Determination of steviol glycosides in commercial extracts of Stevia rebaudiana and sweeteners by ultra-high performance liquid chromatography Orbitrap mass spectrometry

Stevia rebaudiana extracts are used as sweeteners in several countries worldwide. Several extracts of diverse composition are available on the market, and their taste depends on the contents of the various steviol glycosides. This study presents an accurate method for the qualitative and quantitative determination of steviol glycosides in 40 Stevia extracts, 7 sweeteners and 3 Stevia-sweetened beverages by a UHPLC coupled to an Orbitrap mass spectrometer. The sub-2 \u3bcm amide column provided the separation of all the target analytes in a run time of 30 min with high resolution. The effect of different eluent compositions on the ionisation efficiency of the steviol glycosides was studied. The optimal ionisation conditions were achieved in negative mode using 0.05% formic acid. Under this condition, adducts were not found, [M-H]- were the main ions and the spontaneous loss of a glucose residue at C19 was reduced. The %RSD for intra- and inter-day precision for all eleven analytes varied from 2.1\u20134.2% and 3.0\u20135.1%, respectively. The recoveries from spiked Stevia extract samples were greater than 95% for all analytes. Rebaudioside A was the most abundant, ranging from 23\u2013102%. Nine Stevia extracts and one drink were not compliant with the European Regulation. Isosteviol was under the LOD in all samples and steviol was found in four samples in quantities in the range 0.01\u20130.03%

Identification of markers for the authentication of cranberry extract and cranberry-based food supplements

Due to the high cost of the cranberry extract, there have been several reported cases of adulteration. The aim of our study was to find markers to authenticate extracts or cranberry-based food supplements. Cranberry fruits from 7 countries, 17 cranberry extracts and 10 cranberry-based food supplements were analysed by UPLC-DAD-Orbitrap MS. Procyanidins were assessed by DMAC method. Anthocyanin fingerprint and epicatechin/catechin, procyanidin A2/total procyanidin and procyanidin/anthocyanin ratios were used as markers, and PCA carried out to check for similarity. Approximately 24% and 60% of the extracts and food supplements, respectively, differed significantly from the fruits. One seemed adulterated with Morus nigra and two with Hibiscus extract. Six food supplements were non-compliant and five contained mainly cyanidin-glucoside and cyanidin-rutinoside, suggesting adulteration with M. nigra extract. Only four products contained the procyanidin amount declared on the package, and only one provided the daily dose deemed effective for treating a urinary tract infection

Impact of a multistrain probiotic formulation with high bifidobacterial content on the fecal bacterial community and short-chain fatty acid levels of healthy adults

The consumption of probiotic products is continually increasing, supported by growing scientific evidence of their efficacy. Considering that probiotics may primarily affect health (either positively or negatively) through gut microbiota modulation, the first aspect that should be evaluated is their impact on the intestinal microbial ecosystem. In this study, we longitudinally analyzed the bacterial taxonomic composition and organic acid levels in four fecal samples collected over the course of four weeks from 19 healthy adults who ingested one capsule a day for two weeks of a formulation containing at least 70 billion colony-forming units, consisting of 25% lactobacilli and 75% Bifidobacterium animalis subsp. lactis. We found that 16S rRNA gene profiling showed that probiotic intake only induced an increase in a single operational taxonomic unit ascribed to B. animalis, plausibly corresponding to the ingested bifidobacterial strain. Furthermore, liquid chromatography/mass spectrometry revealed a significant increase in the lactate and acetate/butyrate ratio and a trend toward a decrease in succinate following probiotic administration. The presented results indicate that the investigated probiotic formulation did not alter the intestinal bacterial ecosystem of healthy adults and suggest its potential ability to promote colonization resistance in the gut through a transient increase in fecal bifidobacteria, lactic acid, and the acetate/butyrate ratio

R(-)-O-desmethylangolensin is the main enantiomeric form of daidzein metabolite produced by human in vitro and in vivo

