Effectiveness of community hospital post-acute care on mortality, re-admission, institutionalization, and activation of a home care programme in Emilia-Romagna region, Italy.

BACKGROUND: In Italy, there is scant evidence on the impact of Community Hospitals (CHs) on clinical outcomes. AIMS: To assess the effectiveness of CHs versus long-term care hospital or inpatient rehabilitation facilities on mortality, re-admission, institutionalization, and activation of a home care programme in the Emilia-Romagna Region (ERR-Italy) after acute hospitalisation. METHODS: We implemented a cohort study drawing upon the ERR Administrative Healthcare Database System and including hospital episodes of ERR residents subject ≥ 65 years, discharged from a public or private hospital with a medical diagnosis to a CH or to usual care between 2017 and 2019. To control for confounding, we applied a propensity score matching. RESULTS: Patients transferred to CHs had a significantly lower risk of dying but an increased risk of being readmitted to community or acute hospital within 30/90 days from discharge. The hazard of institutionalisation within 30/90 days was significantly lower in the whole population of the CH exposed group but not among patients with cardiac or respiratory chronic diseases or diabetes. The activation of a home care program within 90 days was slightly higher for those who were transferred to a CH. DISCUSSION: The findings of our study show mixed effects on outcomes of patients transferred to CHs compared to those who followed the post-acute usual care and should be taken with cautious as could be affected by the so-called 'confounding by indication'. CONCLUSIONS: The study contributes to the intermediate care available evidence from a region with a well-established care provision through CHs.

Thermally-Induced Lactosylation of Whey Proteins: Identification and Synthesis of Lactosylated ß-lactoglobulin Epitope.

The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, ß-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a ß-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

Continuous production of pectic oligosaccharides from sugar beet pulp in a cross flow continuous enzyme membrane reactor.

Sugar beet pulp pectin is an attractive source for the production of pectic oligosaccharides, an emerging class of potential prebiotics. The main aim of the present work was to investigate a new process allowing to produce pectic oligosaccharides in a continuous way by means of a cross flow enzyme membrane reactor while using a low-cost crude enzyme mixture (viscozyme). Preliminary experiments in batch and semi-continuous setups allowed to identify suitable enzyme concentrations and assessing filtration suitability. Then, in continuous experiments in the enzyme membrane reactor, residence time and substrate loading were further optimized. The composition of the obtained oligosaccharide mixtures was assessed at the molecular level for the most promising conditions and was shown to be dominated by condition-specific arabinans, rhamnogalacturonans, and galacturonans. A continuous and stable production was performed for 28.5 h at the optimized conditions, obtaining an average pectic oligosaccharide yield of 82.9 ± 9.9% (w/w), a volumetric productivity of 17.5 ± 2.1 g/L/h, and a specific productivity of 8.0 ± 1.0 g/g E/h. This work demonstrated for the first time the continuous and stable production of oligosaccharide mixtures from sugar beet pulp using enzyme membrane reactor technology in a setup suitable for upscaling.

Current Trends in Ancient Grains-Based Foodstuffs: Insights into Nutritional Aspects and Technological Applications.

For centuries, ancient grains fed populations, but due to their low yield, they were abandoned and replaced by high-yielding species. However, currently, there is a renewed interest in ancient wheat and pseudocereal grains from consumers, farmers, and manufacturers. Ancient wheat such as einkorn, emmer, spelt, and Kamut®, are being reintegrated because of their low fertilizer input, high adaptability and important genetic diversity. New trends in pseudocereal products are also emerging, and they are mostly appreciated for their nutritional outcomes, particularly by the gluten-free market. Toward healthier lifestyle, ancient grains-based foodstuffs are a growing business and their industrialization is taking 2 pathways, either as a raw ingredient or a functional ingredient. This paper deals with these grain characteristics by focusing on the compositional profile and the technological potential.

Development of a strategy for the total chemical synthesis of an allergenic protein: the peach LTP Pru p 3.

The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Regular monitoring of breast-feeding rates: feasible and sustainable. The Emilia-Romagna experience.

