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INTRODUCTION. Edible insects are considered a promising and sustainable alternative protein source for humans, able to meet the increasing world's nutritional needs. The possible diffusion of insect farming and commercialization in Europe, has prompted the scientific community to investigate the allergenicity risk associated to insect consumption (1,2). Considering that edible insects are generally consumed after thermal processing, it is crucial to assess how processing may modify the protein structure, thus altering the protein cross-reactivity. In this study, we present one of the first investigations on insect cross-allergenicity, in three groups of Italian patients with a convincing history of house dust mite and shrimp allergy and with primary respiratory and food sensitization to mealworm. METHODS. We investigated by immunoblotting, the effect of boiling (5 min at 100 °C) and frying (3 min at 180 °C in sunflower oil) on the IgE-binding capacities towards both soluble and insoluble proteins fractions from five edible insects (mealworm, buffalo worm, silkworm, cricket and grasshopper). The immune-reactive proteins were identified by LC-MS/MS. RESULTS. Our data suggest that, depending on the nature of the reactive protein, the cross-reactivity resulted to be affected in different ways according to the different types of processing. In general, thermal processing influences the protein solubility, resulting in a protein shift from water-soluble to water-insoluble fractions, a shift that is more marked after frying than after boiling. Among the immune-reactive proteins, tropomyosin shows to have an important role as a cross-allergen for house dust mite and shrimp allergic patients and to be heat-stable in both fried and boiled samples. Actually, tropomyosin is considered a pan-allergen in crustaceans, mollusks, mites and insects, due to the high similarity of its amino acid sequences among species (3,4). The larval cuticle protein seems to play a major role in the cross-reactivity of patients primarily sensitized to mealworm. CONCLUSION. On the basis of our results, the effects of processing appeared to be protein-, species- and treatment-specific. Therefore, for patients allergic to house dust mite, shrimp and mealworm the consumption of insects, even after thermal processing, requires some caution.

INTRODUCTION Thermal processing, commonly used during food production, may affect food allergenicity by inducing physical and/or chemical changes in proteins and lipids. However, due to the lack of detailed information on processing procedures, available evidences on this topic are presently contradictory. The aim of this study was to investigate how two different roasting techniques, hot air (HA) and infrared (IR), could modify the hazelnut seed structure, the protein profiles and solubility. Moreover, the ability of the IgEs from hazelnut allergic children to bind hazelnut proteins before and after roasting was evaluated. METHODS Experiments were designed on an Italian variety of C. avellana (Cv. Tonda Gentile delle Langhe) obtained from experimental fields. Two pilot-scale ovens infrared (IR) and hot air (HA) and two combinations of time and temperature for each oven were considered: 140 °C for 25 min (low temperature (LT)) and 170 °C for 17 min (high temperature (HT)) in HA; 140 °C for 12 min (LT) and 170 °C for 10 min (HT) in IR. RESULTS A decrease in hazelnut protein solubility was detectable following LT-IR roasting and it became more evident after HT processing, for both roasting types. The immuno-recognition decreased progressively from raw to high temperature processes. The IR-HT processing caused a quite complete disappearance of immune-recognition of hazelnut proteins. Interestingly, different allergens showed different responses to heat processing. The immune-reactivity of Cor a 9, Cor a 11 and Cor a 14 resulted to be stable until 140 °C, while Cor a 8 immuno-recognition was already reduced at 140 °C and completely lost at 170 °C. Differently, oil body (OB) associated protein immuno-recognition was increased after HA roasting at 140 °C whereas was reduced in the remaining treatments and disappeared in the IR-HT. At structural level, microscopical observations showed a cytoplasmic network disruption with an alteration of the structure of the protein bodies and of the cell wall organization. CONCLUSION IR oven is the least conservative processing, especially at high temperatures, both considering protein solubility and immuno-recognition. Our results could be useful for the development of clinical protocols for the process of oral desensitization of hazelnut allergic patients.

