Luca Laghi

Blood pressure lowering effect of lactotripeptides assumed as functional foods: a meta-analysis of current available clinical trials.

The oral assumption of lactotripeptides Valine–Proline–Proline (VPP) and Isoleucine–Proline–Proline (IPP) as nutraceuticals or functional foods is supposed to improve blood pressure (BP) control by angiotensin-converting enzyme-inhibition. However, data derived from clinical trials have reached conflicting conclusions. To perform a meta-analysis of placebo-controlled clinical trials evaluating the anti-hypertensive effect of lactotripeptides assumed as nutraceuticals or functional foods. Trials identified using a defined search strategy in PubMed were included in the meta-analysis, and their pooled effect was estimated with a random effects model, weighting for the inverse of the variance. Heterogeneity, publication bias, subgroup and meta-regression analyses were performed. A total of 18 trials have been identified, the clinical data of which have been clearly reported. Pooled effect of peptides was a reduction of −3.73u2009mmu2009Hg (95% CI: −6.70, −1.76) for systolic blood pressure (SBP) and 1.97u2009mmu2009Hg (95% CI: −3.85, −0.64) for diastolic blood pressure (DBP). The effect was more evident in Asian patients (SBP=−6.93u2009mmu2009Hg (95% CI: −10.95, −2.94); DBP=−3.98u2009mmu2009Hg(95% CI: −5.38, −2.44)) than in Caucasian ones (SBP=−1.17u2009mmu2009Hg (95% CI: −2.82, 0.72); DBP=−0.52u2009mmu2009Hg (95% CI: −1.39, 0.13)), and apparently not related to age, baseline BP values, dose of lactotripeptides assumed or length of the treatment. VPP and IPP lactotripeptides assumed as functional foods may significantly reduce SBP particularly in Asian subjects. The relevance of this findings in other ethnicities or associated with different dietary pattern should to be further investigated.

Gut microbiota, metabolome and immune signatures in patients with uncomplicated diverticular disease

Objective The engagement of the gut microbiota in the development of symptoms and complications of diverticular disease has been frequently hypothesised. Our aim was to explore colonic immunocytes, gut microbiota and the metabolome in patients with diverticular disease in a descriptive, cross-sectional, pilot study. Design Following colonoscopy with biopsy and questionnaire phenotyping, patients were classified into diverticulosis or symptomatic uncomplicated diverticular disease; asymptomatic subjects served as controls. Mucosal immunocytes, in the diverticular region and in unaffected sites, were quantified with immunohistochemistry. Mucosa and faecal microbiota were analysed by the phylogenetic platform high taxonomic fingerprint (HTF)-Microbi.Array, while the metabolome was assessed by 1H nuclear magnetic resonance. Results Compared with controls, patients with diverticula, regardless of symptoms, had a >70% increase in colonic macrophages. Their faecal microbiota showed depletion of Clostridium cluster IV. Clostridium cluster IX, Fusobacterium and Lactobacillaceae were reduced in symptomatic versus asymptomatic patients. A negative correlation was found between macrophages and mucosal Clostridium cluster IV and Akkermansia. Urinary and faecal metabolome changes in diverticular disease involved the hippurate and kynurenine pathways. Six urinary molecules allowed to discriminate diverticular disease and control groups with >95% accuracy. Conclusions Patients with colonic diverticular disease show depletion of microbiota members with anti-inflammatory activity associated with mucosal macrophage infiltration. Metabolome profiles were linked to inflammatory pathways and gut neuromotor dysfunction and showed the ability to discriminate diverticular subgroups and controls. These data pave the way for further large-scale studies specifically aimed at identifying microbiota signatures with a potential diagnostic value in patients with diverticular disease.

