Influence of adjunct cultures on angiotensin-converting enzyme (ACE)-inhibitory activity, organic acid content and peptide profile of kefir

Abstract

The angiotensin-converting enzyme (ACE)-inhibitory activities, peptide profiles and organic acid contents in kefir produced by kefir grains plus lactic acid bacteria as adjunct cultures were determined. All the kefir samples showed almost similar peptide profiles as detected by RP-HPLC, but quantitative differences were observed during storage. The ACE-inhibitory activities of different lactic cultures did not exhibit a linear tendency during storage period. After 7 days of storage, there was a significant increase in ACE-inhibitory activity of the sample fermented with Lactobacillus helveticus. However, a kefir sample containing Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis exhibited a higher ACE-inhibitory activity (92.23%) compared to the other samples.

Full text

ORIGINAL
RESEARCH Influence of adjunct cultures on angiotensin-converting
enzyme (ACE)-inhibitory activity, organic acid content
and peptide profile of kefir
TUBA S
ßANLI,
1
* HAVVA CEREN AKAL,
1
ATILA YETIS
ßEMIYEN
1
and
ALI ADNAN HAYALOGLU
2
1
Agricultural Faculty, Department of Dairy Technology, Ankara University, 066110, Dıs
ßkapı, Ankara, Turkey, and
2
Engineering Faculty, Department of Food Engineering, In€
on€
u University, 44280, Malatya, Turkey
The angiotensin-converting enzyme (ACE)-inhibitory activities, peptide profiles and organic acid
contents in kefir produced by kefir grains plus lactic acid bacteria as adjunct cultures were deter-
mined. All the kefir samples showed almost similar peptide profiles as detected by RP-HPLC, but
quantitative differences were observed during storage. The ACE-inhibitory activities of different
lactic cultures did not exhibit a linear tendency during storage period. After 7 days of storage,
there was a significant increase in ACE-inhibitory activity of the sample fermented with Lactobacil-
lus helveticus. However, a kefir sample containing Streptococcus thermophilus,Lactobacillus
acidophilus and Bifidobacterium animalis subsp. lactis exhibited a higher ACE-inhibitory activity
(92.23%) compared to the other samples.
Keywords Kefir, Fermented milk, ACE-inhibitory activity, Adjunct culture, Peptide profile.
INTRODUCTION
Kefir is a traditional fermented milk beverage
that is produced by lactic and alcoholic fermen-
tation (Muir et al. 1999). Kefir differs from
other milk products by being a product of fer-
mentation with a mixed microbiota called ‘kefir
grain’(Simova et al. 2002; G€
uzel-Seydim et al.
2011). The kefir grain, which is a unique natural
starter culture for kefir, contains homo- and
heterofermentative lactic acid bacteria (LAB),
acetic acid bacteria and yeasts (Beshkova et al.
2002; G€
uzel-Seydim et al. 2011). The basic
microbiota of kefir grains consists of different
species of yeast such as Saccharomyces cere-
visiae, Sacch. delbrueckii, Candida kefir, Kluy-
veromyces lactis (G€
uzel-Seydim et al. 2011);
LAB such as Lactobacillus kefir, Lb. kefiranofa-
ciens, Lb. helveticus, Lb. casei, S. thermophilus,
S. durans,Lactococcus cremoris,Lac. lactis
(Y€
uksekdağet al. 2004; G€
uzel-Seydim et al.
2011) and natural probiotics mainly Lb. aci-
dophilus,B. bifidum and sometimes acetic acid
bacteria (G€
uzel-Seydim et al. 2011). The micro-
organisms present in kefir grains live symbioti-
cally in equilibrium; however, the population
may change significantly depending on the ori-
gin of grains (Beshkova et al. 2002). Kefiris
recommended for consumption owing to its
potential health benefits, such as antibacterial,
antihypertensive, antimutagenic, antitumor and
antiallergic effects, which are produced by the
metabolic activities of the microbiota in the kefir
grains (Irigoyen et al. 2005; G€
uzel-Seydim
et al. 2011).
Bioactive peptides are considered to be specific
protein fragments, which are liberated from milk
proteins during fermentation of some dairy foods.
