EVALUATION OF A MULTI-STRAIN PROBIOTIC FORMULATION ON GASTROINTESTINAL FUNCTION AND GENERAL WELLNESS IN HEALTHY ADULTS: A RANDOMIZED, DOUBLE-BLIND, DOSE-RESPONSE STUDY By ASMAA M.N. FATANI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2016
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EVALUATION OF A MULTI-STRAIN PROBIOTIC FORMULATION ON GASTROINTESTINAL FUNCTION AND GENERAL WELLNESS IN HEALTHY
ADULTS: A RANDOMIZED, DOUBLE-BLIND, DOSE-RESPONSE STUDY
By
ASMAA M.N. FATANI
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
2 PURPOSE AND HYPOTHESIS .............................................................................. 34
Purpose of the Study .............................................................................................. 34 Hypothesis .............................................................................................................. 34
3 METHODS AND PROCEDURES ........................................................................... 35
Study Design .......................................................................................................... 35 Inclusion and Exclusion Criteria before Attaining Consent ..................................... 35
Randomization and Intervention ............................................................................. 38 Washout and Post Intervention ............................................................................... 39 Compensation ......................................................................................................... 39 Statistical Methods .................................................................................................. 40
Equivalence Testing ......................................................................................... 40 The Frequency and GIMMIX Procedures ......................................................... 41
Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
EVALUATION OF A MULTI-STRAIN PROBIOTIC FORMULATION ON
GASTROINTESTINAL FUNCTION AND GENERAL WELLNESS IN HEALTHY ADULTS: A RANDOMIZED, DOUBLE-BLIND, DOSE-RESPONSE STUDY
By
Asmaa M.N. Fatani
December 2016
Chair: Wendy Dahl Major: Food Science and Human Nutrition
Although many species of bacteria have been evaluated and marketed as
probiotics, little work has examined the dose-response effects of probiotics on
microbiota and host. A probiotic mix, containing eight strains that are selected from 3
genera: Lactobacillus, Pediococcus, and Bifidobacterium., is currently marketed in the
U.S. at 5 billion and 25 billion colony forming units (CFU)/capsule doses. To determine
the effects of the two doses of the multi-strain probiotic on gastrointestinal function and
general wellbeing in healthy adults, a six-week, randomized, double-blind, dose-
response study was conducted with 69 healthy adults (18-50 years old). Participants
were randomly assigned to receive either probiotic supplements or placebo for four
weeks. Daily questionnaires were used to assess the general gastrointestinal symptoms
and general wellness such as gastrointestinal distress, ear-nose-throat, epidermal,
psychological health, cephalic, emetic, diarrhea, constipation, hours of sleep, and bowel
movement frequency. Bristol Stool Form Scale (BSFS) was used in the daily
questionnaire to estimate transit time. In addition, Gastrointestinal Symptoms Rating
Scale (GSRS) was administered weekly to further evaluate gastrointestinal symptoms.
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Equivalence testing showed that the gastrointestinal symptoms and wellness assessed
by the daily questionnaire and GSRS were not affected by the consumption of the multi-
strain probiotic at the two different doses. In addition, there was no significant changes
in the transit time between treatments as assessed by BSFS. No adverse events were
reported. Therefore, the formulation of the multi-strain probiotic is well tolerated by
healthy adults at intakes at dosages up to 25 billion CFU/day.
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CHAPTER 1 LITERATURE REVIEW
Introduction
In the last few years, humans have started to appreciate that their bodies are not
just a home for cells, but a harbor for at least 100 trillion (1014) microbial cells and
quadrillions of viruses. All these taxa make a complex community that interacts within
itself and with the host, significantly impacting human health and physiological function.
The microbial co-inhabitants that exist in and on the human body shape human
microbiota and can be identified by the genes by which they are encoded (Clemente et
al., 2012). According to Fujiya & Kohgo (2010), more than 2,000 species of commensal
bacteria are found in and on the human body. Most of these bacteria are located in the
gut and the majority of them are not pathogens. Recently, whole genome-based
technologies have emphasized the differences in the populations of microbes between
individuals, areas of the gut axis, and various mucosal layers at a single anatomical site.
These findings illustrate the variety and complex role of these microorganisms in human
biological activities.