After ingestion, human intestinal bacteria transform daidzein into dihydrodaidzein, which can be further metabolised to O-desmethylangolensin. This metabolite, unlike daidzein, has a chiral centre and can therefore occur as two distinct enantiomers; however, it is unclear which enantiomer is present in humans. The aim of this study was to define in vitro and in vivo the structure of O-desmethylangolensin and then to evaluate its pharmacokinetic parameters. Daidzein metabolism was preliminarily investigated in anaerobic batch cultures inoculated with mixed faecal bacteria from O-desmethylangolensin producer volunteers. The transformation was monitored by liquid chromatography-mass spectrometry and a chiral column was used to distinguish dihydrodaidzein and O-desmethylangolensin enantiomers. These were purified, analysed by circular dichroism and the results established R(-)-O-desmethylangolensin as the main product (enantiomer excess 91%). However, both dihydrodaidzein enantiomers were detected. Similar results were obtained by in vivo trials. The in vitro formation of O-desmethylangolensin seems to be directly correlated with the number of transforming microorganisms. This correlation was found in vivo for tmax but not for other pharmacokinetic indexes. The pharmacokinetics of daidzein, dihydrodaidzein and O-desmethylangolensin were then evaluated in 11 healthy adult O-desmethylangolensin producers after the single administration of soy milk containing 100mg daidzein. The conjugated forms of daidzein, dihydrodaidzein and O-desmethylangolensin represent more than 90 and 95% of the plasmatic and urinary forms, respectively. The Cmax, tmax and half-life of O-desmethylangolensin in plasma were 62\ub153nM, 28\ub111 and 15\ub16h, respectively. Relevant inter-individual variations were observed as indicated by the high standard deviations

Effect of two different sublingual dosages of vitamin B12 on cobalamin nutritional status in vegans and vegetarians with a marginal deficiency : a randomized controlled trial

Background & Aims: Vegetarians and vegans are more vulnerable to vitamin B12 deficiency with severe risks of megaloblastic anemia, cognitive decline, neuropathy, and depression. An easy and simple method of supplementation consists of taking one weekly dosage of 2000 \ub5g. However, single large oral doses of vitamin B12 are poorly absorbed. The present research evaluates the ability of two different sublingual dosages of vitamin B12 (350 \ub5g/week vs. 2000 \ub5g/week) in improving cyanocobalamin (vitamin B12) nutritional status in vegans and vegetarians with a marginal deficiency. Methods: A 12-week randomized, double-blind, controlled, parallel intervention trial was performed. Forty subjects with marginal vitamin B12 deficiency were enrolled and randomly divided into two groups: test group Ld (low dose, 350 \ub5g/week) and control group Hd (high dose, 2000 \ub5g/week) vitamin B12 supplementation. Blood samples were collected at baseline and after 15, 30, 60, and 90 days from the intervention for the determination of vitamin B12, related metabolic markers, and blood cell counts. Results: Two-way analysis of variance showed a significant effect of time (P < 0.0001) and of time x treatment interaction (P = 0.012) on serum concentration of vitamin B12. In particular, 90 days of supplementation increased the levels of cyanocobalamin (+81.8% in the Ld group and +144.0% in the Hd group) compared to baseline. A significant increase was observed for the levels of holotranscobalamin (+64.5% in the Ld group and +165.2% in the Hd group), while a decrease occurred for the levels of methylmalonic acid (-72.3% in the Ld group and -69.4% in the Hd group), homocysteine (-56.8% in the Ld group and -53.6% in the Hd group), and folate (-22.8% in the Ld group and -17.7% in the Hd group) compared to baseline (time effect, P < 0.0001). No difference was observed between groups (Ld vs. Hd). No effect was detected for the other variables under study. Conclusions: In our experimental conditions, both supplements were able to restore adequate serum concentrations of vitamin B12 and to improve the levels of related metabolic blood markers in subjects with a marginal deficiency. The results support the use of a sublingual dosage of 50 \ub5g/day (350 \ub5g/week) of cobalamin, instead of 2000 \ub5g/week (provided as a single dose), to reach a state of nutritional adequacy of vitamin B12 in this target population

