�������� ����� ��

No difference in fecal levels of bacteria or short chain fatty acids in humanswhen consuming fruit juice beverages containing fruit fiber, fruit polyphenols,and their combination

Alison J. Wallace, Sarah L. Eady, Denise C. Hunter, Margot A. Skinner,Lee Huffman, Juliet Ansell, Paul Blatchford, Mark Wohlers, Thanuja D.Herath, Duncan Hedderley, Douglas Rosendale, Halina Stoklosinski, TonyMcGhie, Dongxiao Sun-Waterhouse, Claire Redman

PII: S0271-5317(14)00259-0DOI: doi: 10.1016/j.nutres.2014.11.002Reference: NTR 7419

To appear in: Nutrition Research

Received date: 15 September 2014Revised date: 18 November 2014Accepted date: 19 November 2014

Please cite this article as: Wallace Alison J., Eady Sarah L., Hunter Denise C., Skin-ner Margot A., Huffman Lee, Ansell Juliet, Blatchford Paul, Wohlers Mark, HerathThanuja D., Hedderley Duncan, Rosendale Douglas, Stoklosinski Halina, McGhie Tony,Sun-Waterhouse Dongxiao, Redman Claire, No difference in fecal levels of bacteria orshort chain fatty acids in humans when consuming fruit juice beverages containingfruit fiber, fruit polyphenols, and their combination, Nutrition Research (2014), doi:10.1016/j.nutres.2014.11.002

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

1

No difference in fecal levels of bacteria or short chain fatty acids in humans when

consuming fruit juice beverages containing fruit fiber, fruit polyphenols, and their

combination

Alison J Wallace1, Sarah L Eady

1, Denise C Hunter

2, Margot A Skinner

2, Lee Huffman

3,

Juliet Ansell3, Paul Blatchford

3, Mark Wohlers

2,Thanuja D Herath

3, Duncan Hedderley

3,

Douglas Rosendale3, Halina Stoklosinski

3,Tony McGhie

3, Dongxiao Sun-Waterhouse

2,

Claire Redman3.

1 The New Zealand Institute for Plant & Food Research Limited, Lincoln, New Zealand

2 The New Zealand Institute for Plant & Food Research Limited, Mount Albert,

Auckland, New Zealand

3 The New Zealand Institute for Plant & Food Research Limited, Palmerston North,

New Zealand

Corresponding author: Dr Alison Wallace, Plant & Food Research, Gerald Street,

Lincoln, New Zealand, Private Bag 4704, Christchurch 8140, New Zealand, telephone

+64 3 3259638, fax +6433252074, email alison.wallace@plantandfood.co.nz.

Running title: Effect of fruit juice beverages on gut health

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

2

Abstract

This study examined the effect of a Boysenberry beverage (750 mg polyphenols), an apple

fiber beverage (7.5 g dietary fiber) and a Boysenberry plus apple fiber beverage (750 mg

polyphenols plus 7.5 g dietary fiber) on gut health. Twenty-five individuals completed the

study. The study was a placebo-controlled crossover study where every individual consumed

one of the four treatments in turn. Each treatment phase was four weeks long and was

followed by a two-week washout period. The trial beverages were 350 g taken in two doses

every day (that is 175mls taken twice daily). The hypothesis for the study was that the

combination of polyphenols and fiber would have a greater benefit on gut health than the

placebo product or the fiber or polyphenols on their own. There were no differences in fecal

levels of total bacteria, Bacteroides-Prevotella-Porphyromonas group, Bifidobacterium spp.,

Clostridium perfringens or Lactobacillus spp. among any of the treatment groups. Fecal short

chain fatty acid concentrations did not vary among treatment groups although PGE2

concentrations were higher after consumption of the Boysenberry juice beverage. No

significant differences were found in quantitative measures of gut health between the

Boysenberry juice beverage, the apple fiber beverage, the Boysenberry juice plus apple fiber

beverage, and the placebo beverage.

Key Words gut health, fruit polyphenols, dietary fiber, bacteria, Boysenberry juice,

apple fiber

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

3

1. Introduction

The human microbiome has a major influence on many metabolic, physiological, nutritional

and immunological processes. The gut contains around 1014

bacteria, many of which may be

categorized as beneficial or potentially pathogenic due to their metabolic activities and

fermentation end products [1, 2]. The gut microbiota is dominated by two bacterial divisions,

Firmicutes and Bacteroidetes. Maintaining a healthy balance of these bacteria contributes to

overall gut health and may have other health promoting effects such as improved immunity,

inhibition of growth or attachment of potential pathogens, improved digestion and absorption

and reduction of inflammatory conditions[3]. The commensal and symbiotic microbes that

reside in the gut vary between individuals and are affected by factors such as age, diet,

antibiotic usage and disease. These changes to the microbial diversity have been linked to

diseases such as obesity, inflammatory bowel disease, diabetes, cardiometabolic

complications and cancer[4]. Thus there is great interest in understanding the mechanisms of

how gut microbes act on the host metabolism and regulation of the composition of the

microbial population. Dietary intake has an important influence on the gut microbiome both

in terms of its composition and metabolism through the provision of nutrients and energy for

the host and the bacteria which in turn improves mucosal immunity and intestinal

permeability[5, 6]. Diet can also be used to manipulate the growth of selected gut microbes

that may result in health benefits, reducing the presence of pathogenic bacteria, and

increasing those that are beneficial [4, 7, 8].

The microbial population of the human gut is essential to the process of breaking down plant

structural polysaccharides, where degradative enzymes are produced by the resident bacteria,

as well as the bacterial transformations of some dietary proteins (collagen and elastin) and

secondary metabolites such as polyphenolic substances[7]. Following fermentation in the

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

4

large bowel, fiber is thought to act as a prebiotic, beneficially affecting the host by selectively

stimulating the growth or activity of one or a limited number of bacteria in the colon that

have the potential to improve health[9]. Prebiotic rich foods have been reported in several

studies to be efficacious through their ability to elevate the number of Bifidobacteria spp.,

Lactobacilli spp., Fecalibacterium prausnitzii and Ruminococcus bromii and to reduce

Clostridium leptum and Clostridium perfringens[4, 8, 10]. Polyphenols are compounds found

in fruits and vegetables that inactivate substances with the potential to damage cells and

tissues in the body[11-13]. As much as 90% of plant polyphenols escape digestion in the

upper gut and persist in the colon, acting as substrates for microbial production of small

phenolic acids[14]. Fermentable carbohydrates and polyphenols both result in the production

of short chain fatty acids (SCFA), which act as an energy source for the colonic epithelium

but also have effects on cell differentiation and gut health[15]. Parkar et al.[16] demonstrated

the ability of polyphenols to stimulate the proliferation of Bifidobacteria spp. as well as

stimulate SCFA production in vitro and Vendrame et al.[17] showed that a beverage

containing wild blueberry and high in polyphenol and fiber was effective at increasing

Bifidobacteria spp. in a clinical study of 20 healthy males when compared with a placebo.