Among various bioactive peptides, the ACE inhi-
bitor or antihypertensive peptides are the most
intermediate products in fermented milks (Donkor
et al. 2007; Gonzalez-Gonzalez et al. 2011).
Antihypertensive peptides play an important role
by inhibiting ACE that is associated with the reg-
ulation of blood pleasure (Nakamura et al. 1995;
Papadimitriou et al. 2007).
The presence of peptides with antihyperten-
sive activity has been described in various fer-
mented milk products including kefir. Most
studies have shown that the antihypertensive
peptides can be produced by starter bacteria dur-
ing fermentation, using the proteins in milk
*Author for
correspondence. E-mail:
tcetin@agri.ankara.edu.tr
©2016 Society of
Dairy Technology
Vol 71, No 1 February 2018 International Journal of Dairy Technology 131
doi: 10.1111/1471-0307.12346

(Nakamura et al. 1995; Donkor et al. 2007; Papadimitriou
et al. 2007; Pihlanto et al. 2010; Gonzalez-Gonzalez et al.
2011). In particular, different strains of lactic acid bacteria
such as Lb. helveticus (Nakamura et al. 1995; Nielsen et al.
2009; Otte et al. 2011), Lb. acidophilus and Lb. casei
(Donkor et al. 2007) exhibited high levels of ACE-inhibi-
tory activity in fermented milk. However, only a few studies
have been published on the ACE-inhibitory activity of kefir
(Maeda et al. 2004; Quiros et al. 2005) and it needs to be
further studied. The aim of this study was to investigate the
effects of traditional kefir grains and some adjunct cultures
on the ACE-inhibitory activity, RP-HPLC peptide profile
and organic acid content of kefir during 28 days of storage.
MATERIAL AND METHODS
Materials
Hippuryl-L-histidyl-L-leucine (Hip-His-Leu), ACE (from
rabbit lung, 0.25 U) and other chemicals were obtained
from Sigma-Aldrich Co. (St. Louis, MO, USA). Kefir grains
were obtained from the Pilot Dairy Plant of Ankara Univer-
sity Agricultural Faculty (Ankara, Turkey). In the labora-
tory, pasteurised milk was inoculated with kefir grains and
incubated at 22 1°C for 22–24 h (until 4.6 pH). At the
end of the fermentation, the resultant product was strained
using a plastic strainer and the kefir grains were separated
after washing under cooled tap water. The grains were kept
at 4 °C for the next production.
The adjunct cultures used in the manufacture of the kefir
samples were Lb. casei (LBC) and Lb. helveticus (LH) and
the PRE1 mixed culture containing S. thermophilus,
Lb. acidophilus and B. animalis subsp. lactis. These cul-
tures [obtained from Maysa Culture Company (Istanbul,
Turkey)] were in freeze-dried form and used as direct vat
inoculation (DVS) at 5 IU per 500 L of milk according to
the manufacturer’s recommendation. For activation, the cul-
tures were kept at room temperature for 25–30 min before
inoculation and each culture was mixed homogeneously in
100 mL sterilised milk.
