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Brief report
Effect of buttermilk consumption on blood pressure in moderately
hypercholesterolemic men and women
q
Val
erie Conway Ph.D.
a
,
b
, Patrick Couture M.D., Ph.D.
b
,
c
, Sylvie Gauthier Ph.D.
a
,
b
,
Yves Pouliot Ph.D.
a
,
b
, Benoı
ˇ
t Lamarche Ph.D.
b
,
*
a
STELA Dairy Research Center, Laval University, Quebec, Canada
b
Institute of Nutrition and Functional Foods, Laval University, Quebec, Canada
c
Lipid Research Center, CHUQ Research center, Quebec, Canada
article info
Article history:
Received 19 April 2013
Accepted 24 July 2013
Keywords:
Buttermilk bioactive peptides
Blood pressure
Renin–angiotensin–aldosterone system
Milk fat globule membrane
abstract
Objectives: Milk fat globule membrane (MFGM) found in buttermilk is rich in unique bioactive
proteins. Several studies suggest that MFGM proteins possess biological activities such as cholesterol-
lowering, antiviral, antibacterial, and anticancer properties, but data in humans are lacking.
Furthermore, to our knowledge, no study has yet investigated the antihypertensive potential of
MFGM proteins from buttermilk. The aim of this study was to investigate the effects of buttermilk
consumption on blood pressure and on markers of the renin–angiotensin–aldosterone (RAS) system
in humans.
Methods: Men and women (N ¼34) with plasma low-density lipoprotein cholesterol <5 mmol/L
and normal blood pressure (<140 mm Hg) were recruited in this randomized, double-blind,
placebo-controlled, crossover study. Their diets were supplemented with 45 g/d of buttermilk
and with 45 g/d of a macro-/micronutrient-matched placebo in random order (4 wk for each diet).
Results: Buttermilk consumption significantly reduced systolic blood pressure (2.6 mm Hg; P¼
0.009), mean arterial blood pressure (1.7 mm Hg; P¼0.015), and plasma levels of the angiotensin
I-converting enzyme (10.9%; P¼0.003) compared with the placebo, but had no effect on plasma
concentrations of angiotensin II and aldosterone.
Conclusion: Short-term buttermilk consumption reduces blood pressure in normotensive
individuals.
Ó2014 The Authors. Published by Elsevier Inc. All rights reserved.
Introduction
Hypertension is a major risk factor for cardiovascular disease
(CVD) and renal dysfunction, and as a leading cause of death
worldwide, is associated with 7.1 million deaths per year [1].A
healthy diet is an important element in the primary prevention
and management of hypertension [2]. The development of dairy-
based functional foods for blood pressure (BP) management has
been of major interest in the past years. Milk peptides have been
shown to lower BP in many clinical trials, and have been
extensively reviewed [3–6]. Indeed, small milk peptides with
hydrophobic and aromatic amino acid residues have been
targeted for the development of functional foods inhibiting
angiotensin-converting enzyme (ACE) activityda key enzyme
involved in BP regulation. Milk fat globule membrane (MFGM)
components, found in large quantities in buttermilk, have been
of growing interest over the past years because of their unique
nutritional properties [7]. However, no study has yet investigated
the antihypertensive potential of MFGM proteins from butter-
milk. The aim of this study was to investigate for the first time
the effects of buttermilk consumption on BP and on the markers
of the renin–angiotensin–aldosterone (RAS) system in normo-
tensive patients.
Materials and methods
Participants
White men and women were recruited in Quebec City at the Institute of
Nutrition and Functional Foods between January and April 2011. Participants’
group age was between 18 and 65 y, with body mass index (BMI) 35 kg/m
2
,
plasma low-density lipoprotein cholesterol concentrations <5 mmol/L, a 10-y
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which
permits non-commercial use, distribution, and reproduction in any medium,
provided the original author and source are credited.
*Corresponding author. Tel.: þ1 418 656 2131; fax: þ1 418 656 5877.
E-mail address: benoit.lamarche@inaf.ulaval.ca (B. Lamarche).
