cardiovascular disease ( cvd ) affects the heart and blood vessels and is the principal cause of death worldwide .
considered the primary risk factor for cvd , high blood pressure , or hypertension , consists of a sustained increase in blood pressure levels . in 2000 , > 25% of the population worldwide ( approximately 1 billion ) suffered from hypertension , a figure predicted to increase to 1.56 billion by 2025 .
angiotensin i - converting enzyme ( peptidyl carboxy peptidase , ec 3.4.15.1 , ace ) belongs to the class of zinc proteases that require zinc and chloride for activation .
ace plays an important role in blood pressure regulation via the renin - angiotensin system ( ras ) and the kallikrein - kinnin system ( kks ) . in the kks
, ace inactivates the vasodilator bradykinin , while in the ras , ace acts as an exopeptidase cleaving his - leu from the c - terminal of decapeptide angiotensin i and producing the potent vasoconstrictor octapeptide angiotensin ii .
use of enzyme technologies for protein recovery and modification has led to production of a broad spectrum of food ingredients and industrial products .
hydrolysis selectivity is commonly manipulated by employing proteases from different sources due to their specificity for peptide bonds adjacent to certain amino acids .
ace - i inhibitory peptides derived from food proteins have attracted particular attention for their ability to prevent hypertension . compared with chemosynthetic drugs , peptides from food proteins
they therefore hold promise as potent functional food additives and represent a healthier and more natural alternative to ace inhibitor drugs .
dietary ace - i inhibitory peptides may be bioavailable . some ace - i inhibitory peptides resist digestion , can be absorbed in the intestine , and are stable in the blood , suggesting they may produce an acute blood pressure - lowering effect after oral administration .
the first ace - i inhibitory peptide was isolated from snake venom , and since then many others have been discovered in enzymatic hydrolysates of different food proteins .
food protein sources used to date include casein , whey protein , fish protein , chicken eggs , and wheat germ .
. the degree of hydrolysis and inhibitory activities of angiotensin i - converting enzyme ( ace ) increased with increasing proteolysis time .
population growth and shrinking food resources are ongoing challenges in developing countries , while excessive animal protein intake and associated unhealthy levels of saturated fats exposure are increasingly common in developed countries . in response ,
research interest has steadily grown in the search for new sources of proteins from nonconventional raw materials .
the genus salvia l. belongs to the lamiaceae family and includes about 900 species found worldwide , most mainly in the mediterranean , southeast africa , and central and south america .
cultivated for culinary , medicinal , and ornamental uses , salvia species form part of ethnopharmacological traditions and are an important crop , especially for small farmers .
recent research has addressed the chemical composition , biological properties , and possible applications of its essential oils , which may be sources for economically promising natural products with uses in the food , pharmaceutical , and cosmetic industries .
the present study objective was to identify and quantify ace - i inhibitory activity in protein hydrolysates from a salvia hispanica protein rich fraction hydrolyzed with an alcalase - flavourzyme sequential system , and in ultrafiltered fractions from this hydrolysate .
chia ( s. hispanica , l. ) seeds were obtained in yucatan state , mexico .
reagents were of analytical grade and purchased from j. t. baker ( phillipsburg , nj , usa ) , sigma ( sigma chemical co. , st .
louis , mo , usa ) , merck ( darmstadt , germany ) , and bio - rad ( bio - rad laboratories inc .
angiotensin - converting enzyme from rabbit lung ( 2  units / mg protein ) was purchased by sigma ( a6778 sigma chemical co. , st .
the alcalase 2.4  l and flavourzyme 500  mg enzymes were purchased from novo laboratories ( copenhagen , denmark ) .  
flour was produced from 6  kg chia seed by first removing all impurities and damaged seeds , crushing the remaining sound seeds ( moulinex dpa139 , zapopan , mexico ) , and milling them ( krups 203 mill , mexico city , mexico ) .