Donor human milk (DHM) represents the best alternative when mother’s own milk is not available,a common occurrence in Neonatal Intensive Care Units. Heat treatment of DHM is mandatory forsafety reasons. Holder pasteurization (HoP, 62.5°C-30’) is recommended by all human milk bankguidelines. Recent studies have demonstrated that HoP affects the digestion profile and behavior ofseveral human milk components. High Temperature-Short Time pasteurization (HTST, 71°C-15’’)is currently under evaluation as a promising alternative technology to limit the denaturation of somebiological compounds of raw human milk.ObjectiveThe aim of the present work was to assess whether the different types of pasteurization (HoP,HTST) impacted the digestive kinetics of human milk during in vitro dynamic digestion.MethodologyPooled raw HM (RHM) was collected and processed by using the two pasteurization techniques.The pasteurized samples and RHM were digested in vitro using preterm gastrointestinal conditions.Samples were collected at different digestion times. Undigested and digested milks samples werecharacterized for their particle size distribution (PSD), triglyceride content, protein and amino acid(AA) profiles.Main findingsDuring gastric digestion, both pasteurization methods modified PSD, as compared to RHM. Caseinswere rapidly hydrolyzed in the gastric phase unlike that for the whey proteins. Lactoferrin was hydrolyzedfaster in the pasteurized samples in comparison to RHM, in which lactoferrin was resistantto gastro-intestinal digestion. Heat-treatments, consequently, affected the intestinal release of someAA, and a higher bioaccessibility of AA was found for HTST, as compared to HoP. Concerning lipolysis,at any time of the intestinal digestion phase, the lipolysis of HoP samples was significantlylower (p < 0.05) than in both RHM and HTST samples.ConclusionThis work provides the first evidences about the differential impact of HoP and HTST pasteurizationtechniques on bioaccessibility of DHM nutrients and biological compounds, for preterm newborns.

IntroductionIn order to minimize the denaturation of bioactive compounds, which is known to affect donor humanmilk (HM) after the mandatory heat treatment (Holder pasteurization at 62.5°C, 30min – HoP), anumber of alternative processing techniques are currently being investigated. Recently, High Temperature-Short Time pasteurization (HTST, 72°C-15s) was found to limit the denaturation of somebioactive components of human milk.ObjectiveThe aim of the study was to investigate whether different types of pasteurization (HoP and HTST)may differently affect peptide release from human milk during preterm infant gastrointestinal digestion,by using a dynamic in vitro systemMethodologyPooled raw HM (RHM) from 5 donors was collected and processed in triplicate according to HoP andHTST. The pasteurized milks and RHM were digested in triplicate using a dynamic in vitro digestionsystem, mimicking the preterm physiology conditions. Samples were collected at different digestiontimes. Peptides from undigested and digested samples were analysed using a Q-Exactive massspectrometer and peptide abundance was subjected to multivariate statistics to unravel specificprofile trends.Main findingsPre-proteolysis occurred mostly on β-casein, from which originated more than 82% of the peptidesfound in undigested milk. During digestion, a differential behaviour between gastric and intestinalpeptide release was found by multivariate statistics. In particular, whereas in the gastric phase thepeptide pattern was more similar for HTST and RHM with respect to HoP, the opposite was found atthe beginning of the intestinal phase, while at the end of digestion no difference was found. Thesedifferences were mostly due to peptides released from β-casein, especially in the gastric phase.Some bioactive peptides from bile salt-stimulated lipase, caseins and lactoferrin presented significantlydifferent abundances between the different samples during intestinal digestion.ConclusionThe results indicate possible consequences of the different pasteurizations on the biological activityof donor’s human milk.

The donkey population in Europe had been experiencing a decline over the past twenty years, due to the growing automation in agriculture, and to the depopulation of rural districts. However, the European donkey population is now increasing, thanks to the interest in donkey's milk (DM). Donkeys as a dairy species have been known since ancient times, also for DM beneficial properties in skin care. In the late nineteenth century, DM was successfully used for feeding orphan infants in Paris (Salimei, 2012). More recently, donkey's milk has been receiving a growing interest as food for sensitive consumers, such as children with cow's milk protein allergy and preterm infants. Clinical studies have indicated that DM can be successfully used as an alternative to the available hypoallergenic formulas for infants suffering from cow milk protein allergy and as a human milk supplement for preterm infants. DM has been shown to be well tolerated in 80-92% of children allergic to cow milk (Monti et al.,2007, Monti et al., 2012, Barni et al. 2018). In preterm infants, the fortification of human milk with DM instead of cow milk, led to a significant improvement of the episodes of feeding intolerance during their stay in the Neonatal Intensive Care Unit (Coscia et al., 2018). The biochemical basis for the DM nutritional adequacy in infants and children is discussed. DM protein and lipid fractions show a substantial similarity to that of human milk (Bertino et al., 2011). DM has a total protein content which is more similar to human milk than cow milk. The comparative analysis of human, donkey and cow milk proteome has revealed that the also protein profile is more similar among human and DM. DM is characterized by a low casein content, with values very close to that found in human milk (Polidori et al., 2015). The major cow milk allergens are either absent, present in traces or they are very different in term of aminoacid sequence in DM. The fat content of DM is lower than the fat content of human milk and this difference is associated with low energy content, thus representing a main limitation to its use as a nutritional source for children who are allergic to cow milk protein during the first year of their life. However, the lipid fraction of DM has shown a more favourable fatty acid composition than that of the milk fat of ruminants, because it is richer in polyunsaturated FA (PUFA). Fat in DM has shown a higher concentration of C18:3n-3 and n-3 FA, and a lower saturated FA (SFA) content than cow milk, as well as a lower n-6 to n-3 FA ratio (Gastaldi et al., 2011).