Characterization of As(III) oxidizing Achromobacter sp. strain N2 : effects on arsenic toxicity and translocation in rice

Achromobacter sp. strain N2 was isolated from a pyrite-cinder-contaminated soil and presented plant growth promoting traits (ACC deaminase activity, production of indole-3-acetic and jasmonic acids, siderophores secretion, and phosphate solubilization) and arsenic transformation abilities. Achromobacter sp. strain N2 was resistant to different metals and metalloids, including arsenate (100 mM) and arsenite (5 mM). The strain was resistant to ionic stressors (i.e., arsenate and NaCl), whereas bacterial growth was impaired by osmotic stress. Strain N2 was able to oxidize 1.0 mmol L-1 of arsenite to arsenate in 72 h. This evidence was supported by the retrieval of an arsenite oxidase AioA gene highly homologous to arsenite oxidases of Achromobacter and Alcaligenes species. Rice seeds of Oryza sativa (var. Loto) were bio-primed with ACCD-induced and non-induced cells in order to evaluate the effect of inoculation on rice seedlings growth and arsenic uptake. The bacterization with ACCD-induced cells significantly improved seed germination and seedling heights if compared with the seeds inoculated with non-induced cells and non-primed seeds. Enhanced arsenic uptake was evidenced in the presence of ACCD-induced cells, suggesting a role of ACCD activity on the mitigation of the toxicity of arsenic accumulated by the plant. This kind of responses should be taken into account when proposing PGP strains for improving plant growth in arsenic-rich soils

Near-Infrared Spectroscopy and Chemometrics for the Routine Detection of Bilberry Extract Adulteration and Quantitative Determination of the Anthocyanins

Consumers must be assured that bought food supplements contain both bilberry extract and the anthocyanin amounts that match the declared levels. *erefore, a Fourier transform near-infrared (FT-NIR) spectroscopic method was validated based on principal component scores for the prediction of bilberry extract adulteration and partial least squares regression model for total anthocyanin evaluation. Anthocyanins have been quantified individually in 71 commercial bilberry extracts by HPLC-DAD, and 6 of them were counterfeit. The anthocyanin content in bilberry extracts was in the range 18\u201334%. Authentic bilberry extracts (n = 65) were divided into two parts: one for calibration (n = 38) and the other for the validation set (n = 27). Spectra were recorded in the range of 4000\u201312500 cm1, and a good prediction model was obtained in the range of 9400\u20136096 and 5456\u20134248 cm1 with r2 of 99.5% and a root-mean-square error of 0.3%. The adulterated extracts subjected to NIR analysis were recognized as noncompliant, thus confirming the results obtained by chromatography. The FT-NIR spectroscopy is an economic, powerful, and fast methodology for the detection of adulteration and quantification of the total anthocyanin in bilberry extracts; above all, it is a rapid, low cost, and nondestructive technique for routine analysis

Nutrients, phytochemicals and botanical origin of commercial bee pollen from different geographical areas

This work evaluated the nutritional, phytochemical composition and botanical origin of commercial bee pollen from three different countries. Fructose (17\u201323%) was the most abundant sugar, followed by glucose (14\u201316%) and sucrose (5\u20136%). The protein content in Colombian (24%) and Italian (22%) pollen was higher than in the Spanish sample (14%). The total lipid contents were higher for the Spanish (6%) and Colombian pollens (6%) than the Italian (2.5%). Twenty-one fatty acids were identified, and the most abundant were palmitic, \u3b1-linolenic, linoleic and oleic acid. Colombian pollen was rich in n\u20123 fatty acids, while Italian and Spanish samples contained high amounts of n\u20126 fatty acids. Polyphenols and carotenoids were identified by UHPLC-DAD-Orbitrap mass spectrometry detection. Thirty-nine polyphenols were identified, and the dominant compounds were tri-caffeoyl- and caffeoyl-di-p-coumaroyl spermidine derivatives. Di-lauryl-zeaxanthin was the main carotenoid detected in all the samples analyzed. Colombian pollen contained traces of lutein, zeaxanthin, \u3b2-carotene and phytoene, while only \u3b2-carotene was present in the Spanish and Italian samples. After saponification, the average total amount of carotenoids was 57, 25 and 221\u202f\u3bcg/g in pollen from Spain, Italy and Colombia, respectively. The free proline to total free amino acid ratio was 53, 59 and 78 for pollen from Spain, Italy and Colombia, respectively

