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International Dairy Journal 19 (2009) 155–165

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International Dairy Journal

journal homepage: www.elsevier .com/locate/ idairy j

Peptide profiles and angiotensin-I-converting enzyme inhibitory activityof fermented milk products: Effect of bacterial strain, fermentation pH,and storage time

Mette S. Nielsen a,1, Torben Martinussen b, Benedicte Flambard c, Kim I. Sørensen c, Jeanette Otte a,*

a Department of Food Science, Centre for Advanced Food Studies, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmarkb Department of Natural Sciences, Statistics, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmarkc Chr. Hansen A/S, Bøge Alle 10-12, DK-2970 Hørsholm, Denmark

a r t i c l e i n f o

Article history:Received 4 March 2008Received in revised form13 October 2008Accepted 13 October 2008

* Corresponding author. Tel.: þ45 3533 3189; fax: þE-mail address: jo@life.ku.dk (J. Otte).

1 Present address: Department of Molecular Biologsity of Aarhus, Denmark.

0958-6946/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.idairyj.2008.10.003

a b s t r a c t

Milk was fermented to defined pH values with 13 strains of lactic acid bacteria. The products wereevaluated after 1 and 7 days of cold storage, and major peptides in selected products were identified. TheStreptococcus thermophilus and Lactobacillus acidophilus strains used did not give rise to products withsignificant angiotensin-1-converting enzyme (ACE)-inhibition. The four Lactococcus lactis strains behavedsimilarly in fermentation, proteolysis and ACE-inhibition. The products made with the seven Lactobacillushelveticus strains varied. The highest ACE-inhibitory activity was obtained with two highly proteolyticstrains of Lb. helveticus and with the Lactococcus strains. Fermentation from pH 4.6 to 4.3 with thesestrains slightly increased the ACE-inhibitory activity, whilst fermentation to pH 3.5 with Lb. helveticusreduced the ACE-inhibitory activity. Cold storage dramatically increased the ACE-inhibitory activity ofsome products. A non-linear correlation was found between peptide amount and ACE-inhibitory activity,and peptides contributing to the ACE-inhibitory activity were identified.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Food products containing hypotensive peptides are of interestfor maintaining good health of humans with moderate hyperten-sion (FitzGerald et al., 2004). In principle, milk products withhypotensive peptides can be produced in two ways, i.e., byenrichment with antihypertensive peptides produced by enzymatichydrolysis of precursor proteins, or by fermentation of milk withlactic acid bacteria (LAB) (FitzGerald et al., 2004, Korhonen & Pih-lanto, 2006; Lopez-Fandino et al., 2006; Otte et al., 2007). Duringfermentation of milk, the cell wall associated proteinases of LABhydrolyse caseins into large peptides, which in turn are transportedinto the cell and broken down by intracellular peptidases, resultingin a range of peptides, among them peptides with hypotensive and/or angiotensin-I-converting enzyme (ACE)-inhibitory activity. SomeACE-inhibitory peptides are products of extracellular proteinasesalone (Yamamoto et al., 1994b), whereas others are the result ofaction of both proteinases and peptidases, e.g., the Tyr-Pro peptideisolated from a yoghurt-like product (Yamamoto et al., 1999).

45 3533 3344.

y, Faculty of Science, Univer-

All rights reserved.

LAB commonly used to ferment milk into yoghurt and otherfermented milk products are thermophilic and mesophilic strainsof Streptococcus, Lactococcus and Lactobacillus species (Walstra,2006). The cell wall proteinases of the Lactococcus strains are serineproteases that are grouped into at least two types; the PI-type thathydrolyses b-CN and has little activity for as1-CN, and the PIII-typethat hydrolyses both caseins (Pritchard & Coolbear, 1993). Lb. hel-veticus proteinases are also serine proteinases that can be groupedinto PI- and PIII-types (Kunji et al., 1996; Pritchard & Coolbear, 1993).The intracellular peptidases so far isolated from Lactococci andLactobacilli are either aminopeptidases or endopeptidases (Chris-tensen et al., 1999; Kunji et al., 1996). Peptidases like PepX involvedin the hydrolysis of proline-containing sequences are important forthe degradation of casein-derived oligopeptides because of theirhigh content of proline.

For the production of fermented milk with hypotensive and/orACE-inhibitory activity, various LAB species have been used,including Lb. helveticus, Lb. casei, Lb. plantarum, Lb. rhamnosus, Lb.acidophilus, Lc. lactis ssp. lactis, Lc. lactis ssp. cremoris and the twoorganisms used in traditional yoghurt production, Lb. delbrueckiissp. bulgaricus and Str. thermophilus (Fuglsang et al., 2003b; Gob-betti et al., 2000; Nakamura et al., 1995; Yamamoto et al., 1994a, b,1999), and recently also Enterococcus faecalis (Muguerza et al.,2006; Quiros et al., 2007). Lb. helveticus has been the preferred

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165156

fermenting organism in the pursuit of an effective ACE-inhibitorymilk product due to its generally higher proteolytic activitycompared with other LAB (Fuglsang et al., 2003b; Kilpi et al., 2007;Sasaki et al., 1995; Yamamoto et al., 1994a), and also due to thespecificity of the proteolytic enzymes resulting in more activepeptides (Nakamura et al., 1995; Seppo et al., 2003; Yamamotoet al., 1999). Milk fermented with one particular strain of Lb. hel-veticus has shown beneficial effects on the heart in rodents inaddition to ACE-inhibitory activity (Flambard, 2004). However, theChr. Hansen collection contains other interesting LAB known to beproteolytic and to produce ACE-inhibitory peptides upon growth inmilk (Flambard, 2003).

Yoghurt is normally fermented at 42 �C until a pH of 4.6 or loweris reached (Donkor et al., 2007a, b; Robinson et al., 2002). Theproducts with ACE-inhibitory peptides have been made byfermentation for a set period of time, e.g., 6, 12, 24 or 48 h, at theoptimum temperature for the species used, reaching pH valuesbetween 4 and 5 in most products (Muguerza et al., 2006; Nakamuraet al., 1995; Seppo et al., 2003; Yamamoto et al., 1994a). However,some of the bacteria may ferment the milk further to a lower pH.Some ACE-inhibitory peptides may be intermediate products ofhydrolysis which upon further fermentation would be degraded intoinactive peptides. Other ACE-inhibitory peptides may be end prod-ucts of hydrolysis, e.g., many di- and tri-peptides, which would beformed upon longer fermentation and/or subsequent cold storage ofthe product. The effect of fermentation pH on the ACE-inhibitoryactivity of such products has received little attention. Nakamuraet al. (1995) found that the ACE-inhibitory activity of the fermentedmilk (Calpis) increased with decreasing pH until pH 3.5 was reached.Also, very little is known about the effect of cold storage on the finalpH, amount of peptides and ACE-inhibitory activity of the products.Donkor et al. (2007a) observed a slight post-acidification in yoghurtduring storage at 4 �C for 28 days. Concomitantly, proteolysiscontinued and the ACE-inhibitory activity of the yoghurt increased.In contrast, the ACE-inhibitory activity of yoghurt containing pro-biotic bacteria, (Lb. acidophilus, Lb. casei and Bifidobacterium lactis)decreased during cold storage due to further break down of ACE-inhibitory peptides formed during fermentation.

