ELOIZE DA SILVA ALVES

Título da Dissertação: OBTENÇÃO DE SORO DE LEITE HUMANO E CARACTERIZAÇÃO DA SUA COMPOSIÇÃO LIPÍDICA POR CG-DIC E ESI-MS

Orientador: Prof. Dr. Jesui Vergilio Visentainer

Data da Defesa: 24/02/2021

RESUMO GERAL

INTRODUCTION. Milk, by definition, originates from the mammary gland of female mammals as a source of food for newborns, aiding in the development of the organism and in survival. Human milk should be the exclusive food source during the first six months of a baby's life, and if breastfeeding is interrupted by some factors, the American Academy of Pediatrics recommends the use of pasteurized donated human milk. Human milk that does not meet the specifications must be disposed of directly in the sewage network, as stipulated in RDC / Anvisa n. 306/2004 (Brazil, 2004). In 2019, all Brazilian states presented reports of statistical data, provided by the database of the Brazilian network of human milk banks, which exposes an average disposal of 20.0 to 30.0%. The authors are not aware of any work that reuses this residual human milk for the development of coproducts; however, studies on this will be promising. Whey is a derivative of milk, light yellow to greenish in color, composed of lactose, soluble proteins, vitamins, lipids and minerals. It can be obtained from the separation / sedimentation of casein from serum milk proteins, by centrifugation (Silva et al., 2007) or ultracentrifugation (Lu et al., 2018). Among several studies in the literature, studies are exposed that assess protein and immunological composition referring to human whey, however there are no reports on its lipid composition.
OBJECTIVES. In order to avoid the disposal of human whey and apply it to infant feeding, the aim of this work was to characterize the human whey derived from human milk discarded by dirt from the Human Milk Bank of Maringá (Paraná, Brazil), through analysis of fatty acid (FA) composition, triacylglycerol (TAG) lipid profile and proximate composition.
MATERIAL AND METHODS. This study was approved by the Research Ethics Committee number 2.797.476, of Universidade Estadual de Maringá. Samples of raw mature human milk discarded by dirt were collected at a cooling temperature of 4 ºC, at the Human Milk Bank of Hospital Universitário de Maringá. Subsequently, human milk was homogenized and a pool was produced. It was packed in polyethylene packaging and stored at -18 °C for further development and analysis of human whey. Obtaining human whey: the milk was centrifuged at 1500 g for 10 minutes at 10 ºC to remove the fat layer. The skimmed human milk was ultracentrifuged at 6000 g for 30 minutes at 30 ºC to sediment casein. Assessment of centesimal composition, analyzes of moisture, ash, protein and carbohydrates by calculating the difference; and lipid content. The energy food value was expressed by the sum of the macronutrients that comprise it. Fatty acid methyl esters (FAME) were prepared by total lipids methylation for composition analysis in FA. And later analyzed in a gas chromatograph (GC, Trace Ultra 3300) with flame ionization detector (FID), the split injection mode was used in the proportion of 1:100. The FAMEs were identified by comparing the retention times of the constituent samples with analytical standards. The FA compositions were expressed as relative percentage. The lipids nutritional quality was assessed by 6 indices: atherogenicity index (AI), thrombogenicity index (TI) and proportion of FA hypocholesterolemic/hypercholesterolemic (H/H). Sum of the omega-6 family due to the omega-3 family, sum of polyunsaturated fatty acids (PUFA) due to the sum of saturated fatty acids (SFA), and sum of eicosapentaenoic (EPA) FA and docosahexaenoic (DHA) FA. The triacyglycerol (TAG) profile was obtained by direct infusion into a mass spectrometry (MS) using electrospray ionization (ESI) source. TAGs were assigned and estimated (%) through the LAMES platform, which is based on the mathematical algorithm that describes the distribution of FA in TAG molecules using the FA percentage determined by GC-FID. With the Lipid maps® database, it was possible to find a molecular formula for TAGs. The data from all other analyzes were submitted to statistical analysis of variance (ANOVA) and Tukey's test (p <0.05). All analyzes were compared to mature human milk (HM).
RESULTS AND DISCUSSION. The sample with the highest moisture was HW (91.56±0.07), while HM (89.11±0.13); the HW value is due to the removal of solid matter (fat and protein) from human milk during skimming. For percentage of ash, the values were: HM (0.15±0.01) and HW (0.15±0.02), there were no significant differences between it, as both samples have the same monerals amount. In the total protein content, the HM sample showed a value of 1.29±0.10, and the HW sample, there presented a decrease in the protein concentration (1.12±0.05), due to the casein precipitation, which caused a reduction in the total crude protein amount. As for lipids, a higher value for HM (3.23±0.13) in comparison to HW (0.93±0.13) was already expected due to the creaming process. For the carbohydrate, the results were HM (6.22±0.23) and HW (6.24±0.20). In the energy value evaluation, losses of the total value were verified, due to the loss of lipids and proteins during obtaining the HW; HM obtained a value of 59.10±0.32 and HW 37.80±0.31; however, for consumption, the minimum recommended amount for feeding infants is 25 Kcal/100 mL. 32 FAs were identified by the GC-FID. Among the FAs analyzed, oleic acid (O, 18:1n-9) was the majority for both samples; HM (31.32±0.49) and HW (32.28±0.42). Then, palmitic acid (P, 16:0) with HW (28.09±0.78) and HM (22.23±0.08). For PUFAs, the FA identified in greater quantity was linoleic acid (L, 18:2n-6), considered a strictly essential FA and precursor of the arachidonic acid (AA, 20:4n-6) also found in the samples. Other long-chain PUFAs have