Orientador: Prof. Dr. Jesuí Vergílio Visentainer

Data da Defesa: 26/05/2020



INTRODUCTION AND AIMS: In the hospital environment, the malnutrition affects approximately 50% of hospitalized patients and classified as deficiency, excess or imbalance of a wide range of nutrients, resulting in various clinical complications such as impairment of healing, impact on immune function, increased rates of postoperative infection, mortality, increased length of hospital stay and high hospital costs. Precocious nutritional diagnosis and adequate nutritional therapy reduce complications associated with malnutrition. Nutritional support can be given orally (oral nutritional supplements), as well as through enteral nutrition (tube feeding) and parenteral nutrition, which offer the possibility of increasing or guaranteeing adequate intake of nutrients in patients with insufficient oral food intake. Oral/enteral nutrition supplements enriched with fish oil are widely used for the recovery of the nutritional status of hospitalized patients, due to the omega-3 fatty acids content, however, they are targets of adulteration. In this way, the techniques traditionally used to evaluate lipid quality and quantity, the authenticity of addition of vegetable and / or animal oils to detect adulteration. Another important theme of our work is highlighted during childhood. In the childhood, o human milk (HM) is considered the gold standard for newborn infant feeding, once it contains all the nutrients, bioactive compounds, antioxidant potential and immunological factors indispensable for the newborn appropriate growth and development. The exclusive breastfeeding recommendation and importance of the NB during the first 6 months of life and continued until 2 years of age or more is well described in the literature due to the many short and long term health benefits However, some conditions may lead to difficulties in establishing and maintaining exclusive breastfeeding. Thus, donated human milk has become an efficient alternative and Human Milk Banks are specialized services responsible for HM collection, processing, quality control, storage and transportation activities to the final recipient. The general objective of this work is to evaluate the lipid composition of commercial enteral diets and the composition of powdered human milk obtained by different technologies. The specific objectives of the articles were: Article 1) To evaluate the lipid quality of five brands of oral / enteral nutritional supplements with the addition of fish oil, available on the Brazilian market (Brazil, Holland and Germany). Article 2) Assess the effect of pasteurization, lyophilization and spray-drying on the lipid, immunological and antioxidant potential of LH. Article 3) Evaluate the concentrations of interleukins (IL) -17A / F, IL-21 and IL-22 in human milk powder.

MATERIAL AND METHODS: Article 1) The fatty acids were quantified by gas chromatography–flame ionization detection, and the lipid profile was acquired by direct electrospray ionization–mass spectrometry, to assess how fatty acids are present in those samples. Article 2) A pool of mature (M), transition (T) and colostrum (C) raw HM underwent pasteurization, lyophilization and spray-drying processes. Fatty acid (FA) composition was evaluated by flame ionization detector gas chromatography (GC-FID), triacylglycerol profile by direct infusion electrospray ionization mass spectrometry (ESI-MS), concentrations of cytokines IL-4, IL-5, IL-10, IL-13, IFN-γ by flow cytometry and antioxidant capacity of the superoxide dismutase (SOD) and catalase (CAT) enzymes and reduced glutathione (GSH) levels in vitro. Article 3) A pool of 900 mL of raw human milk from each phase (colostrum, transitional and mature milk) of nine donors was submitted to pasteurization, lyophilization or spray-drying. The concentrations of the cytokines IL-17A/F, IL-21, IL-22 were analyzed by flow cytometry.

