Impact of microbial cultures on proteolysis and release of bioactive peptides in fermented milkClemencia Chaves-López,Annalisa Serio,Antonello Paparella*,Maria Martuscelli,Aldo Corsetti,Rosanna Tofalo,Giovanna SuzziFaculty of Bioscience and Technology for Food,Agriculture and Environment,University of Teramo,Via C.R.Lerici1,64023Mosciano Sant’Angelo,TE,Italya r t i c l e i n f oArticle history:Received10October2013 Received in revised form28February2014Accepted6March2014 Available online16March2014Keywords:Co-colture starterAngiotensin converting enzyme Bitter tasteFermented milkKumis a b s t r a c tThis study aimed at evaluating co-cultures of selected microorganisms for their proteolytic activity andcapability to produce fermented milk enriched with ACE-inhibitory(ACEI)peptides.Selected yeasts(Torulaspora delbruekii KL66A,Galactomyces geotrichum KL20B,Pichia kudriavzevii KL84A and Kluyver-omyces marxianus KL26A)and lactic acid bacteria strains(Lactobacillus plantarum LAT03,Lb.plantarumKLAT01and the not virulent Enterococcus faecalis KE06)were screened as single cultures for their ca-pacity of releasing ACEI peptides without producing bitter taste.Three strains cultures(yeast,Lb.plan-tarum and E.faecalis)were performed to evaluate the combined impact on microbial growth,lactic acidproduction,citric acid consumption,proteolysis,ACEI activity,and bitter taste after36h of fermentationat28 C.While G.geotrichum KL20B showed a strong stimulating effect on Lb.plantarum strains and theproduction of peptides with ACEI activity,the presence of T.delbruekii KL26A in the cultures was dele-terious both to ACEI activity and product taste.The most effective combination was P.kudriavzevii KL84A,Lb.plantarum LAT3,E.faecalis KL06,which showed the highest ACEI activity(IC50¼30.63Æ1.11m g mlÀ1) and gave no bitter taste for7days at6 C.Our results highlight the importance of choosing the strainscombination carefully,to obtain a high yield of ACEI activity without bitter taste.Ó2014Elsevier Ltd.All rights reserved.1.IntroductionKumis is a natural fermented cow milk,widely consumed in rural and urban areas in south west Colombia.The fermentation is carried out by lactic acid bacteria(LAB)and yeasts at room tem-perature for about two days(Chaves-López et al.,2012).Spontaneous milk fermentation involves a number of metabolic pathways,in which metabolites contribute to confer chemical, biochemical and nutritional attributes to fermented milk.In particular,proteolysis leads to the production of bioactive peptides that have a positive impact on body conditions or functions,and may ultimately influence human health(Kitts and Weiler,2003). The proteolytic system of LAB is a very complex system that con-sists of three major components:a cell-wall bound proteinase that promotes extracellular casein degradation into oligopeptides,then peptide transporters move peptides into the cytoplasm,where finally various intracellular peptidases degrade peptides into smaller molecules and amino acids(Liu et al.,2010).On the other hand,yeasts species also possess both intracellular and extracellular proteases.In particular,extracellular enzymes are known for the genera Candida,Cryptococcus,Rhodotorula,Pichia and Yarrowia.Yeasts peptidases like aminopeptidases and car-boxypeptidases play an important role in the proteolysis of milk proteins(Ferreira and Viljoen,2003).Several anti-hypertensive peptides produced during milk fermentation have a strong activity against Angiotensin I-Con-verting Enzyme(ACE),a dipeptidyl carboxypeptidase that plays a major role in the regulation of blood pressure within the renin e angiotensin system(Riordan,2003),inducing blood pressure in-crease.In vivo studies evidenced a reduction of blood pressure after consumption of fermented milks(Pina and Roque,2008; Hernández-Ledesma et al.,2004).Moreover,in vitro ACE-inhibitory(ACEI)activity of different traditional fermented milks has been reported in the literature(Chaves-López et al.,2011;Sun et al.,2009).Thus,selection of microorganisms to be used in fer-mented products is gaining importance,due to the inherent vari-ations in their ability to produce bioactive peptides,particularly those with specific health claims(Ramchandran and