Ingestion Effect of Formula Milk With Triple Bifidobacteria Strains on Fecal Quality and Metabolites in Healthy Children

Study Overview
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Brief Summary
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The goal of this clinical trial is to compare healthy children between 1 to 3 years old. The main question it aims to answer is Is there an effect towards fecal quality after consuming milk formula supplemented with the triple Bifidobacteria strains (Bifidobacterium longum BB536, Bifidobacterium breve M-16V, and Bifidobacterium longum subsp. infantis M-63)? Is there an effect towards short-chain fatty acid composition after consuming milk formula supplemented with the triple Bifidobacteria strains (Bifidobacterium longum BB536, Bifidobacterium breve M-16V, and Bifidobacterium longum subsp. infantis M-63)? Is there an effect towards clinical profile after consuming milk formula supplemented with the triple Bifidobacteria strains (Bifidobacterium longum BB536, Bifidobacterium breve M-16V, and Bifidobacterium longum subsp. infantis M-63)? The clinical trial period started off with all the participants consuming a milk formula three times a day, for 14 days as a baseline period. Afterwards, the participants were randomly assigned to one of two groups: probiotic and placebo. Following the baseline period, participants in the probiotic group consumed formulated milk supplemented with the triple Bifidobacteria strains, while the placebo group ingested the same formulated milk without adding triple Bifidobacteria strains for 90 days. Researchers will compare the probiotic and placebo groups to see if there are any effects towards the fecal quality, short-chain fatty acid level and clinical profile after consuming the milk formula.

Detailed Description
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This clinical trial is a study of 3 strains of Bifidobacterium was conducted in infants aged 1-3 years in Indonesia. The general objective of this study was to determine the benefits of combined supplementation of Bifidobacteria strains (B. longum BB536, B. breve M-16V, and B. longum subsp. infantis M-63) in healthy children aged 1-3 years. The study is conducted for 104 days with a baseline period of 14 days and continued with an intervention period of 90 days. Participants is divided into two groups, which are the intervention group and the placebo group. The study will be conducted using a double-blind randomized, placebo-controlled trial design. A total of 100 participants will be selected based on inclusion and exclusion criteria. During the screening period (day 0), socialization of the research and signing of informed consent will be carried out for participants who are willing to participate. Participants were asked not to consume fermented milk products, probiotics and prebiotic fortified food/drinks while participating in research activities. During the baseline period (Day 14), the participants were asked to consume control milk and, represented by their parents, were also asked to fill out research diaries in the form of diet records, records of probiotic products and prebiotic fortified food/drinks consumed, type and frequency of bowel movements, health complaints felt (discomfort, headaches, stomachaches, respiratory tract-related illnesses and fever), medication consumption records, days where the participants refused to eat. Participants were asked to collect research documents and collect stool samples on day 14 +/- 1. During the intervention period, from day 15 to day 104, participants consumed 36 grams of the test product or placebo dissolved in 180 ml per serving with a consumption frequency of 3 times per day. Product consumption will be recorded in the product consumption logbook and on the subject's diary record form. Once every 2 weeks a health profile measurement (weight, height, arm circumference, head circumference, skinfold thickness) and sleep quality will be measured. Participants were asked to collect research documents and stool samples on day 104 +/- 1 with the same procedure as in the baseline period.

Official Title
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The Consumption Effect of Formula Milk With Triple Bifidobacteria Strains (Bifidobacterium Longum BB536, Bifidobacterium Breve M-16V, and Bifidobacterium Longum Subsp. Infantis M-63) on Fecal Quality and Metabolites in Healthy Children

Conditions
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Children, Only

Intervention / Treatment
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* Combination Product: Formula milk with triple Bifidobacterium probiotic strain
* Other: Formula milk


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age between 12 - 36 months Normal nutritional status (z-score height-for-age or z-score weight-for-height: -2 SD < X < 2 SD) Has no dairy allergies Does not consume breastmilk anymore during the intervention period Willing to participate through the whole research period Obtain consent from parents or guardians by signing the informed consent form Exclusion Criteria: Has a history of lactose intolerance, congenital and chronic diseases including irritable bowel syndrome, etc. Receive antibiotic or other probiotic supplementation during the 14 day of baseline period.

Ages Eligible for Study
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Minimum Age: 12 Months
Maximum Age: 36 Months

