Common food additive carrageenan inhibits proglucagon expression and GLP-1 secretion by human enteroendocrine L-cells.

Proglucagon mRNA expression and GLP-1 secretion by cultured human L-cells (NCI-H716) were inhibited following exposure to λ-carrageenan, a commonly used additive in processed foods. Carrageenan is composed of sulfated or unsulfated galactose residues linked in alternating alpha-1,3 and beta-1,4 bonds and resembles the endogenous sulfated glycosaminoglycans. However, carrageenan has unusual alpha-1,3-galactosidic bonds, which are not innate to human cells and are implicated in immune responses. Exposure to carrageenan predictably causes inflammation, and carrageenan impairs glucose tolerance and contributes to insulin resistance. When cultured human L-cells were deprived overnight of glucose and serum and then exposed to high glucose, 10% FBS, and λ-carrageenan (1 µg/ml) for 10 minutes, 1 h, and 24 h, mRNA expression of proglucagon and secretion of GLP-1 were significantly reduced, compared to control cells not exposed to carrageenan. mRNA expression of proglucagon by mouse L-cells (STC-1) was also significantly reduced and supports the findings in the human cells. Exposure of co-cultured human intestinal epithelial cells (LS174T) to the spent media of the carrageenan-treated L-cells led to a decline in mRNA expression of GLUT-2 at 24 h. These findings suggest that ingestion of carrageenan-containing processed foods may impair the production of GLP-1, counteract the effect of GLP-1 receptor agonists and induce secondary effects on intestinal epithelial cells.

Personalized nutrition with 3D-printed foods: A systematic review on the impact of different additives.

Three-dimensional (3D) printing is one of the world's top novel technologies in the food industry due to the production of food in different conditions and places (restaurants, homes, catering, schools, for dysphagia patients, and astronauts' food) and the production of personalized food. Nowadays, 3D printers are used in the main food industries, including meat, dairy, cereals, fruits, and vegetables, and have been able to produce successfully on a small scale. However, due to the expansion of this technology, it has challenges such as high-scale production, selection of printable food, formulation optimization, and food production according to the consumer's opinion. Food additives (gums, enzymes, proteins, starches, polyphenols, spices, probiotics, algae, edible insects, oils, salts, vitamins, flavors, and by-products) are one of the main components of the formulation that can be effective in food production according to the consumer's attitude. Food additives can have the highest impact on textural and sensory characteristics, which can be effective in improving consumer attitudes and reducing food neophobia. Most of the 3D-printed food cannot be printed without the presence of hydrocolloids, because the proper flow of the selected formulation is one of the key factors in improving the quality of the printed product. Functional additives such as probiotics can be useful for specific purposes and functional food production. Food personalization for specific diseases with 3D printing technology requires a change in the formulation, which is closely related to the selection of correct food additives. For example, the production of 3D-printed plant-based steaks is not possible without the presence of additives, or the production of food for dysphagia patients is possible in many cases by adding hydrocolloids. In general, additives can improve the textural, rheological, nutritional, and sensory characteristics of 3D printed foods; so, investigating the mechanism of the additives on all the characteristics of the printed product can provide a wide perspective for industrial production and future studies.

Coupling of a streamlined solid-liquid extraction protocol with LC-ESI-MS/MS for the regular oversight of illegal bromate additive use: An accurate and sensitive determination method in preliminary and bakery products.

A reliable solid-liquid extraction protocol coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry in the negative-ion mode was developed and validated for illegal bromate determination in preliminary and bakery products. Crude and dried-treated samples were directly extracted with acetonitrile-water (4:1, v/v). Bromate was determined using a Phenomenex Synergi™ Polar reversed-phase column and MS/MS under multiple reaction monitoring. The chosen solvent efficiently extracted bromate with all applied extraction-assisting techniques (p > 0.05). Although this assay avoids cleanup procedures, matrix effect of <-11% was achieved. Rapid bromate separation in only 8 min was attained by a reversed-phase column. In both commodities, linearity range, R2, recovery%, repeatability, intermediate precision, LOD and LOQ results were 0.05-100 ng mL-1, >0.9999, 88.6-103%, 2.93-9.80% and 9.64-10.10%, 0.015 µg kg-1 and 0.05 µg kg-1, respectively. Out of 288 tested real samples, 13.9% of violations were observed. This high-sensitivity protocol offers effective oversight and consumer protection.