The purpose of the present study was: (i) to test selectedpromising LAB strains from the collections at Chr. Hansen and the

Table 1Source and growth conditions of the Lactobacillus, Lactococcus and Streptococcus species anmid-exponential phase under these conditions.

Species and strain Origin

Lc. lactisssp. lactis CHCC 3906 CHa

ssp. lactis CHCC 3923 CHssp. cremoris F3 KUb – E.W. Nielsen - from ‘‘Filmjolk’’ssp. cremoris W5 KU – E.W. Nielsen - from ‘‘natursyrevækker’’

Lb. helveticusMI 1198 (CH3)c KU – F.K. Vogensen – from SMc

MI 1262 (CIP5715)c KU – F.K. Vogensen from S. LortalMI 1264 (IL430)c KU – F.K. Vogensen from S. LortalMI 1263 (NCDO262)c KU – F.K. Vogensen from S. LortalMI 1169 (CNRZ32)c KU – F.K. Vogensen from J. SteeleCHCC 637 CHCHCC 4080 CHS5 KU – E.W. Nielsen from icelandic ’’skyr’’

Lb. acidophilusCHCC 3777 CH

Str. thermophilusS2 KU – E.W. Nielsen from icelandic ‘‘skyr’’

a CH, The strain collection at Chr. Hansen A/S, Denmark.b KU, The strain collection at Department of Food Science, University of Copenhagen,c The strain numbers of the source collection are given in brackets, NCDO, National Coll

de la Recherche Zootechnique collection, Jouy-en-Josas, France; SM, The National Dairy Rd M17, medium developed by Terzaghi and Sandine (1975); MRS, De Man, Rogosa and

Department of Food Science, which include strains isolated fromNordic dairy products, for their ability to ferment milk andhydrolyse the major milk proteins into peptides with ACE-inhibi-tory activity, (ii) to investigate the effect of pH at which thefermentation was stopped and (iii) the effect of storage at 5 �C onACE-inhibition and peptide profiles. The resulting fermented milksamples were also characterised with respect to number of viablecells and post-acidification.

2. Materials and methods

2.1. Bacterial strains and growth conditions

The LAB strains and growth conditions used in the experimentsare presented in Table 1. All stock cultures were kept at �80 �C inskim milk containing 10% (w/v) glycerol. Lb. strains were streakedon de Man, Rogosa and Sharpe (MRS) medium (Merck, Darmstadt,Germany) and grown under anaerobic conditions overnight. Lac-tococcus and Streptococcus strains were streaked on M17 medium(Oxoid, Hampshire, England) supplemented with 1% (w/v) lactoseand grown overnight under aerobic and anaerobic conditions,respectively. Lactobacillus colonies were inoculated in MRS brothand Lactococcus and Streptococcus colonies in M17 broth supple-mented with 1% lactose and grown to the mid-exponential phase(OD600¼ 0.5–0.8, depending on the strain) at which point theywere harvested and used to ferment milk (Table 1).

2.2. Fermentation of milk and storage

Flasks containing 200 mL of homogenized and pasteurized full-fat milk (Arla Express sødmælk, 3.5%, Arla Foods amba, Slagelse,Denmark) were heated at 95 �C for 30 min and then cooled to 5 �Cand kept overnight. Next day, the pasteurized milk was pre-heatedand inoculated with the LAB culture (1%, v/v), and fermentation wasperformed at the optimal growth temperature (Table 1). For eachstrain, seven flasks were prepared simultaneously, one was used forcontinuous pH-measurement during fermentation by means ofa pH glass electrode (pHC2401-8; Radiometer, Copenhagen, Den-mark) coupled to a data logger (ADC-100; Pico Technology Limited,St. Neots, United Kingdom), and the others were allowed to ferment

d strains used to make overnight cultures, as well as the time (h) needed to reach the

Growth conditions Time to mid exp. phase (h)

30 �CAerobic, M17d 7.5

7.51215

37 �CAnaerobic, MRSd 12

151520212222.515

37 �CAnaerobic, MRS 14

42 �CAnaerobic, M17 12

Denmark.ection of Dairy Organisms, Institute of Food Research Reading; CNRZ, Centre National

esearch Institute previously existing in Hillerød, Denmark.Sharp medium.

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165 157

to approximate pH values of 4.6, 4.3 and 3.5, respectively. When thedesired pH value was reached, the fermented milk product wascooled to room temperature, then vigorously stirred for 1 min (120rpm; Laboratory mixer, Cole-Parmer Instrument Company, VernonHills, Illinois, USA) and divided into 25 mL cups that were stored at5 �C for 1 or 7 days. Pre-heated milk, chemically acidified with 1 M

HCl to pH 4.6, served as a control.

2.3. Viable cell counts and post-acidification

Serial dilutions of the fermented milk samples made in salinewater (0.9%, w/v, NaCl) were spread onto agar plates and incubatedfor 48 h at the relevant growth temperature (Table 1). Lactobacilliwere spread on MRS agar, and Lactococci and Streptococci on M17agar supplemented with 1% (w/v) lactose. Anaerobic conditionswere used for Lactococci and Streptococci. All dilutions were platedin triplicate. Enumeration was performed by manual counting,whenever possible the mean numbers from two different dilutionswere used, and results were expressed as colony forming units permillilitre of fermented milk (cfu mL�1).

The pH of the fermented milk products was measured eachweek during a 4-week storage period at 5 �C.

2.4. Preparation of whey from fermented milk

The fermented milk samples were centrifuged at 5000� g for10 min at 4 �C (Allegra 25R Centrifuge; Beckman Coulter Inc., Full-erton, CA, USA). The pH of supernatants (whey) was adjusted to 7.5with 1 M NaOH, and the resulting whey samples were stored at�20 �C in 2 mL-aliquots. Prior to use, the whey samples were thawedat 5 �C, centrifuged at 11,148 � g for 3 min at room temperature(Sigma centrifuge 113, VWR International Aps, Albertslund, Den-mark), and filtered through a 0.45 mm disposable hydrophilic filter.

