Coupling in vitro food digestion with in vitro epithelial absorption; recommendations for biocompatibility.

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.

Release of multifunctional peptides from kiwicha (Amaranthus caudatus) protein under in vitro gastrointestinal digestion.

BACKGROUND: The multifactorial origin of many chronic diseases provides a new framework for the development of multifunctional foods. In this study, the effect of in vitro simulated gastrointestinal digestion of kiwicha (Amaranthus caudatus) proteins on the release of multifunctional peptides was evaluated. RESULTS: Gastric digest showed higher angiotensin I converting enzyme (ACE) inhibitory activity while 60 min gastroduodenal digest showed the highest antioxidant, dipeptidyl peptidase IV (DPP-IV), α-amylase and Caco-2 cell viability inhibitory activities. Peptides >5 kDa were more effective in inhibiting colon cancer cell viability, whereas peptides <5 kDa were mainly responsible for the antioxidant, ACE, DPP-IV and α-amylase inhibitory activities. Thirteen peptides from amaranth sequenced proteins were identified. Structure-activity relationship analysis of the identified sequences pointed to three amaranth fragments, namely FLISCLL, SVFDEELS and DFIILE, as potential peptides able to concurrently exert antioxidant capacity and ability to inhibit both ACE and α-amylase. CONCLUSIONS: Five of thirteen peptides identified in kiwicha protein digests show high potential to exert multifunctional properties. Thus kiwicha proteins might start to gain importance as ingredients for functional foods for the prevention and/or management of chronic diseases related to oxidative stress, hypertension and/or diabetes. © 2018 Society of Chemical Industry.

Bioaccessible peptides released by in vitro gastrointestinal digestion of fermented goat milks.

In this study, ultrafiltered goat milks fermented with the classical starter bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarus subsp. thermophilus or with the classical starter plus the Lactobacillus plantarum C4 probiotic strain were analyzed using ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) and/or high performance liquid chromatography-ion trap (HPLC-IT-MS/MS). Partial overlapping of the identified sequences with regard to fermentation culture was observed. Evaluation of the cleavage specificity suggested a lower proteolytic activity of the probiotic strain. Some of the potentially identified peptides had been previously reported as angiotensin-converting enzyme (ACE) inhibitory, antioxidant, and antibacterial and might account for the in vitro activity previously reported for these fermented milks. Simulated digestion of the products was conducted in the presence of a dialysis membrane to retrieve the bioaccessible peptide fraction. Some sequences with reported physiological activity resisted digestion but were found in the non-dialyzable fraction. However, new forms released by digestion, such as the antioxidant αs1-casein 144YFYPQL149, the antihypertensive αs2-casein 90YQKFPQY96, and the antibacterial αs2-casein 165LKKISQ170, were found in the dialyzable fraction of both fermented milks. Moreover, in the fermented milk including the probiotic strain, the k-casein dipeptidyl peptidase IV inhibitor (DPP-IV) 51INNQFLPYPY60 as well as additional ACE inhibitory or antioxidant sequences could be identified. With the aim of anticipating further biological outcomes, quantitative structure activity relationship (QSAR) analysis was applied to the bioaccessible fragments and led to potential ACE inhibitory sequences being proposed. Graphical abstract Ultrafiltered goat milks were fermented with the classical starter bacteria (St) and with St plus the L. plantarum C4 probiotic strain. Samples were analyzed using HPLC-IT-MS/MS and UPLC-Q-TOF-MS/MS. After simulated digestion and dialysis, some of the active sequences remained and new peptides with reported beneficial activities were released.

Dynamic gastric digestion of a commercial whey protein concentrate†.