Preparation of kefir samples
Raw milk used in the production of kefir samples was pro-
vided by the Pilot Dairy Plant of Agricultural Faculty
(Ankara University, Ankara, Turkey). The total dry matter,
total protein and fat content of the raw cows’milk used for
the kefir production were 11.54 0.06, 3.38 0.19 and
4.0 0.17 g/100 mL, respectively. After standardisation of
fat (min 2 g/100 mL), the kefir milk was heated at 95 °C
for 5 min, then cooled to 25 °C and divided into two sepa-
rate portions for the production. The first portion of milk
used to produce traditional kefir (A) was inoculated with
3% (w/v) kefir grains. Incubation was carried out at 22 °C
until 4.4–4.5 pH (which takes about 22 h). After fermanta-
tion, the grains were separated from kefir beverage by
filtration. The stirred beverage was portioned into 300-mL
plastic cups and stored. For the second portion, the heated
milk was incubated at 22 °C until it reached pH 4.8 by the
addition of kefir grains at a level of 3% (w/v). After separa-
tion of the kefir grains, the fermented milk (i.e. kefir bever-
age) was divided into four equal batches. Each batch was
inoculated with different bacterial adjunct cultures as fol-
lows: kefir B with Lb. casei,kefir C with Lb. helveticus,
kefir D with Lb. casei plus Lb. helveticus and kefir E with
S. thermophilus,Lb. acidophilus and B. animalis subsp.
lactis (PRE1). The second incubation was carried out at
28 1°C until pH 4.4–4.5 was reached, and the resultant
kefir was portioned into 300-mL plastic cups. Three separate
productions were made at 1-week intervals and all types of
kefir samples were stored at 4 °C for 28 days. Figure 1
shows the flow diagram for kefir production.
Chemical analysis
Titratable acidity, total dry matter and the fat content of
samples were determined by the methods described in Hooi
et al. (2004). Protein content was determined by the micro-
Kjeldahl method (IDF 1993). The pH value was measured
using a digital pH meter (OHAUS, Starter 3000, Germany).
Lactic acid content was determined using a spectrophoto-
metric method as described by Steinsholt and Calbert
(1960).
Determination of ACE-inhibitory activity
For ACE-inhibitory activity, the pH of the kefir was
adjusted to 3.4 using 50% (v/v) lactic acid (Merck Chemi-
cals Ltd, Dorset, UK) and the kefir was then centrifuged at
8000 9g(model 3-18K; SIGMA, Osterode am Harz, Ger-
many) for 10 min at 4 °C. The pH of the supernatant was
adjusted to 8.3 using 1 N NaOH and it was recentrifuged at
the same conditions. The final supernatant was used to
determine ACE-inhibitory activities of the kefir samples.
The ACE-inhibitory activity was measured by a spec-
trophotometric assay according to the method of Cushman
and Cheung (1971) with some minor modifications. A
100 lL of 5 mmol Hip-His-Leu substrate solution (dissolved
in a 0.1 Msodium borate buffer containing 300 mmol NaCl,
pH 8.3) was mixed with 40 lL of the supernatant described
above and then incubated at 37 °C for 3 min. The reaction
was initiated by adding 20 lL of ACE (0.1 U/mL) and the
mixture incubated at 37 °C for 30 min. The reaction was
stopped by the addition of 150 lLof1MHCl. Hippuric acid
formed by ACE action was extracted with 1 mL of ethyl
acetate. After stirring for 30 s, 750 lL of organic phase was
transferred to a glass tube and then the ethyl acetate was
evaporated to dryness in a water bath at 100 °C. The residue
containing hippuric acid was dissolved in 1 mL of distilled
water and the absorbance was measured at 228 nm (UV/VIS
spectrophotometer, Perkin Elmer, Lambda 25, Singapore).
The ACE activity was calculated using the equation:
132 ©2016 Society of Dairy Technology
Vol 71, No 1 February 2018

ACE-inhibitory activity %¼ðACÞ=ðABÞ100
where Ais the absorbance without the whey fraction, Bis
the absorbance without ACE, and Cis the absorbance in the
presence of both ACE and the whey fraction.
RP-HPLC analysis of peptide in kefir samples
A 20 g sample of kefir was mixed with an equal volume of
water and homogenised at 15 000 g (Ultra-Turrax, model
T25 basic; IKA Werke, Staufen, Germany) for 2 min. The
supernatant was centrifuged at 14 000 9g(Hettich model
320 R; Tuttlingen, Germany) for 15 min at +4°C and
Milk
Fat standardisation (min 2 g/100 mL)
Heat treatment (90 oC, 5 min)
Cooling (25 oC)
Inoculation with kefir grains (3%,w/v)
Inoculation with kefir grains (3%,w/v)
Fermentation (22–23 oC, until pH 4.5–4.45)
Primarily fermentation (22– 23 o
C,
until pH 4.8 pH)
Separation of kefir grains
Separationof kefir grains
Distribution into cups
Inoculation (5 U/L)
Storage (+4 oC)
Lb.casei
Lb. helveticus
Lb.casei+Lb.helveticus
PRE1
Kefir B
Kefir C
Kefir D
Kefir E
Kefir A (Traditional kefir)
Secondary fermentation (28 ± 1 oC, until pH 4.5–4.45)
Distribution into cups
Storage (+4oC)
Kefir B, C, D and E
Figure 1 Flow chart of performed experiments.