oil extraction from the milled seeds was done with hexane in a soxhlet system for 2  h. the remaining fraction was milled with 0.5  mm screen ( thomas model 4 wiley , swedesboro , nj , usa ) . the defatted chia flour was dried in a labline stove at 60c for 24  h. extraction of the protein - rich fraction was done by dry fractionation of the defatted flour according to vzquez - ovando et al . .
briefly , 500  g flour was sifted for 20  min using a tyler 100  mesh ( 140  m screen ) and a ro - tap agitation system .  
the chia protein - rich fraction was hydrolyzed in batches by sequential treatment with alcalase and flavourzyme . a predigestion with alcalase for 60  min
hydrolysis conditions were substrate concentration , 2  g/100  g ; enzyme / substrate ratio , 0.3  au  g for alcalase and 50  lapu  g for flavourzyme ; ph , 7 for alcalase and 8 for flavourzyme and temperature , 50c .
hydrolysis was done in a reaction vessel equipped with a stirrer , thermometer , and ph electrode . in all treatments ,
the reaction was stopped by heating to 85c for 15  min , followed by centrifuging at 9880  g for 20  min to remove the insoluble portion .
degree of hydrolysis ( dh ) was calculated by determining free amino groups with o - phthaldialdehyde following the methodology described by nielsen et al .   as follows : dh = h / htot100 , where htot is the total number of peptide bonds per protein equivalent , and h is the number of hydrolyzed bonds .
the htot factor is dependent on the raw material amino acid composition and was determined by reverse - phase high performance liquid chromatography ( rp - hplc ) . samples ( 24  mg protein ) were treated with 4  ml of 6  mol equivalent to l  hcl , placed in hydrolysis tubes , and gassed with nitrogen at 110c for 24  h. they were then dried in a rotavapor and suspended in 1  mol  l sodium borate buffer at ph 9.0 .
amino acids were separated using hplc with a reversed - phase column ( 300  3.9  mm , nova - pak c18 , 4  mm ; waters ) and a binary gradient system with 25  mmol  l sodium acetate containing ( a ) 0.02  g  l sodium azide at ph 6.0 and ( b ) acetonitrile as solvent .
flow rate was 0.9  ml  min , and elution gradient was time 0.03.0  min , linear gradient a  :  b ( 91  :  9 ) to a  b ( 86  :  14 ) ; time 3.013.0  min , elution with a  b ( 8614 ) ; time 13.030.0  min , linear gradient a  b ( 86  :  14 ) to a 
b ( 69  :  31 ) ; time 30.035.0  min , elution with a  b ( 69  :  31 ) . the hydrolysate was fractionated by ultrafiltration , using a high performance ultrafiltration cell ( model 2000 , millipore ) .
five fractions were prepared using four molecular weight cut - off ( mwco ) membranes : 1  kda , 3  kda , 5  kda , and 10  kda .
soluble fractions prepared by centrifugation ( 9880  g for 20  min ) were passed through the membrane starting with the largest mwco membrane cartridge ( 10  kda ) .
retentate and permeate were collected separately , and the retentate recirculated into the feed until maximum permeate yield was reached at this size , as indicated by a decrease in permeate flow rate .
permeate from the 10  kda membrane was then filtered through the 5  kda membrane with recirculation until maximum permeate yield was reached .
the 5  kda permeate was then recirculated through the 3  kda membrane and the 3  kda permeate through the 1  kda membrane .
this process minimized contamination of the larger molecular weight fractions with smaller molecular weight fractions , while producing enough retentates and permeates for the following analyses .
the five ultrafiltered peptide fractions ( upf ) were prepared and designated as > 10  kda ( 10  kda retentate ) ; 510  kda ( 10  kda permeate5  kda retentate ) ; 35  kda ( 5  kda permeate3  kda retentate ) ; 13  kda ( 3  kda permeate1  kda retentate ) ; < 1  kda ( 1  kda permeate ) .  