Other human intervention studies with whole grain cereals containing dietary fiber and

polyphenols, support the fact that increasing consumption of these constituents might

significantly up-regulate groups of commensal bacteria[15] whilst polyphenols may also play

a preventative role in cardiovascular disease, cancers, osteoporosis, neurodegenerative

diseases and diabetes mellitus[18].

Based on the information demonstrating the effectiveness of fiber and polyphenols in the area

of gut health, this study was designed to determine the effect of beverages containing fruit

fiber, fruit polyphenols or fruit fiber combined with polyphenols on gut health. Research has

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

5

shown that fruit beverages rich in fiber and polyphenols are appealing to consumers and the

beverage format is an effective method of delivery of these food components to

individuals[19]. Concentrated Boysenberry juice was selected as a good source of

anthocyanins and other polyphenols that are likely to exert a prebiotic effect whilst evidence

from preliminary animal studies suggest that the addition of the apple fiber may enhance the

bioavailability/uptake of polyphenols enhancing their effectiveness[20, 21]. The hypothesis

for the study was that the combination of polyphenols and fiber would have a greater benefit

on gut health than the placebo product or the fiber or polyphenols on their own. The

objectives were to examine the effect of polyphenols and fiber on measures of fecal gut

bacteria, short chain fatty acids IgA and PGE2.

2. Methods and materials

2.1 Participants

Participants were recruited through advertisements and articles in the local Christchurch

(New Zealand) newspapers in June 2010. Eligible participants were healthy men and women

aged between 40 and 60 years with a body mass index between 18 and 35 kg/m2. Participants

were not eligible if they were smokers, were taking any reflux medications such as Losec or

were regular users of anti-inflammatory drugs. Other exclusion criteria included chronic

diseases such as coronary heart disease, diabetes or compromised gut status (except irritable

bowel syndrome), inflammatory bowel disease, previous gut surgery, gastrointestinal cancer,

renal, hepatic, endocrine or other systemic disease, any known blood-borne disease, untreated

hypertension, pregnancy, history of substance abuse, extreme dietary habits or food allergies

and extreme exercise regimens. Participant demographic characteristics are shown in Table 1.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

6

The present study was conducted according to guidelines laid down in the Declaration of

Helsinki, and all procedures involving human subjects were approved by the Canterbury

Upper South A Ethics Committee. Written informed consent was obtained from all subjects.

The trial was registered with the Australia New Zealand Clinical Trials Registry, registration

number ACTRN12609000907246.

2.2 Experimental design

The study was a placebo controlled, cross-over design where every individual consumed one

of four treatments in turn and acted as their own control over a period of 24 weeks. Each

treatment phase was for the duration of 4 weeks and was followed by a 2-week washout

period where no trial beverages were consumed. Two weeks was considered long enough for

the washout based on a previous study [22]. A total of 30 participants were recruited and

randomly assigned to the three treatments which consisted of three beverages; Boysenberry

juice beverage, apple fibre beverage, Boysenberry juice plus apple fibre beverage and all

participants received a placebo beverage as the fourth treatment. Due to the unstable nature of

the polyphenol components in the fruit beverages, even when frozen, a decision was made to

randomise these beverages to the first three treatment arms of the study to limit the time for

the trial products spent in frozen storage and provide the placebo beverage to all participants

in the last 4 weeks of the study. It was acknowledged that this compromised the study design,

but data from a preliminary unpublished stability trial indicated that this was necessary to

ensure the concentration of total polyphenols in the beverages was at least 750 mg during the

intervention period. A diagram of the study design is shown in Figure 1.

Individuals were asked to continue with their habitual diets over the course of the study but

were asked to include the trial products on a daily basis. Participants were asked to exclude

any fermented probiotic drinks such as Yakult or Activate and fibre supplements such as

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

7

Metamucil and Benefibre from their diet for the duration of the study. The trial beverages

were supplied in 350 g amounts that were to be taken in two doses over the course of the day.

They were supplied frozen on a weekly basis and participants were asked to keep them

refrigerated at all times to help minimise any degradation of the polyphenol products.

Baseline measurements were taken before the allocation of the first intervention. Height and

weight measurements were recorded and the individuals were asked to complete an estimated

3-day diet record of their usual dietary pattern over three consecutive days including one

weekend/non-working day. The participants were also given a colour picture booklet to help

with their estimation of food portion sizes. Nutrient intakes were analysed using the New

Zealand database of Foodworks Professional, version 4, 2005 (Xyris Software, Highgate Hill,

Queensland, Australia) based on the 1999 New Zealand Food Composition Tables from The

New Zealand Institute for Plant & Food Research Limited (Auckland, New Zealand). Trial

participants were asked to attend the clinic in a fasting state (and prior to cleaning their teeth)

and were asked to supply a fresh saliva sample and fecal sample which were kept frozen at -

80oC until processing. These three measurements (diet records, fecal samples and saliva

samples) were collected at each clinic visit at the beginning and end of each 4-week treatment

cycle. Fecal samples were analysed for bacterial populations (Bifidobacteria spp.,

Lactobacilli spp., Bacteroides–Prevotella-Porphyromonas and Clostridium perfringens),

short chain fatty acid (SCFA) concentrations, and immunoglobulin A (IgA) and prostaglandin

E2 (PGE2) concentrations, whilst saliva samples were analysed for IgA and PGE2 as a

measure of inflammation. The saliva sample results were part of another research project and

will be reported separately.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

8

A daily questionnaire tracking the participant’s bowel motions and regularity, and their

physical/mental well being was included throughout the study. Participants completed one for

everyday of the study including during washout periods.

2.3 The Canterbury Earthquake

This study was conducted in 2010 and was at the stage of the end of the second intervention

when a large earthquake (magnitude 7.1) struck the Canterbury region on September 4th

2010

causing significant structural damage to buildings and disruptions to life in this region of

New Zealand. Participants were contacted immediately following this event to assess their

situation and were asked about their willingness to continue in the study. All participants

were willing to continue and did not report extreme levels of stress as a result of this natural

disaster. Following consultation with several clinical trial experts and further approval from

Canterbury Upper South A Ethics Committee, a decision was made to continue with the

study and to include a measure of anxiety in the analysis. The State-Trait questionnaire used

(under licence from www.mindgarden.com) was a two page questionnaire with 40 questions

rated on a scale of 1 to 4. It is a definitive instrument for measuring anxiety in adults and it

clearly differentiates between the temporary condition of “state anxiety” and the more general

and long-standing quality of “trait anxiety”. Participants were asked to complete this

instrument at the visit immediately following the earthquake and at the end of the study.

