins from amaranth were mainly anti-hypertensive. Thiswas the first study to report the presence of a lunasin-likepeptide and other potentially bioactive peptides in amaranthprotein fractions [60]. Further studies on amaranth focusmainly on anti-hypertensive peptides [61,62], but since it is agood source of protein, it is logical to think that it may containother anti-cancer peptides besides lunasin.
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5. Peptide bio-characterisation
5.1. Isolation and fractionation
The analysis of proteins and peptides has been a challenge toresearchers for many years. Initially, attention was focused onthe isolation, characterisation and properties of endogenousproteins, the profile of a healthy system and alterations inpeptides. Each of these approaches requires analytical methodsthat are suitable to its specific problems, such as high specificity,high resolution separation or high sensitivity [63,64]. In thisreview,wepresent a summaryof themost commonand reportedtechniques and strategies in the search for and characterisationof bioactive peptides that may be useful to improve existingmethodologies; new developments in instrumentation andtheory are not covered (Table 3).
A challenge often faced in food protein-derived peptideresearch is the need to obtain high-yield peptide products withpotent bioactivity. This limitation results in the carrying out offurther processing of enzymatic food protein hydrolysates.Therefore, after protein hydrolysis, the resulting peptide productis further processed based on the physicochemical and struc-tural properties of the constituent peptides in a bid to enhancebioactivity. Proteins and peptides can be fractionated intodifferent groups having similar physical and chemical propertiesby a variety of different analytical methods. The peptideproperties often focused on include size, net charge, hydropho-bicity, isoelectric point or affinity depending on the targetedpharmacological uses [65,66].
5.1.1. Enzymatic hydrolysisThe fact that peptides released from food proteins by enzymehydrolysis may exhibit different biological activities is nowgenerally accepted. These peptides are inactive within thesequence of the parent proteins but can be activated whenreleased by the hydrolytic action of commercial enzymatic
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proteolysis or gastro-intestinal digestion [67,68]. Most reportedbioactive peptides are produced by in vitro enzymatic hydrolysis[9,55,65] or fermentation. In most cases, these protein hydroly-sates and peptides have demonstrated better bioactivity com-pared to their parent proteins, which underlines the importanceof the hydrolysis for liberating potent peptides [65,69].
After selection of an appropriate food protein, enzymatichydrolysis is performed using single or multiple specific ornonspecific proteases to release peptides of interest. A simu-lated gastrointestinal enzymatic process has also been used tomimic the normal human digestion of proteins to evaluate thepossibility of releasing peptides. Several factors affect thebioactive properties of the peptides, including the enzymesused for hydrolysis, the processing conditions and the size ofthe resulting peptides, which greatly affects their absorptionacross the enterocytes and their bioavailability in target tissues.Some factors to consider in producing bioactive peptidesinclude hydrolysis time, degree of hydrolysis of the proteins,enzyme–substrate ratios and pre-treatment of the protein priorto hydrolysis [65,70,71].
There are two important obstacles in the industrial gener-ation of peptides by enzymatic hydrolysis. First, small peptidesstand in an extremely complex mixture together with aminoacids, oligopeptides and numerous other substances, such asphenolic compounds and fibres. Second, biologically activepeptides often present a particular physicochemical character-istic, such as the charge, which is essential to their activity. As aconsequence, the development of plant peptides requirespurification and fractionation [72]. In most cases, the enrich-ment is achieved by a tangential ultrafiltration step combinedto at least two low-pressure liquid chromatographic steps [73].
5.1.2. UltrafiltrationThe separation of small peptides from larger compounds suchasoligopeptides by membrane processes, especially ultrafiltration,is a well-known technique. On the other hand, the fractionationof small peptides is classically achieved by chromatographicmethods. These techniques are very efficient to fractionatesmall peptides according to their charge, size or hydrophobicproperties. However, the scale-up issue of a chromatographicmethod generates some high costs because of the use of organicsolvents and because of the low productivity of this technique.For this reason, several studies have focused on small peptidefractionationbynanofiltrationmembranes [72,74,75].Membraneultrafiltration and size-exclusion chromatography can be usedto concentrate peptides of defined molecular weight ranges,especially for obtaining fractions containing low molecularweight peptides that can withstand further in vivo proteolyticdigestion [65]. Chromatographic methods are very efficient tofractionate small peptides according to their charge, size, orhydrophobic properties. However, the scale-up issue of achromatographic method generates high costs because of theuse of organic solvents and because of the low efficacy of thistechnique. Thus, several studies have focused on the smallpeptide fractionation by nanofiltration membranes [72].
