nutrients

Article
Mechanistic Pathways Underlying the
Antihypertensive Effect of Fermented Milk with
Lactococcus lactis NRRL B-50571 in Spontaneously
Hypertensive Rats
Lilia M. Beltrán-Barrientos ID , Adrián Hernández-Mendoza, Aarón F. González-Córdova ID
,
Humberto Astiazarán-García ID , Julián Esparza-Romero and Belinda Vallejo-Córdoba *
Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Carretera a La Victoria Km. 0.6,
Apartado 1735, Hermosillo, Sonora 83304, Mexico; lilia.beltranb@gmail.com (L.M.B.-B.);
ahernandez@ciad.mx (A.H.-M.); aaronglz@ciad.mx (A.F.G.-C.); hastiazaran@ciad.mx (H.A.-G.);
julian@ciad.mx (J.E.-R.)
* Correspondence: vallejo@ciad.mx; Tel.: +52-662-289-2400

Received: 27 January 2018; Accepted: 23 February 2018; Published: 26 February 2018

Abstract: It has been reported that fermented milk (FM) with Lactococcus lactis NRRL B-50571
had an antihypertensive effect in spontaneously hypertensive rats (SHR) and prehypertensive
subjects. Therefore, the objective of the present study was to evaluate the possible mechanisms
involved (angiotensin converting enzyme inhibition (ACEI), enhancement of nitric oxide production,
antioxidant activity and opioid effect), in the antihypertensive effect of FM with SHR. First, twenty
one SHR were randomized into three groups to either receive in a single-oral dose of purified water
(negative control), FM, or naloxone (opioid receptor antagonist) + FM. In a parallel study, twenty
seven SHR were randomized into three groups to either receive ad libitum purified water (negative
control), Captopril or FM. After six weeks of treatment ACEI activity, enhancement of nitric oxide
production, and antioxidant activity were evaluated in plasma. Results indicated that opioid receptors
were not involved in the hypotensive effect of FM. However, ACEI activity (94 U/L), the oxidative
stress index (malondialdehyde/catalase + glutathione peroxidase) 0.9, and nitric oxide in plasma
(4.4 ± 1.3 U/L), were significantly different from the negative control, and not significantly different
from the Captopril group. Thus, these results suggested that these mechanisms are involved in the
hypotensive effect of FM.

Keywords: fermented milk; Lactococcus lactis; angiotensin converting enzyme inhibition; nitric oxide;
antioxidant activity; opioid effect

1. Introduction
Hypertension is an important risk factor for cardiovascular diseases, a leading risk factor for
death and disability. It has been estimated that hypertension affects more than 40% of people over
25 [1]. The high cost and adverse effects associated with pharmacological therapy have encouraged the
scientists to search for new alternatives [2]. Therefore, there has been a rising interest in fermented dairy
foods that, besides being nutritional, may promote health or reduce diseases, such as hypertension [3,4].
The beneficial effects of dairy products are attributed to several bioactive components, such as calcium,
medium-chain fatty acids, lactose, conjugated linoleic acid and bioactive peptides [5]. Bioactive
peptides from milk are liberated from the native protein through proteolysis during gastrointestinal
digestion or food processing, such as fermentation with lactic acid bacteria (LAB) [6]. In fact,
multifunctional properties of milk-derived peptides are increasingly recognized [7].

Nutrients 2018, 10, 262; doi:10.3390/nu10030262 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 262 2 of 15

The antihypertensive effect of fermented milk products has been attributed to bioactive
peptides [8] and/or gamma-amminobutyric acid (GABA) [9] produced during milk fermentation. In
fact, the antihypertensive effect of bioactive peptides is often attributed to angiotensin-I converting
enzyme inhibition (ACEI), an enzyme that plays a crucial role in blood regulation through the renin
angiotensin system (RAS) [10]. Nevertheless, it has been reported that in some cases there is no
correlation between in vitro and in vivo ACEI activity, due to peptides undergoing further degradation
during gastrointestinal digestion, which may cause less bioavailability to reach target organs and cause
the beneficial effect [8]. However, peptides with antioxidant [11], nitric oxide pathway [12], and opioid
receptor binding activities [13] might also exhibit antihypertensive activity. Hence, antihypertensive
bioactive peptides in fermented milks, may be acting via multiple mechanisms [8].
It has been previously reported that a fermented skim milk product with Lactococcus (L.) lactis
NRRL B-50571 had ACEI activity in vitro; and this effect was strain-dependent [14,15]. Furthermore,
fermented milk with L. lactis NRRL B-50571 reduced systolic blood pressure (SBP) and diastolic
blood pressure (DBP), heart rate and had a hypolypidemic effect on spontaneously hypertensive
rats (SHR) [16,17]. Additionally, in a pilot randomized double blind controlled clinical trial with
prehypertensive subjects a blood pressure lowering effect of fermented milk with L. lactis NRRL-B50571
was observed [18]. Afterwards, we assessed that the antihypertensive effect of fermented milk
with L. lactis was not due to the GABA present when it was administered to SHR [19]. Hence, the
antihypertensive effect may be attributed to bioactive peptides present in this fermented milk; yet,
it is not clear which mechanism is involved in the hypotensive effect. Therefore, the aim of the
present study was to determine in SHR if the antihypertensive effect of fermented milk with L. lactis
NRRL B-50571 was through the nitric oxide pathway, the opioid receptor binding, or the ACEI and
antioxidant activities.

2. Materials and Methods

2.1. Strains and Growth Conditions

L. lactis strain NRRL B-50571 was propagated as previously reported by Rodríguez-Figueroa et al. [14]
in 10 mL of sterile lactose (10%, w/v) M17 broth (DIFCO, Sparks, MD) and incubated at 30 ◦ C for 24 h.
Fresh precultures were obtained by repeating the same procedure twice to allow growth until reaching
106 to 107 cfu/mL. To obtain a working culture, a fresh culture was inoculated (3%) in sterile (110 ◦ C,
10 min) nonfat dry milk reconstituted (10%, w/w) and incubated at 30 ◦ C for 12 h.