2.5. ACE-inhibition assay

The ACE-inhibitory activity of the whey samples was measuredaccording to the method described by Shalaby et al. (2006), withsome modifications. The substrate solution was 0.88 mM N-[3-(2-Furyl) acryloyl]-L-phenylalanyl-glycyl-glycine (Sigma Chemical Co.,St. Louis, MO, USA) in 75 mM Tris-HCl buffer containing 0.3 M NaCl,pH 7.5. Angiotensin-I-converting enzyme (ACE; EC 3.4.15.1) fromrabbit lung (Sigma) was prepared immediately before use by adding8 mL of water to a vial containing 2 units (U) of enzyme(0.25 U mL�1) and thorough mixing. The decrease in absorbance at340 nm was recorded every 30 s for 25 min at 37 �C, and the slope(r) over a linear interval of 15 min was taken as a measure of the ACEactivity (rAwhey). The reaction was carried out in 5 replicates for eachwhey sample. Control samples (n¼ 6) giving the initial ACE activitywere prepared with buffer (75 mM Tris-HCl, pH 7.5, containing 0.3 M

NaCl) instead of whey (rAcontrol). In order to avoid any interferencefrom changes in absorbance at 340 nm of the whey or the substrateper se, whey blank samples (n¼ 3) were prepared with waterinstead of ACE solution (rAwhey blank), and substrate blank samples(n¼ 6) were prepared as the control sample, but replacing the ACEsolution with water (rAsubstr. blank). The ACE-inhibition (%) of thewhey samples was calculated according to the formula:

%ACE inhibition ¼h1�

�rAwhey� rAWhey blank

�=ðrAcontrol

� rAsubstr: blankÞi�100% :

Captopril (Fluka Chemie Gmbh, Deisenhofen, Germany) dissolvedat 5 mM in 75 mM Tris-HCL buffer, pH 7.5, containing 0.3 M NaCl,served as a positive inhibitor.

2.6. Analysis of peptide profiles by reversed phase-HPLC

Whey samples (50 mL) were analysed by reversed phase (RP)-HPLC (high-performance liquid chromatography) using a WatersHPLC system consisting of a 700 WISP controller, a 717plus Auto-sampler, a 600E System controller and a 2487 Dual l AbsorbanceDetector operated by Millennium32 Chromatography managerSoftware version 3.2 (all from Waters Corporation, Milford,Massachusetts, USA). Peptides were separated at 30 �C on a Nucle-osil 300-5 C18 column (4.6� 250 mm, 5 mm; Macherey-Nagel,Duren, Germany) using a linear gradient of acetonitrile in 0.1% (v/v)trifluoroacetic acid from 0 to 48% between 10 and 90 min. The flowrate was 1 mL min�1 and detection was at 210 and 280 nm. Theextent of proteolysis in the samples was estimated by the amountof peptides present by integration of the 210 nm peak areas in thepeptide profiles. Total peptide areas as well as peptide areas withinthe retention time (Rt) intervals 10.0–24.0 min, 24.1–38.0 min,38.1–78.0 min, and 78.1–95.0 min were determined.

2.7. Identification of peptides by liquid chromatography-tandemmass spectrometry

Peptides in selected whey samples were identified by liquidchromatography-tandem mass spectrometry (LC-MS/MS) using anAgilent 1100 LC-MSD ion trap system as described by Otte et al.(2007).

2.8. Statistical analyses

The influence of LAB species and strain, fermentation pH, andstorage time on the ACE inhibition of the fermented milk sampleswas tested by two statistical models, both derived from the MixedProcedure in SAS (version 9.1; SAS institute Inc., Cary, NC, USA). Inmodel A, the strain entered as a systematic factor, whereas itentered as a random factor in model B. In both models ACE-datawere transformed according to the formula:

Y ¼ log�h

1��

rAwhey � rAWhey blank

�=ðrAcontrol

� rAsubstr: blankÞiþ 1�:

Since variations in the initial ACE activity (rAcontrol� rAsubstr.

blank) in individual microtiter plates were not significant, this factorwas left out in the final models. The influence of LAB species andstrain, fermentation pH, and storage time on viable cell counts inthe fermented milk samples was tested by a similar model wherestrain entered as a random factor and cfu data were log-trans-formed. The peptide area-profile data were analysed using a linearmixed model with random strain effect and systematic effects ofspecies, pH, and storage time. The four Rt intervals were alsoincluded as a systematic effect. The linear correlation between ACE-inhibition (%) and peptide areas in RP-HPLC chromatograms wastested by the Spearman’s rank test using the statistical packagefrom SPSS for Windows (Release 11.0; SPSS Inc., Chicago, Illinois,USA), and the relationship between ACE-inhibition and totalpeptide area was fitted using the non-linear four-parameter logisticmodel.

3. Results and discussion

3.1. Fermentation of milk by the various LAB species and strains

All LAB strains used were able to grow in the pasteurized milkunder the conditions applied. The fermentation with each strainwas reproducible, except for strain S5 of Lb. helveticus, which wasomitted from further studies. As expected, the acid production

A

3906

3923

W5

S2

3777

1263

1262

1198

4080

1169

1264

637

F3

AC

E in

hib

itio

n (%

)

0

10

20

30

40

50

60

B

Lactic acid bacteria species and strain

AC

E in

hib

itio

n (%

)

0

10

20

30

40

50

LL LB LA ST

Fig. 1. ACE-inhibitory activity of milk samples fermented with strains of Lc. lactis (LL),Lb. helveticus (LB), Lb. acidophilus (LA) and Str. thermophilus (ST), and stored for 1 day(A) or 7 days (B). Milk samples were fermented to pH 4.6 (white), pH 4.3 (grey) or pH3.5 (black). Data are means� SEM.

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165158

stopped around pH 4.4–4.2 during Lactococcus and Streptococcusfermentations, whereas the more acid-tolerant Lactobacillusspecies continued acid production to a pH below 4 with the Lb.helveticus species giving the lowest final pH (3.6–3.4). The Lc. lactisstrains gave the fastest acidification to pH 4.6 (8–13 h), the twolactis subspecies (ssp.) being slightly faster than the two cremorisssp. The Lb. acidophilus strain 3777 gave a very irregular acidifica-tion and long fermentation time (24 h). The Lb. helveticus strainsgave similar acidification curves, all reaching pH 4.6 within 10–15 h. However, two Lb. helveticus strains, 1263 and 1198, weremarkedly slower (26 and 27 h, respectively); after a lag time andinitial fermentation to pH 6.4–6.2, fermentation was halted for 5–10 h before the rapid final fermentation to pH <4. This halt inacidification most probably reflects the time it takes for thesestrains to synthesize a proteolytic system suitable for growth inmilk.

3.2. Characteristics of products fermented to pH 4.6

The average number of viable cells in the products fermentedwith Lc. lactis and Lb. helveticus was w109 cfu mL�1, with theexception of the product made with Lb. helveticus 4080,which contained 2�107 cfu mL�1. The products made with Str.thermophilus and Lb. acidophilus attained populations around108 cfu mL�1.

In all products fermented to pH 4.6, there was a drop in pH(mean¼ 0.15 units) during the first day of refrigerated storage at5 �C, except in that fermented with Str. thermophilus S2. During thesubsequent 27 days of refrigerated storage, post-acidificationoccurred in the products fermented with Lb. helveticus strains(except strain 1169) resulting in a mean decrease of 0.3 units for theLb. helveticus products, revealing metabolic activity in this speciesat 5 �C. Post-acidification also occurred in the product fermentedwith strain F3 of Lc. lactis ssp. cremoris, which was stopped at pH 4.8and reached a pH of 4.4 at day 28.