©2016 Society of Dairy Technology 133
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filtered through Whatman No. 1. A 500 lL sample of fil-
trate was then filtered through an AmiconâUltra 3KDa
membrane (Merck Millipore Ltd. Cork, Ireland). A 80 lL
sample of retentate was injected into a Shimadzu LC 20 AD
Prominence HPLC system (Shimadzu Corporation, Kyoto,
Japan). A Phenomenex Jupiter C18 column with a size of
250 94.6 mm 95lm, 300 
A (Phenomenex Co, Torrance,
CA, USA) was used. The solvents were as follows: (A)
0.1% (v/v) trifluoroacetic acid (TFA, sequencing grade;
Sigma-Aldrich Laborchemikalien GmbH, Seelze, Germany)
in deionised HPLC grade water (Milli–Q system; Waters
Corp., Molsheim, France) and (B) 0.1% (v/v) TFA in ace-
tonitrile (HPLC grade; Merck KGaA, Darmstadt, Germany)
at a flow rate of 0.75 mL/min. Samples were eluted initially
with 100% A for 10 min, then with a gradient from 0% to
50% B and 50% to 60% B over 80 min and 5 min, respec-
tively. It is maintained at 60% B for 5 min, followed by a
linear gradient from 60% to 95% B over 5 min and main-
tained at 95% B for 5 min. Elute was monitored at 214 nm.
RP-HPLC of organic acids
A 5 g sample of kefir was dissolved in 10 mL of mobile
phase (0.005 N sulphuric acid in deionised HPLC grade
water) and homogenised using Ultra-Turrax T15 for 20 s at
14 500 g and then centrifuged for 20 min at 16 000 g.
One mL of supernatant was filtered through an 0.45-mm
PTFE syringe filter and a 20 lL sample was injected into
the HPLC. The analysis was performed using the same
HPLC system that was used for peptide analysis. Organic
acids were identified at 210 nm wavelength and were calcu-
lated by comparison of retention times and peak areas with
authentic standard solutions of each organic acid. Separation
was performed on a Rezex ROA organic acid column
(300 97.8 mm; 00H–0138-K0; Phenomenex Co, Torrance,
CA, USA) for organic acids. The running temperature was a
constant 55 °C, as described in Demir et al. (2014). The
results were expressed as mg/L of kefir and the analysis
was performed in triplicate.
Statistical analysis
Statistical analyses were carried out using the software
Minitab 13.0 (Minitab INC., PA, USA). Experiments were
organised in a randomised complete block design to deter-
mine the influence of adjunct cultures and storage. Duncan’s
multiple comparison test was used to determine the signifi-
cances of differences between means. The level of signifi-
cance was assessed at P<0.05 (Rosner 2006). All
experiments were carried out with three replicates and
parameters were presented as means standard deviation.
