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Abstract In a context where the commercial and nutritional interest in insect chitin is always increasing, an accurate and precise method to quantify this biopolymer, especially in food/feed, is required. In addition, quantification of insect crude protein through nitrogen determination is normally overestimated due to the presence of chitin. In this work, for the first time, an RP-UPLC-ESI/MS method for the simultaneous quantification in insects of chitin, as glucosamine (GlcN), and protein, as total amino acids, is presented. The method is based on acid hydrolysis and derivatization of amino acids and GlcN with the AccQ Tag reagent. Method was optimized and validated in terms of linearity, LOD and LOQ, intraday and inter-day repeatability, and accuracy. A hydrolysed commercial chitin was selected as reference standard for calibration. The instrumental LOD and LOQ correspond respectively to a concentration of 0.00068 mM and 0.00204 mM. The intraday precision satisfied the Horwitz ratio. Data from inter-day precision showed the necessity to perform the analysis within 1 week utilizing standard calibration solutions freshly prepared. A matrix effect was observed, which suggested the necessity to use an internal calibration curve or to work in a particular concentration range of GlcN. The chitin and protein content in black soldier fly (Hermetia illucens) and lesser mealworm (Alphitobius diaperinus) were found in agreement with results obtained by independent methods. The optimized method was also tested on two different commercial food supplements, suggesting its applicability on a wide range of matrices. This newly developed method proved to be simple, more accurate, and faster if compared to methods which separately analyse chitin and protein content.

In this study, biogas and biomethane production during mesophilic anaerobic digestion of wheat substrates coming from national crops and naturally contaminated with deoxynivalenol (DON) and/or T-2/HT-2 toxins was investigated. Biochemical methane potential (BMP) tests of both wholewheat flour and of the main milling fractions were carried out to assess the effect on the concentration of mycotoxins on the anaerobic biological process and their residual presence in the final digestate. The good methane average production achieved (337.0 ± 24.5 NL CH4/kg VS) was substantially comparable with the yields gained from similar biomasses at mesophilic conditions. Moreover, an effective reduction (60.7–100%) of DON concentration was obtained in all the tested substrates (range of contamination in the samples, 368–12,916 μg/kg) whereas the sum of T-2 and HT-2 toxin levels in digestates was always

Metabolic changes occurring in white lupine grain were investigated in response to Plant Growth Promoting Rhizobacteria (PGPR) root inoculation under field condition. We precisely targeted lipids and phenolics changes occurring in white lupine grain in response to Pseudomonas brenneri LJ215 and/or Paenibacillus glycanilyticus LJ121 inoculation. Lipids and phenolic composition were analyzed using an Ultra High-Performance Liquid Chromatography/Tandem Mass Spectrometry Methods. As compared to grain of un-inoculated control plant, Paenibacillus glycaniliticus inoculation highly increased the total lipids content (from 232.55 in seeds of un-inoculated control plant to 944.95 mg/kg) and the relative percentage of several fatty acid such as oleic acid (+20.95%) and linoleic acid (+14.28%) and decreased the relative percentage of glycerophospholipids (- 13.11%), sterol (- 0.2% and - 0.34% for stigmasterol and campesterol, respectively) and prenol (- 17.45%) class. Paenibacillus glycaniliticus inoculation did not affect total phenolic content, while it modulated content of individual phenolic compounds and induced the accumulation of "new" phenolics compounds such as kaempferol. Paenibacillus glycanilyticus LJ121 can be a useful bio-fertilizer to enhance nutritional quality of white lupine grain.

In this work, the anthocyanins’ profile of two hybrid grape varieties—Cabernet Carbon and Prior—grown in two experimental plots in Trentino (Northern Italy) was investigated during 2016 and 2017 harvests. The focus was on the mono- and di-glycoside anthocyanins extraction during the fermentative maceration. It was optimized an HPLC-DAD method, which allows the separation and quantification of mono- and di-glucoside anthocyanin forms (non-esterified, acetylated, and p-coumaroylated) in a single chromatographic run. Furthermore, an HPLC-DAD/HQOMS approach was used to confirm the compound identification. The skin contact fermentation was conducted for 7 days at 24 °C, punching down manually the cap twice a day. Must samples were collected every 24 h. As expected, every anthocyanin showed an initial net extraction phase described by a first-order kinetic, followed by a decrease until the end of the maceration. The analysis of the accumulation rate showed a faster increase of the di-glucoside anthocyanins concentration compared to the corresponding mono-glucoside, due to their richness in the initial grapes. Moreover, the di-glucosides concentration at the end of the alcoholic fermentation underwent a three times minor percentage decrease with respect to the maximum reached. This paper intend to improve the knowledge of the kinetics of each anthocyanin form during the maceration step, providing information about the behavior of di-glucoside compounds, never investigated before in terms of accumulation and percentage decrease.

