Claimed effects, outcome variables and methods of measurement for health claims on foods proposed under European Community Regulation 1924/2006 in the area of appetite ratings and weight management.

All the requests for authorisation to bear health claims under Articles 13(5) and 14 in the context of appetite ratings and weight management have received a negative opinion by the European Food Safety Authority (EFSA), mainly because of the insufficient substantiation of the claimed effects (CEs). This manuscript results from an investigation aimed to collect, collate and critically analyse the information related to outcome variables (OVs) and methods of measurement (MMs) in the context of appetite ratings and weight management compliant with Regulation 1924/2006. Based on the literature review, the appropriateness of OVs and MMs was evaluated for specific CEs. This work might help EFSA in the development of updated guidance addressed to stakeholders interested in bearing health claims in the area of weight management. Moreover, it could drive the applicants during the design of randomised controlled trials aimed to substantiate such claims.

Optimization and validation of Folin-Ciocalteu method for the determination of total polyphenol content of Pu-erh tea.

Pu-erh tea is a post-fermented tea that has recently gained popularity worldwide, due to potential health benefits related to the antioxidant activity resulting from its high polyphenolic content. The Folin-Ciocalteu method is a simple, rapid, and inexpensive assay widely applied for the determination of total polyphenol content. Over the past years, it has been subjected to many modifications, often without any systematic optimization or validation. In our study, we sought to optimize the Folin-Ciocalteu method, evaluate quality parameters including linearity, precision and stability, and then apply the optimized model to determine the total polyphenol content of 57 Chinese teas, including green tea, aged and ripened Pu-erh tea. Our optimized Folin-Ciocalteu method reduced analysis time, allowed for the analysis of a large number of samples, to discriminate among the different teas, and to assess the effect of the post-fermentation process on polyphenol content.

Extraction of Al, Cu, Fe, Mn, Ni and Zn--polyphenol complexes from black tea infusions by Amberlite resins.

BACKGROUND: Metals can be present in tea infusion as ions or as complexes with endogenous bioligands naturally occurring in tea, especially with polyphenols. Their chemical form, i.e. free or metal complexes, can affect their actual bioavailability, gastrointestinal absorption and possible toxicity. RESULTS: The total amount of Al, Cu, Fe, Mn, Ni and Zn in black tea infusions was determined by inductively coupled plasma--atomic emission spectroscopy after mineralization and compared with their fractions occurring as polyphenol complexes, extracted using two different polymeric resins: Amberlite XAD-2 and Amberlite XAD-7. CONCLUSION: The obtained results demonstrated the suitability of Amberlite resins for the extraction of Al, Cu, Fe, Mn, Ni and Zn­polyphenol complexes from black tea infusions and for the evaluation of the actual distribution of the considered metals among different chemical species, i.e. as free metals and as polyphenol complexes.

High-performance liquid chromatographic phenolic compound fingerprint for authenticity assessment of honey.

BACKGROUND: Phenolic compound profiles of 20 honeys of different botanical origin (eucalyptus, citrus, chestnut and linden) were obtained by high-performance liquid chromatography with ultraviolet detection after solid phase extraction, in order to evaluate the effectiveness of the fingerprint method for monofloral honey discrimination. RESULTS: A total of 58 peaks were detected at λ = 280 nm. Distinctive phenolic compound profiles were obtained in which both the nature and the relative amount of the detected compounds were characteristic for different botanical source honeys. In order to detect sample groupings, chromatographic peak areas were submitted to principal component analysis. Then linear discriminant analysis was carried out on the first three principal components. In addition, linear discriminant analysis was carried out on the 58 variables, allowing the selection of five variables able to discriminate honeys of different botanical origin. CONCLUSION: The chemometric evaluation of the phenolic compound profiles yielded classification models able to group honey samples according to their floral source with an excellent degree of agreement. The main advantage of the fingerprint approach with respect to traditional methods is that it does not require time-consuming identification and quantification of the analytes. The method proved to be effective for the assessment of honey authenticity.

An SPME-GC-MS method using an octadecyl silica fibre for the determination of the potential angiogenesis modulators 17beta-estradiol and 2-methoxyestradiol in culture media.

