The effect of yoghurt and its probiotics onlipid prole; a randomised controlled tria

K.L. Ivey a,b,*, J.M. Hodgson c, D.A. Kerr d, P.L. Thomps

ospital, Australiad Curtin University, School of Public Health, Auste Sir Charles Gairdner Hospital, Department of C

Received 12 March 2014; received in revised forAvailable online 1 August 2014

KEYWORDSProbiotics;Yoghurt;Dairy;Blood pressure;Cholesterol;Lipid

Conclusions: The probiotic strains L. acidophilus La5 and B. animalis subsp. lactis Bb12 did not

e (MetS) includeted triglycerides,sterol (HDLC), and

elevated blood pressure [1]. In 2010, 23% of US adults hadMetS [6]. Compared to their non-MetS counterparts, peo-ple with MetS are over 4.6 times more likely to have in-sulin resistance and 5.5 times more likely to have

nology and Diabetes.* Corresponding author. Departments of Nutrition and Epidemiology,

Harvard School of Public Health, 655 Huntington Ave, Boston, MA02115, USA. Tel.: 61 8 9346 3478; fax: 61 8 9346 1317.

E-mail address: kerry.ivey@gmail.com (K.L. Ivey).

http://dx.doi.org/10.1016/j.numecd.2014.07.0120939-4753/ 2014 Elsevier B.V. All rights reserved.

Nutrition, Metabolism & Cardiovascular Diseases (2015) 25, 46e51

Available online at www.sciencedirect.com

Nutrition, Metabolism &

j ournal homepage: www.e lIntroduction

Features of the metabolic syndromincreased waist circumference, elevalowered high density lipoprotein chole

* All work was carried out at the University of Western Australia,School of Medicine and Pharmacology, Sir Charles Gairdner HospitalUnit and the Sir Charles Gairdner Hospital, Department of Endocri-improve cardiovascular risk factors. 2014 Elsevier B.V. All rights reserved.raliaardiovascular Medicine, Australia

m 22 July 2014; accepted 22 July 2014

Abstract Background and aims: Despite strong mechanistic data, and promising results fromin vitro and animal studies, the ability of probiotic bacteria to improve blood pressure andserum lipid concentrations in humans remains uncertain. The aim of this study was to deter-mine the effect of Lactobacillus acidophilus La5 and Bidobacterium animalis subsp lactisBb12, provided in either yoghurt or capsule form, on home blood pressure and serum lipidprole.Methods and results: Following a 3-week washout period, 156 overweight men and womenover 55 y were randomized to a 6-week double-blinded, factorial, parallel study. The fourintervention groups were: A) probiotic yoghurt plus probiotic capsules; B) probiotic yoghurtplus placebo capsules; C) control milk plus probiotic capsules; and D) control milk plus pla-cebo capsules. Each probiotic test article provided a minimum L. acidophilus La5 and B. ani-malis subsp. lactis Bb12 dose of 3.0 109 CFU/d. Home blood pressure monitoring,consisting of 7-day bi-daily repeat measurements, were collected at baseline and week 6.Fasting total cholesterol, low density lipoprotein cholesterol (LDLC), high density lipoproteincholesterol (HDLC), and serum triglyceride were performed at baseline and week 6. Whencompared to control milk, probiotic yoghurt did not signicantly alter blood pressure, heartrate or serum lipid concentrations (P > 0.05). Similarly, when compared to placebo capsules,supplementation with probiotic capsules did not alter blood pressure or concentrations oftotal cholesterol LDLC, HDLC, or triglycerides (P > 0.05).Sir Charles Gairdner Hospital, Department of Endocrinology and Diabetes, Australiac University of Western Australia, School of Medicine and Pharmacology, Royal Perth Ha University of Western Australia, School of Medicine and Pharmacology, Sir Charles Gairdner Hospital Unit, Australiabblood pressure and seruml*

on e, B. Stojceski a,b, R.L. Prince a,b

Cardiovascular Diseases

sev ier .com/ locate/nmcd

Yoghurt and cholesterol 47cardiovascular disease [7]. Therefore it is important toidentify effective, non-pharmacological, population-basedapproaches for prevention of MetS and its co-morbidities.