Discovering the importance of gut microbiota on the host’s health (healthy
digestive tract and healthy immune system) has led to increased impetus on the
research of factors that may affect their numbers and compositions. Arboleya et al.,
(2016) and Vlasova et al., (2016) demonstrated that during the fetal stage of
development, the early colonization of microbiota in the human body initiates. The
process of colonization continues after birth and throughout adulthood. The early
process of colonization is essential for sufficient infant development, forming the
foundation for the later physiological, neurological, and immunological homeostasis of
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the individual. The continuity of this early process is driven by interaction between
genetic factors, mode of delivery, environment, and feeding style and later diet
(Arboleya et al.,2016; Vlasova et al., 2016). However, as Biagi, et al. (2011) mentioned,
during late adulthood the gut microbial ecosystem starts to stabilize until the decline of
the gastrointestinal function starts which causes changes in their numbers and
compositions.
Probiotics are defined by the World Health Organization (WHO) as “live
microorganisms which confer a health benefit on the host”. When these microorganisms
are administered in adequate amounts (Fujiya & Kohgo, 2010; Huang et al., 2015; Patro
et al., 2015), they demonstrate possible benefits for maintaining intestinal health,
nutrition, and treating intestinal and functional disorders. Interestingly, the beneficial
effects of probiotics were first discovered over 100 years ago; however, researchers
started studying the probiotic functions in depth only around 25 years ago (Fujiya &
Kohgo, 2010). The main beneficial effect of probiotics is a reduction in pathogens. This
can be done by stimulating the immune system, modulating the gut microbiota, and/or
producing antimicrobial substances, such as organic acids and bacteriocins (Guerra,
2012; Manuzak et al., 2016).
Because of these benefits and because of the possibility that antibiotics could
cause adverse responses and side effects, researchers have been interested in
focusing on the use of probiotics to prevent or treat different human diseases. As it
mentioned in the Guidelines for the Evaluation of Probiotics in Food report of a Joint
FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in
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Food (2002), probiotics can play an important role for several functions in the human
body: the immunological, digestive, and respiratory functions.
Strains that are selected and used in probiotics must have several
characteristics. Strains must be safe, nonpathogenic, noncarcinogenic, noninvasive,
and nontoxic. To keep these microorganisms alive in the stressful environment of
gastrointestinal (GI) tract, they must be resistant to gastric acidity and bile toxicity. In
addition, it is important that these strains are able to adhere to the host’s epithelial
tissue (mucus and/or gut epithelial tissue) to stimulate the immune system, to inhibit or
decrease adhesion and colonization of pathogens and in some cases, to colonize the
gut (Ahmadova et al., 2013; Dixit et al., 2013). Moreover, probiotic strains must be
suitable for cultivation on an industrial scale, which is essential to obtain cultures with a
high concentration of viable cells. Also, these microorganisms must have the ability to
produce high amounts of antimicrobial substances, such as organic acids and
bacteriocins, which act as antagonists to pathogen growth. Another important
characteristic of probiotics is the ability to survive in delivery vehicles at the time of
intake (Guerra, 2012).
The most common probiotics contain various species of Lactobacillus and
Bifidobacterium, the yeast Saccharomyces cerevisiae, and the bacterium Bacillus
subtilis. These microorganisms have been used in combination or singularly to
formulate diverse nutritional supplements (AlFaleh et al., 2011; Guerra, 2012; Vlasova
et al., 2016).
Lactobacilli
Lactobacillus is a hugely diverse group of Gram-positive, anaerobic bacteria that
produces lactic acid as a major end-product in their metabolism of lactose and other
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fermentable carbohydrates. They have been found to inhabit several areas in the
human body, the human oral, vaginal and intestinal cavities (Patten & Laws, 2015).
There are over 170 species and 17 subspecies of Lactobacillus that are presently
published (Goldstein, Tyrrell, & Citron, 2015). Around 30% of these have been isolated
from fecal sources (Štšepetova et al., 2011). In the human GI tract, there is a plethora of
ecological niches and species of lactobacilli. L. fermentum, L. plantarum, L. casei, and
L. rhamnosus are some of lactobacilli that have been isolated from the gut; however, L.
rhamnosus and L. casei are the most common human clinical isolates (Goldstein et al.,
2015). Due to the special health-promoting properties of lactobacilli, they have received
exceeding amounts of attention, especially when they have shown their association in
digestion, stimulation of immunity and inhibition of pathogens (Štšepetova et al., 2011).
The benefits of probiotics that contain lactobacilli, such as maintaining the intestinal
ecosystem healthy and reducing the occurrence of intestinal disorders such as
antibiotic-associated diarrhea. Efficacy, in part, depends on the main property of this
strain: the ability to survive the tough conditions of the gastrointestinal tract (Kheadr,
2006).