Considering the carcinogenic risk to human health, it is necessary to carry out research into arsenic (As) content in agro-food products and the impact of food processing on the final content. Yeast fermentation may represent a strategy for detoxifying some widespread beverages such as wine, beer and rice wine. A preliminary study of some commercial yeast species showed different viability responses to the presence of As. Yeasts had a noteworthy detoxification capability during fermentation, reducing the initial As content by about 75 % on average (minimum–maximum: 45–92 %), making it possible to produce wines with a considerably reduced content as compared to the corresponding grape juices from naturally As-rich soils. Nevertheless, significant differences between strains were observed in relation to resistance to arsenic toxicity and As removal capability. The choice of yeast strain can determine a difference of 40 % on the As content remaining in the wine after fermentation. Arsenic content of up to 1000 µg/L did not significantly worsen the fermentation of some wine yeasts, suggesting that the use of specific yeasts may represent an effective tool for reducing As in fermented beverages.

The microflora of grapes involved in the production of a traditional Italian straw wine, “Vino Santo Trentino”, was evaluated as a biocontrol agent against Botrytis cinerea, one of the main diseases affecting fruit and grapes. The microbiota was described using plate counts and genotypic characterisation (sequencing of 16S rRNA for bacteria and 26s rRNA for yeast), allowing identification of yeasts belonging to the Hanseniaspora, Metschnikowia, Cryptococcus and Issatchenkia genera and bacteria (Bacillus, Microbacterium, Acetobacter and Gluconobacter spp.). The distribution of these species is related to the extent of B. cinerea infection. 7 isolates were able to halt the growth of B. cinerea in antagonistic cultures grown in Petri plates, using both synthetic growth and grape juice media. Technological characterisation of potential biocontrol agents, performed with the help of flow cytometry and HPLC-ECD, demonstrated that these microorganisms did not represent a risk for wine production due to their low resistance to ethanol, low pH and the absence of off-flavours. This ensures that the biocontrol agents disappear during winemaking and excludes a negative impact on the quality of wines. In conclusion, the microflora associated with dried grapes is a precious source of biocontrol agents against B. cinerea, both in terms of preventing disease in the vineyard and in control of the grape drying process for the production of straw wines.

Storage conditions and duration have a considerable influence on wine quality. Optimum temperature and humidity conditions may improve wine quality through ageing, while incorrect or excessively long storage leads to negative results. In order to evaluate the global effects of storage on red wine composition, 20 Sangiovese wines were stored in two different conditions (cellar or house) for a period of 2 years and analysed every 6 months. Untargeted LC–MS analysis showed various putative markers for the type and length of conservation (i.e. pigments, flavanols, pantothenic acid etc.), while targeted LC–MS confirmed and expanded these results within specific metabolic groups. The results of multivariate analysis showed that wines stored in the cellar changed little even after 2 years of storage, while wines stored in typical domestic conditions (house) developed approximately four times faster, reaching a composition similar to wines stored in the cellar for 2 years after just 6 months. The formation of several monosulfonated flavanols during domestic ageing provided the first evidence in wine of a reaction between wine tannins—both catechins and proanthocyanidins—and the exogenous antioxidant bisulfite. Moreover, ageing in domestic conditions appeared to induce an accelerated decrease in wine pigments, while specifically promoting the formation of pinotin A-like pigments and the hydrolysis of flavonol glycosides.

In this study, a hydrophilic interaction liquid chromatography–mass spectrometry (HILIC-MS/MS) method for the determination of benzalkonium (BAC) homologues and didecyldimethylammonium (DDAC) was developed. A satisfactory chromatographic separation of BAC homologues and DDAC was achieved using, as mobile phase, acetonitrile–aqueous 50 mM ammonium formate (pH 3.2) (93 + 7 v/v) at 0.3 mL min−1. The elution order of BAC homologues was from benzyldimethylhexadecylammonium chloride (C16-BAC) to benzyldimethyldecylammonium chloride (C10-BAC), the exact opposite with respect to separation using reversed liquid chromatography. The instrumental method was successfully applied to powdered and liquid milk for infants (about 50 samples). From powdered milk samples, BAC and DDAC were extracted using 5 % formic acid in methanol for 60 min at 60 °C in an ultrasonic bath; after dilution with water and 5 % NH4OH solution, a purification step using a weak cationic exchange column was performed. Satisfactory limit of detections (LODs) were achieved, below 1.0 μg kg−1 and 0.05 μg L−1 for powdered and liquid milk for infants, respectively. No sample was free of BAC homologues and DDAC, and in one powdered milk sample, the contamination level exceeded 500 μg kg−1, the maximum level recommended by the Standing Committee on the Food Chain and Animal Health for food and feed.