ace - i inhibitory activity in the hydrolysate and its upf was analyzed following the method of hayakari et al .   which is based on the fact that ace - i hydrolyzes hippuryl - l - histidyl - l - leucine ( hhl ) yielding hippuric acid and l - histidyl - l - leucine .
this method relies on the colorimetric reaction of hippuric acid with 2,4,6-trichloro - s - triazine ( tt ) in a 0.5  ml incubation mixture containing 40  mol potassium phosphate buffer ( ph 8.3 ) , 300  mol sodium chloride , 40  mol 3% hhl in potassium phosphate buffer ( ph 8.3 ) , and 100  mu / ml ace - i .
this mixture was incubated at 37c/45  min and the reaction terminated by addition of tt ( 3% v / v ) in dioxane and 3  ml 0.2  m potassium phosphate buffer ( ph 8.3 ) .
after centrifuging the reaction mixture at 10,000  g for 10  min , enzymatic activity was determined in the supernatant by measuring absorbance at 382  nm . all runs were done in triplicate . ace - i inhibitory activity was quantified by a regression analysis of ace - i inhibitory activity ( % ) versus peptide concentration , and ic50 values ( i.e. , the peptide concentration in g protein / ml required to produce 50% ace - i inhibition under the described conditions ) was defined and calculated as follows : 
 ( 1)ace - i inhibitory activity  ( % ) = ( ab)(ac)100 , 
 where a represents absorbance in the presence of ace - i sample , b absorbance of the control , and c absorbance of the reaction blank . consider the following : 
 ( 2)ic50=(50b)m , 
 where b is the intersection and m is the slope .  
amino acid composition was determined in the upf with the highest biological activity , according to the method of alaiz et al .  
samples ( 24  mg protein ) were treated with 4  ml of 6  mol equivalent l  hcl , placed in hydrolysis tubes , and gassed with nitrogen at 110c for 24  h. they were then dried in a rotavapor and suspended in 1  mol  l sodium borate buffer at ph 9.0 .
amino acids were separated using hplc with a reversed - phase column ( 300  3.9  mm , nova - pak c18 , 4  mm ; waters ) and a binary gradient system with 25  mmol  l sodium acetate containing ( a ) 0.02  g  l sodium azide at ph 6.0 and ( b ) acetonitrile as solvent .
flow rate was 0.9  ml  min , and the elution gradient was time 0.03.0  min , linear gradient a  :  b ( 91  :  9 ) to a 
b ( 86  :  14 ) ; time 3.013.0  min , elution with a  b ( 8614 ) ; time 13.030.0  min , linear gradient a  b ( 86  :  14 ) to a 
b ( 69  :  31 ) ; time 30.035.0  min , elution with a  b ( 69  :  31 ) .
after filtration through 10 , 5 , 3 , and 1  kda membranes in a high performance ultrafiltration cell , 10  ml of the fraction with highest ace - i inhibitory activity was injected into a sephadex g-50 gel filtration column ( 3  cm  79  cm ) at a flow rate of 25  ml / h of 50  mm ammonium bicarbonate ( ph 9.1 ) .
peptide molecular masses were determined by referring to a calibration curve running molecular mass markers on the sephadex g-50 under identical conditions and those used for the test samples .
molecular mass standards were thyroglobulin ( 670  kda ) , bovine gamma globulin ( 158  kda ) , equine myoglobin ( 17  kda ) , vitamin b12 ( 1.35  kda ) , and thr - gln ( 0.25  kda ) .
fractions selected for further peptide purification were pooled and lyophilized before rp - hplc .  
one - way anovas were run to evaluate in vitro ace - i inhibitory activity and a duncan multiple range done to identify differences between treatments .
all analyses were done according to montgomery   and processed using the statgraphics plus version 5.1 software .
with a protein content of 46.7% , the chia protein - rich fraction proved to be good starter material for hydrolysis .