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Other
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Triple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: Probiotic Group<br>During the baseline period, all participants consumed milk formula without any addition of probiotics three servings per day for 14 days. One serving of milk formula consists of 36 gram that is diluted in 180 mL lukewarm water. The participants in the probiotic group consumed milk formula added with triple Bifidobacterium strain containing Bifidobacterium longum BB536 (6.9 x 10^7 CFU/serving), Bifidobacterium breve M16-V (6.9 x 10^7 CFU/serving), Bifidobacterium longum subsp. infantis M-63 (6.9 x 10^7 CFU/serving). The milk formula is consumed three servings per day for 90 days during the intervention period. One serving of milk formula consists of 36 gram that is diluted in 180 mL lukewarm water. | Combination Product: Formula milk with triple Bifidobacterium probiotic strain<br>* Formula milk with triple Bifidobacterium strain containing Bifidobacterium longum BB536 (6.9 x 10^7 CFU/serving), Bifidobacterium breve M16-V (6.9 x 10^7 CFU/serving), Bifidobacterium longum subsp. infantis M-63 (6.9 x 10^7 CFU/serving).<br>|
| Placebo Comparator: Placebo Group<br>During the research period (both baseline and intervention period), all participants consumed milk formula without any addition of probiotics three servings per day for 104 days. One serving of milk formula consists of 36 gram that is diluted in 180 mL lukewarm water. | Other: Formula milk<br>* Formula milk without any addition of probiotic strain<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Fecal characteristics | Observed the shape, color and consistency of fecal samples that will be combined to be reported as fecal type according to the Bristol Stool Chart | Before and after intervention period (day 13 and day 105) | 
| Fecal pH | The pH of obtained fecal samples were measured using potential of hydrogen (pH) meter | Before and after intervention period (day 13 and day 105) | 
| Bacterial colony characteristics | From the fecal samples obtained, direct plating using the selective media for Bifidobacterium (Bifidobacterium Selective Medium or BSM) and Enterobacteriaceae (MacConkey Agar) is conducted. Colony bacterial characterization is based on the cell morphology and Gram strain characteristics under the microscope. | Before and after intervention period (day 13 and day 105) | 
| Bifidobacterium's cell count | The cell number is a total of Bifidobacteria cells obtained from DNA extraction of stool samples counted using quantitative PCR (qPCR). Cell count will be expressed in colony forming unit per gram (CFU/g). | Before and after intervention period (day 13 and day 105) | 
| Enterobactericaeae's cell count | The cell number is a total of Enterobacteriaceae's cells obtained from DNA extraction of stool samples counted using quantitative PCR (qPCR). Cell count will be expressed in colony forming unit per gram (CFU/g) | Before and after intervention period (day 13 and day 105) | 
| Short chain fatty acids analysis | The analysis includes the measurement of acetate, propionate, and butyrate. The analysis uses gas chromatography method. | Before and after intervention period (day 13 and day 105) | 
| Biomarker of immune systems | The analysis on immune systems' biomarker uses the fecal water obtained from diluting the fecal samples into phosphate buffer. The fecal water is then added with protease inhibitor to keep fecal samples stable during the analysis. The immune system analyzed are interleukin 6 (IL6), interleukin 8 (IL8), interleukin 10 (IL10) and secretory immunoglobulin A (SIgA). All immune systems are read using the enzyme-linked Immunosorbent Assay (ELISA) Reader BioRad iMark. | Before and after intervention period (day 13 and day 105) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Defecation frequency records | Participants, represented by their parents or guardian, record their defecation frequency in a daily record. | Everyday during the research period (104 days) | 
| Dietary pattern records | Participants, represented by their parents or guardian, record their dietary patterns in a daily record. The dietary pattern recorded includes type and portion of the foods, snacks and beverages. | Four days every two weeks, consists of two weekdays and two weekend days during the research period | 
| Health complaint and medical records | Participants, represented by their parents or guardian, record their health complaint and medical records in a daily record. | Everyday during the research period (104 days) | 
| Health profile record | Participants, represented by their parents or guardian, filled a questionnaire about recent respiratory health conditions. | Before and after intervention period (day 13 and day 105) | 
| Gastrointestinal disorder record | Participants, represented by their parents or guardian, filled a questionnaire about recent gastrointestinal health conditions. | Before and after intervention period (day 13 and day 105) | 
| Sleep quality | Participants, represented by their parents or guardian, filled a questionnaire about recent history of the participant's sleep quality. | Before and after intervention period (day 13 and day 105) | 
| Behaviour assesment | Participants, represented by their parents or guardian, filled a questionnaire about how the participants behave especially how they express their emotions. | Before and after intervention period (day 13 and day 105) | 
| Development screening | Participants, represented by their parents or guardian, filled a questionnaire about recent gastrointestinal health conditions. The Ages and Questionnaires (ASQ-3) were used based on the participant's current age. | Before and after intervention period (day 13 and day 105) | 
| Psychosocial problems | Participants, represented by their parents or guardian, filled a questionnaire about possible psychosocial problems. The Pediatric Symptom Checklist (PSC) was used for this measure. | Before and after intervention period (day 13 and day 105) | 
| Weight | The participants were measured for their weight in kilograms. | Once every two weeks for 105 days | 
| Height | The participants were measured for their height in cm. | Once every two weeks for 105 days | 
| Body Mass Index (BMI) | The measurement of weight and height were combined into BMI and expressed in kg/m^2 | Once every two weeks for 105 days | 
| Arm circumference | The participants were measured for their arm circumference in cm. | Once every two weeks for 105 days | 
| Head circumference | The participants were measured for their head circumference in cm. | Once every two weeks for 105 days | 
| Subcutaneous fat thickness | The participants were measured for their subcutaneous fat thickness in mm. | Once every two weeks for 105 days | 
| Triceps thickness | The participants were measured for their triceps thickness in mm. | Once every two weeks for 105 days | 
| Medical check up | An on-site medical professional will check the health condition of the participant. | Once every two weeks for 105 days | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Children, Fecal sample, Triple Bifidobacterium strains, Probiotic, Formula milk, Indonesia