International Journal of Food Microbiology 159 (2012) 3946

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Yeasts from Colombian Kumis as source of peptides with Angiotensin I converting

enzyme (ACE) inhibitory activity in milk
Clemencia Chaves-Lpez , Rosanna Tofalo, Annalisa Serio, Antonello Paparella,
Giampiero Sacchetti, Giovanna Suzzi
Dipartimento di Scienze degli Alimenti, Universit degli Studi di Teramo, Via C.R. Lerici 1, 64023 Mosciano Sant'Angelo (TE), Italy

a r t i c l e

i n f o

Article history:
Received 8 May 2012
Received in revised form 23 July 2012
Accepted 31 July 2012
Available online 3 August 2012
Keywords:
Yeast
Angiotensin converting enzyme
Proteolysis
Fermented milk
Kumis

a b s t r a c t
This study investigated the possibility of using yeast strains in fermented milks to obtain products with high
Angiotensin I-converting enzyme (ACE) inhibitory activity and low bitter taste. Ninetythree yeast strains
isolated from Colombian Kumis in different geographic regions were molecularly identied, and their milk
fermentation performances were determined. Molecular identication evidenced that Galactomyces
geotrichum, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis, were the dominant species. Eighteen out of 93 strains produced fermented milk with ACE-inhibitory (ACEI) activity values ranging from 8.69
to 88.19%. Digestion of fermented milk samples by pepsin and pancreatin demonstrated an increase in ACEI
activity, with C. lusitaniae KL4A as the best producer of ACEI peptides. Moreover, sensory analysis of the products containing the major ACE-inhibitory activity pointed out that P. kudriavzevii KL84A and Kluyveromyces
marxianus KL26A could be selected as potential adjunct starter cultures in Kumis, since they made a considerable contribution to the ACE inhibitory activity and produced fermented milk without bitter taste. In this
study we observed that Colombian Kumis can be an excellent vehicle for the isolation of yeasts with a potential to enhance bioactive peptides produced during milk fermentation.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Yeasts are involved in a wide range of fermented traditional foods
and beverages, and contribute to the sensory properties that are the
result of combined metabolic activity of single strains or microbial
groups together with process characteristics. The presence of yeasts,
particularly in dairy products, offers potential advantages due to the
production of avour components and/or acceleration of ripening,
by metabolizing milk constituents. The main mechanisms by which
yeast growth can inuence dairy products are: fermentation of
lactose and galactose, assimilation of lactate, lipolytic and proteolytic
activities (Roostita and Fleet, 1996). Yeasts are believed to be
essential in the production of some fermented milks, as Ker, Koumis,
Viili, Longl, Laban, Amasi, Kurut. Recently, their presence in a high
number has been reported in Kumis, a traditional low alcoholic
fermented cow milk produced in rural and urban areas of the South
West Colombia (Chaves-Lpez et al., 2011a). Besides being easily
digestible, fermented milks are a source of functional compounds
that have benecial effects on health (Philanto et al., 2010). In particular, proteolysis involves the production of peptides that may exhibit
Corresponding author at: Dipartimento di Scienze degli Alimenti, Universit degli
Studi di Teramo, Via C.R. Lerici 1, 64023 Mosciano Stazione (TE), Italy. Tel.: +39
0 861 266913; fax: +39 0 861 266915.
E-mail address: chaves@unite.it (C. Chaves-Lpez).
0168-1605/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijfoodmicro.2012.07.028

different biological activities, such as immunomodulating, antihypertensive, osteoprotective, antilipemic, opiate, antioxidative and antimicrobial
activities (Mller et al., 2008). Among the antihypertensive peptides,
ACEI peptides have been proved to reduce blood pressure. It is well
known that Angiotensin I-converting enzyme plays a major role in the
regulation of blood pressure. Within the reninangiotensin system,
ACE catalyzes the conversion from Angiotensin I to Angiotensin II, a
potent vasoconstrictor (Riordan, 2003), which also increases blood pressure. Moreover, ACE hydrolyzes bradykinin, which has vasodilatatory
properties. ACEI activity has been reported in traditional fermented
milks (Chaves-Lpez et al., 2011a; Chen et al., 2010) and in milks
fermented with strains of Lactobacillus delbrueckii subsp. bulgaricus,
Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus casei,
Lactobacillus jensenii, Lactococcus lactis, Lactococcus cremoris, Leuconostoc
mesenteroides spp cremoris, Enterococcus faecalis and Enterococcus
faecium, as a single culture or in combination (Chaves-Lpez et al.,
2011a; Fuglsang et al., 2003; Leclerc et al., 2002; Muguerza et al.,
2006; Philanto et al., 2010; Yamamoto, et al., 1994). However, the
production of peptides with ACEI activity has been documented only
in some yeast species, and namely in K. marxianus, Saccharomyces
cerevisiae and Candida parapsilosis (Didelot et al., 2006; Hamme et al.,
2009; Jang and Lee, 2011; Kuwabara et al., 1995).
Since the dairy industry is keen to explore new possibilities for
enhancing the diversity of its product range, there is an increasing
interest in searching for potential starter organisms from the pool,

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C. Chaves-Lpez et al. / International Journal of Food Microbiology 159 (2012) 3946

Samples of traditional Kumis were collected from 13 different production sites in the Valle del Cauca (Southwest of Colombia). Eight samples
were collected from the valley (average temperature 30 C) and 5 from
the mountains (average temperature 18 C). Each sample (approximately
150 ml) was aseptically transferred to a 250 ml sterile screw-capped
bottle and kept at 4 C until 12 h before the analyses. The pH of each
sample was measured and microbiological analyses were performed.

