Evaluation of GABA Production by Alginate-Microencapsulated Fresh and Freeze-Dried Bacteria Enriched with Monosodium Glutamate during Storage in Chocolate Milk.

Two strains of γ-aminobutyric acid (GABA) producing bacteria, L. brevis Y1 and L. plantarum LM2, were microencapsulated in sodium alginate with two concentrations (1% and 2%) of monosodium glutamate (MSG) by using vibrating technology. The mix of both species was microencapsulated both in fresh and freeze-dried form. After 0, 1, 2, and 4 weeks of storage at 4 °C in quarter strength Ringer's solution, the microcapsules were subjected to cell viable counting and sub-cultured in MRS at 37° for 24 h. The MRS cultures were analyzed for the GABA content. The amount of GABA produced per CFU of MRS inoculum was then calculated. Only the 4-week-old microcapsules were used to inoculate a chocolate milk drink with the aim of obtaining a functionalized drink containing viable probiotic cells and GABA after a 1-week incubation at 4 °C. Therefore, the GABA production in chocolate milk per CFU of the probiotic culture after the incubation time was calculated. Results of the GABA analysis by liquid chromatography mass spectrometry of the MRS sub-cultures showed no significant difference (p > 0.05) in GABA yield between 1% and 2% MSG for the microcapsules containing fresh cells. On the contrary, a significant difference (p < 0.05) in productivity along the storage was registered. Microcapsules containing freeze-dried cells showed significant differences (p < 0.05) in GABA yield between 1% and 2% MSG only after 2 and 4 weeks of storage. A significant difference (p < 0.05) in GABA yield between the storage time was found only for the trials with 2% MSG for freeze-dried cells. The synthesis of GABA in chocolate milk significantly decreased (p < 0.05) only for fresh cells when comparing 2% with 1% MSG. In conclusion, a 1-month storage of microcapsules containing both culture forms, fresh and freeze-dried, did not affect GABA production.

Milk protein digestion and the gut microbiome influence gastrointestinal discomfort after cow milk consumption in healthy subjects.

Many healthy people suffer from milk-related gastrointestinal discomfort (GID) despite not being lactose intolerant; the mechanisms underpinning such condition are unknown. This study aimed to explore milk protein digestion and related physiological responses (primary outcome), gut microbiome and gut permeability in 19 lactose-tolerant healthy nonhabitual milk consumers [NHMCs] reporting GID after consuming cow milk compared to 20 habitual milk consumers [HMCs] without GID. NHMCs and HMCs participated in a milk-load (250 mL) test, underwent blood sample collection at 6 time points over 6 h after milk consumption and collected urine samples and GID self-reports over 24 h. We measured the concentrations of 31 milk-derived bioactive peptides (BAPs), 20 amino acids, 4 hormones, 5 endocannabinoid system mediators, glucose and the dipeptidyl peptidase-IV (DPPIV) activity in blood and indoxyl sulfate in urine samples. Subjects also participated in a gut permeability test and delivered feces sample for gut microbiome analysis. Results showed that, compared to HMCs, milk consumption in NHMCs, along with GID, elicited a slower and lower increase in circulating BAPs, lower responses of ghrelin, insulin, and anandamide, a higher glucose response and serum DPPIV activity. The gut permeability of the two groups was similar, while the habitual diet, which was lower in dairy products and higher in the dietary-fibre-to-protein ratio in NHMCs, possibly shaped the gut microbiome; NHMCs exhibited lower abundance of Bifidobacteria, higher abundance of Prevotella and lower abundance of protease-coding genes, which may have reduced protein digestion, as evidenced by lower urinary excretion of indoxyl sulfate. In conclusion, the findings showed that a less efficient digestion of milk proteins, supported by a lower proteolytic capability of the gut microbiome, may explain GID in healthy people after milk consumption.

The Maillard reaction end product Nε-carboxymethyllysine is metabolized in humans and the urinary levels of the microbial metabolites are associated with individual diet.

