© 2016 Asmaa MN Fatani - University of...

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

To my parents, my husband (Bandar) and my children (Khalid and Omar)

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ACKNOWLEDGMENTS

First, I would like to express my sincere gratitude to my advisor Dr. Wendy J,

Dahl, who supported me limitlessly since day one. I would like to thank her for her

patience, motivation, dedication, and enormous knowledge. Being with Dr. Dahl in this

challenging, joyful, and insightful academic journey has developed me as an emergent

scholar. Words could never do justice to how grateful I am to your efforts and support!

Thank you, Dr. Dahl.

Besides my advisor, I would also like to extend my appreciations to the rest of my

thesis committee members, Dr. Anne Mathews and Dr. Debra Kelly, who always offered

continuous guidance and helpful recommendations for my thesis. Their questions have

helped me to widen my research from diverse perspectives.

I am also very thankful to my colleagues, Amanda Ford and others for their

encouragement, moral support, and making my study at the University of Florida more

enjoyable.

My heartiest gratitude goes to my wonderful husband (Bandar) and my two lovely

sons (Khalid and Omar) for their unconditional love and support, and numerous

sacrifices during my journey. Thank you for being by my side.

I am deeply indebted to my great parents, who are my role model, and whose

passionate encouragement made my completion of this degree possible. I am also very

thankful to my entire family, my mother-in law, father-in-law, my brothers and my sisters.

My joy has no bounds in expressing my warm gratitude to all your support, sacrifices,

and continuous prayers.

Foremost I’m very grateful to God Almighty, who endorsed me with wisdom,

strength, and good health during my journey.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 8

LIST OF ABBREVIATIONS ............................................................................................. 9

ABSTRACT ................................................................................................................... 12

CHAPTER

1 LITERATURE REVIEW .......................................................................................... 14

Introduction ............................................................................................................. 14 Lactobacilli .............................................................................................................. 16

Lactobacillus helveticus .................................................................................... 18

Lactobacillus rhamnosus .................................................................................. 21 Lactobacillus casei ........................................................................................... 22

Lactobacillus plantarum .................................................................................... 25

Bifidobacterium ....................................................................................................... 27 Bifidobacterium breve ....................................................................................... 27

Bifidobacterium longum .................................................................................... 29

Assessment of Gastrointestinal Symptoms and Wellnesses .................................. 31

Gastrointestinal Symptom Rating Scale (GSRS) .............................................. 31 Bristol Stool Form Scale (BSFS) ...................................................................... 32

Conclusions ............................................................................................................ 32

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

Recruitment ............................................................................................................ 37 Baseline .................................................................................................................. 37

Randomization and Intervention ............................................................................. 38 Washout and Post Intervention ............................................................................... 39 Compensation ......................................................................................................... 39 Statistical Methods .................................................................................................. 40

Equivalence Testing ......................................................................................... 40 The Frequency and GIMMIX Procedures ......................................................... 41

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4 RESULTS ............................................................................................................... 45

Participants ............................................................................................................. 45 Daily Questionnaire Analysis .................................................................................. 46

Bristol Stool Form Scale ......................................................................................... 47 Gastrointestinal Symptom Rating Scale Analysis ................................................... 47

5 DISCUSSION ......................................................................................................... 64

Limitations ............................................................................................................... 68 Strengths ................................................................................................................ 68

Suggestion for Future Studies ................................................................................ 69 Conclusion .............................................................................................................. 70

APPENDIX

A IRB APPROVAL ..................................................................................................... 71

B INFORMED CONSENT .......................................................................................... 73

C INTERNATIONAL PHYSICAL ACTIVITY QUESTIONNAIRE ................................. 85

D DAILY QUESTIONNAIRE ....................................................................................... 90

E GASTROINTESTINAL SYMPTOM RESPONSE SCALE ....................................... 94

LIST OF REFERENCES ............................................................................................... 96

BIOGRAPHICAL SKETCH .......................................................................................... 104

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LIST OF TABLES

Table page 3-1 Daily Questionnaire Syndromes and Symptoms ................................................ 43

3-2 Gastrointestinal Symptom Rating Scale Syndromes .......................................... 44

4-1 Participant demographics ................................................................................... 49

4-2 Daily Questionnaire Syndromes Equivalence Testing. ....................................... 50

4-3 Daily Questionnaire Symptoms Equivalence Testing ......................................... 55

4-4 Bristol Stool Form Scale of participants per week .............................................. 58

4-5 Gastrointestinal Symptoms Rating Scale Equivalence Testing as Syndromes .. 59

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LIST OF FIGURES

Figure page 3-1 Study design ....................................................................................................... 42

4-1 Participant flow diagram ..................................................................................... 48

4-2 Mean syndrome score of gastrointestinal distress per week. ............................. 51

4-3 Mean score of cephalic syndrome per week....................................................... 52

4-4 Mean score of epidermal syndrome per week. ................................................... 52

4-5 Mean score of ear-nose-throat syndrome per week. .......................................... 53

4-6 Mean score of emetic syndrome per week. ........................................................ 53

4-7 Mean score of Psychological health per week. ................................................... 54

4-8 Mean score of diarrhea symptom per week. ....................................................... 56

4-9 Mean score of constipation symptom per week. ................................................. 56

4-10 Mean score of hours of sleep per week. ............................................................. 57

4-11 Mean score of bowel movement frequency per week. ........................................ 57

4-12 Mean score of GSRS abdominal pain syndrome per week. ............................... 61

4-13 Mean score of GSRS diarrhea syndrome per week. .......................................... 61

4-14 Mean score of GSRS constipation syndrome per week. .................................... 62

4-15 Mean score of GSRS reflux syndrome per week. ............................................... 62

4-16 Mean score of GSRS indigestion syndrome per week. ...................................... 63

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LIST OF ABBREVIATIONS

AAD Antibiotic-associated diarrhea

ALT Alanine aminotransferase

ANCOVA Analysis of covariance

anti-HCV Hepatitis C antibody

AP Abdominal pain

BMI Body mass index

BSFS Bristol Stool Form Scale

CCL Coping Checklist

CD Crohn’s disease

CFS Chronic fatigue syndrome

CFU Colony forming units

CM Control milk

DAS28 Disease activity score for rheumatoid arthritis

EI Endoscopic index

ENT Ear-nose-throat

FAO Food and Agriculture Organization

FD Functional dyspepsia

FDA Food and Drug Administration

FHEL Facultative heterofermentative lactobacilli

FM Fermented milk

G gram

GERD gastroesophageal reflux disease

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CCMQFjAA&url=http%3A%2F%2Fen.wikipedia.org%2Fwiki%2FCyclin-dependent_kinase_4&ei=KAZJVdqPJMWrNtvcgIgN&usg=AFQjCNHWLbS37Kg-z7siZfQDoaq3TgqwAw&sig2=8mHnGNtpR6yCNM9Mx6YWlA

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CCwQFjAA&url=http%3A%2F%2Fen.wikipedia.org%2Fwiki%2FMitogen-activated_protein_kinase&ei=71lCVZ67N5P_ggT_jIG4Bw&usg=AFQjCNHzgjn6aDWLxE35nRzLeJytfAQpwA&sig2=ZspkeBJg_uHpKyvSQoxM4g