Clustering analysis was performed by means of the UPGMA (unweighted

pair group method with arithmetic mean) method. The repeatability of
RAPD-PCR ngerprints was determined by triplicate loading of independent triplicate reaction mixtures prepared with the same strain. From
every RAPD genotype group showing the same pattern, up to four strains
were selected and sequences of the D1/D2 domain of the 26S rRNA gene
were determined.
PCR amplication of D1/D2 region of the 26S rDNA was achieved by
using primers NL1 (5-GCATATCAATAAGCGGAGGAAAAG-3) and NL4
(5-GGTCCGTGTTTCAAGACGG-3) (Kurtzman and Robnett, 1998). The
reactions were performed in an automatic thermal cycler (GeneAmp
PCR System 9700) under the following conditions: initial denaturation
at 94 C for 5 min, 30 cycles of denaturation at 94 C for 1 min, annealing
at 55 C for 30 s, extension at 72 C for 1 min, nal extension at 72 C for
10 min, holding at 4 C. PCR products were puried by the GFX PCR DNA
and Gel Band Purication Kit (Amersham Biosciences, Uppsala, Sweden),
following the manufacturer's instructions, and delivered to Centro
Ricerca Interdipartimentale Biotecnologie Innovative (Padua University,
Padua, Italy) for sequencing. The D1/D2 domain of the 26S rRNA was
used in a similarity search by means of the BLAST program in GenBank
(http://www.ncbi.nlm.nih.gov). Alignment of sequences obtained for
the isolates and reference sequences retrieved from the database was
performed using CLUSTALX (Thompson et al., 1997)

2.1. Enumeration and isolation of yeast

2.3. Frequency percentage analysis

Samples of Colombian Kumis (10 ml) were mixed with 90 ml of

0.85% (w/v) sterile physiological saline and homogenized in a stomacher
Lab-Blender (400, Seward, UK) for 2 min. A serial dilution in the same
diluents was made. Plates containing Potato Dextrose Agar (PDA)
added with 150 ppm of Chloramphenicol (Oxoid) were used for yeast
enumeration incubating at 25 C for 72 h.
Colonies were randomly selected or all sampled if the plates contained
less than 10 colonies. All isolates were checked for purity by streaking
again and subculturing on the corresponding agar. For long-term maintenance of isolates, stock cultures were stored at 80 C in 20% (v/v)
glycerol, 80% (v/v) YPG (1.0% yeast extract, 2.0% peptone, 2.0% galactose)
broth.

To study distribution species in our samples, the method reported

by Osorio-Cadavid et al. (2008) was used.

which existed at the time of raw milk fermentation (Wouters et al.,

2002). In particular, the selective pressures of tropical environments
may favour yeast biodiversity and highlight a useful technological potential (Chaves-Lpez et al., 2009).
In a previous paper (Chaves-Lpez et al., 2011a), we investigated
the enterococcal population in traditionally manufactured Kumis,
for their role as benecial organisms or opportunistic pathogens.
The tests on ACEI activity showed that nine E. faecalis and two E. faecium
strains produced fermented milk with ACEI activity values ranging
from 39.7% to 84.35%. This paper aims to investigate the ability of
different yeast species, isolated from Colombian Kumis, to release
peptides with ACEI-activity in milk, for potential use as starters in
the dairy industry.
2. Materials and methods

2.2. Molecular identication

Molecular identication at the species level was carried out by
sequencing of D1/D2 region of the 26S rRNA gene and by random
amplication of polymorphic DNA (RAPD-PCR). Total genomic DNA
of the isolates was extracted and puried from 7-ml cultures
according to the protocol of Querol et al. (1992). The RAPD-PCR
screening was performed to reduce the number of isolates to be identied at species level. For primer M13 (5-GAGGGTGGCGGTTCT-3)
(Huey and Hall (1989), with arbitrary chosen sequences, each reaction was carried out in 25 L reaction mix containing 2.5 L 10
PCR buffer (Invitrogen, Life Technologies), 1.5 mM MgCl2, 200 M of
each dNTP, 20 pmol primer, 1 U Taq polymerase and 20 ng extracted
DNA. Amplications were performed in a GeneAmp PCR System 9700
(Perkin-Elmer) with the amplication condition: an initial denaturation at 94 C for 4 min followed by 35 cycles consisting of 30 s at
94 C, 20 s at 45 C, 2 min at 72 C and a nal extension of 7 min at
72 C. Amplication products were separated on a 1.5% agarose gel
in 1 TAE buffer. After electrophoresis, the gels were stained with
ethidium bromide 0.5 g ml 1), washed with deionized water and
photographed under UV transillumination using a Gel Doc 2000 EQ
System (Bio-Rad, Hercules, CA). The molecular sizes of the amplied
fragments were estimated by comparison with the 1-kb plus DNA
molecular size marker (Invitrogen). Conversion, normalization, and
further analysis of the RAPD-PCR patterns were carried out with
Fingerprinting II Informatix software program (Bio-Rad). Similarity
between patterns was evaluated using the Pearson coefcient.