In the present study, the effects of milk fat (0.3% and 3.5% w/w), solids non-fat (8.4% and 13% w/w), and modified tapioca starch (0%, 0.5%, 1.0%, 1.5%, and 2.0% w/w) concentrations on the textural and physicochemical properties as well as the concentration of several endogenous flavor compounds in the headspace of set and stirred yogurts were investigated. The novel proton transfer reaction time-of-flight mass spectrometry technique was implemented for the non-invasive determination of the amounts of volatile organic compounds in the samples headspace. Milk fat and skim milk powder supplementation of the milk samples increased significantly the firmness and adhesiveness of yogurts (p < 0.001) and improved the stability of the formed gels by increasing their water holding capacity and reducing the amounts of expulsed whey (3.94 and 5.1 g for the milk fat and SNF-fortified samples). Acetaldehyde was significantly (p < 0.001) higher in the low fat-unfortified systems (6.15 ± 0.48 and 5.6 ± 0.60 ppmv, respectively). A similar trend was also reported in the case of 2-propanone (0.91 ± 0.11 and 1.13 ± 0.07 ppmv), diacetyl (334 ± 37 and 350 ± 34 ppbv), 2,3-pentanedione (54 ± 6 and 55 ± 6 ppbv), and 2-butanone (56 ± 7 and 68 ± 5 ppbv) for the same systems. In contrast, the concentration of flavor compounds in the headspace with hydroxyl groups (ethanol and acetoin) increased (p < 0.001) by solid non-fat fortification of milk base (350 ± 32 and 206 ± 7 ppbv, respectively, for the systems fortified with skim milk powder). Modified tapioca starch addition improved the textural properties and gel stability of yogurts whereas affected only the ethanol concentration (222 ± 16 and 322 ± 55 for the control and 2.0% w/w containing systems, respectively). Our data suggested that the reinforcement of textural and structural properties combined with the protein binding affinity of the flavor compounds seemed to be responsible for the aforementioned observations. In the case of stirred yogurts, the gel breakdown did not provoke significant changes in the headspace concentration of the most compounds, with the exception of ethanol, acetoin, and 2,3-pentanedione being significantly (p < 0.05) higher in the stirred yogurts (267 ± 29, 153 ± 11, and 38 ± 1 ppbv, respectively) than set style ones (232 ± 19, 134 ± 9, and 45 ± 3 ppbv, respectively).

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.

Background Grapevine berries undergo complex biochemical changes during fruit maturation, many of which are dependent upon the variety and its environment. In order to elucidate the varietal dependent developmental regulation of primary and specialized metabolism, berry skins of Cabernet Sauvignon and Shiraz were subjected to gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) based metabolite profiling from pre-veraison to harvest. The generated dataset was augmented with transcript profiling using RNAseq. Results The analysis of the metabolite data revealed similar developmental patterns of change in primary metabolites between the two cultivars. Nevertheless, towards maturity the extent of change in the major organic acid and sugars (i.e. sucrose, trehalose, malate) and precursors of aromatic and phenolic compounds such as quinate and shikimate was greater in Shiraz compared to Cabernet Sauvignon. In contrast, distinct directional projections on the PCA plot of the two cultivars samples towards maturation when using the specialized metabolite profiles were apparent, suggesting a cultivar-dependent regulation of the specialized metabolism. Generally, Shiraz displayed greater upregulation of the entire polyphenol pathway and specifically higher accumulation of piceid and coumaroyl anthocyanin forms than Cabernet Sauvignon from veraison onwards. Transcript profiling revealed coordinated increased transcript abundance for genes encoding enzymes of committing steps in the phenylpropanoid pathway. The anthocyanin metabolite profile showed F3′5′H-mediated delphinidin-type anthocyanin enrichment in both varieties towards maturation, consistent with the transcript data, indicating that the F3′5′H-governed branching step dominates the anthocyanin profile at late berry development. Correlation analysis confirmed the tightly coordinated metabolic changes during development, and suggested a source-sink relation between the central and specialized metabolism, stronger in Shiraz than Cabernet Sauvignon. RNAseq analysis also revealed that the two cultivars exhibited distinct pattern of changes in genes related to abscisic acid (ABA) biosynthesis enzymes. Conclusions Compared with CS, Shiraz showed higher number of significant correlations between metabolites, which together with the relatively higher expression of flavonoid genes supports the evidence of increased accumulation of coumaroyl anthocyanins in that cultivar. Enhanced stress related metabolism, e.g. trehalose, stilbene and ABA in Shiraz berry-skin are consistent with its relatively higher susceptibility to environmental cues.

The site-specific natural isotope fractionationnuclear magnetic resonance (SNIF-NMR)2H analysis is at present the reference method for determining the origin of ethanol and has been successfully applied for analysing all grape products. The method has also been used to identify the origin of wines and the aim of this study was to verify this application. In all, 3309 samples belonging to the Italian wines data bank were analysed from all 20 Italian regions to study the possibilities of determining and controlling the exact origin and year of the product. The dependency of the deuterium/hydrogen ratio of the methyl site (D/HI) with latitude was confirmed but this was not the case for the D/H ratio of the methylene site (D/HII) and the parameter R [2(D/HII)/(D/HI)] did not add any additional information. The frequency distribution of the NMR parameters follows a regular North-South gradient but they are not sufficiently different in each region to allow unique characterisation. The lack of effectiveness of the method, when applied to identification, depends on interannual and interregional variability. As a consequence, all but some very different regions overlap. The SNIF-NMR method is particularly suitable for detecting simple adulterations but, in the Italian situation, seems not to be so useful for solving problems of geographical origin. For this, it could be helpful to use other isotopic parameters and to ameliorate the sampling technique, to improve the effectiveness of the method.