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GH Global health

GI Gastrointestinal

GRAS Generally Recognized as Safe

GSRS Gastrointestinal Symptoms Rating Scale

HADS Hospital Anxiety and Depression Scale

HbsAg Hepatitis B surface antigen

HSCL Hopkins Symptoms Checklist

hs-CRP high sensitivity C-reactive protein

IBD Inflammatory bowel disease

IBS irritable bowel syndrome

IL Interleukin

IPAQ International Physical Activity Questionnaire

ISAPP International Scientific Association for Probiotic and Prebiotic

LcS Lactobacillus casei Shirota

LcS-FM Lactobacillus casei Shirota-Fermented milk

LP229v Lactobacillus plantarum 229v

LS Least squares

MetS Metabolic syndrome

Mg milligram

mL milliliter

NFM Non-fermented milk

NK Natural Killer

OHEL Obligate heterofermentative lactobacilli


https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CCcQFjAA&url=http%3A%2F%2Fen.wikipedia.org%2Fwiki%2FExtracellular_signal-regulated_kinases&ei=HlpCVdW8BMSqNu2kgJAF&usg=AFQjCNHF3j4kTll4OaVa0Q5B35VXoPT0lg&sig2=7KfhO_IIkH8dbSvZO_gOig

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OHOL Obligate homofermentative lactobacilli

PF Probiotic formulation

QoL Quality of Life

RA Rheumatoid arthritis

TNF-α Tumor necrosis factor alpha

TOST Two one-sided Test

UC Ulcerative colitis

UCDAI Ulcerative colitis disease activity index

UFC Urinary free cortisol

URTIs Upper respiratory tract infections

WHO World Health Organization

γ -GTP γ -glutamyltranspeptidase


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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

homofermentative lactobacilli (OHOL), facultative heterofermentative lactobacilli

(FHEL), and obligate heterofermentative lactobacilli (OHEL). Because of the differences

in process during fermentation in the colon, one lactobacilli strain of a particular species

may use a different pathway than another strain, and as a result provide the energy

differently to the host (Štšepetova et al., 2011).

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Lactobacillus helveticus

Lactobacillus helveticus is one of the lactobacilli species. This microorganism has

a Generally Recognized as Safe (GRAS) status and exhibits some features that make it

suitable for dairy applications. Lactobacillus helveticus is conventionally used in the

production of Swiss-type and Italian-aged cheeses, where it is the most commonly

recovered species in natural lactic starter cultures, and fermented milk drinks.

Lactobacillus helveticus is becoming important as a health-promoting culture in probiotic

and nutraceutical food products (Giraffa, 2014; Griffiths & Tellez, 2013). This

microorganism has been widely studied because of its effects in a variety of biological

systems. It has been shown to prompt anti-inflammatory responses and to decrease

stress in several animal models, as well as in human studies when it is combined with

Bifidobacterium longum (Ohland et al., 2013).

Aihara et al., (2005) studied the efficacy and safety of powdered Lactobacillus

helveticus fermented milk tablets on participants with mild hypertension and high-normal

blood pressure. The study used a placebo-controlled, double-blind, parallel group

comparative design. The study consisted of two groups with 40 participants in each: a

high-normal blood pressure and a mild hypertension group. In the treatment group,

each subject was required to take six test tablets (12 g) that contained powdered

fermented milk with L. helveticus CM4 daily for four weeks. In the placebo group, a

similar number of placebo tablets were taken for four weeks as well. They found that the

tablets containing powdered fermented milk with L. helveticus CM4 reduced high blood

pressure in participants with high-normal blood pressure or mild hypertension without

any adverse effects.

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In addition, Yamamura et al.(2007) evaluated the effect of fermented milk that

contains Lactobacillus helveticus on sleep and health perception in healthy elderly

subjects. The study consisted of 29 healthy elderly participants between 60 to 81 years

old. The study was prospective, randomized, double-blind and placebo-controlled, with

a crossover design. There were two intervention periods, three weeks each, with a

three-week washout period between interventions. Participants were required to take

100 g of either a fermented milk drink or a placebo drink (artificially acidified milk) daily

in the first intervention period and the alternate drink in the second intervention period.

Sleep quality was measured by means of actigraphy and a sleep questionnaire, and the

quality of life (QoL) was evaluated by the SF-36 health survey for each period. They

found a significant improvement in sleep efficiency (P=0.03) and number of wakening

episodes (P=0.007) in actigraph data after taking the fermented milk, while no

significant changes were detected for the placebo.

In another study, Messaoudi et al. (2011) used a probiotic formulation (PF)

containing Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 to study

the beneficial effects of this PF on mood and psychological distress in rat and healthy

human volunteers. For two weeks, rats were administered PF daily and then tested in a

conditioned defensive burying test, a screening model for anti-anxiety agents.

Meanwhile, healthy human volunteers participated in a double-blind, placebo-controlled,

randomized parallel group study. Volunteers took one stick of 1.5 g/d of PF or a placebo

of identical taste during or just after breakfast, for 30 days. One stick of PF (1.5 g)

contained 3 billion CFU of these bacteria. Participants were then assessed with the

Hopkins Symptom Checklist (HSCL-90), the Hospital Anxiety and Depression Scale

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(HADS), the Perceived Stress Scale, the Coping Checklist (CCL) and a 24-hour urinary

free cortisol (UFC) test. Taking the PF significantly reduced anxiety-like behavior in rats

(P<0·05) and decreased psychological distress in the human volunteers, as evidenced

by the HSCL-90 scale (global severity index, P<0·05; somatisation, P<0.05; depression,

P<0.05; and anger–hostility, P<0.05), the HADS (HADS global score, P<0.05; and

HADS anxiety, P<0.06), and by the CCL (problem solving, P<0.05) and the UFC level

(P<0.05). The study determined that taking L. helveticus R0052 and B. longum R0175

in combination potentially led to anxiolytic-like activity in rats and positive psychological

effects in healthy human volunteers.

With another combination of Lactobacillus helveticus, Evans et al., (2016),

studied the effectiveness of probiotic formulation consisted of Lactobacillus helveticus

R0052 and Lactobacillus rhamnosus R0011 on antibiotic-associated diarrhea in healthy

adults. Subjects participated in a 10-week randomized, double-blind, placebo-controlled,

parallel study. Subjects were randomly assigned to receive one week of amoxicillin–

clavulanic acid (875 mg/125 mg) once per day, plus a daily dose of 8 × 109 colony

forming units (CFU) of a multi-strain probiotic (n=80) or placebo (n=80) after completing

the antibiotic. After doing post hoc analysis on the duration of diarrhea-like defecations,

they found that using this probiotic formulation could decrease the length of these

events by one full day (probiotic, 2.70 (SEM 0.36) day; placebo, 3.71 (SEM 0.36) day;

P=0.037; effect size=0.52). This study demonstrated the novel evidence that the

combination of L. helveticus R0052 and L. rhamnosus R0011 significantly decreased

the duration of diarrhea-like defecations in healthy adults receiving antibiotics.

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Lactobacillus rhamnosus

Lactobacillus rhamnosus (L. rhamnosus) is another type of beneficial bacteria

that has been used in probiotics. Barry Goldin and Sherwood Gorbach were the first

scientists who isolated L. rhamnosus in 1983 in the intestines of a healthy human

subject. The bacteria were shown to have incredible tolerance for the acids that are

normally found in the stomach and digestive tract. “GG”, which can be found in the title

of the strain L. rhamnosus, is derived from the last names of these two scientists. L.

rhamnosus has several properties that are beneficial to the intestinal tract and immune

system, mostly in combating intestinal and urinary tract pathogens. L. rhamnosus also

can be used as a natural protective in yogurt-based products to encourage the growth

of helpful organisms that aid in digestion and eliminate or prevent the growth of harmful

bacteria in the intestines (“Lactobacillus Rhamnosus,” n.d.). L. rhamnosus R0011 is

another stain that has been evaluated for probiotic effects, however, studies have

examined a combination of two strains, Lactobacillus helveticus R0052 and

Lactobacillus rhamnosus R0011 in a 95:5 ratio.