BACKGROUND: A dynamic gastrointestinal simulator, simgi® , has been applied to assess the gastric digestion of a whey protein concentrate. Samples collected from the outlet of the stomach have been compared to those resulting from the static digestion protocol INFOGEST developed on the basis of physiologically inferred conditions. RESULTS: Progress of digestion was followed by SDS-PAGE and LC-MS/MS. By SDS-PAGE, serum albumin and α-lactalbumin were no longer detectable at 30 and 60 min, respectively. On the contrary, ß-lactoglobulin was visible up to 120 min, although in decreasing concentrations in the dynamic model due to the gastric emptying and the addition of gastric fluids. Moreover, ß-lactoglobulin was partly hydrolysed by pepsin probably due to the presence of heat-denatured forms and the peptides released using both digestion models were similar. Under dynamic conditions, a stepwise increase in number of peptides over time was observed, while the static protocol generated a high number of peptides from the beginning of digestion. CONCLUSION: Whey protein digestion products using a dynamic stomach are consistent with those generated with the static protocol but the kinetic behaviour of the peptide profile emphasises the effect of the sequential pepsin addition, peristaltic shaking, and gastric emptying on protein digestibility. © 2017 Society of Chemical Industry.

MicroRNA-124 regulates STAT3 expression and is down-regulated in colon tissues of pediatric patients with ulcerative colitis.

BACKGROUND & AIMS: Altered levels and functions of microRNAs (miRs) have been associated with inflammatory bowel diseases (IBDs), although little is known about their roles in pediatric IBD. We investigated whether colonic mucosal miRs are altered in children with ulcerative colitis (UC). METHODS: We used a library of 316 miRs to identify those that regulate phosphorylation of signal transducer and activator of transcription 3 (STAT3) in NCM460 human colonocytes incubated with interleukin-6. Levels of miR-124 were measured by real-time polymerase chain reaction analysis of colon biopsies from pediatric and adult patients with UC and patients without IBD (controls), and of HCT-116 colonocytes incubated with 5-aza-2'-deoxycytidine (5-AZA). Methylation of the MIR124 promoter was measured by quantitative methylation-specific polymerase chain reaction. RESULTS: Levels of phosphorylated STAT3 and the genes it regulates (encoding vascular endothelial growth factor (VEGF), BCL2, BCLXL, and matrix metallopeptidase 9 [MMP9]) were increased in pediatric patients with UC compared with control tissues. Overexpression of miR-124, let-7, miR-125, miR-26, or miR-101 reduced STAT3 phosphorylation by ≥ 75% in NCM460 cells; miR-124 had the greatest effect. miR-124 was down-regulated specifically in colon tissues from pediatric patients with UC and directly targeted STAT3 messenger RNA (mRNA). Levels of miR-124 were decreased, whereas levels of STAT3 phosphorylation increased in colon tissues from pediatric patients with active UC compared with those with inactive disease. In addition, levels of miR-124 and STAT3 were inversely correlated in mice with experimental colitis. Down-regulation of miR-124 in tissues from children with UC was attributed to hypermethylation of its promoter region. Incubation of HCT-116 colonocytes with 5-AZA up-regulated miR-124 and reduced levels of STAT3 mRNA. CONCLUSIONS: miR-124 appears to regulate the expression of STAT3. Reduced levels of miR-124 in colon tissues of children with active UC appear to increase expression and activity of STAT3, which could promote inflammation and the pathogenesis of UC in children.

Peptidomic study of Spanish blue cheese (Valdeón) and changes after simulated gastrointestinal digestion.

It is increasingly evident that digestion can affect the biological activity of cheese by the release of new active peptides from their precursors or, on the contrary, giving rise to fragments without activity. The characterization of the peptidome of a Spanish blue cheese, Valdeón, has been conducted before and after gastrointestinal digestion, and the digests have been compared to those obtained from pasteurized skimmed milk powder (SMP) using a bioinformatics platform. Peptidomic profiling of digests revealed several regions that are especially resistant to digestion (among them ß-casein 60-93, 128-140, and 193-209). Some of them correspond to well-conserved regions between species (human, cow, sheep, and goat) and include peptide sequences with reported bioactivity. The great peptide homology found between both digests, cheese and SMP, suggests that the gastrointestinal digestion could bring closer the profile of products with different proteolytic state. Although most of the biologically active peptides found in cheese after digestion were also present in SMP digest, there were some exceptions that can be attributed to the absence of the relevant precursor peptide before digestion.

Current advances for in vitro protein digestibility.