RESULTS AND DISCUSSION
Table 1 presents some chemical properties of the kefir sam-
ples. The pH values of the kefir samples ranged between
4.46 and 4.52 on day one, and decreased steadily with stor-
age time (P<0.01). It was observed that the same trend
Table 1 Change in titratable acidity, pH and lactic acid contents of kefir samples
Parameter
Storage
time (day)
Kefir Samples
ABCDE
Titratable acidity (SH) 1 37.18 0.58 36.50 2.33 38.65 2.57 36.31 0.22 37.29 0.11
7 39.04 0.44 39.19 0.41 37.72 0.77 37.39 0.54 37.25 1.01
14 40.60 1.56 40.11 0.41 39.78 3.93 38.66 1.55 37.86 1.25
21 41.12 1.12 39.95 0.77 39.67 1.43 40.11 1.56 39.54 1.72
28 44.68 0.39 42.44 0.00 42.32 5.31 43.16 1.65 41.46 0.90
pH 1 4.46 0.06 4.48 0.05 4.52 0.06 4.52 0.02 4.50 0.07
7 4.45 0.06 4.48 0.02 4.51 0.09 4.48 0.04 4.50 0.08
14 4.39 0.03 4.43 0.06 4.48 0.07 4.47 0.03 4.47 0.05
21 4.39 0.09 4.42 0.05 4.43 0.10 4.44 0.05 4.44 0.03
28 4.37 0.08 4.35 0.06 4.37 0.20 4.38 0.08 4.41 0.05
Lactic acid (g/100 g) 1 0.78 0.01 0.75 0.03 0.76 0.03 0.76 0.04 0.71 0.03
7 0.81 0.17 0.78 0.11 0.79 0.11 0.79 0.15 0.75 0.09
14 0.82 0.06 0.81 0.15 0.82 0.07 0.79 0.03 0.80 0.02
21 0.82 0.08 0.87 0.08 0.90 0.13 0.84 0.10 0.88 0.09
28 1.03 0.37 1.01 0.28 1.01 0.38 1.04 0.30 0.97 0.38
Results are expressed as mean SD of three replicates.
A: Control kefir containing solely kefir grains; B: kefir sample containing kefir grains +Lb. casei C: kefir sample containing kefir grains +
Lb. helveticus;D:kefir sample containing kefir grains +Lb. casei +Lb. helveticus;E:kefir sample containing kefir grains +PRE1 (S.
thermophilus,Lb. acidophilus and B. animalis subsp. lactis).
134 ©2016 Society of Dairy Technology
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Table 2 Change in organic acid (mg/L) contents of kefir samples
Organic
acids
Storage
time (day)
Kefir Samples
ABCDE
Hippuric acid 1 286.30 3.65 293.40 1.70 292.4 22.5 309.75 2.02 262.73 2.28
7 246.74 0.48 258.41 1.71 255.67 1.77 296.94 1.44 238.08 0.00
14 251.90 1.47 312.85 1.65 239.55 3.89 275.04 2.29 236.57 5.39
21 257.04 5.94 210.65 0.67 242.61 0.98 265.1 27.5 252.84 0.00
28 329.23 1.68 273.95 0.75 264.17 3.58 281.58 2.91 271.37 1.34
Citric acid 1 1.10 0.00Bab 1.08 0.00Ab 1.12 0.00Aa 1.08 0.00Ab 1.10 10.00Aab
7 1.04 0.00Cc 1.06 0.00Abc 1.08 0.00Bab 1.04 0.00Bc 1.10 0.00Aa
14 1.08 0.00Ba 0.97 0.04Bc 1.04 0.00Cb 1.04 0.02Bb 1.06 0.00Bab
21 1.15 0.01Aa 0.98 0.00Bb 1.00 0.00Db 1.00 0.00Cb 1.13 0.01Aa
28 1.14 0.02Aa 0.98 0.00Bb 0.98 0.00Db 1.00 0.00Cb 1.13 0.01Aa
Pyruvic acid 1 50.58 1.13Ab 41.93 0.01Ac 77.61 1.28Aa 40.20 0.67Bc 44.64 0.42Cc
7 43.51 0.91Bcd 39.23 0.72Ad 50.31 0.27Bb 45.28 0.31Ac 55.18 0.00Aca
14 39.81 0.86Bc 25.47 7.25Bd 45.93 0.32Cab 41.79 0.27ABbc 49.33 0.35Ba
21 16.59 0.04Cb 19.82 0.02Cb 30.63 0.46 Da 28.38 0.25Ca 18.07 0.01Db