During food processing most of the thermally-driven chemical reactions start off on the side chain amino group of lysine generating structurally modified compounds with specific metabolic routes. Upon human digestion, dietary Nε-carboxymethyllysine (CML) may enter the colon and undergo gut microbial metabolism. However, little is known about the in vivo metabolic fate of dietary CML and its relationship with the habitual diet. We explored by hydrophilic interaction liquid chromatography tandem mass spectrometry the metabolites of CML in urine samples collected from 46 healthy subjects and studied the associations with diet. Mean concentration of N-carboxymethylcadaverine (CM-CAD), N-carboxymethylaminopentanoic acid (CM-APA), N-carboxymethylaminopentanol (CM-APO), and the N-carboxymethyl-Δ1-piperideinium ion were 0.49 nmol mg-1 creatinine, 1.45 nmol mg-1 creatinine, 4.43 nmol mg-1 creatinine and 4.79 nmol mg-1 creatinine, respectively. The urinary concentration of CML, its metabolites and lysine were positively correlated. Dietary intake of meat products negatively correlated with urinary excretion of CML and CM-APA; conversely dietary plant-to-animal proteins ratio positively correlated with urinary CML and its metabolites. The identification and quantification of CML metabolites in urine and the associations with diet corroborate the hypothesis that CML, an advanced glycation end-product, can undergo further biochemical transformations in vivo. The gut microbiome may have a major role in human metabolism of dietary CML.

Baobab-Fruit Shell and Fibrous Filaments Are Sources of Antioxidant Dietary Fibers.

Since 2008, baobab-fruit dried pulp is listed as an ingredient on the European Union's Novel Food Catalogue. By pulp production, 80% of the baobab fruit is discarded, forming side streams, namely, shell, fibrous filaments, and seeds. This study explored pulp and side-stream functional properties, including total dietary fiber (TDF), total antioxidant capacity (TAC), polyphenols, and water- (WHC) and oil-holding capacities (OHC), along with endocannabinoids (ECs) and N-acylethanolamines (NAEs) in pulp, seeds, and seed oil. Shell excelled in TDF (85%), followed by fibrous filaments (79%), and showed the highest soluble and direct TAC (72 ± 0.7 and 525 ± 1.0 µmol eq. Trolox/g, respectively). Pulp was the richest in polyphenols, followed by shell, fibrous filaments, and seeds. Quercetin predominated in shell (438.7 ± 2.5 µg/g); whereas epicatechin predominated in pulp (514 ± 5.7 µg/g), fibrous filaments (197.2 ± 0.1 µg/g), and seeds (120.1 ± 0.6 µg/g); followed by procyanidin B2 that accounted for 26-40% of total polyphenols in all the products. WHC and OHC ranged between 2-7 g H2O-Oil/g, with fibrous filaments showing the highest values. ECs were not found, whereas NAEs were abundant in seed oil (2408.7 ± 11.1 ng/g). Baobab shell and fibrous filaments are sources of polyphenols and antioxidant dietary fibers, which support their use as functional food ingredients.

Mediterranean diet consumption affects the endocannabinoid system in overweight and obese subjects: possible links with gut microbiome, insulin resistance and inflammation.

