A Randomized, Double-blind, Placebo-controlled Study to Investigate the Effects of a Bifidobacterium Breve Strain on Fat Loss in Healthy Adults
Phase: NA
Conditions: Obesity, Overweight and Obesity
Sponsor: Morinaga Milk Industry Co., LTD

Page 1 of 48 
 
CLINICAL PROTOCOL COVER PAGE 
 
 
Protocol Title: 
A randomized, double-blind, placebo-controlled study to investigate the 
effects of a Bifidobacterium breve strain on fat loss in healthy adults  
 
 
Protocol Date:  
 
June 26, 2023 
 
Version: 
5 
 
Study Design: 
 
 Randomized, double-blind, placebo-controlled, 2-arm parallel study 
 
Sponsor: 
 
 
 
  
 
Sponsor Contact: 
 
  
 
CRO: 
 
 
Medical Director: 
 
 
 
 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 2 of 48 
 
PROTOCOL SIGNATURE SHEET 
The sponsor and the investigator agree to conduct the study in compliance with the clinical study protocol 
(and amendments), International Conference on Harmonization (ICH) guidelines for current Good 
Clinical Practice (cGCP) and applicable regulatory requirements. 
Name 
Signature 
Date 
Sponsor:  
 
 
 
Contract Research Organization:  
 
 
 
 
 
 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 3 of 48 
 
 
PROTOCOL SYNOPSIS 
Population: Healthy adults  
 
 
 
 
Total number of participants: 100 
 
1.1 
Inclusion Criteria 
1. Male or female between 20 and 65 years of age, inclusive 
2. BMI from 25.0 to 29.9 kg/m2, inclusive 
3. Female participants are not of child-bearing potential, defined as females who have undergone 
a sterilization procedure (e.g. hysterectomy, bilateral oophorectomy, bilateral tubal ligation, 
complete endometrial ablation) or have been post-menopausal (natural or surgically) for at least 
1 year prior to screening 
 
Or,  
Females of child-bearing potential must have a negative baseline urine pregnancy test and agree 
to use a medically approved method of birth control for the duration of the study. All hormonal 
birth control must have been in use for a minimum of three months. Acceptable methods of 
birth control include: 
• 
Hormonal contraceptives including oral contraceptives, hormone birth control patch (Ortho 
Evra), vaginal contraceptive ring (NuvaRing), injectable contraceptives (Depo-Provera, 
Lunelle), or hormone implant (Norplant System) 
• 
Double-barrier method  
• 
Intrauterine devices 
• 
Non-heterosexual lifestyle or agrees to use contraception if planning on changing to 
heterosexual partner(s) 
• 
Vasectomy of partner at least 6 months prior to screening 
4. Self-reported stable body weight for the past 3 months defined as not having gained or lost 
more than 5 kg of body weight throughout the 3 months prior to baseline  
5. Participants with the following body fat percentages:  
• 
Female: ≥ 25% as determined by Bioelectrical Impedance Analysis (BIA) at screening 
and confirmed ≥ 30% by Dual-Energy X-Ray Absorptiometry (DXA) at baseline  
• 
Male: ≥ 15% as determined by BIA at screening and confirmed ≥ 20% by DXA at 
baseline 
6. Agrees to follow the diet and exercise guidelines for the duration of the study 
7. Willingness to complete questionnaires, records, and diaries associated with the study, to 
complete all clinic visits, and provide stool samples 
8. Provide voluntary, written, informed consent to participate in the study 
9. Healthy as determined by medical history, laboratory results and physical exam as assessed by 
the Qualified Investigator (QI) 
1.2 
Exclusion Criteria 
1. Women who are pregnant, breastfeeding, or planning to become pregnant during the trial 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 4 of 48 
 
2. Allergy, sensitivity, or intolerance to the investigational product’s active or inactive 
ingredients 
3. Clinically significant abnormal laboratory results at screening as assessed by the QI 
4. Current or history of any significant gastrointestinal disease requiring medication (e.g. GERD, 
gastroenteritis) 
5. Irregular sleep schedule 
6. Chronic diarrhea or constipation  
7. Participants with hypertension and are on antihypertensive medication 
8. Type I or Type II diabetes 
9. Participants with hyperlipidemia and are on medication 
10. Self-reported sleep apnea 
11. Self-reported current or pre-existing thyroid condition. Treatment on a stable dose of 
medication for at least 3 months will be considered by the QI 
12. Unstable metabolic disease or chronic diseases as assessed by the QI 
13. History of or current diagnosis with kidney and/or liver diseases as assessed by the QI on a 
case-by-case basis, with the exception of history of kidney stones in participants who are 
symptom-free for 6 months 
14. Significant cardiovascular event in the past 6 months. Participants with no significant 
cardiovascular event on stable medication may be included after assessment by the QI on a 
case by case basis 
15. Major surgery in the past 3 months or individuals who have planned surgery during the trial 
period. Participants with minor surgery will be considered on a case-by-case basis by the QI 
16. Cancer, except skin cancers completely excised with no chemotherapy or radiation with a 
follow up that is negative. Volunteers with cancer in full remission for more than five years 
after diagnosis are acceptable 
17. Individuals with an autoimmune disease or are immune-compromised  
18. Self-reported HIV-, Hepatitis B- and/or C-positive diagnosis 
19. Blood/bleeding disorders as determined by laboratory results 
20. Self-reported mental or neuropsychological condition and/or cognitive impairment that, in the 
QI’s opinion, could interfere with study participation 
21. Metal implants that may affect the DXA scan results will be assessed on a case-by-case basis 
by the QI.  
22. Current use of prescribed medications listed in Section Prescribed Medications 7.3.1  
23. Current use of over-the-counter medications, supplements, foods and/or drinks listed in 
Section 7.3.2 
24. Use of cannabinoid products within 60 days of baseline. History of cannabis used will be 
assessed on a case by case basis by the QI 
25. Use of tobacco products within 60 days of baseline 
26. Self-reported alcohol or drug abuse within the last 12 months  
27. High alcohol intake (average of > 2 standard drinks per day or > 10 per week) 
28. Current employment that calls for shift work or have worked shift work in the last 3 weeks 
29. Participation in other clinical research trials 30 days prior to screening  
30. Blood donation 30 days prior to screening, during the study, or a planned donation within 30-
days of the last study visit  
31. Individuals who are unable to give informed consent 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 5 of 48 
 
32. Any other condition, that, in the opinion of the QI, may adversely affect the participant's 
ability to complete the study or its measures, or which may pose a significant risk to the 
participant 
 

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1.3 
Schedule of Assessments 
Procedures/assessments 
Visit 1 
Screen 
(Week -6) 
Run-in 
Period 
Compliance 
Calls 
Visit 2 
Week 0 
Visit 3 
Week 6 
Visit 
4 
Week 
12 
Informed consent 
X 
 
 
 
 
Review inclusion/exclusion 
criteria 
X 
 
X 
 
 
Review medical history 
X 
 
 
 
 
Review daily exercise habits 
X 
 
 
 
 
Review concomitant 
therapies 
X 
 
X 
X 
X 
Biometrics: Height*, weight, 
waist and hip circumference, 
heart rate, blood pressure, 
respiratory rate, and 
temperature 
*only be measured at Visit 1 
X 
 
X 
X 
X 
Urine pregnancy test 
X 
 
X 
 
 
Physical examination 
 
 
X 
 
 
Randomization 
 
 
X 
 
 
CBC, Na, K, Cl, Fe, Mg, Ca, 
phosphate, HbA1c, fasting 
glucose, eGFR, creatinine, 
BUN, AST, ALT, GGT, 
ALP, TSH* and total 
bilirubin 
*only be measured at Visit 1 
X 
 
X 
 
X 
Blood Collection: lipid 
profile and fasting insulin 
X 
 
X 
 
X 
Urinalysis 
X 
 
X 
 
X 
DXA 
 
 
X 
 
X 
BIA 
X 
 
X 
 
 
Stool Collection Kits 
Dispensed 
X 
 
 
X 
 
Stool Collection Kit 
Returned 
 
 
X 
 
X 
Exercise Guideline Review 
X 
X 
X 
X 
 
Daily Bowel Habits Diary 
Dispensed 
X 
 
X 
X 
 
Daily Bowel Habits Diary 
Returned 
 
 
X 
X 
X 
3DFR Dispensed 
X 
 
X 
X 
 
3DFR Returned 
 
 
X 
X 
X 
Exercise Diary Dispensed 
X 
 
X 
X 
 
Exercise Diary Returned 
 
 
X 
X 
X 
Subject Diary Dispensed 
X 
 
X 
X 
 
Subject Diary Returned 
 
 
X 
X 
X 
Placebo Dispensed 
 
X 
 
 
 
Placebo Returned 
 
 
X 
 
 
IP Dispensed 
 
 
X 
X 
 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 7 of 48 
 
Procedures/assessments 
Visit 1 
Screen 
(Week -6) 
Run-in 
Period 
Compliance 
Calls 
Visit 2 
Week 0 
Visit 3 
Week 6 
Visit 
4 
Week 
12 
IP Returned  
 
 
 
X 
X 
Compliance Calculated 
 
 
X 
X 
X 
Adverse Events  
 
X 
X 
X 
X 
 
 
 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 8 of 48 
 
 
LIST OF ABBREVIATIONS 
3DFR  
Three-Day Food Record  
AE 
 
Adverse Event 
ALP 
 
Alkaline phosphatase 
ALT 
 
Alanine Aminotransferase 
AST 
 
Aspartate Aminotransferase 
BIA 
 
Bioelectrical impedance analysis  
BMI 
 
Body Mass Index 
BP 
 
blood pressure 
BUN   
Blood urea nitrogen  
Ca 
 
Calcium 
CBC 
 
Complete Blood Count 
CRF 
 
Case Report Form 
Cl 
 
Chloride 
DXA 
 
Dual-Energy X-Ray Absorptiometry 
EDTA  
Ethylenediaminetetraacetic Acid 
eGFR  
Glomerular Filtration Rate  
etc. 
 
“and so forth” 
e.g. 
 