Risk assessment of feed components of botanical origin - Approaches taken in the European Union.

In view of a continuous trend in replacing synthetic feed additives and especially flavouring compounds by botanical preparations, different aspects of the safety evaluations of plants and plant-derived preparations and components in feed are discussed. This includes risk assessment approaches developed by the European Food Safety Authority (EFSA) for phytotoxins regarding unintentional exposure of target animals and of consumers to animal derived food via carry-over from feed. Relevant regulatory frameworks for feed additives and feed contaminants in the European Union are summarised and the essentials of existing guidelines used in the safety evaluation of botanicals and their preparations and components in feed are outlined. The examples presented illustrate how the safety of the botanicals, their preparations and components present in feed is assessed. An outlook on possible future developments in risk assessment by applying new in vitro and in silico methodologies is given.

Inclusion Complexes of Litsea cubeba (Lour.) Pers Essential Oil into ß-Cyclodextrin: Preparation, Physicochemical Characterization, Cytotoxicity and Antifungal Activity.

The uses of natural compounds, such as essential oils (EOs), are limited due to their instability to light, oxygen and temperature, factors that affect their application. Therefore, improving stability becomes necessary. The objective of this study was to prepare inclusion complexes of Litsea cubeba essential oil (LCEO) with ß-cyclodextrin (ß-CD) using physical mixing (PM), kneading (KN) and co-precipitation (CP) methods and to evaluate the efficiency of the complexes and their physicochemical properties using ATR-FTIR, FT-Raman, DSC and TG. The study also assessed cytotoxicity against human colorectal and cervical cancer cells and antifungal activity against Aspergillus flavus and Fusarium verticillioides. The complexation efficiency results presented significant evidence of LCEO:ß-CD inclusion complex formation, with KN (83%) and CP (73%) being the best methods used in this study. All tested LCEO:ß-CD inclusion complexes exhibited toxicity to HT-29 cells. Although the cytotoxic effect was less pronounced in HeLa tumor cells, LCEO-KN was more active against Hela than non-tumor cells. LCEO-KN and LCEO-CP inclusion complexes were efficient against both toxigenic fungi, A. flavus and F. verticillioides. Therefore, the molecular inclusion of LCEO into ß-CD was successful, as well as the preliminary biological results, evidencing that the ß-CD inclusion process may be a viable alternative to facilitate and increase future applications of this EO as therapeutic medication, food additive and natural antifungal agent.

Computational framework for identifying and evaluating mutagenic and xenoestrogenic potential of food additives.

Food additives are chemicals incorporated in food to enhance its flavor, color and prevent spoilage. Some of these are associated with substantial health hazards, including developmental disorders, increase cancer risk, and hormone disruption. Hence, this study aimed to comprehend the in-silico toxicology framework for evaluating mutagenic and xenoestrogenic potential of food additives and their association with breast cancer. A total of 2885 food additives were screened for toxicity based on Threshold of Toxicological Concern (TTC), mutagenicity endpoint prediction, and mutagenic structural alerts/toxicophores identification. Ten food additives were identified as having mutagenic potential based on toxicity screening. Furthermore, Protein-Protein Interaction (PPI) analysis identified ESR1, as a key hub gene in breast cancer. KEGG pathway analysis verified that ESR1 plays a significant role in breast cancer pathogenesis. Additionally, competitive interaction studies of the predicted potential mutagenic food additives with the estrogen receptor-α were evaluated at agonist and antagonist binding sites. Indole, Dichloromethane, Trichloroethylene, Quinoline, 6-methyl quinoline, Ethyl nitrite, and 4-methyl quinoline could act as agonists, and Paraldehyde, Azodicarbonamide, and 2-acetylfuranmay as antagonists. The systematic risk assessment framework reported in this study enables the exploration of mutagenic and xenoestrogenic potential associated with food additives for hazard identification and management.

Advances in biotin biosynthesis and biotechnological production in microorganisms.

Biotin, also known as vitamin H or B7, acts as a crucial cofactor in the central metabolism processes of fatty acids, amino acids, and carbohydrates. Biotin has important applications in food additives, biomedicine, and other fields. While the ability to synthesize biotin de novo is confined to microorganisms and plants, humans and animals require substantial daily intake, primarily through dietary sources and intestinal microflora. Currently, chemical synthesis stands as the primary method for commercial biotin production, although microbial biotin production offers an environmentally sustainable alternative with promising prospects. This review presents a comprehensive overview of the pathways involved in de novo biotin synthesis in various species of microbes and insights into its regulatory and transport systems. Furthermore, diverse strategies are discussed to improve the biotin production here, including mutation breeding, rational metabolic engineering design, artificial genetic modification, and process optimization. The review also presents the potential strategies for addressing current challenges for industrial-scale bioproduction of biotin in the future. This review is very helpful for exploring efficient and sustainable strategies for large-scale biotin production.

