Marzia Giribaldi

Heard it through the grapevine: Proteomic perspective on grape and wine

Grapevine (Vitis ssp.) is currently considered as the most important fruit plant throughout the world, both due to its economic importance and to its role as a non climacteric model species. The relevance of the studies devoted to the dissection of grapevine biology and biochemistry underlines the great amount of attention that this plant has attracted over the last decade. The milestones among these studies are represented by the accomplishment of the genome sequencing programmes in 2007 [1,2]. Since then, the investigation of grape OMICS has been implemented, and the number of reports published on grape and wine protein investigations using proteomic techniques have significantly improved knowledge in the field.

Effect of two pasteurization methods on the protein content of human milk.

The Holder method is the recommended pasteurization method for human milk banks, as it ensures the microbiological safety of human milk (HM). The loss of some biologically active milk components, due to the heat treatment, is a main limit to the diffusion of donor HM. High-temperature short-time (HTST) pasteurization may be an alternative to maintain the nutritional and immunological quality of HM. The aim of the present study was to compare the impact of Holder and HTST pasteurization on the HM protein profile. The protein patterns of HTST-treated milk and raw milk were similar. The Holder method modified bile salt-stimulated lipase, lactoferrin and components of the immune system. The HTST method preserved the integrity of bile salt-stimulated lipase, lactoferrin and, to some extent, of IgAs. Holder pasteurization decreased the amount of bile salt-stimulated lipase and inactivated the remaining molecules, while the HTST method did not alter its activity. Pasteurization increased the bioavailable lysine quantity. HTST pasteurization seems to better retain the protein profile and some of the key active components of donor HM.

The Effect of Holder Pasteurization on Nutrients and Biologically-Active Components in Donor Human Milk: A Review

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.

Effect of prolonged refrigeration on the lipid profile, lipase activity, and oxidative status of human milk.

Objective: The study was aimed at evaluating the effect of prolonged refrigeration of fresh human milk (HM) on its fatty acid profile, free fatty acid content, lipase activities, and oxidative status. Methods: HM from mothers of preterm newborns was collected, pooled, and placed in the neonatal intensive care unit (NICU) refrigerator. Pooled milk was aliquoted and analyzed within 3 hours of collection, and after 24, 48, 72, and 96 hours of storage. The milk samples were analyzed for pH, total and free fatty acid profile, lipase activity at room temperature and at 4°C, lipase activity at room temperature in presence of sodium cholate (bile salt–dependent lipase), total antioxidant capacity, thiobarbituric acid reactive species, malondialdehyde, and conjugated diene concentration. The experiment was replicated in 3 independent trials. Results: Prolonged refrigeration did not affect the fatty acid composition of breast milk, and preserved both its overall oxidative status and the activity of HM lipolytic enzymes. In particular, bile salt–dependent lipase activity, long-chain polyunsaturated fatty acids, and medium-chain saturated fatty acid concentrations were unaffected for up to 96 hours of refrigerated storage. Conclusions: Prolonged refrigeration of fresh HM for 96 hours maintained its overall lipid composition. The limited lipolysis during storage should be ascribed to the activity of lipoprotein lipase, responsible for the decrease in pH. Our study demonstrates that infants who receive expressed milk stored for up to 96 hours receive essentially the same supply of fatty acids and active lipases as do infants fed directly at the breast.

A multidisciplinary study on the effects of phloem-limited viruses on the agronomical performance and berry quality of Vitis vinifera cv. Nebbiolo

Viral infections are known to have a detrimental effect on grapevine yield and performance, but there is still a lack of knowledge about their effect on the quality and safety of end products. Vines of Vitis vinifera cv. Nebbiolo clone 308, affected simultaneously by Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine virus A (GVA), and Rupestris stem pitting associated virus (RSPaV), were subjected to integrated analyses of agronomical performance, grape berry characteristics, instrumental texture profile, and proteome profiling. The comparison of performance and grape quality of healthy and infected vines cultivated in a commercial vineyard revealed similar shoot fertility, number of clusters, total yield, with significant differences in titratable acidity, and resveratrol content. Also some texture parameters such as cohesiveness and resilience were altered in berries of infected plants. The proteomic analysis of skin and pulp visualized about 400 spots. The ANOVA analysis on 2D gels revealed significant differences among healthy and virus-infected grape berries for 12 pulp spots and 7 skin spots. Virus infection mainly influenced proteins involved in the response to oxidative stress in the berry skin, and proteins involved in cell structure metabolism in the pulp.