“for example” 
et al 
 
“and others” 
EOS 
 
End of the Study 
F/B 
 
Firmicutes/Bacteroidetes 
Fe 
 
Iron 
g 
 
Gram 
GCP 
 
Good Clinical Practice 
GGT 
 
Gamma-glutamyl transferase 
HbA1c  
hemoglobin A1c 
HDL-C  
High-density lipoprotein cholesterol 
HR 
 
Heart Rate 
ICF 
 
Informed Consent Form 
ICH 
 
International Conference of Harmonization 
IP 
 
Investigational product 
ITT 
 
Intent to Treat  
IRB 
 
Institutional Review Board 
K 
 
Potassium 
kg 
 
Kilogram 
LDL-C  
Low-density lipoprotein cholesterol 
MCH  
Mean Corpuscular Hemoglobin 
MCHC  
Mean Corpuscular Hemoglobin Concentration 
MCV  
Mean Corpuscular Volume 
MPV 
 
Mean Platelet Volume 
Mg 
 
Magnesium 
 
Na 
 
Sodium 
NEFA  
non-esterified fatty acids 
NSAIDs 
non-steroidal anti-inflammatory drugs 
OTC  
over-the-counter 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 9 of 48 
 
PP 
Per protocol 
PPIs 
Proton Pump Inhibitors 
QI 
Qualified Investigator 
RBC 
Red Blood Cells 
RDW Red Blood Cell Distribution Width  
SAE 
Serious Adverse Event 
SOP 
Standard operating procedure 
SST 
Serum Separating Tube 
SSRI 
Selective serotonin reuptake inhibitors 
TG 
Triglyceride  
TPD 
Therapeutics Products Directorate 
TSH 
Thyroid stimulating hormone  
ULN 
Upper limit of normal 
VLDL very-low-density lipoprotein 
WBC White Blood Cell 
WHO World Health Organization 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 10 of 48 
 
 
TABLE OF CONTENTS 
 
PROTOCOL SYNOPSIS ................................................................................................................... 3 
1.1 
Inclusion Criteria .......................................................................................................................... 3 
1.2 
Exclusion Criteria ......................................................................................................................... 3 
1.3 
Schedule of Assessments .............................................................................................................. 6 
 
LIST OF ABBREVIATIONS ............................................................................................................ 8 
 
TABLE OF CONTENTS.................................................................................................................. 10 
 
INTRODUCTION ............................................................................................................................. 13 
 
STUDY OBJECTIVES ..................................................................................................................... 14 
 
STUDY DESIGN ............................................................................................................................... 16 
 
SELECTION OF STUDY POPULATION ..................................................................................... 16 
7.1 
Inclusion Criteria ........................................................................................................................ 16 
7.2 
Exclusion Criteria ....................................................................................................................... 17 
7.3 
Concomitant Medications ........................................................................................................... 18 
7.3.1 
Prescribed Medications ...................................................................................................... 18 
7.3.2 
Over-the-counter Medications, Supplements and Foods/Drinks ........................................ 19 
7.4 
Washout Periods ......................................................................................................................... 19 
7.5 
Early Withdrawal ........................................................................................................................ 19 
 
INVESTIGATIONAL PRODUCT .................................................................................................. 20 
8.1 
Manufacturing and Storage ......................................................................................................... 20 
8.2 
Labelling and Coding .................................................................................................................. 21 
8.3 
Investigational Product (Bifidobacterium breve) ........................................................................ 21 
8.4 
Directions .................................................................................................................................... 21 
8.5 
Rescue Medication ...................................................................................................................... 21 
8.6 
Randomization ............................................................................................................................ 22 
8.7 
Blinding and Allocation Concealment ........................................................................................ 22 
 
STUDY ASSESSMENTS ................................................................................................................. 23 
9.1 
Screening (week -6 to -5; Visit 1) * ............................................................................................ 23 
9.2 
Run-in period – 4 weeks (Week -4 to Week -1) ......................................................................... 24 
9.3 
Compliance Call .......................................................................................................................... 24 
9.4 
Baseline (Visit 2, Week 0) .......................................................................................................... 24 
9.5 
Visit 3 (Week 6, 42 ± 3 days) ..................................................................................................... 25 
9.6 
Visit 4 - End of Study (Week 12, 84 ± 3 days) ........................................................................... 26 
9.7 
Follow-up (Week 14) .................................................................................................................. 27 
 CLINICAL ASSESSMENTS AND PROCEDURES ..................................................................... 27 
10.1 
Height and Weight ...................................................................................................................... 27 
10.2 
Circumference Measurements ..................................................................................................... 27 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 11 of 48 
 
10.3 
Blood Pressure and Heart Rate ................................................................................................... 28 
10.4 
Temperature and Respiratory Rate ............................................................................................. 29 
10.5 
Blood Sample Collection ............................................................................................................ 29 
10.6 
Stool Sample Collection.............................................................................................................. 29 
10.7 
Bioelectrical Impedance Analysis (BIA) .................................................................................... 29 
10.8 
Dual-Energy X-Ray Absorptiometry (DXA) Scan ..................................................................... 30 
10.9 
Daily Bowel Habit Diary and Bristol Stool Scale ....................................................................... 30 
10.10 
3-Day Food Record (3DFR) ................................................................................................... 30 
10.11 
Exercise Diary ......................................................................................................................... 30 
10.12 
Compliance ............................................................................................................................. 30 
10.13 
Laboratory Analyses ............................................................................................................... 31 
10.14 
Termination of the Trial .......................................................................................................... 33 
10.15 
Protocol Amendments ............................................................................................................. 33 
 SAFETY INSTRUCTIONS AND GUIDANCE ............................................................................. 33 
11.1 
Adverse Events and Laboratory Abnormalities .......................................................................... 33 
11.1.1 
Adverse Events .................................................................................................................... 33 
11.1.2 
Serious Adverse Event ......................................................................................................... 34 
11.1.3 
Unexpected Adverse Reaction ............................................................................................. 34 
11.1.4 
Laboratory Test Abnormalities ........................................................................................... 34 
11.2 
Treatment and Follow-Up Of AEs And Laboratory Abnormalities ........................................... 35 
11.2.1 
Treatment and Follow-up of AEs ........................................................................................ 35 
11.2.2 
Treatment and Follow-up of Laboratory Abnormalities ..................................................... 35 
11.3 
Reporting of SAEs And Unexpected Adverse Reactions ........................................................... 35 
 STATISTICAL EVALUATION ...................................................................................................... 37 
12.1 
Determination of Sample Size .................................................................................................... 37 
12.2 
Analysis Plan .............................................................................................................................. 37 
12.3 
Statistical Analysis Plan .............................................................................................................. 37 
12.3.1 
Premature Discontinuation Description ............................................................................. 38 
12.3.2 
Safety ................................................................................................................................... 38 
12.4 
Protocol Deviation Description ................................................................................................... 39 
12.5 
Protocol Amendments ................................................................................................................. 39 
 DATA COLLECTION AND STORAGE ....................................................................................... 39 
 ETHICAL ASPECTS OF THE STUDY ......................................................................................... 39 
14.1 
IRB Approval .............................................................................................................................. 39 
14.2 
Volunteer Information and Informed Consent ............................................................................ 40 
14.3 
Potential Risks and Procedures to Minimize Risk ...................................................................... 40 
 QUALITY ASSURANCE AND QUALITY CONTROL .............................................................. 40 
15.1 
Auditing ...................................................................................................................................... 40 
15.2 
Monitoring .................................................................................................................................. 40 
15.3 
Data Management ....................................................................................................................... 41 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 12 of 48 
 
 REFERENCE LIST .......................................................................................................................... 43 
 APPENDICES ................................................................................................................................... 45 
17.1 
Appendix I: Probiotic and Prebiotic Foods to Avoid .................................................................. 45 
17.2 
Appendix II: Daily Bowel Habits Diary with Bristol Stool Scale .............................................. 47 
17.3 
Appendix III: Exercise Guidelines .............................................................................................. 48 
 

Page 13 of 48 
 
 
INTRODUCTION 
In 2016, 1.9 billion adults or 39% of the world’s population were classified as overweight (body mass index 
(BMI) of 25.0 to 29.9 kg/m2) by the World Health Organization (WHO) (1). Overweight has become a 
critical issue in North America, with the prevalence of obesity highest in middle-aged adults (2). In the 
United States (US), the prevalence of overweight and obesity were estimated at 71.6% of the total 
population in 2015-2016 (3). Consequently, weight loss market value has been increasing, with the US 
weight loss market worth approximately $72 billion (4). This value is expected to rise as the population 
becomes more health-conscious and aware of the risks associated with excess body weight.  
In early adulthood, excess body weight is a risk factor associated with several health complications later on 
in life, including type II diabetes, cardiovascular disease, and non-alcoholic fatty liver disease (5, 6). Excess 
weight gain influences several pathways in the body, altering circulating lipids, glucose, insulin, 
hemoglobin A1c (HbA1c) levels, and liver function. Increases in visceral adipose tissue lead to a rise in the 
production of non-esterified fatty acids (NEFA), which decreases insulin sensitivity and play a role in the 
development of insulin resistance (6). As a result, glucose uptake by muscle tissue is decreased while free 
fatty acid and glucose production by adipose tissue and liver are increased, respectively (7). These altered 
pathways increase very-low-density lipoprotein (VLDL) and triglyceride (TG) production in the liver; 
inducing a decrease in high-density lipoprotein cholesterol (HDL-C) and increase in low-density lipoprotein 
cholesterol (LDL-C) and apolipoprotein B (8). Additionally, insulin resistance indirectly contributes to the 
VLDL concentration by lowering lipoprotein lipase activity (7).  
Probiotics have been used for decades for maintaining intestinal health, and in recent years probiotics have 
been proposed for weight management. Previous studies reported a correlation between weight and gut 
Bifidobacterium abundance, showing that the level of bifidobacterium in the gut microbiota was lower in 
obese people (9). Bifidobacterium breve (B. breve) is a probiotic strain known for its beneficial functions 
in weight management (10, 11). It has been shown to improve intestinal barrier function, upregulate fat 
metabolism, and mitigate systemic inflammation (10, 12, 13). 
Several studies have also demonstrated that B. breve may improve parameters associated with obesity. 
Supplementation with B. breve in obese mice was shown to reduce visceral fat gain and to lower serum 
cholesterol, glucose, and insulin compared to the control group (14). In a randomized, double-blind, 
placebo-controlled trial, healthy adults consuming a high-fat diet supplemented with a probiotic mix 
containing B. breve had reductions in body weight and fat mass after 4 weeks of supplementation (15). In 
another randomized placebo-controlled trial overweight adults were supplemented with B. breve for 12 
weeks. Results from the study showed significantly lower fat mass along with improvements in liver 
function and inflammatory markers such as γ-glutamyl transpeptidase (GGT) and high-sensitivity C-
reactive protein (CRP) compared to the placebo group (10, 11).  
Despite the promising results of probiotics and reductions in body weight and fat mass, weight management 
is multifactorial and sustained weight loss often has a low success rate. In conjunction with dietary 
intervention, regular exercise has been proposed to improve body composition, intestinal microbiota 
diversity, and digestion (16, 17). However, prolonged exercise may disrupt the gastrointestinal tract and 
reduce the abundance of beneficial bacteria (17). To counteract this effect, the use of probiotics combined 
with exercise intervention has been proposed for effectively managing weight loss. The combination of 
aerobic exercise and administration of probiotics has been examined in several clinical trials and it was 
found that the combination was effective in improving body composition and inflammatory responses (18-
20). 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 14 of 48 
 
The gut microbiota composition influences the effects of foods and drugs. It is reported that individuals 
who improved their glucose tolerance by taking barley kernel-based bread (responders) had a higher 
Prevotella/Bacteroides ratio as compared with individuals who did not (non-responders) (21). Several 
clinical studies indicate that the gut microbiota affects the therapeutic efficacy of checkpoint blockade 
immunotherapy (22). It is assumed that changes in microbial composition are associated with the anti-
obesity effects of B. breve. 
This study is a randomized, placebo-controlled, clinical trial investigating the effect of Bifidobacterium 
breve supplementation with exercise intervention on fat loss. Participants will be healthy adults between 
the ages of 20 and 65 with a Body Mass Index (BMI) from 25 to 29.9 kg/m2.  
 