production of extensive ( i.e. , > 50% dh ) hydrolysates requires use of more than one protease because a single enzyme can not achieve such high dhs within a reasonable time period . for this reason ,
an alcalase - flavourzyme sequential system was used in the present study to produce an extensive hydrolysate .
alcalase ( ec 3.4.21.62 ) is a proteinase from bacillus licheniformis and the flavourzyme ( ec 3.4.11.1 ) is a fungal protease from aspergillus oryzae with both endo- and exopeptidase activities .
the bacterial endoprotease alcalase is limited by its specificity , resulting in dhs no higher than 20%25% , depending on substrate , but can attain these dhs in a relatively short time under moderate conditions .
when alcalase is used to hydrolyze protein it tends to produce peptides whose c - terminals are amino acids with large side chains and no charge ( aromatic and aliphatic amino acids ) , such as ile , leu , val , met , phe , tyr , and trp .
the fungal protease flavourzyme has broader specificity , which , when combined with its exopeptidase activity , can generate dh values as high as 50% .
flavourzyme is recommended for production of hydrolysates or peptides with biological activity and low bitterness .
both alcalase and flavourzyme tend to generate peptides with hydrophobic amino acid c - terminals , and qsar analyses of ace - i inhibitory peptides have shown that peptides with hydrophobic amino acid c - terminals exhibit potentially strong ace - i inhibition . therefore , alcalase and flavourzyme are probably suitable for preparing high - activity ace - i inhibiting peptides .
in addition , both proteases are suitable for industrial applications and are microbial enzymes , meaning that they are easily obtained and relatively low cost compared to other enzymes such as proteinase k and chymotrypsin c .
chia hydrolysates obtained with the alcalase - flavourzyme sequential system had clear biological activity and are promising prospects for use in new product development .
the highest dh in the present study ( 51.6% ) was attained with flavourzyme at 150  min .
however , it was made possible by alcalase predigestion , which increases the number of n - terminal sites , thus facilitating hydrolysis by flavourzyme .
this dh was higher than reported for hard - to - cook bean hydrolyzed with alcalase - flavourzyme ( 43% ) or pepsin - pancreatin ( 26.2% ) for 90  min .
but it was lower than reported for chickpea ( 65% ) hydrolyzed with alcalase - flavourzyme at 150  min   and for rapeseed ( 60% ) hydrolyzed with the same system at 3  h .
when hydrolyzed sequentially with alcalase and flavourzyme , chia s. hispanica is an appropriate substrate for producing extensive hydrolysates ( dh higher than 10% ) , and a natural source of peptides with potential bioactivity .
a number of natural ace - i inhibitors have been isolated from different organism proteins , including peptides extracted by enzymatic hydrolysis . when added to food systems ,
enzymatic hydrolysates have exhibited advantages such as improved water - binding capacity , emulsifying stability , protein solubility , and nutritional quality .
hydrophobicity of amino - acid side chains is normally due to relatively small peptides with molecular weights between 1,000 and 6,000  da .
enzymatic hydrolysis is an effective way of producing bioactive peptides , which are short peptides released from food proteins by hydrolysis and with biological activities that may be beneficial to the organism .
bioactive peptides usually contain 320 amino acids per molecule and are inactive within the parent protein molecule sequence .  
the protein hydrolysate obtained from the chia protein - rich fraction had 58.5% ace - i inhibition .
this is lower than the 79.5% reported for yak milk casein hydrolyzed with alcalase for 240  min , but higher than the 5%50% obtained for protein hydrolysates from amaranth ( amaranthus hypochondriacus ) albumin 1 and globulin .
ace - i inhibition ranged from 53.8% to 69.3% ( figure 1 ) , with clear increases ( p < 0.05 ) in activity in progressively smaller fractions ; that is , the > 10  kda fraction had the lowest activity and the < 1  kda had the highest .