Food additive emulsifiers and the risk of type 2 diabetes: analysis of data from the NutriNet-Santé prospective cohort study.

BACKGROUND: Experimental studies have suggested potential detrimental effects of emulsifiers on gut microbiota, inflammation, and metabolic perturbations. We aimed to investigate the associations between exposures to food additive emulsifiers and the risk of type 2 diabetes in a large prospective cohort of French adults. METHODS: We analysed data from 104 139 adults enrolled in the French NutriNet-Santé prospective cohort study from May 1, 2009, to April 26, 2023; 82 456 (79·2%) were female and the mean age was 42·7 years (SD 14·5). Dietary intakes were assessed with three 24 h dietary records collected over three non-consecutive days, every 6 months. Exposure to additive emulsifiers was evaluated through multiple food composition databases and ad-hoc laboratory assays. Associations between cumulative time-dependent exposures to food additive emulsifiers and the risk of type 2 diabetes were characterised with multivariable proportional hazards Cox models adjusted for known risk factors. The NutriNet-Santé study is registered at ClinicalTrials.gov (NCT03335644). FINDINGS: Of 104 139 participants, 1056 were diagnosed with type 2 diabetes during follow-up (mean follow-up duration 6·8 years [SD 3·7]). Intakes of the following emulsifiers were associated with an increased risk of type 2 diabetes: total carrageenans (hazard ratio [HR] 1·03 [95% CI 1·01-1·05] per increment of 100 mg per day, p<0·0001), carrageenans gum (E407; HR 1·03 [1·01-1·05] per increment of 100 mg per day, p<0·0001), tripotassium phosphate (E340; HR 1·15 [1·02-1·31] per increment of 500 mg per day, p=0·023), acetyl tartaric acid esters of monoglycerides and diglycerides of fatty acids (E472e; HR 1·04 [1·00-1·08] per increment of 100 mg per day, p=0·042), sodium citrate (E331; HR 1·04 [1·01-1·07] per increment of 500 mg per day, p=0·0080), guar gum (E412; HR 1·11 [1·06-1·17] per increment of 500 mg per day, p<0·0001), gum arabic (E414; HR 1·03 [1·01-1·05] per increment of 1000 mg per day, p=0·013), and xanthan gum (E415, HR 1·08 [1·02-1·14] per increment of 500 mg per day, p=0·013). INTERPRETATION: We found direct associations between the risk of type 2 diabetes and exposures to various food additive emulsifiers widely used in industrial foods, in a large prospective cohort of French adults. Further research is needed to prompt re-evaluation of regulations governing the use of additive emulsifiers in the food industry for better consumer protection. FUNDING: European Research Council, French National Cancer Institute, French Ministry of Health, IdEx Université de Paris, and Bettencourt-Schueller Foundation.

A Gene-Switch Platform Interfacing with Reactive Oxygen Species Enables Transcription Fine-Tuning by Soluble and Volatile Pharmacologics and Food Additives.

Synthetic biology aims to engineer transgene switches for precise therapeutic protein control in cell-based gene therapies. However, off-the-shelf trigger-inducible gene circuits are usually switched on by single or structurally similar molecules. This study presents a mammalian gene-switch platform that controls therapeutic gene expression by a wide range of molecules generating low, non-toxic levels of reactive oxygen species (ROS). In this system, KEAP1 (Kelch-like ECH-associated protein 1) serves as ROS sensor, regulating the translocation of NRF2 (nuclear factor erythroid 2-related factor 2) to the nucleus, where NRF2 binds to  antioxidant response elements (ARE) to activate the expression of a gene of interest. It is found that a promoter containing eight-tandem ARE repeats is highly sensitive to the low ROS levels generated by the soluble and volatile molecules, which include food preservatives, food additives, pharmaceuticals, and signal transduction inducers. In a proof-of-concept study, it is shown that many of these compounds can independently trigger microencapsulated engineered cells to produce sufficient insulin to restore normoglycemia in experimental type-1 diabetic mice. It is believed that this system greatly extends the variety of small-molecule inducers available to drive therapeutic gene switches.