The genus of Lactobacillus is dependent on their fermentative features: obligate
Table 4-4. Bristol Stool Form Scale of participants per week
Transit Time Baseline Intervention* Washout P-values
Placebo Slow 36 (19) 26 (14) 31(15) I: 0.3328
T: 0.7155
I×T: 0.2448
Normal 143 (74) 150 (79) 159 (79)
Fast 13 (7) 14 (7) 11(6)
5 billion
CFU
Slow 39 (21) 32 (16) 24 (13)
Normal 130 (68) 148 (73) 148 (84)
Fast 21(11) 22 (11) 5 (3)
25 billion
CFU
Slow 34 (18) 32 (17) 28 (13)
Normal 136 (74) 144 (75) 154 (72)
Fast 14 (8) 15 (8) 32 (15)
*The average number of each transit time per week during the four weeks of intervention. (%), percentage of stools reported P-values for intervention (baseline, intervention, washout) (I), treatment (placebo, 5 billion, 25 billion) (T)
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Table 4-5. Gastrointestinal Symptoms Rating Scale Equivalence Testing as Syndromes
Data presented as mean±SE.
Syndrome Period Placebo 5 billion CFU 25 billion CFU
Abdominal Pain
Baseline 1.7±0.2 1.6±0.1 1.5±0.1
Week 1 1.5±0.1 1.5±0.1 1.7±0.1
Week 2 1.7±0.2 1.6±0.1 1.5±0.1
Week 3 1.7±0.2 1.5±0.1 1.5±0.1
Week 4 1.6±0.2 1.4±0.1 1.6±0.1
Washout 1.5±0.1 1.4±0.1 1.5±0.1
Reflux Baseline 1.2±0.06 1.1±0.05 1.3±0.2
Week 1 1.1±0.07 1.2±0.07 1.3±0.3
Week 2 1.3±0.1 1.1±0.03 1.3±0.2
Week 3 1.3±0.1 1.4±0.02 1.1±0.1
Week 4 1.2±0.08 1.2±0.1 1.2±0.1
Washout 1.1±0.07 1.2±0.1 1.1±0.04
Indigestion Baseline 2.1±0.2 1.9±0.2 1.6±0.1
Week 1 1.9±0.2 1.7±0.2 1.7±0.2
Week 2 2.1±0.2 1.7±0.2 1.6±0.2
Week 3 1.9±0.2 1.7±0.1 1.6±0.2
Week 4 2.0±0.2 1.8±0.2 1.6±0.1
Washout 1.8±0.1 1.7±0.2 1.7±0.2
Diarrhea Baseline 1.4±0.2 1.5±0.1 1.5±0.2
Week 1 1.4±0.1 1.3±0.1 1.6±0.2
Week 2 1.7±0.3 1.3±0.1 1.3±0.1
Week 3 1.3±0.1 1.4±0.2 1.3±0.1
Week 4 1.5±0.2 1.3±0.1 1.4±0.1
Washout 1.3±0.1 1.3±0.1 1.7±0.3
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Table 4-5. Continued
Syndrome Period Placebo 5 billion CFU 25 billion CFU
Constipation Baseline 1.7±0.2 1.7±0.2 1.5±0.2
Week 1 1.6±0.1 1.5±0.2 1.4±0.2
Week 2 1.8±0.2 1.5±0.2 1.5±0.2
Week 3 1.5±0.1 1.5±0.2 1.5±0.2
Week 4 1.6±0.2 1.5±0.2 1.5±0.1
Washout 1.4±0.2 1.5±0.2 1.4±0.1
Data presented as mean±SE.
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Figure 4-12. Mean score of GSRS abdominal pain syndrome per week. Scale from 1 to 7(1=no discomfort at all, 4=moderate discomfort, 7=very severe discomfort)
Figure 4-13. Mean score of GSRS diarrhea syndrome per week. Scale from 1 to 7(1=no discomfort at all, 4=moderate discomfort, 7=very severe discomfort)
Figure 4-14. Mean score of GSRS constipation syndrome per week. Scale from 1 to 7(1=no discomfort at all, 4=moderate discomfort, 7=very severe discomfort)
Figure 4-15. Mean score of GSRS reflux syndrome per week. Scale from 1 to 7(1=no discomfort at all, 4=moderate discomfort, 7=very severe discomfort)
Figure 4-16. Mean score of GSRS indigestion syndrome per week. Scale from 1 to 7(1=no discomfort at all, 4=moderate discomfort, 7=very severe discomfort)
In this study, a multi-strain probiotic was evaluated for the first time for effects on
gastrointestinal health and wellness. The consumption of the multi-strain probiotic
tested in this study at two doses, 5 and 25 billion CFU/day, was well tolerated by all the
participants as none of them reported any side effects or adverse events. The tolerance
of probiotic strains in healthy people was expected since no adverse effects have been
reported in previous studies on healthy and non-healthy population that consumed
multi-strain probiotics (Chapman et al., 2011; Reid et al., 2003; Bruggencate et al.,
2015).
