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Infectious and toxicological risks are the main potential hazards that operators of Human Milk Banks (HMBs) encounter and must eliminate. HMBs are trying to implement procedures that allow to manage and sanitize human milk without altering significantly its nutritional and biologically protective components, obtaining a product characterized by a valid balance between safety and biological quality. The history of human milk processing is linked to the origins of HMBs themselves. And although other forms of sterilization were used originally, pasteurization soon became the recognized most effective means for sanitizing milk: all the milk that arrives at the HMB must be pasteurized. Holder pasteurization (HoP) is the most used methodology, and it is performed using low temperature and long time (+62.5°C for 30 min). With HoP some bioactive milk components are lost to varying degrees, but many other precious bioactive compounds are completely or partially preserved. To improve the quality of human milk processed by HMBs, maintaining in the meantime the same microbiological safety offered by HoP, new technologies are under evaluation. At present, High-Temperature Short-Time pasteurization (HTST) and High-Pressure Processing are the most studied methodologies. HTST is already utilized in some HMBs for daily practical activity and for research purposes. They seem to be superior to HoP for a better preservation of some nutritional and biologically protective components. Freeze-drying or lyophilization may have advantages for room temperature storage and transportation. The aim of this study is to evaluate the advancement regarding the processing of DHM with a literature search from 2019 to 2022. The effects of the new technologies on safety and quality of human milk are presented and discussed. The new technologies should assure microbiological safety of the final product at least at the same level as optimized HoP, with an improved preservation of the nutritional and bioactive components of raw human milk., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Moro, Girard, Peila, Garcia, Escuder-Vieco, Keller, Cassidy, Bertino, Boquien, Buffin, Calvo, Gaya, Gebauer, Lamireau, Lembo, Picaud, Wesolowska, Arslanoglu, Cavallarin and Giribaldi.)

Background: Since human milk contents does not meet the high need of very low birth weight infants, fortification of breast milk is a standard practice for this population. As donkey milk has been long considered for children allergic to cow's milk proteins due to its low allergic properties, a new donkey milk-derived fortifier (DF) has been recently evaluated as a valid alternative to bovine milk-derived fortifier (BF). It seems to improve feeding tolerance when compared with standard BF, with similar neurodevelopmental and auxological outcome at 18 months of age. The aim of this study is to evaluate the development of allergic manifestations occurring in the population of the "Fortilat Trial" at 6-8 years of age. Methods: Allergic manifestations were assessed by an ad hoc questionnaire administered to families. The occurrence of asthma, allergic rhinitis and oculorhinitis, rashes and atopic dermatitis, food allergies, accesses to an emergency department for allergic reactions, and the need of antihistamine have been investigated. Results: In total, 113 infants were enrolled in the study (BF arm: n  = 60, DF arm: n  = 53). No difference in risk was observed between the two groups for all the considered outcomes. In conclusion, our data suggest that DF does not impact the development of allergic manifestations in the first years of life. Clinical Trial Registration number: ISRCT N70022881.

The nutritional adequacy of human milk (HM) from vegan/vegetarian mothers has been a matter of debate, and a variety of recommendations regarding the eligibility of these mothers as human milk donors exists. According to the latest evidence, HM from vegans/vegetarians is similar in its composition to that from omnivores, however, some differences may be observed regarding vitamin B 12 and omega-3 fatty acids concentrations. Maternal supplementation of these compounds has been proven effective in increasing their HM concentration. With this survey, we aimed to explore the practices currently in use in European human milk banks (HMBs) regarding potential donors following vegan/vegetarian diets. The online survey was distributed to European HMBs between January and July 2022. A total of 188 HMBs were contacted, and 118 replied (response rate 63%). Vegan and vegetarian mothers were recommended supplements of vitamin B 12 to be accepted as donors in 27% and 26% of HMBs, respectively. Additional omega-3 fatty acid supplementation was required in 8% HMBs. In the remaining HMBs, these mothers were either systematically excluded or included regardless of supplementation. The dosage of the recommended supplements was extremely variable. Fifty-one percent of HMBs were following recommendations to guide their practice, national or local recommendations mainly. Great variability in European HMBs practices towards potential donors following vegan/vegetarian diets exists. Some of these practices can result in loss of donors and/or in potential nutritional deficiencies. Standardised evidence-based recommendations on this issue and their implementation in daily HMB practice are needed., (© 2023 The Authors. Maternal & Child Nutrition published by John Wiley & Sons Ltd.)

Human milk (HM) is considered the best source of nutrition for infant growth and health. This nourishment is unique and changes constantly during lactation to adapt to the physiological needs of the developing infant. It is also recognized as a potential route of transmission of some viral pathogens although the presence of a virus in HM rarely leads to a disease in an infant. This intriguing paradox can be explained by considering the intrinsic antiviral properties of HM. In this comprehensive and schematically presented review, we have described what viruses have been detected in HM so far and what their potential transmission risk through breastfeeding is. We have provided a description of all the antiviral compounds of HM, along with an analysis of their demonstrated and hypothesized mechanisms of action. Finally, we have also analyzed the impact of HM pasteurization and storage methods on the detection and transmission of viruses, and on the antiviral compounds of HM. We have highlighted that there is currently a deep knowledge on the potential transmission of viral pathogens through breastfeeding and on the antiviral properties of HM. The current evidence suggests that, in most cases, it is unnecessarily to deprive an infant of this high-quality nourishment and that the continuation of breastfeeding is in the best interest of the infant and the mother., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In Europe, an increasing number of human milk banks (HMBs) collect donor human milk to feed preterm infants when their mother's milk is not available or not enough. Moreover, donor milk is a bridge to breastfeeding, with positive clinical and psychological advantages for both mother and infant. Italy, with 41 HMBs actively operating in 2022, has the highest number of HMBs in Europe. The process of human milk donation is complex, so activity of HMBs must be regulated according to well-established rules. The present recommendations have been prepared as a tool to standardize the organization, management, and procedures of HMBs operating in Italy and to determine the minimal essential requirements to establish new HMBs. This article covers all the aspects of human milk donation and human milk banking, including general recommendations, donor recruitment and screening, expression, handling and storage of donor human milk, milk screening, and milk treatment (pasteurization). A pragmatic approach was taken to drafting the recommendations. Items for which there was consensus or robust published evidence on which to base recommendations were included. When there were differences that could not be resolved by reference to published research, a statement of explanation based on the expert opinion of the authors (all members of the Italian Association of Human Milk Banks) was included. Implementation of these recommendations can contribute to promotion of breastfeeding., (© The Author(s) 2023. Published by Oxford University Press on behalf of the International Life Sciences Institute.)