5.1.3. Chromatographic methodsProblems in the bioanalysis of peptides and proteins areencountered in the concentration stages and the separationsystems. The physicochemical diversity of peptides (charge,
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isoelectric point, hydrophobicity and size) makes them wellsuited to be separated by nearly every liquid-based separationmode. The first attempts to separate peptide mixtures via HPLCappeared in themid-1970s, approximately 10 years after the firstreports on HPLC [76]. The principle of chromatographic fraction-ation is based on the interaction of the proteins or peptides withthe solid support (stationary phase) and the mobile phase. Theinteractionmay be adsorption on silica surfaces, partitioning onreversed-phasematerials or ion exchange based on the effectivecharge of the proteins and peptides. Fractionation is achieved byusing mobile phase gradients whereby proteins or peptides aredifferentially eluted by changing the organic modifier concen-tration with time (RP chromatography) or the salt content withtime (hydrophobic interaction chromatography and ion ex-change, IEX) or by mobile-phase pH gradient (IEX chromatogra-phy) [66,77].
5.1.3.1. Reversed-phase (RP-HPLC). Reversed-phase (RP-HPLC)has been used in a vast number of studies and is a reliable andreproducible method of separating peptides and proteins. Due totheir hydrophobic character, proteins bind differently to thereversed-phase material of the column. The great benefit of thisapproach is that, in principle, every protein of a complexmixtureis accessible for enrichment, in contrast to other methods thatisolate certain proteins due to their affinity to a matrix. Thismethod is, therefore, ideal for a global protein analysis and, dueto its reproducibility, a robust and easily applicable method[63,65,78]. Thanks to column miniaturisation efforts, whichstarted early in the development of HPLC, and the introductionof soft ionisation techniques, such as matrix-assisted laserdesorption ionisation (MALDI) and electrospray ionisation (ESI),RPLC in combination with mass spectrometry (MS) evolved intothe principal analytical technique in the field of proteomics andpeptide analysis [76].
5.1.3.2. Affinity chromatography. Selective separation of aspecific protein or group of proteins can be achieved usingaffinity HPLC. The principle of affinity is based on the ability ofa biologically active molecule to bind specifically and revers-ibly to a complimentary molecule, often bound to a solidsupport. These ligand molecules may include antibodies,metals, lectins, biotin, aptamers, etc. The binding sites of theimmobilised substances must be sterically accessible aftertheir coupling to the solid support and should not bedeformed by immobilisation [66]. The main disadvantage ofthe affinity techniques is the need for a known ligand; inworking with a complex mixture of unknown peptides, thepossible applications are limited.
5.1.3.3. Isoelectric focusing. The principle of isoelectricfocusing (IEF) is very simple to understand and perform. Theprotein sample is mixed with the desired pH range carrierampholyte mixture or other carrier buffers in a focusing cell. Ifan electric potential is applied to the focusing cell, the proteinswill migrate to a position in the established pH gradientequivalent to their respective isoelectric point (pI). If a proteindiffuses away from this pH region, its net charge will change,and the resulting electrophoretic forces will influence itsmigration back to its pI. The net result is the ‘focusing’ ofproteins into narrow bands at their pI values [66].
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An advantage of liquid-phase IEF is the ability to fractionate acomplex mixture of proteins according to their pI in a non-gelmedium. The fractions can be collected and further analysed, ifneeded, by electrophoresis or chromatography. The disadvan-tages of IEF are that high concentrations of ‘neutral’ proteins (e.g.,when focused at their pI) often precipitate from solution (causingoverlapping between factions). Additionally, the ampholytesused to establish the pH gradient may interfere with subsequentanalysis using techniques such as electrospray ionisation massspectrometry (ESI-MS). In addition, highly hydrophobic proteinsmay be lost in sample preparation or during focusing when theproteins reach their isoelectric pI [66,79].