Protein is an essential macronutrient in our diet, source of nitrogen and essential amino acids, but the biological utilization of dietary protein depends on its digestibility and the absorption of amino acids and peptides in the gastrointestinal tract. The methods to define the amount and the quality of protein to meet human nutritional needs, such as the Digestible Indispensable Amino Acid Score (DIAAS), require the use of animal models or human studies. These in vivo methods are the reference in protein quality evaluation, but they are expensive and long-lasting procedures with significant ethical restrictions. Therefore, the development of rapid, reproducible and in vitro digestion methods validated with in vivo data is an old demand. This review describes the challenges of the in vitro digestion methods in the evaluation of the protein nutritional quality. In addition to the technical difficulties to simulate the complex and adaptable processes of digestion and absorption, these methods are affected by similar limitations as the in vivo procedures, i.e., analytical techniques to accurately determine bioavailable amino acids and the contribution of the endogenous nitrogen. The in vitro methods used for the evaluation of protein digestibility, with special attention on those showing comparative data, are revised, emphasizing their pros and cons. The internationally harmonized digestion protocol proposed by the INFOGEST network is being adapted to evaluate protein and amino acid digestibility. The inter-laboratory reproducibility of this protocol was demonstrated for dairy products. The in vivo/in vitro comparability results obtained to date with this protocol for several plant and animal sources are promising, but it requires an extensive validation with a wider range of foods and substrates with known in vivo digestibility. These in vitro methods will probably not be applicable to all foods, and therefore, it is important to identify their limitations, not to elude their use, but to apply them within the limits, by using the appropriate standards and references, and always as a complementary tool to in vivo tests to reduce their number.

Increased susceptibility of melanin-concentrating hormone-deficient mice to infection with Salmonella enterica serovar Typhimurium.

Melanin-concentrating hormone (MCH) was initially identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, the wide distribution of MCH receptors in peripheral tissues suggests additional functions for MCH which remain largely unknown. We have previously reported that mice lacking MCH develop attenuated intestinal inflammation when exposed to Clostridium difficile toxin A. To further characterize the role of MCH in host defense mechanisms against intestinal pathogens, Salmonella enterocolitis (using Salmonella enterica serovar Typhimurium) was induced in MCH-deficient mice and their wild-type littermates. In the absence of MCH, infected mice had increased mortality associated with higher bacterial loads in blood, liver, and spleen. Moreover, the knockout mice developed more-severe intestinal inflammation, based on epithelial damage, immune cell infiltrates, and local and systemic cytokine levels. Paradoxically, these enhanced inflammatory responses in the MCH knockout mice were associated with disproportionally lower levels of macrophages infiltrating the intestine. Hence, we investigated potential direct effects of MCH on monocyte/macrophage functions critical for defense against intestinal pathogens. Using RAW 264.7 mouse monocytic cells, which express endogenous MCH receptor, we found that treatment with MCH enhanced the phagocytic capacity of these cells. Taken together, these findings reveal a previously unappreciated role for MCH in host-bacterial interactions.

Anti-melanin-concentrating hormone treatment attenuates chronic experimental colitis and fibrosis.

Fibrosis represents a major complication of several chronic diseases, including inflammatory bowel disease (IBD). Treatment of IBD remains a clinical challenge despite several recent therapeutic advances. Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide shown to regulate appetite and energy balance. However, accumulating evidence suggests that MCH has additional biological effects, including modulation of inflammation. In the present study, we examined the efficacy of an MCH-blocking antibody in treating established, dextran sodium sulfate-induced experimental colitis. Histological and molecular analysis of mouse tissues revealed that mice receiving anti-MCH had accelerated mucosal restitution and lower colonic expression of several proinflammatory cytokines, as well as fibrogenic genes, including COL1A1. In parallel, they spared collagen deposits seen in the untreated mice, suggesting attenuated fibrosis. These findings raised the possibility of perhaps direct effects of MCH on myofibroblasts. Indeed, in biopsies from patients with IBD, we demonstrate expression of the MCH receptor MCHR1 in α-smooth muscle actin(+) subepithelial cells. CCD-18Co cells, a primary human colonic myofibroblast cell line, were also positive for MCHR1. In these cells, MCH acted as a profibrotic modulator by potentiating the effects of IGF-1 and TGF-ß on proliferation and collagen production. Thus, by virtue of combined anti-inflammatory and anti-fibrotic effects, blocking MCH might represent a compelling approach for treating IBD.