Yoghurt is a complex functional food produced by theprobiotic bacterial fermentation of milk. The Wold HealthOrganisation [18] denes probiotics as live microorgan-isms which, when administered in adequate amounts,confer a health benet on the host. Interestingly, thisdenition does not stipulate that probiotics improvecolonic microora composition, suggesting that metabo-lites of probiotics may exert health benets, independentof gastrointestinal colonisation. The cardiovascular bene-ts of yoghurt have been investigated since the early 1970s[20], with evidence of benecial effects [12,2]. Despite this,there remains little understanding of the role of the wholefood (yoghurt) in these relationships, and the role pro-biotic bacteria can play in improving blood pressure andserum lipid concentrations.

Yoghurt contains biologically active peptides producedin the bacterial fermentation of milk. Some evidencesuggests these bioactive peptides have ACE-inhibitory andantithrombotic activity that may be responsible for thebenecial effects on features of MetS, including reductionin blood pressure [12,22,27,16]. An alternative mode ofaction of probiotic bacteria is through direct colonisationof the gastrointestinal tract. In this regard several mech-anisms have been advanced to explain the hypo-cholesterolemic effect of probiotics including the role ofprobiotic bacteria in increasing: 1) bile acid deconjugationthrough the action of bile salt hydrolase [8,21,25,26]; 2)cholesterol and fatty acid assimilation into probiotic bac-teria membranes [19]; and 3) conversion of cholesterol tocoprostanol in the gastrointestinal tract [19]. Despitepromising and consistent results from in vitro and animalmodels [8,25,26,21,19], the hypocholesterolemic effect ofprobiotics in humans remains uncertain [12,10,23]. Themajor limitation of previous trials is the lack of power todetect small but clinically important effects on serumlipids.

This appropriately powered study aims to determinethe effect of Lactobacillus acidophilus La5 and Bidobacte-rium animalis subsp lactis Bb12, provided in either yoghurtor capsule form, on home blood pressure and serum lipidprole men and women with features of the metabolicsyndrome.

Methods

Subjects

Participants were randomly recruited from the Perthgeneral population aged over 55 y, using the WesternAustralian Electoral Roll [15]. Inclusion criteria included:minimal usual probiotic intake (consuming

Effect of probiotics on blood pressure

48 K.L. Ivey et al.weekly energy expended in physical tasks, as representedby the metabolic equivalent of task (MET) score [9].

Home blood pressure monitoring

Participants performed bi-daily home blood pressuremeasurements during the 7 days preceding both thebaseline and week 6 visits. A fully automated home bloodpressure monitor (UA-767PC, A&D, Japan) was provided toeach participant at visits held one week prior to baseline,and one week prior to week 6. During these visits, par-ticipants were guided through the correct measurementprocedure and were asked to perform a blood pressurereading in front of the study coordinator. This was toensure the reliability of the blood pressure data from homemonitoring.

Participants recorded blood pressure twice daily;approximately 1 h after breakfast and approximately 1 hafter the evening meal. In a quiet room, participantsrelaxed in a seated position for 5 min, and then performed2 measurements over the following 5 min. The bloodpressure cuff was positioned on bare skin, and located2e3 cm above the antecubital position, and the arm wasrested in a lateral position with the cuff in line with theheart.

Systolic blood pressure, diastolic blood pressure, pulsepressure and heart rate measurements were automaticallyrecorded on the home blood pressure monitor, and weredownloaded when monitors were returned to the studycentre at the baseline and week 6 visits. In addition, par-ticipants also kept a diary where they manually recordedresults of each home blood pressure measurement. In thecase of missing or excess data recorded on the device,electronically recorded values were cross referenced withthe manual records.

Measurements of serum lipid prole

At baseline and at 6 weeks, blood samples were collectedfrom the antecubital fossa vein after an overnight fast. Allbiochemical analyses were performed in the PathWestlaboratory at the Royal Perth Hospital, Western Australia.

Serum was analysed for total cholesterol, HDLC andtriglycerides using routine analysis based on an enzymaticcolorimetric test using the fully automated Architectc16000 Analyser (Abbott Diagnostics, Abbott Laboratories,Abbott Park, IL 60064, USA). Low density lipoproteincholesterol (LDLC) concentrations were calculated usingthe Friedewald formula [11].