Human Milk Processing: A Systematic Review of Innovative Techniques to Ensure the Safety and Quality of Donor Milk

ABSTRACT 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.

Effect of Bentonite Characteristics on Wine Proteins, Polyphenols, and Metals under Conditions of Different pH

Studies have yet to evaluate how bentonite properties may affect the protein profile, polyphenol content, metal concentration, and heat stability of a white wine at different pH values. Therefore, this work assessed the proteins, polyphenols, metals, and haze forming tendency when heating white wine samples before and after a fining treatment with four activated sodium bentonites in a typical wine pH range (3.00 to 3.60). Soluble wine proteins were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, and gel images were compared using the Quantity One software package (Bio-Rad Laboratories, Inc., Hercules, CA). The wine haze forming tendency, metals, and polyphenols were measured using heat tests and International Organisation of Vine and Wine (OIV) methods. Low molecular mass proteins were efficiently removed by all of the bentonites, regardless of the pH. High and medium molecular mass proteins were less likely to be removed and the efficiency, which depended on the pH, was variable. Reductions of vacuolar invertase (GIN1) and VVTL1 fractions of the thaumatin-like proteins were induced by bentonites with pH values less than 10. These bentonites were affected to a lesser extent by the negative effect of acidic pH. The reduction in haze forming tendency of the unfined Erbaluce wine was particularly noticeable in bentonite fined samples heated at 50 to 60°C, 60 to 80°C, and 70 to 80°C at pH 3.17, pH 3.30, and pH 3.60, respectively. The poor removal of glycoproteins (YGP1 and Hmp1) at higher pH values contributed to an increased thermal stability. The exchange of cationic species, notably sodium and potassium, between the bentonites and the wine was related more to the wine pH than to the clay type. Finally, the extent of polyphenol removal correlated with the amount of protein removed. When protein removal did not occur, the reduction of polyphenols was driven by the specific surface area and the surface charge density of the bentonite.

A functional approach to the body condition assessment of lactating donkeys as a tool for welfare evaluation

Background The breeding of lactating donkeys is increasing in Western Europe; with it the evaluation of body condition is growing in importance since it is considered a key principle for their welfare. However, assessment of body condition is a complex task, since several factors are involved. The aim of the present study is to investigate which animal-based indicators are the most reliable to describe the body condition of lactating donkeys. For this purpose, new animal-based indicators, which are easy to measure in field conditions (including body measurements, fatty neck score (FNS), dental score), are recorded and their relationship with BCS (a proxy measure for overall adiposity) was assessed. The ones that reveal an association with the BCS are included in an integrated principal component analysis to understand which are the most related to BCS. Methods Fifty-three healthy lactating donkeys of various breeds, including 7 Martina Franca, 10 Ragusano, 2 Romagnolo and 34 crossbreeds, were evaluated. The animal-based indicators that were recorded were: length (OP, olecranon tuber-pinbone and SH, shoulder-hip), heart girth (HG), abdominal circumference (AC), neck length (NL), neck height (NH) and neck thickness (NT) at 0.50 and neck circumference (NC) at 0.25, 0.50 and 0.75, body condition score (BCS) and fatty neck score (FNS). The owners’ evaluation of the BCS was also considered. A dental assessment was performed and the month of lactation and age of each animal was recorded. Results No correlation was found between BCS and the other morphometric body measurements. On the contrary the FNS was correlated with the morphometric measurements of the neck (positive correlation to 0.50 NH and 0.50 NT, 0.50 NC, 0.75 mean NC, and negative correlation to the mean NC:NH and mean NC:NT, 0.50 NC:NT and 0.50 NC:NH ratios). A significant inverse relationship was identified between BCS and dental score. A Principal Component analysis (PCA) separated the BCS classes on the first principal component (PC1). PC1 revealed a meaningful positive correlation between the BCS and the neck measurements (NT, NH and FNS), with high positive loadings, while a negative correlation was found for dental abnormalities. The owners’ evaluation of BCS was different from the expert evaluator’ assessment, since they tended to give higher score that was slightly but significantly correlated to AC. Discussion A new scoring system, called Fatty Neck Score (FNS), has been proposed for the judgement of the adiposity status of donkey neck. The results suggest that caregivers might use the proposed animal based indicators (BCS, FNS and dental scores) together as a tool for the evaluation of the body condition of lactating donkeys. Our findings highlight that caregivers need to be trained in order to be able to properly record these indicators. Ultimately use of these indicators may help to improve the welfare of lactating donkeys.