Shan,2008). Interactions between different microorganisms in fermented milks may contribute to product quality,therefore,the positive or nega-tive effect of the interaction becomes very important(Viljoen,*Corresponding author.Tel.:þ39(0)861266913;fax:þ39(0)861266915.E-mail address:apaparella@unite.it(A.Paparella).Contents lists available at ScienceDirect Food Microbiologyjournal homepage:/locate/fm/10.1016/j.fm.2014.03.0050740-0020/Ó2014Elsevier Ltd.All rights reserved.Food Microbiology42(2014)117e1212001)in fermented milk production.In previous studies,we selected bacteria and yeasts strains able to produce high ACEI ac-tivity in vitro during milk fermentation (Chaves-López et al.,2011,2012);in this study we evaluate co-cultures of selected microor-ganisms for their proteolytic activity and capability to produce fermented milk enriched with ACEI peptides.We also determined product bitterness,which might hamper consumer acceptance of milks containing bioactive peptides as functional ingredients.2.Materials and methods 2.1.StrainsThe strains used in this study,belonging to the Culture Collec-tion of the Faculty of Bioscience and Technology for Food,Agri-culture and Environment of the University of Teramo,were isolated from Colombian Kumis and selected for their capability to produce fermented milk with high ACEI activity.Pure strains of not virulent Enterococcus faecalis KE06(Chaves-López et al.,2011)Lactobacillus plantarum KLAT1,L.plantarum LAT03,Torulaspora delbrueckii KL66A,Galactomyces geotrichum KL20B,Pichia kudriavzevii KL84A,and Kluyveromyces marxianus KL26A,were activated from their frozen forms (stored in 40%glycerol at À80 C)by transferring once in MRS broth (Lb.plantarum )or in M17broth (E.faecalis ),and YPD broth for yeasts.After activation,two successive transfers into the same medium were performed.During this procedure,all micro-organisms were incubated at 30 C.2.2.Preparation of inoculumInoculum of single cultures was obtained as follows:puri fied colonies were transferred into 1ml of MRS,M17or YPD media at 28 C for 24h.Cells were centrifuged at 5000rpm Â10min in a refrigerated centrifuge,the pellet was then suspended in 100ml of sterile reconstituted (10%)skimmed milk (Biolife,Milan,Italy),and incubated at 28 C for 18h for LAB and 48h for yeasts.Each pre-culture sample was used to inoculate (1%v/v)400ml pasteurized whole cow milk in single culture and in co-cultures (Table 1),to obtain approximately 7.0Log CFU ml À1of LAB and 6Log CFU ml À1of yeasts.Un-inoculated milk used as control and inoculated sam-ples were incubated at 28 C for 36h (period used to make tradi-tional fermented Colombian Kumis).Fermentation was stopped by heating at 75 C for 1min.Subsequently,samples inoculated with culture 5,selected for the best performance,were refrigerated at 6 C for 7days to evaluate the stability of ACEI activity and the taste.At the end of fermentation and during the refrigerated storage,samples were analysed to determine pH,microbial growth,lactic and citric acid,proteolysis,ACEI activity and sensory characteristics.2.3.Microbial countsBefore and after fermentation and refrigerated storage,aliquots of 1ml of fermented milks were diluted with sterile saline solution(0.85%NaCl),and 100m l of the samples were plated onto MRS agar for Lb.plantarum ,Kanamicine Azide Agar for E.faecalis and YPD agar with chloramphenicol (150ppm)for yeasts.All the samples were incubated at 30 C (24h for bacteria and 48for yeast).2.4.Determination of lactic and citric acidOrganic acids were quanti fied by high-performance liquid chromatography (HPLC)analysis (ThermoFinnigan Italia Spa,Rodano,Italy),as described by Sarantinopoulos et al.,2001,using a chromatographic system consisted of a Spectra System P4000pump and a Spectra System AS3000autosampler (ThermoFinnigan Italia Spa,Rodano,Italy).2.5.Assay for ACEI activity and peptides pro files and quanti fication The antihypertensive activity of peptides was determined based on ACEI activity and IC 50value.The IC 50value was de fined as the concentration of peptide (mg ml À1)required to reduce 50%of absorbance peak height of the hippuric acid,which was determined by regression analysis of ACE inhibition (%)versus protein concentration.The measurement of ACEI activity was carried out spectropho-tometrically using the pH 4.6soluble fraction of milk obtained by centrifugation (10000g Â10min at 4 C),as previously reported (Pan et al.,2005).The determination of IC 50,proteins,peptides and amino acids content as well as the determination of the stability and formation of new ACEI peptides after hydrolysis with pepsin and pancreatin was performed as reported by Chaves-López et al.