this coincides with a study in which the < 1  kda fraction of an alaska pollock ( theragra chalcogramma ) frame protein hydrolysate had the highest ace - inhibitory activity ( 87.6% , ic50 = 457  g / ml ) . in another study , protein hydrolysate from chinese soft - shelled turtle had a lower ace - i inhibitory effect ( ic50 = 280  g / ml ) than its corresponding < 5000  kda ultrafiltered fraction ( ic50 = 190  5  g / ml ) .
a greater inhibitory effect at smaller molecular weights was also observed in a yak casein hydrolysate fraction , in which the < 6  kda fraction was the most effective ( 85.4% ) .
when taken in conjunction , these results support the suggestion that ultrafiltration is an effective way of enriching ace - i inhibitory peptides from chia proteins .
an amino acid profile was generated for the < 1  kda upf because it had the highest ace - i inhibitory activity . during hydrolysis ,
asparagine and glutamine partially converted to aspartic acid and glutamic acid , respectively ; the data for asparagine and/or aspartic acid were therefore reported as asx while those for glutamine and/or glutamic acid were reported as glx . the high inhibitory activity ( 69.3% ) exhibited by the <
1  kda upf was probably due to its high concentration of hydrophobic amino acids ( 41.68  g/100  g ) , including pro ( 6.11  g/100  g ) , phe ( 11.03  g/100  g ) , leu ( 10.23  g/100  g ) , and ile ( 6.57  g/100  g ) ( table 1 ) . amino acid c - terminal hydrophobicity has the greatest influence on ace - i inhibitory activity , and the higher the hydrophobicity the higher the inhibitory activity .
found that dipeptides could have high ace - i inhibitory activity if c - terminals were aromatic amino acids and proline , and n - terminals were aliphatic amino acid branches .
measured cheung 's peptides samples in the same laboratory , modeled the results with a qsar , and found that dipeptides with positively - charged amino acids at the n - terminal and bulky hydrophobic amino acids at the c - terminal had stronger ace - i inhibitory activity .
using z descriptors to investigate the quantitative structure - activity relationship of ace - inhibitory dipeptides , wu et al .   found that ace - inhibitory activity was strongly affected by the three - dimensional chemical properties and hydrophobicity of c - terminal amino acids ; that is , the higher the volume and the greater the hydrophobicity of the amino acids , the higher the ace - i inhibitory activity .
therefore , some dipeptides with hydrophobic amino acids at the c - terminal , such as phenylalanine , tryptophan , and tyrosine , will have high ace - i inhibitory activity .
wu et al .   also reported that strongly hydrophobic and small n - terminal amino acids , such as valine , leucine , and isoleucine , were more suitable for high - activity tripeptides .
low charge , large size , and weak hydrophobicity in the second amino acid from the n - terminal were more suitable high activity .
finally , for the c - terminal , high charge , large volume , and strongly hydrophobic residues ( e.g. , aromatic amino acids ) were more suitable . in an analysis of ace - i inhibitory
.   found that for peptides with 6 amino acids at the c - terminal , ace - i inhibition was strongly affected by hydrophobicity , positive charge , and volume of amino acids adjacent to the c - terminal , whereas the n - terminal amino acid had no direct relationship .
hydrophobicity and c - terminal amino acid size are apparently the principal aspects affecting ace - inhibitory activity .
it stands to reason that hydrophobic amino acids , aromatic amino acids , and branched - chain amino acids are important components in high - activity peptides , and that proteins with high contents of these amino acid types ( especially aromatic amino acids ) have more potential to produce high activity ace - i inhibitory peptides . therefore , digestion of proteins to produce peptides with hydrophobic amino acids at the c - terminal will tend to increase ace - i inhibitory activity in any derived hydrolysates .
although the structure - activity relationship of ace - i inhibitory peptides has not yet been established , these peptides show some common features . studies of the structure - activity relationships in different ace - i inhibitory peptides indicate that binding to ace is strongly influenced by substrate c - terminal tripeptide sequence .