2.4. Screening of skim milk proteolysis by the yeast strains

Puried yeast strains were inoculated in the YPD medium (10 g/l
yeast extract, 20 g/l peptone and 20 g/l dextrose) and incubated at
28 C for 48 h. The cells were centrifuged at 6000 g for 10 min at 4 C.
The pellet was then suspended in 50 ml of sterile reconstituted (10%
w/v) skimmed milk (Biolife, Milan, Italy) with 100 ppm chloramphenicol, and incubated at 28 C for 48 h. To evaluate the capability of the 93
strains to hydrolyze milk protein and produce peptides with ACEI activity, each pre-culture sample, prepared with a single yeast strain, was inoculated (5% v/v) into triplicate 100 ml of pasteurized skim milk.
Samples were analyzed to determine pH, proteolysis (measured as
free-NH3 groups by the OPA test that was carried out by measuring
the absorbance at 340 nm according to by Church et al. (1983), and
ACEI activity at 52 h of incubation at 28 C. Yeast growth was determined by plating on YPD agar. The fermentation process was stopped
by pasteurization of fermented milk at 75 C for 1 min.
2.5. Assay for ACE inhibitory activity and peptides quantication
The measurement of ACE inhibitory activity was carried out spectrophotometrically using the pH 4.6 soluble fraction obtained by centrifugation (10.000 g 10 min at 4 C) as previously reported (Pan et al.,
2005). Briey, 200 l of HHL buffer (5 mM Hip-His-Leu in 0.1 M borate
buffer containing 0.3 M NaCl, pH 8.3) were mixed with 80 l of sample
solution and pre-incubated for 3 min at 37 C. After addition of 20 l of
ACE (Rabbit lung ACE, dissolved in distilled water, 0.1 units/ml), the
mixture was incubated for 30 min at 37 C. The reaction was stopped
by adding 250 l of 1.0 N HCl and mixed with 1.7 ml of ethyl acetate.
The hippuric acid formed was extracted with ethyl acetate and the
absorbance was measured at 228 nm. Unfermented skim milk was
used as reference.
ACE inhibitory activity index of the samples was calculated according
to the formula:
ACE inhibitory activity index BA=BC  100%;

C. Chaves-Lpez et al. / International Journal of Food Microbiology 159 (2012) 3946

Table 1
Identication of yeast isolates determined by 26S rDNA gene sequence analysis.
Strain

RAPD
Closest relative
pattern

KL68B, KL27A, KL17, KL56, III

KL5, KL6, KL28, KL23A,
KL42B, KL12A, KL36B.
KL38A, C5, L40, 3D, L83A.
VI
KL15, L65, L39, L42,L87A.
VIII
KL47, KL85B, Kl46B, KL2B,
KL62A, KL4, L46A, L50,
L43, C2B, C1B.
KL1, KL36, L20A, L63, L71B,
L44, L18, L13, C43, L54A,
L14, L64, L84B, L51,
L54C, L53, L83B, L87,
L11, L45, L80.
KL7, KL25, KL9, KL58,
KL82, KL2A, KL66B,
KL84A, KL12B, KL2A,
KL52 L70, L27B.
KL57, KL48, Kl34, KL60B,
L26B.
KL35, KL38, KL33,
KL26A, KL2B.
L3A

XXI
XII

Candida tropicalis

99

HM627137.1

Candida glabrata
Candida
pararugosa
Clavispora
lusitaniae

100
99

FN393990.1
AB112430.1

100

AJ539567.1

100

DQ862851.1

Galactomyces
geotrichum

IIIIV

Pichia
kudriavzevii

99

AY529504.1

IX

Kazachstania
100
unispora
Kluyveromyces
98
marxianus
Rhodotorula
99
mucilaginosa
Torulospora
100
delbrueckii
Trichosporon
99
asahii
Trichosporon
99
insectorum
Saccharomyces
100
cerevisiae
Wickerhamomyces
99
pijperi

FN393993.1

XVIII

KL30B, KL66A, C4.

XIII

L59

XVII

C2A, L69

XIX

KL77, KL76, L73.

L16, L37, L55, L8,

L60A, L19, L85A

XX
XXI

10, 24, 38, 52 and 64 h) for the following analyses: cell counts, pH, peptide content and ACEI activity.

Identity Accession
(%)
number

XIV
XV
XVI

VII

41

AJ508567.1
AF335986.1
HE616749.1

2.7. Hydrolysis of the peptides by pepsin and pancreatin

To study both the peptides stability and the formation of new ACEI
peptides, aliquots of 5 ml of fermented skim milk, prepared as
described for the screening of skim milk proteolysis, were subjected
to hydrolysis. The samples were rst hydrolyzed with pepsin (EC
3.4.4.1; 1:60,000, 3400 U mg 1) (Sigma Aldrich) at the following
conditions: 20 mg pepsin g 1 protein, 37 C, pH 3.5, 4 h. The reaction
was stopped by boiling water for 10 min and neutralized to pH 7.0
adding NaOH solution (2 N). The remained neutralized suspension
was further digested with 40 mg g1 of pancreatin (EC 232-468-9;
8002500 units mg1 protein) at 37 C for 4 h, then the enzyme was
inactivated by boiling for 10 min followed by cooling to room temperature and centrifuging (10.000 g30 min). The supernatant was used
for ACEI activity determination.
This activity was also calculated as IC50. The IC50 value was dened as the concentration of peptide (g/ml) required to reduce
50% of absorbance peak height of hippuric acid, which was determined by regression analysis of ACE inhibition (%) versus protein
concentration.