0899-9007/$ - see front matter Ó2014 The Authors. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nut.2013.07.021
Contents lists available at ScienceDirect
Nutrition
journal homepage: www.nutritionjrnl.com
Nutrition 30 (2014) 116–119
calculated Framingham risk <10% [8], normal BP (<140/90 mm Hg) [1], and with
a stable weight. Participants with a previous history of CVD, type 2 diabetes,
monogenic dyslipidemia, endocrine disorders, using medications for hyperlip-
idemia or hypertension, practicing extremenutritional habits (i.e., vegetarianism,
alcohol consumption >2 drinks/d), and elite athletes were not eligible.
Study design
We used a randomized, double-blind, placebo-controlled, crossover study
design, according to which participants were randomly subjected to two
consecutive 4-wk treatments with buttermilk or placebo [9]. During the study
period, participants were instructed to maintain their usual diet, medication,
weight, alcohol consumption, tea/coffee consumption (with a limit of 2 cups/d),
and smoking habits. The use of vitamins and natural health product supplements
was strictly forbidden throughout the trial. Any deviation from those recom-
mendations would lead to exclusion from the study.
The study protocol was approved by The Clinical Research Ethics Committee
of Laval University, and informed consent was obtained from all participants
included (registered at clinicaltrials.gov as NCT01248026).
Study products and diet instructions
The buttermilk and placebo formulations were artificially flavored with
chocolate, and provided in amounts equal to two servings of low-fat milk (45 g/
d of skim milk solids). Sucralose was added to both formulations to improve taste
and acceptability. The placebo product was formulated using dairy ingredients to
match the buttermilk composition, with the exception of the presumed active
components (i.e., MFGM components). The presence of MFGM components in
buttermilk was confirmed by sodium dodecyl sulphate–polyacrylamide gel
electrophoresis (SDS-PAGE) and high-performance liquid chromatograph for
minor proteins and phospholipids, respectively. The general composition of the
test products is presented in Table 1. The major difference between test products
pertained to their content in polar lipids. Indeed, the buttermilk product con-
tained more than 440% more polar lipids than the placebo. The SDS-PAGE profile
also revealed a higher MFGM protein content in the buttermilk product versus
placebo [9].
Participants were instructed to mix 22.5g pouches of chocolate-flavored
buttermilk or a placebo with 250 mL of water, which they consumed just
before breakfast and dinner each day, for a total consumption of 45 g/d of
buttermilk or a placebo. Participants also were required to restrain their dairy
consumption to a maximum of two servings daily.
Biochemical assessments
Blood samples were taken at screening and on two consecutive days at the
end of each 4-wk test period (i.e., wks 4 and 8). The average of the two post-
treatment values was used in all analyses. Plasma levels of ACE were measured
with enzyme-linked immunosorbent assay (ELISA) kits (Quantikine; R&D Sys-
tems, Inc., Minneapolis, MN; coefficient of variation [CV] <10 %). Plasma con-
centrations of angiotensin II (ANG) and aldosterone (ALDOS) were assessed using
ELISA kits (Enzo Life Sciences, Inc., Plymouth Meeting, PA; both CV 18 %).
Anthropometric and blood pressure assessments
Anthropometric measures were taken at the beginning and at the end of each
test period according to standardized procedures. BP was measured on the right
arm using an automated mercury sphygmomanometer after a 10-min rest in a
seated position. Systolic BP (SBP) and diastolic BP (DBP) measures were analyzed
using the mean of three readings at 3-min intervals. Mean arterial pressure
(MAP) was calculated as:
MAP ¼½ð2DBPÞþSBP=3
Statistical analysis
Data are reported as mean (SD). Mixed models for repeatedmeasures in SAS
9.2 (SAS Institute Inc., Cary, NC, USA) wereused to assess the effect of buttermilk on
the study outcomes. Changes in the study outcomes (buttermilk versus placebo)
were analyzed in models using treatment as a fixed effect and adjusting for sex and
values on placebo. There was no treatment bysequence interaction on any of the
outcomes assessed. Significance was accepted at P<0.05.