Background and Aims: The content of fatty acids (FA) in human milk may be influenced by maternal nutrition. We evaluated the role of a Mediterranean diet in influencing the content of FA in human milk among 282 breastfeeding mothers participating in the MEDIDIET study., Materials and Methods: Mediterranean Diet Score (MDS), a composite index, was used to evaluate adherence to the Mediterranean diet. It includes 9 components (i.e., vegetables, fruit, cereals, legumes, monounsaturated to saturated FA ratio - MUFA/SFA, fish, dairy products, meat, and alcohol) and therefore potentially ranges from 0 (no adherence) to 9 (complete adherence) points. None of the mothers obtained the highest score due to the low alcohol consumption in the study population. Mothers were categorized in approximate tertiles of adherence: 0-3 (34.4%), 4 (26.2%), and 5-8 points (39.4%). The mean content of FA across categories of MDS was compared using ANOVA and test for trend., Results: A high adherence of breastfeeding mothers to the Mediterranean diet was associated with lower content of SFA in human milk (42.58 ± 4.36 for MDS = 0-3, 42.58 ± 4.89 for MDS = 4, and 40.92 ± 5.22% of fats for MDS = 5-8 points; p ANOVA and p for trend = 0.02). Conversely, a high adherence was associated with higher content of MUFA in human milk (43.27 ± 4.27 for MDS = 0-3, 43.27 ± 4.47 for MDS = 4, and 45.24 ± 5.22% of fats for MDS = 5-8 points; p ANOVA and p for trend < 0.01), ω-3 FA (1.07 ± 0.25 for MDS = 0-3, 1.22 ± 0.49 for MDS = 4, and 1.31 ± 0.51% of fats for MDS = 5-8 points; p ANOVA and p for trend < 0.01), and the major types of ω-3 FA (i.e., α-linolenic acid - ALA, eicosapentaenoic acid - EPA, docosahexaenoic acid - DHA, docosapentaenoic acid - DPA). These associations were mainly driven by the adherence to the vegetables, MUFA/SFA, fish, and dairy products components of the Mediterranean diet., Conclusion: A high adherence to the Mediterranean diet was associated with human milk showing a lower content of SFA and higher content of MUFA and ω-3 FA, including DHA. The Mediterranean diet may contribute in human milk production with higher content of specific FA which is directly involved in infant's neural and visual development, as reported by previous studies., Competing Interests: The statistical analyses were performed by the Department of Clinical Sciences and Community Health, Branch of Medical Statistics, Biometry and Epidemiology “G.A. Maccacaro”, Università degli Studi di Milano, Milan, Italy (MD, FB, and MF) without the involvement of the Sponsor. SE, EV, and BS were employees of Danone Nutricia Research, Utrecht, Netherlands. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Di Maso, Bravi, Ferraroni, Agostoni, Eussen, Decsi, Quitadamo, Tonetto, Peila, Profeti, Salvatori, Kazmierska, Decarli, Vacca, Bertino, Stahl and Moro.)

Background: The antiviral role of glycosaminoglycans in human milk (HM-GAGs) has been poorly investigated. They are highly sulfated polysaccharides, which were proposed to act as decoy receptors according to their structure. The aim of this study is to evaluate the antiviral potential and the mechanism of action of total and individual HM-GAGs against three pediatric clinically relevant viruses: respiratory syncytial virus (RSV), cytomegalovirus (HCMV), and rotavirus., Methods: HM-GAGs were isolated from HM and a library of individual GAGs, structurally related to HM-GAGs, was prepared. The antiviral activity of HM-GAGs and the impact of thermal treatment were investigated in vitro by specific antiviral assays., Results: We demonstrated that HM-GAGs are endowed with anti-HCMV and anti-RSV activity and that they act by altering virus attachment to cell. We clarified the contribution of individual HM-GAGs, showing a specific structure-related activity. We did not observe any alteration of HM-GAG antiviral activity after thermal treatment., Conclusions: We showed that HM-GAGs contribute to the overall antiviral activity of HM, likely exerting a synergic action with other HM antiviral agents. HM-GAGs can now be added to the list of endogenous factors that may reduce breast-milk-acquired HCMV symptomatic infections and protecting infants from respiratory tract infections by RSV., Impact: HM-GAGs have been poorly investigated for their antiviral action so far. We demonstrated that HM-GAGs are endowed with significant anti-HCMV and anti-RSV activity and that they are able to alter virus binding to the cell. The contribution of individual HM-GAGs is mainly exerted by the FMHep and is not based on a simple charge interaction between the virus and sulfate groups but involves a specific GAG structural configuration. Our results contribute to identifying the multiple factors synergically acting in mediating HM antiviral properties and to clarifying their specific mechanism of action., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Data about the regulatory approaches to donor human milk (DHM) in European countries are lacking. The aim of this study is to describe the various regulations of DHM within European countries, to assess its legislative context and its impact in relation to donor milk banking. We performed a cross-sectional survey using a semistructured online questionnaire addressing 29 national European milk-banking representatives from June 2020 to February 2021. Representatives of 26 national DHM services participated in this study. The legal classification and regulatory status of DHM were defined in 9 out of 26 areas of jurisdiction (35%) as either food product (n = 6), product of human origin according to a blood, tissue, cell regulation (n = 2), or medicinal product (n = 1). In the remainder, DHM remains unclassified. Most legislations did not provide a comprehensive framework concerning DHM and costs to cover milk bank operations were rarely reimbursed. In general, the lack of national legislative governance and the actual legislative regulations in place do not support the use of DHM in European countries. National medical guidelines for the use of DHM have been issued in only 11 countries. The current number and distribution of milk banks (n = 239) within participating countries may not provide an equitable access to DHM for eligible infants. These findings could guide stakeholders aiming to establish a regulatory framework for DHM., (© 2021 The Authors. Maternal & Child Nutrition published by John Wiley & Sons Ltd.)