The ACE-inhibitory activity values of the milk samples fer-mented to pH 4.6 seemed moderate, ranging from 1 to 35% (Fig. 1A,white bars). However, this should be compared with the value of31% for the Danish commercial fermented milk product, A38, whichhas previously been shown to have an ACE-inhibitory activity at thesame level as the antihypertensive Japanese Calpis fermented milk(Otte et al., 2003). The ACE-inhibitory activity of the whey fromchemically acidified milk was only 1.3%, highlighting the impor-tance of proteolysis during fermentation for the production of ACE-inhibitory peptides. The ACE-inhibitory activity of the productsfermented to pH 4.6 with Str. thermophilus was significantly lowerthan that of the products made with Lc. lactis and Lb. helveticus(p< 0.01), otherwise there was no significant difference betweenthe four LAB species with respect to ACE-inhibitory activity. Thehighest activity, between 25 and 33%, was obtained with Lc. lactis,notably the cremoris ssp. (Fig. 1A). It was quite surprising that Lc.lactis, which is generally not considered highly proteolytic, hydro-lysed the milk proteins into peptides with higher bioactivity thanmost of the Lb. helveticus strains. The products fermented by thevarious Lb. helveticus strains varied much with respect to ACE-inhibitory activity (from 11 to 34%). Only four out of the seven Lb.helveticus strains used gave rise to fermented milk samples withACE-inhibitory activities around 25% or higher (Fig. 1A), amongthem strains 1198 and 1263, which had the longest fermentationtimes.

The peptide profiles showed characteristic differences accordingto the LAB species used (not shown). The profiles of the productsmade with the same ssp. of Lc. lactis were identical, but minordifferences were noted between ssp., e.g., a higher concentration ofthe late-eluting peptides in the cremoris ssp. (compare Fig. 2A andC). The peptide profiles of the Lactobacillus products had

similarities to the profiles of the Lactococcus products, except forthe presence of several distinct peaks after 80 min (Fig. 3A–F), butvaried much with the strain used. The strains 1198 and 1263 wereremarkable in that they gave rise to profiles with four marked peaksbetween 80 and 90 min in addition to an enormous amount ofother peaks, seen by the noisy baseline in the profile, confirmingthat substantial hydrolysis had taken place (Figs. 3D and 4A).

The total amount of peptides in the products fermented to pH4.6 as determined from the peptide profiles is given in Table 2. Theproduct made with Str. thermophilus contained mainly one peptideand had a very low peptide area, which is in accordance with thelow ACE-inhibitory activity of this product and the generallylimited proteolytic activity of Str. thermophilus despite many typesof proteolytic enzymes (Hassan & Frank 2001; Robinson et al.,2002). Although Lc. lactis is not generally considered to be veryproteolytic (Hassan & Frank, 2001; Ruas-Madiedo et al., 2005), thefour Lc. strains used in the present study seemed to be somewhatproteolytic during the initial fermentation and produced peptideswith high ACE-inhibitory activity. Accordingly, Lc. lactis strain 3923has been characterised as a ‘‘proteolytic’’ strain of Lc. lactis byFlambard (2003) as opposed to other strains of Lc. lactis that werecharacterised as ‘‘lytic’’. Since the peptide profiles of the Lc. strainsused in this study were similar, the other three strains of Lc. lactisused in the present study must also be more proteolytic thannormal for this species. The two strains of of Lc. lactis ssp. cremoris,

C

Ab

so

rb

an

ce

a

t

21

0 n

m

0.0

0.1

0.2

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E

Retention time (min)

10 20 30 40 50 60 70 80 900.0

0.1

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D

F

Retention time (min)

10 20 30 40 50 60 70 80 90

1 2

5

3 4

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89

1011

1213

1415

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1920

17

A

0.0

0.1

0.2

0.3

0.4

0.5 B

1 25

3

467 8

91011

12 1415

19

20

186a

Fig. 2. Peptide profiles of milk samples fermented with Lc. lactis ssp. cremoris strain F3 (A and B) and ssp. lactis strain 3923 (C–F) to pH 4.6 (A–D) and pH 4.3 (E and F), and stored for1 day (A, C and E) and 7 days (B, D and F). Peaks are numbered consecutively, and major peptides have been identified as presented in Table 3.

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165 159

which gave the highest ACE-inhibition, had a slightly higherpeptide area than the two strains of the lactis ssp. The productfermented with Lb. acidophilus had a peptide amount similar tothose made with Lc. lactis ssp. lactis and most Lb. helveticus strains(Table 2), even though the ACE-inhibitory activity was low (Fig. 1A).Lb. helveticus is generally considered as highly proteolytic, anda high number of peptides were expected in products fermentedwith the strains of Lb. helveticus. Although strains 637 and 4080have been characterised as ‘‘proteolytic’’ by Flambard (2003), thesestrains and most other Lb. helveticus strains used in the presentstudy were not very proteolytic under the conditions applied. Onlythe two Lb. helveticus strains that showed irregular acidificationcurves and high ACE-inhibitory activity (1198 and 1263) gave a veryhigh amount of peptides, higher than the four Lactococcus strains(Table 2), consistent with an up-regulation of their proteolyticsystem during fermentation (Section 3.1).

3.3. Effect of fermentation to lower pH values

The number of viable cells in the products fermented by the fourLactococcus strains and the eight Lactobacillus strains was notsignificantly affected by fermentation to pH w4.3 Str. thermophiluswas more acid sensitive and the cell number significantlydecreased to 3�107 (p¼ 0.004) upon fermentation to pH 4.4,which is in accordance with observations of others (Beal et al.,1999). Upon fermentation to the end pH, the cell number of Lb.acidophilus and Lb. helveticus strains 1169 and 4080 slightlydecreased, whereas no major effect was seen on the cell number ofthe other Lb. helveticus strains.

The products fermented by Lactococcus and Streptococcus strainsto pH 4.1–4.4 did not show post-acidification. A slight post-acidi-fication was noted for the Lb. acidophilus product, from 4.3 to 4.2during 21–28 days of storage at 5 �C. In contrast, the Lb. helveticusproducts, which had not reached their final pH, had the same rate ofpost-acidification as the products stopped at pH 4.6, most reachingthe final pH around 4 after 7 days of storage. Prolonged storage (28days) of the product fermented with 1264 decreased pH to w3.5,whereas the pH of the milk fermented with strain 1169 did notchange during refrigerated storage. Continued fermentation of theLb. helveticus products to the end pH of w3.5, as expected, did notresult in further acidification during storage, except for the productfermented with 1264 (stopped at pH 3.6) which reached pH 3.3after 28 days of storage at 5 �C.