STUDY OBJECTIVES 
The objective of this study is to investigate the effect of Bifidobacterium breve on fat loss in healthy 
adults.  
Primary outcome: 
The difference in change in fat loss from baseline (% or g), as assessed by Dual-Energy X-Ray 
Absorptiometry (DXA), between B. breve and placebo after 12 weeks of supplementation.  
Secondary outcomes: 
The difference in change from baseline between B. breve and placebo in: 
1. Body composition (weight, BMI, android/gynoid fat ratio, and muscle mass (% or g) as 
assessed by DXA after 12 weeks of supplementation 
2. Waist circumference, hip circumference, and waist/hip circumference ratio after 6 and 12 
weeks of supplementation 
3. Lipid profile (total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides) after 12 
weeks of supplementation 
4. Biomarkers of glycemic control (fasting blood glucose, HbA1c, fasting insulin levels) after 
12 weeks of supplementation 
5. Liver markers (aspartate transaminase (AST), alanine aminotransferase (ALT), gamma-
glutamyl transferase (GGT), and alkaline phosphatase (ALP)) after 12 weeks of 
supplementation 
6. Frequency of bowel movements after 6 and 12 weeks of supplementation 
7. Body composition (weight, BMI, total body fat (% or g), android fat (% or g), gynoid fat (% 
or g), android/gynoid fat ratio, and muscle mass (% or g) and waist circumference, hip 
circumference, and waist/hip circumference ratio) in participant groups classified by 
microbiota composition at week 0 
8. Microbiota composition analysis 
 
Safety outcomes: 
1. Incidence of pre-emergent and post-emergent adverse events  

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 15 of 48 
 
2. Vital signs (blood pressure (BP) and heart rate (HR)), respiratory rate, and temperature  
3. Clinical chemistry (total bilirubin, creatinine, blood urea nitrogen (BUN), Na, K, Cl, Ca, Fe, 
Mg, phosphate, and estimated glomerular filtration rate (eGFR))  
4. Hematology (white blood cell (WBC) count with differential (neutrophils, lymphocytes, 
monocytes, eosinophils, basophils), red blood cell (RBC) count, hemoglobin, hematocrit, 
platelet count, RBC indices (mean corpuscular volume (MCV), mean corpuscular 
hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell 
distribution width (RDW), and mean platelet volume (MPV)) 
5. Urinalysis (microscopy, glucose, ketones, specific gravity, blood, protein, nitrite, leukocytes, 
colour, appearance urobilinogen/urine bilirubin, urine pH)  
 
Exploratory analyses 
1. Total body fat (%) change during the run-in period as assessed by bioelectrical impedance 
analysis (BIA)  
2. Comparison of total body fat (%) as assessed by BIA and DXA at baseline 
 
 
 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 16 of 48 
 
 
STUDY DESIGN 
 
This study will be conducted at the KGK Science clinic in London, ON. 
The planned sample size for this study is 100 healthy adults. To evaluate primary, secondary, and safety 
outcomes, study assessments will be conducted as per the Schedule of Assessments in Section 1.3.  
Study Arm 
Number of Participants  
B. breve 
N = 50 
Placebo 
N = 50 
Total 
N = 100 
 
SELECTION OF STUDY POPULATION 
This study will enroll 100 healthy male and/or female adults. Each participant must fulfill the inclusion 
criteria and not meet any of the exclusion criteria as described in Sections 7.1 and 7.2, respectively. 
7.1 
Inclusion Criteria  
1. Male or female between 20 and 65 years of age, inclusive 
2. BMI from 25.0 to 29.9 kg/m2, inclusive 
3. Female participants are not of child-bearing potential, defined as females who have undergone 
a sterilization procedure (e.g. hysterectomy, bilateral oophorectomy, bilateral tubal ligation, 
complete endometrial ablation) or have been post-menopausal (natural or surgically) for at least 
1 year prior to screening 
 
Or,  
Week 12
EOS
(Visit 4)
Placebo
B. breve B-3
Baseline
(Visit 2)
Week 0
Screening
(Visit 1)
Week -6 to -5
12 weeks
Week 6
(Visit 3)
Run-in Period
Week -4 to -1
≤2 weeks
4 weeks
Week 14
Follow up 
call
2 weeks
Biweekly Compliance Call

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 17 of 48 
 
Females of child-bearing potential must have a negative baseline urine pregnancy test and agree 
to use a medically approved method of birth control for the duration of the study. All hormonal 
birth control must have been in use for a minimum of three months. Acceptable methods of 
birth control include: 
• 
Hormonal contraceptives including oral contraceptives, hormone birth control patch (Ortho 
Evra), vaginal contraceptive ring (NuvaRing), injectable contraceptives (Depo-Provera, 
Lunelle), or hormone implant (Norplant System) 
• 
Double-barrier method  
• 
Intrauterine devices 
• 
Non-heterosexual lifestyle or agrees to use contraception if planning on changing to 
heterosexual partner(s) 
• 
Vasectomy of partner at least 6 months prior to screening 
4. Self-reported stable body weight for the past 3 months defined as not having gained or lost 
more than 5 kg of body weight throughout the 3 months prior to baseline  
5. Participants with the following body fat percentages: 
• 
Female: ≥ 25% as determined by Bioelectrical Impedance Analysis (BIA) at screening 
and confirmed ≥ 30% by Dual-Energy X-Ray Absorptiometry (DXA) at baseline 
• 
Male: ≥ 15% as determined by BIA at screening and confirmed ≥ 20% by DXA at 
baseline 
6. Agrees to follow the diet and exercise guidelines for the duration of the study 
7. Willingness to complete questionnaires, records, and diaries associated with the study, to 
complete all clinic visits, and provide stool samples 
8. Provide voluntary, written, informed consent to participate in the study 
9. Healthy as determined by medical history, laboratory results and physical exam as assessed by 
the Qualified Investigator (QI) 
7.2 
Exclusion Criteria 
1. Women who are pregnant, breastfeeding or planning to become pregnant during the trial 
2. Allergy, sensitivity, or intolerance to the investigational product’s active or inactive 
ingredients 
3. Clinically significant abnormal laboratory results at screening as assessed by the QI 
4. Current or history of any significant gastrointestinal disease requiring medication (e.g. GERD, 
gastroenteritis) 
5. Irregular sleep schedule 
6. Chronic diarrhea or constipation  
7. Participants with hypertension and are on antihypertensive medication 
8. Type I or Type II diabetes 
9. Participants with hyperlipidemia and are on medication 
10. Self-reported sleep apnea 
11. Self-reported current or pre-existing thyroid condition. Treatment on a stable dose of 
medication for at least 3 months will be considered by the QI 
12. Unstable metabolic disease or chronic diseases as assessed by the QI 
13. History of or current diagnosis with kidney and/or liver diseases as assessed by the QI on a 
case-by-case basis, with the exception of history of kidney stones in participants who are 
symptom-free for 6 months 

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14. Significant cardiovascular event in the past 6 months. Participants with no significant 
cardiovascular event on stable medication may be included after assessment by the QI on a 
case by case basis 
15. Major surgery in the past 3 months or individuals who have planned surgery during the trial 
period. Participants with minor surgery will be considered on a case-by-case basis by the QI 
16. Cancer, except skin cancers completely excised with no chemotherapy or radiation with a 
follow up that is negative. Volunteers with cancer in full remission for more than five years 
after diagnosis are acceptable 
17. Individuals with an autoimmune disease or are immune-compromised  
18. Self-reported HIV-, Hepatitis B- and/or C-positive diagnosis 
19. Blood/bleeding disorders as determined by laboratory results 
20. Self-reported mental or neuropsychological condition and/or cognitive impairment that, in the 
QI’s opinion, could interfere with study participation 
21. Metal implants that may affect the DXA scan results will be assessed on a case-by-case basis 
by the QI.  
22. Current use of prescribed medications listed in Section Prescribed Medications 7.3.1  
23. Current use of over-the-counter medications, supplements, foods and/or drinks listed in 
Section 7.3.2 
24. Use of cannabinoid products within 60 days of baseline. History of cannabis used will be 
assessed on a case by case basis by the QI 
25. Use of tobacco products within 60 days of baseline 
26. Self-reported alcohol or drug abuse within the last 12 months  
27. High alcohol intake (average of > 2 standard drinks per day or > 10 per week) 
28. Current employment that calls for shift work or have worked shift work in the last 3 weeks 
29. Participation in other clinical research trials 30 days prior to screening  
30. Blood donation 30 days prior to screening, during the study, or a planned donation within 30-
days of the last study visit  
31. Individuals who are unable to give informed consent 
32. Any other condition, that, in the opinion of the QI, may adversely affect the participant's 
ability to complete the study or its measures, or which may pose a significant risk to the 
participant 
7.3 
Concomitant Medications  
Participants who are taking any prescribed medications that are considered not to affect the study outcomes 
must agree to maintain their current dosing regimen during the study unless otherwise recommended by 
their family physician.  
7.3.1 
Prescribed Medications 
Participants on the following prescribed medications and/or treatments will be excluded during enrollment 
unless they have been taken off these therapies by their family physician. In the latter event, the frequency 
of use and/or dosage may be considered by the QI on a case by case basis prior to recommending an 
appropriate washout or their enrollment in the study.  
 