2.2. Sample Preparation

Fermented milk with L. Lactis NRRL B-50571 (FM) was prepared as previously reported [18].
Reconstituted (10%, w/v) commercial skim milk was pasteurized (80 ◦ C for 30 min), inoculated with
3% working culture and fermented at 30 ◦ C for 48 h. To inactivate LAB, fermentation was stopped by
applying heat treatment (75 ◦ C, 15 min), followed by quick cooling; subsequently fermented milk was
frozen (–20 ◦ C), for further analysis.
To obtain the lyophilized water-soluble extracts from L. lactis fermented milk with NRRL
B-50571 (WSE-FM) for the evaluation of the opioid effect, WSE-FM were obtained by centrifugation
(ThermoScientific, Chelmsford, MA, USA) at 5000 rpm for 40 min at 4 ◦ C; then lyophilized with a
freeze-dryer (Labconco, Kansas City, MO, USA), and kept at 4 ◦ C until use for further analysis. Total
protein content (Method 960.52 AOAC, 1998) of the lyophilized extracts was evaluated.

2.3. In Vivo Experimental Protocols

A total of twenty-nine male SHR (4 weeks old; 44.7 ± 5.15 g body weight (BW)) were obtained
from Charles River Laboratories International, Inc. (Wilmington, MA, USA). Rats were housed in
individual cages at 21 ± 2 ◦ C, 12 h light–dark cycles and 52 ± 6% relative humidity, with an ad
libitum intake of a standard diet (Purina, Cd. México, México) and purified water. Blood pressure
Nutrients 2018, 10, 262 3 of 15

was monitored every week until all rats developed hypertension according to Okamoto and Aoki [20].
SBP and DBP were taken 3 times using the non-invasive blood pressure system using a photoelectric
sensor, amplifier, manual inflation cuff and software (Model 229; IITC Life Science Inc., (Woodland
Hills, CA, USA). Once all rats were hypertensive, the possible antihypertensive mechanisms (opioid,
ACEI, antioxidant, and nitric oxide pathway) were evaluated. All procedures involving animals were
approved by the Bioethics Committee of the Research Center for Food and Development (Spanish
acronym, CIAD), Hermosillo, Sonora, Mexico, (CE/009/2015).

2.4. Evaluation of Opioid Effect

When SHR were 16 weeks old (320.8 ± 16 g BW, 187.6 ± 15.6 mmHg SBP and 129.6 ± 16.9 mmHg
DBP); twenty-one SHR were randomized into three groups (Table 1) of seven rats (n = 7). Treatments
were assigned randomly to each group to either receive in a single dose: purified water (negative
control); 35 mg protein of WSE-FM/kg animal BW; or 1 mg/kg animal BW of naloxone (µ-opioid
antagonist receptor) (PiSa Farmacéutica, Cd. México, México) + 35 mg protein of FM-WSE/kg animal
BW. FM-WSE from fermented milk was dissolved in purified water.
In order to prepare the corresponding dose of naloxone and WSE-FM, the SHR were weighed
before administration. Conscious SHR received via subcutaneous (s.c.) naloxone, and afterwards
a single oral dose of WSE-FM through a gastric cannula between 8:00 and 10:00 hours to eliminate
circadian cycles. SBP and DBP were monitored before treatment administration and every 10 min until
60 min post-administration.

2.5. Long-Term Effect of FM on Blood Pressure

Twenty-seven male SHR (19 weeks old, 346.4 ± 17.7 g BW, 201.5 ± 15.4 mmHg SBP and
153.4 ± 24.6 mmHg DBP), were randomized into three groups (Table 2) of nine rats (n = 9). SHR
from the first study had a three-week washout period, before group allocation; during this time, blood
pressure was monitored to assess any residual effect. Treatments were assigned randomly to each
group to either receive: purified water (negative control); Captopril (a proven hypotensive drug,
40 mg/kg of BW, Sigma-Aldrich Co., St. Louis, MO, USA); or FM. All SHR had free access (ad libitum)
to each treatment during the 6 weeks as part of the protocol. SBP and DBP were measured 3 times,
once a week between 8:00 and 10:00 hours to eliminate circadian cycles. After six weeks of treatment,
rats were sacrificed and blood samples were collected to immediately obtain plasma, and freezed
−80 ◦ C for further analysis.

2.6. ACEI Activity in Plasma

Plasma was used for measuring the ACEI activity according to the method of Cushman and
Cheung [21]. Hippuryl-L-histidyl-L-leucine (a substrate for ACE) (Sigma-Aldrich Co., St. Louis,
MO, USA) was dissolved in 0.1 mol/L sodium borate buffer (pH 8.3) containing 0.3 mol/L NaCl.
The reaction was initiated by the addition of 100 µL of the serum and vascular tissue enzyme extract,
then incubated for 30 min at 37 ◦ C and stopped by the addition of 250 µL of 1 mol/L HCl. The hippuric
acid liberated by ACE was extracted with 1.5 mL ethyl acetate, dissolved by addition of 1.0 mL of
Milli-Q water, after removal of ethyl acetate by heating for 20 min at 75 ◦ C, and measured at 228 nm.
One unit (U) of activity was defined as the amount of enzyme, which released 1.0 mmol of hippuric
acid/min under the above conditions. The specific activity of ACE in serum is expressed as U/L.

2.7. Nitric Oxide Pathway

Nitric oxide pathway was estimated through nitric oxide (NO) production. Plasma NO
concentration was determined by the Griess reaction with a colormetric assay kit (Cell Biolabs, Inc.,
San Diego, CA, USA). This assay is based on the conversion of nitrate to nitrite by nitrate reductase,
followed by quantification of nitrate after the Griess reaction. NO concentration in plasma was
expressed as µmol/L.
Nutrients 2018, 10, 262 4 of 15

2.8. Antioxidant Activity

Antioxidant activity from peptides was assessed indirectly by determining oxidative stress
index/ratio. This index indicates the balance between lipoperoxidation (as malondialdehyde, MDA)
and total antioxidant enzyme activity (catalase, CAT, and glutathione peroxidase, GPx) [22].

2.8.1. Superoxide Dismutase Activity (SOD)

The determination of SOD activity in plasma was determined as described by Superoxide
Dismutase assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). In this assay, it determines
the ability to inhibit the reduction of tetrazolium salt induced by xanthine-xanthine oxidase. One unit
of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide
radical; and was expressed as U/mL.

2.8.2. Catalase Activity Determination (CAT)

CAT activity in plasma determination was based on the decomposition of hydrogen peroxide
(30 µM) at 240 nm [23]. CAT activity was defined as the amount of enzyme that removed 1 µmol H2 O2
in 1 min. CAT activity was expressed as µmol H2 O2 /min/L.

2.8.3. Glutathione Peroxidase (GPx) Activity

GPx activity in plasma was evaluated as described as the manufacturer for Glutathione peroxidase
assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). The GPx activity was determined 340 nm
and is expressed as nmol/min/mL.