Effect of Sulodexide on Albuminuria, NAG Excretion and Glomerular Filtration Response to Dopamine in Diabetic Patients

Background: Albuminuria is the best and most readily available marker for glomerular damage and progressive renal function loss in patients with diabetic nephropathy. Recently, administration of the oral glycosaminoglycan sulodexide (a mixture of 80% fast-moving heparin and 20% dermatan sulphate) was shown to effectively decrease albumin excretion rate in diabetics with nephropathy. Aims: To evaluate whether the hypoalbuminuric effect of sulodexide is associated with improvement of the renal vascular or tubule function. Methods: Forty-five type 1 diabetic patients, affected by diabetic nephropathy with albuminuria for at least 5 years, were randomly allocated to sulodexide or untreated. Those allocated to sulodexide were given 100 mg of sulodexide daily for 120 days. Renal vascular function (DIR) and N-acetyl-β-D-glucosaminidase (NAG) excretion were estimated before and at the end of the study, the former in thesulodexide group only. DIR was measured as two Crcl lasting 120 min (before and during 2 µg/kg b.w. i.v. dopamine). Results: The analysis of trends during the study demonstrated a marked reduction of albuminuria in the sulodexide group (from 126.1 ± 15.41 to 93.6 ± 13.7 mg/day). DIR rose from 13.2 ± 2.1% to 15.44 ± 1.9% (relative increase: +16.9%), and NAG excretion showed a decreasing trend decreased in the sulodexide group only (from 5.1 ± 0.62 to 4.7 ± 0.40 U/gcreat). Conclusion: The findings presented in this study indicate for the first time that orally available sulodexide may favorably affect the renal vascular function in type 1 diabetic patients with nephropathy and microalbuminuria. The effect of sulodexide on NAG is strongly influenced by the baseline NAG values, with a significant NAG reduction in the patients with the highest baseline NAG values.

The foodomics approach for the evaluation of protein bioaccessibility in processed meat upon in vitro digestion

The present work describes a foodomics protocol coupling an in vitro static simulation of digestion to a combination of omics techniques, to grant an overview of the protein digestibility of a meat‐based food, namely Bresaola. The proteolytic activity mediated by the digestive enzymes is evaluated through Bradford and SDS‐PAGE assays, combined to NMR relaxometry and spectroscopy, to obtain information ranging from the microscopic to the molecular level, respectively. The simple proteomics tool adopted here points out that a clear increase of bioaccessible proteins occurs in the gastric phase, rapidly disappearing during the following duodenal digestion. However, SDS‐PAGE and the Bradford assay cannot follow the fate of the digested proteins when the products are sized <5 kDa. Conversely, NMR spectroscopy is able to capture the overall molecular profile of small fragments and peptides, which are mainly formed during the duodenal phase, thus giving the kinetics of the whole digestion process. Time domain NMR relaxometry, finally, detects the swelling phenomenon occurring during the gastric phase, when the digestion fluid enters the meat matrix.

Implications of white striping and spaghetti meat abnormalities on meat quality and histological features in broilers

During the past few years, there has been an increasing prevalence of broiler breast muscle abnormalities, such as white striping (WS) and wooden breast conditions. More recently, a new muscular abnormality termed as spaghetti meat (SM) because of the altered structural integrity of the Pectoralis major muscle often associated with WS has emerged. Thus, this study aimed at evaluating the effects of WS and SM conditions, occurring alone or combined within the same P. major muscle, on meat quality traits and muscle histology. In two replications, 96 P. major muscles were classified into four classes: normal (N), WS, SM and WS/SM. The whole fillet was used for weight assessment and morphometric measurements, then each sample was cut in order to separate the superficial layer from the deep one and used to evaluate proximate composition, histological features, nuclear magnetic resonance relaxation times, functional properties and both myofibrillar and sarcoplasmic proteins profile. Fillets affected by WS and SM abnormalities exhibited higher weights and increased thickness and length. SM condition was associated with a relevant decrease in protein content coupled with a significant increase in moisture level, whereas fat content was affected only by the simultaneous presence of WS. Histological evaluations revealed that abnormal samples were characterized by several degenerative aspects that almost completely concerned the superficial layer of the fillets. White striped fillets exhibited necrosis and lysis of fibers, fibrosis, lipidosis, loss of cross striation and vacuolar degeneration. Moreover, SM samples were characterized by poor fiber uniformity and a progressive rarefaction of the endo- and peri-mysial connective tissue, whereas WS/SM fillets showed intermediate histological features. Nuclear magnetic resonance relaxation analysis revealed a higher proportion of extra-myofibrillar water in the superficial section of all the abnormal fillets, especially in SM samples, which consequently led to a reduction of the water holding capacity of meat. As for functional properties, abnormal fillets exhibited a lower protein solubility and higher ultimate pH values on both the superficial and deep sections. Although abnormal fillets exhibited higher yellowness values, no relevant effect on meat color was observed. The occurrence of WS and SM abnormalities led to increased carbonylation levels and more intense proteolytic processes. Overall, muscle abnormalities mainly affect the superficial layer of P. major muscle and particularly the occurrence of SM myopathy seems to implicate a more pronounced modification of meat quality traits than the mere presence of WS.