In 2013, an expert panel was convened by the International Scientific Association
for Probiotics and Prebiotics (ISAPP) to discuss the field of probiotics. In the consensus
statement, they accepted the Health Canada definition to some of bacterial species as
probiotics when they are delivered in food at a level of 1 × 109CFU per serving (Hill et
al., 2014). The formulation used in this study, included probiotics with generally
accepted health benefits such as Bifidobacterium (breve and longum) and Lactobacillus
(casei, plantarum, and rhamnosus). In addition, the formulation included doses above
1x109 CFU per serving.
Several dose-response safety studies have been conducted to evaluate the
safety of consuming different doses of single strain probiotic (Hütt et al., 2011;
Merenstein et al., 2015; Oberhelman et al., 2014). To our knowledge, there is just one
safety study has been carried out on a multi-strain probiotic (Rosenfeldt et al., 2003). In
this study, there were two different formulations of probiotic. The first one was a mixture
of two strains (Lactobacillus rhamnosus 19070-2, and Lactobacillus reuteri DSM 12246)
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and the second formulation was a combination of three commercially used strains,
Lactobacillus casei subsp. alactus CHCC 3137, Lactobacillus delbrueckii subsp. lactis
CH 2329, and Lactobacillus rhamnosus GG. The study was a crossover design. The
participants consumed a dose of 1010 CFU of each strain or placebo twice daily for 18
days, and there was a 17-day washout period between the consumption of the other
probiotic. Participants were asked to report any gastrointestinal symptoms (abdominal
pain, flatulence, nausea), possible adverse events or taking any medication during the
study period. No serious adverse events were reported by the participants during the
consumption of these two multi-strain probiotics. However, the present study could be
the first safety study to evaluate the consumption of a multi-strain probiotic to dose up of
25 billion/CFU per day.
In phase I study, Hibberd et al. (2014) conducted a safety trial on elderly
volunteers to evaluate the safety and tolerability of Lactobacillus rhamnosus GG ATCC
53103 when orally administered at a dose of 1×1010 CFU twice daily for 28 days.
Several laboratory tests were carried out (HIV, Hepatitis B surface antigen (HbsAg),
Hepatitis C antibody (anti-HCV), illicit drugs, and alcohol) before the study started to
ensure of the eligibility to participate in the study. Participants asked to complete a daily
diary of symptoms to assess the adverse events that could be occurred. In addition,
participants were phoned during some days during the intervention period to ensure if
anyone experienced any adverse events as well as discussing their compliance. There
were no serious adverse events (death, life-threatening, hospitalization, disability, or
required intervention to prevent impairment or damage to a volunteer) reported from the
participants, which concluded that Lactobacillus rhamnosus GG ATCC 53103 is safe
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and well tolerated to be consumed in a dose of 1x1010 CFU twice daily by healthy old
adults aged 65 years and older. In our study, the daily questionnaire was similar to the
study’s daily diary of symptoms questionnaire. However, participants had to rate the
symptoms as mild, moderate, severe, or very severe versus a score from 1 to 6 as used
in the present study.
Another safety trial has been done by Hanifi et al. (2015) and demonstrated that
a single probiotic, Bacillus subtilis R0179, is safe and well tolerated by healthy adults
when it is consumed in three different doses, 0.1x109, 1x109, or 10x109 CFU/day. In this
study, they used the same daily questionnaire that we used in our study to assess the
daily gastrointestinal symptoms and general wellness. The main difference between this
study and the present study is that their study did not use the Bristol Stool Form Scale
to evaluate the effect of the probiotic on the transit time while in the present study this
tool was incorporated into the daily questionnaire. Also, these researchers used the
GSRS only three times, on the last day of the baseline, intervention, and washout
periods to evaluate the gastrointestinal symptoms versus weekly in the current study.
There are several studies that have been done on healthy adults and have used
the Bristol Stool Form Scale as a tool to evaluated the transit time, i.e. slow transit (type
1 and 2), normal transit (type 3, 4 and 5), and fast transit (type 6 and 7). The Bristol
Stool Form Scale results in the present study at the baseline were similar to the
baseline results of Dahl et al. (2016) study and lower baseline percentage of the slow
transit time in the current study participants (18-21%) versus the participants in Alyousif
et al. (2016) study (34%). The similarity and difference that we found could be a result
of age range. The range of age in our participants (18-50 y) was similar to the range of
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age in Dahl et al. (2016) study (18-65). While the participants age in Alyousif et al.