When all LAB species and strains were taken together, the pH atwhich the fermentation was stopped did not significantly influencethe ACE-inhibitory activity of the products. However, characteristictrends were noted for each LAB species (Fig. 1A). Fermentation topH 4.3 with Lc. lactis significantly increased the ACE-inhibitoryactivity for 3 out of the 4 strains used (Fig. 1A), mostly with the ssp.lactis. The product made with strain 3906 reached 40% inhibition,the highest among the products evaluated after 1 day of storage. Forall Lb. helveticus products, the ACE-inhibition was not significantlydifferent in pH 4.6 and pH 4.3 products, which was due to differ-ences between strains (Fig. 1A). However, fermentation to pH 3.5with the Lb. helveticus strains caused a significant decrease in ACE-inhibitory activity (p< 0.0001).

The total amount of peptides in the products was significantlyaffected by fermentation pH, and significant interactions between

K

G

H

L

Retention time (min)

10 20 30 40 50 60 70 80 90

I

J

E

B

F

Retention time (min)

10 20 30 40 50 60 70 80 90Ab

so

rb

an

ce at 210 n

m

0.0

0.1

0.2

0.3

C

D

A

Fig. 3. Peptide profiles of milk samples fermented with various Lb. helveticus strains to pH 4.6 and stored for 1 day (A–F) and 7 days (G–L). The strains used were 637 (A, G), 1169 (B,H), 1262 (C, I), 1263 (D, J), 1264 (E, K), and 4080 (F, L).

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165160

pH and species were also noted (p< 0.0001). The products fer-mented with Lc. lactis and Str. thermophilus strains had a slightlyincreased total peptide area after fermentation to pH w4.3 (Table2), in accordance with the slight increase in ACE-inhibitory activityof most of these products (Fig. 1A). For the products fermented withLactobacillus species, there was no consistent effect of pH on thetotal amount of peptides (Table 2). With the Lb. helveticus strains1198 and 1263, a decrease in total peptide area was found (Table 2),despite the slight increase in ACE-inhibition (Fig. 1A). The peptideprofiles of the products fermented to pH 4.3 with the Lc. lactisstrains of both ssp. had less of the late-eluting, but not well resolvedpeaks (80–95 min), and more of the peaks with intermediate Rts(Fig. 2E). This probably reflects degradation of large casein frag-ments into shorter peptides and shows that proteases and/orpeptidases were active during continued fermentation. A similartrend was seen for the products fermented with Str. thermophilus(results not shown). The peptide profiles of the products made withmost Lb. helveticus strains did not significantly change from pH 4.6to pH 4.3, but more so between pH 4.3 and the end pH around 3.5(e.g., Fig. 4A, C and E).

3.4. Effect of cold storage for 6 days

The effect of storage at 5 �C varied with the bacterial speciesused and the pH of the product. The number of viable cells in theproducts fermented to pH 4.6 was not affected by cold storage for 6days. All products fermented to lower pH values with Lb. helveticusstrains also retained a high number of live cells after 7 days ofstorage, except the product fermented to pH 3.5 with 1198, in whichonly 107 cfu mL�1 were recorded. In contrast, the viability of thecocci in the products fermented to pH w4.3 with Lc. strains or Str.

thermophilus decreased significantly by 1–3 log counts uponrefrigerated storage (p¼ 0.002). This is consistent with resultsobtained by others (Beal et al. 1999; Gadaga et al. 2001; Mistry,2001).

There was no overall effect of storage on the ACE-inhibitoryactivity when all strains were taken together, nor when eachspecies was considered separately. However, some clear trendswere noted (compare Fig. 1A and B). All products fermented with Lc.lactis (to pH 4.6 and 4.3) had a markedly higher ACE-inhibitoryactivity after storage for 7 days, the activity of the product fer-mented to pH 4.3 with ssp. lactis strain 3906 reaching 51%, thehighest obtained in this study (Fig. 1B). The products fermented byLb. helveticus strains were affected differently by cold storage. TheACE-inhibition of the products that already had a low ACE-inhibi-tory activity (20% or below) was not affected much by storage(strains 1264, 637 and 1169), and the milk fermented with Lb.helveticus strain 4080 hardly showed any ACE-inhibitory activityafter storage. In contrast, the ACE-inhibitory activity of the fer-mented milk made with the most proteolytic strains (1198 and1263) was increased considerably during storage to reach 40% orhigher, the highest activity measured for the Lb. helveticus products(Fig. 1). Interestingly, the small increase in bioactivity that wasobtained by fermenting from pH 4.6 to pH 4.3 with strains 1198 and1263 was lost during storage. The products fermented to pH 3.5with Lb. helveticus lost much of their remaining activity during coldstorage (Fig. 1).

All products fermented to pH 4.6 (except that produced by Str.thermophilus), and most of those fermented to pH 4.3 had anincreased total peptide area after an additional 6 days of coldstorage (Table 2). This shows that they retained proteolytic activityat 5 �C, and is consistent with increased ACE-inhibitory activity

BA

0.0

0.1

0.2

0.3

0.4

C

Ab

so

rb

an

ce at 210 n

m

0.0

0.1

0.2

0.3

0.4D

E

Retention time (min)

10 20 30 40 50 60 70 80 900.0

0.1

0.2

0.3

0.4F

Retention time (min)

10 20 30 40 50 60 70 80 90

12 3

4

5

67

8911

1315

17

18

19

2021

22

2324

25

Fig. 4. Peptide profiles of milk samples fermented with Lb. helveticus strain 1198 to pH 4.6 (A and B), pH 4.3 (C and D), and pH 3.5 (E and F), and stored for 1 day (A, C and E) and 7days (B, D and F). Peaks are numbered consecutively, and major peptides have been identified in Table 4.

Table 2Peptide amounts determined by the sum of peptide areas in the RP-HPLC peptideprofiles of the fermented milk samples.

Species and strain Peptide amount (arbitrary units)

pH 4.6 pH 4.3 pH 3.5

day 1 day 7 day 1 day 7 day 1

Lc. lactisssp. lactis 3906 68 106 78 87ssp. lactis 3923 68 105 79 89ssp. cremoris F3 79 141 92 133ssp. cremoris W5 86 132 90 110Lb. helveticus

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165 161

after storage (Fig. 1). The peptide profiles also changed duringstorage, mostly in the products fermented to pH 4.6. The milksamples fermented with Lc. lactis and Lb. helveticus to pH 4.6 andstored for 7 days had a higher concentration of the peptides withRts above 40 min, especially of the characteristic late-eluting peaks(Figs. 2–4). This indicates that large casein peptides were formedand thus that proteinases were active during cold storage. The sametrend was seen for the products fermented to pH 4.3, whereas forthe products fermented to the end pH, there was no effect ofstorage on peptide profiles (Fig. 4). The effect of storage on thepeptide profiles of the products fermented with Lb. acidophilus topH 4.6 was a significant increase in all late-eluting peptides,resulting in a peptide profile resembling those produced with Lb.helveticus 1198 and 1263 at pH 4.6 after storage. Due to slowfermentation, this product had a pH of 4.8 when fermentation wasstopped, and upon the subsequent cold storage at the rather highpH, substantial hydrolysis took place, consistent with the high pH-optimum of the proteinases of Lb. acidophilus (Fira et al. 2001).There was no significant effect of storage visible on the peptideprofiles of the products fermented by Str. thermophilus to pH 4.6and 4.3.