Background: Lactation is a demanding period for women, and a good nutrition is crucial for optimal health of mothers and infants., Objectives: To provide new data and summarize the overall evidence on maternal nutrient intakes during lactation in developed countries, we present a systematic review (SR) of the literature and concurrently original results of the Italian MEDIDIET study. We compared nutrient intakes with dietary reference values (DRVs) proposed by the European Food Safety Authority., Methods: Studies were identified searching PubMed/Embase databases up to February 2020. Observational studies reporting at least energy and macronutrient intakes of healthy breastfeeding mothers who followed non-restricted and non-specific diets were included. Studies on populations with severe nutritional deficiencies were excluded. The MEDIDIET study enrolled 300 healthy breastfeeding mothers at 6 ± 1 wk postpartum. Usual diet was concomitantly evaluated through a validated and reproducible FFQ. Nutrient intakes were estimated using a food composition database., Results: Twenty-eight articles regarding 32 distinct study populations were included. Maternal nutrient intakes were generally in agreement across studies included in the SR and conforming to DRVs. Within micronutrients, vitamin D intake was below the recommendation. In the MEDIDIET study, mean intakes of energy (1950 ± 445 kcal/d), carbohydrates (270 ± 20.1 g/d), proteins (87.8 ± 20.1 g/d), and fats (65.6 ± 18.9 g/d) were similar to those observed in the SR. Moreover, observed intakes seemed to reflect the typical Mediterranean diet, with low intakes of carbohydrates, SFAs, and PUFAs and high intakes of MUFAs and vitamins. Conversely, protein intake was mainly derived from animal sources., Conclusions: This SR showed that nutrient intakes of breastfeeding mothers in developed countries are generally in line with DRVs despite different dietary patterns worldwide. Some nutritional deficiencies emerged, highlighting the need for additional nutritional advice. Mothers participating in the MEDIDIET study showed a nutritional profile in agreement with the Mediterranean diet., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Background: Provision of donor human milk is handled by established human milk banks that implement all required measures to ensure its safety and quality. Detailed human milk banking guidelines on a European level are currently lacking, while the information available on the actual practices followed by the European human milk banks, remains limited. The aim of this study was to collect detailed data on the actual milk banking practices across Europe with particular emphasis on the practices affecting the safety and quality of donor human milk., Materials and Methods: A web-based questionnaire was developed by the European Milk Bank Association (EMBA) Survey Group, for distribution to the European human milk banks. The questionnaire included 35 questions covering every step from donor recruitment to provision of donor human milk to each recipient. To assess the variation in practices, all responses were then analyzed for each country individually and for all human milk banks together., Results: A total of 123 human milk banks completed the questionnaire, representing 85% of the European countries that have a milk bank. Both inter- and intra-country variation was documented for most milk banking practices. The highest variability was observed in pasteurization practices, storage and milk screening, both pre- and post-pasteurization., Conclusion: We show that there is a wide variability in milk banking practices across Europe, including practices that could further improve the efficacy of donor human milk banking. The findings of this study could serve as a tool for a global discussion on the efficacy and development of additional evidence-based guidelines that could further improve those practices., Competing Interests: The authors have declared that no competing interests exist.

One of the main concerns in human milk banks (HMB) is the transmission of human cytomegalovirus (HCMV) that could be present in the milk of infected women. There are consistent data showing that this virus is destroyed by Holder pasteurization (62.5°C for 30 min), but there is a lack of information about the response of the virus to the treatment at lower temperatures in strict HMB conditions. In order to analyze the effectiveness of different temperatures of pasteurization to eliminate HCMV in human milk, a preliminary assay was performed incubating HCMV-spiked raw milk samples from donor mothers at tested temperatures in a PCR thermocycler and the viral infectivity was assayed on cell cultures. No signs of viral replication were observed after treatments at temperatures equal or >53°C for 30, 20, and 10 min, 58°C for 5 min, 59°C for 2 min, and 60°C for 1 min. These data were confirmed in a pasteurizer-like model introducing HCMV-spiked milk in disposable baby bottles. No viral infectivity was detected on cell cultures after heating treatment of milk for 30 min at temperatures from 56 to 60°C. Thus, our results show that by using conventional pasteurization conditions, temperatures in the range of 56-60°C are enough to inactivate HCMV. Consequently, we consider that, in order to provide a higher quality product, the current recommendation to pasteurize both mother's own milk and donated milk at 62.5°C must be re-evaluated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gayà, Rittà, Lembo, Tonetto, Cresi, Sottemano, Bertino, Moro, Calvo and Donalisio.)