(2016) were above 30 years old only. In addition, Alyousif et al. (2016) study included
only healthy females which may be at higher risk for constipation than males (Meier et
al., 1995).
The results of the equivalence testing of the daily and GSRS questionnaires
showed that there were no significant changes between all the study groups (placebo, 5
billion, 25 billion) through the intervention periods of the study. Most of the syndromes
and symptoms were concluded equivalent across all period by intervention
comparisons. This is what was expected as the probiotic was tested on a healthy
population. Significant changes could have possibly occurred if participants suffering
from GI problems or diseases had been evaluated. Consequently, the higher dose of
bacteria in probiotic may have a significant effect on those with more GI symptoms.
However, until this time, there is no evidence to conclude whether improvements could
be due to synergistic interactions between strains or a consequence of the higher
probiotic dose (Chapman et al., 2011).
In 2010, a study has been conducted by Jerndal et al. showed how the GSRS
score is higher in unhealthy population who are suffering from GI problems such as IBS.
In this study, they compared the GI symptoms score between healthy participants and
IBS patients. Jerndal et al. found that in addition to the high score of GSRS in IBS
patients, the gastrointestinal-specific anxiety could be an important factor for the
severity of the GI symptom in patients with IBS.
In our study, the results for the gastrointestinal distress showed a trend decrease
of the mean syndrome score with the consumption of the high dose (25 billion CFU)
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across all period compared to those in the placebo and 5 billion treatment group, even
though the equivalence testing showed equivalent results. As mentioned previously, we
did not expect to find any changes (decrease or increase) in the daily and weekly
syndromes and symptoms because of the healthy population that we had. However, the
suggestive decrease in gastrointestinal distress with the consumption of the high dose
of this probiotic generates a hypothesis that there may be an effect and that evaluate
this probiotic formulation should be tested on patients who are suffering from GI
problems such as irritable bowel syndrome.
Limitations
In this study, there are some limitations that could be avoided in future studies.
The small sample is one weakness, although this is common of Phase 1 trials. Our
sample size in each group was similar to the sample size of our recent study which was
a Phase 1 study approved by the FDA (Dahl WJ, personal communication). An increase
the sample size could show the possible positive effects of this mix probiotic on transit
time or other gastrointestinal symptoms. Another limitation in our study could be that the
inclusion and exclusion criteria was less than rigorous. We did not exclude obese and
overweight and we did not carry out medical examinations or check the metabolic profile
to evaluate the health status. In addition, food intake was not recorded, which could be
another influence on our results. However, the cross over design helps to control for this
possible confounder. Although participants were instructed to avoid food that contained
prebiotics or probiotics, their compliances to avoid these foods were not evaluated.
Strengths
This is the first clinical study to evaluate the multi-strain probiotic containing
Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011, Lactobacillus casei
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R0215, Pediococcus acidilactici R1001, Bifidobacterium breve R0070, Bifidobacterium
longum BB536 Lactobacillus plantarum R1012, Lactobacillus lactis ssp. lactis R1058. It
was randomized, double-blinded, dose response trial. Neither the participants nor the
research staff know which supplements each participant had. Also, in this study, we
used two validated questionnaires, GSRS and BSFS.
Suggestion for Future Studies
The classification of the appropriate probiotic for each health benefit is critical
with the increase of the multi-strain probiotic. In addition, each strain of the microbiota
that is used in these probiotics must be studied to identify the benefit of this individual
strain on the host health. As mentioned before, it is not known whether the beneficial
effects of these multi-strain probiotic on some GI problems and diseases is due to the
types of these microbes or to some interactions between them. More studies are
needed to identify the role of and the complex interaction among the microbiota and the
effective proportion of these multi-strain probiotic to achieve the possible benefits. In
addition, the multi-strains which are included in this probiotic should also be evaluated
for their gastrointestinal viability and impact on the intestinal microbiome to ensure that
the possible effects of this probiotic on unhealthy population is due to the bacteria and
not a confounder (Hanifi et al., 2015). This is the next step which is not included in this
thesis. Stool samples were taken from the participants at the baseline, intervention, and
washout periods to assess the effects of consumption the multi-strain probiotic in two
doses, 5 billion and 25 billion CFU/day. Further research will be undertaken to
determine the impact of the multi-strain probiotic on modulation of the intestinal
microbiota including determining the viability of Lactobacillus and Bifidobacterium spp.
tested.