Introduction: It is known that thermal processing, commonly used during food production, may affect food allergenicity by inducing physical and/or chemical changes in macronutrients. However, due to the lack of detailed information on processing procedures, available evidences on this topic are presently contradictory. This study aims to evaluate the effects of different types of roasting on hazelnut protein solubility and immune reactivity. Methods: We investigated the IgE-binding capacities of both soluble and insoluble hazelnut protein fractions from C. avellana (Cv. Tonda Gentile delle Langhe), before and after roasting using two different roasting techniques: hot air (HA) as a ''traditional method," and infrared (IR), as an ''innovative method". Two combinations of time and temperature for each oven were considered: 140°C for 25min (LT) and 170°C for 17 min (HT) in HA; 140°C for 12 min (LT) and 170°C for 10 min (HT) in IR. Total protein and oil body associated protein extracts were used for immunoblotting experiments with allergic patient sera. Results: A decrease in hazelnut protein solubility was detectable following LT-IR roasting and it became more evident after HT processing, for both roasting types. Concerning allergenicity, the raw hazelnut proteins extract was the most immune reactive, followed by LT-HA and LT-IR processed hazelnut that showed a very similar immune reactive pattern. The HA-HT treatment resulted in a low, although detectable, immune reactivity, while the IR-HT processing caused a quite complete disappearance of immune reactivity of hazelnut proteins. The different hazelnut allergens (Cor a 8, 9, 11, 12, 13, 14, 15) were differently affected by thermal treatments, in accordance with their thermal stability, as already reported by literature. Discussion: HA oven is the most conservative processing, especially at low temperatures, both considering protein solubility and immune-reactivity. Both the HT treatments demonstrated to reduce immune-recognition of hazelnut proteins by allergic patient sera.

Introduction. Edible insects are currently being evaluated as an alternative and sustainable protein source for humans in order to meet the nutritional needs of the growing world population. Specific risk assessment for edible insects is necessary in Europe, where the consumption of insects is an emerging practice. The allergenic potential associated to entomophagy has to be investigated since the introduction of this new food source can cause IgE-mediated cross reactions in patients allergic to crustaceans and mites, that belong to the same phylum of insect. Methods. Five edible insects were investigated: buffalo worm (Alphitobius diaperinus), mealworm (Tenebrio molitor), silkworm (Bombyx mori); cricket (Gryllodes sigillatus) and grasshopper (Locusta migratoria). The soluble and insoluble proteins were extracted from raw and processed (boiled and fried) insects and the protein profiles were analyzed by SDS-PAGE. The IgE cross-reactivity of shrimp (n=8) and house dust mite (n=28) Italian allergic patients were investigated by immunoblotting followed by LC-MS/MS analysis for immune-reactive proteins identification. Results. Buffulo worm, mealworm and cricket showed similar protein profiles for raw and boiled extract, while grasshopper and silkworm differed for both processing methods. For all insects, proteins aggregation increased with increasing processing temperatures, causing an enrichment of the insoluble protein fraction. Preliminary results on the immune-recognition of insect proteins by the sera IgEs of patient allergic to shrimp and mites indicate that the major allergens of shrimp and house dust mite, such as tropomyosin and arginine kinase, are cross-reactive in insects. Furthermore, both processing methods show to affect protein reactivity in an insect-dependent manner. Discussion. The evidence of cross-reactivity of IgEs from patients allergic to shrimp and house dust mite towards differently processed edible insect protein extracts confirms the issue of possible risk associated to insects consuming.