A study conducted by Firmesse, et al. (2007) evaluated the ability of L.

rhamnosus R0011 and L. helveticus R0052 strains to survive through the human

digestive tract. Healthy participants were required to take four capsules containing

1.9x109 of R0011 and 1x108 R0052) daily for 12 days, followed by a 12-day washout

period. The researchers found that R0011 survived through the intestinal tract and was

found in stool at high concentrations (7.1x1010 CFU/g of stool), but it showed no lasting

colonization following cessation of the probiotic. R0052, the second strain in the

probiotic, was not present at significant levels in the stool samples, suggesting that this

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lactobacillus strain either did not survive or was associated with the mucosal layer and

not the luminal microbiome because of the highly adherent nature of this bacteria.

In a randomized, controlled, double-blind study of 214 adult patients with

respiratory tract infections being treated with antibiotics by Song et al. (2010),

participants were randomly assigned to receive rhamnosus R0011 and L. helveticus

R0052 or a placebo for 14 days. The probiotic group showed lower incidences of

antibiotic-associated diarrhea (AAD) with four (3.9%) of 103 patients demonstrating

symptoms, compared to the placebo group where eight (7.2%) of 111 patients, (P=0.44)

demonstrated symptoms, but this was not considered a significant difference between

groups. That being said, the probiotic group experienced less changes in bowel

frequency and consistency (50/103, 48.5%) compared to the placebo group (35/111,

31.5%)(P=0.01).

Lactobacillus casei

Lactobacillus casei produces lactic acid via fermentation which usually occurs in

the human intestinal tract. L. casei is also one of the more popular strains that can be

used as a probiotic supplement. Shirota (Lactobacillus casei Shirota) is the most viable

strain of this bacteria that can survive within the intestinal tract and recover balanced

gut microbiota (Alipour et al., 2014; Shida et al., 2015). In the last several years, animal

and human studies have been done to evaluate the immune-regulatory effects of this

type of microbiota (Shida et al., 2015; Spanhaak, Havenaar, & Schaafsma, 1998). In

addition, bowel habits and behavior associated with emotion and anxiety were other

targets for these type of studies (Benton, Williams, & Brown, 2006; Rao et al., 2009;

Tilley et al., 2014).

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A controlled study of 17 healthy volunteers (nine in the experimental group and

eight in the control group) (NAGAO et al., 2000) revealed that the consumption of

fermented milk containing Lactobacillus casei Shirota (LcS) daily for three weeks

caused a significant increase in the NK cell activity for six weeks after starting the

treatment. It is important to note that natural killer (NK) cells have an important role in

immune surveillance against tumor development and viral infection (NAGAO et al.,

2000). In another study, Shida et al. (2015) conducted a randomized, controlled study of

96 male workers to evaluate the effects of the Lactobacillus casei strain Shirota-

fermented milk (LcS-FM) on the frequency of upper respiratory tract infections (URTIs).

The healthy office workers consumed LcS-FM or control milk (CM) once daily for 12

weeks during the winter season. A questionnaire of URTI symptoms was used by a

physician to evaluate the URTI episodes. The LcS-FM group experienced lower URTIs

during the intervention than the CM group (22.4 vs. 53.2 %, P=0.002). Also, the LcS-FM

group showed an inhibition of reduction in NK cell activity in peripheral blood

mononuclear cells and a rise in salivary cortisol levels. The researchers suggested that

the daily consumption of fermented milk with Lactobacillus casei strain Shirota may

decrease the risk of URTIs in healthy middle-aged office workers, possibly through

modulation of the immune system.

Results from a controlled study of 46 female patients with established rheumatoid

arthritis (RA) for more than one year (Alipour et al., 2014) also showed an improvement

in inflammatory biomarkers when L. casei 01, was added to conventional treatment.

Serum high-sensitivity C-reactive protein (hs-CRP) levels, tender and swollen joint

counts, global health (GH) score and disease activity score for rheumatoid arthritis

24

(DAS28) decreased with the consumption of L. casei 01 supplementation (P<0.05). At

the end of the study, a significant difference was detected between the two groups for

Interleukin (IL)-10, IL-12 and tumor necrosis factor alpha (TNF-α) changes through the

study (P<0.05), supportive the probiotic group.

L. casei Shirota also showed a positive effect on the bowel habit (stool

consistency) in subjects suffering from hard stools. Tilley et al. (2014) evaluated the

ability of L. casei Shirota to improve the stool consistency in subjects who were

suffering from hard stools. In addition, they tested the ability for the microbiota to survive

through the gastrointestinal tract. In a double-blind, placebo-controlled, randomized

study design, Tilley et al. had 120 subjects with symptoms of constipation for over an

eight-week period. All subjects had to consume 65 ml/day of a probiotic fermented milk

drink containing L. casei Shirota or a placebo. In addition, all subjects had to complete a

questionnaire to assess the consistency of their stools. They found that there was a

significant reduction in stool hardening in the experimental group, and the number of L.

casei Shirota bacteria in the stools increased when consuming the L. casei Shirota

fermented milk and decreased during washout.

Rao et al. (2009) conducted a pilot study of 39 Chronic Fatigue Syndrome (CFS)

patients to determine the possible effects of L. casei Shirota on depression and anxiety

symptoms in these patients. The participants were randomized to receive either L. casei

Shirota or a placebo daily for two months. Before and after the study, patients had to

provide stool samples and were required to complete the Beck Depression and Beck

Anxiety Inventories. A significant increase observed in both Lactobacillus and

Bifidobacterium in the experimental group, in addition; there was also a significant

25

decrease in anxiety symptoms among participants who were taking the probiotic vs

controls (p=0.01).

Lactobacillus plantarum

Lactobacillus plantarum (L. plantarum) is one of lactic acid bacterium that can be

found in a range of environmental niches. There are several studies have been done on

this strain that show the ability of L. plantarum to survive gastric transit and colonize the

intestinal tract of human and other mammals. In addition, numerous studies show the

effects of L. plantarum consumption on human physiology (de Vos et al., 2006; Zago et

al., 2011).

Irritable bowel syndrome (IBS) was one of the disorders that with which

researchers tried to examine the effect of L. plantarum. In a study done by Niedzielin et

al. (2001), forty patients (20 patients in each group) were randomly assigned to receive

either L. plantarum 299V in liquid suspension or a placebo over a period of four weeks.

All the participants had to complete clinical examination at the baseline and at the end

of the study, in addition to complete a self-assessment to their symptoms every week.

All patients who received L. plantarum 299V reported a reduction of their abdominal

pain compared to 11 patients from the placebo group (P═0.0012). Six out of the 10

patients who used to be constipated but were treated with L. plantarum 299V

experienced a normalization of stools frequency in comparison to just two out of 11

treated with placebo (P=0.17). Additionally, 95% of patients in the L. plantarum 299V

group reported an improvement in all IBS symptoms in contrast to 15% of patients in the

placebo group (P<0.0001).

In another study, Barreto et al. (2014) evaluated the effect of fermented milk with

L. plantarum in the parameters associated with metabolic syndrome (MetS), as well as

26

other parameters linked to cardiovascular risk in postmenopausal women. Twenty-four

participants were divided in two groups: those given non-fermented milk (NFM) at 80

mL/d and those given fermented milk (FM) at 80 mL/d. Researchers measured the

anthropometric and blood pressure measurements, as well as biochemical,

inflammatory, and immunologic biomarkers. There was a significant decrease in total

cholesterol, γ -glutamyltranspeptidase (γ -GTP), and IL-6 (P ═ 0.010, P ═ 0.018, and P

═ 0.001, respectively). In addition, glucose and homocysteine levels showed a

significant decrease in the FM group compared with the NFM group (P ═ 0.037 and P ═

0.019, respectively).