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Conclusion
This is the first assessment of this multi-strain probiotic formulation at two
dosages in a double-blind, randomized, clinical trial. Consumption of this probiotic
supplement in two different doses for a period of four weeks was well tolerated by
participants. The results of this study demonstrate that the regular consumption of the
multi-strain probiotic is well tolerated in healthy individuals and has no effects on
gastrointestinal function and wellness at dosages up to 25 billion CFU/day. In addition,
the consumption of the multi-strain probiotic has no effect on the transit time.
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APPENDIX A IRB APPROVAL
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APPENDIX B INFORMED CONSENT
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APPENDIX C INTERNATIONAL PHYSICAL ACTIVITY QUESTIONNAIRE
INTERNATIONAL PHYSICAL ACTIVITY QUESTIONNAIRE We are interested in finding out about the kinds of physical activities that people do as part of their everyday lives. The questions will ask you about the time you spent being physically active in the last 7 days. Please answer each question even if you do not consider yourself to be an active person. Please think about the activities you do at work, as part of your house and yard work, to get from place to place, and in your spare time for recreation, exercise or sport. Think about all the vigorous and moderate activities that you did in the last 7 days. Vigorous physical activities refer to activities that take hard physical effort and make you breathe much harder than normal. Moderate activities refer to activities that take moderate physical effort and make you breathe somewhat harder than normal.
PART 1: JOB-RELATED PHYSICAL ACTIVITY The first section is about your work. This includes paid jobs, farming, volunteer work, course work, and any other unpaid work that you did outside your home. Do not include unpaid work you might do around your home, like housework, yard work, general maintenance, and caring for your family. These are asked in Part 3. 1. Do you currently have a job or do any unpaid work outside your home?
Yes No Skip to PART 2: TRANSPORTATION The next questions are about all the physical activity you did in the last 7 days as part of your paid or unpaid work. This does not include traveling to and from work. 2. During the last 7 days, on how many days did you do vigorous physical activities like
heavy lifting, digging, heavy construction, or climbing up stairs as part of your work? Think about only those physical activities that you did for at least 10 minutes at a time.
_____ days per week
No vigorous job-related physical activity Skip to question 4 3. How much time did you usually spend on one of those days doing vigorous physical
activities as part of your work?
_____ hours per day _____ minutes per day
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4. Again, think about only those physical activities that you did for at least 10 minutes at a time. During the last 7 days, on how many days did you do moderate physical activities like carrying light loads as part of your work? Please do not include walking.
_____ days per week
No moderate job-related physical activity Skip to question 6 5. How much time did you usually spend on one of those days doing moderate physical
activities as part of your work?
_____ hours per day _____ minutes per day
6. During the last 7 days, on how many days did you walk for at least 10 minutes at a time
as part of your work? Please do not count any walking you did to travel to or from work.
_____ days per week
No job-related walking Skip to PART 2: TRANSPORTATION 7. How much time did you usually spend on one of those days walking as part of your
work?
_____ hours per day _____ minutes per day
PART 2: TRANSPORTATION PHYSICAL ACTIVITY These questions are about how you traveled from place to place, including to places like work, stores, movies, and so on. 8. During the last 7 days, on how many days did you travel in a motor vehicle like a train,
bus, car, or tram?
_____ days per week No traveling in a motor vehicle Skip to question 10 9. How much time did you usually spend on one of those days traveling in a train, bus,
car, tram, or other kind of motor vehicle?
_____ hours per day _____ minutes per day
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Now think only about the bicycling and walking you might have done to travel to and from work, to do errands, or to go from place to place. 10. During the last 7 days, on how many days did you bicycle for at least 10 minutes at a
time to go from place to place?
_____ days per week No bicycling from place to place Skip to question 12 11. How much time did you usually spend on one of those days to bicycle from place to
place?
_____ hours per day _____ minutes per day
12. During the last 7 days, on how many days did you walk for at least 10 minutes at a time
to go from place to place?
_____ days per week No walking from place to place Skip to PART 3: HOUSEWORK,
HOUSE MAINTENANCE, AND CARING FOR FAMILY
13. How much time did you usually spend on one of those days walking from place to
place?
_____ hours per day _____ minutes per day
PART 3: HOUSEWORK, HOUSE MAINTENANCE, AND CARING FOR FAMILY This section is about some of the physical activities you might have done in the last 7 days in and around your home, like housework, gardening, yard work, general maintenance work, and caring for your family. 14. Think about only those physical activities that you did for at least 10 minutes at a time.
During the last 7 days, on how many days did you do vigorous physical activities like heavy lifting, chopping wood, shoveling snow, or digging in the garden or yard?