Metabolic Variability of a Multispecies Probiotic Preparation Impacts on the Anti-inflammatory Activity.

Background: In addition to strain taxonomy, the ability of probiotics to confer beneficial effects on the host rely on a number of additional factors including epigenetic modulation of bacterial genes leading to metabolic variability and might impact on probiotic functionality. Aims: To investigate metabolism and functionality of two different batches of a probiotic blend commercialized under the same name in Europe in models of intestinal inflammation. Methods: Boxes of VSL#3, a probiotic mixture used in the treatment of pouchitis, were obtained from pharmacies in UK subjected to metabolomic analysis and their functionality tested in mice rendered colitis by treatment with DSS or TNBS. Results: VSL#3-A (lot DM538), but not VSL#3-B (lot 507132), attenuated “clinical” signs of colitis in the DSS and TNBS models. In both models, VSL#3-A, but not VSL#3-B, reduced macroscopic scores, intestinal permeability, and expression of TNFα, IL-1β, and IL-6 mRNAs, while increased the expression of TGFβ and IL-10, occludin, and zonula occludens-1 (ZO-1) mRNAs and shifted colonic macrophages from a M1 to M2 phenotype (P < 0.05 vs. TNBS). In contrast, VSL#3-B failed to reduce inflammation, and worsened intestinal permeability in the DSS model (P < 0.001 vs. VSL#3-A). A metabolomic analysis of the two formulations allowed the identification of two specific patterns, with at least three-folds enrichment in the concentrations of four metabolites, including 1–3 dihydroxyacetone (DHA), an intermediate in the fructose metabolism, in VSL#3-B supernatants. Feeding mice with DHA, increased intestinal permeability. Conclusions: Two batches of a commercially available probiotic show divergent metabolic activities. DHA, a product of probiotic metabolism, increases intestinal permeability, highlighting the complex interactions between food, microbiota, probiotics, and intestinal inflammation.

Efficacy and Safety of a Multistrain Probiotic Formulation Depends from Manufacturing

Background Variability in probiotics manufacturing may affect their properties, with potential implications for their efficacy and safety. This is of particular concern with probiotic products destined for use in patients with serious medical conditions, including human immunodeficiency virus (HIV) infection. The purpose of the study was to carry out a series of experiments comparing the properties of the US-made probiotic formulation originally commercialized under the brand name VSL#3®, with those of the Italian-made formulation now commercialized under the same name. The US-made formulation has previously shown beneficial effects at the intestinal and neurological levels in HIV-infected subjects as well as in patients with inflammatory bowel diseases and hepatic encephalopathy. Methods Eleven subjects receiving combined antiretroviral therapy for HIV-1 were treated for 6u2009months with the US-made VSL#3 formulation. At baseline and 6u2009months, T-cells were analyzed for phenotype and activation markers, and fecal samples were analyzed for bifidobacteria, lactobacilli, and their metabolites. The fecal metabolome was assessed using 1H-NMR spectroscopy. Production of metabolites of interest by bacteria obtained from sachets of the two formulations was compared in vitro and their effects on a rat intestinal epithelial cell line (IEC-6) were assessed. Particular attention was paid to the metabolite 1,3-dihydroxyacetone (DHA). Results At 6u2009months, fecal samples showed a significant increase in the specific bacterial genera contained in the probiotic supplement. Immune activation was reduced as shown by a significant reduction in the percentage of CD4+CD38+HLA-DR+ T-cells at 6u2009months. Fecal concentrations of DHA decreased significantly. In vitro, significant differences in the production and metabolism of DHA were found between bacteria from the US-made and Italian-made formulations: the US-made formulation was able to metabolize DHA whereas the bacteria in the Italian-made formulation were producing DHA. DHA reduced the viability of Streptococcus thermophilus, reduced IEC-6 cell viability in a dose-dependent manner, and also led to a lower rate of repair to scratched IEC-6 cell monolayer. Conclusion Our data, in conjunction with previously published findings, confirm that the new Italian-made formulation of VSL#3® is different from the previous US-made VSL#3 and therefore its efficacy and safety in HIV-infected subjects is still unproven.

