The Tolerance of Organic Formula Milk and Its Fecal Microbiome Characteristic in Infants
Phase: NA
Conditions: Food Allergy in Infants
Sponsor: Hasanuddin University

 
 
 
 
 
 
 
 
 
 
 
The Tolerance of Organic Formula Milk and Its Fecal 
Microbiome Characteristic in Infants  
  
 
Study Protocol 
 
 
April 29th, 2021 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

The Tolerance of Organic Formula Milk and Its Fecal 
Microbiome Characteristic in Infants  
 
Protocol 
 
Responsible Personnel 
 
Principal Investigator Contact Information 
Name: Bahrul Fikri 
Title: MD, PhD 
Institution: Allergy and Immunology Division, Department of Pediatrics, Faculty of Medicine, 
Hasanuddin University 
Address: Jln. Perintis Kemerdekaan KM 11 Makassar, South Sulawesi, Indonesia, 90245 
Telephone: +62(411)584461 
Email: bahrulfikri@unhas.ac.id 
 
Other Contact   
Name: Conny Tanjung 
Title: MD, PhD 
Institution: Graduate School of Medical Sciences, Faculty of Medicine, Hasanuddin University 
Address: Jln. Perintis Kemerdekaan KM 11 Makassar, South Sulawesi, Indonesia, 90245 
Telephone: +62(411)581666 
Email: mfconnytanjung@yahoo.com 
 
Summary: 
An experimental study aims to investigate the tolerance of organic formula milk on 
infants supplemented with organic formula milk. This study also observes gut microbiota, short 
chain fatty acids, nutritional status, and atopic manifestation on infants supplemented with 
organic formula milk. This study will be done on 50 subjects, with an age of 6-7 months old, 
38-42 weeks of gestation, had a birth weight ranging from 2700 grams to 4200 grams, not 
suffering from any major congenital anomaly, not severely stunted at birth, has a normal 
thyroid function, not suffering any prominent gastrointestinal disease, not having a severe 
disease at the beginning of study, and has an approval from their parents. Subjects’ diet will 
be added an organic formula for infant for 3 months, and they will be monitored regularly, 
since this study starts, at each month, and at the end of this study. The subjects’ gut 
microbiomes will be calculated at every session of monitoring by collecting their fecal samples, 
and brought to laboratory. Anthropological data (weight, height, body mass index), atopic 
manifestation, IL-6 and IL-10 will also be collected.  
 
Introduction: 
An enormous number of microbiotas inhabit the human intestinal tract.  The microbiota 
forms a unique and complex gut ecosystem by interacting with the host, and this affects the 
health and diseases of the host in various ways, including the modification of the immune 
system. Butyrate produced by the gut microbiota promotes differentiation of peripherally 
derived regulatory T cell (pTreg) in colonic lamina propria through epigenetic modification. 
Besides, it induces B cell Lymphocyte to secrete IgA (sIgA) which will be transferred to 
intestinal lumen. sIgA plays an important role in antigen binding and clearance. Additionally, 
gut microbiota can inhibit histone deacetylases (HDACs) as well. All these functions may have 
the inflammation control not only in the gut but also in other parts of the body. [1].  
 
The microbiota hypothesis states that the disruption of mucosal regulatory immune 
responses caused by the perturbation of microbiota due to Western lifestyle factors leads to 
the development of both Th1- and Th2- mediated disease. It is necessary to have a complex 

microbiota in order to prevent some diseases such as allergy, diarrhea, autoimmune disorder, 
etc. The depletion or deletion of bacterial communities, as associated with an elevated serum 
IgE level, increases the circulating basophil population, thereby exaggerating the Th2 cell 
responses and allergic inflammation. It has been clarified that gut microbiota plays a critical 
role in oral tolerance in mouse model. Various studies have revealed that the microbiota plays 
an important role in pathogenesis of allergic diseases. It remains unclear how the differences 
in age, region, dietary intake or other environmental factors such as allergen exposure or 
infection, affect this role [2,3]. Candidate bacterial taxa have been reported it still remain 
unclear which bacteria (or other microbes), in which numbers and combinations, and when 
during the gut colonization process may prevent allergic diseases and asthma [4]. Some 
different microbiota may produce the same metabolome. For example, one of SCFA (short 
chain fatty acid), pyruvate acid is produced by Bacteroides and Clostridium as well. 
 
Imbalance of gut microbiota or dysbiosis may start in early life. The increasing in 
cesarean section, formula feeding and the use of antibiotics during pregnancy or infancy have 
the significant impacts on microbial homeostasis. 
 
 
 
Objectives of Trial: 
1. Primary Objective 
a. To know the acceptability of organic formula milk on infants 
2. Secondary Objective:  
a. To determine gut microbiota characteristics on infants supplemented with 
organic formula milk 
b. To determine gut short chain fatty acids on infants supplemented with 
organic formula milk 
c. Nutritional status including body mass index. 
d. Atopic manifestation 
 
Hypothesis: 
 
H0 : Organic formula milk is not well accepted by infants 
 
H1 : Organic formula milk is well accepted by infants  
Trial Design:  
Observational study 
 
Study Population: 
 
Number of samples: 
50 subjects 
 
Inclusion criteria: 
- 
Healthy infants 6-7 months of age. 
- 
Gestational age 38-42 weeks 
- 
Birth weight >2700 and <4200 gram 
- 
Not suffering from a major congenital anomaly, severely stunted at birth, 
not having a thyroid problem, not suffered from prominent gastrointestinal 
diseases, severe diseases at the time of inclusion (severe pneumonia, 
severe dehydration, etc) 
- 
Parents want to follow the study by signing the informed consent  
 
Exclusion Criteria: 
- 
Subjects are in the severe disease condition at the time of recruitment 
- 
Severe acute malnutrition 

- 
Have conditions that will influence the nutritional status such as moderate 
to severe dehydration, organomegaly, edema. 
 