ondetti and cushman   proposed a binding model for interactions between the substrate and active ace site .
c - terminal tripeptide residues may interact with the s1 , s1 , and s2 subsites at the active ace site .
ace appears to prefer substrates or competitive inhibitors that contain hydrophobic amino acid residues at the three c - terminal positions .
captopril owes its potency and selectivity to chemical design guided by a hypothetical active site model based on the observed properties of ace and on an analogy to the known active site of a related zinc - containing peptidase .
ace 's zinc ion is appropriately located between s1 and s1 , allowing it to participate in hydrolytic cleavage of the substrate peptide bond and resulting in release of a dipeptide product .
studies of the structure - activity relationship in ace - i inhibitory peptides have shown that those with potent inhibitory activity have proline , phenylalanine , or tyrosine at the c - terminal , as well as hydrophobic amino acids in their sequence . in light of this previous research ,
the ace - i inhibitory activity observed here in the chia hydrolysate was probably due to amino acid composition ( figure 2 ) .
of the upfs , the < 1  kda fraction exhibited the highest ace - i inhibitory activity and was selected for further fractionation .
a molecular weight profile was generated of this upf using gel filtration chromatography ( sephadex g-50 column ) .
this profile was typical of a protein hydrolysate formed by a pool of peptides , with gradually decreasing molecular masses .
elution volumes between 406 and 518  ml included free amino acids and peptides with molecular masses ranging from 0.4 to 3.6  kda .
this range was fractionated into three fractions and ace - i inhibitory activity determined for each .
fractions with elution volumes smaller than 406  ml and greater than 518  ml were not analyzed because they largely included peptides with high molecular weights , as well as free amino acids .
ace - i inhibitory activity ( % ) in the < 1  kda upf ranged from 48.4% to 62.6% ( figure 3 ) .
the highest ace - i inhibitory activity was observed in fraction f1 ( 62.6% ; 427455  ml elution volume ) . its molecular mass was approximately 1.52.5  kda , indicative of 712 amino acid residues . the ic50 value for f1 ( 3.97  g / ml ) was lower than those of gel filtration ( sephadex g-25 ) peptide fractions from tuna broth hydrolysate ( 210 to 25,260  g / ml )   or from buckwheat fagopyrum esculentum moench ( sephadex c-25 = 25,715.1  g / ml ; sephadex g-10 = 21,315.1  g / ml ) .
other studies suggest that ace inhibition by hydrolysates depends on the source species and hydrolysate purity level .
for instance , production of a 13  kda protein hydrolysate from alaska pollock frame using an ultrafiltration membrane bioreactor system resulted in high ace - i inhibition ( ic50 = 110  g / ml ) , but further purification using consecutive chromatographic methods in a sp - sephadex c-25 column and hplc in an octadecylsilane column resulted in a still stronger effect ( ic50 = 14.7  g / ml ) . in another example , an enzymatic hydrolysate from cuttlefish ( sepia officinalis ) muscle protein was found to have high ace - i inhibitory activity ( 87.1  0.9% at 200  g / ml ) . however , size exclusion chromatography with a sephadex g-25 produced a fraction ( p6 ) with yet higher ace - i inhibition ( ic50 = 11.6  mol / l ) , which , when fractionated by reverse - phase ( rp)-hplc , was found to contain ala - his - ser - tyr , gly - asp - ala - pro , ala - gly - ser - pro , and asp - phe - gly . in ace - i inhibitory peptides from chia , the protein - rich fractions are not as potent as hypertension treatment drugs but hold promise as a safe , natural therapeutic agent without adverse side effects .
the potential of chia protein - derived peptides as antihypertension agents depends on the ability of these peptides to reach their target site without suffering degradation and consequent inactivation by intestinal or plasma peptidases .