Food peptides as inducers of CCK and GLP-1 secretion and GPCRs involved in enteroendocrine cell signalling.

The strong effect of protein digestion products on gastrointestinal-released hormones is recognised. However, little is known about the specific peptide sequences able to induce gastrointestinal hormone secretion and the receptors involved. Our objective was to identify peptides able to induce the secretion of cholecystokinin (CCK) and glucagon like peptide-1 (GLP-1) in the enteroendocrine cell line STC-1, and to evaluate the involvement of the calcium-sensing receptor and G-protein coupled receptor-93 in this cell signalling. The key role of the amino acidic sequence on CCK and GLP-1 secretion is demonstrated. Removing Ser from the N-terminus of κ-casein 33SRYPS37, or the N-terminal Trp-Ile in lysozyme 123WIRGCRL129 decreased the secretion of both hormones. However, substituting Tyr by Ala in peptide αs1-CN 90RYLG93 enhanced the CCK secretion levels but not the GLP-1 ones. In addition, the involvement of CaSR and GPR93 was evidenced, but our results pointed to the contribution of additional receptors or transporters.

CCK and GLP-1 response on enteroendocrine cells of semi-dynamic digests of hydrolyzed and intact casein.

A semi-dynamic gastrointestinal device was employed to explore the link between protein structure and metabolic response upon digestion for two different substrates, a casein hydrolysate and the precursor micellar casein. As expected, casein formed a firm coagulum that remained until the end of the gastric phase while the hydrolysate did not develop any visible aggregate. Each gastric emptying point was subjected to a static intestinal phase where the peptide and amino acid composition changed drastically from that found during the gastric phase. Gastrointestinal digests from the hydrolysate were characterized by a high abundancy of resistant peptides and free amino acids. Although all gastric and intestinal digests from both substrates induced the secretion of cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) in STC-1 cells, GLP-1 levels were maximum in response to gastrointestinal digests from the hydrolysate. The enrichment of protein ingredients with gastric-resistant peptides by enzymatic hydrolysis is proposed as strategy to deliver protein stimuli to the distal gastrointestinal tract to control food intake or type 2 diabetes.

Peptidomic Characterization and Amino Acid Availability after Intake of Casein vs. a Casein Hydrolysate in a Pig Model.

It is known that casein hydrolysis accelerates gastrointestinal transit in comparison to intact casein, although the effect of the protein hydrolysis on the composition of the digests is not fully understood. The aim of this work is to characterize, at the peptidome level, duodenal digests from pigs, as a model of human digestion, fed with micellar casein and a previously described casein hydrolysate. In addition, in parallel experiments, plasma amino acid levels were quantified. A slower transit of nitrogen to the duodenum was found when the animals received micellar casein. Duodenal digests from casein contained a wider range of peptide sizes and a higher number of peptides above five amino acids long in comparison with the digests from the hydrolysate. The peptide profile was markedly different, and although ß-casomorphin-7 precursors were also found in hydrolysate samples, other opioid sequences were more abundant in the casein digests. Within the same substrate, the evolution of the peptide pattern at different time points showed minimal changes, suggesting that the protein degradation rate relies more on the gastrointestinal location than on digestion time. Higher plasma concentrations of methionine, valine, lysine and amino acid metabolites were found in animals fed with the hydrolysate at short times (<200 min). The duodenal peptide profiles were evaluated with discriminant analysis tools specific for peptidomics to identify sequence differences between both substrates that can be used for future human physiological and metabolic studies.

A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds.

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Carotid resistive index in treated hypertensive patients: relationship with target organ damage.