(1) Background: Several studies have reported associations between maternal diet in terms of single foods or nutrients and human milk compounds, while the overall role of maternal diet and related dietary patterns has rarely been investigated. (2) Methods: Between 2012 and 2014, we enrolled 300 healthy Italian mothers, who exclusively breastfed their infant. During a hospital visit at 6 weeks postpartum, a sample of freshly expressed foremilk was collected and information on maternal dietary habits in the postpartum period was obtained through an interviewer-administered food frequency questionnaire. We applied principal component factor analysis to selected nutrients in order to identify maternal dietary patterns, and assessed correlations in human milk macronutrients and fatty acids across levels of dietary patterns. (3) Results: Five dietary patterns were identified, named "Vitamins, minerals and fibre", "Proteins and fatty acids with legs", "Fatty acids with fins", "Fatty acids with leaves", "Starch and vegetable proteins". These dietary patterns were correlated with some milk components, namely fatty acids, and in particular ω-3 and its subcomponents. (4) Conclusions: This study showed that overall maternal dietary habits during breastfeeding may influence human milk composition, suggesting the importance of adequate maternal nutrition during lactation not only for the mother herself but also to provide the infant with milk containing adequate amount and quality of nutrients for a balanced nutrition.

Background: It is known that breastfeeding protects the infant from enteric and respiratory infections; however, the antiviral properties of human milk against enteric and respiratory viruses are largely unexplored., Research Aims: To explore the antiviral activity of human preterm colostrum against rotavirus and respiratory syncytial virus and to assess whether the derived extracellular vesicle contribute to this activity., Methods: We used a cross-sectional, prospective two-group non-experimental design. Colostra were collected from mothers of preterm newborns ( N = 10) and extracellular vesicles were purified and characterized. The antiviral activity of colostra and derived extracellular vesicles were tested in vitro against rotavirus and respiratory syncytial virus and the step of viral replication inhibited by extracellular vesicles was investigated., Results: Each sample of colostrum and colostrum-derived extracellular vesicles had significant antiviral activity with a wide interpersonal variability. Mechanism of action studies demonstrated that extracellular vesicles acted by interfering with the early steps of the viral replicative cycle., Conclusion: We demonstrated the intrinsic antiviral activity of human colostrum against rotavirus and respiratory syncytial virus and we showed that extracellular vesicles substantially contribute to the overall protective effect. Our results contribute to unravelling novel mechanisms underlying the functional role of human milk as a protective and therapeutic agent in preterm infants.

Adequate nutrition is fundamental to neonatal survival and short-term outcomes, but it also has long-term consequences on quality of life and neurologic development of preterm infants. Donkey milk has been suggested as a valid alternative for children allergic to cows' milk proteins, due to its biochemical similarity to human milk; we, hence, hypothesized that donkey milk could be a suitable basis for developing an innovative human milk fortifier for feeding preterm infants. The aim of the current study was to extend the findings and to evaluate the neurodevelopmental outcomes at 18 months of corrected age of the infants enrolled in the clinical trial named "Fortilat". Infants born ≤1500 g and <32 weeks of gestational age were randomized to receive either a combination of bovine milk-based multicomponent fortifier and protein supplement or a combination of a novel multicomponent fortifier and protein supplement derived from donkey milk. The followed fortification protocol was the same for the two groups and the two diets were designed to be isoproteic and isocaloric. All infants enrolled were included in a developmental assessment program. The neurodevelopmental assessment was performed at 18 ± 6 months of corrected age. Minor and major neurodevelopmental impairment and General Quotient (GQ) at the Griffiths-II Mental Development Scale were considered. The GQ was considered both in continuous and as two classes: lower than and higher than (or equal to) a defined cutoff (GQcl). The difference in GQ and GQcl between the two arms was estimated using Mann-Whitney-Wilcoxon test or Fischer exact test, respectively, on the assumption of casual loss at follow-up. A further analysis was performed using generalized linear models. There were 103 children (bovine milk-derived fortifier arm = 54, donkey milk-derived fortifier arm = 49) included for the neurodevelopmental follow-up. All observations were included in the interval of 18 ± 6 months of corrected age. No significant difference was observed between the two arms in the incidence of neurologic sequelae and the GQs were similar in the two arms. Our results demonstrated no difference for the donkey milk-derived fortifier compared to standard bovine-derived fortifier regarding long-term neurodevelopmental outcomes.

Human milk fortification is a routine clinical practice for feeding preterm infants. We hypothesized that donkey milk can be a suitable basis for developing an innovative human milk fortifier. Our randomized controlled single-blind clinical trial, named "Fortilat", evaluated the feeding tolerance, growth and clinical short-term outcomes in a population of preterm infants fed with a novel multi-component fortifier and a protein concentrate derived from donkey milk. The aim of the current study is to extend the previous findings and to evaluate the auxological outcomes of the infants enrolled in the "Fortilat" trial at 18 months of age. In the previous trial "Fortilat", the fortification protocol followed was the same for the two groups, and the two diets were designed to be isoproteic and isocaloric. All infants enrolled in the trial were included in a premature infant developmental evaluation program consisting of hospital visits at 40 ± 1 weeks of postmenstrual age, and at 6, 12 and 18 months of corrected age. Weight, head circumference and length were expressed in z-score using neonatal Intergrowth21st and INeS charts at birth, and WHO 0-5 years growth charts at 18 months. 122 children (Bovine-arm = 62, Donkey-arm = 60) were included in this study. All the observations were recorded in the interval of 18 ± 3 months of the correct age. The two groups did not differ for head circumference, length or weight at 18 months of age. Our data show that fortifiers derived from donkey milk had not different long term auxological outcomes of standard bovine-derived fortifier, but the new donkey milk fortifier was well tolerated in our population.