A simple and easily automable method based on solid-phase microextraction followed by gas chromatographic-mass spectrometric analysis was developed for the determination of two potential angiogenesis modulators 17beta-estradiol (17-BE) and 2-methoxyestradiol (2-MEOE) in culture media. Trifluoroacetic anhydride was used as the derivatising agent. A homemade octadecyl silica coating, characterised by a coating thickness of 72 +/- 10 microm and a good thermal stability until 250 degrees C, was prepared. Experimental design was used to optimise the extraction conditions in terms of derivatisation time, derivatisation temperature and time of extraction. As for method validation, lower limits of quantification of 0.17 and 0.015 microg/l for 17beta-estradiol and 2-methoxyestradiol, respectively, were obtained. Finally, the capabilities of the developed fibres were evaluated for the analysis of the investigated analytes developed by granulosa cells in culture media maintained under normoxic, hypoxic and anoxic conditions, in order to better elucidate their possible role in the angiogenic process. An increase of the production of both 17-BE and 2-MEOE in hypoxic and anoxic conditions seems to be related to the effect of oxygen deprivation.

Volatile fingerprint of unroasted and roasted cocoa beans (Theobroma cacao L.) from different geographical origins.

The aroma characterization of 58 unroasted cocoa beans from 22 different geographical origins was performed by head space solid phase micro-extraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). Sampling is representative of the average world production (America, Africa, and Southeast Asia). Analysis of cocoa beans before and after roasting were performed to follow the aroma modification with the aim to achieve a cocoa volatile fingerprint and a discrimination model based on beans origin. A total of 57 volatiles was identified in unroasted cocoa beans, while 71 volatiles were identified in roasted cocoa beans. The compounds belong to several chemical groups including esters, alcohols, organic acids, aldehydes, ketones and pyrazines. Datasets were submitted to multivariate statistical analysis (Principal Component Analysis, PCA). Results allowed to discriminate unroasted cocoa beans based on their geographical origin: samples coming from African countries were separated from samples of American regions, whereas samples from Southeast Asia lie between the other two continents suggesting that Asian samples have intermediate characteristics between African and South American cocoa beans. PCA, applied on the corresponding roasted samples, showed that although the same roasting treatment has been applied to all the samples, the differences among the unroasted samples were also maintained in the aromatic profile after roasting. The discrimination model based on volatile fingerprint combined with chemometric tools, showed interesting potential for origin authentication of both unroasted and roasted cocoa beans.

Little Difference in Milk Fatty Acid and Terpene Composition Among Three Contrasting Dairy Breeds When Grazing a Biodiverse Mountain Pasture.

In the mountains, autochthonous and robust breeds are often used to valorize biodiverse grasslands. Along with their lower nutrient requirements, compared to specialized dairy breeds, they are expected to be better adapted to complex environments and valorize grasslands into dairy products of high quality. Therefore, the aim of the present study was to investigate the grazing selection of three contrasting dairy breeds on a biodiverse mountain pasture, and its consequences on milk fatty acid (FA) profile and prevalence of individual terpenes. A dual-purpose breed from the Italian Alps, the Valdostana Red Pied (Va), was compared to Montbéliardes (Mo), more specialized in milk production, and the highly specialized Holsteins (Ho). Diet selection was measured by scan-sampling, calculating selectivity indexes, and collecting simulated bites during two consecutive days in June (end of first grazing cycle) and July (second grazing cycle). Milk samples were collected at each milking during these experimental periods. Yield of milk and its fat and protein contents were measured. Milk FA and terpenes were analyzed by gas chromatographic methods. We tested the effects of breed, period and their interaction in a repeated mixed model, and calculated Pearson's correlations between behavioral data and milk FA as well as terpenes. The Va grazed less mature vegetation than Ho, but this difference was not sufficient to lead to a major breed effect on milk FA profile and prevalence of terpenes. However, the proportion of α-linolenic acid (C18:3 n-3) was always higher in the milk fat of Va than Ho (Mo were intermediary), but this without any correlation to grazing selection. This could be a consequence from a different metabolism concerning ruminal biohydrogenation, but must be further investigated. Finally, we confirmed previous studies that highlighted a link between milk quality and cows' grazing behavior, but here without differences among breeds. All cows adapted their behavior to the herbage evolution during the season, leading to higher proportions of unsaturated FA in July than June milks. Our study suggests that under mountain grazing conditions (biodiverse pasture and cows in late lactation), milk quality depends more on herbage composition than on cow breed.

Innovative sol-gel coatings for solid-phase microextraction. Development of fibers for the determination of polycyclic aromatic hydrocarbons at trace level in water.