2.8.4. Determination of Lipid Peroxidation

The degree of lipid peroxidation in plasma was determined as described by Todorova et al. [24], with
some modifications. This method uses thiobarbituric acid (TBA), which measures malondialdehyde
(MDA) reactive products. 200 µL of plasma were mixed with 200 µL of PBS, and 200 µL trichloroacetic
acid (25%). Afterwards, mixtures were centrifuged (2000 g, 20 min) and supernatants were mixed with
150 µL of TBA (1%), followed by heated at 95 ◦ C for 1 h. After cooling absorbance was determined at
532 nm. The MDA concentration was calculated by using an extinction coefficient of 155 (1/mM·cm).
MDA was expressed as µmol/L.

2.9. Statistical Analysis

Baseline systolic and diastolic blood pressures were defined as the mean of the values measured in
the first run-in period. The blood pressure outcomes were presented as the mean value with standard
deviations (SD) for all SHR in each group. For the evaluation of opioid effect the blood pressure (min
50 and 60 post-treatment) between groups were analyzed with one-way ANOVA. Differences among
means were assessed by Scheffe multiple comparison test and considered significant when p < 0.05.
For the long-term effect of FM on blood pressure, the outcomes for each week between groups
were analyzed with one-way ANOVA. Differences among means were assessed by Scheffe multiple
comparison test and considered significant when p < 0.05. The outcomes for ACEI activity, superoxide
dismutase, GPx activity, MDA, and oxidative stress index, were analyzed with Kruskal–Wallis test
since they did not presented a normal distribution; data are presented as medians and were considered
significant when p < 0.05. The outcomes for nitric oxide and CAT activity were analyzed with one-way
ANOVA. Differences among means were assessed by Scheffe multiple comparison test and considered
significant when p < 0.05. Data are presented as means ± SEM.

3. Results and Discussion

The use of Lactococcus lactis NRRL B-50571 as a starter for fermented milk with antihypertensive
effect has been reported in SHR [16,17] and prehypertensive subjects [18], and this hypotensive effect
Nutrients 2018, 10, 262 5 of 15

has been attributed to ACEI. Moreover, 21 identified peptides in this fermented milk possessed ACEI
activity [15]. However, in several studies it has been reported that there is a lack of correlation
between the in vitro and in vivo ACEI activity. This may be due to peptide degradation throughout
gastrointestinal digestion, and this may implicate difficulty to reach the target organs in a sufficient
amount to exert ACEI effect. However, it should not be disregarded that other mechanisms may be
involved in the antihypertensive effect mediated through the interaction with receptor located at the
gut [25]. Hence, to the best of our knowledge this is the first study that evaluates the possible
mechanisms where peptides may be involved in the hypotensive effect; such as opioid, ACEI,
antioxidant and NO pathway (by NO production).

3.1. Opioid Effect

Twenty one SHR with blood pressure higher than 186/126 mmHg for systolic and diastolic blood
pressures were eligible for randomization (Table 1). As expected, there were no significant differences
(p > 0.05) between groups on clinical characteristics (systolic blood pressure, diastolic blood pressure,
heart rate and weight).

Table 1. Clinical characteristics of SHR.

Groups Negative Control (Purified Water) WSE-FM NRRL B-50571 Naloxone + WSE-FM NRRL B-50571 p Value
Weight 319 ± 20.1 321.9 ± 16 322.6 ± 10.24 0.97
SBP (mmHg) 188.1 ± 21.8 186.3 ± 20 189 ± 8.8 0.99
DBP (mmHg) 126.2 ± 24.3 127 ± 13.2 131.6 ± 10.7 0.83
Heart rate (beats/min) 488.8 ± 35.1 465.2 ± 27.7 453.7 ± 28.9 0.13

SHR: spontaneously hypertensive rats; SBP: systolic blood pressure; DBP: diastolic blood pressure; WSE: water
soluble extract; FM: fermented milk.

Figures 1 and 2 depicts the changes of SBP and DBP every 10 min for 60 min after a single oral
dose of purified water (negative control); WSE-FM; or naloxone + WSE-FM. After 50 and 60 min
post-treatments, reductions on SBP from WSE-FM group and naloxone + WSE-FM group were
13.8 ± 26.1 and 7.7 ± 9.3 mmHg; and 15.2 ± 29.8 and 12.7 ± 17.7 mmHg, respectively; and were
significantly different (p < 0.05) from the negative control. Though, DBP were not significantly different
(p > 0.05) between all groups, DBP from groups receiving WSE-FM or naloxone + WSE-FM, tended to
be slightly lower.
It has been previously reported that milk derived peptides possess opioid-like effect, and that
this effect may exert a hypotensive effect through binding a specific µ opioid receptor [13]. In fact,
the common structural feature for opioid milk peptides is the presence of tyrosine at the N-terminal
end, and the presence of another aromatic residue [26]. Meanwhile, it was previously reported that
three peptides with tyrosine at the N-terminal (YPSYGL, YPSYG and YIPIQYVLS) where present in
the fermented milk with L. lactis NRRL B-50571 [15], therefore, we evaluated if the antihypertensive
effect may be due to milk peptides binding to opioid receptors.
In the present study µ-opioid receptors were blocked with antagonist opioid receptor (naloxone),
and blood pressure reduction was not significantly different (p > 0.05) in WSE-FM group and naloxone
+ WSE-FM group. Hence, in this study the blood pressure lowering effect may not be attributed to
peptides binding to opioid receptors.
On the other hand, it was reported that the mechanism of the blood pressure lowering effect of
a tetrapeptide from milk whey, after a single subcutaneous administration to SHR, was by opioid
receptors, since the response was antagonized with naloxone [13]. Thus, it should not be disregarded
that if SHR were administered subcutaneously with specific peptides present in the fermented milk
with L. lactis NRRL B-50571) [15], the antihypertensive effect could be via binding opioid receptors.
Nutrients 2018, 10, 262 6 of 15
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Figure 1. Change in systolic blood pressure in spontaneously hypertensive rats with different
Figure 1. Change in systolic blood pressure in spontaneously hypertensive rats with different
treatments. Negative
Figure 1. Change in control:
systolicpurified water; FM
blood pressure in L.spontaneously
lactis NRRL B-50571: lyophilized
hypertensive water-soluble
rats with different
treatments. Negative control: purified water; FM L. lactis NRRL B-50571: lyophilized water-soluble
extract (35 mg protein/kg body weight) of fermented milk with Lactococcus lactis
treatments. Negative control: purified water; FM L. lactis NRRL B-50571: lyophilized water-soluble NRRL B-50571;
extract (35 mg(1protein/kg
Naloxone: mg/kg bodybody weight)
weight) of fermented
Antagonist μ opioid milk with +Lactococcus
receptor FM L. lactislactis
NRRL NRRL B-50571;
B-50571:
extract (35 mg protein/kg body weight) of fermented milk with Lactococcus lactis NRRL B-50571;
Naloxone: (1
lyophilized mg/kg body
water-soluble weight) Antagonist µ opioid receptor + FM L. lactis NRRL B-50571:
Naloxone: (1 mg/kg bodyextract (35 mg
weight) protein/kg
Antagonist μ body
opioidweight) of fermented
receptor milk with
+ FM L. lactis NRRL Lactococcus
B-50571:
lyophilized
lactis water-soluble
NRRL
lyophilized water-soluble extract
B-50571. Data extract (35(35
mg mg
are presented as protein/kg
means
protein/kg ± body body
SEM.weight)
Data ofweight)
points sharingof
fermented the fermented
milksame
withletter milk with
within
Lactococcus
Lactococcus
alactis
week lactisnot
was
NRRL NRRL B-50571.
significantly
B-50571. Data Data are
aredifferent
presented (p >aspresented
0.05).
means ± SEM. as means ± SEM.
Data points Datathe
sharing points
samesharing the same
letter within
lettera within
week wasa week was not significantly
not significantly different (p >different
0.05). (p > 0.05).