OBJECTIVE: An efficient breast-feeding monitoring system should be in place in every country to assist policy makers and health professionals plan activities to reach optimal breast-feeding rates. Design/Setting/Subjects From March to June 2015, breast-feeding rates at 3 and 5 months of age were monitored in Emilia-Romagna, an Italian region, using four questions added to a newly developed paediatric immunization database with single records for each individual. Data were collected at primary-care centres. Breast-feeding definitions and 24 h recall as recommended by the WHO were used. Direct age standardization was applied to breast-feeding rates. Record linkage with the medical birth database was attempted to identify maternal, pregnancy and delivery factors associated with full breast-feeding rates at 3 and 5 months of age. RESULTS: Data on breast-feeding were collected for 14044 infants. The mean regional full breast-feeding rate at 3 months was 52 %; differences between local health authorities ranged from 42 to 62 %. At 5 months of age, the mean regional full breast-feeding rate dropped to 33 % (range between local health authorities: 26 to 46 %). Record linkage with the birth certificate database was successful for 93 % of records. Total observations more than doubled with respect to the previous regional survey. CONCLUSIONS: The new monitoring system implemented in 2015 in Emilia-Romagna region, totally integrated with the immunization database, has proved to be feasible, sustainable and more efficient than the previous one. This system can be a model for other regions and countries where the vast majority of mothers obtain vaccinations from public health facilities and that already have an immunization database in place.

Phlorotannin Composition of Laminaria digitata.

INTRODUCTION: Phlorotannins are complex mixtures of phloroglucinol oligomers connected via C-C (fucols) or C-O-C (phlorethols) linkages. Their uniformity in subunits and large molecular weight hamper their structural analysis. Despite its commercial relevance for alginate extraction, phlorotannins in Laminaria digitata have not been studied. OBJECTIVE: To obtain quantitative and structural information on phlorotannins in a methanolic extract from L. digitata. METHODOLOGY: The combined use of 13 C and 1 H NMR spectroscopy allowed characterisation of linkage types and extract purity. The purity determined was used to calibrate the responses obtained with the colorimetric 2,4-dimethoxybenzaldehyde (DMBA) and Folin-Ciocalteu (FC) assays. Using NP-flash chromatography, phlorotannin fractions separated on oligomer size were obtained and enabled structural and molecular weight characterisation using ESI-MS and MALDI-TOF-MS. RESULTS: The fucol-to-phlorethol linkage ratio was 1:26 and the extract was 60.1% pure, determined by NMR spectroscopy. For DMBA, the response of the extract was 12 times lower than that of phloroglucinol, whereas there was no difference for FC. By accounting for differences in response, the colorimetric assays were applicable for quantification using phloroglucinol as a standard. The phlorotannin content was around 4.5% DM. Fucol- and phlorethol-linkage types were annotated based on characteristic MSn fragmentations. Structural isomers of phlorotannins up to a degree of polymerisation of 18 (DP18) were annotated and identification of several isomers hinted at branched phloroglucinol oligomers. With MALDI-TOF-MS phlorotannins up to DP27 were annotated. CONCLUSION: By combining several analytical techniques, phlorotannins in L. digitata were quantified and characterised with respect to fucol-to-phlorethol linkage ratio, molecular weight (distribution), and occurrence of structural isomers. Copyright © 2017 John Wiley & Sons, Ltd.

How Looking for Celiac-Safe Wheat Can Influence Its Technological Properties.

Because of the continuous increase in the prevalence of gluten-related disorders, selection of wheat with a low content of immunogenic gluten epitopes could be an innovative alternative for prevention. In this review, the focus is on literature data concerning the deallergenization tools of wheat, which are mainly related to breeding approaches (classic and advanced) and processing operations (germination and fermentation). Until now, no safe wheat genotype has been identified, whereas decreasing wheat allergenicity is possible. On the other hand, the decrease of gluten or some of its epitopes can strongly affect technological properties. Thus, obtaining celiac-safe gluten without affecting the technological properties of wheat could be considered as a new challenge that scientists will be facing. Celiac-safe wheat-based product development could be a great revolution in the market of foods for special medical purposes. The present paper is aiming to: (a) review the strategies and the approaches used, or that can be used, for developing low allergenic wheat: their utilities and limits were also discussed and (b) screen the impact of gluten reduction or removal on the quality of wheat end-use products.