2.4 Trial Products

The composition of the beverages is shown in Table 2. The drinks were formulated to deliver

the equivalent of 750 mg of polyphenols in the Boysenberry & apple fiber and the

Boysenberry beverages. The beverages were delivered to the study participants weekly.

Accordingly, characterization of the beverages was also conducted on a weekly basis (i.e. 3-

day intervals) including analysis of their phytochemical composition and evaluation of the

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

9

stability of the phytochemicals under conditions of frozen storage (at -18±2ºC) throughout

the study and refrigerated storage (2±1ºC) for 7 days after distribution of the drinks to the

study participants. Sample beverages of each type were removed from the freezer (at -

18±2ºC) each Friday and transferred to a cold room (2±1ºC). On the Monday (termed “0 h”),

Thursday (termed “78 h”) and following Monday (termed “168 h”), aliquots (~2.0 g) of each

beverage were collected and diluted to 10 mL with ethanol/water/formic acid (80:20:1) in a

volumetric flask. After overnight at -18ºC, samples were transferred to screw-capped test

tubes and stored at -18ºC until analysis. Samples were analyzed using Ultra High

Performance Liquid Chromatography (UHPLC) Analysis (Dionex Ultimate 3000 RS UPLC

system, separation column SB-C18 Rapid Resolution (2.1 x 150 mm, 1.8 m) from Agilent

Technologies).

2.5 Compliance

Compliance was measured by asking participants to record their daily consumption of the test

beverage on the Daily Questionnaires. Overall the level of compliance for this study was 87%

with the study beverages.

All four trial beverages were formulated to have a balanced total sugar of 22.9 g per 350 g

serve or 6.55 g per 100 g of beverage. To account for the losses of polyphenols during

production and storage the Boysenberry juice plus apple fiber and the Boysenberry juice

beverages were formulated with an average of 802 mg per serve of polyphenols to achieve

the target of 750 mg polyphenols per 350 g serve.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

10

2.6 Trial Beverage Preparation

The trial beverages were produced at Xenos Aseptic Beverages Systems, Palmerston North,

New Zealand, using the scaled up formulations based on Table 3 (i.e. a 400 kg batch (~1100

x 350 g bottles)) for each of the four trial beverages.

The placebo beverage was produced by dissolving 6.6g/100g sugar in water (70°C) then the

0.13g/100g elderberry concentrate and 0.06g/100g citric acid was added into a 500 L tank

with overhead mixing. The obtained mixture was ultra high temperature (UHT) treated at 120

°C for 3 sec with a flow rate of 800 L/hr, using a UHT unit immediately cooled to 25ºC and

filled inline. The placebo beverage (25°C) was filled into PET bottles that were pre-sterilised

with steam and UV light (Xenos Aseptic Filler, X2000). After filling, the beverages were

labelled, cooled and stored at -18°C.

2.7 Quantification of microbiota from fecal samples

2.7.1 DNA Sample processing

The fecal samples were shipped frozen to Plant & Food Research, Palmerston North, where

they were stored at -80°C until required. Approximately 2 g of each sample was weighed out

and diluted in the appropriate volume of phosphate buffered saline (PBS) buffer (MP

Biomedicals) to make a 1:4 dilution. The suspension was vigorously vortexed to ensure a

homogenous solution and 200 µL was dispensed into a sterile microtube and stored at -80°C

until testing. DNA was extracted from each of the dispensed suspension samples using the

Qiagen QIAamp DNA Stool Mini Kit (cat no. 51540), following manufacturers’ instructions

for bacterial DNA extraction.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

11

2.7.2 Standard preparation

DNA from pure bacterial cultures of Bacteroides fragilis NZ RM964, Enterococcus fecalis

AGR 991, Clostridium perfringens ATCC 13124 and Lactobacillus reuteri DPC 16 were

obtained from stocks kindly donated by Gunaranjan Paturi (Plant & Food Research,

Auckland); these were stored at -20°C. DNA obtained was extracted in the same manner

using the same kit as above.

Bifidobacterium adolescentis NCTC 11814 was grown in MRS broth (Oxoid) + 0.05%

cysteine at 37°C for 2 days under anaerobic conditions. This suspension was adjusted to a

final concentration of 1 x 109 cfu/mL. DNA was extracted as above.

qPCR

Following DNA extraction of the samples and all standard bacterial DNA was prepared,

absolute quantification of the resultant samples was performed using a Roche Lightcycler

Real-Time PCR (RT-PCR) instrument and detected using the Roche Lightcycler 480 SYBR

Green I Master Mix (04707516001), according to the manufacturer’s Product Instructions.

Primers used in the study were obtained from the Invitrogen custom oligonucleotide service.

Each qPCR run included one activation cycle (95°C), 30 run cycles (including the

denaturation step at 95°C, the annealing step (temperature 55ºC - 63ºC) and the extension

step at 72°C), and one melt curve cycle followed by a cooling cycle. The melt curve Tm

cooling cycle enabled the differentiation between the target product and non-specific double

stranded product such as primer-dimers, etc. Primers were diluted in PCR grade water to a

concentration of 50 µM. This was then diluted at a ratio of 1:10 to obtain a 5 µM solution for

the PCR reaction.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

12

For the universal primers, a sample from each of the other four bacterial group standards was

mixed to obtain a representative of total bacteria in the sample. Samples were tested in

triplicate[23].

2.7.3 Short Chain Fatty Acid Quantification by Gas Chromatography

Short chain fatty acids (SCFA) in wet fecal matter were measured by gas chromatography

(GC) – flame ionization detection according to a modified method of Richardson and

colleagues[24].

Wet fecal matter from the -80°C freezer was weighed into 15 mL tubes. SCFA were

extracted into an aqueous phase by diluting 10-fold with phosphate buffered saline (Sigma 10

mM PBS) containing 2-ethyl butyrate (Merck) as an internal standard (at a 5.0 mM final

concentration). The sample was mixed and centrifuged (Juan CR4i) at 2000 x g for 10 min at

4°C, and the supernatant (0.5 mL) was collected and acidified with concentrated HCl (0.25

mL) before diethyl ether (1.0 mL) was added. The mixture was well mixed by vortexing and

then centrifuged (Thermo Electron Corporation IEC Micro CL 17R) at 10,000 x g for 10 min.