Preterm and low birth weight infants require specific nutrition to overcome the accumulated growth deficit, and to prevent morbidities related to postnatal growth failure. In order to guarantee an adequate nutrient-intake, mother's milk, when available, or donated human milk, are usually fortified with additional nutrients, in particular proteins. Fortification with processed ingredients may result in additional intake in oxidative compounds, deriving from extensive heat treatments that are applied during processing. This study aims to compare the in vitro antioxidant activity and oxidative compound content conveyed by different preterm infant foods and fortifiers, namely raw and pasteurized human milk, two different preterm infant formulas, two bovine milk-based fortifiers and two experimental donkey milk-based fortifiers. The antioxidant activity of the infant foods and human milk fortifiers considered in the present research were evaluated as Trolox equivalent antioxidant capacity (TEAC), radical scavenging activity on the radical DPPH and in vitro antioxidant activity using red blood cells as indicators (CAA-RBC). As far as the oxidative content is concerned, the formation of malondialdehydes and protein carbonyls were assessed by HPLC and UV-spectrophotometric method respectively. Univariate and multivariate statistical analyses revealed significant differences between the different products. In particular, the choice of the protein source (hydrolysed vs. whole proteins) may greatly impact the resulting total antioxidant capacity of the preterm meal. The addition of fortifiers to human milk may result in higher antioxidant capacity; among bovine milk proteins, hydrolysed whey proteins resulted to provide higher antioxidant capacity than whole proteins. In general, the use of human milk minimizes the amount of oxidative compound in the diet in comparison to infant formulas, irrespectively of pasteurization or fortification, especially when malondialdehyde content is taken into account. The characterization of macro and micronutrients, bioactive compounds, as well as contaminants, deriving from food alteration, is of primary importance in order to guarantee high quality food products and adequate protein intake, especially when intended for infants, as formulas and fortifiers.

The in vivo and invitro digestion protocol for assessign human milk digestiblity are discussed.

Thirty-nine Balb-c mice (20 months old) were randomly allotted to three nutritionally balanced, isocaloric and isoproteic diets. Mice were fed for 30 days, with a 22% w/w substitution of the protein content (AA mix from casein hydrolysate - diet A) or with cow's milk containing either the A1/A2 ?-casein (diet B) or the A2/A2 ?-casein (diet C). Following gut inflammatory status and gastrointestinal function were evaluated: intestinal lymphocyte subpopulations quantitation (by flow cytometry targeting CD3, CD4, CD8, CD19, CD45, panNK, TCR??, TCR??); DPP IV activity in jejunal extracts (by fluorimetric activity assay kit). Samples of duodenum and ileum were processed for histomorphological investigations. Gut morphology was evaluated through morphometric measurements of villus height (Vh), crypt depth (Cd) and villus height to crypt depth ratio (Vh/Cd). Histopathological alterations were scored using a semiquantitative scoring system (0-3). Fecal SCFA content was assessed by HPLC-UV. Data were analysed by univariate and multivariate statistics. The study gives preliminary evidences of a positive effect of diet supplementation with A2/A2 ?-casein milk on some gut health associated parameters, in aging mice. The results also suggest intriguing insights on the role of microbial SCFA on the differentiation of intestinal lymphocytes sub-populations.

Description of ongoing research and first evidences on the antiviral role of exosomes derived from human colostrum

Introduction: Due to increasing request of consumers for non-bovine milks, and to the increase in the number of dairy donkey farms in Italy, farmers require more sophysticated and reliable processing devices, in order to increase their market share beyond local, and to guarantee a safe product of high quality. To this aim, a new small-scale continuous-flow High-Temperature Short-Time (HTST) pasteurizer (72°C for 15 sec), that was initially prototyped by the authors for treating small volumes of human milk, was tested on donkey milk, in order to evaluate its efficacy in producing high quality milk. Materials & Methods: The efficacy of the new HTST device on raw donkey milk microflora was tested by Total Aerobic Bacteria, Enterobacteriaceae and Bacillus cereus enumeration. In addition, the biochemical quality of donkey milk after HTST pasteurization was assessed by determining the protein profile in both reducing and non reducing conditions and by measuring lysozyme activity, and compared to that achieved by standard Holder pasteurization (63°C for 30 min). Results and Discussion: The new HTST apparatus was shown to be able to reduce the total bacteria count to undetectable level, as well as to completely eradicate Enterobacteriaceae. Bacillus cereus, when present, was eradicated with variable efficiency, mainly depending on the bacterial loads of the starting raw milk. Changes in the protein profile were observed in the milk pasteurized according to both processes. In detail, HTST seems to preserve the protein profile more than the traditional Holder method, limiting casein degradation. Nevertheless, both methods reduced significantly lysozyme activity with respect to raw donkey milk, thus indicating that structural changes may occurr also in less intense methods. In conclusion, the HTST method should be applied to safely pasteurize donkey milk, despite the low volumes involved in the standard routine of small dairy donkey farms. Moreover, this new device could represent an interesting strategy to ensure a higher retention of some protein fractions. Further evaluations and efficient device prototypation could thus contribute to both consumer safety, in particular for baby nutrition, and to increase of farmers' market consistency.