Background: In the current SARS-Coronavirus-2 (SARS-CoV-2) pandemic little is known about SARS-CoV-2 in human milk. It is important to discover if breast milk is a vehicle of infection. Objective: Our aim was to look for the presence of SARS-CoV-2 RNA in the milk of a group of SARS-CoV-2 positive mothers from North-West Italy. Methods: This is a prospective collaborative observational study where samples of human milk from 14 breastfeeding mothers positive for SARS-CoV-2 were collected. A search of viral RNA in breast milk samples was performed by RT-PCR (Real-Time reverse-transcriptase-Polymerase-Chain-Reaction) methodology tested for human milk. All the newborns underwent a clinical follow up during the first month of life or until the finding of two sequential negative swabs. Results: In 13 cases the search for SARS-CoV-2 RNA in milk samples resulted negative and in one case it was positive. Thirteen of the 14 newborns were exclusively breastfed and closely monitored in the first month of life. Clinical outcome was uneventful. Four newborns tested positive for SARS-CoV-2 and were all detected in the first 48 h of life, after the onset of maternal symptoms. Also the clinical course of these 4 infants, including the one who received mother's milk positive for SARS-CoV-2, was uneventful, and all of them became SARS-CoV-2 negative within 6 weeks of life. Conclusion: Our study supports the view that SARS-CoV-2 positive mothers do not expose their newborns to an additional risk of infection by breastfeeding., (Copyright © 2020 Bertino, Moro, De Renzi, Viberti, Cavallo, Coscia, Rubino, Tonetto, Sottemano, Campagnoli, Soldi, Mostert, Cresi, Lembo and Collaborative Research Group on SARS-CoV-2 in Human Milk.)

The benefits of human milk are mediated by multiple nutritional, trophic, and immunological components, able to promote infant's growth, maturation of its immature gut, and to confer protection against infections. Despite these widely recognized properties, breast-feeding represents an important mother-to-child transmission route of some viral infections. Different studies show that some flaviviruses can occasionally be detected in breast milk, but their transmission to the newborn is still controversial. The aim of this study is to investigate the antiviral activity of human milk (HM) in its different stages of maturation against two emerging flaviviruses, namely Zika virus (ZIKV) and Usutu virus (USUV) and to verify whether HM-derived extracellular vesicles (EVs) and glycosaminoglycans (GAGs) contribute to the milk protective effect. Colostrum, transitional and mature milk samples were collected from 39 healthy donors. The aqueous fractions were tested in vitro with specific antiviral assays and EVs and GAGs were derived and characterized. HM showed antiviral activity against ZIKV and USUV at all the stages of lactation with no significant differences in the activity of colostrum, transitional or mature milk. Mechanism of action studies demonstrated that colostrum does not inactivate viral particles, but it hampers the binding of both flaviviruses to cells. We also demonstrated that HM-EVs and HM-GAGs contribute, at least in part, to the anti-ZIKV and anti-USUV action of HM. This study discloses the intrinsic antiviral activity of HM against ZIKV and USUV and demonstrates the contribution of two bioactive components in mediating its protective effect. Since the potential infectivity of HM during ZIKV and USUV infection is still unclear, these data support the World Health Organization recommendations about breast-feeding during ZIKV infection and could contribute to producing new guidelines for a possible USUV epidemic., Competing Interests: The authors have declared that no competing interests exist.

Fortification of human milk (HM) for preterm and very low-birth weight (VLBW) infants is a standard practice in most neonatal intensive care units. The optimal fortification strategy and the most suitable protein source for achieving better tolerance and growth rates for fortified infants are still being investigated. In a previous clinical trial, preterm and VLBW infants receiving supplementation of HM with experimental donkey milk-based fortifiers (D-HMF) showed decreased signs of feeding intolerance, including feeding interruptions, bilious gastric residuals and vomiting, with respect to infants receiving bovine milk-based fortifiers (B-HMF). In the present ancillary study, the urinary metabolome of infants fed B-HMF ( n = 27) and D-HMF ( n = 27) for 21 days was analyzed by 1 H NMR spectroscopy at the beginning (T0) and at the end (T1) of the observation period. Results showed that most temporal changes in the metabolic responses were common in the two groups, providing indications of postnatal adaptation. The significantly higher excretion of galactose in D-HMF and of carnitine, choline, lysine and leucine in B-HMF at T1 were likely due to different formulations. In conclusion, isocaloric and isoproteic HM fortification may result in different metabolic patterns, as a consequence of the different quality of the nutrients provided by the fortifiers.

Breast milk is a complex biofluid that nourishes infants, supports their growth and protects them from diseases. However, at the same time, breastfeeding is a transmission route for human cytomegalovirus (HCMV), with preterm infants being at a great risk of congenital disease. The discrepancy between high HCMV transmission rates and the few reported cases of infants with severe clinical illness is likely due to the protective effect of breast milk. The aim of this study was to investigate the anti-HCMV activity of human preterm colostrum and clarify the role of colostrum-derived extracellular vesicles (EVs). Preterm colostrum samples were collected and the EVs were purified and characterized. The in vitro anti-HCMV activity of both colostrum and EVs was tested against HCMV, and the viral replication step inhibited by colostrum-purified EVs was examined. We investigated the putative role EV surface proteins play in impairing HCMV infection using shaving experiments and proteomic analysis. The obtained results confirmed the antiviral action of colostrum against HCMV and demonstrated a remarkable antiviral activity of colostrum-derived EVs. Furthermore, we demonstrated that EVs impair the attachment of HCMV to cells, with EV surface proteins playing a role in mediating this action. These findings contribute to clarifying the mechanisms that underlie the protective role of human colostrum against HCMV infection.