 
Product Description: 
 
Organic formula milk + solid food/breast feeding 
 
Arla Baby&Me Organic Infant Formula 6-12 months, contains: 
Skimmed milk, Lactose, Vegetable oils (Palm oil, Rapeseed oil, Soy bean oil, 
Coconut oil), Galactooligosaccharide (GOS), Concentrated whey protein 
powder, Fructooligosaccharide (FOS), Arachidonic acid from M. alpina, 
Docosahexaenoic acid from C. cohnii, Minerals (Tricalcium disphosphate, 
Calcium carbonate, Calcium hydroxide, Potassium chloride, Tripotassium 
citrate, Trisodium citrate, Sodium chloride, Magnesium chloride, Ferrous 
sulphate, Zinc sulphate, Copper sulphate, Potassium iodide, Manganese 
sulphate, Sodium selenite), Vitamins (Retinyl acetate, Cholecalciferol, D-alpha 
tocopherol, Phytomenadione, L-ascorbic acid, Ascorbyl palmitate, Thiamine 
hydrochloride, 
Nicotinamide, 
Calcium-D-pantothenate, 
Pyridoxine 
hydrochloride, D-biotin, Folic acid), Emulsifier (Lecithin E322). 
         *Contains milk and soy ingredients. 
 
 
 
Outcomes: 
The tolerance will be the primary outcome measured by clinical examination and 
questionnaire. We will measure the outcome every month.  The secondary outcomes, gut 
microbiomes which will be obtained through fecal samples collection. Fecal DNA extraction, 
polymerase chain reaction amplification and library construction for bar-coded 16S rRNA gene 
amplicon sequencing, using the Illumina MiSeq platform will be examined. The 16SrRNA gene 
amplicon sequences will be analyzed using QIIME1.9.0 and operational taxonomic units 
(OTUs). Fecal SCFAs will be measured by LC-MS/MS. Data will then be calculated to see if 
there is a significant difference before and after organic formula feeding. Additionally, we also 
will measure nutritional status, including body mass index, atopic manifestation, IL-6, and IL-
10. Measurements of weight, length, and head circumference will be monitored at every visits. 
Anthropometric data will be collected at the beginning of study, first visit (4 weeks after) and 
at second visit (end of study) to evaluate the subjects growth.  
 
Conduct of Trial: 
All the guardians or parents of the subjects who participate in this study will be 
interviewed in order to record the dietary and sign for inform consent. Parents will be given a 
journal to record the subject’s diet, any sign of colic on subjects, their quality of sleep, and 
wellbeing. All the subjects will be fed organic formula milk as a part of their dietary for 12 
weeks. Before feeding the formula, the nutritional status will be measured and the subject’s 
fecal will be collected for microbiome and short chain fatty acid (SCFA) examination. Every 
month the parent will be called in order to assess for child’s sickness, allergy, body weight, 
length, head circumference, and to review their journal. 
 

 
 
Figure 1. Study Procedure 
 
 
Description of Visits: 
A visit follow up will be done before and after  the study, the tolerance, nutritional status 
(including body mass index) and fecal collection for all subjects will be performed. We will also 
collect the allergy related data. Each fecal examination will follow a strict cold chain procedure, 
each sample will be collected directly, bottled, and frozen to minimize error. The frozen 
samples will be transported in insulated bags with frozen ice blocks before being transferred 
to -80°C for storage.  
 
 
Statistical Aspects and Considerations: 
A statistical analysis will be performed using r program and STATA 13.0. The Normality 
of data distribution will be tested using the Shapiro–Wilk test. To evaluate potential 
associations between factors in relation to fecal microbiome or SCFAs, Spearman's rank 
correlations will be calculated. A p-value <0.05 will be considered significant.  
The model will be adjusted for potential confounders. Estimated associations will be 
described as odds ratios (ORs) with 95% confidence intervals (CIs). The non-parametric 
Wilcoxon test for paired samples will be used to compare different laboratory parameters at 
different times of collecting samples. The Mann–Whitney U test for unpaired samples will be 
applied to compare different categorical parameter between infants before and after treatment. 
Multivariable linear regressions will be done to find out the association of different parameters 
change before and after treatment.  
 
Ethical Aspects: The institutional ethics committee of each participating Hasanuddin 
University Hospital and Wahidin Sudirohusodo Hospital must be approved the study protocol 
 
 
 
 
 
 
 
 
 

References: 
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Y, 
Shimojo 
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and 
allergies.Semin 
Immunopathol.2015;37:57-64. 
3. Sakurai K, Miyaso H, Eguchi A, et al. Chiba study of Mother and Children`s Health (C-
MACH): cohort study with omics analyses. BMJ Open 2016;6:1-7. 
4. Sjödin KS, Vidman L, Rydén P, et al. Emerging evidence of the role of gut microbiota 
in the development of allergic diseases. Cur Opin Allergy Clin Immunol 
2016;16:390-395.