RESULTS AND DISCUSSION: Article 1) The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) contents found ranged from 1.67–19.23% and 2.90–25.88%, respectively, in the form of triacylglycerols. However, brand 1 presented the lowest levels of EPA + DHA in its lipid composition, and in three brands, the addition of medium chain triglycerides was identified. From the GC–FID data, it was possible to identify 12 fatty acids in samples 1, 2 and 3, 13 fatty acids in sample 4, and 9 fatty acids in sample 9. The content of the n-3 fatty acids EPA and DHA varied among the samples, in the range of 8.39–35.78%. Besides, among the five samples, three were exclusively sources of the 8:0 and 10:0 fatty acids, as a result of the addition of the medium-chain triacylglycerols (MCT) ingredient. Based on the ESI–MS results, it was observed that the fatty acids of those three samples were present in triacylglycerols (TAG) form. The lipids of the samples analyzed did not present a pattern of quality despite all the samples indicated to assist in the nutritional recovery of hospitalized patients. Article 2) Fatty acids (FAs), triglycerides, cytokines, CAT, SOD and GSH profiles were conserved after processing. A total of 35 FAs were identified and quantified in the samples, and the same classes of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) were found in the raw, pasteurized, lyophilized and spray-drying milk at all stages. Palmitic acid (16:0) presented the highest concentration in all samples. Hence, we observed that oleic acid (18:1n-9) was the highest concentration MUFA in all samples. In particular, the most important FAs in the HM are the PUFAs. The samples of linoleic acid (18:2 n-6; LA) after processing were larger in comparison to raw HM. Regarding arachidonic acid (20:4 n-6; AA), there was no significant difference between the processed samples and raw HM in the T and M phases. α-linolenic acid concentrations (18:3n-3, ALA), eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) indicated no statistical difference between the processed samples and raw HM in all phases. In our study, there was no statistically significant difference between the processed samples and raw HM in all phases of the conjugated linoleic acid (CLA-9c, 11t-18: 2 and 10t, 12c-18: 2). The TAGs with the highest intensities in the samples of C, T and M in the different processing in the range of m/z 878-904. The results are in agreement with the results obtained by the GC-FID analysis, where the 16:0, 18:1n-9, 18:2n-6, 20:0 and 22:0 FAs showed higher concentrations in the HM samples analyzed. This is the first study to evaluate the Th1 (IFN-γ) and Th2 (IL-4, IL-5, IL-10, IL-13) comparing its concentrations in pasteurized, lyophilized and spray drying HM. Highlighting the main finding, it was observed the stability of the levels of these cytokines after processing, in which no statistically significant difference was observed in the concentrations of cytokines between raw and processed HM. This study is a pioneer in the evaluation of antioxidant capacity in raw, pasteurized, lyophilized and spray-drying milk in the different phases of HM, that there was no significant difference in SOD activity of lyophilized and spray-dryied samples of all phases when compared to raw HM. There was no statistical difference between CAT activity of lyophilized samples from all phases when compared with raw HM. Article 3) This work is a pioneer in the evaluation of the Th17 cytokine profile (IL-17A/F, IL-21 and IL-22) comparing its concentrations in pasteurized, lyophilized and spray dried HM. Highlighting the main finding, the stability of these cytokines after processing, in which no statistically significant difference was observed in the concentrations of these cytokines between raw HM and the processed colostrum, transitional milk and mature milk.

CONCLUSIONS: Article 1) The lipid quality assessment of these supplements allows an informed choice of appropriate supplements for the nutritional support of patients. This work evaluated the lipid quality of five brands of oral/enteral nutrition supplements enriched with fish oil and available on the Brazilian marketThus, the lipid quality evaluation of these supplements by analytical methods are valuable since the identification and quantification of the fatty acids present in oral/enteral nutrition supplements favor a choice of a supplement for the adequate treatment of hospitalized patients. Article 2) In conclusion, HM powder ensures stability after processing. Article 3) Human milk powder may be an immunologically safe alternative to the human milk bank, since there was stability of the cytokines IL-17A/F, IL-21 and IL-22 even after pasteurization, lyophilization and spray-drying processes.

KEY-WORDS: docosahexaenoic acid, malnutrition, nutritional therapy, eicosapentaenoic acid, newborn, powdered human milk, human milk bank, immune response, processing, fatty acids, triacylglycerols, antioxidants, cytokines s, protein structure


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