Relazione istruttoria per la concessione di agevolazione Progetto Rif. N. Pos. 59 Bando Industria Sostenibile FRI - MISE in qualità di esperto CNR

RELAZIONE ISTRUTTORIA PER LA CONCESSIONE DI AGEVOLAZIONI FINANZIARIE A VALERE SUL FONDO PER LA CRESCITA SOSTENIBILE (F.C.S.)Bando Horizon 2020 - PON 2014/2020, Progetto Rif. N. 435 in qualità di esperto CNR.

Edible insects can be a source of sustainable protein with low environmental impact. Among them, the silkworm (Bombyx mori) pupae (SWP) has been included in the EFSA (European Food Safety Authority) list of insect species with a interesting food application. Insects are a traditionally food in East and Southeast Asia and every year in China 1000 people have allergic reaction after SWP ingestion. In the Europe the introduction of insect into the diet will take place after study of specific risk assessment, including the evaluation of the allergenic potential. The aim of this work was to evaluate the allergenicity of SWP by immunoblotting performed with the sera of two patient sensitized during manipulation of insect flour and to identify the immuno-reactive proteins with mass spectrometry approach. Total proteins extract were separated by SDS-PAGE and transferred into nitrocellulose membrane. The membranes were incubated o.n. with the two sensitized patient sera. The immune-complex was detected with anti-human IgE AP Conjugate antibody. The reactive band was cut from the gel, destained and digested by trypsin (o.n., at 37°C) and the resulting peptides were analyzed by MALDI-TOF/TOF. On the three more relevant piques were performed the Post Source Decay(PSD) analysis. The mass spectra were analyzed by Flex Analysis software and the m/z list were used by Protein Prospector search software (MS-Fit and MS-Tag)to query UniProt data base. The Immunoblotting results show only one reactive band for both the sensitized patients. The identification show different protein belonging of 30Kda Lipoprotein family, one of the most abundant protein in SWP hemolymph. This is a defense protein and it's already known to be a food allergen. This preliminary study about SWP allergenicity could improve the state-of-the-art about edible insect safety from the allergenic point of view. It's one of the first evaluation of SWP allergenic potential using the sera of two Italian patient sensitized during manipulation of insect flour; a rare case of allergy to insect proteins in Europe.

transmission of some viral pathogens by breast-feeding is an important risk for pre-term infants since they are more susceptible to develop severe clinical symptoms. Despite human cytomegalovirus (HCMV) is commonly excreted in breast milk from seropositive women, only a low percentage of exposed infants will acquire postnatal CMV infection. this observation points towards a protective effect of breast milk components against HCMV transmission to the breastfed child. the aims of our study were: 1) to assess the anti-HCMV potency of breast milk from a cohort of pre-term mothers at different stages of lactation 2) to evaluate the antiviral role of immune factors of colostrum, like secretory iga, and non immune factors, like exosomes. 3) to evaluate the impact of Holder and HtSt pasteurizations on the antiviral properties of breast milk. the study revealed that all milk samples exerted significant anti-HCMV activity in vitro, although to a different extent and with interpersonal variability. interestingly, colostra from HCMV igg-positive mothers exhibited higher antiviral activity than samples from seronegative mothers. We demonstrated a partial contribution of siga to antiviral activity of colostrum and identified colostrum-derived exosomes as new anti-HCMV constituents of human milk. preliminary data indicate the ability of exosomes to inhibit the attachment of HCMV to cells. finally, we evidenced that Holder pasteurization significantly reduced anti-HCMV activity of breast milk, whereas HtSt did not affect it.