The diethyl ether extract (100 µL) and the derivatizing reagent N-tert-butyldimethylsilyl-N-

methyltrifluoroacetamide containing 1% tert-Butyldimethylcholorosilane (MTBSTFA + 1%

TBDMSCI, Sigma-Aldrich) (20 µL) were placed into a 2.5 mL glass vial and capped.

Following a brief vortex mixing, the mixture was incubated at 80°C for 20 min, and then left

at room temperature for a further 48 h to ensure complete derivatization. The mixture was

again vortexed and transferred to vial inserts, capped, and then quantified by GC. Standard

mixtures containing 5 mM 2-ethyl butyrate for calibration were extracted and derivatized

following the same steps as those for the fecal matter samples.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

13

Analysis was performed on a Shimadzu gas chromatograph (GC-17A) equipped a with flame

ionization detector (FID) and an Agilent HP-1 (methyl silicone gum) column (10 m length ×

0.53 mm internal diameter × 2.65 µm film thickness). The carrier gas was helium with a total

flow rate of 37 mL/min and pressure of 7 kPa. The temperature program began at 70°C, and

elevated to 80°C at an increasing rate of 10°C/min and then to 255°C at an increasing rate of

20°C/min. The pressure program was initially set to 7 kPa, then elevated to 15 kPa at an

increasing rate of 0.8 kPa /min, and held at 15 kPa for 4 min. The injector and detector

temperatures were set at 260°C. The test samples were injected (1 µL) in a splitless mode.

The instrument was monitored and the chromatograms were acquired using GC solution

software (Shimadzu). Based on the acquired GC data including standard curves, the SCFA in

wet fecal matter is expressed as “µmol SCFA/g wet fecal matter”.

2.7.4 Fecal IgA and PGE2 analysis

All fecal samples were weighed separately into 15 mL falcon tubes (approximately 1 g each)

and vortexed (Maxi Mix II, Thermolyne, USA) with equal volumes of 1.0% (w/v) bovine

serum albumin solution in 50 mM Tris buffer (BDH Laboratories, Poole, England), pH7.5,

containing 0.15 M- NaCl for approximately 60 min at room temperature. Approximately 1

mL of each treated fecal sample was then transferred into a 2 mL Eppendorf® tube, vortexed

and centrifuged (IEC MicroCL17R, Thermofisher, Germany) at 17,000 x g for 15 min. The

supernatant fractions were then carefully separated into new Eppendorf®

tubes for PGE2 and

IgA analysis.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

14

2.7.4.1 PGE 2 Analysis

PGE2 Enzyme Immunometric Assay kit monoclonal (#514010, Cayman Chemical Company,

Michigan, USA) was used to quantify fecal PGE2, according to the manufacturer’s

instructions.

2.7.4.2 IgA Analysis

Human IgA ELISA quantitation (E80-102) and ELISA starter accessory kit (E101) from

Bethyl Laboratories, Texas, USA, were used for the IgA analysis, according to the

manufacturer’s instructions.

2.8 Statistical Analyses

A total sample size of 30 participants in this cross over design enabled effect sizes of >0.1 to

be detected as significantly significant (two tailed α <0.05) with 80 % power.

Data are presented as means (standard deviation). The dietary analysis was carried out in

SAS 9.2 (Cary, North Carolina, USA) with time, treatment group and their interaction as

variables. Subject within treatment group was treated as a random effect with a first-order

auto-regressive heterogeneous covariance structure also used. Statistically significant main

effects were further investigated using pair-wise comparisons with Tukey’s HSD to control

for inflated type 1 error due to multiple testing. Additionally some intakes needed to be log-

transformed prior to analysis to stabilize the variance.

The analysis of the bacteria results was performed using two-way analysis of variance

(ANOVA) on GenStat software (version 11.1, VSNi Ltd, Hemel Hempsted, United

Kingdom). Data were first log transformed before analysis.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

15

All SCFA analyses were carried out using log-transformed data SAS 9.2’s Mixed Procedure.

Fitted means were back transformed onto the original scale with letters to indicate which

treatment means significantly differed (means with no letters in common are statistically

different at the 5% level). These letters were only used when the overall F-test was significant

at the 5% level.

Changes in IgA and PGE2 concentrations relative to baseline were analyzed using ANOVA

in GenStat (version 11.1), with subjects as blocks and treatment order (1st, 2

nd, 3

rd or 4

th) and

beverages as variables. The absolute change was analyzed for IgA; the percentage change for

PGE2 as these gave more consistent results.

3. Results

Of the 30 people who started the study, 25 people completed the 24-week study. The five

individuals who withdrew from the study were four males and one female. One withdrew due

to non-compliance and four due to illness caused by reasons unrelated to the study beverages.

3.1 Dietary Intake

In general, there was no evidence of statistical differences amongst the treatments in terms of

dietary intake (Table 4). Energy intake was lower during the Boysenberry juice plus apple

fiber beverage intervention but not significantly. Fiber intake also was higher but was not

significant during each of the three interventions compared with baseline or during placebo

consumption.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

16

3.2 Fecal sample analysis

Fecal samples were analyzed for total bacteria, Bacteroides-Prevotella-Porphyromonas ,

Bifidobacterium spp., Clostridium perfringens and Lactobacillus spp. No trends were

observed for any of the bacteria using scatter plots and ANOVA, suggesting no significant

differences in bacteria with any of the treatment groups (Figures 2a-d). However, it was

found that total bacteria populations increased irrespective of group after the first treatment,

that is, whether individuals consumed the Boysenberry juice beverage, the apple fiber

beverage or the Boysenberry juice plus apple fiber beverage (Figure 3).

Fecal samples were also analyzed for SCFAs including acetate, butyrate, isobutyrate,

propionate, succinate and lactate (Table 5). No significant differences in fecal SCFA

concentrations were observed between the treatment groups.

The changes in fecal IgA concentrations varied considerably across participants before and

after treatments. Therefore, the fecal IgA concentrations after each treatment were examined

as the percentage change from the baseline value.

ANOVA was performed for the treatment order with subjects as blocks, and treatment order

(1st, 2

nd, 3

rd or 4

th) and beverage as factors. This assigned a p value of 0.326 for the treatment

effect, indicating no significant difference between the mean changes in fecal IgA

concentrations after consumption of the different beverages (Figure 4).

