Projects / Programmes source: ARRS

The role of human milk in development of breast fed child's intestinal microbiota

Research activity

Code Science Field Subfield
4.02.00  Biotechnical sciences  Animal production   

Code Science Field
B420  Biomedical sciences  Nutrition 
human milk, nutrition, breastfed child, microbiota, fatty acids, intestinal microbiota, bifidobacteria, lactobacilli, probiotics
Evaluation (rules)
source: COBISS
Researchers (15)
no. Code Name and surname Research area Role Period No. of publications
1.  19258  PhD Tadej Avčin  Human reproduction  Researcher  2010 - 2013  437 
2.  33338  PhD Evgen Benedik  Human reproduction  Junior researcher  2012 - 2013  502 
3.  11150  PhD Bojana Bogovič Matijašić  Animal production  Researcher  2010 - 2013  362 
4.  02254  PhD Borut Bratanič  Human reproduction  Researcher  2010 - 2013  328 
5.  15659  PhD Andreja Čanžek Majhenič  Animal production  Researcher  2010 - 2012  227 
6.  15312  PhD Nataša Fidler Mis  Human reproduction  Researcher  2010 - 2013  415 
7.  34722  Janja Gržinić    Technician  2012 - 2013 
8.  33150  PhD Anja Mavrič    Junior researcher  2011 - 2013  12 
9.  25516  PhD Petra Mohar Lorbeg  Animal production  Researcher  2010 - 2013  98 
10.  28205  Tanja Obermajer    Technician  2011 - 2013  48 
11.  05325  PhD Darja Paro  Neurobiology  Researcher  2011 - 2013  330 
12.  14020  PhD Barbka Repič Lampret  Human reproduction  Researcher  2010 - 2013  140 
13.  08857  PhD Irena Rogelj  Animal production  Principal Researcher  2010 - 2013  703 
14.  31910  PhD Primož Treven  Animal production  Junior researcher  2010 - 2013  62 
15.  34337  PhD Tina Tušar  Biotechnical sciences  Junior researcher  2012 - 2013  15 
Organisations (2)
no. Code Research organisation City Registration number No. of publications
1.  0312  University Medical Centre Ljubljana  Ljubljana  5057272000  71,385 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  63,013 
The ideal food for normal infants is human milk, a complex blend of nutrients and bioactive substances that nourish and protect the baby. In addition, breast milk has been shown to be a continuous source of commensal, and/or probiotic bacteria to the infant gut where they play a key role in the initiation and development of the gut microbiota. Intestinal colonization is essential for maturation of the gut-associated lymphoid tissue (GALT) and the homeostasis of the intestinal epithelium. Colonization begins immediately after birth, while later the composition of the gut microbiota is affected by feeding practices. The microbiota of breast-fed and formula-fed infants is different in the levels of particular bacterial groups as well as in the species composition. Results of some studies suggest that long-chain polyunsaturated fatty acids (LCPUFAs) promote the adhesion of probiotics to mucosal surfaces and along with probiotic bacteria contribute to the regulation of innate and adaptive immune responses and present a link among mother’s diet, and microbes. It was observed that specific changes in the initial establishment and species composition of bifidobacteria in neonates took place as consequence of the consumption of probiotics by the mothers. There is still surprising lack of studies of human milk microbiota of healthy women including potential probiotic bacteria and the mechanisms which enable bacteria to colonize mother's milk, are quite unclear. In the current study, we intend to evaluate the predominant bacteria existing in breast milk of healthy women and to screen for bacteriocin producers, which may affect the qualitative and quantitative composition of milk and neonate gut microbiota. The possibility and mechanisms of endogenous transfer of probiotic child’s faeces isolate Lactobacillus gasseri K7 from the maternal gut to the mammary gland will be studied in vitro in a trans-well co-culture system. Additionally the transfer of probiotics, orally consumed during the pregnancy, into mothers' milk or infant’s intestine and consequently their possible effects on the infant’s microbiota will be examined in pilot randomized placebo-controlled double blind trial. The main goal of the proposed research is to establish the link among mothers’ nutrition, human milk LCPUFAs composition and microbiota and their potential influence on child’s gut microbiota development. Pregnant women from three different regions of Slovenia will be involved into the study. Dietary intakes will be assessed during pregnancy, at the beginning of the 3rd trimester of pregnancy, and during lactation, at 4 weeks post partum, by 7-day weighed dietary protocol (7DP). Human milk (colostrum, and mature human milk) and infant’s faeces will be sampled twice: at 2 or 3rd day post partum (meconium) and at 4 weeks post partum for the determination of human milk LCPUFAs composition and the assessment of human milk (colostrum und mature) and faeces microbiota. The fatty acid composition of human milk will be analysed by capillary gas-liquid chromatography (GC). Microbiota of milk and faeces will be studied using conventional microbiological and modern molecular approach such as qualitative and quantitative PCR amplification of informative genomic regions, DGGE/TGGE and/or t-RFLP and sequencing. In order to successfully recruit volunteers participating in clinical studies, an innovative approach will be established consisting of preparative meeting including the research protocol information, practical instructions and demonstration about writing 7-day weighed dietary protocols. Regular monthly meetings with the researchers will be organised for discussion and advising. Basic health parameters of infants will be followed. Data will be gathered from maternity department medical charts, from regular paediatric health care visits and from especially designed health care diary each mother will keep until the end of her child’s first year.
Significance for science
