Casein phosphopeptides : from milk to nutraceutical

Milk and dairy products are known sources of bioavailable calcium for its association with casein, whose proteolysis produces caseinphosphopeptides (CPPs). CPPs are phosphorylated peptides able to bind and solubilise calcium. In human intestinal tumor cells differentiated in vitro toward an enterocityc phenotype, they also induce a calcium uptake. Moreover, in human in vitro osteoblasts, CPPs favour the mineralization of the extracellular matrix. CPPs can differently affect proliferation and apoptosis in differentiated and /or tumor intestinal cells. Due to all these properties, CPPs may be considered as potential nutraceutical/functional food

Preliminary study of the effects of ultrasound on red wine polyphenols

The article evaluates the effect of high-power ultrasound (20 kHz) on the phenolic structure of red wines, to study the possible applications of this innovative technique in wine aging. Different treatment conditions, with times of 1, 3 and 5 minutes and amplitudes of 51, 102 and 153 \u3bcm, were applied. In the experimental conditions the main parameters related to the evolution of red wine phenolic compounds show interesting variations; in particular, we found significant differences on tannins evaluated with some indices of tannins reactivity. On the other hand, free anthocyanins do not undergo changes due to ultrasound application. The preliminary results, which definitely need a depth, nonetheless allow us to hypothesize the application of ultrasound technology to accelerate the aging of red wines, which normally requires (needs) long times in the cellar condition

Tourist and Viral Mobilities Intertwined: Clustering COVID-19-Driven Travel Behaviour of Rural Tourists in South Tyrol, Italy

Travel patterns have dramatically changed during the COVID-19 pandemic. Tourism has been both a vector and a victim of the disease. This paper explores the pandemic’s impact on rural tourism, using the theoretical framework of the “mobilities turn” to investigate issues of corporeal and communicative travel found between the first and second waves of the COVID-19 pandemic. A sample of 874 guests visiting the Italian region of South Tyrol, where rural tourism is the norm, identified different patterns of physical travel and approaches to collecting on-site information on COVID-19. Results from a principal component analysis (PCA) and a cluster analysis highlighted at least two different approaches from visitors to the region: the first is more cautious, mostly practiced by domestic tourists, with limited mobility on-site, coupled with a need for information; the second is instead a more adventurous approach, with higher on-site mobility, more use of sustainable forms of transport and less interest in data evidence on COVID-19. Implications for rural tourism and its future are discussed. The hypothesis of an inverse relationship between corporeal and communicative travel needs further exploration in future research

High power ultrasound treatments of red young wines: Effect on anthocyanins and phenolic stability indices

Polyphenols, especially anthocyanins, play an important role on red wine sensory qualities and their evolution during storage. High Power Ultrasound (HPU) has been recognized as one of the most promising technologies which can be applied in winemaking processes for several purposes, and it is recently officially approved for crushed grapes treatments. The effect of ultrasound amplitude (41 and 81%) and treatment time (1, 3, and 5 min) has been studied on anthocyanins, flavan-3-ols, tannins, polymerized pigments, HCl index, and the color intensity of two finished red young wines. Anthocyanins and phenols compounds were not degraded with an increase in amplitude and sonication time, and the chromatic properties of the selected wines were preserved. Amplitude and ultrasound time were also evaluated considering their effect on evolution of anthocyanin content and phenolic stability indices during the first thirty days of storage. The higher level of amplitude (81%) induced a higher percentage decrease in tannins, 15% and 40% after 15 and 30 days of storage, respectively, compared to untreated wine which did not show a significant change during storage. HPU shows a possible chemical effect on the evolution of some analytical indices during bottling maturation, but their effectiveness could be strictly linked with the initial phenolic profile and ratios between polyphenol classes

In vitro antioxidant properties of digests of hydrolyzed casein and caseinophosphopeptide preparations in cell models of human intestine and osteoblasts

Three commercial samples consisting of enriched calcium-free caseinophosphopeptides (CPP), enriched calcium-bound caseinophosphopeptides (Ca-CPP) and an enzymatically hydrolyzed casein (hCN) were in vitro digested according to COST-Infogest protocol. As assessed by UPLC-HR-MS/MS, the digests contained 207\u2013235 unique caseinophosphopeptides, and the species presenting the cluster sssEE were more abundant in CPP digest. The antioxidant activity at three different doses of each digest was firstly evaluated on human intestinal Caco-2/HT-29 70/30 co-culture. In presence of AAPH, hCN and CPP digests displayed a dose-dependent antioxidant activity equal or even greater than Vitamin C. In presence of Fe2+, the digests exerted an antioxidant activity mainly at the highest dose. Antioxidant activities of the intestinal metabolized digests was then evaluated on human osteoblast (Saos-2) cells. The digests exerted an antioxidant activity in presence of AAPH, but not in presence of Fe2+. These results highlight milk-derived peptides as potential dietary supplements for gut and bone health

New methodological approach to induce a differentiation phenotype in Caco-2 cells prior to post-confluence stage

BACKGROUND: Various differentiation-inducing agents or harvesting of spontaneously late post-confluence cultures have been used to differentiate the human colon carcinoma Caco-2 cell line. We report a new procedure to generate pre-confluent subcultures of Caco-2 population at various stages of differentiation without altering culture conditions. MATERIALS AND METHODS: Ultrastructural analysis, cell proliferation activity and biochemical markers of differentiation were evaluated at different passages. RESULTS: Subcultures of Caco-2 cells at pre-confluence, exhibiting progressive acquisition of a more benign differentiation phenotype, were generated. Early passages of Caco-2 cells showed a well-developed brush border and incomplete junctional apparatus; subsequent subcultures yielded cell populations with well-developed junctions similar to those of small intestinal cells. CONCLUSION: These culture conditions represent a new versatile model not only to progressively induce the differentiation program in Caco-2 cells at pre-confluence without changes of culture media, but also to explore mechanistic modes of drug transport and tumor development