AIM: The resistive index (RI) is a hemodynamic parameter that reflects local wall extensibility and related vascular resistance. We analyze the relationship between common carotid RI and target organ damage in treated hypertensive patients. METHODS: We analyzed 265 consecutive hypertensive patients. Risk factors, cardiovascular history and treatments were collected; blood test, urinary albumin excretion (UAE), echocardiography to determine left ventricular mass index (LVMI), ankle-brachial index (ABI) and carotid echo-Doppler ultrasound to calculate the carotid intima-media thickness (IMT) and RI of both common carotids arteries were performed. RESULTS: A positive correlation was found between carotid RI and age, systolic blood pressure, heart rate, carotid IMT, LVMI, UAE and a negative correlation was found with diastolic blood pressure and ABI. Subjects at the top quartile of carotid RI showed a higher prevalence of left ventricular hypertrophy and peripheral artery disease (increased IMT, carotid plaques and lower ABI) compared with those with low RI (p < 0.05). Multiple regression analysis demonstrated that age, systolic and diastolic blood pressure and LVMI independently influence carotid RI. CONCLUSION: Carotid RI is related with age, systolic-diastolic blood pressure and LVMI in hypertensive patient. This evaluation could predict the presence of early cardiovascular damage and provide an accurate estimation of overall risk in this population.

In vitro digestion of milk proteins including intestinal brush border membrane peptidases. Transepithelial transport of resistant casein domains.

The use of enzymes from the brush border membrane (BBM) in simulated gastrointestinal digestion of milk proteins has been evaluated. With this purpose, the resistant sequences from casein and milk whey proteins after INFOGEST in vitro digestion with and without BBM have been analyzed by tandem mass spectrometry. The use of BBM revealed additional cleavages to those found with pancreatic enzymes, although the number of total identified peptides decreased due to the reduction of the peptide size. These new cleavages were mainly attributed to the activity of amino- and carboxy-peptidases, which was also reflected in the higher concentration of free amino acids found in the gastrointestinal digests with BBM. The peptidome of the simulated gastrointestinal digests was compared with that previously obtained in digests aspirated from human jejunum after oral administration of the same substrates. The addition of BBM did not change significantly the peptide profile, although it allowed the identification of peptides found in human digests. However, none of the models was able to reproduce the large variety of peptides found in vivo. In addition, in vitro transepithelial transport of six ß-casein derived peptides resistant to gastrointestinal digestion, including the opioid ß-casomorphin-7, was also evaluated. The results point to the importance of the nature of the N- and C-terminal end for the transport rate through the Caco-2 cell monolayer. Therefore, the use of BBM as a supplementary step after simulated pancreatic digestion can be considered in bioavailability studies since the final sequence can determine the absorption of peptides.

Gastrointestinal co-digestion of wine polyphenols with glucose/whey proteins affects their bioaccessibility and impact on colonic microbiota.

Interactions between food components during their gastrointestinal digestion are constant and could affect compounds digestibility and bioaccessibility. These interactions could have a key role in the bioactivity of dietary polyphenols. This study aimed to investigate the food matrix effects during the co-digestion of red wine with glucose and whey proteins using the gastrointestinal dynamic simulator simgi®. Bioaccessibility of wine polyphenols and nutrients and the effect of co-digestion on colonic microbiota composition and metabolism were evaluated. Co-digestion with red wine led to a reduction of over 50% of glucose bioaccessibility and lowered α-lactalbumin gastric degradation. Still, co-digestion with the food matrices modified polyphenols profiles, including their bioaccessible and non-bioaccessible fractions. For instance, the (-)-epicatechin bioaccessible fraction increased 70% when the wine was co-digested with glucose. Hence, the combined feeding of wine and each food matrix affected microbiota composition and functionality at colonic level. Glucose and whey proteins reduced bacterial diversity, but homogenization of beta-diversity by wine was observed. Moreover, wine presence favoured intestinal health-related taxa as Akkermansia or Bifidobacterium, and the co-digestion of wine and food matrices significantly increased total short- and medium-chain fatty acids production, especially butyric acid. Overall, this study provides evidence of the convenience of the simgi® system to evaluate the effects of co-digestion and highlights the importance of food matrix effects on our understanding of polyphenol bioactivity.