Figure 2. Change in diastolic blood pressure in spontaneously hypertensive rats with different
treatments.
Figure Negative
2. Change control:
diastolicpurified water; FM L. lactis NRRL B-50571: lyophilized water-soluble
Figure 2. Change in indiastolic blood pressure
blood pressure in
in spontaneously
spontaneously hypertensive
hypertensiverats rats
with different
with different
extract (35
treatments. mg protein/kg
Negative body
control: weight)
purified of fermented
water; FM L. milk
lactis with
NRRL L. lactis
B-50571: NRRL B-50571;
lyophilized Naloxone: (1
water-soluble
treatments. Negative control: purified water; FM L. lactis NRRL B-50571: lyophilized water-soluble
mg/kg body
extract (35 mgweight)
protein/kgAntagonist μ opioid
body weight) receptormilk
of fermented + FM withL.L.lactis
lactis NRRL B-50571:Naloxone:
NRRL B-50571; lyophilized
(1
extract (35 mg protein/kg body weight) of fermented milk with L. lactis NRRL B-50571; Naloxone:
water-soluble extract (35
mg/kg body weight) mg protein/kg
Antagonist body weight)
μ opioid receptorof+fermented milkNRRL
FM L. lactis with L.B-50571:
lactis NRRL B-50571.
lyophilized
(1 mg/kg
Data body
are weight)
presented as(35Antagonist
means ± SEM. µ opioid receptor + FM L. lactis NRRL B-50571: lyophilized
water-soluble extract mg protein/kg body weight) of fermented milk with L. lactis NRRL B-50571.
water-soluble extract (35 mg protein/kg body weight) of fermented milk with L. lactis NRRL B-50571.
Data are presented as means ± SEM.
Data are presented as means ± SEM.
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3.2. Long-Term Effect of FM on Blood Pressure
3.2. Long-Term Effect of FM on Blood Pressure
In a parallel study, twenty seven SHR with blood pressure higher than 200/150 mmHg for
In a parallel study, twenty seven SHR with blood pressure higher than 200/150 mmHg for
systolic and diastolic blood pressures were selected for randomization (Table 2). As expected, there
systolic and diastolic blood pressures were selected for randomization (Table 2). As expected, there
were no significant differences (p > 0.05) between groups on clinical characteristics (systolic blood
were no significant differences (p > 0.05) between groups on clinical characteristics (systolic blood
pressure, diastolic blood pressure, heart rate and weight). In this study, we evaluated the other possible
pressure, diastolic blood pressure, heart rate and weight). In this study, we evaluated the other
mechanisms involved ininvolved
possible mechanisms the antihypertensive effect of effect
in the antihypertensive fermented
of fermented milkL.
milk with lactis,
with afterafter
L. lactis, six weeks
six of
administration. Changes in Changes
weeks of administration. SBP andinDBPSBP for
andevery week
DBP for everyareweek
represented in Figures
are represented 3 and3 4.
in Figures Both
and 4. SBP
and DBP values
Both SBP andfrom
DBP the Captopril
values from thegroup and group
Captopril FM group
and FMwere significantly
group differentdifferent
were significantly from thefrom
negative
the group
control negative(p control
> 0.05),group (p > were
but they 0.05), not
but significantly
they were notdifferent
significantly
(p <different (p < 0.05)
0.05) between between
them. Maximal
them. Maximal SBP decreases from the FM group was of 49.9 ± 14.2 mmHg,
SBP decreases from the FM group was of 49.9 ± 14.2 mmHg, after 6 weeks of treatment, while after 6 weeks of the
treatment, while the Captopril group SBP decreased 45.2 ± 23.6 mmHg on the
Captopril group SBP decreased 45.2 ± 23.6 mmHg on the same week of intervention. After six weeks same week of
intervention. After six weeks of intervention, SHR were sacrificed to obtain plasma and evaluate
of intervention, SHR were sacrificed to obtain plasma and evaluate ACEI activity, NO production and
ACEI activity, NO production and antioxidant effect.
antioxidant effect.
Table 2. Clinical characteristics of SHR.
Table 2. Clinical characteristics of SHR.
Groups Negative Control (Purified Water) Captopril FM NRRL B-50571 p Value
Groups
Weight Negative Control (Purified
341.5Water)
± 18.5 Captopril
357.2 ± 12.3 FM340.5
NRRL±B-50571
17.9 p Value
0.70
SBP (mmHg)
Weight 341.5 ± 18.5
200.8 ± 16.1 357.2 ± 12.3
201.8 ± 18.3 340.5 ±±17.9
201.8 13.1 0.98
0.70
SBP (mmHg) 200.8 ± 16.1 201.8 ± 18.3 201.8 ± 13.1 0.98
DBP
DBP (mmHg) (mmHg) 152 ± 23.3 152 ± 23.3 158 ± 30.2
158 ± 30.2 150.1
150.1 ±±21.8
21.8 0.78
0.78
Heart
Heart rate (beats/min)
rate (beats/min) 461.7 ± 33.5
461.7 ± 33.5 442.6 ± 40.1
442.6 ± 40.1 433.7±±3838
433.7 0.28
0.28

SHR:SHR: spontaneously
spontaneously hypertensive
hypertensive rats;rats; SBP:
SBP: systolicblood
systolic bloodpressure;
pressure; DBP:
DBP: diastolic blood
bloodpressure;
pressure; FM:
fermented milk.
FM: fermented milk.