Nutrient excess in a stabilized co-culture system of Caco2/HT-29 cells: an ultrastructural and functional time-course study

The intestine represents one of the most important barriers of our body and interacts with several exogenous substances, among which nutrients. Today, the effects due to an excess of nutrients on intestinal morpho-functional changes, similar to the ones found in obesity, have been studied only in in vivo animal models. Many experimental difficulties hampered in establishing a physiological long-term experimental model starting from primary cultures of normal small intestinal and colon cells. For this reason, an intestinal Caco2/HT-29 (70/30) co-culture was set up in our lab starting from the differentiated parental cell populations to mimic the human intestinal epithelium. Co-culture was harvested at confluence (T0) and at 3, 7, and 15 days (T3, T7, and T15, respectively) post-confluence. Ultrastructural (TEM) and functional analysis (Alkaline Phosphatase, ALP; Aminopeptidase N, APN; Dipeptidyl Peptidase-IV , DPPIV; Transepithelial Electrical Resistance, TEER) were carried out. In the present study, two parallel experimental groups were cultured: the standard group and the excess group. In the standard group, the culture medium was changed every four days, whilst in the excess group on alternate days from T0. Transmission electron microscopy revealed that the excess of nutrients drives co-cultures towards a less differentiated absorptive phenotype. On the other hand, mucus granule presence was more and more evident from T3. The specific activity of ALP and APN, known markers of intestinal differentiation, and that of DPPIV, a specific marker of enterocyte differentiation, progressively increased. TEER, indicative of the barrier properties of the co-culture, increased at post confluence up to T15. In conclusion, data here presented show that the excess of nutrients can directly modify both morphology and function of the intestinal cells, opening the way to study at the effects due to specific nutrients on cell proliferation and differentiation involved in the acquisition of an obese human phenotype

The influence of caseinphosphopeptides on intracellular calcium changes in primary human osteoblasts : a nutrient dependent modulation of bone cell metabolism

Caseinphosphopeptides (CPPs) are a family of peptides originating from in vivo and in vitro hydrolysis of casein. They possess a sequence of three phosphorylated serines followed by two glutamic acids, the acidic motif, able to bind minerals such as calcium. These nutritional compounds display the ability to increase calcium solubility in the digestive tract. Thus, CPPs were hypothesized to increase the calcium absorption and retention in vivo, with potential effects on bone mineralization. Notwithstanding, there are controversial reports on CPP action. The methodological approach used by different laboratories to study calcium absorption and bone mineralization resulted unable to out light whether the peptides have a specific effect on bone metabolism besides the enhancement of calcium availability. We have therefore designed the following study to evaluate a possible direct role of CPPs in bone cell metabolism. Primary human osteoblasts were established in culture using trabecular bone samples obtained from waste materials during orthopedic surgery of patients without metabolic or malignant bone disease. Cytosolic calcium changes were measured by video-microscopy using the fura-2 method on single cells. A mixture of CPPs of commercial origin as well as pure synthetic CPPs were used. The administration of CPPs to human osteoblasts caused an immediate but transient intracellular calcium change in a dose dependent manner. This CPP-induced effect, analogous to that reported for human intestinal cells, is not cytotoxic and is triggered by an influx of the extracellular ions through the cell plasma membrane. The osteoblast pre-treatment with the active form of vitamin D, known to differentiate human osteoblast, does not affect the cell responsiveness to CPP administration. The 24 hours cell incubation with CPPs induced the increase of the activity of alkaline phosphatase, a marker of osteoblast differentiation, reaching a level similar to that produced by vitamin D. The same CPP treatment caused a small but significative reduction in cell rate proliferation and a slight increase in apoptosis activity. Taken together these results indicate that CPPs are endowed of a bone specific effect which underlying mechanism requires further evaluation. CPPs may act not only as a mere carrier for improving calcium absorption and utilization, but also as a trophyc compound for bone health by enhancing osteoblast differentiation and activity

Co-culture of Caco2 and HT-29 cells as an innovative method to mimic in vitro the morphology and permeability properties of human intestinal epithelium

For investigating the complexity of the human intestinal epithelium, a valid experimental approach is represented by co-culture. In the present study an intestinal co-culture Caco2/HT-29 (70/30) was set up starting from the parental populations of differentiated cells as previously described [1, 2]. Co-culture was harvested at 0 (T0), 6 (T6), and 14 (T14) days of post confluence after plating. Transmission electron microscopy was carried out to monitor the morphological features of cell differentiation. Alkaline Phosphatase (ALP), Aminopeptidase N (APN) and Dipeptidyl Peptidase IV (DPP IV) activity were assayed as known markers of intestinal cell differentiation. The measure of TEER and the apparent permeability of Lucifer Yellow allows to monitor the integrity of the tight junctions and the permeability of the cell layer formed. At T0 a classical monolayer is present, with a mixed population of immature absorptive elements and secretive cells. At T6 and T14, cells are progressively organized in a multilayer with a parallel growth of microvilli. At T6, co-culture demonstrates good properties of permeability and barrier components, such as mucus, representing an appropriate model for absorption study. At T14, the brush border is even more developed respect to T6 and, together with the increase of the specific activity of ALP, APN, and DPP IV, indicate co-culture as a good model for digestion study. The advantage of this co-culture described is the use of the whole cell population without particular inducers of subclones and growth supports. In conclusion, the morphological and biochemical features of co-cultured parental cells change with time, strongly supporting i) an active interaction between the two parental cell lines and ii) the versatility of this model, with more than one prevalent cell type depending on the post confluent stage