Enzymatic pectic oligosaccharides (POS) production from sugar beet pulp using response surface methodology.

Pectic oligosaccharides (POS) have been indicated as novel candidate prebiotics. Traditionally, POS are produced from pectin-rich by-products using a two-step process involving extraction of the pectin, followed by its hydrolysis into POS. A one-step approach, in which the POS is directly produced from the raw material, might provide a more efficient alternative. Thus, the main aim of this paper was to investigate a one-step enzymatic hydrolysis approach to directly produce POS from sugar beet pulp (SBP). The POS yield was investigated as a function of the process parameters, as well as raw material characteristics. A statistically-based response surface methodology, using a central composite design was applied, to investigate the individual as well as the combined influences of the diverse parameters. The model was confirmed by a validation experiment, carried out at 135 g/l substrate concentration, 0.75 FPU/g SBP enzyme concentration, 0.8 mm particle size and 3 h hydrolysis time. Under these conditions, a POS-rich hydrolysate was obtained, containing rhamnose, arabinose, galactose, xylose and galacturonic acid, at 0.9, 15.2, 5.1, 1.4, and 13.2 g/l, respectively, enzymes were added each at 20 FPU/g dry matter (DM).

Peroxidase induced oligo-tyrosine cross-links during polymerization of α-lactalbumin.

Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α-lactalbumin (α-LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Yn, n=3-8) was found in addition to di-tyrosine (Y2). Two stages of cross-linking of α-LA were identified: a) 1-2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3-4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α-LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates.

Spontaneous, non-enzymatic breakdown of peptides during enzymatic protein hydrolysis.

It is expected that during the hydrolysis of proteins with specific enzymes only peptides are formed that result from hydrolysis of the specific cleavage sites (i.e. specific peptides). It is, however, quite common to find a-specific peptides (i.e. resulting from a-specific cleavage), which are often ignored, or explained by impurities in the enzyme preparation. In recent work in a whey protein isolate (WPI) hydrolysate obtained with the specific Bacillus licheniformis protease (BLP), 13 peptides of 77 identified were found to be the result of a-specific cleavage. These were formed after degradation of 6 specific peptides, after 5 different types of amino acids. The fact that other peptides were not hydrolyzed after these 5 amino acids suggests that the cleavages were not the result of a contamination with a different enzyme. In other systems, certain peptide sequences have been described to degrade chemically, under relatively mild conditions. This process is referred to as spontaneous cleavage. To test if the a-specific peptides observed in the WPI hydrolysis are the results of spontaneous cleavages, the parental peptides were synthesized. Surprisingly, 4 of the 5 synthesized peptides were indeed spontaneously cleaved under the mild conditions used in this study (i.e. 40°C and pH 8) showing that peptides are less stable than typically considered. The rate of cleavage on the a-specific bonds was found to be enhanced in the presence of BLP. This suggests that the formation of a-specific peptides is not due to side activity but rather an enhancement of intrinsic instability of the peptides.

Pectic oligosaccharides from agricultural by-products: production, characterization and health benefits.

Pectin containing agricultural by-products are potential sources of a new class of prebiotics known as pectic oligosaccharides (POS). In general, pectin is made up of homogalacturonan (HG, α-1,4-linked galacturonic acid monomers) and rhamnogalacturonan (RG, alternate galacturonic acid and rhamnose backbone with neutral side chains). Controlled hydrolysis of pectin containing agricultural by-products like sugar beet, apple, olive and citrus by chemical, enzymatic and hydrothermal can be used to produce oligo-galacturonides (GalpOS), galacto-oligosaccharides (GalOS), rhamnogalacturonan-oligosaccharides (RGOS), etc. However, extensive research is needed to establish the role of POS, both as a prebiotic as well as therapeutic agent. This review comprehensively covers different facets of POS, including the nature and chemistry of pectin and POS, potential agricultural residual sources of pectin, pre-treatment methods for facilitating selective extraction of pectin, identification and characterization of POS, health benefits and important applications of POS in food and feed. This review has been compiled to establish a platform for future research in the purification and characterization of POS and for in vivo and in vitro studies of important POS, so that they could be commercially exploited.