Research on human milk, colostrum, meconium and feces of infants with the state of the art methods have led to important new insights about the diversity of microbial communities that inhabit this environment. Results will be useful in future studies aimed to explain the colonization of the gut early in life and long-term role of microbiota in the maintaining of health. Important scientific value of the results of this research at the international level is also due to the relatively high number of volunteers and their children involved in the study, compared to most of other related studies. In the literature, there is almost no reports where so many parameters were followed in the same subjects; from the data on long-term dietary habits and nutrition during pregnancy and lactation (questionnaires, 4-day weighted dietary protocol), nutritional status (fatty acid composition of blood serum and milk of mothers, vitamin D in blood serum and milk), the microbiota of biological samples - colostrum, milk, meconium, stool (quantity and ratio of selected groups of bacteria), to the health status of mothers and children and many morphometric measurements (body mass index, body fat, body length, skin folds, bone density) . From the complex processing of data, which, because of the abundance of data is ongoing, we expect that we can explain the important links between diet, fatty acids and microbial composition of milk, children’s microbiota and health status of children, which was also closely monitored by pediatricians. Studies of microbiota and the mechanisms of possible transfer of bacteria from the gut to the mammary gland (in vitro model of pregnant mice) have required the introduction or modification of a number of molecular, genetic, genomic and immunological methods. These methods include real-time PCR for different groups of bacteria; three-dimensional model of the intestinal epithelium, which comprises dendritic cells derived from human peripheral blood monocytes, human intestinal epithelial cells and probiotic bacteria. This represents a good starting point for further work of our group on a similar topic, as well as researchers at the global level. Also the results of studying the mechanisms of transmission of lactobacilli (Lb. gasseri K7 , Lb . rhamnosus GG) from the intestine into the mammary gland deserve attention at the international level.
Significance for Slovenia
The project represents one of the largest interdisciplinary clinical studies, dealing with the initial development of the child in terms of diet and establishment gut microbiota. In addition to the researchers of Biotechnical Faculty and University Medical Centre, Division of Pediatrics, who were members of the project team, many experts and researchers from various fields, from gynecologists and personnel who carry out schools for parents (recruitment of pregnant women), medical staff in 10 maternity hospital (collection of the first samples - meconium, colostrum, the first faeces), pediatricians and anthropologists (help with examination of children, bone density measurement, anthropometric measurements), analysts (determination of D vitamine, fatty acid composition), statisticians and bioinformatics, young researchers and students volunteers were involved in the research and promotion of the study. All have gained new knowledge and experience in research work. The participants of My-milk symposium, which was organized by project team upgraded their knowledge with the latest discoveries in the field of early development. Lectures by eminent foreign professors involved in the project team as consultants were presented at the symposium as well. The symposium was recognized by Medical Chamber as important for education and therefore credits were conferred to participating physicians and nurses. In the frame of the project we gained a lot of valuable biological samples that demanded specific protocols of preparation and analysis, so researchers gained new research skills and experiences. Especially the part of the research in which microbiomes of the samples of 13 mothers and their children were analysed by new generation sequencing required new knowledge by the involved researchers (development of protocols, bioinformatic and statistical analysis), which will be useful in future research. New approaches for the evaluation of nutrition and dietary diaries were introduced and evaluated, which will be useful for nutritionists. Acquired knowledge and analytical results have been shared with professionals and the general public through various seminars and presentations (i.e. Bedenik E, Fidler Mis N. New recommendations for the intake of vitamin D. Zdravniški vestnik, 2013; Fidler Mis N, Benedik E. Diet of vegetarian pregnant women and lactating mothers. Celiakija, 2012; Carli T, Jarc K, Benedik E. Recently revealed secrets of a fraction of a magic potion: a study of breast milk. Medicina danes, 2011; Gregoric M, Benedik E. The role of breast milk in the development of infant gut microbiota. Bambino.si , 2012) . In the frame of the project, 2 doctoral thesis and 3 diploma thesis were elaborated, while some of them are still in progress. The results obtained represent the first so complex study in the field of initial child development in Slovenia and we believe they are an excellent basis for further work. New knowledge, new approaches and methods (eg. evaluation of nutritional status, anthropometric measurement in pregnant women and the determination of body fat), may contribute to better medical treatment of the most sensitive groups of the population and thus improved health status. Since we maintain contacts with the study participants, we consider that the study should be continued on the same children, in order to further follow their development. Slovenian research work in the field of early development has become recognizable also abroad.
Most important scientific results Annual report 2010, 2011, 2012, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2010, 2011, 2012, final report, complete report on dLib.si
Views history