Background: Feeding intolerance is a frequent diagnosis in very preterm infants. As seen in the FortiLat trial, human milk fortification with the new donkey milk-derived human milk fortifier (DF) seems to improve feeding tolerance in these infants. The aim of this study was to evaluate the effects of using the DF compared with bovine milk-derived fortifier (BF) on gastroesophageal reflux (GER) in very low birth weight (VLBW) infants., Methods: Over a total of 156 preterm infants were enrolled into the FortiLat trial (GA <32 weeks and birth weight <1500 g) and randomized into the BF arm or DF arm, and we selected all infants with clinical signs of GER and cardiorespiratory (CR) symptoms. All the infants underwent CR and multichannel intraluminal impedance and pH (MII/pH) monitoring associated with gastric ultrasound to evaluate GER and gastric emptying time., Results: 10 infants were enrolled, and 5 were in the DF arm. At MII/pH, infants enrolled into the DF arm showed a lower GER frequency than BF arm infants ( p = 0.036). Half gastric emptying time was similar in DF and BF arm infants ( p = 0.744)., Conclusion: The use of donkey-derived human milk fortifier reduced the GER frequency and consequently should be recommended in infants with feeding intolerance.

Oxysterols are cholesterol oxidation derivatives. Those containing an additional hydroxyl group on the side chain of the cholesterol molecule result from a physiological enzymatic synthesis and include the majority of oxysterols present in the circulation. Among these, 25-hydroxycholesterol (25OHC) and 27-hydroxycholesterol (27OHC) are characterized by a broad antiviral activity and are now considered involved in the innate immune response against viruses. Despite the emerging role of these sterols in the innate antiviral defences, no data are available on their presence in human breast milk (BM) to date. In this study, we investigated the content of oxysterols of enzymatic synthesis in BM of twelve donor mothers at different stages of lactation (i.e. in colostrum, transitional milk, and mature milk) by gas chromatography-mass spectrometry analysis. The side-chain oxysterols 25OHC, 27OHC, and 24S-hydroxycholesterol (24SOHC) were actually present in BM in all stages of lactation, but the concentration of 27OHC showed a remarkable peak in colostrum. Antiviral assays revealed that all the colostrum samples contained 27OHC concentrations that were active in vitro against two relevant pediatric viral pathogens: the human rotavirus and the human rhinovirus. Overall, this study discloses new antiviral components of BM and suggests a passive transfer of these protective factors to the infant via breastfeeding, especially in the first few days of lactation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Evidence indicates that human milk (HM) is the best form of nutrition uniquely suited not only to term but also to preterm infants conferring health benefits in both the short and long-term. However, HM does not provide sufficient nutrition for the very low birth weight (VLBW) infant when fed at the usual feeding volumes leading to slow growth with the risk of neurocognitive impairment and other poor health outcomes such as retinopathy and bronchopulmonary dysplasia. HM should be supplemented (fortified) with the nutrients in short supply, particularly with protein, calcium, and phosphate to meet the high requirements of this group of babies. In this paper the European Milk Bank Association (EMBA) Working Group on HM Fortification discusses the existing evidence in this field, gives an overview of different fortification approaches and definitions, outlines the gaps in knowledge and gives recommendations for practice and suggestions for future research. EMBA recognizes that "Standard Fortification," which is currently the most utilized regimen in neonatal intensive care units, still falls short in supplying sufficient protein for some VLBW infants. EMBA encourages the use of "Individualized Fortification" to optimize nutrient intake. "Adjustable Fortification" and "Targeted Fortification" are 2 methods of individualized fortification. The quality and source of human milk fortifiers constitute another important topic. There is work looking at human milk derived fortifiers, but it is still too early to draw precise conclusions about their use. The pros and cons are discussed in this Commentary in addition to the evidence around use of fortifiers post discharge.

Objectives: To develop recommendations from the European Milk Bank Association (EMBA) for the establishment and operation of human milk banks (HMB) in Europe. Method: A working group comprising members of the EMBA was convened in 2015 to develop Europe-wide recommendations for milk banks. Each member had experience of guideline development and/or milk banking operations. An initial survey was agreed using collated published global recommendations. A total of 108 potential recommendations were included in the survey; responders noted which were included in their national guidelines. The responses were collated, compared, and discussed and the group determined where there was consensus and where substantial or minor differences were identified. Where there was consensus or robust published evidence on which to base recommendations these were included. When there was no consensus and no clear evidence base, a statement of explanation based on collective expert opinion was agreed. Results: Published, internationally available guidelines with recommendations for human milk banks from France, Italy, and the UK, together with guidelines from Austria, Denmark, Germany, Norway, Slovakia, Spain, Sweden, and Switzerland were included as source materials. These covered: General recommendations; Donor recruitment and screening; Expression, handling, and storage of donor human milk (DHM); Pooling of DHM; Milk screening; Milk treatment (pasteurization); Delivery of DHM to recipients. Conclusions: Evidence based recommendations and consensus statements from the EMBA will now be published on the EMBA website to assist in the safe establishment and operation of HMBs throughout Europe. These have also been used to inform the chapter on human milk to be included in the 2019 edition of the Guide to the quality and safety of tissues and cells for human application , published by the European Directorate for the Quality of Medicines & HealthCare (EDQM).