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1. Beta-blockers and thiazide diuretics (within 4 weeks of baseline) 
2. Weight loss medication (within 4 weeks of baseline) 
3. Lipid-lowering medications (within 4 weeks of baseline) 
4. Anticoagulants and coagulants (within 4 weeks of baseline) 
5. Sleep medication  
6. Selective serotonin reuptake inhibitors (SSRI) 
7. Antibiotics  
8. Non-steroidal anti-inflammatory drugs (NSAIDs) 
9. Proton pump inhibitors (PPIs) 
10. Metformin (unless on a stable dose for the last 6 months) 
 
7.3.2 
Over-the-counter Medications, Supplements and Foods/Drinks 
Participants who are currently consuming the following over-the-counter (OTC) medications, supplement, 
and food/drinks will not be allowed to participate unless willing to undergo the specified washout period 
prior to their baseline visit and agree not to take the supplements during the study. Other OTCs supplement 
and food/drinks will require a case-by-case assessment by the QI based on dose and/or frequency of use to 
determine adequate washout.  
1. Probiotics, prebiotics, and synbiotics (30 days prior to baseline and throughout the study) (see 
appendix 17.1 for full lists) 
2. OTC NSAIDs (PRN use is acceptable) 
3. OTC blood pressure medication or supplements (within 4 weeks of baseline) 
4. Lipid metabolising supplements (within 4 weeks of baseline) 
a. Fish oil and omega-3 supplements 
b. Red yeast rice 
c. Plant sterols and stanols  
5. OTC medication or supplements marketed for weight loss (within 4 weeks of baseline) 
6. Vitamin E supplements (within 4 weeks of baseline) 
7. Coagulant/anticoagulant supplements (within 4 weeks of baseline) 
8. PPIs 
7.4 
Washout Periods  
Please refer to section 7.3.2 for washout periods for OTC medications, suppliments and drinks.   
7.5 
Early Withdrawal 
Personal reasons 
As stated in the Informed Consent Form, a participant may withdraw from the study for any reason at any 
time. 
Removal by Qualified Investigator 

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Participant discontinuation should be considered at the discretion of the qualified investigator.  The 
circumstances of any discontinuation must be documented in detail in the participant file and final report.  
If possible, the evaluations planned for the end of treatment will be carried out at the time when the 
participant is withdrawn from the study.   
Criteria for removal of participants from the study includes: 
Clinical reasons 
A participant may be withdrawn from the study if, in the opinion of the qualified investigator, it is not in 
the participant's best interest to continue.  Any participant who experiences a serious adverse event (SAE) 
may be withdrawn from the trial at the discretion of the qualified investigator.  A participant will also be 
withdrawn due to adverse events causing clinically significant illness or the need for prohibited 
medication(s) during the trial. Any female participant who becomes pregnant during the course of the trial 
will be withdrawn and will be followed up until giving birth.  
Protocol violation 
Any participant found to have entered this study in violation of the protocol will be discontinued from the 
study at the discretion of the qualified investigator.  This will include any participant found to have been 
inappropriately enrolled (did not meet eligibility criteria).  Participant non-compliance includes failure to 
show up for study visits, failure to take the investigational product as directed, or refusal to undergo study 
visit procedures.  Participants who are found to be taking prohibited medications or supplements without 
the knowledge of the qualified investigator will also be withdrawn. Any major protocol deviations (i.e. 
those that increase the risk to participants and/or compromise the integrity of the study or its results) will 
result in participant discontinuation. 
Particiapnt Replacement  
For all early withdrawals, a participant leaving the study prematurely or in the event of participant removal 
will NOT be replaced by another.  It is understood by all concerned that an excessive rate of withdrawals 
can render the study un-interpretable, thus unnecessary withdrawal of participants should be avoided. 
 
INVESTIGATIONAL PRODUCT 
8.1 
Manufacturing and Storage 
The investigational product will be provided to KGK by the Sponsor.  The investigational product will be 
carefully stored at the study site in a lockable, limited access area, accessible only to study team personnel 
in compliance with pertinent regulations.  Only authorized persons will have access to the investigational 
product.  The products will be stored at room temperature and will not be exposed to direct sunlight or heat. 
The investigational products will be kept in a locked investigational product storage room at KGK Science 
Inc. on receipt.  An accountability log will be kept for investigational products.  

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All unused investigational product will be returned to the study sponsor by KGK (at the sponsor’s expense) 
or destroyed on receipt of written confirmation from the sponsor at study closeout (within one month of 
last participant visit). 
Manufactured by: 
 
Morinaga Nutritional Foods, Inc.  
8.2 
Labelling and Coding 
The investigational product will be labeled according to the requirements of ICH-GCP guidelines and 
applicable local regulatory guidelines. The investigational product will be randomized and coded by an 
unblinded person at KGK who is not involved in data collection or analysis.  
8.3 
Investigational Product (Bifidobacterium breve) 
Dietary Ingredient 
Quantity (Qty) 
Bifidobacterium breve 
10 billion/capsule 
Non-medical ingredients: corn starch, milk protein hydrolysate, hypromellose, sodium alginate, calcium 
carbonate, and calcium phosphate 
 
Placebo 
Ingredients: corn starch, milk protein hydrolysate, hypromellose, sodium alginate, calcium carbonate, and 
calcium phosphate 
8.4 
Directions 
Participants will be instructed to take 2 capsules of placebo for 4 weeks during the run-in period. On day 1 
participants will be instructed to take either 2 capsules of placebo or B. breve (20 billion) in the morning 
after breakfast for 12 weeks. Clinic staff will instruct participants to save all unused and open packages and 
return them to KGK for a determination of compliance. If a dose is missed participants will be instructed 
to take their dose when they remember with their next meal. If an entire day is missed, participants are 
instructed to record it as missed and continue on with the dose as instructed the next day. Participants will 
be advised not to exceed 2 capsules daily. 
8.5 
Rescue Medication  
Rescue medication is not applicable for this study.  

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8.6 
Randomization 
A randomization schedule will be created and provided to the Investigator indicating the order of 
randomization. Each participant will be assigned a randomization code according to the order of the 
randomization list generated using www.randomization.com. Enrolled participants will be randomized to 
the different study arms at Day 0.  
8.7 
Blinding and Allocation Concealment 
Concealment of the allocation of treatment will be employed through the use of opaque sealed envelopes, 
each labeled with a randomization number. Each envelope will contain information regarding the treatment 
associated with each randomization number. These envelopes will be readily available for the investigator 
to open in the event that it becomes necessary to know which product a participant is taking for the sake of 
the participant's health care.  
Unblinding should not occur except in the case of emergency situations. In the event that a serious adverse 
event occurs, for which the identity of the investigational product administered is necessary to manage the 
participant’s condition, the treatment received by the participant will be unblinded and the investigational 
product identified. The sponsor must be notified of any unblinding within 24 hours. Details of participants 
who are unblinded during the study will be included in the Final Report.  
 
 

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STUDY ASSESSMENTS 
9.1 
Screening (week -6 to -5; Visit 1) * 
* At the discretion of the Qualified Investigator, any participants falling outside of the screening window 
(week -6 to -5) due to scheduling issues will be asked to repeat eligibility/screening procedures prior to 
randomization at baseline. 
 
Participants will arrive at the clinic for screening assessments in a 12-hour or greater fasted state. 
An informed consent form will be given to the potential volunteer. They will be required to read the 
information and will be given the opportunity to seek more information if needed, or the option of taking 
the consent form home to review prior to making their decision. If agreeable, the volunteer will sign the 
consent form and receive a duplicate of the signed copy. Once consent has been obtained, the screening 
visit will proceed. Each volunteer will be sequentially assigned a screening number to be entered in the 
screening and enrollment log.   
Screening assessments include: 
1. Review medical history, concomitant therapies, and daily exercise habits  
2. Assess inclusion and exclusion criteria  
3. Urine collection 
a. Urine pregnancy test for female potential participants that are of child-bearing potential 
b. Urinalysis (microscopy, chemical, urobilinogen/urine bilirubin) 
4. Seated resting blood pressure, heart rate, respiratory rate, and temperature measurements 
5. Weight, height (BMI calculation), waist circumference and hip circumference measurements  
6. Body fat composition as determined by BIA  
7. Collect fasted blood samples for analysis of CBC, Na, K, Cl, Fe, Mg, Ca, phosphate, HbA1c, 
fasting glucose, eGFR, creatinine, BUN, AST, ALT, GGT, ALP, TSH, total bilirubin, lipid 
profile, and fasting insulin  
8. Dispense Three-Day Food Record (3DFR) 
9. Dispense Daily Bowel Habits Diary  
10. Dispense Exercise Diary  
11. Dispense Subject Diary 
12. Dispense stool collection kits 
The next visit will be scheduled for potentially eligible participants for baseline (Week 0). 
Reminders for participants prior to their next in-clinic visit: 
1. Return to the clinic in a fasted state (12 hours or greater) 
2. Abide by instructions for prescribed medications as per Section 7.3.1 and OTC medications, 
supplements, and food/drinks as per Section 7.3.2 
3. Limit alcohol intake (<2 standard drinks per day or <10 per week) 
4. Return stool collections 
5. Complete and return Daily Bowel Habits Diary  
6. Complete and return 3DFR Diary  
7. Complete and return Exercise Diary 
8. Complete and return Subject Diary 

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9.2 
Run-in period – 4 weeks (Week -4 to Week -1)  
Eligible participants will be contacted to undergo a 4 week (28 days) run-in period, during which they will 
be consuming the placebo and following an exercise intervention. Participants will come to the clinic to 
pick up the placebo. Compliance calls will be completed throughout the run-in period at fixed intervals.  
Instructions during the run-in period: 
1. Begin consuming placebo according to dosing instructions provided 
2. Begin exercise regimen according to instructions provided (20 min aerobic exercise/day; 3 
days/week) and record exercise in an exercise diary 
3. Complete Daily Bowel Habits Diary, 3DFR and subject diary  
4. Record adverse events 
9.3 
Compliance Call  
Participants will receive bi-weekly calls throughout the run-in and study period to confirm compliance on 
exercise intervention, concomitant therapies, and IP consumption. Participants will also be reminded to 
bring in their stool samples at baseline visit.  
9.4 
Baseline (Visit 2, Week 0) 
Eligible participants (compliant with the exercise intervention and placebo consumption during the run-in 
period) will return to the clinic fasted for 12 hours with completed subject diaries, food records, daily bowel 
habit diaries, exercise diaries, and stool collections, for baseline assessments. Compliance calls will be 
made at fixed intervals (bi-weekly). Participants who have not provided a stool sample within 3 days of 
visit 2 and participants who have significant weight loss (> 0.7 kg/week) during the run-in period will be 
withdrawn from the study. 
Baseline (Day 0) assessments include: 
1. Review concomitant therapies and adverse events 
2. Assess inclusion and exclusion criteria  
3. Collect remaining placebo and calculate compliance 
4. Physical examination  
5. Collect and store stool samples  
6. Collect and review Daily Bowel Habits Diary  
7. Collect and review 3DFR 
8. Collect and review Exercise Diary 
9. Collect and review Subject Diary 
10. Urine collection 
a. Urine pregnancy test for female participants that are of child-bearing potential 
b. Urinalysis (microscopy, chemical, urobilinogen/urine bilirubin) 
11. Seated resting blood pressure, heart rate, respiratory rate, and temperature measurements 
12. Weight (BMI calculation), waist and hip circumference measurements (waist/hip 
circumference ratio) 

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13. Randomization of eligible participants  
14. Body fat composition as determined by BIA 
15. DXA scan 
16. Collect fasted blood samples for analysis of CBC, Na, K, Cl, Fe, Mg, Ca, phosphate, HbA1c, 
fasting glucose, eGFR, creatinine, BUN, AST, ALT, GGT, ALP, total bilirubin, lipid profile, 
and fasting insulin 
17. Dispense investigational product and instruct participants on use 
18. Dispense Daily Bowel Habits Diary  
19. Dispense 3DFR 
20. Dispense Exercise Diary 
21. Dispense Subject Diary 
The next visit will be scheduled for week 6. 
Reminders for participants prior to their next in-clinic visit: 
1. Return to the clinic in a fasted state (12 hours or greater) 
2. Maintain exercise intervention (20 min aerobic exercise x 3 days/week) 
3. Abide by instructions for prescribed medications as per Section 7.3.1 and OTC medications, 
supplements, and food/drinks as per Section 7.3.2 
4. Limit alcohol intake (<2 standard drinks per day or <10 per week) 
5. Complete and return Daily Bowel Habits Diary  
6. Complete and return 3DFR Diary  
7. Complete and return Exercise Diary 
8. Complete and return Subject Diary 
9. Return unused IP 
9.5 
Visit 3 (Week 6, 42 ± 3 days) 
Participants will return to the clinic fasted for 12 hours, with the unused product, in addition to completed 
subject diaries, food records, daily bowel habits diaries andexercise diaries, for visit 3. Compliance calls 
will be made at fixed intervals. 
Visit 3 assessments include: 
1. Review concomitant therapies and adverse events 
2. Collect unused investigational product; calculate compliance 
3. Collect and review Subject Diary 
4. Collect and review 3DFR 
5. Collect and review Daily Bowel Habits Diary 
6. Collect and review Exercise Diary  
7. Seated resting blood pressure, heart rate, respiratory rate, and temperature measurements 
8. Weight (BMI calculation), waist and hip circumference measurements (waist/hip 
circumference ratio) 
9. Dispense investigational product and instruct participants on use 
10. Dispense Subject Diary 
11. Dispense 3DFR 
12. Dispense Daily Bowel Habits Diary 