Food additives impair gut microbiota from healthy individuals and IBD patients in a colonic in vitro fermentation model.

Intestinal fibrosis is a long-term complication of inflammatory bowel diseases (IBD). Changes in microbial populations have been linked with the onset of fibrosis and some food additives are known to promote intestinal inflammation facilitating fibrosis induction. In this study, we investigated how polysorbate 80, sucralose, titanium dioxide, sodium nitrite and maltodextrin affect the gut microbiota and the metabolic activity in healthy and IBD donors (patients in remission and with a flare of IBD). The Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) with a static (batch) configuration was used to evaluate the effects of food additives on the human intestinal microbiota. Polysorbate 80 and sucralose decreased butyrate-producing bacteria such as Roseburia and Faecalibacterium prausnitzii. Both compounds, also increased bacterial species positively correlated with intestinal inflammation and fibrosis (i.e.: Enterococcus, Veillonella and Mucispirillum schaedleri), especially in donors in remission of IBD. Additionally, polysorbate 80 induced a lower activity of the aryl hydrocarbon receptor (AhR) in the three groups of donors, which can affect the intestinal homeostasis. Maltodextrin, despite increasing short-chain fatty acids production, promoted the growth of Ruminococcus genus, correlated with higher risk of fibrosis, and decreased Oscillospira which is negatively associated with fibrosis. Our findings unveil crucial insights into the potential deleterious effects of polysorbate 80, sucralose and maltodextrin on human gut microbiota in healthy and, to a greater extent, in IBD patients.

Extraction, purification, structural characteristics, bioactivity and potential applications of polysaccharides from Avena sativa L.: A review.

Avena sativa L. (A. sativa L.), commonly known as oat, is a significant cereal grain crop with excellent edible and medicinal value. Oat polysaccharides (OPs), the major bioactive components of A. sativa L., have received considerable attention due to their beneficial bioactivities. However, the isolation and purification methods of OPs lack innovation, and the structure-activity relationship remains unexplored. This review emphatically summarized recent progress in the extraction and purification methods, structural characteristics, biological activities, structure-to-function associations and the potential application status of OPs. Different materials and isolation methods can result in the differences in the structure and bioactivity of OPs. OPs are mainly composed of various monosaccharide constituents, including glucose, arabinose and mannose, along with galactose, xylose and rhamnose in different molar ratios and types of glycosidic bonds. OPs exhibited a broad molecular weight distribution, ranging from 1.34 × 105 Da to 4.1 × 106 Da. Moreover, structure-activity relationships demonstrated that the monosaccharide composition, molecular weight, linkage types, and chemical modifications are closely related to their multiple bioactivities, including immunomodulatory activity, antioxidant effect, anti-inflammatory activity, antitumor effects etc. This work can provide comprehensive knowledge, update information and promising directions for future exploitation and application of OPs as therapeutic agents and multifunctional food additives.

The food additive xylitol enhances the butyrate formation by the child gut microbiota developed in a dynamic colonic simulator.

The gut microbiota should be included in the scientific processes of risk assessment of food additives. Xylitol is a sweetener that shows low digestibility and intestinal absorption, implying that a high proportion of consumed xylitol could reach the colonic microbiota. The present study has evaluated the dose-dependent effects of xylitol intake on the composition and the metabolic activity of the child gut-microbiota. The study was conducted in a dynamic simulator of the colonic microbiota (BFBL Gut Simulator) inoculated with a child pooled faecal sample and supplemented three times per day, for 7 days, with increasing xylitol concentrations (1 g/L, 3 g/L and 5 g/L). Sequencing of 16S rRNA gene amplicons and group-specific quantitative PCR indicated a xylitol dose-response effect on the abundance of Lachnospiraceae, particularly the genera Blautia, Anaerostipes and Roseburia. The microbial changes observed with xylitol corresponded with a dose-dependant effect on the butyrate concentration that, in parallel, favoured an increase in epithelial integrity of Caco-2 cells. The study represents a detailed observation of the bacterial taxa that are the main contributors to the metabolism of xylitol by the child gut microbiota and the results could be relevant in the risk assessment re-evaluation of xylitol as a sweetener.

Validating Enteroid-Derived Monolayers from Murine Gut Organoids for Toxicological Testing of Inorganic Particles: Proof-of-Concept with Food-Grade Titanium Dioxide.