Osmotic dehydration of organic kiwifruit pre-treated by pulsed electric fields and monitored by NMR

Osmotic dehydration (OD) is a widely used preservation technique that consists in the reduction in food water activity by the immersion of the biological tissue in hypertonic solutions. The aim of this work was to analyze the effect of pulsed electric fields (PEF) in mass transfer as a pre-treatment of the OD using NMR. In this sense, PEF pre-treatments were done using three different voltages (100, 250 and 400V/cm) and 60 number of pulse. The OD of kiwifruit was carried out in 61.5% of sucrose solution at 25°C, for a contact period from 0 to 120min. The water distribution into the cellular tissue was studied by NMR relaxometry. In conclusion, NMR is an excellent technique for quantifying water molecules according to their interactions in the fruit tissue, obtaining the adsorbed water and opening the possibility to apply the BET model to fit the adsorbed isotherm over the whole range of water activity.

Novel approaches for the taxonomic and metabolic characterization of lactobacilli: Integration of 16S rRNA gene sequencing with MALDI-TOF MS and 1H-NMR.

Lactobacilli represent a wide range of bacterial species with several implications for the human host. They play a crucial role in maintaining the ecological equilibrium of different biological niches and are essential for fermented food production and probiotic formulation. Despite the consensus about the ‘health-promoting’ significance of Lactobacillus genus, its genotypic and phenotypic characterization still poses several difficulties. The aim of this study was to assess the integration of different approaches, genotypic (16S rRNA gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (1H-NMR), for the taxonomic and metabolic characterization of Lactobacillus species. For this purpose we analyzed 40 strains of various origin (intestinal, vaginal, food, probiotics), belonging to different species. The high discriminatory power of MALDI-TOF for species identification was underlined by the excellent agreement with the genotypic analysis. Indeed, MALDI-TOF allowed to correctly identify 39 out of 40 Lactobacillus strains at the species level, with an overall concordance of 97.5%. In the perspective to simplify the MALDI TOF sample preparation, especially for routine practice, we demonstrated the perfect agreement of the colony-picking from agar plates with the protein extraction protocol. 1H-NMR analysis, applied to both culture supernatants and bacterial lysates, identified a panel of metabolites whose variations in concentration were associated with the taxonomy, but also revealed a high intra-species variability that did not allow a species-level identification. Therefore, despite not suitable for mere taxonomic purposes, metabolomics can be useful to correlate particular biological activities with taxonomy and to understand the mechanisms related to the antimicrobial effect shown by some Lactobacillus species.

A first step towards a consensus static in vitro model for simulating full-term infant digestion

In vitro alternatives to clinical trials are used for studying human food digestion. For simulating infant digestion, only a few models, lacking physiological relevance, are available. Thanks to an extensive literature review of the in vivo infant digestive conditions, a gastrointestinal static in vitro model was developed for infants born at term and aged 28days. The model was applied to the digestion of a commercial infant formula. Kinetics of digestion, as well as the structural evolution, were compared with those obtained while submitting the same formula to the adult international consensus protocol of in vitro static digestion. The kinetics of proteolysis and lipolysis differed according to the physiological stage resulting mainly from the reduced level of enzymes and bile salts, as well as the higher gastric pH in the infant model. This in vitro static model of infant digestion is of interest for scientists, food or pharmaceutical manufacturers.