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13. Dispense Exercise Diary  
14. Dispense stool collection kits 
The next visit will be scheduled for week 12 (EOS). 
Reminders for participants prior to their next in-clinic visit: 
1. Return to the clinic in a fasted state (12 hours or greater) 
2. Maintain exercise intervention (20 min aerobic exercise x 3 days/week) 
3. Abide by instructions for prescribed medications as per Section 7.3.1 and OTC medications, 
supplements, and food/drinks as per Section 7.3.2 
4. Limit alcohol intake (<2 standard drinks per day or <10 per week) 
5. Complete and return stool collections 
6. Complete and return Daily Bowel Habits Diary  
7. Complete and return 3DFR   
8. Complete and return Exercise Diary 
9. Complete and return Subject Diary 
10. Return unused IP 
9.6 
Visit 4 - End of Study (Week 12, 84 ± 3 days) 
Participants will return to the clinic fasted for 12 hours or greater with the unused product, in addition to 
completed subject diaries, food records, daily bowel habits diaries, exercise diaries, and stool collections, 
for assessments. Compliance calls will be made at fixed intervals.   
Visit 4 assessments include: 
1. Review concomitant therapies and adverse events  
2. Return the unused investigational product in the original packaging and remnants; calculate 
compliance 
3. Collect and store stool samples  
4. Collect and review Subject Diary 
5. Collect and review 3DFR 
6. Collect and review Daily Bowel Habits Diary 
7. Collect and review Exercise Diary  
8. Urine collection 
a. 
Urinalysis (microscopy, chemical, urobilinogen/urine bilirubin) 
9. Seated resting blood pressure, heart rate, respiratory rate, and temperature measurements  
10. Weight (BMI calculation), waist and hip circumference measurements (waist/hip 
circumference ratio) 
11. Collect fasted blood samples for analysis of CBC, Na, K, Cl, Fe, Mg, Ca, phosphate, HbA1c, 
fasting glucose, eGFR, creatinine, BUN, AST, ALT, GGT, ALP, total bilirubin, lipid profile, 
and fasting insulin 
12. DXA scan 

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9.7 
Follow-up (Week 14) 
Participants will receive a follow-up call at week 14 for adverse event reporting.  
 CLINICAL ASSESSMENTS AND PROCEDURES 
Calculations or measurements of specific parameters are required, as indicated in the schedule of 
assessments in Section 1.3. Instructions for determining these parameters are provided in the following 
sections. 
10.1 
Height and Weight 
Weight measurements will be performed with shoes removed and bladder empty on calibrated scales at all 
visits.   
At least two separate measurements will be taken at each visit.  If the two measurements are more than 0.5 
kg (1.1 lbs) apart, a third measurement will be taken. The two closest values will be selected and entered 
into the database.   
Measurement of height will be performed with the participant’s shoes removed.  The participant’s knees 
will be straightened, and head held upright. 
 
10.2 
Circumference Measurements 
Waist circumference 
The waist circumference of the participant is to be measured when standing at the part of the trunk located 
midway between the lower costal margin (bottom of lower rib) and the iliac crest (top of pelvic bone). The 
measurer is to stand beside the participant and fit the tape tightly against the skin without compressing 
underlying soft tissues. The circumference should be measured at the end of a normal expiration.  At least 
two separate measurements are to be taken at each visit. If the two measurements differ by more than 10%, 
a third measurement is to be taken. The two closest values will then be entered into the database. All 
measurements will be recorded to the nearest 0.1 cm. 
The height of the waist should be recorded at baseline, and then used to determine the waist throughout the 
study in order to ensure a consistent measurement at follow-up visits.  
Hip circumference 
While the participant is standing upright, a measuring tape will be placed around the greater trochanteric 
prominence (the widest part of the hips). The tape will be held firmly, but not pressing the tape into the 
skin. When the tape is parallel to the floor, the reading will be recorded. At least two separate measurements 

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investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
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are to be taken at each visit. If the two measurements differ by more than 10% a third measurement will be 
taken.  The two closest values are to be entered into the database.   
To ensure consistent measurement at follow-up visits, the height of the identified hips will be recorded at 
the first visit and then used at subsequent visits to determine the hip circumference throughout the study. 
Waist/hip ratio 
The waist-to-hip ratio will be assessed by comparing the waist circumference to hip circumference at each 
study visit.  
10.3 
Blood Pressure and Heart Rate 
In-office, seated, resting blood pressure assessment:  
The participant should be seated comfortably with their back supported and the upper arm bared 
without restrictive clothing. Feet should be flat on the floor and legs will not be crossed. The 
participant will rest in this position for at least 5 minutes prior to the first reading.  
At screening: 
Seated blood pressure will be checked in both arms and if different the arm with the higher systolic 
blood pressure reading will be taken for measurements. If the systolic blood pressure is the same 
in both arms, the arm with the higher diastolic blood pressure will be used. If both are equal, then 
the left arm will be used. In the chosen arm, a second measurement will be taken at least 1 minute 
from the first measurement. If a difference of more than 8 mmHg exists between the two readings 
a third reading will be taken. An average of the two lowest readings from the chosen arm will be 
taken for the determination of inclusion into the study. As per the QI’s opinion, a high office blood 
pressure should be rechecked manually after the participant is given a glass of water and is seated 
for 15 min. Also, participant should be queried about their usual blood pressure.  
The arm chosen for use at the initial visit will be documented in the study file and used in all 
subsequent visits.  
At study visits:  
Once enrolled in the study, BP will be measured in the chosen arm. Three readings will be made, 
averaged and recorded in the chart. 
Heart Rate (beats/min) will be measured using the reading on the automated blood pressure monitor, or 
manually by the clinical coordinator placing their index finger on the participant's radial artery while 
observing a timer and counting the number of beats over 30 seconds and then multiplying the number by 
two. This is repeated for a total of three measurements. 

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10.4 
Temperature and Respiratory Rate  
Oral temperature will be measured after ensuring that the participant did not consume any hot or cold 
beverages for a minimum of 20 minutes. The temperature will be taken by placing the thermometer with 
probe cover under the participant's tongue within the sublingual pocket. The participant will be instructed 
to lower his/her tongue and close their lips during the measurement. The participant will be instructed to 
not talk during the measurement until the thermometer indicates that the final temperature has been 
recorded. Once the recording is complete the thermometer will be removed from the participant's mouth 
and probe cover discarded.  
Respiratory rate will be measured and recorded by counting the number of breaths a participant takes in 
one minute with the participant at rest.  
10.5 
Blood Sample Collection 
An appropriately trained and qualified phlebotomist will perform the venipuncture procedure to collect the 
necessary blood samples. Participants will be placed in a comfortable seated position with their desired arm, 
at the phlebotomist’s discretion, fully extended and supported with a pillow. A tourniquet will be applied 
3-4 inches above the elbow with the participants opening and closing their fist a few times to allow the 
phlebotomist to manually determine the approximate size, depth, and location of the vein. Following the 
site of the venipuncture being appropriately sterilized, the phlebotomist will collect the sample using the 
vacutainer system according to the relevant laboratory order requirement. Once the collection is complete 
a cotton ball will be immediately placed on the venipuncture site which will be periodically checked to 
ensure clotting has begun at which point a clean cotton ball will be applied and secured with surgical tape. 
All collected sample tubes will be labeled with subject identification codes, visit number, project code, 
DOB (Date of Birth), gender, date and time of draw.   
10.6 
Stool Sample Collection 
Participants will be instructed on stool sample collection at screening. They will be provided with a stool 
sample collection kit and instructions at screening and at visit 3. All collected samples tubes will be labeled 
with subject identification codes, visit number, project code, DOB (Date of Birth), gender, date and time of 
collection.   
10.7 
Bioelectrical Impedance Analysis (BIA) 
The Bioelectrical Impedance Analysis will be conducted at KGK clinic (London, ON) at the screening and 
baseline visits. The BIA assessment will be carried out and fully explained by trained clinic coordinators 
upon participants arrival at the clinic. The total body water will be determined from the resistance of the 
alternate electric current through the body’s water pool. The value obtained will be used to estimate the 
body fat mass percentage.  

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investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
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10.8 
Dual-Energy X-Ray Absorptiometry (DXA) Scan 
The Dual-Energy X-Ray Absorptiometry Scan will be conducted at KGK clinic (London, ON) at visits 2 
(week 0), and 4 (week 12) by trained technicians. Body tissue density will be measured by a form of X-ray 
radiation and converted into body fat and muscle mass percentage for assessment. Prior to the day of the 
DXA assessment, the coordinators will remind the participants to limit the amount of water and food 
consumption accordingly. A full body composition DXA scan will expose the participant to a total dose of 
<10uSV, depending on the size of the participant. This amount is less than a person is exposed to on an east 
to west coast flight within the USA and similar to the normal background radiation received over the course 
of a single day level (23, 24). 
 