Background: A mother's own milk (MOM) is the gold standard for the feeding and nutrition of preterm and full term infants. When MOM is not available or there is not enough, donor human milk (DHM) should be used. Milk delivered to Human Milk Banks (HMBs) should be pasteurized to inactivate viral and bacterial agents. Currently, a pasteurization process at 62.5°C for 30 min (Holder pasteurization, HoP) is recommended in all international HMBs guidelines. State of the art: It is known that HoP affects some of the nutritional and biological components of human milk. Studies have demonstrated that temperature cycle in HoP is not always controlled or calibrated. A better check of these parameters in the pasteurizers on the market today may contribute to an improvement of the quality of HM, still maintaining some of the negative effects of the heat treatment of human milk. So, food industry, and dairy industry in particular, are evaluating innovative methodologies alternative to HoP to better preserve the nutritional and biological properties of fresh human milk, while assuring at least the same microbiological safety of HoP. The most studied processing techniques include High-Temperature-Short-Time (HTST) pasteurization, High Pressure Processing (HPP), and Ultraviolet-C (UV-C) irradiation. HTST is a thermal process in which milk is forced between plates or pipes that are heated on the outside by hot water at a temperature of 72°C for 5-15 s. HPP is a non-thermal processing method that can be applied to solid and liquid foods. This technology inactivates pathogenic microorganisms by applying a high hydrostatic pressure (usually 300-800 MPa) during short-term treatments (<5-10 min). UV irradiation utilizes short-wavelength ultraviolet radiation in the UV-C region (200-280 nm), which is harmful to microorganisms. It is effective in destroying the nucleic acids in these organisms, so that their DNA is disrupted by UV radiation. Aim: The aim of this paper is to present the EMBA recommendations on processing of HM, based on the most recent results obtained with these new technologies. Conclusions: Although research on the most promising technologies that will represent an alternative to HoP (HTST, HPP, UV-C) in the future is progressing, it is now important to recognize that the consistency and quality assurance of the pasteurizers on the market today represent a fundamental component that was previously lacking in the Holder approach.

Objectives: The purpose of the present randomized controlled clinical trial was to compare the use of donkey milk-derived fortifier (DF) with commercial bovine milk-derived fortifier (BF) in very preterm or very-low-birth-weight newborns, in terms of feeding tolerance., Methods: This trial included 156 newborns born at <32 weeks of gestational age and/or with a birth weight ≤1500 g. Newborns were randomized 1:1 to receive enteral feeding with either a BF-arm, or a new, DF-arm for 21 days. The fortification protocol was the same for both study arms, and the 2 diets were designed to be isoproteic and isocaloric. Feeding tolerance was assessed by a standardized protocol., Results: The risk of feeding intolerance tended to be lower in DF-arm than in BF-arm, with a relative risk reduction of 0.63 (95% confidence interval: -0.29, +0.90). The mean number of episodes per newborn of feeding intolerance and feeding interruptions (any duration) were consistently lower in the DF-arm than in the BF-arm. Episodes of bilious gastric residuals and vomiting were significantly lower in the DF-arm. Time needed to reach full enteral feeding (150 mL · kg · day) and daily weight increase between the first day of exclusive enteral feeding (ie, without administering intravenous fluids) and discharge were similar in the BF- and DF-arms., Conclusions: These results suggest that DF improve feeding tolerance when compared with standard bovine-derived fortifiers, with a similar auxological outcome.

Objectives: This study aimed to investigate the anti-human cytomegalovirus (CMV) activity of milk from seropositive and seronegative mothers of preterm infants and to analyze its changes throughout the different stages of lactation and after Holder pasteurization, a procedure adopted by donor human milk banks., Methods: Eighteen mothers of preterm infants were enrolled in the study. Colostrum, transitional milk, and mature milk samples were collected and tested for anti-CMV activity. Depletion of immunoglobulins A from milk samples was carried out by jacalin resin. Pools of milk samples were pasteurized according to Holder technique., Results: All samples were endowed with anti-CMV activity, although to a different extent. In CMV IgG-positive mothers, colostra were significantly more active than the transitional milk and mature milk samples. Moreover, they were more potent than colostra from seronegative mothers. Immunoglobulins A depletion in colostra from IgG-positive mothers resulted in a partial loss of anti-CMV activity. Holder pasteurization significantly reduced the antiviral activity., Conclusions: Human milk is endowed with anti-CMV activity and its potency may vary depending on the stage of lactation and the serological status of the mother. This biological property could partially neutralize CMV particles excreted in the milk of CMV IgG-positive mothers thus reducing the risk of transmitting infectious viruses to the infant.

Holder pasteurization (62. 5°C for 30 min) is recommended by all international human milk bank guidelines to prevent infections potentially transmitted by donor human milk. A drawback is that it affects some human milk bioactive and nutritive components. Recently, High Temperature-Short Time (HTST) pasteurization has been reported to be a valuable alternative technology to increase the retention of some biological features of human milk. Nevertheless, to date, few data are available about the impact of pasteurization methods other than Holder on the antiviral activity of human milk. The present study was aimed at evaluating the antiviral activity of human milk against a panel of viral pathogens common in newborns and children (i.e., herpes simplex virus 1 and 2, cytomegalovirus, respiratory syncytial virus, rotavirus, and rhinovirus), and at assessing the effect of Holder and HTST pasteurization on milk's antiviral properties. The results indicate that human milk is endowed with antiviral activity against all viruses tested, although to a different extent. Unlike the Holder pasteurization, HTST preserved the inhibitory activity against cytomegalovirus, respiratory syncytial virus, rotavirus and herpes simplex virus type 2. By contrast, both methods reduced significantly the antiviral activities against rhinovirus and herpes simplex virus type 1. Unexpectedly, Holder pasteurization improved milk's anti-rotavirus activity. In conclusion, this study contributes to the definition of the pasteurization method that allows the best compromise between microbiological safety and biological quality of the donor human milk: HTST pasteurization preserved milk antiviral activity better than Holder.