Results
Participants
Of the 40 participants randomized, 6 did not complete the
intervention and were excluded from statistical analysis. No
change in body weight and in hip and waist circumferences was
recorded during the duration of the trial, and there were no side
effects associated with the consumption of buttermilk compared
with a placebo. Self-reported compliance was >97% for both
treatments. There was no difference in sodium intake between
the two dietary phases (not shown). As shown previously [9],
buttermilk consumption in these participants reduced plasma
cholesterol and triglycerides (TG) concentrations by 3.1% (P¼
0.019) and 10.7% (P¼0.007), respectively, compared with
placebo.
Table 1
Composition of the ready-to-use buttermilk and placebo products (for a daily dose of 45g) and data on blood pressure and biomarkers of the RAS system in 34
normotensive men and women
Placebo
*
Buttermilk
*
Difference
y
P
z
Mean SD Mean SD
Composition (45 g of powder)
x
Energy (kcal) 179.2 177.8 0.8%
Lactose (g) 23.0 1.0 22.8 1.0 0.9%
Total proteins (g) 13.0 0.8 12.8 1.0 1.5%
Total fat (g) 4.0 0.4 4.0 0.4 0.0%
SFA (g) 2.30 2.08 9.6%
MUFA (g) 0.98 0.84 14.3%
PUFA (g) 0.14 0.12 14.3%
Total phospholipids (mg) 34.6 187.5 þ441.9%
Ashes (g) 3.6 0.4 4.0 0.0 þ11.1%
Water (g) 1.4 0.4 1.4 0.0 0.0%
Systolic blood pressure (mm Hg) 110.9 11.2 108.3 11.6 2.3% 0.009
Diastolic blood pressure (mm Hg) 65.6 8.6 64.4 8.3 1.8% 0.069
Mean arterial pressure (mm Hg) 80.7 8.8 79.0 8.9 2.1% 0.015
ACE level (ng/mL)
k
157.5 48.0 138.8 42.1 10.9% 0.003
ANG-II level (pg/mL) 29.3 51.1 27.8 46.5 5.1% 0.126
ALDOS level (pg/mL)
k
224.6 154.2 226.6 129.0 þ0.9% 0.780
ACE, angiotensin I converting enzyme; ALDOS, aldosterone; ANG, angiotensin; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; RAS, renin–
angiotensin–aldosterone; SFA, saturated fatty acid
*
Values are means SD.
y
Values are expressed as percentages of change compared with placebo.
z
P-values were obtained using the PROC MIXED procedure in SAS.
x
Lactose, total proteins, total fat, ashes, and water values are presented as means and SD (n ¼3).
k
Analyses were performed on log-transformed values.
V. Conway et al. / Nutrition 30 (2014) 116–119 117
SBP and MAP both decreased significantly following butter-
milk treatments compared with the placebo (Table 1). Re-
ductions in SBP and MAP averaged 2.6 mm Hg (2.3%; P¼
0.009) and 1.7 mm Hg (2.1%; P¼0.015), respectively. The
reduction in DBP (1.2 mm Hg) did not reach statistical signifi-
cance (1.8%; P¼0.069).
Plasma concentration of ACE, ANG II, and ALDOS were
analyzed as markers of the RAS system (Table 1). Buttermilk
consumption significantly reduced plasma levels of ACE
compared with placebo (10.9%; P¼0.003), but had no effect on
ANG II (P¼0.126) and ALDOS concentrations (P¼0.780). No
significant correlation was found between the change (%) in
plasma concentration of ACE, ANG II, and ALDOS and the percent
change in SBP, DBP, and MAP.