also been obtained, such as alpha-linolenic acid (aLn, 18:3n- 3), which is a precursor of the eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic (DHA, 22:6n-3). Therefore, both HM and HW maintain strictly essential FAs, its precursors and essential FAs. However, it is important to note that the essential FA content has increased in the co-product. FA C9,t11 and t10,C12, which are named conjugated linoleic acid, remained present in all samples. Once the serum was obtained, an increase in C9,t11 and a decrease in t10,C12 was identified. The HW sample showed the highest level of the saturated fatty acids sum (ΣSFA) (49.34±0.49), and HM (43.58±0.47). For monounsaturated fatty acids sum (ƩMUFA), the HW sample was predominant, with a value of 37.03±0.17%. Finally, for the polyunsaturated fatty acids sum (ƩMUFA) the HM sample stood out, obtaining a result of 22.43±0.04%. The atherogenicity index (AI) had the highest value for HM sample (0.98±0.02) and HW sample presented a value of 0.91±0.01; because of the ƩMUFA and n-3 values in the HW samples are higher in relation to HM; the 12:0 and 14:0 concentrations were also higher in the HM sample, corroborating with the result. The thrombogenicity index (TI) presented a value of 1.41±0.09 for HW sample and a lowest value for HM sample (1.07±0.01). There are no reference values in the literature for AI and TI, these indices indicate potential for platelet aggregation, therefore, low levels are desirable. The hypocholesterolemic/hypercholesterolemic (H/H) proportion, the value found were: HM sample (1.51±0.04) and a lowest value for the HW sample (1.35±0.04);
the H/H proportionality indicates the FA specific effects on cholesterol metabolism, values above 2.0 are desirable, as it leads to greater health benefits, preventing cardiovascular diseases. The values found are below 2.0 for all samples, becausa the ƩSFA were higher than the ƩMUFA, both values related to the maternal diet. The ratio of Ʃn-6 to n-3, has an acceptable proportion for the proper functioning of the organism is between 5 and 10; HM obtained 13.01 ± 0.17, outside the acceptable proportion, while the HW sample presented values within the indicated parameter (5.95 ± 1.02). This relationship is important because these FAs compete for the metabolic pathways of stretching and desaturation. PUFA/ SFA showed HM (0.51 ± 0.01) and HW (0.28 ± 0.01) results. Foods with an PUFA/SFA ratio below 0.45 were considered unhealthy because of their potential to induce increases in blood cholesterol; only the HM presented values above the mentioned value. Ʃ (EPA) + (DHA), HW (0.73 ± 0.13) and HM (0.20 ± 0.02); however, the presence of both FAs in the samples is extremely important. The Ʃn-6 toƩ n-3 ratio has an acceptable proportion for the proper organism functioning between 5 and 10; HM obtained 13.01±0.17, outside the acceptable proportion, while the HW sample presented values within the indicated parameter (5.95±1.02). This relationship is important because these FAs compete for the metabolic pathways of stretching and desaturation. PUFA/SFA showed HM (0.51±0.01) and HW (0.28±0.01) results. Foods with PUFA/SFA ratio below 0.45 are considered unhealthy because of its potential to induce increases in blood cholesterol; only the HM presented values above the mentioned value. Ʃ(EPA)+(DHA), HW (0.73±0.13) and HM (0.20±0.02); however, the presence of both FAs in the samples is extremely important. The TAG results show the HM and HW spectra obtained by direct infusion in ESI-MS in the m/z range from 530 to 1100, with the most intense ionic spectral peak present between m/z 876 and 877. In the HM spectrum, the ionic peaks are more intense compared to HW, since the lipids percentage in the HM sample is higher (3.23±0.13) than HW (0.93 ± 0.13). The 21 largest indexes m/z were found, with its respective TAGs, found in the region between m/z 792 to 916, by the LAMES Platform. Comparing the results obtained by the FAs composition, with the presented TAGs, it was possible to observe the frequency of oleic (O, 18:1n-9) and palmitic (P, 16:0) FAs in TAGs, both with highest concentrations in relation to the FAs. The TAGs percentage in the HW sample in relation to the HM sample varied due to its distribution in the fat globules, as the ultracentrifugation was performed, those present in the casein decreased its percentage [TAG+NH4]+ LaOP, MOP, PLP, MOO , PLO and OLO. While those associated with albumin, fraction soluble in serum ([TAG+NH4]+ PPP, POP, SPP, PVcO, POO, SLP, SOP, SPS, SOS, OOO, SLO, SOO, BhOP) showed a high percentage, justified because the lipids that remained in the liquid fraction had its percentage rebalanced.
CONCLUSIONS. It is possible to obtain a human milk co-product from milk discarded by the human milk banks; the human whey (HW). The results obtained revealed that the chemical composition underwent significant modifications since the HW was obtained from the HM, except for the percentage of ash and carbohydrates. Regarding the fatty acids composition, it was observed that strictly essential fatty acids, essential fatty acids and all other FAs found in HM remained present in HW, being extremely important, since these FAs are responsible for several benefits in the infants' health, as already demonstrated. Considering the lipid nutritional quality, both the AI and the TI presented adequate values for both samples, indicating a lipid food quality and its potential effects on the development of coronary diseases. Finally, the TAGs lipid profile showed variation in the samples analyzed, with higher percentage of saturated and monounsaturated fatty acids, which is important, as it assists in the infants’ digestion. Therefore, HW has the potential to be applied isolated or to be used in other foods.
Keywords: Human milk; Human whey; GC-FID; Fatty acids; ESI-MS; Triacylglycerol.

 

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