(2011;2012).2.6.Sensory analysisSamples for sensory analysis were tasted at room temperature and analysis was performed according to Chaves-López et al.(2012).2.7.Statistic analysisAll the experiments were carried out in triplicate,and the an-alyses in duplicate.Experimental data were subjected to analysis of variance (ANOVA),and pair-comparison of treatment means was achieved applying Tukey ’s procedure at p <0.05,using Statistica for Windows.3.Results3.1.Co-cultures growth and milk acidi ficationIn co-culture trials (Table 2),yeasts growth was lower than in single culture:the lactose fermenting K.marxianus KL26A (mix 7and 8)increased only by about 0.5Log CFU ml À1,P.kudriavzevii KL84A did not grow in mix 5,whereas T.delbrueckii KL66A and G.geotrichum KL20B decreased in all the mixes.E.faecalis KE06counts were similar in all the mixes with an increase of approxi-mately 1.5Log CFU ml À1and with slightly signi ficant differences with respect to single cultures.On the contrary,both Lb.plantarum strains grew signi ficantly (p <0.05)in association with E.faecalis KE06and G.geotrichum KL20B (Mixes 3and 4)compared to single cultures,with an increase of about 1and 1.5Log CFU ml À1.Growth of the co-cultures was associated to accumulation of organic acids,leading to medium acidi fication from pH 6.6to 4.1after 36h of fermentation.Acidi fication was highly correlated with lactic acid production,which was higher in co-cultures 3and 4with G.geotrichum KL20B (from 11.4to 13.1mg ml À1)probably due to anTable 1Microorganisms used as starters to produce fermented milks.Co-culture YeastsEnterococcus faecalis Lactobacillus plantarum 1T.delbrueckii KL66A KE06LAT32T.delbrueckii KL66A KE06KLAT13G.geotrichum KL20B KE06LAT34G.geotrichum KL20B KE06KLAT15P.kudriavzevii KL84A KE06LAT36P.kudriavzevii KL84A KE06KLAT17K.marxianus KL26A KE06LAT38K.marxianus KL26AKE06KLAT1C.Chaves-López et al./Food Microbiology 42(2014)117e 121118increase of Lb.plantarum counts,and in co-cultures7and8(from 13to14mg mlÀ1)probably because K.marxianus KL26A is a lactose fermenting yeast(Fig.1).Moreover,the minor accumulation of citrate was registered in presence of the association of LAT3with Torulaspora delbruekii KL66A and P.kudriavzevii KL84A.3.2.Proteolysis of fermented milksAs reported in Table3,T.delbrueckii KL66A and P.kudriavzevii KL84A were the most proteolytic yeasts in single culture.The as-sociation with LAB determined an increase of peptides content that could not be attributed to any particular strain.In detail,the activity enhancement was higher in mixes1e2with T.delbrueckii KL66A, and5-6with P.kudriavzevii KL84A.3.3.ACEI activity in fermented milks after36hFermentation with E.faecalis KE06,G.geotrichum KL20B and K.marxianus KL26A in single cultures,produced milks with ACEI activity higher than70%(Table3).In spite of the highest peptides content,the interaction of Tor-ulaspora debruekii KL66A,E.faecalis KE06and both strains of Lb. plantarum did not result in any detectable ACEI activity.Moreover, the co-culture with P.kudriavzevii KL84A or K.marxianus KL26A and Lb.plantarum strains,showed a very low ACEI activity particularly in presence of KLAT1.On the other hand,the co-culture with E.faecalis KE06,Lb.plantarum LAT03and G.geotrichum KL20B, showed the highest ACEI activity(70.6%).Fermented milk samples obtained with co-cultures number3,4 and5,showing significant ACEI activity(70%,49%and33% respectively)after simulated physiological digestion increased their activity to75%,53%and40%respectively.According to IC50values after digestion,the most powerful peptides were present in co-cultures5,with values of30.63Æ1.11m g mlÀ1followed by co-cultures3and4(48.12Æ0.73and54.25Æ1.0m g mlÀ1respectively).3.4.Impact of the co-cultures on product bitterness after36hAs shown