Discussion
The small but significant reduction in SBP (2.6 mm Hg; P¼
0.009) after 4 wk of buttermilk consumption in participants with
normal BP is close in magnitude to changes that have been
observed after consumption of dairy peptides [5,6]. Indeed, a
meta-analysis of available studies showed that supplementation
with lactotripeptides reduces SBP by an average of 3.7 mm Hg
over a mean duration of approximately 7 wk, with effects being
slightly higher among Asians (6.9 mm Hg) than among white
subjects (1.2 mm Hg ) [5]. Study duration appears to influence the
magnitude of the effect of milk tripeptide supplementation on BP.
Indeed, a meta-analysis showedreduction in SBP of 1.3, 2.2,and 3.3
mm Hg after 2, 4, and 8 wk of supplementation, respectively, with
milk tripeptide [6].Therefore,itispossiblethatlongersupple-
mentation with buttermilk might also lead to more important re-
ductions in BP. This needs to be verified in future studies.
We observed a significant reduction in the plasma concen-
tration of ACE (11%; P¼0.003) after 4-wk supplementation
with buttermilk compared with placebo, although there was no
change in the plasma levels of ALDOS and ANG II levels. The
significant reduction observed in plasma concentration of ACE is
of interest because elevated levels of soluble ACE reflect
enhanced ACE activity [10], and have been associated with an
increased risk for coronary heart disease [11]. This data provides
indirect evidence that the small but significant reduction in BP
with buttermilk may be partly attributable to down-regulated
ACE activity. However, other mechanisms such as direct relaxa-
tion of vascular muscles, as well as opioid action and antioxidant
activities, have been proposed as alternative mechanisms un-
derlying the BP-lowering effects of milk peptides, and may be
partly responsible for the BP-lowering effect of buttermilk con-
sumption [12]. For that matter, in our previous work, we also
demonstrated the antioxidant capacity of specific buttermilk
peptides compared with milk and whey hydrolysates [13].
Some [14], but not all [15,16] studies have shown that in-
dividuals with a higher baseline SBP tend to respond more
favorably to BP-lowering treatments than normotensive in-
dividuals. Further studies are required to examine if patients
with higher BPs also would show benefits from buttermilk
consumption. To the best of our knowledge, studies have yet to
document the effect of milk peptides on BP in normotensive
individuals [3].
The short duration of the study and the use of a sphygmo-
manometer to measure BP, as opposed to ambulatory BP mea-
sures, are two important limitations of this study. As indicated
previously, the data are restricted to individuals with normal
BP, and further studies are required to replicate these data in
hypertensive populations. This study represents a secondary
analysis of a trial designed to investigate the effect of buttermilk
supplementation the plasma lipid levels [9]. Although the BP-
lowering effect seen in the present study is most likely due to
the MFGM components found in buttermilk, other minor com-
pounds of buttermilk may have contributed to these effects.
In conclusion, data from this randomized trial reports that
short-term supplementation with buttermilk (45 g/d)dpossibly
through its high MFGM content and its effect on the ACE sys-
temdlowers SBP in normotensive individuals. Combined with
the buttermilk-induced reduction in plasma levels of total
cholesterol and TGs in the same individuals [9], these data indi-
cate that buttermilk may represent a new safe food modality to
manage blood cholesterol and BP as part of healthy eating. Further
studies are required to validate these observations.
Acknowledgments
We acknowledge all the nurses, laboratory staff, and partici-
pants at the Institute of Nutrition and Functional Foods. We also
acknowledge Pharmalab Inc. for helping us with the formulation
and packaging of the ready-to-use test products (buttermilk and
placebo) consumed in the present study. This project was funded
by a grant from the AAFC/Dairy Research Cluster. Patrick Couture
and Benoı
ˇ
t Lamarche have received funding in the past from the
Dairy Farmers of Canada, Patrick Couture and Benoı
ˇ
t Lamarche
also have received funding from Dairy Australia, and Agriculture
and Agri-Food Canada. Benoı
ˇ
t Lamarche is the chair of the Expert
Scientific Advisory Committee (ESAC) that reviews funding ap-
plications as part of the funding program of the Dairy Farmers of
Canada. He also has received funding from Danone. Sylvie
Gauthier and Yves Pouliot have received funding from the Dairy
Farmers of Canada.
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