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. 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, Tony
McGhie, Dongxiao Sun-Waterhouse, Claire Redman
PII:
DOI:
Reference:

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

To appear in:

Nutrition Research

Received date:
Revised date:
Accepted date:

15 September 2014
18 November 2014
19 November 2014

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

This is a PDF le of an unedited manuscript that has been accepted for publication.
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ACCEPTED MANUSCRIPT
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

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Alison J Wallace1, Sarah L Eady1, Denise C Hunter2, Margot A Skinner2, Lee Huffman3,
Juliet Ansell3, Paul Blatchford3, Mark Wohlers2,Thanuja D Herath3, Duncan Hedderley3,

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Douglas Rosendale3, Halina Stoklosinski3,Tony McGhie3, Dongxiao Sun-Waterhouse2,

Claire Redman3.

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

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

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Auckland, New Zealand

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

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New Zealand

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

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

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

Running title: Effect of fruit juice beverages on gut health

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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

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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

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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

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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

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

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

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chain fatty acid concentrations did not vary among treatment groups although PGE2
concentrations were higher after consumption of the Boysenberry juice beverage. No

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

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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

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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

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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,

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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,

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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,

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antibiotic usage and disease. These changes to the microbial diversity have been linked to
diseases such as obesity, inflammatory bowel disease, diabetes, cardiometabolic

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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

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

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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

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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

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studies to be efficacious through their ability to elevate the number of Bifidobacteria spp.,
Lactobacilli spp., Fecalibacterium prausnitzii and Ruminococcus bromii and to reduce

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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

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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

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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

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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

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

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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

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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

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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

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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

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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

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bacteria, short chain fatty acids IgA and PGE2.

Participants

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2.1

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2. Methods and materials

Participants were recruited through advertisements and articles in the local Christchurch

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(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.

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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.

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The trial was registered with the Australia New Zealand Clinical Trials Registry, registration
number ACTRN12609000907246.
Experimental design

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2.2

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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

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

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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

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

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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

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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

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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

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

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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,

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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

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

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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

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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., BacteroidesPrevotella-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.

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A daily questionnaire tracking the participants bowel motions and regularity, and their
physical/mental well being was included throughout the study. Participants completed one for

The Canterbury Earthquake

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2.3

everyday of the study including during washout periods.

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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

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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

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

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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

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(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

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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. 3day intervals) including analysis of their phytochemical composition and evaluation of the

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stability of the phytochemicals under conditions of frozen storage (at -182C) throughout
the study and refrigerated storage (21C) for 7 days after distribution of the drinks to the

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

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182C) each Friday and transferred to a cold room (21C). On the Monday (termed 0 h),
Thursday (termed 78 h) and following Monday (termed 168 h), aliquots (~2.0 g) of each

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

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

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tubes and stored at -18C 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

Compliance

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2.5

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Technologies).

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

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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.

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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

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

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The placebo beverage was produced by dissolving 6.6g/100g sugar in water (70C) 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 25C and

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filled inline. The placebo beverage (25C) was filled into PET bottles that were pre-sterilised
with steam and UV light (Xenos Aseptic Filler, X2000). After filling, the beverages were

2.7

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labelled, cooled and stored at -18C.

Quantification of microbiota from fecal samples

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2.7.1 DNA Sample processing

The fecal samples were shipped frozen to Plant & Food Research, Palmerston North, where
they were stored at -80C 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 L was dispensed into a sterile microtube and stored at -80C
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.

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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

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obtained from stocks kindly donated by Gunaranjan Paturi (Plant & Food Research,
Auckland); these were stored at -20C. DNA obtained was extracted in the same manner

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using the same kit as above.

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Bifidobacterium adolescentis NCTC 11814 was grown in MRS broth (Oxoid) + 0.05%
cysteine at 37C for 2 days under anaerobic conditions. This suspension was adjusted to a

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

qPCR

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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

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

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Green I Master Mix (04707516001), according to the manufacturers Product Instructions.