AF189882.1

2.8. Sensory analysis

FJ196728.1
FN393983.1
AB449697.1

Bold strains were sequenced and subsequently compared with those available in the
EMBL nucleotide sequence database.

where A is the absorbance in presence of ACE and in presence of the ACEI

component, B is the absorbance with ACE and without the ACEI component, C is the absorbance without the ACE or ACEI component.
The protein content of the samples was determined using BC protein
assay (Bio-Rad Laboratories, CA, USA) with bovine serum albumin as
standard. Peptide content was measured using o-phthaldialdehyde
method as previously described by Church et al. (1983).
Peptide proles of the pH 4.6-soluble fractions for the most active
ACE inhibitors producers were also analyzed by RP-HPLC using an
ACTA Basic Instrument (Amersham Pharmacia Biotech, Milan, Italy)
and a Source 5RPC ST 4.6/150 column (Amersham Pharmacia Biotech).
The conditions of analysis were the same used by Chaves-Lpez et al.
(2006) The elution system consisted of A: 0.1% (v/v) of triuoroacetic
acid (TFA, sequential grade, Sigma) in HPLC grade water (Milli-Q
system, Waters Corp.) and B: 0.1% (v/v) TFA in 80% of acetonitrile
(HPLC grade) and HPLC grade water. Eluates were monitored at a
214 nm (Stepaniak and Fox, 1995). A gradient starting with 100% A for
5 min1 and continuing with a linear gradient to 70% B over 40 min1
and 100% B after 10 min1. The column was washed with 100% B for
10 min1, followed by equilibration with 100% A for 10 min1 before
injecting the following sample.

2.6. Dynamics of peptides with ACE inhibitory activity production in vitro

Among 18 yeast isolates able to produce peptides ACEI, six were
selected to evaluate the dynamics of ACEI activity production. These
strains were chosen for their ability to produce fermented milk with
high ACEI activity (>63%). Samples (250 ml of skim milk) were prepared
as above described, and aliquots of 4 ml were periodically collected (0,

A panel of six members who were previously trained to recognize

the basic tastes, conducted the sensory analysis. Training sessions
were carried out with standard solutions of lactose (sweet), L-leucine
(bitter) prepared in mineral water. The standard solutions were
presented to the untrained assessors, who were requested to identify
the tastes.
The assessors were requested to assess the taste of the fermented
skim milk by placing 2 ml aliquots directly onto their tongues and evaluating the presence and intensity of each basic taste (sweet and bitter).
Each attribute was rated by the assessors using a intensity scale of 1 to 5.

2.9. Statistic analysis

All the experiments were performed in triplicate and all the analyses
were carried out in duplicates.
The average and standard deviations were calculated for the experimental data, using analysis of variance (ANOVA). One-way ANOVA
was used to compare mean values of the data following Tukey test.
Spearman's rank correlation coefcient was used to evidence the possible
correlation between ACE inhibition and free NH3 groups production during skim milk fermentation. The relationship between the area of peaks
detected by HPLC analysis and ACE activity was tested by multiple partial least regression MPLSR which was applied to study cause-effect relationship between individual peaks (independent variables) and ACE
activity (response variable).
MPLSR was carried out using the SIMPLS algorithm (de Jong,
1993) and choosing a sigma restricted procedure and a model
with no intercept. All the independent variables were used to calculate the nal model which was eventually validated by adding 2
external samples to the data set, ve samples were used for computation and four samples for validation. Cross validation was carried
out and the optimal level of extracted factors was calculated by the
root mean square error of prediction (RMSEP). The adequacy of the
nal model was expressed by the optimum number of extracted
factors, the determination coefcient R 2 and the RMSEP. Data
were processed using the STATISTICA for Windows (StatSoft,
Tulsa, OK) package.

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C. Chaves-Lpez et al. / International Journal of Food Microbiology 159 (2012) 3946

3. Results
3.1. Identication and characterization of the yeast isolates
A total of 93 yeast isolates from Colombian Kumis were subjected
to RAPD-PCR followed by cluster analysis, using a conventional coefcient of similarity of 80% to characterize the identical biotypes and
thus narrow the number of isolates to identify. From RAPD-PCR analysis, we recognized 21 different electrophoretic proles (Table 1).
Moreover, to deduce the correct species assignment, the D1/D2 domain
of the 26S rRNA gene from 31 isolates, from 1 up to 4 representative of
the RAPD groups, were sequenced and subsequently compared with
those available in the EMBL nucleotide sequence database. Most of the
sequences obtained displayed similarity values ranging from 98 to
100% to reference sequences. In particular for KL35, KL38, KL33,
KL26A and KL2B, PCR-RFLP analysis of 5.8S-ITS was performed (data
not shown). As observed, the analysis evidenced three different biotypes for Galactomyces geotrichum and Clavispora lusitaniae and two
for Pichia kudriavzevii, Candida tropicalis, Wickerhamomyces pijperi. For
K. marxianus, Candida glabrata and S. cerevisiae no signicant differences
were determined.
The strains occurring as dominant yeasts were found to belong to 14
species shared among 11 genera. As shown in Table 2, G. geotrichum was
the most isolated species, representing 22.58% of the total yeasts
followed by P. kudriavzevii (13.9%), C. lusitaniae (11.8%) and C. tropicalis
(11.8%). W. pijperi (7.5%), K. marxianus, Candida glabrata, Candida
pararugosa and Kazachstania unispora (5.31%). The other species were
found less frequently. The species found in the samples, commonly observed in Kumis, were represented by K. marxianus and G. geotrichum
(7/13 samples) or C. tropicalis.