1198 146 225 122 138 861262 69 94 73 125 71264 66 113 65 88 451263 252 280 156 166 1541169 58 90 53 81 83637 58 86 88 76 634080 80 119 84 113 61Lb. acidophilus3777 69 305 42 78 58Str. thermophilusS2 26 28 29 30

3.5. Relationship between peptide amount and ACE-inhibition

As presented in the previous sections, an increase in peptideamount was sometimes accompanied by an increase in ACE-inhibitory activity of the product; however, this was not always thecase. Accordingly, the correlation coefficient for the linear correla-tion between the peptide amount and ACE-inhibitory activity for allproducts (Fig. 5) was only 0.25. In other studies, correlating the

ACE-inhibition to the peptide amount in dairy products andingredients, a simple linear relationship was also not obtained(Fuglsang et al., 2003b; Hernandez-Ledesma et al., 2004; Nakamuraet al., 1995; Pripp et al., 2006; Robert et al., 2004; Ryhanen et al.,2001). As in some of these studies (Fuglsang et al., 2003b; Robertet al., 2004; Ryhanen et al., 2001), the ACE-inhibitory activity in ourstudy seemed to reach its maximum at a certain peptide amount.Fitting our data by a non-linear four-parameter logistic model,

Total peptide area (arbitrary units)

0 50 100 150 200 250 300

AC

E-in

hib

ito

n (%

)

0

10

20

30

40

50

Fig. 5. Correlation between peptide amount as measured from RP-HPLC peptideprofiles (210 nm) and ACE-inhibitory activity of all fermented products stored for 1 day(white) or 7 days (grey). The milk samples were fermented with Lc. lactis (6), Str.thermophilus (7), Lb. helveticus (,) or Lb. acidophilus (B).

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165162

a very good correlation (r2¼ 0.68) was obtained for the productsfermented to pH 4.6 and stored for 1 day. For all products (Fig. 5)the correlation coefficient though was only 0.34. This is probablydue to differences between species and strains, especially withrespect to the ratio of peptides that inhibit ACE and peptides that donot, and emphasises the need to identify the individual peptides, todistinguish the bioactive from the non-active.

3.6. Identification of major peptides and proteolytic activity inselected fermented products

Three products with high ACE-inhibitory activity, all fermentedto pH 4.6 and stored for 7 days, were selected for identification ofmajor peptides; two products fermented with each of the ssp. of Lc.lactis and one product fermented with Lb. helveticus strain 1198. Thepeptide profiles of these products are shown in Figs. 2B and D, and

Table 3Masses and tentative identification of peptides in milk samples fermented with Lc. lactis ss

Peak no.a Rtb (min) Mass obser-ved Mass theore-tical Fragment tentatively id

4 36 600 598.7 as1-CN 107–1115 37 1140 1140.4 as1-CN 1–9

6a 40 1216 1217.5 b-CN 155–1656 42 802 801.9 b-CN 176–1827 45 905 904.0 as1-CN 24–318a 47a 856 855.0 b-CN 80–878 47b 1184 1184.3 b-CN 47–569 49 998 996.2 b-CN 167–175

117510 51 756 755.9 b-CN 47–52

11 128312 57 1564 1564.9 b-CN 169–18214 67 297515 130216 71 1152 1151.4 b-CN 199–20917 75 1781 1782.1 b-CN 78–93

120319 76 1718 1717.1 b-CN 194–20920 78 1881 1880.3 b-CN 193–209

a Peak numbers refer to the peptide profiles in Fig. 2B and D.b Retention time in RP-HPLC.c CN, casein.d ACE-inhibition is in most references given as the concentration needed to inhibit AC

4 B, respectively. The identified peptides comprised a few N-terminal fragments of as1-CN and several fragments from b-CN(Tables 3 and 4). Fragments from as1-CN were not identified in theproduct made with Lb. helveticus 1198, probably due to the multi-tude of peptides eluting at the same Rt in the first part of thechromatogram (Fig. 4B), preventing the identification of thesepeptides. Peptides derived from the N-terminal, central and the C-terminal parts of b-CN were found in both Lc. and Lb. products,however, only one fragment, b-CN 194–209, was found in bothproducts. The peptides identified as originating in b-CN are alignedto the primary sequence in Fig. 6.

The presence of as1-CN f1–9 in the products fermented with theLc. lactis strains, which has been shown to be produced with the PI-type proteinases but not with the PIII-type proteinases of Lc. lactissubsp. cremoris origin (Exterkate et al., 1993), and of peptidesformed by cleavage of the bonds 165–166 and 166–167 in b-CN thatare not cleaved by the PIII-type (Pritchard & Coolbear, 1993),suggests that the active cell wall proteinases in both Lactococcalstrains used in this study belong to the PI-type (Kunji et al., 1996;Pritchard & Coolbear, 1993). The N-terminal as1-CN fragment (f1–9)is commonly found in fermented dairy products produced withLactococcus and/or Lb. helveticus (Ardo et al., 2007; Broadbent et al.,2002; Oberg et al., 2002; Ryhanen et al., 2001; Saito et al., 2000;Yamamoto et al., 1994b; Zevaco & Gripon, 1988). Fragment 24–31 ofas1-CN has not previously been observed after fermentation of milkwith Lc. lactis strains, but has been found as a product fromdegradation of caseinate by an extracellular proteinase from Lb.helveticus CP790 (Yamamoto et al., 1994b), whereas as1-CN f107–111 has not previously been detected in fermented milk or caseinhydrolysates produced with LAB proteinases. The higher proteo-lytic activity of the Lactococcus strains used in the present studycould thus partly stem from the ability of the cell wall proteinase tocleave the bonds 23–24, 31–32, 106–107 and 111–112 in addition tothe bond 8–9 in as1-CN. In accordance with the observations ofJuillard et al. (1995), who used an extracellular Lc. lactis proteinaseto hydrolyse b-CN, most of the b-CN derived peptides identified inthe Lc. lactis products originated in the C-terminal part of b-CN(Table 3), suggesting that most of the b-CN fragments identified inthe Lc. products in the present study result from the action of thecell wall proteinase alone (Fig. 6; Gobbetti et al., 2000; Juillard et al.,

p. lactis strain 3923 and ssp. cremoris strain F3 to pH 4.6 and stored for 7 days at 5 �C.

entifiedc Remark ACE-inhibitiond and reference

w15 mM, Ryhanen et al. (2001);13 mM, Saito et al. (2000)

absent or lower in strain 3923higher in strain 3923 >1000 mM, Robert et al. (2004)

higher in strain 3923f164–175: 39 mM, Robert et al. (2004)

higher in strain F3 257 mM, Robert et al. (2004);>1000 mM, Quiros et al. (2007)

higher in strain F3

absent in strain F3absent in strain F3

higher in strain F3100 mM, Robert et al. (2004)

E by 50%; ACE, Angiotensin-Converting Enzyme.