PURPOSE: To investigate whether a Mediterranean diet (MD) affected the plasma concentrations of endocannabinoids (ECs), N-acylethanolamines (NAEs) and their specific ratios in subjects with lifestyle risk factors for metabolic diseases. To identify the relationship between circulating levels of these compounds and gut microbiome, insulin resistance and systemic inflammation. METHODS: A parallel 8-week randomised controlled trial was performed involving 82 overweight and obese subjects aged (mean ± SEM) 43 ± 1.4 years with a BMI of 31.1 ± 0.5 kg/m2, habitual Western diet (CT) and sedentary lifestyle. Subjects were randomised to consume an MD tailored to their habitual energy and macronutrient intake (n = 43) or to maintain their habitual diet (n = 39). Endocannabinoids and endocannabinoid-like molecules, metabolic and inflammatory markers and gut microbiome were monitored over the study period. RESULTS: The MD intervention lowered plasma arachidonoylethanolamide (AEA, p = 0.02), increased plasma oleoylethanolamide/palmitoylethanolamide (OEA/PEA, p = 0.009) and OEA/AEA (p = 0.006) and increased faecal Akkermansia muciniphila (p = 0.026) independent of body weight changes. OEA/PEA positively correlated with abundance of key microbial players in diet-gut-health interplay and MD adherence. Following an MD, individuals with low-plasma OEA/PEA at baseline decreased homeostatic model assessment of insulin resistance index (p = 0.01), while individuals with high-plasma OEA/PEA decreased serum high-sensitive C-reactive protein (p = 0.02). CONCLUSIONS: We demonstrated that a switch from a CT to an isocaloric MD affects the endocannabinoid system and increases A. muciniphila abundance in the gut independently of body weight changes. Endocannabinoid tone and microbiome functionality at baseline drives an individualised response to an MD in ameliorating insulin sensitivity and inflammation. Clinical Trial Registry number and website NCT03071718; www.clinicaltrials.gov.

Endocannabinoids, endocannabinoid-like molecules and their precursors in human small intestinal lumen and plasma: does diet affect them?

PURPOSE: To determine the small intestinal concentration of endocannabinoids (ECs), N-acylethanolamines (NAEs) and their precursors N-acylphosphatidylethanolamines (NAPEs) in humans. To identify relationships between those concentrations and habitual diet composition as well as individual inflammatory status. METHODS: An observational study was performed involving 35 participants with an ileostomy (18W/17M, aged 18-70 years, BMI 17-40 kg/m2). Overnight fasting samples of ileal fluid and plasma were collected and ECs, NAEs and NAPEs concentrations were determined by LC-HRMS. Dietary data were estimated from self-reported 4-day food diaries. RESULTS: Regarding ECs, N-arachidonoylethanolamide (AEA) was not detected in ileal fluids while 2-arachidonoylglycerol (2-AG) was identified in samples from two participants with a maximum concentration of 129.3 µg/mL. In contrast, mean plasma concentration of AEA was 2.1 ± 0.06 ng/mL and 2-AG was 4.9 ± 1.05 ng/mL. NAEs concentrations were in the range 0.72-17.6 µg/mL in ileal fluids and 0.014-0.039 µg/mL in plasma. NAPEs concentrations were in the range 0.3-71.5 µg/mL in ileal fluids and 0.19-1.24 µg/mL in plasma being more abundant in participants with obesity than normal weight and overweight. Significant correlations between the concentrations of AEA, OEA and LEA in biological fluids with habitual energy or fat intakes were identified. Plasma PEA positively correlated with serum C-reactive protein. CONCLUSION: We quantified ECs, NAEs and NAPEs in the intestinal lumen. Fat and energy intake may influence plasma and intestinal concentrations of these compounds. The luminal concentrations reported would allow modulation of the homeostatic control of food intake via activation of GPR119 receptors located on the gastro-intestinal mucosa. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE: NCT04143139; www.clinicaltrials.gov .

N-Acylphosphatidylethanolamines and N-acylethanolamines increase in saliva upon food mastication: the influence of the individual nutritional status and fat type in food.