In a single-blind, cross-over design of two trials applying the double-isotope (⁵⁵Fe

and ⁵⁹Fe) technique, Hoppe et al. (2015) investigated the effect of Lactobacillus

plantarum 299v (Lp299v) on the non-heme iron absorption from a fruit drink. Healthy

women of reproductive age were divided into two groups. In Trial 1, the experimental

group drank a fruit drink containing 10⁹ CFU Lp299v, while the control group had a

control drink without Lp299v. Trial 2 had the same design but the fruit drink contained

1010 CFU of Lp299v. There was around 5 mg of iron as ferrous lactate in the test and

control drinks, and they were labelled with ⁵⁹Fe (B) and ⁵⁵Fe (A), respectively. Each

were consumed on four repeated days in the order AABB. Mean iron absorption from

the test drink containing 10⁹ CFU Lp299v (28.6±12.5%) was significantly higher than

that from the control drink (18.5±5.8%) (n=10, P<0.028). The test fruit drink with 1010

CFU Lp299v presented a mean iron absorption of 29.1±17.0%, while the control drink

presented an absorption of (20.1±6.4%) (n=11, P<0·080). They concluded that with that

27

the consumption of probiotics, Lp299v can increase the absorption of iron by almost

50% from a fruit drink that contains high iron bioavailability.

Bifidobacterium

Bifidobacterium is one of the lactic acid bacteria which are found in fermented

foods like yogurt. It is also one of the many genus bacteria among the enormous gut

bacterial community that controls the gut of healthy breast-fed infants (80% of the

cultivable fecal bacteria). In adulthood the levels of this bacteria are lower (up to 25% of

the cultivable fecal bacteria) but relatively stable (Picard et al., 2005). The presence of

different species of Bifidobacterium in human body changes with age. In infants,

Bifidobacterium longum, B. breve, and B. bifidum are normally the predominant species,

while in adults, B. catenulatum, B. adolescentis and B. longum are more dominant.

Researchers have studied the efficacy of Bifidobacterium in the prevention and

treatment of a wide range of animal and/or human gastrointestinal disorders (AlFaleh et

al., 2011; Arboleya et al., 2016; Picard et al., 2005).

Bifidobacterium breve

Bifidobacterium brave has been studied to investigate the effect of this species

on human health. There are several studies that have been done in constipated adults

and children with a Bifidus yoghurt containing Bifidobacterium breve, Bifidobacterium

bifidum and Lactobacillus acidophilus that have shown an improvement in defecation

frequency without any side effects. Because of this, Tabbers et al. (2011) conducted a

pilot study to evaluate the effect of Bifidobacterium breve as a treatment of childhood

constipation. Twenty children between the ages of 3 to 16 years with functional

constipation participated in this study. Children received one sachet of powder daily,

containing 108 - 1010 CFU Bifidobacterium breve, through four weeks. Additionally, after

28

each meal, children were asked to try to defecate on the toilet for 5-10 minutes and to

complete a standardized bowel diary daily. The defecation frequency per week

significantly improved from 0.9 at baseline to 4.9 in week 4 (p<0.01). Also, after 4 weeks

of Bifidobacterium breve consumption, the mean stool consistency score increased from

2.6 at baseline to 3.5 in week 4 (p=0.03). The number of fecal incontinence episodes

per week significantly reduced from 9.0 at baseline to 1.5 in week 4 (p<0.01). Every

week, the abdominal pain episodes significantly reduced from a rating of 4.2 at baseline

to 1.9 in week 4 (p=0.01). Moreover, no side effects occurred.

Several studies showed that a low number of Bifidobacterium spp. is associated

with the development of obesity and/or diabetes (Turnbaugh et al., 2009; Wu et al.,

2010). In the other side, Bifidobacterium breve B-3 showed an anti-obesity effect on

high-fat diet-induced obesity mice. Therefore, Minami et al. (2015) conducted a

randomized, double-blind, placebo-controlled study to evaluate the effect of the

consumption of Bifidobacterium breve B-3 on body compositions and blood parameters

in adults with possibility for obesity. After a 4-week run-in period, 44 participants were

randomly assigned to receive either placebo or a Bifidobacterium breve B-3 capsule

(about 5x1010 CFU) daily for 12 weeks. The results showed a significant decrease in the

fat mass in the intervention group compared with the placebo group at week 12. In

addition, there were Improvements in γ-GTP and high-sensitivity C-reactive protein

which are related to liver functions and inflammation. Moreover, significant correlations

were found between the changed values of some blood parameters and the changed fat

mass in the intervention group compared with the placebo group, positive correlation

29

with alanine aminotransferase (ALT) and γ-GTP; negative correlations with 1,5-

anhydroglucitol.

Bifidobacterium longum

Bifidobacterium longum is another strain of Bifidobacteria that has been used in

several studies to investigate the effect of this stain on numerous gastrointestinal tract

diseases. In child study by Giannetti et al. (2016), 48 children with irritable bowel

syndrome between the age of 8 to 17 years, and 25 with functional dyspepsia (FD) in

the range of 8 to 16 years, were randomly assigned to receive either a mixture of three

Bifidobacteria or a placebo for six weeks. Bifidobacteria contains 3 billion

Bifidobacterium longum BB536, one billion Bifidobacterium infantis M-63, and one billion

Bifidobacterium breve M-16V. After a two-week "washout" period, each participant was

switched to the other group and followed up for additional six weeks. At baseline and

follow-up periods, participants completed a symptom diary and a quality of life

questionnaire. Abdominal pain (AP) resolution was the primary outcome parameter.

Bifidobacteria showed a complete resolution of AP in IBS but not in FD, when it was

compared with placebo (P=0.006), as well as significantly improved AP frequency

(P=0.02). There was a significant increase in the number of IBS children who

experienced an improvement in QoL after consuming the probiotics compared with the

consumption of placebo (48% vs. 17%, P= 0.001), but this outcome was not found in

FD.

According to Saez-Lara et al. (2015), inflammatory bowel disease (IBD) can be

characterized by chronic inflammation of the gastrointestinal tract. It classically consists

of Crohn’s disease (CD) and ulcerative colitis (UC). The differences between CD and

UC are in the intestinal localization and some inflammation features. CD inflammation

30

can occur in any part of the gastrointestinal tract, but UC inflammation starts in the

rectum and is limited to the colon. There are several studies that confirm the ability of

probiotic containing lactobacillus and Bifidobacteria to improve the quality of life of IBD

patients.

In addition, Saez-Lara et al., (2015) also reported some proposed mechanisms of

how these microorganisms may exert the beneficial effect for IBD patients. Short chain

fatty acids (SCFAs) and lactate production may inhibit the growth of possibly pathogenic

organisms, in addition to having an anti-inflammatory effect on the gut. Another

mechanism could be the increase of transit time by the net flow of water from the blood

to the intestinal lumen, which may affect the adherence of bacteria to the intestinal wall.

In addition, the consumption of probiotics may help to reduce the production of noxious

substances that may be involved in the pathogenesis of IBD.

Tamaki et al. (2016) evaluated the efficacy of Bifidobacterium longum 536 (B.

longum 536) supplementation for induction of remission in Japanese patients with active

ulcerative colitis. Fifty-six patients with mild to moderate UC participated. Twenty-eight

participants were randomly assigned to receive 2–3x1011 freeze-dried viable B. longum

536 while 28 other participants were given placebo for 8 weeks. A total of 63% of

participants in the treatment group showed clinical remission (UC disease activity index

(UCDAI) ≤2) at week 8 compared to 52% in the placebo group (P=0.395). In addition,

there was a significant decrease of UCDAI scores (3.8±0.4 at baseline to 2.6±0.4 at

week 8) in the treatment group (P<0.01); however, no significant decrease was

observed in the placebo group (P=0.88). The treatment group showed a significant

decrease in the Rachmilewitz endoscopic index (EI) and the Mayo sub score at week 8,

31

whereas the placebo group did not. One participant in the treatment group complained

of a mild side effect, but no other adverse effects were noticed.