Table 4Masses and tentative identification of peptides in milk samples fermented with Lb. helveticus strain 1198 to pH 4.6 and stored for 7 days at 5 �C.

Peak no.a Rtb (min) Mass observed Mass theoretical Fragment tentatively identifiedc ACE-inhibitiond and reference

6 34 681 680.7 b-CN 52–577 35 760 759.9 b-CN 95–101 80%, Gomez-Ruiz et al. (2002)19 65 2172 2172.7 b-CN 102–119 F106–119: >1000 mM, Robert et al. (2004)20 75 1364 1363.7 b-CN 197–209 >700 mM, Quiros et al. (2007)

210821 76 1718 1717.1 b-CN 194–20922 79 1994 1993.4 b-CN 192–20923 81 3280 3279.9 b-CN 58–87 A1 21 mM, Yamamoto et al. (1994b)

2107 2106.6 b-CN 191–2093748 3747.4 b-CN 58–91 A1

24 84 3240 3239.9 b-CN 58–87 A23708 3707.4 b-CN 58–91 A24035 4034.8 b-CN 58–94 A1

25 86 3995 3994.8 b-CN 58–94 A2

a Peak numbers refer to the peptide profile shown in Fig. 4B.b Retention time in RP-HPLC.c CN, casein.d ACE-inhibition is given either in % relative to a control, or as the concentration needed to inhibit ACE by 50%; ACE, Angiotensin-Converting Enzyme.

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165 163

1995; Kunji et al., 1996; Pritchard & Coolbear, 1993). The exceptionsare b-CN f155–165 and f199–209 (Fig. 6), of which fragment 155–165 could result from endopeptidase and/or aminopeptidaseprocessing of f153–168 or perhaps f153–165. The bond Leu198-Gly199 in b-CN has been reported as a cleavage site for one Lb.helveticus proteinase (Kunji et al., 1996), and it is possible that theproteinase from the Lc. lactis ssp. lactis strain used in this study hasaffinity for this site. The proteinase of the ssp. cremoris lacked theability to hydrolyse f194–209 at the cleavage site 198–199, sincef194–209 occurred at a higher concentration in the product madewith the cremoris ssp., and the peptide b-CN f199–209 was uniqueto the product made with the lactis ssp. Similarly, the almostcomplete absence of b-CN f169–182 and therefore the presenceof f176–182 in the products made with the lactis ssp., suggeststhat the proteinase of this ssp. also hydrolysed the site 175–176 inb-CN.

The peptides identified as originating from the first part of b-CNin the milk fermented by Lb. helveticus 1198 (Table 4, Fig. 6) have notpreviously been found in milk fermented with Lactobacillus or otherspecies of LAB (Hernandez-Ledesma et al., 2004; Juillard et al.,

R-E-L-E-E-L-N-V-P-G-E-I-V-E-S-L-S-S-S-E-E-S-I-T-R-I-N-K-1 10 20

-P-F-A-Q-T-Q-S-L-V-Y-P-F-P-G-P-I-H/P-N-S-L-P-Q-N-I-P-P-51 60 A1/A2 70

-A-M-A-P-K-H-K-E-M-P-F-P-K-Y-P-V-E-P-F-T-E-S-Q-S-L-T-101 110 120

-L-P-P-T-V-M-F-P-P-Q-S-V-L-S-L-S-Q-S-K-V-L-P-V-P-Q-K-A151 160 170

-V-R-G-P-F-P-I-I-V201 209

12

Fig. 6. Identified peptides originating in b-CN from milk samples fermented with Lc. lactis s1198 (solid lines). Peptide bonds in b-CN cleaved by most LAB proteinases are marked withstrains are marked with dashed line arrows and full line arrows, respectively. Cleavage site

1995; Kunji et al., 1996; Quiros et al., 2007; Robert et al., 2004; Saitoet al., 2000; Yamamoto et al., 1994b; Zevaco & Gripon, 1988).However, since the sites 57–58, 87–88, 94–95 and 101–102 in b-CNhave been cleaved by some Lc. lactis strains (Gomez-Ruiz et al.,2002; Kunji et al., 1996; Quiros et al., 2007), and the site 119–120has been cleaved by several Lb. helveticus proteinases (Robert et al.,2004; Yamamoto et al., 1994b; Zevaco & Gripon, 1988), we expectthe proteinase of this strain to possess a broad specificity andcatalyse the formation of all these peptides. Two of the C-terminalb-CN fragments identified had been formed by cleavage of peptidebonds normally cleaved by LAB (Fig. 6; Kunji et al., 1996; Robertet al., 2004; Yamamoto et al., 1994b; Zevaco & Gripon, 1988). Itcannot be concluded from our experiments, though, whether thefragments f194–209 and f197–209 result from aminopeptidasecleavage of f190/191–209, or from a unique specificity of theproteinase towards the bonds Tyr193-Glu194 and Pro196-Val197 inb-CN. Interestingly, the Lb. helveticus profiles did not contain manylarge unspecified casein fragments at Rt w90 min, indicatinga more complete degradation of caseins than by the Lactococcusstrains.

K-I-E-K-F-Q-S-E-E-Q-Q-Q-T-E-D-E-L-Q-D-K-I-H30 40 50

L-T-Q-T-P-V-V-V-P-P-F-L-Q-P-E-V-M-G-V-S-K-V-K-E80 90 100

L-T-D-V-E-N-L-H-L-P-L-P-L-L-Q-S-W-M-H-Q-P-H-Q-P130 140 150

-V-P-Y-P-Q-R-D-M-P-I-Q-A-F-L-L-Y-Q-E-P-V-L-G-P180 190 200

7

sp. lactis strain 3923 and ssp. cremoris strain F3 (dashed lines) and Lb. helveticus strainarrow heads. Peptide bonds cleaved by some Lc. lactis strains and some Lb. helveticus

s not previously identified as targets for LAB proteinases are marked with asterisks.