This study aimed to evaluate the influence of the individual nutritional status on the salivary concentration of N-acylethanolamines (NAEs), including linoleoylethanolamide (LEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA), and their precursors N-acylphosphatidylethanolamines (NAPEs) upon mastication of biscuits containing different fats. Three types of biscuits were formulated with 10% extra-virgin olive oil (EVOB), 10% palm oil (PALMB) or 10% paraffin oil (0% lipids, CONB). Twenty-five healthy subjects, 12 normal weight (NW, 9 F, 30.4 ± 8.7 years) and 13 obese (OB, 8 F, 35.5 ± 10.7 years) participated in a randomized crossover study. Fasting subjects collected unstimulated saliva (US) and stimulated saliva by masticating a parafilm piece (PP), and CONB, EVOB and PALMB. NAPEs, LEA, OEA and PEA were quantified in saliva samples by liquid chromatography-high-resolution mass spectrometry. The results showed that salivary NAPE and NAE concentrations in OB were higher than in NW in both US (NAPEs: 280.0 ± 45.4 ng mL-1vs. 121.8 ± 24.4 ng mL-1, p = 0.015; NAEs: 10.8 ± 1.4 ng mL-1vs. 4.8 ± 0.8 ng mL-1, p = 0.002, respectively) and PP (NAPEs: 259.8 ± 47.1 ng mL-1vs. 121.7 ± 16.9 ng mL-1, p = 0.049; NAEs: 6.1 ± 0.8 ng mL-1vs. 3.8 ± 0.4 ng mL-1, p = 0.03, respectively). NAPE and LEA levels were similar in US and PP, while the levels of OEA and PEA were lower in PP vs. US. Compared to PP, biscuit mastication increased the salivary NAPEs, LEA, OEA and overall NAEs in NW and OB. NAPEs increased in the order of EVOB = CONB > PALMB in NW and EVOB > CONB = PALMB in OB. LEA, OEA and overall NAEs increased similarly with all the biscuits in NW and in the order of EVOB > PALMB > CONB in OB. In contrast, the PEA concentration did not vary in saliva upon biscuit mastication in NW and neither with EVOB in OB, while it lowered with CONB and PALMB in OB. In conclusion, OB showed higher salivary levels of NAPEs and NAEs than NW. Mastication itself did not vary salivary NAPEs and LEA but reduced OEA, PEA and overall NAEs. Biscuit mastication increased salivary NAPEs and all NAEs, but PEA. Altogether, the data suggested that NAPEs and NAEs were released in saliva from biscuits at levels influenced by the individual nutritional status and biscuit type. These findings may have implications in molecular mechanisms underpinning gustatory processes in humans.

Genome-Wide HMG Family Investigation and Its Role in Glycoalkaloid Accumulation in Wild Tuber-Bearing Solanum commersonii.

Steroidal glycoalkaloids (SGAs) are a class of nitrogen-containing glycosides occurring in several plant families and biosynthesized through a specific pathway. HMG-CoA reductase is the first enzyme of this pathway, and its transcription can be regulated by biotic and abiotic stressors and even in a tissue-specific manner. This study aimed to characterize the HMG genes family in a tuber-bearing potato species, Solanum commersonii, using transcriptional and functional approaches. Our results provided evidence that four ScHMGs with different tissue-specificities represent the HMG gene family in S. commersonii and that they originated from ScHMG1 through segmental duplications. Phylogenetic analysis suggests that ScHMG1 is the direct ortholog of AtHMG1, which is associated with SGAs accumulation in plants. Its overexpression in S. commersonii revealed that this gene plays a key role in the accumulation of glycoalkaloids regulating the production of dehydrocommersonine.

Mediterranean diet intervention in overweight and obese subjects lowers plasma cholesterol and causes changes in the gut microbiome and metabolome independently of energy intake.

OBJECTIVES: This study aimed to explore the effects of an isocaloric Mediterranean diet (MD) intervention on metabolic health, gut microbiome and systemic metabolome in subjects with lifestyle risk factors for metabolic disease. DESIGN: Eighty-two healthy overweight and obese subjects with a habitually low intake of fruit and vegetables and a sedentary lifestyle participated in a parallel 8-week randomised controlled trial. Forty-three participants consumed an MD tailored to their habitual energy intakes (MedD), and 39 maintained their regular diets (ConD). Dietary adherence, metabolic parameters, gut microbiome and systemic metabolome were monitored over the study period. RESULTS: Increased MD adherence in the MedD group successfully reprogrammed subjects' intake of fibre and animal proteins. Compliance was confirmed by lowered levels of carnitine in plasma and urine. Significant reductions in plasma cholesterol (primary outcome) and faecal bile acids occurred in the MedD compared with the ConD group. Shotgun metagenomics showed gut microbiome changes that reflected individual MD adherence and increase in gene richness in participants who reduced systemic inflammation over the intervention. The MD intervention led to increased levels of the fibre-degrading Faecalibacterium prausnitzii and of genes for microbial carbohydrate degradation linked to butyrate metabolism. The dietary changes in the MedD group led to increased urinary urolithins, faecal bile acid degradation and insulin sensitivity that co-varied with specific microbial taxa. CONCLUSION: Switching subjects to an MD while maintaining their energy intake reduced their blood cholesterol and caused multiple changes in their microbiome and metabolome that are relevant in future strategies for the improvement of metabolic health.

