Breast milk is the mainstay of newborn nutrition, providing all the nutrients and vitamins necessary for optimal growth of newborn infants. Beside its nutritional properties, breast milk also contains bioactive factors such as soluble immune factors, antimicrobial proteins, functional fatty acids, hormones, oligosaccharides, stem cells and microbiota. Although fresh breast milk is the optimal source of nutrition for newborn infants, breast milk must be expressed and stored in some conditions.

Breast milk was thought to be sterile for many years. This idea has changed with the isolation of live bacteria in the breast milk of healthy mothers in the recent past. Thus, it has been shown that breast milk is not actually a sterile body fluid, but has a microbiota of its own. This study aimed to examine the effect of storage conditions of breast milk at different temperatures on the microbial composition of the breast milk.

The prospective experimental study will be conducted under the supervision of Istanbul University, Istanbul Medical Faculty, Department of Pediatrics, Neonatology Department. Informed consent of mothers will be taken. Thirty mililiter (30 mL) breast milk that was obtained from volunteer mothers will be divided into 3 samples, each 10 mL of breast milk will be stored at different temperatures. Ten mililiter will be studied for microbial composition with 16S rRNA amplicon sequencing and bioinformatics analysis immediately, 10 mL will be stored at +4'C for 3 days and will be studied at the end of 3rd day, other 10 mL will be stored at -20'C for 3 months and will be studied at the end of 3rd months. 16S rRNA amplicon sequencing and bioinformatic analysis studies will be performed at Istanbul Medical Faculty Clinical Nutrition and Microbiota Research Laboratory.

This present study is planned to be carried out for 24 months between January 2023 and December 2024

Fresh breast milk is the mainstay of newborn nutrition and provides all the nutrients and vitamins needed for optimal growth of newborn babies. In addition to the nutritional properties of breast milk, there are some bioactive factors such as soluble immune factors, antimicrobial proteins, functional fatty acids, hormones, oligosaccharides, stem cells and microbiota in breast milk. Breastfeeding also strengthens the bond between mother and baby in postpartum period and provides nutrition to the newborn infant. Breast milk improves microbial colonization, improves immune system maturation and supports the metabolic activities of the newborn infant.

Although fresh breast milk is the optimal food source for newborn babies, in some cases, breast milk must be expressed and stored.It is reported that breast milk can be stored in the refrigerator for 3 days without being frozen at +4 degrees Celsius, and can be stored as frozen for 3 months if it is frozen within the first 24 hours after milking. The North American Breast Milk Banking Association, on the other hand, reports that the storage period can be extended up to 12 months at temperatures below -20 degrees Celsius.

While breast milk was thought to be a sterile fluid, this idea has changed with the isolation of live bacteria in the milk of healthy mothers in the recent past. The presence of many bacteria such as Lactobacillus, Lactococcus, Bifidobacterium, Streptococcus, Enterococcus and Staphylococcus has been demonstrated by traditional culture methods of breast milk. The new generation sequencing technologies obtained higher microbial diversity of breast milk and detected mouth-dwelling bacteria such as Veillonella and Prevotella, skin bacteria such as Propionobacterium and gram-negative bacteria such as Pseudomonas, Ralstonia, Klebsiella. Thus, it has been shown that breast milk is not actually a sterile body fluid, but has a microbiota of its own.

There are few studies evaluating the effect of breast milk storage conditions on microbiota. In a study, it was shown that the number of bacterial colonies in breast milk decreased as the freezing period increased. It has been shown that 98-100% of bacteria in frozen breast milk are lost after 12 weeks of storage.

Although studies evaluating fresh breast milk microbiota have been reported in the literature, there are few studies evaluating the effect of milk storage conditions on microbial diversity. In existing studies, microbial growth was ensured by culture method and diversity was defined by traditional microbiological methods. No study was found that evaluated the microbial composition of breast milk with next-generation sequencing based on the 16S rRNA amplicon sequencing strategy.

This study aimed to evaluate how the generally recommended breast milk storage conditions and duration of storage affect the microbiota composition of breast milk. This study will provide new data and new recommendations about storage of breast milk.

Inclusion Criteria:

• Women who had vaginal delivery
• Women who gave delivery at gestational age above 37 weeks
• Women who have a child older than 1 weeks of age
• Mothers with a body mass index between 18.5-25
• Women who gave consent

Exclusion Criteria:

• Women who had cesarean delivery
• Women who gave premature delivery (<37 weeks of gestation)
• Women who gave birth within prior 7 days
• Women with premature rupture of membranes
• Women with chorioamnionitis
• Women who had antibiotherapy within last 1 week
• Presence of mastitis
• Presence of maternal co-morbid diseases such as; pregestational diabetes, pregestational hypertension, connective tissue diseases, inflammatory bowel syndrome, solid organ transplantation
• Presence of evidenced congenital infections
• Women who didn't give consent

Healthy young mothers who had vaginal delivery at least one week ago will be included

It is planned to share individual participant data in terms of changes in microbiota composition of breast milk with different storage conditions. Special data about identity of participants will be kept blinded.

Data about Clinical Study Report will become available after all samples are obtained, analyzed and study completed. The data will be available for 12 months period.

The IPD will be uploaded to the registration system, any investigator will be able to access IPD