Human exposure to foodborne inorganic nanoparticles (NPs) is a growing concern. However, identifying potential hazards linked to NP ingestion often requires long-term exposure in animals. Owing these constraints, intestinal organoids are a promising alternative to in vivo experiments; as such, an in vitro approach should enable a rapid and reliable assessment of the effects of ingested chemicals on the gut. However, this remains to be validated for inorganic substances. In our study, a transcriptomic analysis and immunofluorescence staining were performed to compare the effects of food-grade TiO2 (fg-TiO2) on enteroid-derived monolayers (EDMs) from murine intestinal organoids to the known impacts of TiO2 on intestinal epithelium. After their ability to respond to a pro-inflammatory cytokine cocktail was validated, EDMs were exposed to 0, 0.1, 1, or 10 µg fg-TiO2/mL for 24 h. A dose-related increase of the muc2, vilin 1, and chromogranin A gene markers of cell differentiation was observed. In addition, fg-TiO2 induced apoptosis and dose-dependent genotoxicity, while a decreased expression of genes encoding for antimicrobial peptides, and of genes related to tight junction function, was observed. These results validated the use of EDMs as a reliable model for the toxicity testing of foodborne NPs likely to affect the intestinal barrier.

[Route to the Adoption of Quantitative NMR in the Japanese Pharmacopoeia].

Quantitative NMR (qNMR) is employed to determine the purity of reagents used as standards for HPLC quantification in the Japanese Pharmacopoeia (JP) and has become recognized as a new absolute quantification method in various fields such as pharmaceuticals, foods, and food additives. This report outlines how and why qNMR has been adopted as an official method in the JP and introduces its progression from JP16 to JP18. The results of a survey of companies in the Japan Pharmaceutical Manufacturers' Association regarding how and when to use qNMR from development to manufacturing stages are introduced. The issues involved in the expansion of the use of qNMR in the field of chemical pharmaceuticals in 2017 are discussed and how these were resolved.

[Standardization and Practical Application of Quantitative NMR (qNMR)].

In Japan, quantitative NMR (qNMR) has already been recognized as a standard method for determining the purity of quantitative samples not only in the Japanese Pharmacopoeia and the Japanese Standards and Specifications for Food Additives but also in the Japanese Industrial Standard (JIS K 0138: 2018). However, since there was no consensus on the establishment of a standard method, the international standardization of qNMR was initiated based on a proposal from Japan. After three years of discussion among experts, International Organization for Standardization/Technical Committee on Food (ISO/TC34) published ISO 24583: 2022 "Quantitative nuclear magnetic resonance spectroscopy-Purity determination of organic compounds used for foods and food products-General requirements for 1H-NMR internal standard method." Publication of this standard has resulted in an internationally agreed upon set of requirements for purity determination using qNMR. New technologies emerge from the cycle of basic research, practical use, and standardization, and qNMR is no exception. A novel chromatographic quantification method based on relative molar sensitivity (RMS) is now being put into practical use. The RMS of an analyte with respect to a different reference substance can be determined by using qNMR to accurately determine the molar ratio and then introducing it into the chromatographic system. This method uses the RMS determined by combining qNMR and chromatography instead of the analyte's reference material to determine its content in sample. This method has been adopted in the Japanese Pharmacopoeia, and the development of a general rule in the Japanese Agricultural Standards (JAS) is also under consideration.

Improved biocatalytic activity of steapsin lipase in supercritical carbon dioxide medium for the synthesis of benzyl butyrate: A commercially important flavour compound.

Enzymatic synthesis of flavours, fragrances and food additives compounds have great demand and market value. Benzyl butyrate is commercially important flavour and food additive compound having global use around 100 metric tons/year and widely used in various industrial sectors. However, industrial synthesis of food additive benzyl butyrate is carried out by conventional chemical process which demands for the green biobased sustainable synthetic process. The present work reports steapsin catalyzed synthesis of benzyl butyrate for the first time in supercritical carbon dioxide (Sc-CO2) reaction medium. All reaction variables are optimized in details to obtain competent conversion of 99% in Sc-CO2 reaction medium. The developed steapsin catalyzed synthesis in Sc-CO2 medium offered almost four-fold higher conversion to benzyl butyrate than organic (conventional) solvent. The steapsin biocatalyst was effectually recycled up to five reaction cycles in Sc-CO2 medium. Moreover, the developed steapsin catalyzed protocol in Sc-CO2 medium was extended to synthesize different ten industrially significant flavour fragrance compounds that offers 99% conversion and three to five-folds higher conversion than organic medium. Thus, the present steapsin catalyzed protocol offered improved synthesis of various commercially significant flavour compounds in Sc-CO2. medium.