Primers used in the study were obtained from the Invitrogen custom oligonucleotide service.
Each qPCR run included one activation cycle (95C), 30 run cycles (including the
denaturation step at 95C, the annealing step (temperature 55C - 63C) and the extension
step at 72C), 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 M. This was then diluted at a ratio of 1:10 to obtain a 5 M solution for
the PCR reaction.

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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

Short Chain Fatty Acid Quantification by Gas Chromatography

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2.7.3

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triplicate[23].

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

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

colleagues[24].

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Wet fecal matter from the -80C freezer was weighed into 15 mL tubes. SCFA were
extracted into an aqueous phase by diluting 10-fold with phosphate buffered saline (Sigma 10

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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

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

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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 L) and the derivatizing reagent N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide containing 1% tert-Butyldimethylcholorosilane (MTBSTFA + 1%
TBDMSCI, Sigma-Aldrich) (20 L) were placed into a 2.5 mL glass vial and capped.
Following a brief vortex mixing, the mixture was incubated at 80C 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.

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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 m film thickness). The carrier gas was helium with a total

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flow rate of 37 mL/min and pressure of 7 kPa. The temperature program began at 70C, and
elevated to 80C at an increasing rate of 10C/min and then to 255C at an increasing rate of

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20C/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

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temperatures were set at 260C. The test samples were injected (1 L) 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

Fecal IgA and PGE2 analysis

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2.7.4

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wet fecal matter is expressed as mol SCFA/g wet fecal matter.

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

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

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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.

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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 manufacturers

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instructions.

2.7.4.2 IgA Analysis

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

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

2.8

Statistical Analyses

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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 <0.05) with 80 % power.

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

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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

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

effects were further investigated using pair-wise comparisons with Tukeys HSD to control
for inflated type 1 error due to multiple testing. Additionally some intakes needed to be logtransformed 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.

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All SCFA analyses were carried out using log-transformed data SAS 9.2s 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

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

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at the 5% level.

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

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in GenStat (version 11.1), with subjects as blocks and treatment order (1st, 2nd, 3rd or 4th) and
beverages as variables. The absolute change was analyzed for IgA; the percentage change for

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PGE2 as these gave more consistent results.

3. Results

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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

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

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.

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3.2

Fecal sample analysis

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

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

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

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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,

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that is, whether individuals consumed the Boysenberry juice beverage, the apple fiber
beverage or the Boysenberry juice plus apple fiber beverage (Figure 3).

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Fecal samples were also analyzed for SCFAs including acetate, butyrate, isobutyrate,
propionate, succinate and lactate (Table 5). No significant differences in fecal SCFA

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concentrations were observed between the treatment groups.

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

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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, 2nd, 3rd or 4th) 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).

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

(1st, 2nd, 3rd or 4th) and beverage as factors. This suggested a significant order effect (p <

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0.001), with the amount of PGE2 increasing from the 1st to 2nd to 3rd treatments indicating a
cumulative effect, but no significant difference between beverages (p = 0.339). When the

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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

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(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

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not significant (p = 0.874).

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

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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

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

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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).

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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

3.3

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

Stability of the polyphenol content of the trial products

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Each of the three trial products contained a complex phytochemical composition. Beverages
with Boysenberry fruit contained anthocyanins, ellagitannins and ellagic acid derivatives

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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 4C, and were found to be stable over this time

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(Figure 6).

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4. Discussion

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

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

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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

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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

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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

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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

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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

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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

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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

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

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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

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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

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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

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

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

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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

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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

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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

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

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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

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

interactions may have been an important factor for this study.

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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

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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

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consumption may still not be a true reflection of the actual beverage consumption. Noncompliance 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,

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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

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September 2010. Although anxiety levels following the earthquake were monitored via the
State/Trait anxiety Questionnaire and results showed no significant differences in anxiety

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

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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 proinflammatory 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

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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

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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

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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

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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

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beverages are required.

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

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

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

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

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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

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decided to give participants the placebo beverage last, in order to reduce the storage time for
those polyphenol-containing beverages in the -18C freezer after beverage manufacture. Any

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

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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

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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

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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

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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

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polyphenol viability in this study.