Background: Fortification of human milk is a standard practice for feeding very low birth weight infants. However, preterm infants often still experience suboptimal growth and feeding intolerance. New fortification strategies and different commercially available fortifiers have been developed. Commercially available fortifiers are constituted by a blend of ingredients from different sources, including plant oils and bovine milk proteins, thus presenting remarkable differences in the quality of macronutrients with respect to human milk. Based on the consideration that donkey milk has been suggested as a valid alternative for children allergic to cow's milk proteins, due to its biochemical similarity to human milk, we hypothesized that donkey milk could be a suitable ingredient for developing an innovative human milk fortifier. The aim of the study is to evaluate feeding tolerance, growth and clinical short and long-term outcomes in a population of preterm infants fed with a novel multi-component fortifier and a protein concentrate derived from donkey milk, in comparison to an analogous population fed with traditional fortifier and protein supplement containing bovine milk proteins., Methods: The study has been designed as a randomized, controlled, single-blind clinical trial. Infants born <1500 g and <32 weeks of gestational age were randomized to receive for 21 days either a combination of control bovine milk-based multicomponent fortifier and protein supplement, or a combination of a novel multicomponent fortifier and protein supplement derived from donkey milk. The fortification protocol followed is the same for the two groups, and the two diets were designed to be isoproteic and isocaloric. Weight, length and head circumference are measured; feeding tolerance is assessed by a standardized protocol. The occurrence of sepsis, necrotizing enterocolitis and adverse effects are monitored., Discussion: This is the first clinical study investigating the use of a human milk fortifier derived from donkey milk for the nutrition of preterm infants. If donkey milk derived products will be shown to improve the feeding tolerance or either of the clinical, metabolic, neurological or auxological outcomes of preterm infants, it would be an absolute innovation in the field of feeding practices for preterm infants., Trial Registration: ISRCTN - ISRCTN70022881 .

Pasteurization, performed at 62.5°C for 30 minutes (holder pasteurization), is currently recommended in all international human milk banks guidelines, but it affects some human milk bioactive and nutritive components. The present systematic review is aimed at critically reviewing evidence on the suitability of human milk processing techniques other than holder pasteurization, both thermal and nonthermal, to ensure microbiological safety, and on the effects of these techniques on biologically active donor milk components. A systematic review of English and non-English articles using Medline, PubMed, Embase, SCOPUS, and CAB Abstracts, with no restriction in publication date was performed. Search terms included: human, breast, donor, or banked milk, breastmilk, breast fed, breastfed, breastfeed; HTST, Flash, High Pressure, UV, ultrasonic or nonthermal; process, pasteuris, pasteuriz. Only primary research articles published in peer-reviewed journals were included, providing or not a comparison with holder pasteurized human milk, provided that the pasteurization technique was clearly described, and not intended for domestic use. Additional studies were identified by searching bibliographies of relevant articles. Twenty-six studies were identified as being relevant. Two examined both High Pressure Processing and High-Temperature-Short-Time pasteurization; 10 only examined High Pressure Processing; 10 only examined High-Temperature-Short-Time; 2 articles examined ultraviolet irradiation; 2 articles examined (thermo-)ultrasonic processing. The results indicate that data about safety for microbiological control are still scarce for most of the novel technologies, and that consensus on processing conditions is necessary for nonthermal technologies, before any conclusions on the qualitative and nutritional advantages of these techniques can be drawn.

When a mother's milk is unavailable, the best alternative is donor milk (DM). Milk delivered to Human Milk Banks should be pasteurized in order to inactivate the microbial agents that may be present. Currently, pasteurization, performed at 62.5 °C for 30 min (Holder Pasteurization, HoP), is recommended for this purpose in international guidelines. Several studies have been performed to investigate the effects of HoP on the properties of DM. The present paper has the aim of reviewing the published papers on this topic, and to provide a comparison of the reported variations of biologically-active DM components before and after HoP. This review was performed by searching the MEDLINE, EMBASE, CINHAL and Cochrane Library databases. Studies that clearly identified the HoP parameters and compared the same DM samples, before and after pasteurization, were focused on. A total of 44 articles satisfied the above criteria, and were therefore selected. The findings from the literature report variable results. A possible explanation for this may be the heterogeneity of the test protocols that were applied. Moreover, the present review spans more than five decades, and modern pasteurizers may be able to modify the degradation kinetics for heat-sensitive substances, compared to older ones. Overall, the data indicate that HoP affects several milk components, although it is difficult to quantify the degradation degree. However, clinical practices demonstrate that many beneficial properties of DM still persist after HoP.

Background: At present, the Italian Association of Donor Human Milk Banks (Associazione Italiana Banche del Latte Umano Donato, AIBLUD) consists of 31 milk banks, whose management is based on Italian Guidelines. In 2013, AIBLUD performed a systematic survey to collect data on the activities of banks operating in Italy in the years previous to this date., Objective: The purpose of this survey was to evaluate the operational procedures of Italian Human Milk Banks in order to identify both areas of strength and room for improvement., Methods: A questionnaire was utilized to obtain national data from the 28 banks active in the year 2012 in order to evaluate the number of donors, volume of human milk collected, and other information relating to the period 2007 to 2012., Results: In all, 89% of the banks (25/28) responded to the survey. Data received primarily concerned the number of donors, volume of milk collected, and average amount of milk from each donor in the period 2007 to 2012. It was evident that in 2012 human milk banks collected a higher volume of milk than in 2007. Further, the average amount of milk from each donor was higher. Most of the milk banks were following the Italian Guidelines for traceability, control of donors, bacteriological checks, method of pasteurization, storage, thawing, type of containers, and utilization of the Hazard Analysis and Critical Control Points system., Conclusion: This survey identified both areas of strength and room for improvement in the Italian human milk banks., (© The Author(s) 2015.)