Enzymatic production and degradation of cheese-derived non-proteolytic aminoacyl derivatives.

Gamma-glutamyl-amino acids, lactoyl-amino acids and pyroglutamyl-amino acids, collectively named Non-Proteolytic Aminoacyl Derivatives (NPADs) are unusual aminoacyl derivatives of non-proteolytic origins found in consistent amount in several cheeses. Although their enzymatic origin arising from lactic acid bacteria has been demonstrated, the exact enzymes originating them, the ones eventually degrading them and also their resistance to digestive enzymes in the human gastrointestinal tract and in the blood serum after eventual absorption are still unknown. In this paper, pure enzymes and biological media were tested on NPAD and their aminoacidic precursors, for identifying the conditions favoring bioproduction and biodegradation of these compounds. Pure gamma-glutamyl-phenylalanine and its precursor (glutamic acid and phenylalanine), also in the isotopically labeled forms, were tested with Parmigiano-Reggiano extracts, blood serum and different pure enzymes, including typical digestion enzymes (pepsin, trypsin and chymotrypsin), gamma-glutamyl transpeptidase and carboxypeptidase. The data suggested that their production in cheese, and also their partial degradation, might be due to the action of peptidases and gamma-glutamyl transpeptidase. Anyway, under simulated gastrointestinal digestion and in blood serum these compounds turned out to be perfectly stable, suggesting a potential to be absorbed as such and possibly being transported to the body tissues.

Introducing enzyme selectivity: a quantitative parameter to describe enzymatic protein hydrolysis.

Enzyme selectivity is introduced as a quantitative parameter to describe the rate at which individual cleavage sites in a protein substrate are hydrolyzed relative to other cleavage sites. Whey protein isolate was hydrolyzed by Bacillus licheniformis protease, which is highly specific for Glu and Asp residues. The molar concentration of all peptides (58) from ß-lactoglobulin formed during hydrolysis was determined from the UV214 signal. The quality of identification and quantification of the peptides were described by newly defined parameters: the peptide sequence coverage (on average 94 %) and the molar sequence coverage (on average 75 %). The selectivity was calculated from the rate of hydrolysis of each cleavage site, and showed differences of up to a factor of 5,000. The ability to quantitatively discriminate the enzyme preference towards individual cleavage sites is considered essential to the understanding of enzymatic protein hydrolysis.

Qualitative and quantitative determination of peptides related to celiac disease in mixtures derived from different methods of simulated gastrointestinal digestion of wheat products.

During wheat digestion, gluten-derived proteolytic resistant peptides are generated, some of them involved in celiac disease. In vitro digestion models able to mimic the peptides generated in the human gastrointestinal tract are extremely useful to assess the pathogenicity of wheat-derived products. In this paper, samples belonging to three different durum wheat varieties were taken at six different steps of the pasta production chain and two different digestion models present in the literature were assessed on the different samples: a more complex one using artificial fluids simulating the exact composition of digestive juices, and a simplified method based on a peptic-tryptic/chymotryptic treatment of wheat ethanolic extract. An extensive characterization of the peptides generated using two in vitro digestion models was performed through LC-MS/MS techniques and the two methods were compared in order to evaluate qualitative and quantitative differences and their possible implications for varietal screening. Strong differences in the type of peptides produced with the two methods were detected, indicating that the simplified method can still be used for a varietal screening but is not representative of the peptides really generated after physiological human digestion. Results indicate a clear necessity of physiologically accurate models for simulating human gastrointestinal digestion of wheat products.

How D-amino acids embedded in the protein sequence modify its digestibility: Behaviour of digestive enzymes tested on a model peptide used as target.

D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH2 (belonging to ß-lactoglobulin, position 48-60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved.

Exploitation of bones-rich poultry by-products to produce protein hydrolysates: optimization of hydrolysis parameters and chemical characterization.