Assessment of Gastrointestinal Symptoms and Wellnesses

Gastrointestinal Symptom Rating Scale (GSRS)

GSRS is a questionnaire that has been developed to evaluate the presence and

severity of common symptoms of gastrointestinal disorders. It has been validated and

used in a number of gastrointestinal illnesses such as, gastric ulcers, irritable bowel

syndrome, and gastroesophageal reflux disease (GERD) (Kulich et al., 2008; Revicki,

Wood, Wiklund, & Crawley, 1997). However, healthy individuals may also experience

gastrointestinal symptoms due to diet changes and GSRS may be relevant to healthy

populations and not limited to gastrointestinal diseases event it has not been validated

in healthy adults.

The GSRS contains 15 questions with a 7-grade Likert scale (1 = none, 2 =

minor, 3 = mild, 4 = moderate, 5 = moderately severe, 6 = severe, and 7 = very severe

discomfort) that was originally created to evaluate a wide range of GI symptoms by

interview. Later it became a self-reported questionnaire. The GSRS breaks down 15

items into five symptom clusters depicting abdominal pain (three symptoms), reflux (two

symptoms), indigestion (four symptoms), diarrhea (three symptoms), and constipation

(three symptoms) syndromes. The measurement of the final score is usually calculated

as the mean value of the items that are below specific syndrome with a minimum value

of 1 (absence of troublesome symptoms) and a maximum value of 7 (very troublesome

symptoms) (Dong, Guo, Ding, Zhou, & Wu, 2014; Kulich et al., 2008).

32

Bristol Stool Form Scale (BSFS)

Stool form scale can be used as a simple visual tool to measure stool

consistency dependent on the shape and apparent texture of the stool. It can be

considered as a standardized and inexpensive method of classifying stool form. The

Bristol Stool Form Scale (BSFS) is the most widely stool form scale used throughout the

world in both clinical and research settings. It has been validated by several studies as

a proxy tool for transit time in healthy and chronic constipation adults (Blake et al., 2016;

Lewis & Heaton, 1997; Saad et al., 2009). Bristol Stool Form Scale can be used by

doctors, health care professionals, researchers, patients and the general population to

identify the form of the stool using seven different images with written descriptors. It

ranges from Type 1 (the hardest stool) to Type 7 (the softest stool) (Blake, Raker, &

Whelan, 2016; Malhotra et al., 2016).

Conclusions

Although limited research has been carried out each strain of bacteria found in

probiotics, the usage of probiotics has increased in the last several years as a form of

alternative and supplemental health care. It seems that each strain of beneficial bacteria

has specific impact on gut health. Therefore, it is important to do more research to

identify the appropriate type of these bacteria for certain situations such as constipation

or side effects associated with some drugs.

In the study discussed in this thesis, a formulation of the Jarro-Dophilus EPS®,

consisting of Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011,

Lactobacillus casei R0215, Pediococcus acidilactici R1001, Bifidobacteria breve R0070,

Bifidobacteria longum BB536, Lactobacillus plantarum R1012 and Lactobacillus lactis

ssp. lactis R1058 was used to determine the effect of this combination on

33

gastrointestinal symptoms and wellbeing. Jarro-Dophilus EPS® is marketed in the U.S.

for intestinal, immune function, and health. Most of the species in this probiotic have not

been studied to evaluate the safety and side effects of each individually or with other

strains, and this is the reason why the literature review does not include all of them.

Interestingly, this multi-strain probiotic can be found in pharmacies and online although

there no safety trail has been done on this probiotic. This was the main reason to

conduct our safety clinical trial on this multi-strain probiotic. Gastrointestinal Symptom

Rating Scale questionnaire and daily questionnaires (paper or electronic) of bowel

movement frequency, gastrointestinal and wellness symptoms were used to evaluate

the symptoms and to ensure of the safety of this probiotic to be taken with all different

strains that included in it.

34

CHAPTER 2 PURPOSE AND HYPOTHESIS

Purpose of the Study

This is the first study to evaluate the multi-strain formulation containing

Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011, Lactobacillus casei

R0215, Pediococcus acidilactici R1001, Bifidobacterium breve R0070, Bifidobacterium

longum BB536 Lactobacillus plantarum R1012, Lactobacillus lactis ssp. lactis R1058.

As probiotics need to be delivered alive to the gut and remain alive in the gut, probiotics

must resist other microbes, gastric acid, and bile salts throughout gastrointestinal

transit. Probiotics must also demonstrate efficacy, i.e. a positive impact on human

health. The purpose of this study is to determine the effect of administering the multi-

strain probiotic on gastrointestinal function and general wellness in healthy adults.

Hypothesis

We hypothesized that the consumption of the multi-strain probiotic will not have

any effects on gastrointestinal health and wellness when it is consumed by healthy

adults at 5 billion or 25 billion CFU/ day. Also, the consumption of the multi-strain

probiotic may cause a decrease in the diarrhea and constipation in participants who

experience these symptoms.

35

CHAPTER 3 METHODS AND PROCEDURES

Study Design

A six-week, randomized, double-blind, dose-response study was conducted with

75 healthy adults between the ages of 18 and 50 years old. All participants were

randomized in parallel into one of three groups, two dosage groups: 1) 5 billion

CFU/capsule, or 2) 25 billion CFU/capsule or 3) placebo (Figure 3-1). Institutional

Review Board approval was obtained from the University of Florida’s IRB-01 on

04/06/2015 (Appendix A).

Inclusion and Exclusion Criteria before Attaining Consent

To be included in the study, participants needed to be between 18-50 years of

age. They were required to be willing and able to complete the Informed Consent

(Appendix B) in English as well as completing the International Physical Activity

Questionnaire (IPAQ) (questions regarding physical activity including intensity, duration

and frequency) (Appendix C).

The purpose of using the IPAQ as a screening criteria is to exclude highly

sedentary individual and extreme athletes (i.e. professional athletes, individuals training

for long distance running, etc.) and not to exclude those individuals who had a manual

labor component to their job. Thus, data cleaning and analysis were used consistent

with IPAQ guidelines and where necessary those individuals scoring as “highly active”

were further evaluated for inclusion.

Participants were required to be willing to take height and weight measurements

as well as providing demographic information (e.g. age, race, sex). They were required

to be willing to consume 5 billion CFU/capsule or 25 billion CFU/capsule of the multi-

36

stain probiotic or placebo for a 28-day period (1 capsule per day). They were required to

have Internet access for the duration of the study in order to complete a daily

questionnaire (Appendix D) regarding general and gastrointestinal wellness

electronically. The daily questionnaire contains around 30 questions, 4 questions about

gastrointestinal distress symptoms, 4 questions about ear-nose-through symptoms, 3

questions about epidermal symptoms, 3 questions about psychological health

symptoms, 2 question about cephalic symptoms, 2 questions about emetic symptoms,

and one question for each of these symptoms diarrhea, constipation, fatigue, and

hungry. The daily questionnaire also included the Bristol Stool Form Scale and other

questions about visiting doctors, taking the probiotic, taking antibiotic or any other

medications in that day. In addition, participants were required to complete the

Gastrointestinal Symptom Rating Scale (GSRS) every week (Appendix E). Three stool

samples were required to be provided through the duration of the study (baseline, week

4 of intervention, and during washout).