in Table3,different co-cultures produced varying levels of bitterness in thefinal ks fermented with T.delbrueckii KL66A in co-cultures had similar bitterness,which was equivalent to1.4mM of caffeine.G.geotrichum KL20B showed bitterness values close to0.7mM of caffeine in single culture but yielded bitter products in co-cultures,although with different in-tensity:in presence of Lb.plantarum KLAT01intensity was higher (between0.7and1.4mM of caffeine)than in samples inoculated with Lb.plantarum LAT03,whose bitterness was close to zero.Co-cultures7and8with K.marxianus KL26A,not producing bitter-ness in single culture,showed bitterness of about0.7mM of caffeine.Conversely,in milks produced by P.kudriavzevii KL84A co-Table2Inoculum size andfinal counts for yeasts and lactic acid bacteria in single culture and co-culture at the start and the end of fermentation(36h).Mix Yeasts(log CFU mlÀ1)Enterococcus faecalis(log CFU mlÀ1)Lactobacillus plantarum(log CFU mlÀ1)Inoculum Single culture Co-culture Inoculum Single culture Co-culture Inoculum Single culture Co-culture1 6.5Æ0.08a 6.3Æ0.14b 5.7Æ0.3c7.0Æ0.05a8.3Æ0.10b8.4Æ0.1b7.0Æ0.05a8.6Æ0.1b8.6Æ0.2b2 6.5Æ0.08a 6.3Æ0.14b 6.0Æ0.1c7.0Æ0.05a8.3Æ0.10b8.5Æ0.1c7.2Æ0.1a8.4Æ0.1b8.5Æ0.1b3 6.1Æ0.10a 6.2Æ0.15a 5.8Æ0.2b7.0Æ0.05a8.3Æ0.10b8.5Æ0.1c7.0Æ0.05a8.6Æ0.1b9.5Æ0.1c4 6.1Æ0.10a 6.2Æ0.15a 5.5Æ0.3b7.0Æ0.05a8.3Æ0.10b8.4Æ0.1b7.2Æ0.1a8.4Æ0.1b8.9Æ0.1c5 6.3Æ0.16a 6.5Æ0.18a 6.2Æ0.1a7.0Æ0.05a8.3Æ0.10b8.5Æ0.1c7.0Æ0.05a8.6Æ0.1b8.4Æ0.06b6 6.3Æ0.16a 6.5Æ0.18a 6.8Æ0.2b7.0Æ0.05a8.3Æ0.10b8.6Æ0.3c7.2Æ0.1a8.8Æ0.1b8.2Æ0.04b7 6.3Æ0.07a 6.9Æ0.14b 6.9Æ0.1b7.0Æ0.05a8.3Æ0.10b8.3Æ0.1c7.0Æ0.05a8.6Æ0.1b8.3Æ0.1b8 6.3Æ0.07a 6.9Æ0.14b 6.9Æ0.1b7.0Æ0.05a8.3Æ0.10b8.5Æ0.05c7.2Æ0.1a8.4Æ0.1b8.6Æ0.05bMean log CFU mlÀ1within each block and the same row with different superscript letters are significantly different(p<0.05).ctic and citric acids accumulation and standard deviation of the samples during milk fermentation of the different co-cultures incubated at28 C for36h.Black bars:lactic acid;grey bars:citric acid.Table3ACEI activity(%),peptide content and specific bitterness intensity detected in fer-mented milks inoculated with co-cultures of LAB and yeasts after36h of fermen-tation at28 C.ACEIactivity(%)Peptidescontentmeasuredas OPAindex(mg mlÀ1)Specific bitternessintensity*Single culturesE.faecalis KE0670.3Æ5.2a0.8Æ0.2a 1.3Æ0.5aG.geotrichum KL20B81.2Æ7.7b 1.2Æ0.1b 2.1Æ0.4bK.marxianus KL26A73.3Æ6.4a 1.3Æ0.2b 1.6Æ0.5cT.delbrueckii KL66A57.9Æ3.7c 1.5Æ0.1c 1.0Æ0.0dP.kudriavzevii KL84A50.1Æ4.9c 1.4Æ0.1d 1.0Æ0.0dLb.plantarum KLAT165.2Æ4.4ac 1.7Æ0.1d 1.0Æ0.0dLb.plantarum LAT350.4Æ5.3c 1.5Æ0.1c 1.0Æ0.0dCo-cultures1n.d. 2.0Æ0.4a 3.3Æ0.5a2n.d. 2.0Æ0.1a 3.0Æ0.0a370.6Æ7.4a 1.5Æ0.2a 1.1Æ0.4b449.5Æ5.0b 1.4Æ0.2c 2.5Æ0.5c533.3Æ3.7c 2.0Æ0.2a 1.0Æ0.0b6 5.1Æ1.0d 1.9Æ0.3a 1.0Æ0.0b722.1Æ4.3e 1.5Æ0.2c 1.8Æ0.4cb8 3.8Æ3.4d 1.6Æ0.4c 2.5Æ0.5cbMeans with a superscript in common are not statistically different at a level of p<0.05over all comparisons.n.d.¼not detectable.ACEI activity¼[(BÀA)/(BÀC)]Â100%where A is the absorbance in presence of ACE and ACEI component,B is the absorbance with ACE and without ACEI component and C is the absorbance without ACE and ACEI component.Peptide content was measured using o-phthaldialdehyde(OPA)method as previ-ously described by Church et al.(1983).*(1)null;(2)light(0.7mM caffeine);(3)medium(1.4mM caffeine);(4)strong (2.8mM caffeine)and(5)very strong(4.6mM caffeine).C.Chaves-López et al./Food Microbiology42(2014)117e121119cultures,no bitterness was detected by panellists and this charac-teristic was maintained up to 4days of storage at 6 C;however ACEI activity increased up to 75%,accompanied with a decrease of 2Log CFU ml À1in P.kudriavzevii KL84A cell counts (Table 4).No signi ficant changes in IC 50and taste (data not shown)were observed during storage time.4.DiscussionStarter cultures with desirable properties are of particular in-terest to get reproducible and improved quality of fermented milks,where milk provides the nutrients for the development of speci fic active microbial populations,and the interactions within micro-biota have pronounced effects on product characteristics.In fact,individual metabolic activities result in a de fined outcome that can