10.9 
Daily Bowel Habit Diary and Bristol Stool Scale 
The participants will be instructed to fill and complete the Daily Bowel Habit Diary for assessment at visits 
2 (week 0), 3 (week 6), and 4 (week 12). The diary is a 7-item questionnaire that evaluates each bowel 
movement experienced. The Bristol Stool Scale is a 7-item image scale that measures the shape and 
consistency of stool. 
10.10 3-Day Food Record (3DFR) 
The participants will be instructed to complete an online 3-day food record called Libro, by Nutritics, for 
assessment on visits 2 (week 0), 3 (week 6), and 4 (week 12). Clinic coordinators will create an online 
access for each participant void of all personal information to protect any confidential participant 
information. The recorded food and drink consumption will be used for daily calories, macronutrients, 
micronutrients, and compliance calculation. Participants will be counselled accordingly after their food 
record has been review by a trained clinic staff. In the event the access to Libro is disrupted, participants 
will be instructed to record their food consumption on a paper food record, and once access is restored data 
will be incorporated into the database. 
10.11 Exercise Diary 
The participants will be instructed to complete an online weekly exercise diary. Clinic coordinators will 
create an online access for each participant void of all personal information to protect any confidential 
participant information. The recorded exercise will be used for compliance calculation. Participants will be 
counselled accordingly after exercise record has been reviewed by a trained clinic staff. In the event that 
online access is disrupted, participants will be instructed to record their exercise in a paper diary, and once 
access is restored data will be incorporated into the database. 
10.12 Compliance  
Compliance to supplement 

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Compliance will be assessed by counting the returned unused study product at each visit. Compliance is 
calculated by determining the number of dosage units taken divided by the number of dosage units expected 
to have been taken multiplied by 100. 
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑜𝑠𝑎𝑔𝑒 𝑢𝑛𝑖𝑡𝑠 𝑡𝑎𝑘𝑒𝑛
𝑛𝑢𝑚𝑏e𝑟 𝑜𝑓 𝑑𝑜𝑠𝑎𝑔𝑒 𝑢𝑛𝑖𝑡𝑠 𝑒𝑥𝑝𝑒𝑐𝑡𝑒𝑑 𝑡𝑜 ℎ𝑎𝑣𝑒 𝑏𝑒𝑒𝑛 𝑡𝑎𝑘𝑒𝑛 × 100% 
In the event of a discrepancy between the information in the treatment diary and the amount of study product 
returned, compliance will be based on the product returned unless an explanation for loss of product has 
been provided. Participants found to have compliance of <80% or >120% will be counseled.  
Compliance to exercise  
Aerobic exercise 20 minutes per day, 3 days a week 
Weekly maximum score = 3 (compliant 3 days/week) 
Compliance is calculated weekly by dividing the actual weekly score by the maximum weekly score 
multiplied by 100. 
𝑎𝑐𝑡𝑢𝑎𝑙 𝑠𝑐𝑜𝑟𝑒
3
 × 100% 
Participants found to have compliance of <80% will be counseled. Compliance of <70% will be considered 
as non-compliant and any participant demonstrating non-compliance for two consecutive weeks will be 
withdrawn from the study. 
10.13 Laboratory Analyses 
12 hours fasting blood samples will be drawn from the participants at screening (Visit 1), baseline visit 
(Visit 2), and at the End of Study visit (Visit 4) as indicated in the schedule of assessments in Section 1.3.   
Protection of subject confidentiality will extend to all data generated from the assaying of these samples.  
These samples will be alphanumerically coded and the persons performing the analysis will not be aware 
of the subject’s identity or the product they received. 
At screening (Visit 1), 18 mL of 12 hour fasting whole blood will be collected in: 
1. Two 4 mL EDTA vacutainer tubes for: 
a. CBC analysis (One tube) 
b. HbA1c analysis (one tube) 
 
2. Two 5 mL SST vacutainer tubes to generate serum for: 
a. Na, K, Cl, Fe, Ca, Mg, phosphate, fasting glucose, TSH, Lipid Panel, creatinine, eGFR, 
BUN, GGT, ALP, AST, ALT, and total bilirubin analysis (one tube) 
b. Fasting Insulin (One tube) 

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3. Urine will be collected for urine pregnancy test of women of childbearing potential and 
urinalysis 
 
At baseline (Visit 2), 18 mL of 12 hours fasting blood will be collected in: 
1. Two 4 mL EDTA vacutainer tubes for: 
a. CBC analysis (one tube) 
b. Hb1-Ac analysis (one tube) 
 
2. Two 5 mL SST vacutainer tubes to generate serum for: 
a. Na, K, Cl, Fe, Ca, Mg, phosphate, fasting glucose, Lipid Panel, creatinine, eGFR, BUN, 
GGT, ALP, AST, ALT, and total bilirubin analysis (one tube) 
b. Fasting Insulin (One tube) 
3. Urine will be collected for urine pregnancy test of women of childbearing potential and 
urinalysis. 
 
4. Fecal Sample also will be collected for analysis 
 
At the end of the study (Visit 4), 18 ml of 12 hours fasting blood will be collected in: 
1. Two 4 mL EDTA vacutainer tubes for: 
a. CBC analysis (one tube) 
b. Hb1-Ac analysis (one tube) 
 
2. Two 5 mL SST vacutainer tubes to generate serum for: 
a. Na, K, Cl, Fe, Ca, Mg, phosphate, fasting glucose, Lipid Panel, creatinine, eGFR, BUN, 
GGT, ALP, AST, ALT, and total bilirubin analysis (one tube) 
b. Fasting Insulin (One tube) 
3. Urine will be collected for urinalysis 
 
4. Fecal Sample also will be collected for analysis 
The total blood volume collection for the laboratory assessments listed above will be approximately 54 mL, 
over the period from screening to end of study. At any study visit, blood loss per volunteer is not expected 
to exceed 18 ml.  Additional blood samples may be collected during the study in order to perform or repeat 
laboratory tests outlined in the Schedule of Assessments if needed.  
Dynacare a central laboratory will be used in this study to measure blood parameters and urinalysis. Another 
lab will be used to process fecal samples. 
Urine pregnancy test will be performed at the KGK Science clinic site.  

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10.14 Termination of the Trial 
In the case of premature termination of the trial, participating investigators/participants, and the Institutional 
Review Board must be promptly informed of the termination. In the event of early termination, as many 
assessments will be completed as agreed upon by participant 
10.15 Protocol Amendments 
If amendments to the study protocol are required after approval such changes will be captured in writing 
the reasons for the change documented and signed and dated by the sponsor.  Any such amendments may 
be subject to IRB and Health Canada review/approval prior to implementation. Exception: if it becomes 
necessary to alter the protocol to eliminate an immediate hazard to participants, an amendment may be 
implemented prior to IRB approval. In this circumstance, the Investigator must notify IRB and Health 
Canada in writing within five (5) working days of the implementation. 
 SAFETY INSTRUCTIONS AND GUIDANCE  
11.1 
Adverse Events and Laboratory Abnormalities 
11.1.1 Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical investigation participant who has 
been administered an investigational product and does not necessarily have a causal relationship with this 
treatment.  An AE can be any unfavorable and unintended sign (including an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of a product, whether or not it is considered related 
to that product.  Pre-existing conditions that worsen during a study are to be reported as AEs. 
During the study, participants should record any adverse effects in their diary.  At each visit the participant 
will be asked, "Have you experienced any difficulties or problems since I saw you last?".  Any adverse 
events (AEs) will be documented in the study record and will be classified according to the description, 
duration, intensity, frequency, and outcome.  The qualified investigator will assess any AEs and decide 
causality. 
Intensity of AEs will be graded on a three-point scale (mild, moderate, severe) and reported in detail in the 
study record. 
Mild:  
Awareness of event but easily tolerated 
Moderate:          Discomfort enough to cause some interference with usual activity 
Severe:  
Inability to carry out usual activity 
The causality relationship of investigational product to the adverse event will be assessed by the qualified 
investigator as either: 

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Most probable: There is a reasonable relationship between the investigational product and AEs. 
The event responds to withdrawal of investigational product (dechallenge) and 
recurs with rechallenge when clinically feasible. 
Probable: 
There is a reasonable relationship between the investigational product and AEs.  
The event responds to dechallenge when clinically feasible. 
Possible: 
There is a reasonable relationship between the investigational product and AEs.  
Dechallenge information is lacking or unclear. 
Unlikely: 
There is a temporal relationship to the investigational product administration but 
there is no reasonable causal relationship between the investigational product and 
the AEs. 
Not related: 
There is no temporal relationship to investigational product administration or there 
is a reasonable causal relationship between non-investigational product, 
concurrent disease or circumstance and the AEs.  
11.1.2 Serious Adverse Event 
A serious adverse event (SAE) is any experience that suggests a significant hazard, contraindication, side 
effect or precaution.  It is any AE that results in any of the following outcomes: 
1. Death 
2. A life-threatening adverse event 
3. Inpatient hospitalization or prolongation of existing hospitalization 
4. A persistent or significant disability or incapacity 
5. A congenital anomaly/birth defect in the offspring of a participant who received the study 
treatment 
Important medical events that may not be immediately life-threatening or result in death or hospitalization 
but may jeopardize the participant or may require intervention to prevent one of the outcomes listed above.  
Examples of such events are intensive treatment in an emergency room or at home for allergic 
bronchospasm; blood dyscrasias or convulsions that do not result in hospitalization; or the development of 
drug dependency or drug abuse. 
11.1.3 Unexpected Adverse Reaction 
An unexpected adverse reaction is an adverse reaction, the nature and severity of which is not consistent 
with the applicable product information (e.g., Investigator’s Brochure for an unapproved investigational 
product or package insert/summary of product characteristics for an approved product). 
11.1.4 Laboratory Test Abnormalities 
The investigator must assess the clinical significance of all abnormal laboratory values as defined by the 
compendium of normal values for the reference laboratory.  

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Any treatment emergent abnormal laboratory result which is clinically significant, i.e., meeting one or more 
of the following conditions, should be recorded as a single diagnosis on the AEs form in the study record: 
1. Accompanied by clinical symptoms 
2. Leading to interruption or discontinuation of the investigational product 
3. Requiring a change in concomitant therapy 
This applies to any protocol and non-protocol specified laboratory result from tests performed after the first 
dose of the investigational product, which falls outside the laboratory reference range and meets the clinical 
significance criteria for liver and kidney tests as well as for hematology and clinical chemistry, etc. (i.e. 
AST and/or ALT > 2 x ULN). 
This does not apply to any abnormal laboratory result which falls outside the laboratory reference range but 
which does not meet the clinical significance criteria or those which are a result of an AE which has already 
been reported. 
Any laboratory result abnormality fulfilling the criteria for a serious adverse event (SAE) should be reported 
as such, in addition to being reported as an AE in the study record. 
11.2 
Treatment and Follow-Up Of AEs And Laboratory Abnormalities 
11.2.1 Treatment and Follow-up of AEs 
AEs, especially those for which the relationship to the investigational product is suspected, should be 
followed up until they have returned to baseline status or stabilized. 
If after follow-up, return to baseline status or stabilization cannot be established, an explanation should be 
recorded in the study record. 
11.2.2 Treatment and Follow-up of Laboratory Abnormalities 
In the event of participant-initiated withdrawal or clinically significant unexplained abnormal laboratory 
test values, the participant will be withdrawn from the treatment and will remain in the study and be required 
to attend all remaining study visits as part of a safety arm.  
11.3 
Reporting of SAEs And Unexpected Adverse Reactions 
The qualified investigator will be responsible for classification of an AE as an SAE within 24 hours of 
notification. Causality should be signed off by the qualified investigator prior to reporting to ethics and 
regulatory bodies.  Notification of any serious adverse events must be made in writing to the study sponsor. 
The IRB will be notified of all product related SAEs and unexpected adverse reactions.  All blinded SAE's 
or unblinded-participant-on-active product SAE's will be reported to the Therapeutics Products Directorate 
(TPD) in an expedited manner. 

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KGK Science must notify the TPD of all blinded or unblinded-participant-on-active product serious adverse 
events and reactions as follows: 
If it is neither fatal or life threatening, within 15 calendar days after the day on which the sponsor 
becomes aware of the information; and 
If it is fatal or life threatening, must be reported as soon as possible, but not later than seven (7) 
days after the day on which the sponsor becomes aware of the information. 
 