Food database of N-acyl-phosphatidylethanolamines, N-acylethanolamines and endocannabinoids and daily intake from a Western, a Mediterranean and a vegetarian diet.

The contents of N-acylphosphatidylethanolamines (NAPEs), N-acylethanolamines (NAEs) and endocannabinoids (ECs) in 43 food products were assessed and daily intakes, based on consumption of Mediterranean, vegetarian and Western diets, were simulated. NAPEs and NAEs were more abundant in plant-based foods than in animal food products; NAPEs were in the ranges 0-4032 vs 4-398 µg/g dw and NAEs were in the ranges 0-35 vs 0.1-0.7 µg/g dw, respectively while ECs were in the range 0-0.1 vs 0-34 µg/g dw. Daily intakes of NAPEs and NAEs were higher from Mediterranean (263 and 0.25 mg/day) and vegetarian (242 and 0.28 mg/day) diets than the Western diet (163 and 0.08 mg/day). Conversely, ECs intakes were higher from Western and Mediterranean diets (0.17 mg/day) than the vegetarian diet (0.01 mg/day). Future studies will evaluate the physiological role of dietary NAPEs, NAEs and ECs in humans.

Effect of heat and pectinase maceration on phenolic compounds and physicochemical quality of Strychnos cocculoides juice.

Strychnos cocculoides fruit is an important food source for rural populations in Zimbabwe in times of scarcity. Its thick pulp tightly adheres to its seeds, causing pulp extraction constraints and waste during processing, leading to underutilisation. Therefore, pectinase maceration combined with heat treatments was studied to improve juice yield and juice quality. Metabolite profiling according to the heat map, FancyTile chromatic scale approach and phenolic compound content were used to compare the identified compounds. Prior to treatments, 16 known phenolic compounds, predominantly belonging to the phenolic acids, flavonoids and iridoid glucoside classes, were tentatively characterized for the first time in S. cocculoides using High Resolution Mass Spectrometry and LC/MS/MS. Overall, results showed that enzymatic treatments increased pulp yield (by 26%), physicochemical quality (38% increase in juice clarity), content of phenolic compounds (predominantly kaempferol, quercetin, caffeic acid, protocatechuic acid, iridoids) and antioxidant activity.The improved extraction of S. cocculoides pulp increases juice yield as well as juice quality by supplying larger amounts of phenolic compounds that have potential health benefits and act as dietary sources of antioxidants for the prevention of diseases caused by oxidative stress.

Salivary endocannabinoids and N-acylethanolamines upon mastication of a semisolid food: implications in fat taste, appetite and food liking.

This study aimed to evaluate whether salivary endocannabinoid (EC) and N-acylethanolamine (NAE) concentrations upon mastication of a semisolid food were involved in the sensory perception of fat taste, food liking and appetite in humans. A fat-enriched (FEP) and a low-fat control (CP) pudding were developed and used in a randomized cross-over study with 19 healthy volunteers. The study protocol combined a Modified Sham-Feeding (MSF) with a multiple-spoon Temporal Dominance of Sensations method. Subjects masticated and expectorated 10 spoons of the pudding and selected the dominant sensations among a list of attributes. Saliva samples, appetite and food liking scores were collected at baseline, immediately after the MSF of the pudding and every 5 min until 20 min after MSF. Salivary concentrations of all monitored ECs and NAEs increased during pudding mastication compared to baseline (except for palmitoylethanolamide with FEP). The raise was lower with FEP than with CP for all compounds except for 2-arachidonoylglycerol whose increase was higher than the other compounds and independent of pudding type. Salivary N-arachidonoylethanolamine, linoleoylethanolamide and palmitoylethanolamide were significantly lower at 10 and 20 min after MSF of FEP than CP. Fatty taste at the 2nd spoon and creaminess at the 5th spoon were perceived as dominant with FEP whereas only wateriness was dominant with CP at the 2nd spoon. No difference between puddings for individual appetite or food liking over the 20 min of the protocol was recorded. During mastication of a semisolid fat-enriched food, the fatty taste and the creaminess were perceived as dominant. Salivary ECs and NAEs were not associated with the individual perception of fatty taste, pudding liking and appetite sensations.