The changes in PGE2 concentrations (end minus corresponding baseline) against the baseline

concentrations were also plotted (Figure 5).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

17

ANOVA was performed for the treatment order with subjects as blocks, and treatment order

(1st, 2

nd, 3

rd or 4

th) and beverage as factors. This suggested a significant order effect (p <

0.001), with the amount of PGE2 increasing from the 1st to 2

nd to 3

rd treatments indicating a

cumulative effect, but no significant difference between beverages (p = 0.339). When the

order effect was ignored, and apple fiber and Boysenberry juice ingredients were considered

as separate factors, the beverages containing Boysenberry juice showed an increase in PGE2

(p = 0.014). In fact, the apple fiber × Boysenberry juice interaction was marginally significant

(p = 0.073) suggesting the effect is stronger without apple fiber. The apple fiber effect was

not significant (p = 0.874).

A Daily Gut Health Questionnaire completed by participants raised questions related to the

frequency of bowel movement, description of bowel motion, unpleasant bowel symptoms

such as pain, bloating, constipation, wind or diarrhea, and beneficial symptoms such as less

flatulence, bloating and feeling more energetic. Amongst the questions, two showed positive

results; people felt they had an increase in beneficial symptoms relating to gut health such as

less flatulence and bloating after the Boysenberry juice beverage treatment in comparison to

the baseline, and after the apple fiber beverage and the Boysenberry juice plus apple fiber

beverage (p < 0.0001). However, these beneficial effects were not significantly greater than

the symptoms felt during the washout periods and on the placebo beverage. People felt they

generally had better mental well-being after baseline, each washout period, placebo and the

apple fiber beverage than after the Boysenberry juice beverage and the Boysenberry juice

plus apple fiber beverage (p = 0.045).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

18

The State -Trait Anxiety Questionnaire was administered as a result of the 2010 Canterbury

earthquake. No significant differences in anxiety between the time when the earthquake

occurred and some 4 months later when the study ended (Table 6).

3.3 Stability of the polyphenol content of the trial products

Each of the three trial products contained a complex phytochemical composition. Beverages

with Boysenberry fruit contained anthocyanins, ellagitannins and ellagic acid derivatives

whilst beverages containing apple fibre contained phloridzin, quercetin glycosides and

chlorogenic acid. The total concentrations of polyphenols were averaged for all sampling

dates and over the 7-day storage period at 4°C, and were found to be stable over this time

(Figure 6).

4. Discussion

This study found that the levels of total bacteria, Bacteroides-Prevotella-Porphyromonas ,

Bifidobacterium spp., Clostridium perfringens and Lactobacillus spp. in faeces did not differ

significantly between treatment groups. However, total bacteria numbers in faeces increased

irrespective of group after the first treatment; that is, whether individuals consumed the

Boysenberry juice beverage, the apple fiber beverage or the Boysenberry juice plus apple

fiber beverage. This could be a response to a general change in consumption of fiber and/or

polyphenols. It might be possible that the bacteria in the gut “reacted’ to a change in diet and

then stabilized as they adapted to it, suggesting an initial reaction period exists when the diet

changed. There have been no studies that directly examined the effect of a Boysenberry juice

plus apple fiber beverage on gut health; however, other studies have examined the effect of

polyphenols and dietary fiber from other fruits and in beverage formats on gut health.

Consumption of red wine that was rich in polyphenols by 10 healthy male volunteers for 4

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

19

weeks significantly increased the numbers of Enterococcus, Prevotella, Bacteroides,

Bifidobacterium, Bacteroides uniformis, Egerthella lenta and Blautia coccoides-Eubacterium

rectale group[25]. Vendrame et al.[17] found a significant increase in the quantity of

Bifidobacterium spp. after the consumption of a wild blueberry drink (containing 375 mg

anthocyanins and 127.5 mg chlorogenic acid) for 6 weeks by 20 healthy males[17]. Tzounis

et al.[26] found a significant increase in Bifidobacteria and Lactobacillus bacteria and

decreased Clostridia bacteria in 22 healthy volunteers after consumption of a high cocoa

flavanol beverage (494 mg cocoa flavanols/day) compared with a low cocoa flavanol

beverage (23 mg cocoa flavanols/day) for 4 weeks [26]. Yamakoshi et al.[22] investigated the

effect of proanthocyanadin-rich extract containing 38.5% proanthocyanidin on nine healthy

adults at a dose of 0.5 g/day (0.19 g/day as proanthcyanidin) for 2 weeks. Green tea extract

and champignon extract were administered in a similar manner to that for the controls. After

2 weeks of proanthocyanidin-rich extract intake, the number of Bifidobacterium had

increased significantly[22]. Studies have also shown that the inclusion of dietary fibre into

the diet can result in favourable changes to the gut microbiota promoting diversity and

leading to possible health benefits[27]. This shift towards a healthier bacterial population is

caused by the availability of fibre as a substrate for bacterial fermentation which results in an

increase in SCFA such as butyrate, acetate and proprionate that help maintain colonic

health[28]. Different types of fibre such as inulin has been shown to stimulate the growth of

Bifidobacteria and restrict the growth of the potentially pathogenic bacteria such as E.coli,

Salmonella and Listeria [29] and diets containing xylo-oligisaccharides result in lower levels

of Bacteriodes fragilis, the sulphate reducing bacteria[28]. Hooda et al, 2012 showed that the

consumption of soluble corn fibre and polydextrose both result in favourable changes to the

abundance of bacteria such as Clostridiaceae, Veilloellaceae and Faecalibacterium which is

thought to have anti-inflammatory properties[30]. Turner et al, 2013 also showed the role

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

20

dietary fibre may play in the reduction of colon cancer risk through manipulation of the gut

bacterial population [31] whilst Alenberg & Wu, 2014 demonstrated a similar effect in the

reduction of coronary vascular disease and inflammatory bowel disease risk[32].

While there is evidence supporting the efficacy of polyphenols in modulating gut bacteria, the

concentrations, bioavailability and method of delivery varied considerably between studies.

There were no significant changes in the bacterial populations in the present study, which

could have been due to several factors. The food matrix used to deliver the polyphenols can

significantly influence their bioavailability[33, 34]. In this study, the Boysenberry juice

concentrate was the source of anthocyanins and ellagitannins/ellagic acid and so it would be

expected that the concentrations of these polyphenols in the Boysenberry juice beverage

would be the same as in the Boysenberry juice plus apple fiber beverage. However, although

the overall levels of polyphenols remained stable over the study period, the concentration of

ellagitannin/ellagic acid in the Boysenberry beverage was higher than in the combined

ingredients in the Boysenberry juice plus apple fiber beverage. Similarly, the anthocyanin

concentrations in the Boysenberry beverage were lower than in the Boysenberry juice plus

apple fiber beverage. These results suggest that the presence of apple fiber affected the

extractability and availability of the polyphenols in different ways although the underlying

mechanism remains unknown[34-36]. Food matrix effects and the interactions between

polyphenols and co-existing food components such as fiber, other polysaccharides and

proteins during storage and after ingestion, which could have impacts upon absorption, have

not been fully explored[37]. More research is required to determine how polyphenols may

interact with other dietary components in the gut, such as the binding of polyphenols to

macromolecules like different polysaccharide or protein biopolymers and the formation of

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

21

various complexes, and how such interactions affect their bioavailability. Nevertheless, such

interactions may have been an important factor for this study.