Innovative solid-phase microextraction coatings synthesized by sol-gel technology were developed for the determination of environmental pollutants, i.e. polycyclic aromatic hydrocarbons at trace level in water. The fibers obtained, based on the use of (3-aminopropyl)triethoxysilane and diethoxydiphenylsilane were characterised in terms of film thickness, porosity, thermal stability and pH resistance. An excellent thermal stability was obtained in the case of diethoxydiphenylsilane-based coating, with a negligible weight loss until 400 degrees C. A very good fiber-to-fiber and batch-to-batch repeatability was also obtained with RSD lower than 6% using a mixture of aromatic hydrocarbons in the low ng/l range. Finally, the capabilities of the developed coating for sampling polycyclic aromatic hydrocarbons in water at trace levels were proved obtaining limits of detection about twofold lower than those achieved by using the commercially available polydimethylsiloxane 7 microm fiber.

Claimed effects, outcome variables and methods of measurement for health claims proposed under European Community Regulation 1924/2006 in the area of blood glucose and insulin concentrations.

Most requests for authorization to bear health claims under Articles 13(5) and 14 related to blood glucose and insulin concentration/regulation presented to the European Food Safety Authority (EFSA) receive a negative opinion. Reasons for such decisions are mainly ascribable to poor substantiation of the claimed effects. In this scenario, a project was carried out aiming at critically analysing the outcome variables (OVs) and methods of measurement (MMs) to be used to substantiate health claims, with the final purpose to improve the quality of applications provided by stakeholders to EFSA. This manuscript provides a position statement of the experts involved in the project, reporting the results of an investigation aimed to collect, collate and critically analyse the information relevant to claimed effects (CEs), OVs and MMs related to blood glucose and insulin levels and homoeostasis compliant with Regulation 1924/2006. The critical analysis of OVs and MMs was performed with the aid of the pertinent scientific literature and was aimed at defining their appropriateness (alone or in combination with others) to support a specific CE. The results can be used to properly select OVs and MMs in a randomized controlled trial, for an effective substantiation of the claims, using the reference method(s) whenever available. Moreover, results can help EFSA in updating the guidance for the scientific requirements of health claims.

Antiangiogenesis in swine ovarian follicle: a potential role for 2-methoxyestradiol.

Inhibition of angiogenesis is an important new approach for cancer treatment and the research on this topic deserve special attention. 2-Methoxyestradiol (2-ME), a molecule shown to possess antiangiogenic activity, is a naturally occurring derivative of estradiol which can be potentially produced in the ovarian follicle. This study was therefore aimed firstly to asses 2-ME content in swine follicular fluid. Moreover, we evaluated the effect of this substance on VEGF production, superoxide anion generation (O(2)(-)) and superoxide dismuatase activity in granulosa cells. Our data evidence that 2-ME is present in follicular fluid where it potentially acts as a physiological inhibitor of angiogenesis by reducing VEGF production by granulosa cells: this effect could be mediated by a decrease of O(2)(-) generation.

Claimed Effects, Outcome Variables and Methods of Measurement for Health Claims Proposed Under European Community Regulation 1924/2006 in the Framework of Maintenance of Skin Function.

Evidence suggests a protective role for several nutrients and foods in the maintenance of skin function. Nevertheless, all the requests for authorization to use health claims under Article 13(5) in the framework of maintenance of skin function presented to the European Food Safety Authority (EFSA) have received a negative opinion. Reasons for such failures are mainly due to an insufficient substantiation of the claimed effects, including the choice of inappropriate outcome variables (OVs) and methods of measurement (MMs). The present paper reports the results of an investigation aimed at collecting, collating and critically analyzing the information with relation to claimed effects (CEs), OVs and MMs related to skin health compliance with Regulation 1924/2006. CEs, OVs and MMs were collected from both the EFSA Guidance document and from the authorization requests of health claims under Article 13(5). The critical analysis of OVs and MMs was based on a literature review, and was aimed at defining their appropriateness (alone or in combination with others) in the context of a specific CE. The results highlight the importance of an adequate choice of OVs and MMs for an effective substantiation of the claims.

Development and validation of a solid phase micro-extraction-gas chromatography-mass spectrometry method for the determination of furan in baby-food.

An efficient and simple method for the determination of furan in baby-food (vegetables and fruits) by solid phase micro-extraction-gas chromatography-mass spectrometry (SPME-GC-MS) was developed and validated. Experimental design was used to investigate the effects of temperature and time of extraction. The calculated regression model was used to find the experimental conditions providing the optimal SPME extraction yield. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low ngkg(-1) were achieved, whereas linearity was established over two order of magnitude. Good precision was obtained both in terms of intra-day repeatability and between-day precision on two concentration levels (RSD% lower than 3.6%). Recovery values of 91.5+/-6.2% and of 96.1+/-1.3% (n=3) were calculated at 75 ngkg(-1) and 75 microgkg(-1) level. Finally, the applicability of the method to the determination of furan in a number of commercial and home-made baby-food samples was demonstrated.