Statistical analysis

A pre-specied sample size calculation determined that 68participants in each main effect treatment arm (probioticyoghurt or probiotic capsules) would be sufcient todetect a 3.6% change in LDLC (w0.13 mmol/L), with 80%power at P Z 0.05. Given the factorial study design andanalysis approach adopted by this study, this sample sizeequated to 34 participants in each of the 4 interventionWhen compared to control milk, probiotic yoghurt did notsignicantly alter blood pressure or heart rate (P > 0.05).Similarly, when compared to placebo capsules, supple-mentation with probiotic capsules did not alter bloodpressure or heart rate (P > 0.05); Table 4.

Effect of probiotics on serum lipid prole

When compared to control milk, probiotic yoghurt did notsignicantly alter lipid concentrations (P > 0.05). Similarly,when compared to placebo capsules, supplementationwith probiotic capsules did not alter concentrations oftotal cholesterol LDLC, HDLC, or triglycerides (P > 0.05);Table 5.groups. With P Z 0.05, 68 participants per main effecttreatment arm was also calculated to provide >80% powerto detect a 3.5 mmHg difference in mean systolic bloodpressure (measured twice daily over 7 days). This numberwas increased to allow for a predicted drop-out fromtreatment of around 10%.

All data was analysed according to a pre-speciedprotocol using SPSS (PASW version 18; IBM Corp., NewYork, NY, USA). Outcome measurements were comparedacross intervention groups using a multivariableregression model, with adjustment for the baselinelevels of each outcome, and for the effect of the otherintervention [24]. As a secondary analysis, the interac-tion between probiotic yoghurt and probiotic capsuleswas explored.

Results

Baseline cohort characteristics

A total of 60 women and 96 men were randomised, with amean age of 67 8 years and a mean BMI of 31 4 kg/m2.The mean baseline total cholesterol of the cohort was 5.33(1.15) mmol/L. Mean 7-day home systolic and diastolicblood pressures at baseline were 131 (12) and 74 (9)mmHg, respectively.

Treatment groups were well matched, and there wereno signicant differences between groups for baselinecharacteristics (P > 0.05); Table 1. Similarly, the bloodpressure, heart rate (Table 2), and serum lipid prole(Table 3) of participants were similar between interven-tion groups at baseline (P > 0.05).

Physical activity, energy and nutrient intakes

The level of physical activity as well as intakes of energyand nutrients were similar between the interventiongroups at baseline (Table 1) and week 6 (P > 0.05).Furthermore, we did not observe any signicant (P > 0.05)differences across treatment groups in change (Week 6 eBaseline) of these measures (data not shown).

Table 1 Baseline characteristics of participants by treatment group.

Dairy test article Probiotic yoghu

Capsule test article Probiotic

Group A

Number 40Baseline characteristicsAge (years) 68 8Gender (M:F) 25:15Body mass index (kg/m2) 31 4Hypolipidaemic medication use [n(%)] 25 (62)Hypolipidaemic supplement usea [n(%)] 14 (35)History of Cigarette smoking [n(%)] 21 (52)Prevalent cardiovascular disease [n(%)] 12 (30)Physical activity (MET) 111 7

Dietary intakeEnergy (kJ/d) 7.6 2.6Saturated fat (g/d) 27 12Cholesterol (g/d) 303 133Fibre (g/d) 20 8Sugar (g/d) 80 34

Results are mean SD or n(%) where appropriate.No signicant between group differences were identied by ANOVA or cha Hypolipidaemic supplements included sh oil and omega 3 suppleme

Yoghurt and cholesterol 49Secondary analysis

In a secondary analysis, we found no evidence for aninteraction between probiotic yoghurt and probiotic cap-sules (P > 0.05). As such, the lack of signicant effects ofprobiotic yoghurt and probiotic capsules did not appear tobe inuenced by the presence or absence of the otherprobiotic test article.

DiscussionWe assessed the effects of 6 week probiotic supplemen-tation on blood pressure and serum lipid prole in 156

Table 2 Seven day home blood pressure parameters stratied bytreatment group.