A Preliminary Assessment of HTST Processing on Donkey Milk

Due to increasing attention from consumers on non-bovine milk types, and to the increase in the number of small dairy donkey farms in Italy, farmers require more advanced and reliable processing devices, in order to guarantee a safe product of high quality. To this aim, a new small-scale High-Temperature Short-Time (HTST) pasteurizer (72 °C for 15 s), prototyped by the authors, was tested on donkey milk. The efficacy of the HTST device was tested on raw donkey milk microflora by enumeration of total aerobic bacteria, Enterobacteriaceae and Bacillus cereus. The biochemical quality was assessed by determining the protein profile by monodimensional electrophoresis and by measuring lysozyme activity. The HTST apparatus was able to reduce the total bacteria count, and to completely eradicate Enterobacteriaceae. Bacillus cereus, when present, was decreased with low efficiency. Changes in the protein profile were observed in milk pasteurized in accordance with both processes, although HTST seemed to limit casein degradation. Lysozyme activity was not substantially affected in comparison to raw donkey milk. In conclusion, a tailored small-volume HTST device could be safely applied to pasteurize donkey milk in on-farm pasteurization processes on small dairy donkey farms.

Observations of the haematological, haematochemical and electrophoretic parameters in lactating donkeys ( Equus Asinus )

&NA; A cross‐sectional study was conducted on 92 female donkeys. Blood samples were collected, and the following parameters were evaluated: red blood cell (RBC), white blood cell, neutrophil, lymphocyte, monocyte (MON), eosinophil (EOS) and basophil counts, hemoglobin concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and the hematocrit (HCT), alanine aminotransferase (GPT), aspartate aminotransferase, total proteins, &ggr;‐glutamyl‐transferase, alkaline phosphatase, creatinine, urea, and blood urea nitrogen and electrophoretic profile. Age (≥2 years ≤ 3 [very young], >3 years ≤ 10 [young], and >10 years ≤ 17 [adult]) and lactation (early lactation [≤3 months], middle [>3 months ≤ 6], and late lactation [>6 months]). Groups were independently analyzed using one‐way analysis of variance or Kruskal–Wallis (post hoc test: Bonferronis or Dunns multiple test) tests; P was set as <.05. Very young animals had lower EOS than young and adult animals; in addition, they showed the highest MON and RBC and the lowest MCV and GPT; MCHC was lower in adult than that in the very young group; MCH was higher in the adult than that in the very young group; Alpha 2‐globulin values were greater in young than those in very young animals; MCH was higher in the late lactation group than that in early lactation; alpha 1 and alpha 2‐globulins showed a significant increase from the early to the late lactation period. Values reported herein could provide a useful clinical guide and represent a basis for further research into monitoring the health status of lactating donkeys. HighlightsObservations of blood chemistry parameters in lactating donkeys (Equus asinus).Very young donkeys had lower eosinophil, highest monocyte and red blood cell than young and adult.Mean corpuscular hemoglobin (MCH) was higher in adult compared to very young.Alpha 2‐globluin values were greatest in young donkeys compared to very young MCH was highest late lactation, alpha1, 2 globulins increased from early to late.