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The beneficial relationship between the host health and its gut microbiota has led in the last decades to the enormous increase of research and commercial interest in the development of probiotic preparations to manipulate microbiome in helping host physiology or preventing diseases [1]. Producers of functional foods are exploring technological solutions for developing foods supplemented with additional functional benefits but with high level of consumer satisfactions (health-orientated as well as taste-orientated). Many strains belonging to lactic acid bacterial (LAB) species are widely used as probiotics in commercial products since, additionally to functional properties conferred to foods, they may also act as starters establishing a mild fermentation that protects products from deterioration. Particularly, a L. paracasei strain (IMPC 2.1) has been studied and used for the development of innovative patented functional foods based on the association of that probiotic strain with vegetables (olives, artichokes, cabbage etc.) whose commercialization has been authorized by the Italian Ministry of Health [2]. Clinical trials performed on subjects suffering from constipation and healthy subjects demonstrated the efficacy of L. paracasei IMPC 2.1 carried by ready-to-eat artichokes in transiently colonize the human gut thus modulating potentially harmful bacteria, faecal enzyme activity and short chain fatty acid production as well as symptom profile [3]. Currently among probiotic foods, fish products have been rarely investigated as vehicles for probiotic strains in humans, even if they are suitable to sustain viable LAB populations [4]. Therefore our investigation aimed to evaluate the ability of the probiotic L. paracasei strain IMPC 2.1 to survive in marinated ready-to-eat swordfish fillets and to reach, viable, the human gut in an alternate day- based study. Probiotic ready-to-eat swordfish fillets (PR-RTE) were prepared by brining (3% NaCl) fillets for 2 days with live cells of the probiotic strain at 7 log CFU/g; then fillets were drained, seasoned with sunflower seed oil, spiced with parsley and stored under vacuum packing at 4°C in polyethylene trays (shelf life 4 months). The final product, containing more than 7 log CFU/g of the probiotic strain, showed physicochemical characteristics (protein profile and total free amino acid content, pH, aw) similar to those of control-RTE fillets (traditionally processed with an acidic marinade) along shelf life. The probiotic fillets were used in a human feeding study involving 8 volunteers to assess the suitability of the fish matrix in delivering viable cells of the probiotic strain [5]. Participants integrated their dietary intakes throughout the study period with a portion (100 g) of PR-RTE fillets containing about log 9 CFU of probiotic cells. The protocol was approved by a local Scientific and Ethics Committee. The dietary intervention study lasted 27 days and subjects consumed PR-RTE fillets on alternate days for 20 days, with a washout period of 7 days. A good compliance of PR-RTE fillets was registered from all participants. To evaluate the transient gut colonization of the strain, its presence was molecularly ascertained in faecal samples of subjects. Results demonstrated that after consuming 10 portions during 20 days, the probiotic strain was recovered in the faeces of all subjects at concentrations ranging from 6.15 to 7.47 log CFU/g. Interestingly, five subjects resulted to be colonized by the probiotic strain, at levels ranging from 6.30 to 7.74 log CFU/g of faeces, after eating 5 portions of the product (day 10). The probiotic colonization led to an increase, with respect to the start of the study, in the genetic diversity of faecal LAB based on the Shannon diversity index, even though no differences in LAB counts were observed. We can conclude that swordfish fillet is a suitable food carrier for the probiotic strain L. paracasei IMPC 2.1which was able to survive on the fish matrix during a 4-month storage period and to transiently colonize the gut of volunteers after thus providing consumers with a non conventional probiotic food to achieve a Functional Diet.

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Il latte umano è la prima scelta nell'alimentazione di tutti neonati, compresi i prematuri. Tuttavia, in questa ultima categoria, dati i particolari fabbisogni nutrizionali, il latte umano deve essere fortificato per alcuni nutrienti, soprattutto proteine e sali minerali. Fornire un maggior apporto proteico richiede un supplemento semplice, ben tollerato, costituito da proteine di alta qualità. Sfortunatamente questo è un obiettivo solo parzialmente raggiunto dai prodotti forniti dall'industria alimentare, che contengono proteine derivate dal latte bovino. E' ormai noto che il profilo biochimico del latte d'asina sia molto più simile al latte umano di quanto non sia il latte vaccino dal punto di vista sia quantitativo sia qualitativo, soprattutto per quanto attiene al contenuto proteico. Scopo dell'attività é stata la progettazione e la produzione su scala pilota di un fortificatore del latte umano derivato dal latte di asina. A questo fine é stato messo a punto di un processo di ultrafiltrazione del latte di asina che consentisse di ottenere due prodotti: (i) un fortificatore multicomponente con contenuto in proteine pari al 20%; , (ii) un concentrato proteico, con contenuto in proteine pari al 45%. I due prodotti sperimentali sono attualmente in uso nell'ambito trial prospettico randomizzato, condotto in un gruppo di nati pretermine presso la Terapia Intensiva Neonatale dell'Università di Torino. Il trial ha lo scopo di valutare se neonati pretermine alimentati con latte umano fortificato con supplementi derivati da latte d'asina possano migliorare la qualità della crescita e l'outcome clinico, auxologico, metabolico, e neurologico a breve e lungo termine, se confrontati con neonati cui viene somminitrato un supplemento disponibile nel commercio derivato dal latte bovino.