Figure
Figure 3. Changeininsystolic
3. Change systolic blood
blood pressure
pressure in
in spontaneously
spontaneously hypertensive rats rats
hypertensive with with
different
different
treatments. Negative control: purified water; Captopril: 40 mg/kg body weight; FM
treatments. Negative control: purified water; Captopril: 40 mg/kg body weight; FM L. lactis L. lactis NRRLNRRL
B-50571 (ad libitum): fermented milk with L. lactis NRRL B-50571. Data are presented as means ±
B-50571 (ad libitum): fermented milk with L. lactis NRRL B-50571. Data are presented as means ± SEM.
SEM. Data points sharing the same letter within a week was not significantly different (p > 0.05).
Data points sharing the same letter within a week was not significantly different (p > 0.05).
Nutrients 2018, 10, 262 8 of 15
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Figure
Figure 4. Change
4. Change in in diastolicblood
diastolic blood pressure
pressure in in spontaneously
spontaneously hypertensive
hypertensive rats rats
withwithdifferent
different
treatments. Negative control: purified water; Captopril: 40 mg/kg body weight; FM L. lactis NRRL
treatments. Negative control: purified water; Captopril: 40 mg/kg body weight; FM L. lactis NRRL
Figure 4.(adChange
B-50571 libitum):in fermented
diastolic blood pressure
milk with in spontaneously
L. lactis NRRL B-50571.hypertensive rats with
Data are presented different
as means ±
B-50571 (ad libitum):
treatments. fermented
Negative control: milk
purified L. lactis
withwater; NRRL B-50571.
Captopril: 40 not
mg/kg Data
bodyare presented
weight; FM L. as means ± SEM.
SEM. Data points sharing the same letter within a week was significantly different (p lactis NRRL
> 0.05).
Data B-50571
points sharing the same
(ad libitum): letter milk
fermented withinwitha week was
L. lactis not significantly
NRRL B-50571. Data different (p > 0.05).
are presented as means ±
SEM.Activity
3.3. ACEI Data points sharing the same letter within a week was not significantly different (p > 0.05).
3.3. ACEI Activity
One Activity
3.3. ACEI method to determine in vivo ACEI activity indirectly from peptides is through the
determination
One methodoftocirculatingdetermine ACEIin activity
vivo ACEI in plasma [27].indirectly
activity Figure 5 represents the ACEIisactivity
from peptides through in the
plasmaOneof method
SHR to six
after determine
weeks in vivo ACEI Plasma
post-treatment. activityACEI
indirectly from
activity was peptides
reduced is SHR
in through the
treated
determination of circulating ACEI activity in plasma [27]. Figure 5 represents the ACEI activity
determination
with Captopril of circulating
(a potent ACEACEI activity
inhibitor) andin FMplasma
groups,[27].
and Figure
was not5 represents thedifferent
significantly ACEI activity in
in plasma of SHR after six weeks post-treatment. Plasma ACEI activity was reduced inbetween
SHR treated
plasma
them (p of SHR Moreover,
> 0.05). after six weeks
ACEIpost-treatment.
activity in plasma Plasma
fromACEI activity was
the negative reduced
control groupinwas
SHR 5.5treated
times
with with
Captopril (a potent
Captopril ACE inhibitor) and and FMFM groups, andwaswas not significantly different between
higher than the (aFM potent
group, ACE
andinhibitor)
was significantly groups,
differentand
(p < 0.05)not significantly
from the groupsdifferent between
treated with FM
themthem
(p > (p
0.05). Moreover,
> 0.05). Moreover, ACEI activity in plasma from the negative control group was timestimes
5.5
or Captopril. Thus, the effectACEI activity
may be in plasma
attributed to thefrom theACE
in vivo negative control
inhibition fromgroup was 5.5
bioactive peptides
higher thanthan
higher
in
the FM
FM, because
group,
the circulating
FM group,and
and
ACE
waswas significantly
significantly
activity
different
different (p(p<<0.05)
was reduced.
0.05) from
from thethe groups
groups treated
treated with with
FM FM
or Captopril. Thus,
or Captopril. thethe
Thus, effect may
effect maybebeattributed
attributed to thethein
invivo
vivoACEACE inhibition
inhibition from
from bioactive
bioactive peptides
peptides
in because
in FM, FM, because circulating
circulating ACEACE activitywas
activity wasreduced.
reduced.

Figure 5. Angiotensin converting enzyme activity (U/L) in plasma from spontaneously hypertensive
rats after long-term treatments. Negative control: purified water; Captopril: 40 mg/kg body weight;
FigureFigure
FM 5. Angiotensin
5.L.Angiotensin
lactis converting
NRRL converting
B-50571 enzymeactivity
(adenzyme
libitum): activity (U/L)
(U/L)
fermented ininplasma
milk plasma
with L.from
fromspontaneously
lactis hypertensive
spontaneously
NRRL B-50571. hypertensive
Data are
rats
rats after after long-term
long-term
presented treatments.
treatments.
as median; and was Negative
Negative control:
analyzedcontrol: purified water;
purified water;
by non-parametric Captopril: 40 mg/kg
Captopril: 40 mg/kg
test (Kruskal–Wallis body weight;
body
p < 0.05). weight;
Data
FM L. lactis NRRL B-50571 (adsignificantly
libitum): fermented
FM L.sharing the same
lactis NRRL letter
B-50571 was not
(ad libitum): fermented milk(pmilk
different with L. lactis NRRL B-50571. Data are
> 0.05).
with L. lactis NRRL B-50571. Data are presented
presented as median; and was analyzed by non-parametric test (Kruskal–Wallis p < 0.05). Data
as median; and was analyzed by non-parametric test (Kruskal–Wallis p < 0.05). Data sharing the same
sharing the same letter was not significantly different (p > 0.05).
letter was not significantly different (p > 0.05).
Nutrients 2018, 10, 262 9 of 15