Potential bioaccessibility and functionality of polyphenols and cynaropicrin from breads enriched with artichoke stem.

In this study, an artichoke stem powder (ASP) was used at three concentrations (3%, 6% and 9%) in the formulation of new breads. The bioaccessibility of polyphenols and cynaropicrin from the ASP-enriched breads was evaluated in vitro by using a digestion model combined to high resolution mass spectrometry analysis. The overall total antioxidant capacity of the bioaccessible and unsolubilized fractions obtained during the intestinal steps and the potential ability to modulate α-glucosidase activity were tested. Data showed that 82% of totally bioaccessible polyphenols and 74% of cynaropicrin were released during the duodenal digestion whereas 88% of caffeic acid was released in the colon step. The antioxidant capacity and the α-glucosidase inhibitory activity of the duodenal extract correlated with the amount of ASP in the bread. Data demonstrated that ASP might be a valuable functional ingredient to create a reducing environment in the intestine and to partially modulate glucose metabolism.

Brassica rapa hairy root extracts promote skin depigmentation by modulating melanin production and distribution.

BACKGROUND: Skin whitening products, used for ages by Asian people for cultural and esthetic purposes, are very popular nowadays in Western countries as well, where the need to inhibit skin spots after sun exposure has become not only a cosmetic but also a health-related issue. Thus, the development of effective and safe depigmenting agents derived from natural products gets continuous attention by cosmetic brands and consumers. OBJECTIVES: The aim of this study was to determine the effects of two preparations, obtained from the hairy root cultures of the species Brassica rapa, on melanogenesis and the expression of the extracellular matrix proteins involved in a correct pigment distribution. METHODS: The two preparations, obtained by water-ethanol extraction and by digestion of cell-wall glycoproteins of the root cells, were chemically characterized and tested on skin cell cultures and on human skin explants to investigate on their dermatological activities. RESULTS: Both the extracts were able to decrease melanin synthesis pathway in melanocytes and modulate the expression of genes involved in melanin distribution. One of the extracts was also effective in inducing the expression of laminin-5 and collagen IV, involved into the maintenance of tissue integrity. The two extracts, when tested together on human skin explants, demonstrated a good synergic hypopigmenting activity. CONCLUSIONS: Taken together, the results indicate that the extracts from B. rapa root cultures can be employed as cosmetic active ingredients in skin whitening products and as potential therapeutic agents for treating pigmentation disorders.

Microencapsulated bitter compounds (from Gentiana lutea) reduce daily energy intakes in humans.

Mounting evidence showed that bitter-tasting compounds modulate eating behaviour through bitter taste receptors in the gastrointestinal tract. This study aimed at evaluating the influence of microencapsulated bitter compounds on human appetite and energy intakes. A microencapsulated bitter ingredient (EBI) with a core of bitter Gentiana lutea root extract and a coating of ethylcellulose-stearate was developed and included in a vanilla microencapsulated bitter ingredient-enriched pudding (EBIP). The coating masked bitterness in the mouth, allowing the release of bitter secoiridoids in the gastrointestinal tract. A cross-over randomised study was performed: twenty healthy subjects consumed at breakfast EBIP (providing 100 mg of secoiridoids) or the control pudding (CP) on two different occasions. Blood samples, glycaemia and appetite ratings were collected at baseline and 30, 60, 120 and 180 min after breakfast. Gastrointestinal peptides, endocannabinoids (EC) and N-acylethanolamines (NAE) were measured in plasma samples. Energy intakes were measured at an ad libitum lunch 3 h after breakfast and over the rest of the day (post lunch) through food diaries. No significant difference in postprandial plasma responses of gastrointestinal hormones, glucose, EC and NAE and of appetite between EBIP and CP was found. However, a trend for a higher response of glucagon-like peptide-1 after EBIP than after CP was observed. EBIP determined a significant 30 % lower energy intake over the post-lunch period compared with CP. These findings were consistent with the tailored release of bitter-tasting compounds from EBIP along the gastrointestinal tract. This study demonstrated that microencapsulated bitter secoiridoids were effective in reducing daily energy intake in humans.