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

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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.

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Table 4. Dietary data at baseline and during consumption of each of the four trial beverages.
Table 5. Concentrations (mol SCFA/g wet faecal matter) of faecal SCFA at baseline,

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

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Table 6. State Trait Analysis.

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Figures
Figure 1. Schematic of the trial design

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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.

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

determined at baseline and after each phase.

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

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percentage of initial IgA concentration.

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

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Figure 6. Average concentrations for total polyphenols for all sampling weeks during the 7day storage at 4C.

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.

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Fig. 1

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Fig. 2

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Fig. 3

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Table 1. Baseline characteristics of the study participants.

Mean

Standard Deviation

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Variable

Gender (n)
9

Female

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Age (years)

50

5.9

Weight (kg)

76

13.7

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Male

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BMI (kg/m2)

27

3.7

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* Values are mean (standard deviation)

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

Nutrition /350g

Juice

fibre

Energy (kJ)

479

497

Protein (g)

1.1

Fat (g)

Boysenberry juice plus

Apple

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Boysenberry

Placebo

589

383

0.7

1.8

0.0

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

0.2

5.8

5.8

0.0

0.0

1.8

1.7

0.0

802.8

0.2

802.8

0.2

320.4

316.8

310.5

326.8

0.9

0.2

1.1

0.2

350.0

350.0

350.0

350.0

Insoluble(g)

Ash (g)
Total (g)

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Water (g)

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Polyphenols (mg)

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Soluble (g)

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apple fibre

* Values are means

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Table 3. Formulation of the trial beverages.
Ingredient

Boysenberry Apple
fibre

Placebo

juice plus

Juice

Boysenberry

Water (g) (Potable water from

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Apple fibre
88.2

90.2

85.2

93.3

8.3

4.2

4.0

3.5

5.5

2.5

6.6

0.13

0.13

0.06

100

100

100

100

Boysenberry Juice Concentrate (g)

8.3

(65Brix) Berry Fruit NZ, Nelson,

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Palmerston North municipal supply)

New Zealand.
Apple Fibre (g) (Herbacel Classic

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AF01, Herbafood Ingredients GmBH,

Werder, Germany; purchased through

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Hawkins Watts Ltd, Auckland New

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Zealand)

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Sucrose (g) (Chelsea White sugar,

Auckland New Zealand)
Elderberry Juice concentrate (g)
(7568; Zymus, Auckland New
Zealand)
Citric Acid (g) (Anhydrous; Hawkins
Watts Ltd, Auckland New Zealand)
Total
* Values are means

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

Boysenberry

Apple fibre

Boysenberry

Placebo

Juice plus

Energy (kJ)

8932

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apple fibre
8563 (2216)

8584 (3112)

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(3096)

8091 (2439)

8273 (2532)

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)

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Protein (%)

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# Values are means (standard deviation)

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

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

Treatment
Period
Boysenberry
Juice

Apple
Fiber

Boysenberry
+ Apple
Fiber

Placebo

Washout

P
value

34.64
9.16
1.19
7.50
1.77
0.72

36.04
8.63
1.69
7.61
2.36
0.50

37.47
10.69
1.70
7.77
2.35
0.73

37.81
10.69
1.70
8.49
2.70
0.94

52.96
13.44
1.56
9.42
1.97
0.87

39.82
10.90
1.70
8.44
2.25
0.73

0.15
0.24
0.08
0.92
0.70
0.20

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Values are fitted means.

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Baseline

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Short chain fatty acid

species
(mol SCFA/g wet faecal
matter)
Acetate
Propinate
Isobutyrate
Butyrate
Lactate
Succinate

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Table 6. State Trait Analysis.
Y1 State

Y2 Trait

Questionnaire

Questionnaire

Questionnaire

Questionnaire

September 2010

September 2010

December 2010

37.76 (14.96)

39.6 (11.86)

35.08 (13.87)

Y2 Trait

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Mean

Y1 State

38.48 (10.77)

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

December 2010

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