In vitro simulated semi-dynamic gastrointestinal digestion: evaluation of the effects of processing on whey proteins digestibility and allergenicity.

The effect of thermal processing on digestibility of milk proteins should be better understood as this can greatly affect their immunoreactivity. The aim of this study was to evaluate the effects of thermal processing and lactosylation on digestibility and allergenicity, by comparing non heat-treated with industrially processed whey proteins. A semi-dynamic model was used to mimic the kinetics of digestion, and ELISA inhibition tests against human specific serum IgE were performed on the mass-spectrometry characterized products. A quicker gastric digestion of the industrially treated sample produced a lower immunogenic response in comparison with the raw sample, where intact conformational epitopes remained. In later digests, greater IgE reactivity was shown in the heat treated product, probably due to the release of linear epitopes, while at intestinal level the immunogenic response was negligible. Moreover, transepithelial transport of a reported ß-lactoglobulin-derived allergen, KIDALNENVLVL, produced during digestion was assayed. It was found that the epitope-belonging peptide could be transported through the cell monolayer, both in the native and mono-lactosylated forms, with a favored passage of the native peptide.

Suitability of a colorimetric method for the selective determination of chitosan in dietary supplements.

A colorimetric method previously described for the determination of chitosan has been evaluated because lack of linearity had been observed at certain concentrations. Calibration curves of varied-characteristic chitosans, recovery studies and chitosan quantification in seven commercial dietary supplements have been performed. Some analysis conditions including the solvent of the samples have been studied and optimised. Different data combinations have been checked in order to select the widest range of concentrations where no serial correlation was found. With the selected conditions the method is linear, reproducible and provides reliable results in the analysis of the chitosan content in capsules. Its selectivity has been proved by the lack of interference with other compounds present in the dietary supplements. But in the case of tablet products, the presence of cellulose and magnesium stearate may produce an underestimation of the chitosan content.

Composition of Brazil Nut (Bertholletia excels HBK), Its Beverage and By-Products: A Healthy Food and Potential Source of Ingredients.

The consumption of plant-based beverages is a growing trend and, consequently, the search for alternative plant sources, the improvement of beverage quality and the use of their by-products, acquire great interest. Thus, the purpose of this work was to characterize the composition (nutrients, phytochemicals and antioxidant activity) of the Brazil nut (BN), its whole beverage (WBM), water-soluble beverage (BM-S), and its by-products of the beverage production: cake, sediment fraction (BM-D), and fat fraction (BM-F). In this study, advanced methodologies for the analysis of the components were employed to assess HPLC-ESI-QTOF (phenolic compounds), GC (fatty acids), and MALDI-TOF/TOF (proteins and peptides). The production of WBM was based on a hot water extraction process, and the production of BM-S includes an additional centrifugation step. The BN showed an interesting nutritional quality and outstanding content of unsaturated fatty acids. The investigation found the following in the composition of the BN: phenolic compounds (mainly flavan-3-ols as Catechin (and glycosides or derivatives), Epicatechin (and glycosides or derivatives), Quercetin and Myricetin-3-O-rhamnoside, hydroxybenzoic acids as Gallic acid (and derivatives), 4-hydroxybenzoic acid, ellagic acid, Vanillic acid, p-Coumaric acid and Ferulic acid, bioactive minor lipid components (ß-Sitosterol, γ-Tocopherol, α-Tocopherol and squalene), and a high level of selenium. In beverages, WBM had a higher lipid content than BM-S, a factor that influenced the energy characteristics and the content of bioactive minor lipid components. The level of phenolic compounds and selenium were outstanding in both beverages. Hydrothermal processing can promote some lipolysis, with an increase in free fatty acids and monoglycerides content. In by-products, the BM-F stood out due to its bioactive minor lipid components, the BM-D showed a highlight in protein and mineral contents, and the cake retained important nutrients and phytochemicals from the BN. In general, the BN and its beverages are healthy foods, and its by-products could be used to obtain healthy ingredients with appreciable biological activities (such as antioxidant activity).