Compliance with the storage and consumption rules of the study beverages were reported as

high as 87% because of the effort made to ensure that participants complied with the study

protocol. The study participants reported that they found the beverages appealing and most

liked the taste and texture. However, we were aware that these reports on beverage

consumption may still not be a true reflection of the actual beverage consumption. Non-

compliance to study products may be due to reasons such as failure to advise of the exact

instruction for consumption, failure to follow required storage instruction, forgetfulness,

aversion to the taste of the study drink(s), limits related to time constraint due to

life/work/stress, and undetectable disruption caused by the Canterbury Earthquake in

September 2010. Although anxiety levels following the earthquake were monitored via the

State/Trait anxiety Questionnaire and results showed no significant differences in anxiety

level for our trial participants, undetectable anxiety or other influencing factors could still

exist given the significant damage to living environment and varied interruptions to daily

routines within the community. These are considered potential interfering factors for this

study.

Following the consumption of the Boysenberry juice beverage, there was a significant

increase in the level of PGE2 (p < 0.05). PGE2 is a bioactive lipid that exerts a wide range of

biological effects particularly in relation to inflammation. Whilst PGE2 assumes a pro-

inflammatory role in driving acute inflammation, it is also able to elicit an anti-inflammatory

response leading to a reduction in inflammation and tissue repair[38]. Previous studies[39]

have shown that the polyphenols from berry fruits were effective in modulating enzymatic

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

22

pathways that are active in the inflammatory process which suggests that the Boysenberry

beverage may have a positive effect in this aspect. The levels of PGE2 increased from

treatment 1 to treatments 2 and 3, indicating a treatment effect over the time period of this

study, although the possible association of this finding to the effects of the Canterbury

earthquake and derived stress could not be completely excluded. It is known that PGE2

increases under certain situations related to stress[40]. This study did not include the

measurement of any related inflammatory biomarker except for conducting a questionnaire

follow-up to address increased anxiety; thus, the latter result of increased PGE2

concentrations is not of major significance and further studies with Boysenberry juice

beverages are required.

It is difficult to ascertain the relevance of the results from the subjective measures of feelings

of gut wellbeing collected throughout the study. Participants experienced a reduction in

feelings of gut discomfort (bloating/flatulence) when consuming the Boysenberry juice

beverage in comparison to baseline, and the apple fiber or the Boysenberry juice plus apple

fiber beverages. This improvement was not significantly different when participants were

consuming the placebo or during washout period. The Boysenberry beverage was anecdotally

reported as the most desirable drink overall among the study products. The comfort effects

found here may have been influenced by this sensory appreciation of the beverage. The

unpleasant feelings of bloating and flatulence could be associated with the fiber content in the

beverages containing apple fiber because dietary fiber is well documented to cause bloating

and flatulence effects.

A limitation of this study was that the clinical trial was not fully randomized. This was a

trade-off for the possible instability of the polyphenols in the study beverages. That is, it was

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

23

decided to give participants the placebo beverage last, in order to reduce the storage time for

those polyphenol-containing beverages in the -18ºC freezer after beverage manufacture. Any

changes that occurred as a result of the placebo beverage may have been negated due to the

carry-over effects of the previous three treatments that may not have had sufficient time to

washout from the body. Effects from this placebo treatment may also have been affected by

seasonal timing of the beverage consumption over the 24-week period, which would have

occurred over the same 4 weeks for all trial participants, and as it occurred at the end of the

study, compliance may have also been an associated issue. Randomization of clinical trials is

an essential factor in reducing bias, minimizing variability, avoiding confounding from other

factors and forming a basis for the statistical analysis of the data. Due to the challenges of

confounding variables of storage time vs. single batch manufacturing of beverages, the

decision to give the placebo in one treatment phase was the best option for retaining

polyphenol viability in this study.

The hypothesis for this study was that the combination of polyphenols and fiber would have a

greater benefit on gut health than the placebo product or the fiber or polyphenols on their

own. In conclusion, with the proviso that the study was not fully randomized, no significant

differences were found in quantitative measures of gut health between the Boysenberry juice

beverage, the apple fiber beverage, the Boysenberry juice plus apple fiber beverage, and the

placebo beverage.

Tables

Table 1. Baseline characteristics of the study participants.

Table 2. Nutritional composition of the trial beverages per 350 g serve.

Table 3. Formulation of the trial beverages.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

24

Table 4. Dietary data at baseline and during consumption of each of the four trial beverages.

Table 5. Concentrations (µmol SCFA/g wet faecal matter) of faecal SCFA at baseline,

following consumption of each test beverages and after the washout period.

Table 6. State Trait Analysis.

Figures

Figure 1. Schematic of the trial design

Figure 2 Scatterplot demonstrating the population of a) Lactobacillus b) Bifidobacteria c)

Bacteroides and d) Clostridium perfringens in fecal samples as determined at baseline and

after consumption of each of the test beverages.

Figure 3. Scatterplot demonstrating the population of total bacteria in fecal samples as

determined at baseline and after each phase.

Figure 4. Changes to fecal IgA concentrations after consumption of beverages, expressed as a

percentage of initial IgA concentration.

Figure 5. Changes in PGE2 concentrations after consumption of test beverages.

Figure 6. Average concentrations for total polyphenols for all sampling weeks during the 7-

day storage at 4ºC.

Acknowledgments

The authors have no conflicts of interest to declare. The study was funded by the Foundation

for Research, Science and Technology. The Foundation for Research, Science and

Technology had no role in the design, analysis or writing of this article.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

25

References

1. Qin J, Ruiqiang L, Raes J, Arumugam M, Burgdorf S, Manichanh C, et al. A human

gut microbial gene catolgue established by metagenomic sequencing. Nature 2010;

464:59-65.

2. Wong JMW, de Souza R, Kendall CWC, Azadeh E, Jenkins DJA. Colonic health:

fermentation and short chain fatty acids. J Clin Gastroenterol 2006; 40:235-43.