Participants were excluded from the study if they did not meet any of the above

criteria and if they had a known allergy to soy, milk, or milk protein. They were excluded

if they were taking any medications for constipation or diarrhea, if they had taken

antibiotics within the past four weeks prior to randomization, or if they were already

taking a probiotic supplement that they did not want to discontinue at least two weeks

prior to the beginning of the study. In addition, they were excluded if they had been

diagnosed or were currently being treated for any diseases or illnesses such as the

gastrointestinal diseases: gastric ulcers, Crohn’s disease and ulcerative colitis or other

chronic diseases such as diabetes and kidney disease or immune-compromising

37

diseases/conditions such as HIV, AIDS, hepatitis, and cancer, or if they were a

transplant patient. Smokers were excluded from the study in addition to the pregnant

and lactating women.

Recruitment

Healthy adults were recruited by flyers and posters that were posted around the

University of Florida and the Gainesville community. Also, some participants were

recruited by word of mouth. Following an initial screening of participants using the

inclusion and exclusion criteria listed above, participants were consented by trained

study coordinators and then they were asked to complete the IPAQ. If the participant

still met the inclusion/exclusion criteria, they were scheduled to begin a 7-day baseline

period. Recruitment began on 02/28/2016 and ran intermittently until 03/16/2016.

Baseline

Following informed consent, participants were provided with a folder including

their study numbers the study calendar, list of the important study days, stool collection

instructions, and list of some food that they should avoid since the study started such as

any food has probiotic or prebiotic. They were informed about the day that the study

started on which was the first day of receiving the daily questionnaire. Participants

received the daily questionnaires every day at 5:00 p.m., and they had to be completed

in the morning before 4:59 a.m. of the next day. Participants were also instructed on the

proper procedure for the collection of a whole stool sample by trained research staff.

The stool collection kits were provided (Fisher Scientific) to the participants. In addition,

participants were instructed to bring the sample in for processing on ice within four

hours of defecation. They had a window of two days to bring each stool sample.

Following the completion of the baseline week, participants attended their first clinic visit

38

in which research coordinators took weight measurements on a Seca® scale (model

874) and height measurements on a Seca® portable stadiometer (model 217).

Participants also completed a demographic information questionnaire evaluating age,

gender, race and ethnicity. Participants were then randomly assigned to receive the

multi-strain probiotic at 5 billion CFU/capsule, 25 billion CFU/capsule or placebo.

The completion of the daily questionnaires for each participant was monitored by

trained research staff. If the questionnaire had not been completed by 9:59 p.m., an

email reminder was sent to the participant to ensure full compliance to study protocol.

Participants who failed to comply with study protocol were withdrawn from the study

prior to randomization.

Randomization and Intervention

A randomization scheme was completed by an unaffiliated researcher in the

Food Science and Human Nutrition department. Each treatment group was doubled

coded to ensure that a complete treatment group was not unblinded during the

intervention period, if an adverse event were to cause the researchers to unblind. No

restrictions were applied to the randomization sequence. Each random allocation was

arranged to fit on a single sheet of white paper, indicating the coded intervention group,

the date, the number of capsule bottles given to the participants and the participant’s

study number. These sheets were placed in envelopes and sealed to ensure the

integrity of the blinding process. The envelopes were separated into four stacks, on top

of each envelope was printed: Stack # and Envelope #. Participants were enrolled by

trained research coordinators during the consenting process. On randomization day,

research coordinators unsealed the envelopes and assigned the participants to their

intervention group.

39

Post randomization, participants were instructed to take one capsule per day at

the end of a meal. They were provided with enough capsules for the entire 4-week

intervention period. The bottles were clearly marked with a specialized six-digit code (for

the purposes of randomization), the title of the study (Evaluation of Jarro-Dophilus

EPS® probiotic Formulation), and the instructions, “Take one capsule daily with a meal,”

“Keep refrigerated.” Participants were informed of these instructions and that they

should begin consumption of the capsules that day (Day 8 of the study). They also were

informed to continue taking the capsules for 28 days. Participants were asked to retain

the bottles with any extra capsules and return at their second appointment. In the last

week of the intervention phase, the second fecal samples were collected.

All research staff were blinded. Lallemand Health Solutions (Montreal, QC)

provided the study coded capsules. They were identical in size, shape and color, and

they kept inside white, sealed pill bottles.

Washout and Post Intervention

Following the intervention phase, participants attended their second clinical visit

where they returned the pill bottles and any remaining capsules. Weight measurements

were taken. Participants were instructed to continue completing their daily

questionnaires for the next seven days. They were also instructed to bring in a final

stool sample before their last appointment.

Compensation

Following completion of the entire study, participants had their last visit. In this

visit, weight measurements were taken and the compensation was provided.

Participants were compensated a maximum of $171 USD for six weeks of completing

the daily questionnaire, consumption of the capsules, and providing three fecal samples

40

($15 per stool) and $14 per week for the baseline and washout weeks and $24.50 per

week for the intervention weeks.

Statistical Methods

Equivalence Testing

Equivalence testing was conducted for both the daily and weekly questionnaires.

For the daily questionnaire, the daily values were averaged into weekly values for

individual variables (bowel movement frequency, hours of sleep, diarrhea, and

constipation) and combinations of variables (Gastrointestinal Distress, Cephalic,

Epidermal, Ear-nose-throat, Emetic, and Psychological Health). The combinations

variables were daily averages averaged over the week. Most of the variables were

skewed right and had some zeros; therefore, the equivalence tests among treatments

within a time period were run on the square roots of the weekly means. A tolerance of

0.4 was used for the equivalence of square roots. For hours of sleep, the weekly means

were normally distributed; therefore, no transformation was needed. An equivalence of

0.5 was used rather than 1 here because the categories of hours of sleep were

attenuated between 1 and 5 which representing 5 to 10 or more hours of sleep. The

equivalence tests rejected 1 or 2 pairs of baseline values in psychological health and

hours of sleep variables. Consequently, analysis of covariance (ANCOVA) was run with

treatment and baseline (psychological health and hours of sleep variables) values as a

covariate. Then, the equivalence test was conducted on the treatment Least Squares

means (LSmeans) for each time period from the ANCOVAs (which are adjusted to

average of the baseline values).

Participants completed the GSRS every week, at baseline, intervention, and post

washout periods. Individual symptoms evaluated by the GSRS were averaged into

41

syndrome scores (Table 3-2). Two One-Sided Tests (TOST) conducted to assess

whether there is a practical difference in means during the study and between the

intervention groups. The test looked at the difference between logged values of the

syndromes which is exactly the same as testing the log of the ratio between 2 numbers.

The equivalence tests were run at 0.4 for abdominal pain, diarrhea, constipation,

indigestion, while at 0.5 for reflux.

The Frequency and GIMMIX Procedures

For data analysis, BSFS was divided into three categories to detect the transit

time as it has been done by Dahl et al. (2016). Type 1 and 2 represent slow transit time

(hard stool after removing water), type 3, 4, and 5 represent normal transit time, and

type 6 and 7 represent fast transit time (diarrhea or watery stool). The frequency

procedure was used to detect the frequency of each transit type during the whole study

period in each group. Then, GIMMIX procedure was conducted to exam the effect of

treatments on the transit time.

