Foodstuffs of both plant and animal origin contain a wide range of bioactive compounds. Although human intervention studies are mandatory to assess the health effects of bioactives, the in vitro approach is often used to select the most promising molecules to be studied in vivo. To avoid misleading results, concentration and chemical form, exposure time, and potential cytotoxicity of the tested bioactives should be carefully set prior to any other experiments. In this study the possible cytotoxicity of different bioactives (docosahexaenoic acid, propionate, cyanidin-3-O-glucoside, protocatechuic acid), was investigated in HepG2 cells using different methods. Bioactives were supplemented to cells at different concentrations within the physiological range in human blood, alone or in combination, considering two different exposure times. Reported data clearly evidence that in vitro cytotoxicity is tightly related to the exposure time, and it varies among bioactives, which could exert a cytotoxic effect even at a concentration within the in vivo physiological blood concentration range. Furthermore, co-supplementation of different bioactives can increase the cytotoxic effect. Our results underline the importance of in vitro cytotoxicity screening that should be considered mandatory before performing studies aimed to evaluate the effect of bioactives on other cellular parameters. Although this study is far from the demonstration of a toxic effect of the tested bioactives when administered to humans, it represents a starting point for future research aimed at verifying the existence of a potential hazard due to the wide use of high doses of multiple bioactives.

As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier.

Nowadays the high nutritional value of whole grains is recognized, and there is an increasing interest in the ancient varieties for producing wholegrain food products with enhanced nutritional characteristics. Among ancient crops, einkorn could represent a valid alternative. In this work, einkorn flours were analyzed for their content in carotenoids and in free and bound phenolic acids, and compared to wheat flours. The most promising flours were used to produce conventional and sourdough fermented breads. Breads were in vitro digested, and characterized before and after digestion. The four breads having the best characteristics were selected, and the product of their digestion was used to evaluate their anti-inflammatory effect using Caco-2 cells. Our results confirm the higher carotenoid levels in einkorn than in modern wheats, and the effectiveness of sourdough fermentation in maintaining these levels, despite the longer exposure to atmospheric oxygen. Moreover, in cultured cells einkorn bread evidenced an anti-inflammatory effect, although masked by the effect of digestive fluid. This study represents the first integrated evaluation of the potential health benefit of einkorn-based bakery products compared to wheat-based ones, and contributes to our knowledge of ancient grains.

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein–protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named “Applied Proteomics” has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer—the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named “applied proteomics”. It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, “in vitro” and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined.

Background: Creatine is one of the most widespread dietary supplements among athletes and weightlifters. It is most commonly administered as creatine monohydrate, which transforms into phosphocreatine once within the muscle, which leads to the production of adenosine triphosphate (ATP). Clonapure® is a functional ingredient which consists of a combination of creatine monohydrate, phosphate salt and phosphocreatine. It is believed that Clonapure® has an impact on muscular ATP synthesis due to its phosphocreatine content. A comparative in vitro study was carried to evaluate the potential effect of Clonapure® on ATP synthesis versus creatine monohydrate. Methods: A C2C12 myoblast cell line finally differentiated into myotubes was used. Bioaviability of samples was measured at 24h through biocompatibility studies. Myotubes were also exposed to samples at different concentrations for 2h and intracellular ATP levels were evaluated througha ergular validated luminescence methos. Glucose was used as positive control. Results: The increase in intracellular ATP levels induced by Clonapure® was 10% higher than the caused by creatine monohydrate and glucose, which both showed similar effects in ATP synthesis. The combination of creatine-phosphate phosphate and creatine monohydrate contained in Clonapure® triggers the intracellular ATP production cascade more efficiently in muscle cells. Dose response analysis revealed that Clonapure® supplementation could boost conveniently physical activities related to endurance and performance.