with particular attention to the fate of peptides with ACE-inhibitory

activity.
Based on the average of the OPA index, the strains were grouped
into three classes depending on their value of total proteolytic activity
(class I: from 0 to 1.0 mg/ml 1; class II: from 1.1 to 1.5 mg/ml 1,
class III: from 1.51 to 2.0 mg/ml 1). Most of the microorganisms
assayed (47 strains), belonging mainly to the species C. tropicalis,
K. marxianus, C. lusitaniae, Trichosporon insectorum and Trichosporon
asakii, showed OPA index higher than milk and were clustered in
class III, indicating a hydrolytic action against milk proteins. Class II
included mainly P. kudriavzevii, K. unispora, C. glabrata, C. pararugosa,
W. pijperi, while S. cerevisiae and Rodothorula mucilaginosa clustered
in class I. The greatest increase in total free amino acids (FAA) concentration was detected in samples inoculated with S. cerevisiae
(0.23 mM of leucine), C. glabrata, R. mucilaginosa and T. insectorum
(0.19 mM of leucine) (Table 2).
All strains were analyzed for the production of ACEI peptides.
Eigteen out of 93 strains were able to produce ACE inhibitors that reduced the activity of the enzyme from 8.69% to 89.19% (Table 3); apparently, this characteristic was not conned to a single species of
yeast but it was strain-dependent. However, P. kudriavzevii strains
were the most efcient to hydrolyze milk proteins into peptides
with higher ACEI activity, with inhibition values ranging from 72%
(strain KL84A) to 89% (strain KL52). Other strains such as K. marxianus
KL26A, C. lusitaniae KL4A and Torulaspora delbrueckii KL66A also
showed high values (80%, 76% and 74% respectively). Spearman's
rank correlation coefcient, used to evaluate the possible correlation
between ACEI activity and free NH3 groups production during skim
milk fermentation, did not show any correlation.
3.3. ACE-inhibitory activity dynamics in skim milk

3.2. Proteolysis of skim milk by the yeast species

To verify the potential role of the yeasts in Kumis proteolysis, all the
93 strains were evaluated for their ability to hydrolyze milk proteins,
Table 2
Frequency of yeast species isolated from Colombian Kumis samples and mean of peptide
content and free amino acids detected in fermented milks, inoculated with a single culture, after 52 h of fermentation at 28 C.
Species

Presence of the
species in the
samplesa

Candida tropicalis
7/13
Candida glabrata
4/13
Candida
5/13
pararugosa
Clavispora
7/13
lusitaniae
Galactomyces
13/13
geotrichum
Pichia kudriavzevii
7/13
Kazachstania
5/13
unispora
Kluyveromyces
3/13
marxianus
Rhodotorula
1/13
mucilaginosa
Torulospora
3/13
delbrueckii
Trichosporon
1/13
asakii
Trichospoon
2/13
insectorum
Saccharomyces
2/13
cerevisiae
Wickerhamomyces
7/13
pjiperi
a

Percentages Free amino

acids (mM of
of isolates
leucine)
(%)

OPA index
(mg/ml)

11.82
5.37
5.37

0.13 0.05A
0.19 0.09B
0.13 0.09A

1.68 0.23A
1.30 0.45B
1.43 0.19B

11.82

0.14 0.03A

1.67 0.32A

22.58

0.13 0.08A

1.43 0.90B

13.97
5.37

0.16 0.07B
0.14 0.05A

1.36 0.36B
1.38 0.30B

5.37

0.19 0.08B

1.69 0.21A

1.08

0.19 0.03B

0.94 0.28C

3.2

0.13 0.06A

1.70 0.21A

1.08

0.16 0.02B

1.72 0.10A

2.1

0.19 0.07B

1.69 0.41A

3.2

0.23 0.05C

0.96 0.08C

7.5

0.17 0.09B

1.40 0.21B

Referred to the total number of samples.

The results described above indicate that some yeast strains

inuenced the release of peptides with ACEI activity from skim
milk. For this reason, we investigated the dynamics of ACEI peptides
production in strains possessing an activity greater than 68%. Fig. 1
shows the yeast growth kinetics, as determined by cell counts. Cell
Table 3
ACE-inhibitory activity (%) of the milk fermented by different isolates from Kumis.
Species

Candida tropicalis
Candida glabrata
Candida
pararugosa
Clavispora
lusitaniae
Galactomyces
geotrichum
Pichia kudriavzevii
Kazachstania
unispora
Kluyveromyces
marxianus
Trichosporon
insectorum
Trichosporon asakii
Torulospora
delbrueckii
Rhodotorula
mucilaginosa
Saccharomyces
cerevisiae
Wickerchamomyces
pjiperi
Total

No. of
No. of
isolates positive
isolates

ACE inhibitory activity (%)

11
5
5

2
0
1

8.69

10.11

67.34

9.41 1.00

67.34 5.21

11

68.26

78.09

73.17 6.95

21

56.64

76.44

61.86 4.88

13
5

3
1

72.25

89.19
38.72

80.17 10.57

66.12

80.38

73.24 10.09

1
3

0
1

74.27

74.27 4.29

48.26

56.06

52.16 5.51

15.35

15.35 2.72

93

18

Minimum Maximum Mean and S.D.