Metabolic and Molecular Changes of the Phenylpropanoid Pathway in Tomato (Solanum lycopersicum) Lines Carrying Different Solanum pennellii Wild Chromosomal Regions.

Solanum lycopersicum represents an important dietary source of bioactive compounds including the antioxidants flavonoids and phenolic acids. We previously identified two genotypes (IL7-3 and IL12-4) carrying loci from the wild species Solanum pennellii, which increased antioxidants in the fruit. Successively, these lines were crossed and two genotypes carrying both introgressions at the homozygous condition (DHO88 and DHO88-SL) were selected. The amount of total antioxidant compounds was increased in DHOs compared to both ILs and the control genotype M82. In order to understand the genetic mechanisms underlying the positive interaction between the two wild regions pyramided in DHO genotypes, detailed analyses of the metabolites accumulated in the fruit were carried out by colorimetric methods and LC/MS/MS. These analyses evidenced a lower content of flavonoids in DHOs and in ILs, compared to M82. By contrast, in the DHOs the relative content of phenolic acids increased, particularly the fraction of hexoses, thus evidencing a redirection of the phenylpropanoid flux toward the biosynthesis of phenolic acid glycosides in these genotypes. In addition, the line DHO88 exhibited a lower content of free phenolic acids compared to M82. Interestingly, the two DHOs analyzed differ in the size of the wild region on chromosome 12. Genes mapping in the introgression regions were further investigated. Several genes of the phenylpropanoid biosynthetic pathway were identified, such as one 4-coumarate:CoA ligase and two UDP-glycosyltransferases in the region 12-4 and one chalcone isomerase and one UDP-glycosyltransferase in the region 7-3. Transcriptomic analyses demonstrated a different expression of the detected genes in the ILs and in the DHOs compared to M82. These analyses, combined with biochemical analyses, suggested a central role of the 4-coumarate:CoA ligase in redirecting the phenylpropanoid pathways toward the biosynthesis of phenolic acids in the pyramided lines. Moreover, analyses here carried out suggest the presence in the introgression regions of novel regulatory proteins, such as one Myb4 detected on chromosome 7 and one bHLH detected in chromosome 12. Overall our data indicate that structural and regulatory genes identified in this study might have a key role for the manipulation of the phenylpropanoid metabolic pathway in tomato fruit.

In vitro bioaccessibility and functional properties of polyphenols from pomegranate peels and pomegranate peels-enriched cookies.

Obesity is an urgent social problem and new functional foods providing polyphenols and dietary fibers (DF) may be promising tools to modulate oxidative stress, inflammation and energy homeostasis. Pomegranate peels (PPe) are an agro-industrial by-product containing polyphenols such as ellagitannins (ETs), gallic acid (GA), ellagic acid (EA) and its derivatives (EAs), as well as DF. In this study, PPe enriched cookies (PPeC) were developed, and the bioaccessibility as well as the ability of their polyphenols to exert antioxidant activity along the Gastro-intestinal Tract (GiT) and to modulate digestive enzymes were evaluated in vitro. Data showed that the potential bioaccessibility of ETs was 40% lower from PPeC than PPe whereas EAs' and GA bioaccessibility increased by 93% and 52% for PPeC compared to PPe. The concentration of the polyphenols at each digestion step was associated with the total antioxidant capacity of the potentially bioaccessible material. Moreover the polyphenols released in the simulated duodenal phase upon PPeC digestion exhibited inhibitory activity towards α-glucosidase, α-amylase and lipase, being α-glucosidase > α-amylase > lipase. In conclusion, the data demonstrated that the inclusion of PPe at 7.5% in a bakery product potentially led to a high bioaccessibility of ETs' degradation products (mainly EA and EAs) in the duodenum, with a consequent antioxidant protection along the GiT and modulation of glucose metabolism. Further human studies are warranted to evaluate whether these effects also occur in vivo.

