Tiziana Nardin

Biodiversity and γ-aminobutyric acid production by lactic acid bacteria isolated from traditional alpine raw cow's milk cheeses.

“Nostrano-cheeses” are traditional alpine cheeses made from raw cows milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of “Nostrano-cheeses” and evaluated their potential to produce γ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters (n = 97) were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated were Lactobacillus paracasei, Streptococcus thermophilus, and Leuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers were Lactobacillus paracasei but other GABA producing species included Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosaceus, and Streptococcus thermophilus. No Enterococcus faecalis or Sc. macedonicus isolates produced GABA. The isolate producing the highest amount of GABA (80.0±2.7 mg/kg) was a Sc. thermophilus.

Free and glycosylated simple phenol profiling in Apulian Italian wines

Free simple phenols have a significant role in defining the sensory and nutritional characteristics of wines, affecting the organoleptic profile and having positive effects on health, but glycosidically bound phenols can also be hydrolysed during the winemaking process, releasing the corresponding volatile compounds and making a possible contribution to the final sensory profile. In this work, application of on-line SPE liquid chromatography-high resolution mass spectrometry, operating in negative polarity with heated electrospray, allowed to detect over eighty free and glycosylated simple phenols in Primitivo di Manduria and Negroamaro wines. Sixty-one phenols, four of which phenolic glucosidic precursors, were quantified as having quantification limits ranging from 0.001 to 0.1μgmL(-1), calibration R(2) of 0.99 for over 92% of compounds, and precision (R.S.D.%) always lower than 12%. Twenty-four simple phenolic precursors were tentatively identified as hexoside, pentoside and hexoside-hexoside derivatives, on the basis of accurate mass, isotopic pattern and MS/MS fragmentation.

Verifying the botanical authenticity of commercial tannins through sugars and simple phenols profiles.

Commercial tannins from several botanical sources and with different chemical and technological characteristics are used in the food and winemaking industries. Different ways to check their botanical authenticity have been studied in the last few years, through investigation of different analytical parameters. This work proposes a new, effective approach based on the quantification of 6 carbohydrates, 7 polyalcohols, and 55 phenols. 87 tannins from 12 different botanical sources were analysed following a very simple sample preparation procedure. Using Forward Stepwise Discriminant Analysis, 3 statistical models were created based on sugars content, phenols concentration and combination of the two classes of compounds for the 8 most abundant categories (i.e. oak, grape seed, grape skin, gall, chestnut, quebracho, tea and acacia). The last approach provided good results in attributing tannins to the correct botanical origin. Validation, repeated 3 times on subsets of 10% of samples, confirmed the reliability of this model.

Targeted and untargeted profiling of alkaloids in herbal extracts using online solid-phase extraction and high-resolution mass spectrometry (Q-Orbitrap)

The biological activity of alkaloids (ALKs) and the different content of these natural products in herbs and plants have made them an attractive field for chemical studies. A screening method automatically combining online solid-phase purification and concentration of samples with analysis using ultra-high performance liquid chromatography coupled with a hybrid quadrupole orbitrap mass spectrometer was developed and is reported in this paper. The proposed quantification method was validated for 35 ALKs with reference to pure analytical standards. A further 48 ALKs were identified on the basis of their accurate mass and characterised for chromatographic retention time and fragmentation profile, following their confirmation in extracts of herbs already well documented in the literature. More than 250 other untargeted ALKs were also tentatively identified using literature information, such as exact mass and isotopic pattern. The mass spectrometer operated in positive ion mode and mass spectra were acquired, with full MS-data-dependent MS/MS analysis (full MS-dd MS/MS) at a resolution of 140 000. The method was linear up to an ALK concentration of 1000/3000 µg l(-1) , with R(2) always >0.99 and limits of detection ranging between 0.04 and 10 µg l(-1) . Accuracy, expressed as the recovery relative error, had a median value of 7.4%, and precision (relative standard deviation %) was generally lower than 10% throughout the quantitation range. The proposed method was then used to investigate the targeted and untargeted ALK profile of a selection of 18 alpine herbal plants, establishing that pyrrolizidine, pyrrolidine and piperidine ALKs were the most well represented. Copyright