 

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 STATISTICAL EVALUATION 
12.1 
Determination of Sample Size 
With a total sample size of 100 (50 per group; 40 completers), it is possible to detect a difference in mean 
change in percent body fat or body fat mass of 0.76% or 0.79 kg respectively, in a parallel design at a two-
sided unadjusted 5% significance level, 80% power and 20% attrition rate. This calculation is based on 
standard deviations of 1.19% and 1.24 kg for percent body fat and body fat mass respectively, estimated 
from a study by Minami et al (2018).  
12.2 
Analysis Plan 
The Safety Population will consist of all participants who received any amount of either product, 
and on whom any post-randomization safety information is available. 
The Intent-to-Treat (ITT) Population consists of all participants who received either product 
and on whom any post-randomization efficacy information is available. 
The Per Protocol (PP) Population consists of all participants who consumed at least 80% of 
treatment or placebo doses, do not have any major protocol violations and complete all study 
visits and procedures connected with measurement of the primary variable. 
12.3 
Statistical Analysis Plan 
For each continuous endpoint including percent body fat, body fat mass, visceral fat area, weight, BMI, 
total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, waist circumference, and hip 
circumference, fasting blood glucose, HbA1c, fasting insulin levels, AST, ALT, GGT, ALP, a summary 
table will be prepared with a variety of summary statistics including mean, standard deviation, median, 
min-max range and number of participants analyzed for each time point-treatment pairing. These data will 
be classified in subgroups based on the similarity of microbiota composition. 
To account for any a priori differences between groups, the similar summary statistics of the changes 
from baseline will also be provided. Mean values will be displayed as graphs, with a separate line for each 
product, and error bars indicating ± SEM. Mean changes from baseline will also be graphed similarly. 
Changes in continuous endpoints from screening/baseline will be calculated as:  
Change to Vi = Value at Vi – Value at V screening/baseline 
Continuous variables will be tested for normality and log-normality using histograms, box plots and Q-Q 
plots. Log-normally distributed variables will be analyzed in the logarithmic domain. Summary statistics 
will be provided as non-transformed values. Intractably non-normal variables will be analyzed by 
appropriate non-parametric tests. 

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The primary endpoint - change in fat loss from baseline (% or g), as assessed by Dual-Energy X-Ray 
Absorptiometry (DXA), between B. breve and placebo after 12 weeks of supplementation - will be assessed 
for between-group differences using ANCOVA, with the baseline measurements as covariate. 
Continuous secondary endpoints will be tested for between-group differences using repeated measures 
linear mixed-effects ANCOVA. This will allow for the random effects associated with each subject to be 
accounted for. The dependent variable will be the change at each visit, the fixed effects will be the study 
group * visit number, and the covariate will be the dependant variable at baseline (Day 0).  
Baseline values for continuous variables will be tested for between-group differences using the independent 
Student’s t-test. Within-group differences will be assessed using the Student’s paired t-test or, in the case 
of intractable non-normality, the Wilcoxon sign rank test. The frequency of BMs will be calculated as the 
average number of BMs per day over the 7 days prior to baseline and 7 days prior to week 6 and week 12. 
For categorical variables, counts and percentages will be presented. The denominator for each percentage 
will be the number of subjects within the study group for that visit/week unless otherwise specified. Possible 
differences between groups will be assessed by using the two-tailed Chi-squared or Fisher’s exact test, as 
appropriate. 
 
Microbiota analysis will be performed at Morinaga Milk Co., Ltd. The subgroups will be determined based 
on the similarity of microbiota composition by hierarchical clustering. These subgroups will be defined 
before analyzing week 12 data. 
All missing efficacy/effectiveness values analysis will be imputed using the most recent previously-
available value (LOCF, or “last-observation-carried-forward” imputation) or multiple imputation. No 
imputation will be performed for missing values of safety variables. 
All statistical analysis will be completed using R Statistical Software version 3.5.3 (R Core Team, 2019) 
for Microsoft Windows or SAS software, Copyright © 2019 SAS Institute Inc. SAS and all other SAS 
Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., 
Cary, NC, USA. 
12.3.1 Premature Discontinuation Description 
For each premature discontinuation, the following parameters will be listed: participant number, dates of 
start and end of treatment, and the reason of premature discontinuation. 
12.3.2 Safety 
For adverse events, a descriptive analysis will be given.  Adverse events will be presented in a frequency 
table by category and treatment.  Furthermore, description, frequency, severity, and causality will be 
reported for each adverse event. 
Continuous safety parameters (e.g. hematology, clinical chemistry, heart rate and blood pressure, urine 
specific gravity) will be summarized using a table including mean, standard deviation, median, minimum 
value, and maximum value for each measurement point. These will be compared to normal clinical ranges 
and any abnormalities will be discussed. 

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Categorical safety parameters (e.g. incidence of adverse events and urinalysis (microscopy, glucose, 
ketones, blood, protein, colour)) will be summarized as counts and percentages. The denominator for each 
percentage will be the number of subjects within the study group for that visit/week unless otherwise 
specified. Possible differences between groups will be assessed by using the two-tailed Chi-squared or 
Fisher’s exact test, as appropriate. 
12.4 
Protocol Deviation Description 
Protocol deviations will be listed in the final study report. 
12.5 
Protocol Amendments 
Once the protocol has been approved by the IRB and Health Canada, any changes to the protocol must be 
documented in the form of an amendment. All amendments will be documented in the final study report. 
 
 DATA COLLECTION AND STORAGE 
All data collection and record storage will be done in compliance with ICH GCP Guidelines and applicable 
local regulatory guidelines. 
 
 ETHICAL ASPECTS OF THE STUDY 
This study will be conducted with the highest respect for the individual participants (i.e., participants) 
according to the protocol, the ethical principles that have their origin in the Declaration of Helsinki, and the 
ICH Harmonised Tripartite Guideline for GCP.  
14.1 
IRB Approval 
KGK Science Inc. will supply relevant documents for submission to an IRB for the protocol’s review and 
approval. The following must be submitted to the IRB: this protocol, a copy of the informed consent form, 
and, if applicable, volunteer recruitment materials and/or advertisements and other documents required by 
all applicable laws and regulations. The IRB’s written approval of the protocol and volunteer informed 
consent must be obtained before commencement of the study.  The IRB approval must refer to the study by 
exact protocol title, number, and version date; identify versions of other documents (e.g., informed consent 
form) reviewed; and state the approval date. 

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KGK must adhere to all requirements stipulated by the IRB. This may include notification to the IRB 
regarding protocol amendments, updates to the informed consent form, recruitment materials intended for 
viewing by volunteers, local safety reporting requirements and submission of the investigator’s annual/final 
status report to the IRB. 
14.2 
Volunteer Information and Informed Consent 
Written consent documents will embody the elements of informed consent as described in the declaration 
of Helsinki and the ICH Guidelines for GCP and will be in accordance with all applicable laws and 
regulations. The informed consent form describes the planned and permitted uses, transfers, and disclosures 
of the volunteer’s personal and personal health information for purposes of conducting the study. The 
informed consent form further explains the nature of the study, its objectives, and potential risks and 
benefits, as well as the date informed consent is obtained. The informed consent form will detail the 
requirements of the volunteer and the fact that he or she is free to withdraw at any time without giving a 
reason and without prejudice to his or her further medical care.  
14.3 
Potential Risks and Procedures to Minimize Risk 
All potential risks are disclosed to study participants prior to their participation.  The potential risks 
associated with this study include venipuncture and the associated risks.  Risks associated with venipuncture 
include pain, bruising, and infection at the site.  Alcohol swabs and proper venipuncture procedures will be 
followed to minimize the risk of infection. 
 
 QUALITY ASSURANCE AND QUALITY CONTROL 
15.1 
Auditing 
All materials used in clinical studies are subjected to quality control. Quality assurance audits may be 
performed by the sponsor or any health authority during the course of the study or after its completion.  
The Investigator agrees to comply with the sponsor and regulatory requirements in terms of auditing of the 
study. This includes access to the source documents for source data verification. 
15.2 
Monitoring 
An initiation meeting will be conducted by the sponsor or an approved representative (CRO).  At this 
meeting, the protocol and logistical aspects of the study will be reviewed with the Investigator and all study 
staff. 

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Source documents will be reviewed to ensure that all items have been completed and that the data provided 
are accurate and obtained in the manner specified in the protocol.  The participant files will be reviewed to 
confirm that: 
1. Informed consent was obtained and documented 
2. Enrolled participants fulfilled all inclusion criteria and did not meet any exclusion 
criteria; 
3. AE/SAE reporting has been performed as applicable 
4. Study visits have been conducted as per protocol and information has been recorded in 
the appropriate place in the source document 
5. The study product is being stored correctly and an accurate record of its dispensation to 
the study participants is being maintained (accountability) 
Incorrect, inappropriate, or illegible entries in the participant files will be returned to the Investigator or 
designee for correction.  No data disclosing the identity of participants will leave the study center. The 
Investigator and any designees will maintain confidentiality of all participant records. 
The Investigator will permit trial-related monitoring, audits, IRB/IEC review, and regulatory inspections 
and will allow direct access to source data and documents for these purposes. 
15.3 
Data Management 
Data required for the analysis will be acquired from source documentation (including laboratory reports) 
and entered into Open Clinica Enterprise Version study instance designed specifically for this study. The 
two instances for the database would be created, test instance and production instance. A UAT (User 
Acceptance Testing) of the database would be performed in the test instance and then moved to the 
production instance. A password protected user id’s will be created which would give access to the limited 
authorized personnel. Only properly trained Data management staff will be granted access to perform 
database designing, according to SOP - Designing Database in Open Clinica Enterprise Version and SOP - 
Creating user in Open Clinica Enterprise Version. A study-specific Data Management Plan will be 
generated after the finalization of the database.   
The standard data validation and edit checks would be performed on the production instance of the study 
by designing study specific rules and restrictions as defined in the eCRF. The discrepancies will be queried 
and managed according to the SOP - Discrepancy Management SOP in Open Clinica Enterprise Version. 
Data tables will be created, queried and exported during and at the end of study using PostgreSQL tool 
(pgadminIII 9.5) and MS Access.  
For Statistical analysis, the validated soft-lock copy of the blinded study database will be sent to the 
Statistician to perform the analysis. The study database would be a read-only file to ascertain changes in 
the data are not made during or after the analysis.  
High safety standards for the transfer and storage of study data are guaranteed by the use of technologies 
such as password protection, firewalls and periodic backup to protect stored data.  

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All study data is archived for a period not less than 25 years from the date of completion of the study in 
accordance with Health Canada regulatory requirements. 
 