42

Figure 3-1. Study design

43

Table 3-1. Daily Questionnaire Syndromes and Symptoms

Gastrointestinal Distress

Cephalic

ENT

Psychological Health

Emetic

Epidermal

Unclassified Symptoms

Bloating Headache Sore throat

Feeling anxious Nausea Skin rash Diarrhea

Flatulence Dizziness Blocked ear canal

Feeling depressed

Vomiting Skin redness / flushing

Constipation

Abdominal cramping

Nasal congestion

Feeling stressed

Hours of sleep

Abdominal noises

Runny eyes

44

Table 3-2. Gastrointestinal Symptom Rating Scale Syndromes

Abdominal Pain Reflux Indigestion Diarrhea Constipation

Hunger pains in

the stomach or

belly

Stomach ache or

pain

Rumbling in the

stomach or belly

Diarrhea Constipation

Nausea Heartburn Stomach felt bloated Loose stools Hard stools

Acid Reflux Burping Urgent need to have

a bowel movement

Feeling of not

completely emptying

bowels

Passing gas or flatus

45

CHAPTER 4 RESULTS

Participants

Sixty-eight participants completed the six-week study (Figure 4-1). Six

participants were excluded before the randomization (two extreme athletes, one highly

sedentary individual, one smoker, two no longer interested). One participant in the 25

billion CFU intervention group withdrew during the study due to being no longer being

interested in completing the study. No serious adverse event was reported. Participants

did not differ in age, and the ages ranged from 18 to 34 across all intervention groups.

However, there were significant differences between the three groups in gender. The

placebo group differed from the other intervention groups in the race, while the 25 billion

CFU/ group differed from the other groups in ethnicity. Although most of the participants

were in the normal Body Mass Index (BMI) range, there were significant differences

between the BMI in 5 billion CFU/ group and placebo, 5 billion CFU/ group and 25 billion

CFU/ group. However, no significant difference was obtained between placebo and 25

billion CFU/ group. The 25 billion CFU/group differed from the other intervention groups

in the supplements compliance category, % total of capsules consumed during

intervention, (94.9% compliance vs. 96.4 and 96.1 in the placebo, and 5 billion CFU/

groups respectively, p<0.01). The compliance for completing the questionnaires

significantly differed between the all three, p<0.01(92.9% in placebo, 95.2% in 5 billion

CFU/ group, and 91.2% in 25 billion CFU/ group). The 5 billion CFU/ group differed from

the other intervention groups in the compliance of providing the stool samples (100%

compliance vs. 97.1% and 98.6% in 25 billion group in the placebo, and 25 billion CFU/

groups respectively, p<0.01) (Table 4-1).

46

Daily Questionnaire Analysis

A total of 2360 daily questionnaires were completed and analyzed. Table 4-2

presents the syndrome data of the daily questionnaire. The gastrointestinal distress

syndrome scores of the daily questionnaire (symptoms: bloating, flatulence, abdominal

cramping, abdominal noises) (Figure 4-2), the cephalic syndrome scores (symptoms:

headache, dizziness) (Figure 4-3), epidermal syndrome (symptoms: itching, skin rash,

skin redness/flushing) (Figure 4-4), ear-nose-throat (symptoms: sore throat, blocked ear

canal, nasal congestion, runny eyes) (Figure 4-5), emetic (symptoms: nausea, vomiting)

(Figure 4-6), psychological health (symptoms: feeling anxious, feeling depressed,

feeling stressed) (Figure 4-7), were concluded equivalent across all intervention groups

by period comparisons. All mean syndrome scores were below the clinically significant

threshold of 0.4. The mean of syndromes scores for each group during the whole study

period are presented in Table 4-3.

Individual symptoms (diarrhea, constipation, hours of sleep, bowel movement

frequency) were also tested using the equivalence testing method. All the individual

symptoms were concluded equivalent across all intervention groups by period

comparisons. The equivalence value of 0.4 was used with all individual symptoms

except hours of sleep the equivalence value was 0.5. The mean of symptoms scores for

each group during the whole study period are presented in Table 4-3, Figure 4-8, 4-9, 4-

10, and 4-11.

The score of hours of sleep of the daily questionnaire of the placebo, 5 billion and

25 billion CFU groups were concluded equivalent with equivalence value of 0.5. The

placebo (3.1±0.1) group was concluded not equivalent to the 5 billion (3.3±0.2) and 25

billion (3.6±0.2) CFU groups in the baseline week. In addition, the scores of hours of

47

sleep were non-equivalent between the 5 billion (3.5±0.2) (3.3±0.2) and the 25 billion

groups (3.6±0.2) (3.9±0.2) in week 4 of treatment and washout period respectively.

Bristol Stool Form Scale

The transit time frequencies (slow, normal, fast) are presented in Table 4-4. The

GIMMIX procedure test results in Table 4-4 showed that neither treatment nor period

were statistically significant.

Gastrointestinal Symptom Rating Scale Analysis

A total of 390 GSRS questionnaires were completed and analyzed. The

abdominal pain syndrome (symptoms: hunger pains in the stomach or belly, nausea)

(Figure 4-12), diarrhea syndrome (symptoms: diarrhea, loose stools, urgent need to

have a bowel movement) (Figure 4-13), constipation syndrome (symptoms:

constipation, hard stools, feeling of not completely emptying your bowels) (Figure 4-14)

were concluded equivalence across all intervention groups and periods with

equivalence value 0.4. Syndromes testing by using GSRS questionnaire are presented

in Table 4-5.

Similar results were produced for the reflux syndrome (symptoms: stomach ache

or pain, heartburn, acid reflux) using equivalence value 0.5. The scores of reflux

syndrome were concluded equivalence between all the study groups during the whole

time of the study (Figure 4-15).

Indigestion syndrome (symptoms: rumbling in stomach or belly, stomach felt

bloated, burping, passing gas or flatus) was also tested using equivalence testing with

value 0.4. The score of indigestion symptom of the GSRS questionnaire of the placebo,

5 billion and 25 billion CFU groups were concluded equivalent. The placebo (2.1±0.2)

48

(2.1±0.2) group was concluded not equivalent to the 5 billion (1.9±0.2) (1.7±0.2) CFU

group in the baseline and the second week of treatment, respectively (Figure 4-16).

Figure 4-1. Participant flow diagram

49

Table 4-1. Participant demographics

Placebo (n=23)

5 billion CFU (n=22)

25 billion CFU

(n=23)

P value

Gender(M/F), n 6/17* 8/14* 11/12* 0.01

Age, years Median (range) 27 (18-31) 22 (18-30) 23 (19-34) NS

Race, n (%) * 0.01

Asian 2 (9) 6 (27) 7 (31)

African-American 2 (9) 2 (9) 1 (4)

White 18 (78) 13 (59) 13 (57)

More than One Race 1 (4) 1 (5) 1 (4)

Ethnicity, n (%) * 0.01

Hispanic 7 (30) 6 (27) 3 (13)

Non-Hispanic 16 (70) 16 (73) 19 (83)

Unknown - - 1 (4)

BMI * 0.01

Under Weight (under 18.5) - 1 -

Normal (18.5 to 24.9) 15 15 15

Over Weight (25 to 29.9) 7 5 7

Obese (over 30) 1 1 1

Compliance (%)

Supplement protocol 96.4% 96.1% 94.9%* 0.01

Questionnaire protocol 92.9%* 95.2%* 91.2%* 0.01

Stool protocol 97.1% 100%* 98.6% 0.01

50

Table 4-2. Daily Questionnaire Syndromes Equivalence Testing.