_____ days per week
No vigorous activity in garden or yard Skip to question 16 15. How much time did you usually spend on one of those days doing vigorous physical
activities in the garden or yard?
_____ hours per day _____ minutes per day
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16. Again, think about only those physical activities that you did for at least 10 minutes at a
time. During the last 7 days, on how many days did you do moderate activities like carrying light loads, sweeping, washing windows, and raking in the garden or yard?
_____ days per week
No moderate activity in garden or yard Skip to question 18 17. How much time did you usually spend on one of those days doing moderate physical activities in the garden or yard?
_____ hours per day _____ minutes per day
18. Once again, think about only those physical activities that you did for at least 10 minutes
at a time. During the last 7 days, on how many days did you do moderate activities like carrying light loads, washing windows, scrubbing floors and sweeping inside your home?
_____ days per week
No moderate activity inside home Skip to PART 4: RECREATION,
SPORT AND LEISURE-TIME PHYSICAL ACTIVITY
19. How much time did you usually spend on one of those days doing moderate physical
activities inside your home?
_____ hours per day _____ minutes per day
PART 4: RECREATION, SPORT, AND LEISURE-TIME PHYSICAL ACTIVITY This section is about all the physical activities that you did in the last 7 days solely for recreation, sport, exercise or leisure. Please do not include any activities you have already mentioned. 20. Not counting any walking you have already mentioned, during the last 7 days, on how
many days did you walk for at least 10 minutes at a time in your leisure time?
_____ days per week No walking in leisure time Skip to question 22 21. How much time did you usually spend on one of those days walking in your leisure
time?
_____ hours per day _____ minutes per day
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22. Think about only those physical activities that you did for at least 10 minutes at a time.
During the last 7 days, on how many days did you do vigorous physical activities like aerobics, running, fast bicycling, or fast swimming in your leisure time?
_____ days per week
No vigorous activity in leisure time Skip to question 24 23. How much time did you usually spend on one of those days doing vigorous physical
activities in your leisure time?
_____ hours per day _____ minutes per day
24. Again, think about only those physical activities that you did for at least 10 minutes at a
time. During the last 7 days, on how many days did you do moderate physical activities like bicycling at a regular pace, swimming at a regular pace, and doubles tennis in your leisure time?
_____ days per week
No moderate activity in leisure time Skip to PART 5: TIME SPENT
SITTING 25. How much time did you usually spend on one of those days doing moderate physical
activities in your leisure time? _____ hours per day _____ minutes per day
PART 5: TIME SPENT SITTING The last questions are about the time you spend sitting while at work, at home, while doing course work and during leisure time. This may include time spent sitting at a desk, visiting friends, reading or sitting or lying down to watch television. Do not include any time spent sitting in a motor vehicle that you have already told me about. 26. During the last 7 days, how much time did you usually spend sitting on a weekday?
_____ hours per day _____ minutes per day
27. During the last 7 days, how much time did you usually spend sitting on a weekend
day?
_____ hours per day _____ minutes per day
This is the end of the questionnaire, thank you for participating.
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APPENDIX D DAILY QUESTIONNAIRE
1. Did you experience any of the following symptoms in the last 24 hours? Please circle one choice on the scale from 0 to 6 (0= none, 3=moderate, 6= very severe). Note: If you are a female, please do not rate menstrual cramping and bloating. Please rate the severity by circling/indicating the appropriate number below. Bloating 0 1 2 3 4 5 6 none moderate very severe Flatulence 0 1 2 3 4 5 6 none moderate very severe Abdominal cramping 0 1 2 3 4 5 6 none moderate very severe Abdominal noises 0 1 2 3 4 5 6 none moderate very severe Headache 0 1 2 3 4 5 6 none moderate very severe Dizziness 0 1 2 3 4 5 6 none moderate very severe
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Sore throat 0 1 2 3 4 5 6 none moderate very severe Blocked ear canal 0 1 2 3 4 5 6 none moderate very severe Nasal congestion 0 1 2 3 4 5 6 none moderate very severe Runny eyes 0 1 2 3 4 5 6 none moderate very severe Nausea 0 1 2 3 4 5 6 none moderate very severe Vomiting 0 1 2 3 4 5 6 none moderate very severe Diarrhea 0 1 2 3 4 5 6 none moderate very severe Constipation 0 1 2 3 4 5 6 none moderate very severe
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Itching 0 1 2 3 4 5 6 none moderate very severe Fatigue 0 1 2 3 4 5 6 none moderate very severe Skin rash 0 1 2 3 4 5 6 none moderate very severe Skin redness/flushing 0 1 2 3 4 5 6 none moderate very severe Feeling anxious 0 1 2 3 4 5 6 none moderate very severe Feeling depressed 0 1 2 3 4 5 6 none moderate very severe Feeling stressed 0 1 2 3 4 5 6 none moderate very severe In general, how hungry did you feel today? 0 1 2 3 4 5 6 none moderate very severe
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2. How many stools (bowel movements) did you have today? 0 1 2 3 4 5 6 7 8 9 10 >10 3. Please rate the stool consistency (select one). Note : We will try to include clip art/drawings/pictures for each, see below) ___ Type 1: Seperate hard lumps, like nuts (hard to pass) ___ Type 2: Sausage-shaped but lumpy ___ Type 3: Like a sausage but with cracks on the surface ___ Type 4: Like a sausage or snake, smooth and soft ___ Type 5: Soft blobs with clear-cut edges ___ Type 6: Fluffy pieces with ragged edges, a mushy stool ___Type 7: Watery, no solid pieces. Entirely Liquid
4. How many hours did you sleep last night? Do not include the time it took you to fall asleep or anytime you were awakened during the night. Please indicate the time that most closely matches your sleep.