Dairy test article Probiotic yoghurt Control milk

Capsule test article Probiotic Placebo Probiotic Placebo

Group A Group B Group C Group D

Number 40 37 39 40Systolic blood pressureBaseline (mmHg) 131 13 130 12 132 12 130 12Week 6 (mmHg) 131 14 129 11 130 12 129 13

Diastolic blood pressureBaseline (mmHg) 74 11 74 7 76 10 74 7Week 6 (mmHg) 74 10 75 7 75 9 73 8

Pulse pressureBaseline (mmHg) 57 12 57 11 56 14 56 12Week 6 (mmHg) 57 12 55 10 56 13 56 14

Heart rateBaseline (bpm) 71 9 71 9 70 14 72 12Week 6 (bpm) 70 9 70 9 70 14 72 11

Results are mean SD.No signicant between group differences were identied by ANOVA(P > 0.05).overweight or obese men and women. There was no evi-dence that blood pressure, heart rate, or lipid concentra-tions were altered as a result of consuming whole-fatyoghurt or capsules containing the probiotic strains L. ac-idophilus La5 and B. animalis subsp. lactis Bb12.

We found that combination therapy of L. acidophilusLa5 and B. animalis subsp. lactis Bb12 had no effect onblood pressure. Our ndings of lack of effect are sup-ported by results of other randomised controlled trialswhich supplemented with the same combination ofprobiotic strains [4,29]. As the effects of probiotic bac-

rt Control milk

Placebo Probiotic Placebo

Group B Group C Group D

37 39 40

68 8.7 65 7 65 825:12 23:16 23:1730 4 31 4 31 421 (57) 20 (51) 27 (68)14 (36) 11 (28) 16 (40)21 (57) 21 (46) 21 (52)13 (35) 16 (41) 18 (45)109 8 109 8 111 6

7.4 2.4 8.2 2.5 7.4 2.329 12 33 14 30 13297 122 335 159 308 13421 8 22 8 20 774 26 82 23 73 31

i squared test, where appropriate (P > 0.05).nts.teria are highly strain specic [17], it is not surprisingthat studies which supplemented with different probioticstrains and species found different results. Specically,numerous randomised controlled trials of Lactobacillushelveticus have demonstrated that supplementation withthis species has an antihypertensive effect in humans[12,27,16]. As such, the results of our study support thestrain specicity of probiotic actions by conrming thatunlike other probiotic preparations, L. acidophilus La5and B. animalis subsp. lactis Bb12 co-therapy does notimprove blood pressure.

Similar to results from other clinical trials [12,10,23],our study found that daily probiotic supplementation hadno effect on serum lipid concentrations. However, thesendings are in contrast to other human trial datademonstrating that probiotic supplementation improvesserum lipid concentrations [3,5]. Our results are furtheropposed by strong in vitro and animal model data whichclearly dene mechanisms supporting the hypocholester-olaemic effect of probiotics [8,25,26,21,19].

The lack of cholesterol lowering effect in our, and otherstudies, may be explained by subject parameters. Themean baseline cholesterol concentration in our cohortcholesterol was 5 mmol/L. The mean baseline total

Table 3 Serum lipid concentrations stratied by treatment group.

Dairy test article Probiotic yoghurt Control milk

Capsule test article Probiotic Placebo Probiotic Placebo

Group A Group B Group C Group D

Number 40 37 39 40Total cholesterolBaseline (mmol/L) 5.52 0.95 5.35 1.40 5.20 1.12 5.22 1.12Week 6 (mmol/L) 5.37 0.88 5.32 1.33 5.15 1.10 5.21 1.09

LDLCBaseline (mmol/L) 3.31 0.77 3.22 1.16 3.07 0.92 3.10 0.95Week 6 (mmol/L) 3.18 0.75 3.20 1.18 3.03 0.87 3.10 0.95

HDLCBaseline (mmol/L) 1.42 0.37 1.36 0.32 1.41 0.33 1.42 0.35Week 6 (mmol/L) 1.40 0.34 1.36 0.32 1.39 0.33 1.44 0.36

TriglyceridesBaseline (mmol/L) 1.70 0.77 1.70 0.94 1.58 0.74 1.53 0.79Week 6 (mmol/L) 1.71 0.75 1.65 0.77 1.59 0.82 1.47 0.62