A significant quantity of bone-rich poultry by-products must be disposed of by poultry processors. These products still contain a significant amount of nutritionally valuable animal proteins. In the present work, a hydrolysis protocol was optimized to recover the protein fraction of bone-rich poultry by-products while simultaneously minimizing the amount of water required for hydrolysis (thus reducing drying costs) and recycling the hydrolytic broth up to 3 times, to reduce the cost of the proteolytic enzyme. The final hydrolysis conditions involved the use of (protease from B. licheniformis, ≥2.4 U/g; 0.5 V/w of raw material) and a hydrolysis time of 2 h at 65°C. The protein hydrolysate obtained has a high protein content (79-86%), a good amino acid profile (chemical amino acid score equal to 0.7-0.8) and good gastric digestibility (about 30% of peptide bonds are already hydrolyzed before digestion). This supports its use as an ingredient in food, pet food or animal feed formulations.

Assessment of Protein Quality and Digestibility in Plant-Based Meat Analogues.

In this first work, commercial steak-like (n = 3) and cured meat (n = 3) analogues with different legume and cereal formulations were studied and compared to their animal-based (n = 3) counterparts. Plant-based products showed lower protein content than meat controls but a good amino acidic profile even though the sum of essential amino acids of plant-cured meats does not fulfill the requirements set by the Food and Agriculture Organization for children. A comparable release of soluble proteins and peptides in the digestates after in vitro digestion was observed in meat analogues as meat products, whereas the digestibility of proteins was lower in plant-based steaks and higher in plant-based cured meats than their counterparts. The overall protein quality and digestibility of products are related to both the use of good blending of protein sources and processes applied to produce them. An adequate substitution of meat with its analogues depends mostly on the quality of raw materials used, which should be communicated to consumers.

PNA-NLS conjugates as single-molecular activators of target sites in double-stranded DNA for site-selective scission.

Artificial DNA cutters have been developed by us in our previous studies by combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with Ce(IV)-EDTA-promoted hydrolysis. The pcPNAs have two modified nucleobases (2,6-diaminopurine and 2-thiouracil) instead of conventional A and T, and can invade double-stranded DNA to activate the target site for the scission. This system has been applied to site-selective scissions of plasmid, λ-phage, E. coli genomic DNA, and human genomic DNA. Here, we have reported a still simpler and more convenient DNA cutter obtained by conjugating peptide nucleic acid (PNA) with a nuclear localization signal (NLS) peptide. This new DNA cutter requires only one PNA strand (instead of two) bearing conventional (non-pseudo-complementary) nucleobases. This PNA-NLS conjugate effectively activated the target site in double-stranded DNA and induced site-selective scission by Ce(IV)-EDTA. The complex formation between the conjugate and DNA was concretely evidenced by spectroscopic results based on time-resolved fluorescence. The target scission site of this new system was straightforwardly determined by the Watson-Crick base pairing rule, and mismatched sequences were clearly discriminated. Importantly, even highly GC-rich regions, which are difficult to be targeted by a previous strategy using pcPNA, were successfully targeted. All these features of the present DNA cutter make it promising for various future applications.

Microbial origin of non proteolytic aminoacyl derivatives in long ripened cheeses.

Cheese ripening involves a complex series of biochemical events that contribute to the development of each cheese characteristic taste, aroma and texture. Proteolysis, which has been the subject of active research in the last decade, is the most complex of these biochemical events. However, also aminoacyl derivates of non-proteolytic origin (γ-glutamyl-amino acids and lactoyl-amino acids) with interesting sensory properties have been identified in cheeses. In the present work, an enzymatic activity producing γ-glutamyl-phenylalanine in Parmigiano-Reggiano water soluble extracts was observed. It was hypothesized that γ-glutamyl-amino acids and lactoyl-amino acids could be originated by enzymes of bacterial origin. In order to confirm this hypothesis, Lactobacillus helveticus and Lactobacillus rhamnosus were chosen as representative of starter and non starter microbiota of Parmigiano Reggiano cheese. They were used as model bacteria, in the presence of suitable precursors, to verify their ability to produce γ-glutamyl-phenylalanine and lactoyl-phenylalanine. The eventual abilities of these strains were tested both during growth and after cell lyses. While γ-glutamyl-phenylalanine was produced only by lysed cells, lactoyl-phenylalanine was produced either by growing or lysed cells in different amount depending on the species, the cells condition and the time of incubation.