Nutrients 2018, 10, x FOR PEER REVIEW 9 of 15

A similar finding of ACEI activity in vivo after long-term effect of lactoferrin hydrolysates in
SHR wasAevaluated [28]. ofInACEI
similar finding another study
activity where
in vivo afterthere was aeffect
long-term single intake of lactoferrin-derived
of lactoferrin hydrolysates in
peptides,
SHR there were also
was evaluated reductions
[28]. In another instudy
circulating
where ACEthere activity (40%)intake
was a single afterof
1 hlactoferrin-derived
post-administration;
furthermore,
peptides, these effects
there were were
also similarintocirculating
reductions the SHR group, which
ACE activity received
(40%) after 1Captopril [29], henceforth
h post-administration;
furthermore,
angiotensin these effects
converting enzyme wereis similar
an enzymeto thethat
SHRplays
group, which received
a crucial Captopril
role in blood [29], henceforth
regulation through the
angiotensin converting enzyme is an enzyme that plays a crucial role in blood regulation
renin angiotensin system (RAS), thus its inhibition exerts antihypertensive effects [30]. In the present through
study,the renin
since angiotensin
reductions ofsystem
the ACEI(RAS), thus its
activity in inhibition
circulating exerts antihypertensive
plasma were similareffects
in the[30]. In the and
FM group
present study, since reductions of the ACEI activity in circulating plasma were similar in the FM
Captopril group; henceforth we may assume that this mechanistic pathway may be involved in the
group and Captopril group; henceforth we may assume that this mechanistic pathway may be
hypotensive effect of FM with L. lactis NRRL B-50571.
involved in the hypotensive effect of FM with L. lactis NRRL B-50571.
3.4. NO Pathway
3.4. NO Pathway
In our In present study,
our present NONO
study, concentration
concentrationinin plasma wasdetermined
plasma was determined (Figure
(Figure 6). 6). After
After long-term
long-term
effecteffect
of each treatment, plasma NO concentrations were significantly (p <
of each treatment, plasma NO concentrations were significantly (p < 0.05) higher in the0.05) higher inFMthe FM
group group and Captopril group than in the negative control group. Actually, plasma NO was 1.6 timestimes
and Captopril group than in the negative control group. Actually, plasma NO was 1.6
higher in the
higher in FM group
the FM group than
thanininthe
thenegative
negative control group;and
control group; andwaswas significantly
significantly different
different (p < 0.05).
(p < 0.05).
RecentRecent studies
studies havehave reported
reported that
that someantihypertensive
some antihypertensive peptides
peptides may
mayimprove
improveNONO bioavailability
bioavailability
through
through antioxidant
antioxidant effects,effects, but certain
but certain peptides
peptides mayenhance
may also also enhance NO production,
NO production, improve improve
endothelial
endothelial function and improve blood pressure [31]. NO is an important bioregulatory
function and improve blood pressure [31]. NO is an important bioregulatory molecule, which improves molecule,
which improves endothelial function, improves vasodilatation and controls blood pressure;
endothelial function, improves vasodilatation and controls blood pressure; therefore it is considered
therefore it is considered to be the main vasodilator. SHR may develop endothelial dysfunction by
to be the main vasodilator. SHR may develop endothelial dysfunction by reducing bioavailability
reducing bioavailability of NO. Therefore, increased bioavailability of NO may also improve
of NO. Therefore,and
vasodilation increased bioavailability
reduce blood of NOOur
pressure [32,33]. mayfindings
also improve vasodilation
are similar with those and reduce
by Kim et al.blood
pressure [32,33]. Our findings are similar with those by Kim et al. [33]; they reported
[33]; they reported that fermented milk had an antihypertensive effect in SHR, had less ACEI activity that fermented
milk in
had an antihypertensive
plasma, effect inofSHR,
and more concentration NO, had less ACEI
compared activity
to their controlingroup.
plasma, and more
Hence, concentration
NO production
of NO,maycompared to their control
also be considered group. involved
a mechanism Hence, NO production
in the hypotensivemay alsoofbe
effect FMconsidered a mechanism
with L. lactis NRRL
B-50571.
involved in the hypotensive effect of FM with L. lactis NRRL B-50571.

Figure 6. Nitric
Figure oxide oxide
6. Nitric (U/L) (U/L)
in plasma from spontaneously
in plasma from spontaneouslyhypertensive rats after
hypertensive ratslong-term treatments.
after long-term
Negative control:
treatments. purified
Negative water;purified
control: Captopril:
water;40Captopril:
mg/kg body weight;
40 mg/kg bodyFM L. lactis
weight; FMNRRLL. lactisB-50571
NRRL (ad
B-50571
libitum): (ad libitum):
fermented milk fermented milk with
with Lactococcus Lactococcus
lactis lactis NRRL
NRRL B-50571. Data B-50571. Data are as
are presented presented
means ± as SEM.
means ± SEM. Data sharing the same letter are not significantly
Data sharing the same letter are not significantly different (p > 0.05). different (p > 0.05).
Nutrients 2018, 10, 262 10 of 15