resistance to peptidase degradation is a probable prerequisite for any ace inhibitory hydrolysates / peptides to exercise an antihypertensive effect after oral or intravenous administration .
proline - containing peptides are generally resistant to degradation by digestive enzymes , and tripeptides containing a pro - pro c - terminal are resistant to proline - specific peptidases . however , peptide degradation or fragmentation results in smaller peptides and therefore in potentially more potent ace - i inhibitory activity . clearly , in vivo studies are needed to confirm the effect of these peptides since it is both difficult and unwise to extrapolate directly from in vitro to in vivo activity .
the main challenges in doing this are determining bioavailability of ace - i inhibitory peptides after oral administration and the fact that peptides may influence blood pressure by mechanisms other than ace - i inhibition . to exert an antihypertensive effect after oral ingestion
, ace - i inhibitory peptides must reach the cardiovascular system in an active form , meaning that they need to remain active during digestion by human proteases and transport through the intestinal wall into the blood .
bioavailability has been studied for some ace - i inhibitory peptides , and it is known that proline - containing peptides are generally resistant to degradation by digestive enzymes .
peptides can be absorbed intact through the intestine by paracellular and transcellular routes , although postabsorption bioactivity potency is inversely correlated to chain length .
all the chia derivatives studied here had ic50 values far higher than the 0.0013  g / ml of captopril , a synthetic ace - i inhibitor . nonetheless , the chia purified peptides have biological potential , and the f < 1  kda fraction had high ace - i inhibitory activity , suggesting that ace - i inhibitory peptides are rich in hydrophobic amino acids ( aromatic or branched chains ) and in proline .
ace - i inhibiting peptides from food sources have garnered increasing attention in recent years as promising natural biofunctional alternatives to synthetic drugs .
many of these peptides have been discovered in enzymatic hydrolysates of different food - source proteins and subsequently applied in the prevention of hypertension and initial treatment of mildly hypertensive individuals . the ongoing search for natural ace inhibitors may eventually help to create safer and less costly alternatives to synthetic pharmaceutical treatments .
chia proteins hydrolyzed with the alcalase - flavourzyme sequential enzyme system resulted in hydrolysates with ace - i inhibitory activity .
ultrafiltration produced a very low molecular weight fraction ( < 1  kda ) which had the highest activity .
enzymatic hydrolysis and ultrafiltration are promising bioprocesses for production of new bioactive food ingredients such as ace inhibitory peptides purified from chia hydrolysate .
 synthetic angiotensin i - converting enzyme ( ace - i ) inhibitors can have undesirable side effects , while natural inhibitors have no side effects and are potential nutraceuticals . a protein - rich fraction from chia ( 
 salvia hispanica l. ) seed was hydrolyzed with an alcalase - flavourzyme sequential system and the hydrolysate ultrafiltered through four molecular weight cut - off membranes ( 1  kda , 3  kda , 5  kda , and 10  kda ) . 
 ace - i inhibitory activity was quantified in the hydrolysate and ultrafiltered fractions . 
 the hydrolysate was extensive ( dh = 51.64% ) and had 58.46% ace - inhibitory activity . 
 inhibition ranged from 53.84% to 69.31% in the five ultrafiltered fractions and was highest in the < 1  kda fraction ( 69.31% ) . 
 this fraction 's amino acid composition was identified and then it was purified by gel filtration chromatography and ace - i inhibition measured in the purified fractions . 
 amino acid composition suggested that hydrophobic residues contributed substantially to chia peptide ace - i inhibitory strength , probably by blocking angiotensin ii production . 
 inhibitory activity ranged from 48.41% to 62.58% in the purified fractions , but fraction f1 ( 1.52.5  kda ) exhibited the highest inhibition ( ic50 = 3.97  g / ml ; 427455  ml elution volume ) . 
 the results point out the possibility of obtaining bioactive peptides from chia proteins by means of a controlled protein hydrolysis using alcalase - flavourzyme sequentional system . 