of positive
strains

C. Chaves-Lpez et al. / International Journal of Food Microbiology 159 (2012) 3946

Fig. 1. Viable counts of yeast cells and pH during the fermentation of skim milk by K.
marxianus 26A (), T. delbrueckii KL66 (), C. lusitaniae KL4 (), P. kudriavzevii KL52
(), P. kudriavzevil KL84A (), G. geotrichum KL20A (). Solid line: log CFU ml1, Dotted line: pH.

growth was higher in C. lusitaniae KL4, T. delbrueckii KL66A and

K. marxianus KL26A with respect to P. kudriavzevii KL 84A and
G. geotrichum KL20A after 52 h of fermentation. During fermentation,
an increase in OPA index was observed (data not shown), often accompanied by an increase in ACEI activity of the product. In fact, in
all samples, the ACEI activity increased until stationary phase (52 h) although with different production rates; after that, a signicant activity
decrease was determined (Fig. 2). The best ACEI peptide production,
with a signicantly higher average content (pb 0.05), was observed in
skim milk fermented by the strains C. lusitaniae KL4A, G. geotrichum
KL20B and P. kudriavzevii KL52, whereas the lowest values were
determined for T. delbrueckii KL66A.
The degree of proteolysis at the higher ACEI activity (52 h) was
conrmed by RP-HPLC analysis of pH 4.6 soluble fractions (Fig. 3).
The peptide proles showed characteristic differences among species
and strains; K. marxianus KL26A, followed by C. lusitaniae KL4 and
T. delbrueckii KL66, showed the most efcient peptidases. In particular,
a remarkable proteolytic activity was observed in K. marxianus
KL26A with several marked peaks between 6 and 30 min (Fig. 3B),
conrming that substantial hydrolysis had taken place. On the
other hand, in C. lusitaniae KL4 (Fig. 3D), instead, the production of
several peptides was detected between 20 and 32 min. G. geotrichum
KL20B consumed peptides with a shorter retention times, whereas
peptides with a retention time between 18 and 30 min appeared at
the same time (Fig. 3C). The peptide proles of the two products
obtained with P. kudriavzevii strains (Fig. 3E, G), previously classied
in class II for proteolytic activity, were denitely different. In fact, in

43

the samples inoculated with the strain KL52, a clear reduction of the
peaks was observed between 18 and 25 min, with a higher concentration of the late-eluting peptides; in contrast, in the samples inoculated with the strain KL84A, an increase of peaks was detected
between 10 and 12 min. T. delbrueckii KL 66A was characterized by
a production of peptides with a retention time between 10 and
22 min (Fig. 3F). Relationships between the area of individual
peaks and ACE activity data was studied by MPLSR and the results
of the analysis were reported in Table 4 where peaks were dened
according to their retention time for practical purposes. The coefcient of determination of the calibration regressions was of 0.97
and the coefcient of determination of the validation regression
was of 0.85, indicating that the models has a good predictive ability.
Some peaks which were present in the blank sample showed a negative correlation with ACE activity but other peaks showed a positive
correlation with the dependent variable and are likely to be responsible for ACE activity. As shown MPLSR separated a set of peptides
whose amount is positively correlated to ACE activity thus ACE activity seems to be determined not only for one particular peptide but by
several peptides. Further studies should be carried out to isolate and
identify these compounds in order to better understand cause-effect
relationships.
3.4. Hydrolysis under simulated gastrointestinal conditions
ACEI activity of the fermented milks was assessed after a process
simulating gastrointestinal digestion. With this aim, pepsin and pancreatin digestion of supernatant of fermented skim milk was
performed. After digestion (Table 5), the ACEI activities were similar
to that of undigested crude fermented milks of C. lusitaniae KL4,
P. kudriavzevii KL52 and KL84 and G. geotrichum KL20, whereas an increase in ACEI activity was observed in the milk samples fermented
by the other strains.
ACE inhibitory activity of the six different fermented milks was
compared by determining the IC50 after simulated gastrointestinal digestion. Although this value can be overestimated in presence of free
amino acids interfering with the calculation of the peptide concentration and, more generally, because of the possible breakdown of large
peptides resulting from the proteolytic activity (Chaves-Lpez et al.,
2011a), this value can be considered a useful tool to compare the different fermented milks. As observed in Table 5, the most potent ACEI
peptides after digestion were evidenced in milks fermented by
K. marxianus KL26A (IC50 =9.59 g ml1) and C. lusitaniae KL4 (IC50 =
10.26 g ml1).
3.5. Selection of strains producing low bitterness peptides
Recent studies (Cheung and Li-Chan, 2010; Pripp and Ard, 2007)
associated peptide bitterness with ACEI activity. For this reason, sensory analysis was conducted on the fermented milks obtained in this
study. All fermented milks had good appearance and a pleasant
odour. However, a bitter taste was perceived with high intensity in
milks fermented by the strains with the most potent ACEI activity,
namely C. lusitaniae KL4 and P. kudriavzevii KL52. A slight bitter
taste was perceived for fermented milks produced by G. geotrichum
KL20A and K. marxianus KL26A. On the contrary, in samples inoculated with P. kudriavzevii KL84A no bitter taste was detected (Table 6).
4. Discussion

Fig. 2. Evolution of ACE inhibitory activity during the fermentation of skim milk by
K. marxianus 26A (), T. delbrueckii KL66 (), C. lusitaniae KL4 (), P. kudriavzevii
KL52 (), P. kudriavzevil KL84A (), G. geotrichum KL20A ().