28 12.24 0.02Dc 14.84 0.17 Da 25.62 0.25Ea 19.36 0.65Db 17.47 0.01Dbc
Succinic acid 1 1.62 0.08Ab 1.54 0.00Ab 1.35 0.09Ac 1.12 0.05Bd 1.80 0.00Aa
7 1.55 0.07Aa 1.19 0.09Bc 1.25 0.04Ac 1.31 0.01Ab 1.12 0.00Bc
14 1.35 0.07Ba 0.76 0.11Cc 1.10 0.00Bb 1.34 0.00Aa 0.80 0.02Cc
21 0.98 0.00Cb 0.70 0.00Cd 0.84 0.05Cc 1.17 0.01Ba 0.58 0.00Dd
28 0.68 0.00Dd 0.80 0.00Cdc 0.78 0.00Cb 1.08 0.02Ba 0.54 0.00Dc
Uric acid 1 1.56 0.08BCb 1.51 0.01Ab 1.69 0.07ABb 1.32 0.06Ab 2.42 0.00ABa
7 1.50 0.08Cbc 1.23 0.09ABcd 1.72 0.06ABb 1.02 0.03ABd 2.72 0.00Aa
14 1.70 0.08ABCb 1.09 0.01Bc 1.92 0.00Aab 1.07 0.01Ac 2.22 0.02BCa
21 1.99 0.15Aa 1.02 0.00Bb 1.63 0.07ABa 1.14 0.00Ab 1.98 0.06CDa
28 1.93 0.07ABa 0.24 0.00Cd 1.52 0.00Bb 0.67 0.58Bc 1.70 0.03Dab
Acetic acid 1 5784.1 118.8A 5552.0 1.30B 5612.6 40.2B 5487.4 17.5C 5557.4 22.1B
7 5635.9 7.96A 5764.3 105.4B 5819.9 72.4AB 5798.5 18.5BC 5991.8 0.00A
14 5807.6 10.6Aab 5598 208Bb 5693.0 7.00ABab 6038 200Ba 5794.6 82.4ABab
21 58880.4 93.7Aa 5387.7 2.33Bb 6075 175Aa 5769.5 4.85BCa 5983 341Aa
28 6035.8 1.43Abc 6166 253Aab 5737.7 9.69ABc 6584 300Aa 6182 158Aab
Propionic acid 1 119.3 1.90A 116.0 0.69A 128.2 4.36A 110.2 3.04B 123.16 9.48AB
7 115.8 1.37A 124.6 1.13A 131.3 0.04A 114.3 0.42B 133.16 0.00A
14 120.1 0.06A 66.8 23.7AB 120.6 0.18AB 65.9 20.2BC 135.67 0.81A
21 167.5 112.7Aa 80.7 1.94ABb 57.7 19.1BCb 231.4 2.63Aa 194.9 108.1Aa
28 31.90 0.76B 31.4 2.02B 22.0 1.13C 20.39 0.91C 59.8 20.3B
Results are expressed as mean SD of 3 replicates.
Uppercases indicate that the values in the same column differ significantly (P<0.01).
Lowercases indicate that the values in the same line differ significantly (P<0.01).
A: Control kefir containing solely kefir grains; B: kefir sample containing kefir grains +Lb. casei; C: kefir sample containing kefir grains +Lb. helveticus;D:kefir sample containing
kefir grains +Lb. casei +Lb. helveticus;E:kefir sample containing kefir grains +PRE1 (S. thermophilus,Lb. acidophilus and B. animalis subsp. lactis).
©2016 Society of Dairy Technology 135
Vol 71, No 1 February 2018

occurred in other fermented milk products like yoghurt
(Beshkova et al. 2002; Irigoyen et al. 2005). Use of adjunct
cultures in the manufacture of the kefir did not significantly
change the pH value of the kefir samples (P>0.05). Simi-
lar results were obtained by Beshkova et al. (2002) who
concluded that the pH of kefir samples made with starter
culture and kefir grains was almost at the same level
(P>0.05). Parallel results were obtained for the titratable
acidity and lactic acid content of the kefir samples. Titrat-
able acidity values of the kefir samples ranged from 0.71%
to 1.04% at 1 and 28 days of storage, respectively, and
these values were in accordance with other studies (Muir
et al. 1999; Beshkova et al. 2002).