3. Saulnier DM, Kolida S, Gibson GR. Microbiology of the human intestinal tract and

approaches for its dietary modulation. Current Pharm Design 2009; 15:1403-14.

4. Chen J, He X, Huang J. Diet effects in gut microbiome and obesity. J Food Sci 2014;

79:R442-51.

5. Hooper LV, Gordon JI. Commensal host-bacterial relationships in the gut. Science

2001; 292:1115-18.

6. Martins dos Santos V, Muller M, De Vos WM. Systems biology of the gut: the

interplay of food, microbiota and host at the mucosal interface. Curr Opin Biotech

2010; 21:539-50.

7. Power SE, O'Toole PW, Stanton C, Ross RP, Fitzgerald GF. Intestinal microbiota,

diet and health. Brit J Nutr 2014; 11:387-402.

8. Umu OCO, Oostinjer M, Pope PB, Svihus B, Egelandsdal B, Nes IF, et al. Potential

applications of gut microbiota to control human physiology. Anton Leeuw Int J G

2013; 104:609-18.

9. Cummings JH, Antione J-M, Azpiroz F, Bourdet-Sicard R, Brandtzaeg P, Calder PC,

et al. PASSCLAIM - Gut health and immunity. Eur J Clin Nutr 2004; 43:II/118 - 73.

10. Slavin J. Fiber and prebiotics: mechanisms and health benefits. Nutrients 2013;

5:1417-35.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

26

11. Ovaskainen ML, Torronen R, Koponen JM, Sinkko H, Hellstrom J, Reinivuo H, et al.

Dietary intake and major sources of polyphenols in Finnish adults. J Nutr 2008;

138:562-66.

12. Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols. J Nutr

2000; 130:2073S-85S.

13. Saura-Calixto F, Goni I. Antioxidant capacity of the Spanish Mediterranean diet.

Food Chem 2006; 94:442-7.

14. Parkar SG, Trower TM, Stevenson DE. Fecal microbial metabolism of polyphenols

and its effects on human gut microbiota. Anaerobe 2013; 23:12-9.

15. Tuohy KM, Conterno L, Gasperotti M, Viola R. Up-regulating the human intestinal

microbiome using whole plant foods, polyphenolas and/or fiber. J Agric Food Chem

2012; 60:8776-82.

16. Parkar SG, Trower TM, Stevenson DE. Fecal microbial metabolism of polyphenols

and its effect on human gut microbiota. Anaerobe 2013; 23:12-9.

17. Vendrame S, Guglielmetti S, Riso P, Arioli S, Klimis-Zacas D, Porrini M. Six-week

consumption of a wild blueberry powder drink increases Bifidobacteria in the human

gut. J Agric Food Chem 2011; 59:12815-20.

18. Landete JM. Updated knowledge about polyphenols:functions, bioavailability,

metabolism and health. Crit Rev Food Sci 2012; 52:936-48.

19. Sun-Waterhouse D, Nair S, Wibisono R, Wadhwa SS, Massarotto C, Hedderley DI,

Zhou J, Jaeger SR, Corrigan V. Insights into smoothies with high levels of fibre and

polyphenols: factors influencing chemical, rheological and sensory properties. World

Academy of Science, Engineering and Technology 2010; 4:1046-55.

20. D'Archivio M, Filesi C, Vari R, Scazzocchio B, Masella R. Bioavailability of the

polyphenols: status and controversies. Int J Mole Sci 2010; 11:1321-42.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

27

21. Ghosh D, McGhie TK, Zhang J, Adaim A, Skinner M. Effects of anthocyanins and

other phenolics of boysenberry and blackcurrant as inhibitors of oxidative stress and

damage to cellular DNA in SH-SY5Y and HL-60 cells. J Sci Food Agric 2006;

86:678-86.

22. Yamakoshi J, Tokutake S, Kikuchi M, Kubota H, Mitsuoka T. Effect of

proanthicyanidin-ricj extract from grape sees on human fecal flora and fecal odour.

Microb Ecol Health D 2001; 13:25-31.

23. Ciric L, Pratten J, Wilson M, Spratt D. Development of a novel multi-triplex qPCR

method for the assessment of bacterial community structure in oral populations.

Environ Microbiol Reports 2010; 2:770-74.

24. Richardson AJ, Calder AG, Stewart CS, Smith A. Simultaneous determination of

volatile and non-volatile acidic fermentation products of anaerobes by capillary gas

chromatography. Lett Appl Microbiol 1989; 9:5-8.

25. Queipo-Ortuno MI, Boto-Ordonez M, Mirri M, Gomez-Zumaquero JM, Clemente-

Postigo M, et al. Influence of red wine polyphenols and ethanol on the gut micobiota

ecology and biochemical biomarkers. Am J Clin Nutr 2012; 95:1323-34.

26. Tzounis X, Rodriguez-Mateos A, Vulevic J, Gibson GR, Kwik-Uribe C, Spencer JPE.

Prebiotic evaluation of cocoa-derived flavanols in healthy humans by using a

randomized, controlled, double-blind, crossover intervention study. Am J Clin Nutr

2011; 93:62-72.

27. Hugenholtz F, Mullaney JA, Kleerebezem M, Smidt H, Rosendale D. Modulation of

the microbial fermentation in the gut by fermentable carbohydrates. Bioactive

Carbohydrates and Dietary Fibre 2013; 2:133-42.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

28

28. Zeng H, Lazarova DL, Bordonaro M. Mechanisms linking dietary fibre, gut

microbiota and colon cancer prevention. World Journal of Gastrointestinal Oncology

2014; 6:41-51.

29. Bosscher D, Breynaert A, Pieters I, Hermans N. Food –based strategies to modulate

the compostion of the intestinal microbiota and their associated health effects. J

Physiol Pharmacol 2009; 60:5-11.

30. Hooda S, Boler BM, Serao MC, Brulc JM, Staeger MA, Boileau TWD, et al. 454

pyrosequencing reveals a shift in fecal micorbiota of healthy adult men consuming

polydextrose or soluble corn fiber . J Nutr 2012; 142:1259-65.

31. Turner ND, Ritchie LE, R.S. B, Chapkin RS. The microbiome and colorectal

neoplasia: environmental modifiers of dysbiois. Current Gastroenterological Reports

2013; 15:346.

32. Alenburg LG, Wu GD. Diet and the intestinal microbiome: associations, functions

and implications for health and disease. Gastroenterology 2014; 146:1564-72.

33. Manach C, Williamson G, Morand C, Scalbert A, Remesy C. Bioavailability and

bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J

Clin Nutr 2005; 81:230S-42S.