Genotoxicity evaluation of food additive titanium dioxide using a battery of standard in vivo tests.

The increasing use of titanium dioxide (TiO2) nanoparticles (NPs) has raised concern about the safety of food additive TiO2. TiO2 has been considered no longer safe by EFSA due to concerns over genotoxicity, however, there are conflicting opinions upon the safety of TiO2 as a food additive, and the number of in vivo genotoxicity studies conducted on food additive TiO2 was limited. In order to investigate the potential genotoxicity of food additive TiO2, we evaluated the genotoxicity of a commercial food additive TiO2 (average size of 135.54 ± 41.01 nm, range from 60.83 to 230.16 nm, NPs account for 30% by number) using a battery of standard in vivo tests, including mammalian erythrocyte micronucleus test, mammalian bone marrow chromosomal aberration test and in vivo mammalian alkaline comet test. After 15 days of consecutive intragastric administration at doses of 250, 500, and 1000 mg/kgBW, food additive TiO2 neither increased the frequencies of bone marrow micronuclei or chromosomal aberration in mice, nor induced DNA strand breakage in rat liver cells. These results indicate that under the condition of this study, food additive TiO2 does not have genotoxic potential although it contains a fraction of NPs.

Subatomic structure of orthorhombic thaumatin at 0.89 Å reveals that highly flexible conformations are crucial for thaumatin sweetness.

Thaumatin is a sweet-tasting protein that elicits a sweet taste at a threshold of approximately 50 nM. Structure-sweetness relationships in thaumatin suggest that the basicity of two amino acids residues, Arg82 and Lys67, are particularly responsible for sweetness. Using tetragonal crystals, our structural analysis suggested that flexible sidechain conformations of these two residues play an important role in sweetness. However, in tetragonal crystals, Arg82 is adjacent to symmetry-related residues, and its flexibility is relatively restrained by the crystal packing. To reduce and diminish these symmetry-related effects, orthorhombic crystals were prepared, and their structures were successfully determined at a resolution of 0.89 Å. Within the orthorhombic lattice, two alternative conformations were more clearly visible at Lys67 than in a tetragonal system. Interestingly, for the first time, three alternative conformations at Arg82 were only found in an orthorhombic system. These results suggest the importance of flexible conformations in sweetness determinants. Such subtle structural variations might serve to adjust the complementarity of the electrostatic potentials of sweet receptors, thereby eliciting the potent sweet taste of thaumatin.

Oral exposure to food grade titanium dioxide (E171) induces intestinal and behavioural alterations in adult mice but limited effects in young mice.

BACKGROUND: Food-grade titanium dioxide (E171), a white colourant widely used in ultra-processed food products, has been banned in the European Union. However, its usage is still permitted in medicines, and in several other countries. The estimated intake of E171 in children is higher than in adults, which led us to hypothesise that E171 induces differential effects depending on age, with adult mice being the most susceptible due to age, despite the lower dose. AIM: To evaluate the effects of oral administration of E171 on intestinal permeability, ileum, and colon histology, and how these effects impact anxious and depressive behaviour in young and adult mice of both sexes. METHODS: Young and adult mice of both sexes C57BL/6 mice received 10 mg/kgbw E171/3 times per week for 3 months. E171 was administered orally in water by pipetting, while control groups only received drinking water, then intestinal permeability, histology and animal behaviour were analysed. RESULTS: E171 showed an amorphous shape, primary particles sized below 1 µm and anatase crystalline structure. Oral administration of E171 disrupted the intestinal permeability in adult male and female mice, but no effects were observed in young mice of both sexes. E171 promoted ileal adenoma formation in half of the adult female population, moreover hyperplastic crypts, and hyperplastic goblet cells at histological level in adult mice of both sexes. The colon presented hyperplastic goblet cells, hyperchromatic nuclei, increased proliferation and DNA damage in adult mice of both sexes. The anxiety and depressive behaviour were only altered in adult mice treated with E171, but no changes were detected in young animals of both sexes. CONCLUSIONS: Adult mice displayed higher susceptibility in all parameters analysed in this study compared to young mice of both sexes.