Results are mean SD.P >

on

50 K.L. Ivey et al.No signicant between group differences were identied by ANOVA (

Table 4 Main effect model of yoghurt and probiotic supplementation

Treatment armacholesterol concentration of participants in the otherrandomised controlled trials that did not observe a bene-cial effect was less than 5.4 mmol/L [12,10,23].Conversely, the mean baseline total cholesterol concen-tration of participants in trials which observed a

Yoghurt supplementationNo Yes

Systolic blood pressure (mmHg) 129 1 130Diastolic blood pressure (mmHg) 74 1 75Pulse pressure (mmHg) 56 1 56Heart rate (bpm) 71 1 70

Probiotic supplementationNo Yes

Systolic blood pressure (mmHg) 129 1 130Diastolic blood pressure (mmHg) 74 1 75Pulse pressure (mmHg) 56 1 56Heart rate (bpm) 70 1 70a Baseline and treatment adjusted week 6 values (SE).b Mean difference between yes and no (SE).

Table 5 Main effect model of yoghurt and probiotic supplementation on

Treatment arma

Yoghurt supplementationNo Yes

Total cholesterol (mmol/L) 5.29 0.06 5.23 LDLC (mmol/L) 3.16 0.05 3.10 HDLC (mmol/L) 1.42 0.02 1.38 Triglyceride (mmol/L) 1.58 0.05 1.63

Probiotic supplementationNo Yes

Total cholesterol (mmol/L) 5.29 0.06 5.24 LDLC (mmol/L) 3.16 0.05 3.10 HDLC (mmol/L) 1.41 0.02 1.39 Triglyceride (mmol/L) 1.57 0.06 1.64 a Baseline and treatment adjusted week 6 values (SE).b Mean difference between yes and no (SE).0.05).

home blood pressure parameters.

Baseline adjusted differenceb P valuehypocholesterolaemic effect of probiotics was 5.7 mmol/L [3,5]. These ndings suggest that the hypocholester-olaemic benets of probiotics may be limited to pop-ulations with borderline high/high baseline totalcholesterol levels.

1 1.1 1.8 0.548 1 1.1 1.3 0.422 1 0.1 1.8 0.933 1 0.6 1.7 0.703

1 0.9 1.8 0.601 1 0.8 1.3 0.543 1 0.9 1.8 0.615 1 0.1 1.7 0.950

serum lipid parameters.

Baseline adjusted differenceb P value

0.06 0.06 0.09 0.4770.05 0.06 0.07 0.4100.02 0.0.4 0.02 0.1260.06 0.05 0.08 0.490

0.06 0.06 0.08 0.5130.05 0.06 0.07 0.3770.02 0.02 0.02 0.3560.05 0.07 0.08 0.376

The salary of JMH is supported by the National Health and

powdered fermented milk with Lactobacillus helveticus on subjectswith high-normal blood pressure or mild hypertension. J Am Coll

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containing Lactobacillus acidophilus L1 on serum cholesterol inhypercholesterolemic humans. J Am Coll Nutr 1999;18:43e50.

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[5] Ataie-Jafari A, Larijani B, Alavi Majd H, Tahbaz F. Cholesterol-lowering effect of probiotic yogurt in comparison with ordinaryyogurt in mildly to moderately hypercholesterolemic subjects.Ann Nutr Metab 2009;54:22e7.

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In conclusion, the probiotic strains L. acidophilus La5and B. animalis subsp. lactis Bb12 did not improve 7-dayhome blood pressure. The lack of antihypertensive effectis likely due to the strain specicity of probiotic actions.Furthermore, probiotic supplementation did not improveserum lipid parameters. This lack of effect may be a resultof the relatively good baseline cholesterol levels of thecohort. As such, future replication studies supplementingwith L. acidophilus La5 and B. animalis subsp. lactis Bb12 inhypercholesterolemic subjects are indicated.

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The effect of yoghurt and its probiotics on blood pressure and serum lipid profile; a randomised controlled trialIntroductionMethodsSubjectsStudy designBaseline and week 6 measurementsHome blood pressure monitoringMeasurements of serum lipid profileStatistical analysis

ResultsBaseline cohort characteristicsPhysical activity, energy and nutrient intakesEffect of probiotics on blood pressureEffect of probiotics on serum lipid profileSecondary analysis

DiscussionSources of supportReferences