5.1.3.4. Ion exchange. Ion-exchange chromatography is anestablished technique used in the separation of chargedmolecules across a breadth of applications and industries.Chemically, ion exchange involves the exchange of solutes oflike charge from a solid support bearing the opposite charge(adsorbent). Ion exchange is a widely used technique inbioseparation since peptides, proteins, nucleic acids and relatedbiopolymershave ionisable chemicalmoieties that render themsusceptible to charge enhancement or reversion as a function ofpH [80]. Onedisadvantageof this technique is the lowselectivityand the need for further processing of the fractions obtained.
5.1.3.5. Centrifugal partition chromatography. Support-freeliquid–liquid chromatographic techniques, in its hydrostatic(centrifugal partition chromatography or CPC) and its hydrody-namic (counter-current chromatography or CCC) versions areboth based on the use of biphasic solvent systems. They haveemerged as interesting alternative tools for the purification ofbiomolecules such as peptides. Different development modesdefining differentways to implement such processes have beenproposed [73].
5.1.3.6. Capillary electrophoresis. Capillary electrophoresis(CE) has become a powerful separation tool and iswidely used inthe analysis of biomolecules, such as peptides, proteins, andpolynucleotides, due to its high separation efficiency, highresolution and fast speed. However, the poor detection limit ofCE caused by the short optical path length across the capillaryand small injection volume is still a serious problem. Therefore,dedicated sample preparation schemes to enrich the targetcomponents before separation are usually necessary for realsample analysis. However, the commonly used procedures, suchas solvent–solvent extraction and solid-phase extraction, areoften laborious and time-consuming. In addition, a number ofsensitive detectors, such as electrochemical detectors, fluores-cence detectors and mass spectrometry (MS), have also beensuccessfully developed. Nevertheless, they are sophisticated,expensive, selective and difficult to automate compared withabsorption detection. On-line concentration of sample is analternative in CE to improve the concentration detection limits.Up to now, two distinctly different methods for on-columnsample enrichment have been developed, namely electropho-retic stacking and chromatographic concentration [66,80,81].
To identify bioactive peptides from food proteins, studieshave been carried out to fractionate and purify the activepeptides. Despite these previous investigations, the relation-ship between the structure of the isolated peptides and their
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specific activity has not been fully elucidated. Most of theattention has been focused on ACE inhibitor peptides evenwhen the most relevant peptides from food sources are theBowman–Birk inhibitors and Lunasin, both of which areinvolved in the prevention and treatment of cancer. A singleprotein may include in their sequence a variety of peptidicregions, which once isolated can exert biological activity bybinding to cellular components directly related to replicationand cell survival. A protein enzymatic approach is preferredover chemical methods for obtaining peptides from cereal,where extraction by enzymatic hydrolysis yielded substantialamounts of protein when preparing protein isolates; however,a combination of several parameters for a substrate contrib-ute to different types of biological activities [8,36].
While the sample loading capacity of CE is often men-tioned as a serious drawback, it can be significantly improvedby on-line pre-concentration techniques. In addition, thislower sample capacity turns into an advantage when dealingwith limited sample quantities. The electrospray interfacingis clearly the key component required for the successfuldeployment of CE/MS in practice [82].
5.2. Characterisation of peptides
Complex mixtures of peptides are analysed by HPLC. Peptidebonds absorb UV light in a range of 210–220 nm, while aromaticamino acids absorb it between 250 and 290 nm, which enablesthe use of a UV detector. Reversed-phase (RP) columns aremostfrequently used for peptide separation. However, for certainapplications, ion exchange (IEX), size-exclusion (SEC) or mixed-mode (HILIC/CEX) columns are possible options [83]. HPLCchromatograms allow the evaluation of the purity of certainpeptides and their isoforms, as well as the estimation of theirabundance. Colorimetric assays with UV-based determinationare used to measure the peptide concentration in fractions [84].For higher sensitivity, the peptides can be hydrolysed, withsubsequent quantification of the amino acids [85]. In any case,the compatibility of involved reagents, for example in thechromatography solvent, with thequantificationmethodneedsto be confirmed to avoid over- or underestimated values.
