Orientador: Jesuí Vergílio Visentainer

Data da Defesa: 18/02/2022

INTRODUCTION. In exceptional situations where the neonate cannot be breastfed or receive their mother’s own milk (MOM), among the main recommendations is human milk (HM) donated from the Human Milk Bank (HMB). In Neonatal Intensive Care Units (NICU), MOM can be extracted and given for immediate use, without the quality control requirement. In order to reduce the risks of neonatal infection, the most common treatment in HM is Holder Pasteurization (HoP, 62.5 °C for 30 min). When HM is heated in temperatures higher than its physiological temperatures, the nutritional and immunological properties could be changed. Thus, in HMB, freeze-drying is seen as a promising alternative in the storage and preservation of HM, being effective in preserving nutritional properties.
AIMS. This study aiming at comparing the effect of three methods of conservation (pasteurization, freeze-drying, and pasteurization followed by freeze-drying) into the HM composition. Furthermore, evaluating the possibility of using freeze-dried raw HM when donated MOM, as well as using HM that would be discarded for scientific purposes.
MATERIAL AND METHODS. Mature HM was donated by the HMB. Subsequently, a pool was made and divided into 4 treatments: control, untreated human milk (HMC), pasteurized human milk (HMP), freeze-dried human milk (HMF), pasteurized and freeze-dried human milk (HMPF). HoP (62.5 °C for 30 min) process was performed on the raw HM to obtain the HMP and HMPF. HMF and HMPF were obtained with a SLH-50 lyophilizer, under vacuum of up to 50 μHg, at -55 °C condenser temperature and heating plate temperature of 40 °C. Subsequently, the powdered HM was reconstituted. The moisture, crude protein and total ash determinations were performed according to AOAC (1995). Total lipid content was analyzed according to Folch et al. (1957), and carbohydrate content was obtained by difference. The energy value was calculated by the equation of Fischer Fumeaux et al. (2019). The detection of coliform bacteria was performed according to Novak and Almeida (2002). The extraction of fatty acid methyl esters (FAME) from HM was performed by direct methylation according to the methodology described by Cruz-Hernandez et al. (2013). Subsequently, the FAME were separated using a gas chromatograph (GC) and a flame ionization detector (FID), and the identification was performed by comparing retention times with analytical standards. In relation to cytokines, GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNF-α contents were determined using the ProcartaPlex 10-plex Human Custom Kit. The results were subjected to statistical analysis of variance (ANOVA), and the triplicates of the samples were compared by Tukey's Test (P < 0.05) probability level.
RESULTS AND DISCUSSION. The data presented in this study indicate that pasteurization (HMP) and freeze drying (HMF), compared to raw HM (HMC), do not affect the macronutrient value and energy value. In the present research, proteins were less preserved with heat treatment followed by freeze-drying (HMPF), with a significant reduction (P < 0.05). Regarding the analysis of coliform bacteria, HMC and HMF were positive for total coliforms, while HMP and HMPF were negative. The results showed that the use of the equipment maintained the microbiological quality in relation to total coliforms of HM before the process. Thus, in case coliforms are detected, as in the study, it is necessary to perform heat treatment before processing, as done in HMPF. About the FA composition, oleic acid was the majority FA, with values ranging from 633.38 to 910.02 mg 100 g-1 of HM. The results indicate that pasteurization and pasteurization followed by freeze-drying did not significantly alter the concentration of any FA (P < 0.05). However, the fact that one sample had been pasteurized before freeze-drying (HMPF) and another sample had been freeze-dried only (HMF) may have caused changes in the internal structures and, consequently, in the physical properties of the fat molecules. According to the results, freeze-drying at 40 °C shelf temperature destabilized the fat and caused the separate and adherence to the bottle when reconstituted. About cytokines, temperature, storage and freeze-thaw cycles are factors that can affect the stability of them, since some cytokines are more unstable to these factors. Thus, the data suggest that the removal of water through the freeze-drying (HMF) did not affect the biological structure, being able to maintain the cytokine content. Moreover, the storage of HM in powder form also proved favourable in the conservation of cytokine content, as occurred with IL-4 and TNF-α in HMPF, while when submitted to the HoP process (HMP), it caused a significant decrease in IL-6, IFN-γ, TNF-α and IL-4 (P < 0.05).
CONCLUSIONS. This study is the first to evaluate the effects of each treatment on HM composition by comparing the effect of freeze-drying on raw HM. According to the results, each treatment caused a change in the composition of raw HM, with a significant decrease in total protein (HMPF), fatty acids (HMF) and some cytokines (HMP and HMPF). In relation to microbiological results, it is suggested that pasteurization remains the most viable alternative. Although MOM submitted only to freeze-drying maintains important bioactive compounds for the neonate, further studies using different shelf plate temperatures would be necessary to find optimal process conditions that maintain the FAs composition. In addition, it would be necessary to ensure that milking and handling of MOM were performed free of microbiological contamination, following the microbiological quality standards of HMB and with result of absence of coliform bacteria.
Key words: breast milk, processing, macronutrient, fatty acid, total coliforms.

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