Metabolite Profiling of Italian Tomato Landraces with Different Fruit Types.

Increased interest toward traditional tomato varieties is fueled by the need to rescue desirable organoleptic traits and to improve the quality of fresh and processed tomatoes in the market. In addition, the phenotypic and genetic variation preserved in tomato landraces represents a means to understand the genetic basis of traits related to health and organoleptic aspects and improve them in modern varieties. To establish a framework for this approach, we studied the content of several metabolites in a panel of Italian tomato landraces categorized into three broad fruit type classes (flattened/ribbed, pear/oxheart, round/elongate). Three modern hybrids, corresponding to the three fruit shape typologies, were included as reference. Red ripe fruits were morphologically characterized and biochemically analyzed for their content in glycoalkaloids, phenols, amino acids, and Amadori products. The round/elongate types showed a higher content in glycoalkaloids, whereas flattened types had higher levels of phenolic compounds. Flattened tomatoes were also rich in total amino acids and in particular in glutamic acid. Multivariate analysis of amino acid content clearly separated the three classes of fruit types. Making allowance of the very low number of genotypes, phenotype-marker relationships were analyzed after retrieving single nucleotide polymorphisms (SNPs) among the landraces available in the literature. Sixty-six markers were significantly associated with the studied traits. The positions of several of these SNPs showed correspondence with already described genomic regions and QTLs supporting the reliability of the association. Overall the data indicated that significant changes in quality-related metabolites occur depending on the genetic background in traditional tomato germplasm, frequently according to specific fruit shape categories. Such a variability is suitable to harness association mapping for metabolic quality traits using this germplasm as an experimental population, paving the way for investigating their genetic/molecular basis, and facilitating breeding for quality-related compounds in tomato fruits.

Salivary concentration of N-acylethanolamines upon food mastication and after meal consumption: Influence of food dietary fiber.

The primary objective of this study was to evaluate the impact of the amount and type of food dietary fiber on salivary concentrations of N-acylethanolamines (NAEs) and glucose upon food mastication and in the post-prandial phase. Three types of biscuits enriched with 3% barley ß-glucan (ßGB) or whole-wheat bran (WWBB) or without dietary fiber (control, CB) were developed. A crossover randomized human study was carried out by collecting saliva samples from eighteen healthy and fasting participants in a resting condition, upon mastication of parafilm and one of the three biscuits. Subsequently, the amount of biscuits consumed in an ad-libitum breakfast was measured and post-prandial saliva samples, blood glucose, appetite, and food liking were collected over the following 2h. Salivary concentration of oleoylethanolamide (OEA) and linoleoylethanolamide (LEA) significantly increased during all biscuits mastication compared to food-free conditions, with OEA increasing more (~138 folds) than LEA (~7 folds). Subjects consumed always 75g of biscuits at breakfast. Salivary OEA peaked at 15min and returned to baseline concentration at 60min after consumption of all types of biscuits whereas LEA peaked only after WWBB. Fifteen minutes after ßGB consumption all NAE levels were significantly lower than those after WWBB. No difference of biscuit type on post prandial blood glucose was recorded. Results demonstrated that NAEs were released in saliva during biscuit mastication, independently from dietary fiber composition. The type of dietary fiber could influence the persistence of NAEs in saliva over 30min after consumption. Future studies will clarify the mechanisms behind these findings and the role of salivary NAEs in food liking and appetite cues after food consumption.