 

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World Health Organization. Internet: https://www.who.int/gho/ncd/risk_factors/overweight/en/. 
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Public Health Ontario. Obesity A Burden Across The Life Course. 2014. 
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Centers for Disease Control and Prevention. Internet: https://www.cdc.gov/nchs/fastats/obesity-
overweight.htm. 
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Research and Markets. Internet: 
https://www.researchandmarkets.com/research/qm2gts/the_72_billion?w=4. 
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Houston DK, Ding J, Nicklas BJ, Harris TB, Lee JS, Nevitt MC, Rubin SM, Tylavsky FA, 
Kritchevsky SB. Overweight and obesity over the adult life course and incident mobility 
limitation in older adults: the health, aging and body composition study. Am J Epidemiol 
2009;169(8):927-36. doi: 10.1093/aje/kwp007. 
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Al-Goblan AS, Al-Alfi MA, Khan MZ. Mechanism linking diabetes mellitus and obesity. 
Diabetes Metab Syndr Obes 2014;7:587-91. doi: 10.2147/dmso.S67400. 
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Miller WM, Nori-Janosz KE, Lillystone M, Yanez J, McCullough PA. Obesity and lipids. Curr 
Cardiol Rep 2005;7(6):465-70. 
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Antonio, Kenyon, Ellerbroek A, Carson, Burgess, Tyler P, Mike, Roberts J, Angeli, Peacock. 
Comparison of Dual-Energy X-Ray Absorptiometry (DXA) versus a Multi-frequency 
Bioelectrical Impedance (InBody 770) Device for Body Composition Assessment after a 4-Week 
Hypoenergetic Diet. Journal of Functional Morphology and Kinesiology 2019;4:23. doi: 
10.3390/jfmk4020023. 
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Angelakis E, Armougom F, Million M, Raoult D. The relationship between gut microbiota and 
weight gain in humans. Future Microbiol 2012;7(1):91-109. doi: 10.2217/fmb.11.142. 
10. 
Minami J, Kondo S, Yanagisawa N, Odamaki T, Xiao JZ, Abe F, Nakajima S, Hamamoto Y, 
Saitoh S, Shimoda T. Oral administration of Bifidobacterium breve modifies metabolic functions 
in adults with obese tendencies in a randomised controlled trial. J Nutr Sci 2015;4:e17. doi: 
10.1017/jns.2015.5. 
11. 
Minami J, Iwabuchi N, Tanaka M, Yamauchi K, Xiao JZ, Abe F, Sakane N. Effects of 
Bifidobacterium breve on body fat reductions in pre-obese adults: a randomized, double-blind, 
placebo-controlled trial. Biosci Microbiota Food Health 2018;37(3):67-75. doi: 
10.12938/bmfh.18-001. 
12. 
Kondo S, Kamei A, Xiao JZ, Iwatsuki K, Abe K. Bifidobacterium breve exerts metabolic 
syndrome-suppressing effects in the liver of diet-induced obese mice: a DNA microarray 
analysis. Benef Microbes 2013;4(3):247-51. doi: 10.3920/bm2012.0019. 
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Kurose Y, Minami J, Sen A, Iwabuchi N, Abe F, Xiao J, Suzuki T. Bioactive factors secreted by 
Bifidobacterium breve enhance barrier function in human intestinal Caco-2 cells. Benef Microbes 
2019;10(1):89-100. doi: 10.3920/bm2018.0062. 
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Kondo S, Xiao JZ, Satoh T, Odamaki T, Takahashi S, Sugahara H, Yaeshima T, Iwatsuki K, 
Kamei A, Abe K. Antiobesity effects of Bifidobacterium breve strain B-3 supplementation in a 
mouse model with high-fat diet-induced obesity. Biosci Biotechnol Biochem 2010;74(8):1656-
61. doi: 10.1271/bbb.100267. 
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Osterberg KL, Boutagy NE, McMillan RP, Stevens JR, Frisard MI, Kavanaugh JW, Davy BM, 
Davy KP, Hulver MW. Probiotic supplementation attenuates increases in body mass and fat mass 
during high-fat diet in healthy young adults. Obesity (Silver Spring) 2015;23(12):2364-70. doi: 
10.1002/oby.21230. 

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investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
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16. 
Monda V, Villano I, Messina A, Valenzano A, Esposito T, Moscatelli F, Viggiano A, Cibelli G, 
Chieffi S, Monda M, et al. Exercise Modifies the Gut Microbiota with Positive Health Effects. 
Oxid Med Cell Longev 2017;2017:3831972. doi: 10.1155/2017/3831972. 
17. 
Bermon S, Petriz B, Kajeniene A, Prestes J, Castell L, Franco OL. The microbiota: an exercise 
immunology perspective. Exerc Immunol Rev 2015;21:70-9. 
18. 
Georges J, Lowery RP, Yaman G, Kerio C, Ormes J, McCleary SA, Sharp M, Shields K, Rauch 
J, Silva J, et al. The effects of probiotic supplementation on lean body mass, strength, and power, 
and health indicators in resistance trained males: a pilot study. Journal of the International Society 
of Sports Nutrition 2014;11(1):P38. doi: 10.1186/1550-2783-11-S1-P38. 
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Huang WC, Hsu YJ, Li H, Kan NW, Chen YM, Lin JS, Hsu TK, Tsai TY, Chiu YS, Huang CC. 
Effect of Lactobacillus Plantarum TWK10 on Improving Endurance Performance in Humans. 
Chin J Physiol 2018;61(3):163-70. doi: 10.4077/cjp.2018.Bah587. 
20. 
Huang WC, Wei CC, Huang CC, Chen WL, Huang HY. The Beneficial Effects of Lactobacillus 
plantarum PS128 on High-Intensity, Exercise-Induced Oxidative Stress, Inflammation, and 
Performance in Triathletes. Nutrients 2019;11(2). doi: 10.3390/nu11020353. 
21. 
Kovatcheva-Datchary P, Nilsson A, Akrami R, Lee YS, De Vadder F, Arora T, Hallen A, 
Martens E, Bjorck I, Backhed F. Dietary Fiber-Induced Improvement in Glucose Metabolism Is 
Associated with Increased Abundance of Prevotella. Cell Metab 2015;22(6):971-82. doi: 
10.1016/j.cmet.2015.10.001. 
22. 
Li W, Deng Y, Chu Q, Zhang P. Gut microbiome and cancer immunotherapy. Cancer Lett 
2019;447:41-7. doi: 10.1016/j.canlet.2019.01.015. 
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Shepherd JA, Ng BK, Sommer MJ, Heymsfield SB. Body composition by DXA. Bone 
2017;104:101-5. doi: 10.1016/j.bone.2017.06.010. 
24. 
Prevention CfDCa. Internet: https://www.cdc.gov/nceh/radiation/air_travel.html (accessed July 
2020. 
 
 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 45 of 48 
 
 APPENDICES 
17.1 
Appendix I: Probiotic and Prebiotic Foods to Avoid  
Dietary Guidelines  
 
Please use the following guidelines to help you make meal and snack choices between study 
visits that are appropriate for the study.   
*** Please refrain from ingesting any foods or beverages with live bacteria or yeast  
present during the course of the trial. *** 
Supplements to Avoid: 
Probiotic supplements – any food, capsule or orally administered product containing live bacteria 
or yeast, often labeled as bacteria or yeast fermentation products (such as: Lactobacillus, 
Bifidobacteria, Bacillus, saccharomyces) 
Prebiotic supplements – any orally administered product with the specific function of increasing 
bacterial populations.  
Foods to Avoid: 
• Yoghurt 
• Naturally fermented cabbage (including but not limited to sauerkraut, kimchi) 
• Naturally fermented pickles, cucumbers, and other vegetables 
• Fermented soy products (including but not limited to tempeh, miso, natto) 
• Uncooked buttermilk products 
• Soft cheeses, live culture cheeses (including but not limited to blue cheese, gouda, goat 
cheese) 
• Cold or uncooked sausage products 
• Foods marketed as “Probiotic” or “Live Culture” 
Beverages to Avoid: 
• Kefir 
• Kombucha 
• Apple cider vinegar  
• Lambic beers 
• Sour ales 
• Wheat beers 
• Other beverages labeled as probiotic 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 46 of 48 
 
Foods to Limit: (~2 servings per week) 
• Onions, leeks, garlic 
• 
Asparagus, artichokes, chicory root, dandelion greens 
 
 
 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 47 of 48 
 
17.2 
Appendix II: Daily Bowel Habits Diary with Bristol Stool Scale 
Daily Bowel Habits Diary with Bristol Stool Scale will be completed every day by the participants. 
 
Date:
Comments/Therapies/Medication (dose, frequency, reason for use)/Changes in Health:
For each bowel movement you experienced today record the time (in 24-hour format) for question 1 and circle
Yes or No (Y/N) for questions 2-7.
For question 5 please refer to the Bristol Stool Scale and record the type that best describes the bowel
movement.
If you do not have any bowel movements, circle 0 and complete question 2, 6 and 7.
Bowel Movement Number  
* If required, additional movements and details can be added to the back of the diary
0
1
2
3
4
5
1. Time of Bowel 
Movement (hh:mm)
N/A
___ : ___
___ : ___
___ : ___
___ : ___
___ : ___
2. Straining to start 
defecation?
Y / N 
or N/A
Y / N
Y / N
Y / N
Y / N
Y / N
3. Straining to stop 
defecation?
N/A
Y / N
Y / N
Y / N
Y / N
Y / N
4. Feeling of 
incomplete 
defecation?
N/A
Y / N
Y / N
Y / N
Y / N
Y / N
5. Bristol Stool Form 
Type (1-7) see below?
N/A
6. Were laxatives, 
enemas or 
suppositories used in 
the 24 hours prior to 
the bowel 
movement?
Y / N
Y / N
Y / N
Y / N
Y / N
Y / N
7. For Females: Do you feel that your bowel movements today were affected by your menstrual cycle?  Y / N
 

Protocol 20BWHM:  
A randomized, double-blind, placebo-controlled study to 
investigate the effects of a Bifidobacterium breve strain 
on fat loss in healthy adults 
 
June 26, 2023 
 
 
-Confidential- 
Page 48 of 48 
 
17.3 
Appendix III: Exercise Guidelines 
Please use the following guidelines to help you select the type of exercise that is appropriate for 
this study 
*** Please ensure that you exercise 3 times per week for 20 minutes at a moderate intensity 
(can be in bouts of 5-10 minutes)*** 
 
Examples of moderate and high-intensity exercise 
Moderate-intensity Physical Activity 
 
Activities elevate the heart rate and cause light 
sweating after about 10 minutes. Breathing is 
heavier than normal, but you can carry a 
conversation. 
High-intensity Physical Activity 
 
Activities elevate the heart rate substantially and 
cause significant sweating. You cannot carry a 
conversation due to heavy breathing. 
• 
Brisk walking 
• 
Bicycling (easy pace) 
• 
Light to moderate workouts on gym 
equipment  
• 
Water aerobics 
• 
Swimming (leisure pace) 
• 
Carrying moderate loads 
• 
Participating in active games with 
children (eg. Hopscotch, dodgeball) 
• 
Dancing (social) 
• 
Running 
• 
Bicycling (brisk pace) 
• 
Swimming (fast pace) 
• 
Carrying heavy loads 
• 
Playing competitive sports 
• 
Cross-country skiing 
• 
Dancing (competitive)