The organic acid content of the kefir samples is shown in
Table 2. The hippuric and citric acid levels were similar in
all samples. The concentrations of pyruvic, succinic and uric
acid in the kefir samples significantly decreased during stor-
age (P<0.05). The concentration of acetic acid, which is
one of the main organic acids in kefir beverage, was found
to be quite high compared to the other organic acids and
was therefore considered to be principal organic acid in the
kefir. This was probably due to the presence of acetic and/
or lactic acid micro-organisms in the kefir grains, which use
the heterofermentative pathway during fermentation of kefir
milk (Alvarez-Martin et al. 2008; Magalhaes et al. 2010).
The use of different adjunct cultures did not cause any con-
siderable change in the propionic acid content.
Significant differences were observed in the ACE-inhibi-
tory activities of kefir samples produced by different adjunct
cultures and the changes were not associated with the
duration of storage period. Some fluctuations were observed
in the levels of ACE-inhibitory activities of some samples
as shown in Figure 2. These may be linked to differences in
the peptidase specificities of the adjunct cultures (Gonzalez-
Gonzalez et al. 2011) and the rate of degradation of pep-
tides by the cultures during fermentation (Donkor et al.
2007). The changes in the levels of ACE activities in the
samples may be linked to the duration of storage period
with dependence of the type of peptides produced during
storage. The highest ACE-inhibitory activity was deter-
mined on the first day of storage in the traditional kefir sam-
ple (Kefir A). However, the ACE-inhibitory activity in the
control sample decreased sharply after the first week
(P<0.01). In other samples, the ACE-inhibitory activity
increased over the storage period and reached a maximum
level at the end of storage regardless of treatment, as shown
in Figure 2. This indicates a modification of peptides by the
microbiota and the release of new peptides during the pro-
longed storage (Gobetti et al. 2000; Ryhanen et al. 2001).
Among the lactic acid bacteria, it has been reported that
Lb. helveticus can produce peptides with high ACE-inhibi-
tory activity due to this species’strong proteolytic activity
(Leclerc et al. 2002; Lopez-Fandino et al. 2006; Nielsen
et al. 2009). This study also found that prolonged storage
of kefir samples fermented with L. helveticus (sample C)
resulted in high ACE-inhibitory activity, as shown in
Figure 2. After 7 days of storage, an increase was observed
in the ACE-inhibitory activity associated with a decline in
pH values from 4.48 to 4.37 (see Table 1 and Figure 2).
This result indicated that the ACE-inhibitory activity of milk
fermented with Lb. helveticus increased with a decrease in
pH from pH 4.6 to 4.3 during fermentation (Otte et al.
2011). However, Nielsen et al. (2009) reported that ACE-
inhibitory activity of milk products fermented by Lb.
helveticus varied significantly with strain.
A lower level of ACE-inhibitory activity was obtained
in kefir (sample B) fermented with L. casei (<50% at
48 h, Figure 2) when compared to the other samples. In
contrast with this result, Gonzalez-Gonzalez et al. (2011)
reported that Lb. casei exhibited more than 90% of ACE-
inhibitory activity after 48 h of milk fermentation. How-
ever, Lb. casei showed an increase in ACE-inhibitory
activity in kefir during storage, which reached a level of
72.75% after 28 days storage. This value was similar to
those obtained by Pihlanto et al. (2010) who reported that
Lb. casei-17 produced 74% ACE-inhibitory activity after
44 h fermentation. This may be due to the fact that the
strain Lb. casei-17 produced a lower level of ACE-
Figure 2 CE-inhibitory activity of kefir samples. A: Control kefir contain-
ing solely kefir grains; B: kefir sample containing kefir grains +L. casei C:
kefir sample containing kefir grains +Lb. helveticus;D:kefir sample con-
taining kefir grains +Lb. casei +Lb. helveticus;E:kefir sample containing
kefir grains +PRE1 (S. thermophilus,Lb. acidophilus and B. animalis
subsp. lactis). [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 3 RP-HPLC peptide profile of kefir samples after 1 and 28 days of storage. A: Control kefir containing solely kefir grains; B: kefir sample
containing kefir grains +Lb. casei; C: kefir sample containing kefir grains +Lb. helveticus;D:kefir sample containing kefir grains +Lb. casei +Lb.
helveticus;E:kefir sample containing kefir grains +PRE1 (S. thermophilus,Lb. acidophilus and B. animalis subsp. lactis). [Colour figure can be
viewed at wileyonlinelibrary.com]
136 ©2016 Society of Dairy Technology
Vol 71, No 1 February 2018

©2016 Society of Dairy Technology 137
Vol 71, No 1 February 2018

inhibitory peptides. Furthermore, none of changes were
observed in kefir samples fermented with adjunct cultures
of Lb. helveticus and Lb. casei (sample D).