Peptides of unknown sequence can be sequenced by Edmandegradation [86]. This chemical procedure was alreadyautomatised in the 1960s and could determine sequences of upto 60 amino acids [87]. Initially, the individual amino acids weredetected by thin-layer chromatography. The derivatisation tophenylthiohydantoin (PTH) of amino acids, in combination withRP-HPLC-UV, lowered the detection limits to pico- or evenfemtomolar [88]. Introducing isotope labels andmass spectrom-etry further improved the detection to attomole levels [89].Indeed, mass spectrometry became one of the most versatilemethods for the characterisation of food-derived peptides [90].Soft ionisation techniques such as electrospray ionisation (ESI)and matrix assisted desorption/ionisation (MALDI) made theanalysis of intact peptides without causing their fragmentationpossible [91,92]. Isobaric peptides can be distinguished by ionmobility spectrometry (IMS) [93]. This technology is available insome high performance mass spectrometers. Further, in MS/MSinstruments, the peptides may be fragmented to analyse theiramino acid sequence, for example by collision-induced dissoci-ation (CID) [94].
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Discriminating between the isobaric fragments of leucine andisoleucine is a technical challenge in mass spectrometry-basedpeptide sequencing, but suitable protocols exist even for low-resolution devices [95]. The fragmentation of peptides alsoenables the assessment of post-translational modifications(PTMs), which are important for biological function [96]. Mostfrequently, CID is employed to reveal the nature and localisationof peptidemodifications [97]. However, the investigation of labilePTMs such as phosphorylation or glycosylation may requireadditional techniques such as electron transfer dissociation(ETD) [98]. Often, both CID and ETD analyses are performed forthe same sample because the information provided by thedifferent fragmentation spectra are complementary [99]. Never-theless, one must be aware of possible PTM artefacts that mayoriginate from measurement conditions or sample treatment,such as oxidation events [100]. Since a plethora of excellent freemass spectrometry software is available (see http://www.ms-utils.org), wewill present only two examples:mMass is a generalMS data analysis programme that can be used for the manualanalysis of MS and MS/MS spectra of linear or cyclic peptides.The programme assists in de-novo sequencing and supports thesearch for PTMs [101]. Automated high throughput de-novosequencing, needed for the analysis of complex biologicalmixtures with numerous peptides, can be performed withPepNovo+ and UniNovo [102,103]. MASSyPup, a Linux distribu-tion for the analysis of mass spectrometry data, contains acollectionof free programmes that canbeused for the evaluationof HPLC and MS(/MS) data of peptides [104].
The three-dimensional structure of crystallised peptides canbe investigated by X-ray diffraction experiments [105]. Detailedstructural studies of peptides in solution can be performed bynuclear magnetic resonance (NMR) spectrometry [106,107].Contrary to X-ray analysis, NMR provides information aboutthe dynamics of peptides, and their biological function can bestudied under physiological conditions.
6. Conclusions
Areviewof the literaturehas shown that food-basedproteins area relevant source of bioactive peptides. There is significantevidence that the enzymatic hydrolysis of food proteins is anefficient way to isolate those peptides. Ultrafiltration has beenwidely used to enrich and pre-concentrate the obtained extracts,and in some cases, fractions of a certain molecular weight canbe probed before proceeding to the further purification andidentification step. The principal methodology used in thepurification and identification of the peptides consists of acombination of two techniques, HPLC and mass spectrometry;their inherent versatility comes from their variants, each basedon different properties of the sample.
The techniques and methods reported for the isolation,characterisation and evaluation of the bioactivity of peptideswill provide a backbone from which to continue the search forthese biological compounds in a more systematic way toanalyse their mechanism or even find new sources for them.Likewise, adding new techniques or modifying the order inwhich these are used can create a search led by the bioactivityand matrix characteristics. Actual studies tend to understandthe mechanisms through which the peptides exert their
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bioactivity, besides structure–activity studies; a useful tool forthis is the growing number of databases due to the fastadvancement of proteomics.
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Conflict of interest statement
The authors haveno conflicts of interest, financial or otherwise.
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