in fluence the overall metabolism;in particular,interactions be-tween yeasts and LAB are deemed important for the quality of natural fermented milks (Narvhus and Gadaga,2003).Although growth of LAB is believed to be promoted in co-culture with yeasts,in this study we observed that this phenomenon could be a species-speci fic characteristic.In fact,among four different yeast species studied,only the cultures with G.geotrichum increased Lb.planta-rum cells number.In this type of cultures,the yeast count was signi ficantly lower than in singles cultures.The main reason for this result could be attributed to yeast autolysis;in this way,yeasts can provide several compounds that contribute to bacterial growth,such as vitamins,amino acids,and growth factors.The speci ficity of the yeast species in enhancing LAB growth has been also evidenced in fermented milks by Gadaga et al.(2001)and Liu and Tsao (2009).The signi ficant differences in proteolysis in the various cultures here studied can be attributed to possible synergistic effects among the three species (E.faecalis,Lb.plantarum and yeast species).In particular,T.delbrueckii KL66A and P.kudriavzevii KL84A seemed to contribute to a higher accumulation of peptides in the co-cultures where they were present,whereas the presence of G.geotrichum KL20B and K.marxianus KL26A did not seem to exert any in fluence.Very few yeasts species are strongly proteolytic and some have caseinolytic activity;in particular the last above-mentioned species were not strongly proteolytic in single culture,in agreement with Roostita and Fleet (1996).However,interactions between yeasts and LAB,e.g.co-operation,had some impact on proteolysis in both co-cultures with T.delbrueckii KL66A and P.kudriavzevii KL84A.This result was in line with other studies of Hamme et al.(2009).The production of bioactive peptides with multifunctional ac-tivities,in situ ,is an appealing approach,since this confers an additional positive health effect to milk products,which already possess a healthy image and have a long history of safe production (Philanto et al.,2010).In this study,the impact of the mixed starter on the accumulation of peptides with ACEI activity was evidenced;among eight co-cultures,only the co-culture number 3promoted a high ACEI activity.These results could be explained by a lowproduction of ACEI peptides or by a decrease of ACEI activity in co-cultures,due to a reduction of the metabolic activity or to changes in metabolic pathways.The absence of ACEI activity in milks fer-mented with T.delbrueckii KL66A and the strong differences be-tween other co-cultures suggest that yeast strain and Lb.plantarum were determinant for the accumulation of peptides with this bioactivity.There are few reports regarding the ability of LAB and yeasts co-cultures to produce fermented cow milks with ACEI activity (Nakamura et al.,1995;Takano,1995;Hamme et al.,2009;Didelot et al.,2006;Vermeirssen et al.,2003),however they did not study the relationship between ACE activity and bitter taste.In our study,we observed a varying impact of the different starters on product taste,with bitterness ranging from zero to 1.4mM of caffeine.In particular,G.geotrichum KL20B and K.marxianus KL26A,in co-cultures with Lb.plantarum KLAT1,had a negative impact on milk taste.Although G.geotrichum is known to produce aminopepti-dases and carboxypeptidases and to reduce cheese bitterness by the breakdown of bitter peptides (Wyder and Puhan,1999;Molimard et al.,1994),it did not show the same effect in fer-mented milks,probably due to the short time of milk fermentation.5.ConclusionsIn this study,we showed that mixed cultures of LAB and yeasts have a great impact on the ACEI activity of fermented milks.In fact,the interactions between the different strains here studied under-line the importance of carefully choosing the strains combination,to obtain a high yield of ACEI activity without bitter taste.After 36h of fermentation at 28 C in milk,no changes of ACEI activity and taste have been observed during refrigerated storage of fermented milk for 7days at 6 C.Our results prove that our formulation number 5has good properties to be used 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