Natural fermented milk beverages are produced in many countries of

the world and yeasts are involved in the production process of some of
them (Abdelgadir et al., 2001; Gran et al., 2003; Fleet, 2006). In different
Colombian Kumis many yeast species have been found, and most of them
have been commonly reported in indigenous Asian or African fermented
milks, contributing considerably to the development of the nal avours

44

C. Chaves-Lpez et al. / International Journal of Food Microbiology 159 (2012) 3946

457.95

457.95

406.55

406.55

355.15

355.15

303.75

303.75

252.35

252.35

200.95

200.95

149.55

149.55

98.15

98.15

46.75

46.75

-4.65

-4.65

-56.05

-56.05

-107.45

-107.45
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

D
457.95

457.95
406.55

406.55

355.15

355.15

303.75

303.75

252.35

252.35

200.95

200.95

149.55

149.55

98.15

98.15

46.75

46.75

-4.65

-4.65

-56.05

-56.05

-107.45

-107.45
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

F
457.95

457.95
406.55

406.55

355.15

355.15

303.75

303.75

252.35

252.35

200.95

200.95

149.55

149.55

98.15

98.15

46.75

46.75

-4.65

-4.65

-56.05

-56.05
-107.45

-107.45
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

G
457.95
406.55
355.15
303.75
252.35
200.95
149.55
98.15
46.75
-4.65
-56.05
-107.45
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

Fig. 3. Reverse phase liquid chromatography proles of water-soluble extract of the fermented milks obtained by using A: control; B: K. marxianus KL26A, C: G. geotrichum KL 20B,
D: C. lusitaniae KL4A, E: P. kudriavzevii KL84A, F: T. delbrueckii KL66A, G: P. kudriavzevii KL 52 A.

of the products (Fleet, 2006; Kebede et al., 2007; Lore et al., 2005; Njage et
al., 2011; Shuangquan et al., 2004). However, the predominant yeast consortium depends on its source of production with mixtures of lactose and
non-lactose fermenting species. In Asian fermented milks, the most prevalent species is K. marxianus often associated with Saccharomyces spp.

(cerevisiae, unisporus) or Pichia fermentans (Bai et al., 2010; Ni et al.,

2007; Watanabe et al., 2008), as well as Saccharomycopsis crataegensis associated with Candida castellii (Dewan and Tamang, 2006). Instead, in
African fermented milks the prevalence of S. cerevisiae, Candida spp.
(inconspicua, ker, crusei, zeilanoides), Pichia spp. (Gadaga et al., 2007;

C. Chaves-Lpez et al. / International Journal of Food Microbiology 159 (2012) 3946

Table 4
Results of partial least squares (PLS) regression analysis between ACE and the pick
areas of the peptides produced during fermentation.
Attribute Retention
times of the
peaks with
positive
correlation

Retention
times of the
peaks with
negative
correlation

Calibration Validation RMSEP Aopt

ACE

33.60*;
13.47;
11.86*;
26.22; 25.02;
28.59; 19.08;
29.86; 24.45;
17.10; 8.04.

0.97

23.72*; 19.76*;
33.73*; 14.37;
30.95; 28.38*;
26.57; 6.94*;
29.2; 31.16*;
20.54; 39.58;
22.28*; 18.84;
31.97; 27,32*

0.85

10

Signicant variables are marked (*). RMSEP: root mean square error prediction; Aop:
optimum number of component.

Jespersen, 2003; Njage et al., 2011) and also of D. hansenii, T. delbrueckii

and K. marxianus (Narvhus and Gadaga, 2003) has been reported. In this
study on Colombian Kumis, G. geotrichum and P. kudriavzevii were
among the main species, together with C. lusitaniae. It is important to
underline that the selective pressure exerted by the environmental conditions encountered by microbial cells during Kumis fermentation, accounts for the consolidated dominance of selected species of yeasts.
Moreover, the impact of the process variables and their changes during
continuous propagation enable the association of some yeast species to
endure also for years.
The ability of yeast proteases to release peptides from food proteins
has already been reported (Addis et al., 2001; Chaves-Lpez et al.,
2011b; Chen et al., 2011). In our study, yeasts from Kumis possessed a
clear proteolytic activity although at lower levels if compared with the
enterococci isolated from the same samples (Chaves-Lpez et al.,
2011a). Roostita and Fleet (1996) reported that K. marxianus showed
greater proteolytic activity than S. cerevisiae, as evidenced by the increase of the amino acids content in milk. However, according to our results, only two out of eight strains of K. marxianus released higher
quantities of FAA in comparison with those released by the three strains
of S. cerevisiae. This suggests a certain intra-species variability, as
reported by Hansen and Jakobsen (2001). In this study, differences in
proteolytic activity were also detected in C. lusitaniae and C. tropicalis.
As regards G. geotrichum, it has been proposed to discriminate two
groups, depending on the weak or strong proteolytic activity (Boutrou
et al., 2006; Gueguen and Jacquet, 1982). These differences, both in
terms of FAA content and OPA index, were determined also for the
strains isolated from Colombian Kumis. A similar behavior was
observed in P. kudriavzevii strains isolated from Kumis, with two different proteolytic patterns at low or high OPA index; in this respect, Chen
et al. (2011) suggested that P. kudriavzevii plays a role in promoting

Table 5
Angiotensin converting enzyme inhibition activity and IC50 (g/ml) values of the selected yeast after simulated physiological digestion.
Strain

Clavispora lusitaniae KL4

Pichia kudriavzevii KL52
Pichia kudriavzevii KL84A
Kluyveromyces marxianus KL26A
Torulaspora delbrueckii KL66A
Galactomyces geotrichum KL20B

Undigested

After digestion with

pepsin and pancreatin

% inhibition

IC50

% inhibition

IC50

82.59 3.46A
78.75 7.76A
67.25 4.01A
73.03 4.48A
65.72 2.72A
81.74 3.28A

11.21
20.32
29.10
10.37
22.54
11.21

90.70 5.74B
78.70 4.85A
60.28 5.60A
77.70 3.21A
76.59 4.76B
79.72 4.08A

10.26
19.39
31.28
9.59
21.13
12.25

Mean and standard deviation of three repetitions. Different letters in the same row mean
signicant differences (pb 0.05) among the treatments. IC50 (g/ml) is the concentration
of an ACE-inhibitor needed to inhibit 50% of ACE activity and the coefcient of IC50 variation was always lower than 5%.