Syndrome Period Placebo 5 billion CFU 25 billion CFU


(Bloating, Flatulence,

Abdominal cramping,

Abdominal noises)

Baseline 0.7±0.1 0.7±0.1 0.6±0.1

Week 1 0.6±0.1 0.6±0.1 0.5±0.1

Week 2 0.7±0.1 0.6±0.1 0.5±0.1

Week 3 0.6±0.1 0.6±0.1 0.4±0.1

Week 4 0.6±0.1 0.6±0.1 0.4±0.1

Washout 0.6±0.1 0.6±0.1 0.4±0.1

Cephalic (Headache,

Dizziness)

Baseline 0.4±0.04 0.4±0.05 0.4±0.05

Week 1 0.3±0.03 0.3±0.03 0.3±0.04

Week 2 0.4±0.05 0.4±0.05 0.4±0.04

Week 3 0.5±0.06 0.4±0.04 0.4±0.05

Week 4 0.4±0.05 0.4±0.05 0.4±0.05

Washout 0.3±0.03 0.4±0.05 0.3±0.04

Ear- nose- throat (Sore

throat, Blocked ear

canal, Nasal congestion,

Runny eyes)

Baseline 0.3±0.07 0.3±0.09 0.3±0.09

Week 1 0.2±0.06 0.2±0.07 0.3±0.08

Week 2 0.2±0.07 0.2±0.08 0.2±0.08

Week 3 0.2±0.08 0.3±0.07 0.2±0.07

Week 4 0.3±0.1 0.3±0.07 0.3±0.07

Washout 0.2±0.07 0.4±0.09 0.2±0.06

Data presented as mean±SE.

51

Table 4-2. Continued.


Emetic (Nausea,

Vomiting)

Baseline 0.1±0.05 0.1±0.05 0.06±0.03

Week 1 0.1±0.04 0.2±0.05 0.07±0.04

Week 2 0.06±0.03 0.2±0.07 0.09±0.04

Week 3 0.2±0.07 0.1±0.05 0.05±0.04

Week 4 0.1±0.05 0.07±0.04 0.2±0.07

Washout 0.1±0.04 0.1±0.04 0.1±0.05

Psychological Health (Feeling anxious, Feeling depressed, Feeling stressed)

Baseline 0.6±0.1 0.7±0.2 0.4±0.1

Week 1 0.5±0.2 0.7±0.2 0.3±0.1

Week 2 0.4±0.1 0.6±0.2 0.5±0.1

Week 3 0.4±0.1 0.6±0.2 0.4±0.1

Week 4 0.6±0.1 0.6±0.1 0.4±0.1

Washout 0.5±0.1 0.6±0.1 0.6±0.2

Data presented as mean±SE

Figure 4-2. Mean syndrome score of gastrointestinal distress per week. Scale from 0 to 6 (0=none, 3=moderate, 6=very severe)

0

0.2

0.4

0.6

0.8

1

Baseline Week 1 Week 2 Week 3 Week 4 Washout

Mean S

yndro

me S

core


Placebo 5 billion CFU/ capsule 25 billion CFU/ capsule

52

Figure 4-3. Mean score of cephalic syndrome per week. Scale from 0 t0 6 (0=none, 3=moderate, 6=very severe)

Figure 4-4. Mean score of epidermal syndrome per week. Scale from 0 to 6 (0=none, 3=moderate, 6=very severe)

0

0.2

0.4

0.6

0.8

1


Mean S

yndro

me S

core

Cephalic


0

0.2

0.4

0.6

0.8

1


Mean S

yndro

me S

core

Epidermal


53

Figure 4-5. Mean score of ear-nose-throat syndrome per week. Scale from 0 to 6 (0=none, 3=moderate, 6=very severe)

Figure 4-6. Mean score of emetic syndrome per week. Scale from 0 to 6 (0=none, 3=moderate, 6=very severe)

0

0.2

0.4

0.6

0.8

1


Mean S

yndro

me S

core

Ear-nose-throat


0

0.2

0.4

0.6

0.8

1


Mean S

yndro

me S

core

Emetic


54

Figure 4-7. Mean score of Psychological health per week. Scale from 0 to 6 (0=none, 3=moderate, 6=very severe)

0

0.2

0.4

0.6

0.8

1


Mean S

yndro

me S

core

Psychological Health


55

Table 4-3. Daily Questionnaire Symptoms Equivalence Testing


Diarrhea Baseline 0.1±0.1 0.1±0.1 0.2±0.1

Week 1 0.2±0.1 0.1±0.04 0.2±0.1

Week 2 0.2±0.1 0.1±0.04 0.1±0.04

Week 3 0.2±0.1 0.1±0.1 0.1±0.1

Week 4 0.2±0.1 0.1±0.1 0.2±0.1

Washout 0.2±0.1 0.2±0.1 0.2±0.1

Constipation Baseline 0.4±0.1 0.3±0.1 0.2±0.1

Week 1 0.3±0.1 0.2±0.1 0.1±0.1

Week 2 0.3±0.1 0.2±0.1 0.2±0.1

Week 3 0.3±0.1 0.2±0.1 0.2±0.1

Week 4 0.2±0.1 0.2±0.1 0.3±0.1

Washout 0.2±0.1 0.2±0.1 0.2±0.1

Hours of Sleep

Baseline 3.1±0.1 3.3±0.2 3.6±0.2

Week 1 3.3±0.2 3.5±0.2 3.7±0.2

Week 2 3.4±0.1 3.4±0.2 3.7±0.2

Week 3 3.3±0.1 3.5±0.2 3.7±0.2

Week 4 3.3±0.2 3.5±0.2 3.6±0.2

Washout 3.4±0.2 3.3±0.2 3.9±0.2

Bowel Movement Frequency

Baseline 1.1±0.04 1.1±0.05 1.1±0.06

Week 1 1.1±0.05 1.1±0.05 1.1±0.05

Week 2 1.1±0.05 1.1±0.04 1.2±0.05

Week 3 1.1±0.05 1.1±0.05 1.1±0.04

Week 4 1.1±0.04 1.2±0.06 1.1±0.05

Washout 1.1±0.04 1.02±0.05 1.2±0.04


56

Figure 4-8. Mean score of diarrhea symptom per week. Scale from 0 to 6 (0=none,

3=moderate, 6=very severe)

Figure 4-9. Mean score of constipation symptom per week. Scale from 0 to 6 (0=none, 3=moderate, 6=very severe)

0

0.2

0.4

0.6

0.8

1


Mean S

ym

pto

m S

core

Diarrhea


0

0.2

0.4

0.6

0.8

1


Mean S

ym

pto

m S

core

Constipation


57

Figure 4-10. Mean score of hours of sleep per week. Scale from 1 to 5 (1= 5-6 hours, 2= 6-7 hours, 3= 7-8 hours, 4= 8-9 hours, 5 >9 hours)

Figure 4-11. Mean score of bowel movement frequency per week. Scale from 1 to ≥11

0

1

2

3

4

5


Mean S

ym

pto

m S

core

Hours of Sleep


0

1

2

3

4

5


Mean S

ym

pto

m S

core

Bowel Movement Frequency


58

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)

59

Table 4-5. Gastrointestinal Symptoms Rating Scale Equivalence Testing as Syndromes



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

60

Table 4-5. Continued


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


61

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)

0

1

2

3

4

5

6

7


Mean S

yndro

me S

core

Abdominal Pain


0

1

2

3

4

5

6

7


Mean S

yndro

me S

core

Diarrhea


62

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)

0

1

2

3

4

5

6

7


Mean S

yndro

me S

core

Constipation


0

1

2

3

4

5

6

7


Mean S

yndro

me S

core

Axis Title

Reflux


63

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)

0

1

2

3

4

5

6

7


Mean S

yndro

me S

core

Indigestion


64

CHAPTER 5 DISCUSSION

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)

65

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

66

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

67

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)

68

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

69

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.

70

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.

71

APPENDIX A IRB APPROVAL

73

APPENDIX B INFORMED CONSENT

85

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

86

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?


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?


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?


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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?


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?


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?


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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?


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?


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?


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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?


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?


27. During the last 7 days, how much time did you usually spend sitting on a weekend

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

91

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 hours 5-6 hours 6-7 hours 7-8 hours 8-9 hours >9 hours

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

degree in nutritional sciences.

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