5. Did you take your probiotic capsule today? Yes No 6. Did you visit a doctor today? Yes No 7. Did you take an antibiotic today? Yes No 8. Did you take any other medications today? Yes No If yes, please list the medication(s) you took today. ________________________
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APPENDIX E GASTROINTESTINAL SYMPTOM RESPONSE SCALE
(1) No discomfort at all (5) Moderately severe discomfort (2) Slight discomfort (6) Severe discomfort (3) Mild discomfort (7) Very severe discomfort (4) Moderate discomfort Questions: Answer each question using the response scale above. _____Have you been bothered by stomach ache or pain during the past week?
(Stomach ache refers to all kinds of aches or pains in your stomach or belly)
_____Have you been bothered by heartburn during the past week?
(By heartburn we mean a burning pain or discomfort behind the breastbone in your chest)
_____Have you been bothered by acid reflux during the past week?
(By acid reflux we mean regurgitation or flow of sour or bitter fluid into your mouth)
_____Have you been bothered by hunger pains in the stomach or belly during the past
week?
(This hallow feeling in the stomach is associated with the need to eat between meals.)
_____Have you been bothered by nausea during the past week?
(By nausea we mean a feeling of wanting to be sick)
_____Have you been bothered by rumbling in your stomach or belly during the past
week?
(Rumbling refers to vibrations or noises in the stomach)
_____Has your stomach felt boated during the past week?
(Feeling bloated refers to swelling in the stomach or belly)
_____Have you been bothered by burping during the past week?
(Burping refers to bringing up air or gas through the mouth)
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_____Have you been bothered by passing gas or flatus during the past week?
(Passing gas refers to the release of air or gas from the bowels)
_____Have you been bothered by constipation during the past week?
(Constipation refers to a reduced ability to empty the bowels)
_____Have you been bothered by diarrhea during the past week?
(Diarrhea refers to frequent loose or watery stools)
_____Have you been bothered by loose stools during the past week?
(If your stools have been alternately hard and loose, this question only refers to the extent
you have been bothered by the stools being loose)
_____Have you been bothered by hard stools during the past week?
(If your stools have been alternately hard and loose, this question only refers to the extent
you have been bothered by stools being hard)
_____Have you been bothered by an urgent need to have a bowel movement during the
past week?
(This urgent need to open your bowels makes you rush to the toilet)
_____When going to the toilet during the past week, have you had the feeling of not
completely emptying your bowels? (The feeling that after finishing a bowel movement, there
is still some stool that needs to be passed)
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BIOGRAPHICAL SKETCH
Asmaa Fatani was born in 1984, in Jeddah, Saudi Arabia. After graduation in
2001 from Al-Rabaah High School, Jeddah, Saudi Arabia, she attended King Abdul Aziz
University (KAU) for her undergraduate. She earned a bachelor’s degree of education
with first honor in food science and nutrition in 2006. By 2007 she was nominated by the
Food Science and Nutrition Department of KAU to join the university as a Teacher
Assistant (TA). After being TA, she started teaching in the department for four years. In
2012, she earned a scholarship from KAU to get her master’s degree in human
nutrition. She traveled to the USA and in 2014, she was accepted into Human Nutrition
Master’s Program in Department of Food Science and Human Nutrition at University of
Florida under the supervision of Dr. Wendy Dahl. After graduation, she will continue the
studying in the same department at University of Florida to get a Doctor of Philosophy