45

Table 6
ACE-inhibitory activity (%) and panelist attribution of the milk fermented by different
yeast isolates after 52 h of fermentation at 30 C.
STRAIN

ACE inhibitory activity

IC50
(g/ml)

Intensity of bitter taste

Clavispora lusitaniae
KL4
Pichia kudriavzevii
KL52
Pichia kudriavzevii
KL84A
Kluyveromyces
marxianus KL26A
Torulaspora
delbrueckii KL66A
Galactomyces
geotrichum KL20B

82.53 5.21a

11.21

4.0*A

78.83 3.65b

20.32

3.75 0.41A

67.32 3.11c

29.10

1C

73.28 5.25b

10.37

1.66 0.59C

65.98 3.89c

22.54

1C

81.21 5.52ab

11.21

2.91 0.51D

Mean and standard deviation of three repetitions. IC50 is the concentration of an

ACE-inhibitor needed to inhibit 50% of ACE activity and the coefcient of IC50 variation
was always lower than 4%.
Different letters in the same column mean signicant differences (p b 0.05) among the
samples. * (1) null; (2) light; (3) medium; (4) strong and (5) very strong.

proteolytic activity in cheese. In our study, no generalization is possible

for the other yeast species, which were detected at low levels. However,
in the seven strains of Wickerhamomyces pjiperi, a similar proteolytic activity was observed, classied in class II, while no data were previously
reported on milk protein hydrolysis by this species.
A large variety of peptides with potential biological activities are
released during milk fermentation. In particular, the production of
ACEI peptides received special attention because of their benecial effects for hypertension control. In the present study, C. lusitaniae KL4,
G. geotrichum KL20A and P. kudriavzevii KL52 induced higher ACEI activity (from 78% to 82%) during skim milk fermentation after 52 h of
fermentation. Hamme et al. (2009) reported an ACE-inhibitory activity of 45% for K. marxianus in fermented whey after 168 h, while Jang
and Lee (2011) found 67.8% of ACE inhibition in red wine fermented
with S. cerevisiae. To our knowledge, this is the rst report of ACEI activity in C. lusitaniae.
As demonstrated by our data on the production kinetics of ACEI
peptides, the fermentation of milk by yeasts is prone to a dynamic
system where peptides are constantly released; some of them are
subsequently hydrolyzed and probably utilized for cell growth,
while others accumulate over fermentation. A similar trend has
been evidenced in milks fermented by lactic acid bacteria (ChavesLpez et al;, 2011a; Quirs et al., 2007; Ramachandran and Shah,
2008), although the maximum production was detected during the
rst 30 h. Moreover, Didelot et al. (2006) reported a sharp decrease
after 120 h of fermentation of C. parapsilosis in co-culture with
Lactobacillus paracasei. In addition, our results clearly evidenced an increase of ACEI activity in the samples after simulated gastrointestinal digestion; these skim milks may contain ACE-inhibitor precursors, which
can lead to the release of ACEI peptides during the simulated digestion.
It is well known that the production of high quality fermented
dairy products depends on the proteolytic system of the starter
used, since peptides and the amino acids formed have a direct impact
on avour or act as avour precursors (Williams and Banks, 1997). In
this respect, we observed the impact of proteolysis in the production
of bioactive peptides and its contribution to increase bitterness in the
fermented milks. As reported above, the most potent ACE inhibitors
in products fermented by C. lusitaniae KL4 and P. kudriavzevii KL52
produced also the most bitter taste. Pripp and Ard (2007) provided
experimental evidence of the relationship between bitterness and
ACEI activity of protein hydrolysates. In addition, Kawakami et al.
(1995) reported that the most potent bitterness was found when
phenylalanine was present in the C-terminal of peptides with a potent ACEI activity. This potential correlation between ACEI activity
and bitterness might be a challenge to the consumer acceptance of

46

C. Chaves-Lpez et al. / International Journal of Food Microbiology 159 (2012) 3946

milks containing these bioactive peptides as functional food ingredients. In fact, if yeasts producing bioactive peptides are used in milk
fermentation, the sensory attributes as taste should be taken into
account.
Results from this study allow us to conclude that strains P. kudriavzevii
KL84A and K. marxianus KL26A could be selected to be included as
adjunct starter cultures in kumis, since they exert a considerable contribution to the ACEI activity in fermented milk. Moreover they did not produce milk with a bitter taste. These activities should be considered also in
co-culture with lactic acid bacteria to understand the role of microbial interaction on ACEI activity in fermented milk. For this reason, the yeast
strains will be further evaluated in pilot scale trials as a part of several
mixed starter cultures, in order to determine their inuence on avour
formation and ACEI activity.

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