34. Palafox-Carlos H, Ayala-Zavala JF, Gonzalez-Aguolar GA. The role of dietary fiber

in the bioaccessibility and bioavailability of fruit and vegetable antioxidants. J Food

Sci 2011; 76:R6-15.

35. Quiros-Sauceda AE, Palafox-Carlos H, Sayago-Ayerdi SG, Ayala-Zavala JF, Bello-

Perez LA, Alvarez-Parilla E, et al. Dietary fibre and phenolic compounds as

functional ingredients: interaction and possible effect after ingestion. Food and

Function 2014; 5:1063-72.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

29

36. Tourino S, Perez-Jimenez J, Mateos-Martin ML, Fuguet E, Vinardell MP, Cascante

M, et al. Metabolites in contact with rat digestive tract after ingestion of a phenolic-

rich dietary fibre matrix. J Agric Food Chem 2011; 59:5955-63.

37. Nozaki A, Kimura T, Ito H, Hatano T. Interaction of polyphenolic metabolites with

human serum albumin: a circular dichroism study. Chem Pharm Bull 2009; 57:1019-

23.

38. Nakanishi M, Rosenberg DW. Multifaceted roles of PGE2 in inflammation and

cancer. Seminars in Immunopathology 2013; 35:123-37.

39. Wu X, Schauss AG. Mitigation of inflammation with foods. J Agric Food Chem

2012; 60:6703-17.

40. Furuyashiki T, Narumiya S. Roles of prostaglandin E receptors in stress responses.

Curr Opin Pharmacol 2009; 9:31-8.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

30

Fig. 1

Beginning and end of treatment phase, faeces, saliva samples collected. Three day diet record completed; colon transit time tracked with carmine red and daily questionnaire completed

Beginning and end of treatment phase, faeces, saliva samples collected. Three day diet record completed; colon transit time tracked with carmine red and daily questionnaire completed

Beginning and end of treatment phase, faeces, saliva samples collected. Three day diet record completed; colon transit time tracked with carmine red and daily questionnaire completed

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

31

Fig. 2

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

32

Fig. 3

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

33

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

34

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

35

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

36

Table 1. Baseline characteristics of the study participants.

Variable Mean Standard Deviation

Gender (n)

Male

Female

9

21

Age (years) 50 5.9

Weight (kg) 76 13.7

BMI (kg/m2) 27 3.7

* Values are mean (standard deviation)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

37

Table 2. Nutritional composition of the trial beverages per 350 g serve.

Nutrition /350g

Boysenberry

Juice

Apple

fibre

Boysenberry juice plus

apple fibre Placebo

Energy (kJ) 479 497 589 383

Protein (g) 1.1 0.7 1.8 0.0

Fat (g) 0.1 0.6 0.7 0.0

Saturated (g) 0.1 0.1 0.1 0.0

Carbohydrates , Total (g) 27.3 24.1 28.4 22.9

Sugars Total(g) 22.9 22.9 22.9 22.9

Fibre Total (g) 0.2 7.6 7.5 0.0

Soluble (g) 0.2 5.8 5.8 0.0

Insoluble(g) 0.0 1.8 1.7 0.0

Polyphenols (mg) 802.8 0.2 802.8 0.2

Water (g) 320.4 316.8 310.5 326.8

Ash (g) 0.9 0.2 1.1 0.2

Total (g) 350.0 350.0 350.0 350.0

* Values are means

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

38

Table 3. Formulation of the trial beverages.

Ingredient Boysenberry

Juice

Apple

fibre

Boysenberry

juice plus

Apple fibre

Placebo

Water (g) (Potable water from

Palmerston North municipal supply)

88.2 90.2 85.2 93.3

Boysenberry Juice Concentrate (g)

(65°Brix) Berry Fruit NZ, Nelson,

New Zealand.

8.3 0 8.3 0

Apple Fibre (g) (Herbacel Classic

AF01, Herbafood Ingredients GmBH,

Werder, Germany; purchased through

Hawkins Watts Ltd, Auckland New

Zealand)

0 4.2 4.0 0

Sucrose (g) (Chelsea White sugar,

Auckland New Zealand)

3.5 5.5 2.5 6.6

Elderberry Juice concentrate (g)

(7568; Zymus, Auckland New

Zealand)

0 0.13 0 0.13

Citric Acid (g) (Anhydrous; Hawkins

Watts Ltd, Auckland New Zealand)

0 0 0 0.06

Total 100 100 100 100

* Values are means

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

39

Table 4. Dietary data at baseline and during consumption of each of the four trial beverages.

Baseline Boysenberry Apple fibre Boysenberry

Juice plus

apple fibre

Placebo

Energy (kJ) 8932

(3096)

8563 (2216) 8584 (3112) 8091 (2439) 8273 (2532)

Protein (%) 17.8 (4.1) 17.8 (4.8) 17.3 (3.8) 17.9 (4.1) 16.4 (3.5)

Fat (%) 31.4 (7.2) 31.2 (7.9) 30.7 (8.5) 33.1 (7.2) 33.8 (7.7)

Carbohydrate (%) 46.9 (7.6) 47.4 (8.2) 46.6 (10.0) 44.2 (8.7) 44.6 (7.2)

Fibre (g) 24.8 (9.2) 30.0 (14.1) 28.8 (16.5) 28.7 (14.0) 24.8(10.1)

# Values are means (standard deviation)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

40

Table 5. Concentrations (µmol SCFA/g wet faecal matter) of faecal SCFA at baseline,

following consumption of each test beverages and after the washout period.

Treatment Period

Short chain fatty acid species (µmol SCFA/g wet faecal matter)

Baseline Boysenberry Juice

Apple Fiber

Boysenberry + Apple Fiber

Placebo Washout P value

Acetate 34.64 36.04 37.47 37.81 52.96 39.82 0.15 Propinate 9.16 8.63 10.69 10.69 13.44 10.90 0.24 Isobutyrate 1.19 1.69 1.70 1.70 1.56 1.70 0.08 Butyrate 7.50 7.61 7.77 8.49 9.42 8.44 0.92 Lactate 1.77 2.36 2.35 2.70 1.97 2.25 0.70 Succinate 0.72 0.50 0.73 0.94 0.87 0.73 0.20 Values are fitted means.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

41

Table 6. State Trait Analysis.

Y1 State

Questionnaire

September 2010

Y2 Trait

Questionnaire

September 2010

Y1 State

Questionnaire

December 2010

Y2 Trait

Questionnaire

December 2010

Mean 37.76 (14.96) 39.6 (11.86) 35.08 (13.87) 38.48 (10.77)

Results are expressed as Mean (SD) with p > 0.05.