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High Temperature-Short Time (HTST) pasteurization was proposed as an alternative to Holder pasteurization (HOP) to increase the retention of specific human milk (HM) bioactive proteins. The present study explored whether HTST and HOP differently affect peptide release during simulated preterm infant gastrointestinal digestion. Raw (RHM), HOP- and HTST- pasteurized HM were digested using an in vitro dynamic system, and the identified peptides were analyzed by mass spectrometry and multivariate statistics. Before digestion, 158 peptides were identified in either RHM, HTST- or HOP- HM, mostly (84.4%) originating from ?-casein (CASB). During gastric digestion, HOP-HM presented a greater number and more abundant specific CASB peptides. A delayed release of peptides was observed in RHM during the intestinal phase, with respect to both pasteurized HM. Although limited to gastric digestion, the HM peptidomic profile differed according to the pasteurization type, and the pattern of the HTST peptides showed a greater similarity with RHM.

baCkgrounD: Very preterm newborns (gestational age lower than 32 weeks) and Very low birthweight (VlbW) infants (birthweight lower than 1500 grams) currently represent the majority of patients cared in neonatal intensive Care units (niCu). the increase of the survival rate for these newborns, due to improvements in perinatal care, has opened new perspectives regarding their outcome and has a significant impact on their health status in adulthood. in these groups of infants, nutrition represents a fundamental factor not only for neonatal survival and short-term outcome, but also for long-term consequences and quality of life. the main issue is to ensure an adequate qualitative and quantitative nutrition, particularly in terms of protein intake, which is the main cause of post-natal growth deficit [1]. Human milk is the recommended food for all neonates including preterm infants [2]. breast milk alone, however, does not meet the recommended nutritional needs in preterm infants [3]. the most common strategy is to cope with potential nutrient deficits by supplementing breast milk with additional nutrients (mainly proteins and minerals) to satisfy the special nutritional requirements of these infants[4]. at present commercially available fortifiers are based on bovine milk (bM), whose protein intake has raised concerns because of its association with allergies [5] and a possible role as a trigger of intestinal inflammation in preterm neonates [6]. in previous studies we observed that donkey milk (DM) was well tolerated in a group of highly problematic cow's milk allergic children [7]. our hypothesis is that feeding preterm infants with HM supplemented with fortifiers derived from DM will improve the feeding tolerance. aiM. the purpose of the present randomized clinical trial is to compare the use of DM-derived fortifiers with commercial bMbased fortifiers in infants with birthweight =80ml/kg/day reached within the first 4 weeks of life. the neonates affected by severe gastrointestinal pathologies, chromosomal abnormalities or major malformations, metabolic diseases, intravascular disseminated coagulopathy, shock, patent ductus arteriosus requiring medical care or surgery at time of randomization, and severe renal failure were excluded. before the beginning of this study, approximately 45% VlbWi or preterm infants admitted to our niCu had at least one episode of feeding intolerance. a 25% reduction in the frequency of the primary endpoint was regarded as the minimum clinically important difference (MCiD): 62 infants per arm (planned study) had to be recruited to ensure an 80% study power, if the risk of type i error is set to the usual level of 5%. Since the occurrence of primary endpoint in our study population resulted to be much lower than that assumed in the protocol, and no adverse effect was observed, when the planned study size was achieved, it was decided to continue the enrollment until the end of the stock of fortilat (extended study). infants were randomized 1:1 by a software-generated list in two arms: bf-arm (bovine fortifier) and Df-arm (Donkey fortifier). observation period was defined as 21 days since the beginning of fortification. in bf-arm, the human milk was fortified with commercial multi-component fortifier (fM85 nestlè) and protein concentrate (protifar nutricia), derived from bovine milk, while in Df-arm with multi-component fortifier and protein concentrate derived from donkey milk (fortilat), produced by an ultrafiltrationprocess of pasteurized donkey milk in a pilot stainless steel. advancing of enteral feeds was strictly regulated according to the feeding protocol adopted in our niCu, based on the evaluation of signs of feeding intolerance. the primary endpoint was the occurrence of at least one episode of feeding intolerance, defined as interruption of enteral feeding for at least 8 consecutive hours during the observation period. We also evaluated the total number of feeding intolerance episodes and the number of feeding interruption episodes of any duration. Statistical analysis. in the intention-to-treat population (itt, all randomized infants) failure included the occurrence of at least one episode of feeding intolerance, neC, death, and transfered before 21 days of observation. the difference in the occurrence of failure (primary endpoint) between the two arms of the trials was tested with fisher's exact test. reSultS. a total of 124 neonates were enrolled in planned study (bf-arm n= 62; Df-arm n=62). further 32 infants were enrolled in extended study (bf-arm n= 79, Df-arm n=77). the primary risk of failure in the planned sample has resulted lower in Df-arm than in bf-arm with a relative risk reduction (rrr) [Ci(95%)] of 0.40 [-0.27; +0.72]. the trend of these results was similar in the extended sample: 0.46 [ 0.09; +0.73]. the mean [iC(95%)] of the total episodes of feeding intolerance and the mean [iC(95%)] of enteral feeding interruptions of any duration was lower in Df-arm (0.11 [0.01; 0.21] and 0.27 [0.12; 0.42] respectively) than in bf-arm (0.20 [0.07; 0.34] and 0.39 [0.20; 0.57] respectively). DiSCuSSion. our first results about donkey milk based fortifiers are promising. Compared to the bovine fortifiers, the donkey milk based fortifier seems to have a better feeding tolerance even if these results are not significant. When we extended the study including more patients (extended sample), we observed a further slight improvement in feeding tolerance in favor of the Df-arm. We can therefore hypothesize that increasing the sample size we would observe a significant better feeding tolerance in Df-arm. to confirm our data it will be useful to perform a multicenter study in order to increase the sample size. it will be interesting to analyze metabolic, auxological and neurodevelopmental outcomes in order to determine if the use of donkey milk fortifiers will have also long-term benefits.

Given its unique nutritional and functional advantages, human milk (HM) should be considered as the first choice for the nutrition of all infants, including preterm newborns. Since its protein, mineral and energy contents are not suitable to meet the high needs of very-low-birth-weight (VLBW) infants, HM should be fortified for these components. Fortification of HM is an important nutritional intervention in order to provide appropriate nutritional intake and appropriate growth. The standard fortification strategy has yielded inadequate protein intakes, resulting in slower growth as compared to preterm formulas. Improvement of outcomes depends on new fortification strategies, considering the large variability of HM composition. Individualized fortification, either targeted or adjustable, has been shown to be effective and practical in attaining adequate protein intakes and growth. Most commercially available multi-nutrient fortifiers and protein concentrates are derived from bovine milk (BM), which has a protein composition very different from that of HM. The use of BM proteins has been recently questioned for possible association with intestinal inflammation in VLBW infants. Recently, one HM-based fortifier was shown to be associated with lower necrotizing enterocolitis rates and lower mortality in extremely premature infants, compared to BM-based products. Other milk sources are currently under evaluation: a randomized, controlled, single-blind clinical trial, coordinated by the Neonatal Unit of the University of Turin in collaboration with the Italian National Research Council of Turin and the University of Cagliari, is being carried out to evaluate the adequacy of fortifiers derived from donkey milk for the nutrition of preterm infants.