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165164

3.7. ACE-inhibitory peptides in the selected fermented products

Several of the major peptides identified in the milk samplesfermented with Lc. lactis strains have been shown to possess ACE-inhibitory activity, i.e., the highly potent as1-CN fragment 1–9 andthe b-CN fragments 47–52, 176–182 and 193–209 with loweractivity (Table 3). The fragment b-CN f167–175 has not beenreported to possess ACE-inhibitory activity. However, some frag-ments with the same C-terminal (which is important for binding toACE) have been reported to inhibit ACE, e.g., f164–175(IC50¼ 39 mM; Robert et al., 2004), f168–175 (IC50¼ 25 mM, Yama-moto et al., 1994b), and f169–175 (IC50¼1000 mM; Maeno et al.,1996). Despite the high IC50 value for the latter peptide, it reducedthe systolic blood pressure in spontaneously hypertensive rats(Maeno et al., 1996). This suggests that the peptide correspondingto b-CN f167–175 might also possess a hypotensive effect. Theultimate C-terminal fragment of b-CN, f194–209, has not beentested for ACE-inhibitory activity, however, since it has the same C-terminal as f193–209, it could be expected to possess a similarbioactivity. Being the only major ACE-inhibitory peptides eluting inthe Rt interval between 78 and 95 min, which correlated best withACE-inhibition, these C-terminal fragments of b-CN probablycontribute to the ACE-inhibitory activity of the products fermentedwith Lc. lactis, in addition to as1-CN f1–9. For the cremoris ssp. therewas a correlation (r2¼ 0.56) between ACE-inhibition and thepeptide amount of the Rt interval 38–78 min, in accordance withthe higher abundance of b-CN f194–209 eluting at 76 min in thisssp. For the lactis ssp. the best correlation of ACE-inhibitory waswith the Rt interval 24–38 min (r2¼ 0.68) in accordance with thehigher concentration of as1-CN f1–9 in this ssp. The increased ACE-inhibitory activity after storage of the products fermented with Lc.lactis to pH 4.6 occurred concomitantly with an increase of allpeptides with Rt> 42 min, among them the C-terminal b-CNfragments f194–209, 193–209 and f176–182. In contrast, theincreased ACE-inhibitory activity obtained during storage of the Lc.products fermented to pH 4.3 was accompanied by only slightchanges in peptide profiles (Figs. 2 and 3). Thus, additional minorpeptides with ACE-inhibitory activity, not visible in the profile, mayalso have been generated during storage of these products. Thedecrease in the Lactococcus cell number during cold storage of thisproduct suggests that lysis of cells occurred, which would result inrelease of intracellular peptidases and formation of small peptides,many of which might have ACE-inhibitory activity (Fuglsang et al.,2003a).

For all Lb. helveticus products a good linear correlation (r2> 0.6)was obtained with peptide amounts within each of the Rt intervals24–38 min, 38–78 min, and 78–95 min, indicating that peptides ofvarious sizes and hydrophobicity contributed to the bioactivity ofthese products. Accordingly, the three peptides identified in theproduct fermented with Lb. helveticus 1198 as ACE-inhibitorypeptides, i.e., b-CN f95–101, f197–209 and f191–209, had Rts of35 min, 75 min and 81 min, respectively (Table 4). Interestingly, thelate-eluting C-terminal b-CN f191–209 identified in this productwas even more potent (21 mM, Yamamoto et al., 1994b) than theshorter C-terminal fragments identified in the products fermentedwith Lactococcus (Table 3). This peptide probably is primarilyresponsible for the correlation of ACE-inhibitory activity with thepeptide amount of the late Rt interval. Accordingly, the loss ofactivity during storage of the product fermented to pH 4.3 withstrain 1198 (Fig. 1) is consistent with a decrease in all peaks withRt> 80 min to a level lower than in the original product (Fig. 4). Theother ACE-inhibitory C-terminal fragment of b-CN found in thisproduct (b-CN f197–209) was less potent (>700 mM, Quiros et al.,2007). However, this product also contained other C-terminalfragments of b-CN, f192–209 and f194–209, which might possessACE-inhibitory activity. It is quite intriguing that the ultimate

C-terminal b-CN fragments identified in this study, differing only inthe N-terminal, have such varying inhibitory activity, from 21 mM forthe longest one, f191–209, to more than 700 mM for the shorterfragment f197–209. These fragments are all rather large andhydrophobic, and with the same C-terminal sequence, which is themost important determinant of the ACE-inhibitory activity. Part ofthe discrepancy might be due to the use of different substrates andACE concentrations in the assay (Murray et al., 2004). The increasein ACE-inhibitory activity during storage of the milk samples fer-mented with strains 1198 and 1263 to pH 4.6 was accompanied byan increased concentration of all peaks with Rt> 65 min, includingthe C-terminal fragments of b-CN, e.g., b-CN f197–209, f192–209and f191–209 (Figs. 3 and 4), suggesting that these peptides areprimarily responsible for the bioactivity of these products.However, as indicated by correlation of the ACE-inhibitory activitywith the peptide amounts of the shorter Rt intervals, b-CN f95–101and some of the unidentified peptides might contribute to the ACE-inhibitory activity of the products made with strains 1198 and 1263.

4. Conclusions

All strains from the 4 species of LAB tested fermented thepasteurized milk to pH 4.6 in 8–27 h at the conditions used,however the number of peptides and ACE-inhibitory activity of thefermented milk samples varied significantly. The four Lc. lactisstrains behaved similarly in fermentation and proteolysis and gavehigh ACE-inhibitory activities, with slight differences between ssp.The peptide profiles and ACE-inhibitory activity of the productsfermented by Lb. helveticus varied significantly with strain; thehighest activity was obtained with the two proteolytic strains (1263and 1198).

Fermentation to the end pH (4.3) with Lc. lactis increased theACE-inhibitory activity, whereas continued fermentation to the endpH (3.5) with Lb. helveticus decreased the ACE-inhibitory activity,showing that the optimal bioactivity was obtained at an interme-diate fermentation time (pH) with strains of the latter species.

A period of cold storage improved the bioactivity of productsmade with Lc. lactis and with Lb. helveticus strains 1198 and 1263,depending on pH. The products fermented by Lb. helveticus shouldbe fermented to pH 4.6 only before storage, showing that proteol-ysis should not be too extensive, whereas with the Lc. lactis strains,fermentation should be continued to the end (pH w4.3) beforestorage in order to obtain the optimal bioactivity.

The ACE-inhibitory activity correlated non-linearly with thetotal peptide peak area, due to the presence of both active andinactive peptides. Several peptides were identified showing a broadspecificity of the investigated Lc. lactis and Lb. helveticus proteolyticsystems. Peptides contributing to the ACE-inhibitory activityinclude C-terminal fragments of b-CN, as1-CN f1–9, and other earlyeluting, unidentified peptides.

Acknowledgements

The financial support from the FØTEK programme through theCentre for Advanced Food Studies at the Faculty of Life Sciences(LIFE), University of Copenhagen, and from the Velux Foundation of1981, Denmark, as well as from the Centre for Advanced FoodStudies is highly appreciated. Egil W. Nielsen and Finn K. Vogensen,Department of Food Science, LIFE, are gratefully acknowledged forthe kind supply of lactic acid bacterial strains. We are also gratefulto Mila Zakora, Mona Østergaard, Bashir Y. Aideh and Anders OlaKarlsson at the Department of Food Science and Marie Koefoed atChr. Hansen for their excellent technical assistance. Furthermore,Richard Ipsen, Ylva Ardo, Are Hugo Pripp and in particular Finn K.Vogensen from Department of Food Science and Anne Skriver fromChr. Hansen A/S are highly recognised for fruitful discussions.

M.S. Nielsen et al. / International Dairy Journal 19 (2009) 155–165 165

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