The highest ACE-inhibitory activity (>90%) was deter-
mined after 7 days of storage in sample E containing
S. thermophilus,Lb. acidophilus and B. animalis subsp.
lactis. A sharp decrease was observed in ACE-inhibitory
activity of sample E during storage: it declined from
92.23% (day 1) to 44.25% (day 28) (of storage
(P<0.01). This indicates that the ACE-inhibitory peptides
had broken down into lower MW peptides with possibly
lower ACE-inhibitory activity, and amino acids depending
on peptidase specificity of the micro-organisms during the
prolonged storage time (Donkor et al. 2007; Nielsen et al.
2009).
RP-HPLC peptide profiles of the kefir samples were
explored after 1, 7, 14, 21 or 28 days of storage (Figure 3).
There were only small differences in the RP-HPLC chro-
matograms of the samples; substantial age-related changes
were also observed. Almost the same peptide profiles with
only small age-related changes were observed for all sam-
ples during storage therefore, only the RP-HPLC peptide
profiles of the 1- and 28-day-old samples are shown in
Figure 3. However, the changes occurring during storage
were discussed with citations for all sampling times. High
ACE-inhibitory activity was determined in samples A
(control) and C (74.07% and 62.02%, respectively) on the
first day of storage (Figure 3). Early eluting peptides with
retention time of 10 min were found to be of higher peak
areas than the peptides with later retention times. It can be
considered that the peaks might be related to the peptides
with ACE-inhibitory activity that hydrolysed faster. The
peptide profiles of kefir samples were similar over 7 and
14 days of storage (chromatograms not shown), expect for
sample E. The peptide profile of sample E changed mark-
edly after 7 days storage. The increase in the height of
peaks at retention time of 40 min and late eluting peaks
(between 80 and 90 min) indicate a significant increase in
ACE-inhibitory activity (92.23%).
The peak heights between retention times of 40 and 50
min in the chromatograms of samples of kefir fermented
with Lb. casei plus Lb. helveticus (sample D) declined after
2 days of storage, which might be related to low ACE-
inhibitory activity (38.48%). Interestingly, peptide profiles
of samples B, C and D which showed higher ACE activity
(72.75%, 87.53% and 81.93%, respectively) were signifi-
cantly different after 28 days of storage. According to these
results, the variation of time-dependent peptide profiles of
the samples indicates differences in the peptidase activity of
the lactic acid bacteria. However, it was considered that the
levels of ACE-inhibitory activity of the kefir samples were
directly related to height of peaks with intermediate reten-
tion time (between 40 and 50 min).
CONCLUSION
The results showed that ACE-inhibitory peptides naturally
formed in kefir prepared traditionally by inoculation of kefir
grains. These findings suggest that kefir containing ACE-
inhibitory peptides may have potential as functional foods
for the prevention of hypertension. The production of kefir
containing Lb. helveticus, Lb. casei and Lb. acidophilus
strains as adjunct cultures did not significantly contribute to
the level of ACE-inhibitory peptides although small
differences were observed among the samples. Some differ-
ences were found in RP-HPLC peptide profiles of kefir sam-
ples fermented with adjunct lactic acid bacteria and these
may indicate the proteinase specificity of the adjunct
cultures. Similarly, the observed differences in organic acid
production may be due to variations in the ratio and types
of micro-organisms in the adjunct cultures. In conclusion,
lactic acid bacteria used as adjunct cultures in kefir produc-
tion contributed in different ways to the accumulation of
organic acid and peptides and also slightly contributed to
the formation of ACE-inhibitory peptides.
ACKNOWLEDGEMENT
This project was financially supported by the Scientific
Research Projects Coordination Unit of Ankara University
(Project No: 12B4347003).
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