Evidence of dysbiosis in the intestinal microbial ecosystem of children and adolescents with primary hyperlipidemia and the potential role of regular hazelnut intake

Hyperlipidemia starts at a pediatric age and represents an unquestionable risk factor for cardiovascular disease. Modulation of the intestinal microbial ecosystem (IME), in principle, can ameliorate lipid profiles. In this study, we characterized the IME of children and adolescents with primary hyperlipidemia by analyzing fecal samples through 16S rRNA gene profiling (n\ua0=\ua015) and short chain fatty acid (SCFA) quantification (n\ua0=\ua032). The same analyses were also carried out on age-matched normolipidemic controls (n\ua0=\ua015). Moreover, we evaluated the modulatory effect of regular hazelnut intake (approximately 0.43 g of hazelnuts with skin per kg of body weight) on the IME of 15 children and adolescents with hyperlipidemia for eight weeks. We found alterations of numerous operational taxonomic units potentially associated with SCFA-producing bacteria and reductions in the fecal levels of acetate, butyrate and propionate in hyperlipidemic subjects. Furthermore, we observed that an eight-week hazelnut intervention may induce limited changes in fecal microbiota composition but can significantly modulate the fecal levels of predominant intestinal SCFAs, such as acetate. Finally, correlation analyses indicated that changes in lipidemic parameters are linked to modifications of the abundance of specific bacterial taxa, such as the families Lachnospiraceae and Ruminococcaceae and the genera Akkermansia, Bacteroides, Roseburia, and Faecalibacterium. This study suggests that children and adolescents with primary hyperlipidemia possess an altered IME. The promising results presented here support the need for future dietary interventions aimed at positively modulating the IME of hyperlipidemic subjects

Validation and application of an ultrahigh-performance liquid chromatographic-Orbitrap mass spectrometric method for the simultaneous detection and quantification of volatile and non-volatile organic acids in human faecal samples

A simple and selective ultrahigh-performance liquid chromatographic-Orbitrap mass spectrometric (UHPLC-HR-MS) method was developed and validated for the simultaneous detection and quantification of short-chain fatty acids (SCFAs) such as acetic, propionic, butyric, isobutyric, valeric, isovaleric, 2-methyl-butyric (IS) and lactic, pyruvic and succinic acid in human faecal samples. A simple extraction procedure with 0.001% formic acid in water was performed on 40 samples. The extracts were centrifuged and analyzed by UHPLC-HR-MS on a sub-2\u3bcm column using gradient elution; meanwhile, the same samples were analyzed by GC-FID and HPLC-UV as reference methods The UHPLC-HR-MS method showed a recovery of 83-105%, a repeatability of 2.2-8.3% and an intermediate precision of 2.9-9.4%. The LOD and LLOQ were in the range of 0.04-0.23 and 0.2-0.5\u3bcg/ml, respectively. Regarding the SCFAs, statistical analysis showed a good correlation between the data obtained by UHPLC-HR-MS and those provided by GC-FID (p>0.05). On the contrary, the LC-UV data were not in agreement with those obtained by UHPLC-HR-MS determination (p<0.05). To the best of our knowledge, this is the first method available for the simultaneous extraction and quantification of SCFAs, lactic, pyruvic and succinic in faecal samples by UHPLC-HR-MS