3.5. Antioxidant Effect

Nutrients 2018, 10, x FOR PEER REVIEW 10 of 15

To date, there is growing evidence that oxidative stress is one of the main responsible factors
3.5. Antioxidant Effect
for the initiation or evolution of hypertension and its complications. Increased oxidative stress is
To date,to
also considered there is growing
be an important evidence that oxidative
causative factor ofstress is one of the
the vascular main responsible
endothelial factors for
dysfunction, causing
the initiation or evolution of hypertension and its complications. Increased oxidative stress is also
decrement of NO production [34]. Therefore, there has been a rising interest on the pursuit for
considered to be an important causative factor of the vascular endothelial dysfunction, causing
bioactive peptides with antioxidant activity, which may provide additional benefit to the endogenous
decrement of NO production [34]. Therefore, there has been a rising interest on the pursuit for
antioxidant
bioactivedefense
peptides system
with [35]. Moreover,
antioxidant lipid which
activity, peroxidation plays a additional
may provide major rolebenefit
in oxidative
to thestress.
Hence, bioactive components that may reduce lipoperoxidation may help
endogenous antioxidant defense system [35]. Moreover, lipid peroxidation plays a major role decrease oxidative stress
in [34].
It hasoxidative
been reported
stress. Hence, bioactive components that may reduce lipoperoxidation may help decrease side
that food bioactive peptides have strong antioxidant effect without significant
effects [36], and
oxidative that[34].
stress milk derived
It has peptidesthat
been reported are food
the most studied
bioactive [25].have
peptides In fact, it has
strong been reported
antioxidant effect that
wheywithout
proteinsignificant side effects
has beneficial effects[36], and that
through milk derivedofpeptides
enhancement are the
antioxidant most studied
enzymes [25]. Inregulation
and down fact,
it has been reported that whey protein
of oxidative markers such as lipoperoxidation [37]. has beneficial effects through enhancement of antioxidant
enzymes and down
In the present regulation
study, of oxidative
we evaluated themarkers such as lipoperoxidation
concentration of three antioxidant[37]. enzymes in plasma
In the present study, we evaluated the concentration of three antioxidant enzymes in plasma
post-treatments. Results indicated that SOD (Figure 7) and CAT (Figure 8) were not significantly
post-treatments. Results indicated that SOD (Figure 7) and CAT (Figure 8) were not significantly
different (p > 0.05) in either group; nevertheless, values were higher in the Captopril group and the FM
different (p > 0.05) in either group; nevertheless, values were higher in the Captopril group and the
group FMthan
groupin the
thannegative controlcontrol
in the negative group.group.
Moreover, GPxGPx
Moreover, activity (Figure
activity 9) in
(Figure 9) the Captopril
in the Captoprilgroup
was significantly
group was significantly higher (p < 0.05) than in the FM group and negative control group; however, GPx
higher (p < 0.05) than in the FM group and negative control group; however,
activity
GPxvalue from
activity the FM
value fromgroup
the FM was slightly
group washigher
slightlythan in the
higher negative
than control group.
in the negative controlAdditionally
group.
we evaluated
Additionallylipoperoxidation in plasma through
we evaluated lipoperoxidation TBA, by
in plasma detecting
through TBA,MDA productsMDA
by detecting (Figure 10). In this
products
(Figure 10). In this study, although we did not detect differences between all
study, although we did not detect differences between all groups (p > 0.05), MDA values from the FM groups (p > 0.05), MDA
group values
and from the FM
Captopril groupwere
group and Captopril
lower than group were
in the lower than
negative in the negative control.
control.

Figure 7. Superoxide
Figure 7. Superoxidedismutase
dismutase (U/mL)
(U/mL) inin plasma
plasmafrom
fromspontaneously
spontaneously hypertensive
hypertensive rats after
rats after
long-term treatments.
long-term treatments.Negative
Negative control: purifiedwater;
control: purified water; Captopril:
Captopril: 40 mg/kg
40 mg/kg body weight;
body weight; FM L. FM
L. lactis NRRL
lactis NRRLB-50571
B-50571 (ad
(ad libitum): fermentedmilk
libitum): fermented milkwith
with Lactococcus
Lactococcus lactis
lactis NRRLNRRL B-50571.
B-50571. Data Data
are are
presented as median;
presented and
as median; were
and wereanalyzed
analyzedbybynon-parametric test(Kruskal–Wallis
non-parametric test (Kruskal–Wallis p < p0.05).
< 0.05).
Nutrients
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2018, 10,
10, x262
FOR PEER REVIEW 11
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of15
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Figure
Figure 8.8.Catalase
Catalase activity
activity (CAT)(CAT) (micromol
(micromol H2O2/min/L)
H2 O2 /min/L) in plasmainfrom
plasma from spontaneously
spontaneously hypertensive
Figure 8. Catalase
hypertensive rats after activity
long-term (CAT) (micromol
treatments. H2Ocontrol:
Negative 2/min/L) in plasma from spontaneously
purified water; Captopril: 40 weight;
mg/kg
rats after long-term treatments. Negative control: purified water; Captopril: 40 mg/kg body
hypertensive
body weight; rats
FM after
L. long-term
lactis NRRL treatments.
B-50571 (ad Negative fermented
libitum): control: purified
milk water;
with L. Captopril:
lactis NRRL 40 mg/kg
B-50571.
FM L. lactis NRRL B-50571 (ad libitum): fermented milk with L. lactis NRRL B-50571. Data are presented
body
Data weight; FM
are presented L. lactis
as means NRRL B-50571
± SEM, and (ad libitum):
were analyzed fermented milk
by one-way ANOVA.with L. lactis NRRL B-50571.
as means ± SEM, and were analyzed by one-way ANOVA.
Data are presented as means ± SEM, and were analyzed by one-way ANOVA.

Figure 9. Glutathione peroxidase activity (nmol/min/mL) in plasma from spontaneously hypertensive

Figure 9. Glutathione peroxidase activity (nmol/min/mL) in plasma from spontaneously
rats after9.long-term
Figure treatments.
Glutathione Negative
peroxidase control:
activity purified water; in
(nmol/min/mL) Captopril:
plasma 40 mg/kg body weight;
hypertensive rats after long-term treatments. Negative control: purified water;from spontaneously
Captopril: 40 mg/kg
FM L. lactis NRRL
hypertensive B-50571 (ad libitum): fermented milk with Lactococcus lactis NRRL B-50571.40Data are
body weight; FM L. lactis NRRL B-50571 (ad libitum): fermented milk with Lactococcus lactis mg/kg
rats after long-term treatments. Negative control: purified water; Captopril: NRRL
presented
body weight; as median; and was analyzed by non-parametric test (Kruskal–Wallis p <0.05). Data sharing
B-50571. Data FM L. lactis NRRL
are presented B-50571
as median; and(adwas
libitum): fermented
analyzed milk with Lactococcus
by non-parametric lactis NRRLp
test (Kruskal–Wallis
the sameData
B-50571. letterare
arepresented
not significantly
as different
median; and (p > analyzed
was 0.05). by non-parametric test (Kruskal–Wallis p
<0.05). Data sharing the same letter are not significantly different (p > 0.05).
<0.05). Data sharing the same letter are not significantly different (p > 0.05).
Nutrients 2018, 10, 262 12 of 15
Nutrients 2018, 10, x FOR PEER REVIEW 12 of 15
Nutrients 2018, 10, x FOR PEER REVIEW 12 of 15