Botanical origin characterisation of tannins using infrared spectroscopy

Different approaches to analysing the botanical origin of tannins have been proposed in the last fifteen years, but are generally time consuming and require the use of advanced instrumentation. This study aims to suggest an effective, easy, rapid and cheap method based on the acquisition of FT-IR spectra of 3g/L hydroalcoholic tannin solutions, overcoming possible disadvantages due to sample or particle size inhomogeneity. 114 commercial powder tannins from 7 different botanical sources (oak, chestnut, gall, quebracho, tea, grape skin and grape seed) were collected and the FT-IR spectra were acquired in the region 926-5011cm-1. Partial Least Squares regression, Discriminant Analysis and Artificial Neural Networks were applied to FT-IR spectra to investigate the possibility of differentiating the 7 botanical origins. The best results were obtained using Discriminant Analysis, with 95% correct re-classification, and 97% grouping of grape skin and seed in a single source.

Targeted and untargeted high resolution mass approach for a putative profiling of glycosylated simple phenols in hybrid grapes

Vitis vinifera is one of the most widespread grapevines around the world representing the raw material for high quality wine production. The availability of more resistant interspecific hybrid vine varieties, developed from crosses between Vitis vinifera and other Vitis species, has generated much interest, also due to the low environmental effect of production. However, hybrid grape wine composition and varietal differences between interspecific hybrids have not been well defined, particularly for the simple phenols profile. The dynamic of these phenols in wines, where the glycosylated forms can be transformed into the free ones during winemaking, also raises an increasing health interest by their role as antoxidants in wine consumers. In this work an on-line SPE clean-up device, to reduce matrix interference, was combined with ultra-high liquid chromatography-high resolution mass spectrometry in order to increase understanding of the phenolic composition of hybrid grape varieties. Specifically, the phenolic composition of 4 hybrid grape varieties (red, Cabernet Cantor and Prior; white, Muscaris and Solaris) and 2 European grape varieties (red, Merlot; white, Chardonnay) was investigated, focusing on free and glycosidically bound simple phenols and considering compound distribution in pulp, skin, seeds and wine. Using a targeted approach 53 free simple phenols and 7 glycosidic precursors were quantified with quantification limits ranging from 0.001 to 2mgKg-1 and calibration R2 of 0.99 for over 86% of compounds. The untargeted approach made it possible to tentatively identify 79 glycosylated precursors of selected free simple phenols in the form of -hexoside (N=30), -pentoside (21), -hexoside-hexoside (17), -hexoside-pentoside (4), -pentoside-hexoside (5) and -pentoside-pentoside (2) derivatives on the basis of accurate mass, isotopic pattern and MS/MS fragmentation.

Glycosylated simple phenolic profiling of food tannins using high resolution mass spectrometry (Q-Orbitrap)

Tannins are polyphenolic compounds extensively present in plants and used by food industry as processing aids. Due to the heterogeneity of plant sources, actions involved in food processing and tannin commercial costs can be different. In the last years different approaches aimed at correctly identifying the tannin botanical origin have been developed, in order to satisfy the industrys request to verify product labels. This work aimed to define the glycosidic simple phenolic profile of a large selection of monovarietal commercial tannins of different origin, using a high-resolution untargeted approach. Using accurate mass, isotopic pattern and MS/MS fragmentation, 167 precursors, 89 as monoglycosylated and 78 as diglycosylated derivatives were tentatively identified in tannins, validating the untargeted approach with 3 custom-synthesized glycosidic precursors. Almost all tannin botanical varieties were shown to be characterised by a specific glycosylated phenolic profile, providing possible tools for tannin classification in the case of glycosylphenol standard availability.