The use of insects as an alternative source of protein in animal feed is becoming more globally appealing, also in poultry. Soybean or fish meal in broiler chicken or laying hen have been successfully replaced at low inclusion levels by black soldier flies (Hermetia illucens), housefly (Musca domestica), mealworms (Tenebrio molitor, TM) and silkworm (Bombix mori) pupae. In this experiment, female Label chickens were reared in free range condition (5 replicates per treatment and 12 birds per replicate). Birds were housed at the age of 42 days (average weight 715 g). Two experimental diets were designed: 72.0% corn, 17.0% soybean meal, 7.5% gluten meal (C-group) or mealworm meal (TM-group) and 3.5% mineral-vitamin premix. Diets were formulated using AMEn values calculated for broiler chickens by our research group (Schiavone et al., 2015). C-group and TM group diets were isoenergetic (AMEn: 12.18 and 12.22 MJ/kg, respectively) and isonitrogenous (crude protein: 16.9% and 16.8%, respectively). The experiment lasted 60 days. Birds were weighted at the beginning and at the end of the experiment. At slaughtering, blood samples were taken and the main commercial cuts were evaluated (carcass for grilling, breast and thigh) as well as liver, spleen and Bursa of Fabricius weights. In addition foot pad dermatitis score was evaluated according to the EU Commission recommendations. Data were analysed by Student's t-Test for independent samples. C-group and TM-group displayed similar final body weight (2131 vs. 2162 g), feed conversion ratio (4.37 vs. 4.36 g), carcass for grilling (1459 vs. 1545 g), breast (347 vs. 371 g), thigh (497 vs. 503 g), abdominal fat (41 vs. 45 g), liver (37 vs. 39 g), spleen (4.0 vs. 3.8 g) and Bursa of Fabricius (4.3 vs. 4.2 g). By comparing blood analysis of C-group vs. TMgroup data of red (2.4 vs. 2.6 millions) and white blood cells (9.4 vs. 9.9 thousands), H/L ratio (0.55 vs. 0.51), total protein (3.98 vs. 4.05 d/dl), AST (190 vs. 198), ALT (14.5 vs. 14.0 UI/l), uric acid (5.0 vs. 3.9 mg/dl), triglycerides (43.4 vs. 47.8 mg/dl) and glucose (262 vs. 244 mg/dl) no significant differences were found. Birds did not displayed any foot pad dermatitis lesion. In conclusion TM meal can be safely used as feed ingredient for Label chicken at the inclusion level of 7.5% without affecting growth and slaughter performance as well as blood and welfare parameters.

Introduzione: Il latte materno, se non usato immediatamente, va conservato in maniera adeguata in frigorifero o in freezer. Attualmente vi è incertezza per quanto tempo il latte materno possa permanere in frigorifero e il conseguente impatto sulla qualità biologica. Obiettivi: Valutare gli effetti della conservazione per 96 ore in frigorifero sul profilo degli acidi grassi, sul contenuto degli acidi grassi liberi, sull'attività lipasica e sullo stato ossidativo del latte umano. Metodi: E' stato raccolto il latte di madri pretermine in 3 differenti giornate. Dal latte raccolto in ogni singolo giorno è stato formato un pool e quindi suddiviso in 5 aliquote (T0,T1,T2,T3,T4); lo stesso esperimento è stato ripetuto 3 volte. A T0: le analisi sono state eseguite immediatamente, T1-T2-T3-T4 sono stati conservati in frigorifero rispettivamente per 24-48-72 e 96 ore e quindi analizzati. La temperatura del frigorifero è stata monitorizzata ogni 5 minuti. I campioni di latte sono stati analizzati valutando: pH, il profilo degli acidi grassi liberi e totali, l'attività lipasica a temperatura ambiente e a 4°C, l'attività lipasica a temperatura ambiente in presenza di sodio colato (BSDL), capacità antiossidante totale, test acido tiobarbiturico, concentrazione di malondialdeide e dieni coniugati. Risultati: La refrigerazione prolungata del latte materno non determina effetti sulla composizione degli acidi grassi, sullo stato ossidativo complessivo e sull'attività degli enzimi lipolitici. Il significativo incremento di specifici acidi grassi liberi, come LC-PUFA n-6, potrebbe dipendere dall'attività stereo specifica delle lipoproteinlipasi. Conclusioni: il latte umano conservato in frigorifero per 96 ore mantiene la composizione globale lipidica, con un danno ossidativo contenuto. La limitata lipolisi durante la conservazione potrebbe essere dovuta all'attività delle lipoproteinlipasi, a cui consegue il decremento del ph. Importanti marker della qualità nutritiva del latte materno ( BSDL attività, LC-PUFAs e MCSFAs) sono preservati in frigorifero fino a 4 giorni.