Figure
Figure 10. 10. Lipid
Lipid peroxidation represented
peroxidation represented as as malondialdehyde
malondialdehyde (MDA)
(MDA)content in plasma
content from from
in plasma
spontaneously
Figure 10. Lipid
spontaneously hypertensive
peroxidation
hypertensive rats
rats after long-term
after long-term
represented treatments.
treatments.Negative
as malondialdehyde (MDA)
Negativecontrol:
content purified
in
control:plasma water;
fromwater;
purified
Captopril:
spontaneously40 mg/kg body weight;
hypertensive rats FM L.long-term
after lactis NRRL B-50571 (ad
treatments. libitum):control:
Negative fermented milk with
purified water;L.
Captopril: 40 mg/kg body weight; FM L. lactis NRRL B-50571 (ad libitum): fermented milk with
lactis NRRL B-50571. Data are presented as median; and were analyzed by non-parametric
Captopril: 40 mg/kg body weight; FM L. lactis NRRL B-50571 (ad libitum): fermented milk with L. test
L. lactis NRRL B-50571. Data are presented as median; and were analyzed by non-parametric test
(Kruskal–Wallis p < 0.05).
lactis NRRL B-50571. Data are presented as median; and were analyzed by non-parametric test
(Kruskal–Wallis p < 0.05).
(Kruskal–Wallis p < 0.05).
Nonetheless, after the evaluation of the oxidative stress index (Figure 11), which indicates the
balance between
Nonetheless,
Nonetheless, lipoperoxidation
after thethe
after evaluation
evaluation (as
ofmalondialdehyde,
of the
the oxidative MDA)
stress
oxidative stress index
index and total
(Figure
(Figure 11),antioxidant
11), which
which enzyme
indicates
indicates the the
activity
balance (CAT and
between GPx), results
lipoperoxidationdemonstrated
(as that the
malondialdehyde, Captopril
MDA) group
and and the
total
balance between lipoperoxidation (as malondialdehyde, MDA) and total antioxidant enzyme activity FM group
antioxidant were not
enzyme
(CATsignificantly
activity
and GPx), different
(CATresults
and GPx), (p results
> 0.05),demonstrated
demonstrated yetthat
theythewere significantly
that groupdifferent
the Captopril
Captopril and the (p
group and
FM < group
0.05)
the FMfrom the
group
were negative
notwere not
significantly
control group.
significantly Since FM
different (p > decreased
0.05), yet oxidative
they were stress in SHR,
significantly antioxidant
different (p < activity
0.05) from may
the also be
negative
different (p > 0.05), yet they were significantly different (p < 0.05) from the negative control group. Since
considered
control group.as anSince
underlying mechanism
FM decreased pathwaystress
oxidative on theinantihypertensive
SHR, antioxidant effect of FM.may
activity Thus, daily
also be
FM decreased oxidative stress in SHR, antioxidant activity may also be considered as an underlying
consumption
considered asof anfermented
underlying milk with L. lactis
mechanism NRRLon
pathway B-50571 may help lowereffect
the antihypertensive high of
blood
FM. pressure, as
Thus, daily
mechanism
well as pathway on the
MDAof levels and antihypertensive
oxidative effect
index,ofB-50571
FM. Thus, daily consumption of fermented milk
consumption fermented milk with L.stress
lactis NRRL ACEI activity,
may helpand loweranhigh
enhancement of NO
blood pressure, as
L. lactis
withproduction.
well as NRRL B-50571
MDA levels andmay help lower
oxidative stress high blood
index, pressure,
ACEI as well
activity, and as an MDA levels and
enhancement of oxidative
NO
stressproduction.
index, ACEI activity, and an enhancement of NO production.

Figure 11. Oxidative stress index represented as the balance between lipid peroxidation (MDA) and
antioxidant enzymes stress
Figure 11. Oxidative (Catalase and
index Glutathione
represented as peroxidase), from spontaneously
the balance between hypertensive
lipid peroxidation (MDA) rats
and
Figure 11. Oxidative stress index represented as the balance between lipid peroxidation (MDA) and
after long-term
antioxidant treatments.
enzymes Negative
(Catalase control: purified
and Glutathione water; Captopril:
peroxidase), 40 mg/kg body
from spontaneously weight; FM
hypertensive L.
rats
antioxidant enzymes (Catalase and Glutathione peroxidase), from spontaneously hypertensive rats
lactis NRRL B-50571
after long-term (ad libitum):
treatments. fermented
Negative control: milk withwater;
purified L. lactis NRRL B-50571.
Captopril: 40 mg/kgData areweight;
body presented
FM as
L.
aftermedian;
long-termand treatments.
was analyzed Negative
by control: purified
non-parametric test water; Captopril:
(Kruskal–Wallis p < 40Data
0.05). mg/kg body
sharing theweight;
same FM
lactis NRRL B-50571 (ad libitum): fermented milk with L. lactis NRRL B-50571. Data are presented as
L. lactis NRRL
letter
median; B-50571
wasand
not (ad libitum):
significantly
was analyzed fermented
different (p > 0.05). milk
by non-parametric with L. lactis NRRL
test (Kruskal–Wallis B-50571.
p < 0.05). Data Data arethe
sharing presented
same as
median;
letterand
waswas analyzed by
not significantly (p > 0.05). test (Kruskal–Wallis p < 0.05). Data sharing the same
non-parametric
different
letter was not significantly different (p > 0.05).
Nutrients 2018, 10, 262 13 of 15

Similarly, it was reported that the antihypertensive effect of whey protein concentrate
hydrolysates in SHR was through ACEI activity, oxidative damage reduction, and enhancement
of NO production [38].

4. Conclusions
The antihypertensive effect of fermented milk with Lactococcus lactis NRRL B-50571 in SHR and
prehypertensive subjects was previously reported. Nonetheless, this is the first study that elucidates the
basic mechanistic pathways underlying the hypotensive effect of fermented milk with Lactococcus lactis
NRRL B-50571. Results indicated that fermented milk with Lactococcus lactis NRRL B-50571 seem to act
as angiotensin converting enzyme inhibitor, nitric oxide production enhancer and as an antioxidant;
which overall helped reduce blood pressure in SHR.

Acknowledgments: The authors would like to thank Alejandro Santos-Espinosa, Anahí Gaxiola-Villa and Lourdes
Santiago-López for their technical assistance.
Author Contributions: The author’s responsibilities were as follows: Belinda Vallejo-Córdoba, Aarón
F. González-Córdova, Adrián Hernández-Mendoza and Lilia María Beltrán-Barrientos designed the study.
Lilia María Beltrán-Barrientos conducted the study and wrote the manuscript. Belinda Vallejo-Córdoba revised the
manuscript and had primary responsibility for the final content of the manuscript. Aarón F. González-Córdova,
Adrián Hernández-Mendoza, Humberto Astiazarán-García and Julián Esparza-Romero supplied valuable knowledge
and scientific consultation throughout the study; and all authors read and approved the final manuscript.
Conflicts of Interest: The authors hereby declare no conflict of interests. This study was supported by the
Mexican Council of Science and Technology (CONACYT; México City, Mexico) research project 240338 CONACYT.
CONACYT had no role in the design of the study; in the collection, analyses, or interpretation of data; in the
writing of the manuscript, or in the decision to publish the results.

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