Bioremediation of toluene and naphthalene: development and validation of a GC-FID method for their monitoring.

Bioremediation of toluene and naphthalene in liquid cultures of bacteria grown in the presence of these aromatic compounds as unique sources of carbon was investigated by gas chromatography (GC). For this purpose, a method based on the use of GC with flame ionization detection was developed and validated. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. In the case of naphthalene, LOD and LOQ values of 0.43 and 0.72 mg kg(-1) were achieved. Linearity was established over one order of magnitude in the range of interest, i.e. 10-100 mg kg(-1). Excellent precision was obtained both in terms of intra-day repeatability and between-day precision on two concentration levels (RSD% lower than 0.5%). A recovery of 97.9 +/- 0.2% (n=3) was calculated by addition of 640 mg kg(-1) of naphthalene to the Bushnell & Haas mineral salts basal solution containing the micro-organisms. Findings clearly showed a reduction of the naphthalene content equal to 50% and 75% after two and four weeks of contact with the micro-organisms, whereas a lower degradation was shown in the case of toluene. Finally bioremediation activity was ascribed to two different microbial populations, Bordetella Petrii and Bacillus Sphericus, which survived in the polluted medium.

Hypoxia stimulates the production of the angiogenesis inhibitor 2-methoxyestradiol by swine granulosa cells.

We previously demonstrated the presence of 2-methoxyestradiol (2-ME) in swine follicular fluid. Present study was aimed first of all to investigate if swine granulosa cell produce 2-ME; in addition, we tried to assess a potential effect of hypoxia in modulating 2-ME output. Finally, we explored the effect of 2-ME in an angiogenesis bioassay set up in our lab. Our data show that cultured granulosa cells are able to produce 2-ME; interestingly, the secretion of the hormone appeared to be stimulated by hypoxia. Angiogenesis bioassay points out that 2-ME displays an inhibitory effect on neovascularisation. Therefore our data suggest that 2-ME could be a local effector in determining the fine tuning responsible for follicle angiogenesis. These data deserve special attention since the ovary is a valuable experimental model in angiogenesis research.

Comparison of comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry and gas chromatography-mass spectrometry for the qualitative characterisation of roasted barley by solid-phase microextraction.

Volatile compounds of roasted barley used in the production of barley coffee, the most common coffee substitute, were analysed by using solid-phase microextraction (SPME) followed by GC-MS and comprehensive GC x GC-TOF-MS, respectively. The optimised SPME extraction conditions in terms of selection of the fibre coating, extraction time and extraction temperature allowed to obtain the highest GC response, thus enhancing the identification capabilities of the developed method. As for the SPME-GC x GC-TOF-MS analysis, 64 compounds with similarity, reverse and probability values above 800, 900 and 6000, respectively, were identified, by using a polar x apolar column set combination; in contrast, GC-MS was able to identify a lower number of compounds, i.e. 40 volatiles.

Retention indices in the analysis of food aroma volatile compounds in temperature-programmed gas chromatography: database creation and evaluation of precision and robustness.

A retention index (RI) database containing 250 volatile compounds was created on a polar stationary phase column and validated for food aroma characterisation. Precision of the retention indices (RIs) was assessed by performing replicated injections of a representative number of volatiles under the same experimental conditions: differences lower than 1 U were observed for all the compounds. Robustness was evaluated by carrying out injections of the same set of volatile compounds under different experimental conditions, i.e. program temperature, column batches and instrumentation. Excellent results were obtained with a maximum difference in the RI values of 10 U. The capabilities of the created database for food aroma characterisation were finally evaluated by analysing the volatile fractions of different food matrices such as dry sausages, cheese and bread. A great number of volatile compounds were identified in the analysed samples on the basis of their RI, thus proving the usefulness of the RI collections in the field of food analysis.

Multivariate calibration on NIR data: development of a model for the rapid evaluation of ethanol content in bakery products.

A new NIR method based on multivariate calibration for determination of ethanol in industrially packed wholemeal bread was developed and validated. GC-FID was used as reference method for the determination of actual ethanol concentration of different samples of wholemeal bread with proper content of added ethanol, ranging from 0 to 3.5% (w/w). Stepwise discriminant analysis was carried out on the NIR dataset, in order to reduce the number of original variables by selecting those that were able to discriminate between the samples of different ethanol concentrations. With the so selected variables a multivariate calibration model was then obtained by multiple linear regression. The prediction power of the linear model was optimized by a new "leave one out" method, so that the number of original variables resulted further reduced.