In the past, several studies investigated the effects of different storage times and temperatures on different human milk components, including the abundance and/or the activity of single selected proteins. Nevertheless, extensive information on how prolonged refrigeration may modify the milk protein pattern is currently lacking. In the present study, skimmed breast milk was collected, aliquoted, and stored in a refrigerator for 24, 48, 72, and 96 hours before analysis. The experiment was independently replicated three times. The refrigerator temperature was constantly monitored. The resulting samples were then analysed for available lysine and secretory IgA contents, and their protein profile was obtained by monodimensional electrophoresis. Most abundant proteins were identified by MALDI TOF/TOF mass spectrometry. The refrigeration process did not affect either the total protein content or the HM protein profile. Markers of milk nutritional quality (dietary protein content and available lysine content), milk bioactive proteins (including the antimicrobial components lysozyme and lactoferrin) and immunological components (sIgAs, heavy chain of immunoglobulin and polymeric immunoglobulin receptor), were preserved up to 96h of refrigeration. In conclusion, refrigeration of expressed human milk in controlled conditions for 96 hours preserved protein bioactivity and nutritional quality.

Due to the limited knowledge on which common advice on human milk storage is based, current recommendations concerning safe human milk conservation are far from being univocal. In an attempt to clarify some of the uncertainties existing on the storage of HM, we designed a comprehensive study to investigate the effects of prolonged refrigeration on the nutritive and non-nutritive characteristics of human milk, and on its microbiological composition. Human milk was collected and divided into 5 aliquots. One aliquot underwent analyses immediately, while the other aliquots were stored in the refrigerator for 24, 48, 72, and 96 hours before analysis. The temperature was constantly monitored. The samples were analysed for pH, free fatty acid (FFA) content, bile salt-dependent lipase (BSDL) activity, oxidative status, and bacterial profile. The experiment was replicated three times. The continuous temperature monitoring of the refrigerator showed a mean value of 6.8 ± 1.1°C. Limited lipolysis was seen during prolonged refrigerated storage, consistently with a slight decrease in milk pH. Refrigeration preserved milk overall oxidative status and the activity of BSDL. Moreover, the overall bacterial composition remained stable, with mean values for total bacteria, Enterobacteriaceae and coagulase positive Staphylococci counts under the recommended concentrations set for acceptability of milk in milk banks. In conclusion, refrigeration of expressed human milk in controlled conditions for 96 hours allowed to maintain its bioactivity and nutritional quality, without compromising its microbiological safety.

Introduzione: Il latte materno, se non usato immediatamente, va conservato in maniera adeguata in frigorifero o in freezer. Attualmente vi è incertezza per quanto tempo il latte materno possa permanere in frigorifero e il conseguente impatto sulla qualità biologica. Obiettivi: Valutare gli effetti della conservazione per 96 ore in frigorifero sul profilo degli acidi grassi, sul contenuto degli acidi grassi liberi, sull'attività lipasica e sullo stato ossidativo del latte umano. Metodi: E' stato raccolto il latte di madri pretermine in 3 differenti giornate. Dal latte raccolto in ogni singolo giorno è stato formato un pool e quindi suddiviso in 5 aliquote (T0,T1,T2,T3,T4); lo stesso esperimento è stato ripetuto 3 volte. A T0: le analisi sono state eseguite immediatamente, T1-T2-T3-T4 sono stati conservati in frigorifero rispettivamente per 24-48-72 e 96 ore e quindi analizzati. La temperatura del frigorifero è stata monitorizzata ogni 5 minuti. I campioni di latte sono stati analizzati valutando: pH, il profilo degli acidi grassi liberi e totali, l'attività lipasica a temperatura ambiente e a 4°C, l'attività lipasica a temperatura ambiente in presenza di sodio colato (BSDL), capacità antiossidante totale, test acido tiobarbiturico, concentrazione di malondialdeide e dieni coniugati. Risultati: La refrigerazione prolungata del latte materno non determina effetti sulla composizione degli acidi grassi, sullo stato ossidativo complessivo e sull'attività degli enzimi lipolitici. Il significativo incremento di specifici acidi grassi liberi, come LC-PUFA n-6, potrebbe dipendere dall'attività stereo specifica delle lipoproteinlipasi. Conclusioni: il latte umano conservato in frigorifero per 96 ore mantiene la composizione globale lipidica, con un danno ossidativo contenuto. La limitata lipolisi durante la conservazione potrebbe essere dovuta all'attività delle lipoproteinlipasi, a cui consegue il decremento del ph. Importanti marker della qualità nutritiva del latte materno ( BSDL attività, LC-PUFAs e MCSFAs) sono preservati in frigorifero fino a 4 giorni.