Simultaneous Determination of Squalene, α-Tocopherol and β-Carotene in Table Olives by Solid Phase Extraction and High-Performance Liquid Chromatography with Diode Array Detection

Olives, the fruit of the Olea europaea tree, are highly appreciated in olive oil and table olives (20 % of crops) not only for their flavor but also for their nutritional properties, especially for antioxidant compounds such as squalling (SQ), α-tocopherol (TH) and β-carotene (BC). This paper presents a new analytical method for simultaneously determining SQ, TH and BC in table olives by using solid phase extraction (SPE) and high performanceliquid chromatography with diode array detection (HPLCDAD), avoiding the classic saponification process. The correlation coefficients of calibration curves of the analyzed compounds ranged from 0.998 to 0.999, and the recoveries were in the range of 89.4–99.6 %. The validated method was used to analyze 30 table olive samples from Italy for their content of SQ (537–1,583 mg kg−1), TH (21–90 mg kg−1) and BC (0.4–2.6 mg kg−1). Finally, experiments with HPLC-MS were conducted to compare this novel method with the classic saponification procedure

Biogenic amines as freshness index of meat wrapped in a new active packaging system formulated with essential oils of Rosmarinus officinalis

Biogenic amines (BAs) are considered as an important indicator of freshness and quality of food. In this work, a new active packaging (AP) system for meat that, incorporating essential oil of Rosmarinus officinalis at 4% (w/w), inhibits the increase of BAs and the bacteria involved into their production was developed. BAs were analyzed by a SPE-HPLC-DAD method during the storage time of meat (0–7 d, 4 C). Results showed that, in each monitored day, Biogenic Amine Index (BAI) expressed in mgkg1 is lower in meat wrapped in AP with respect to that packed in polycoupled packaging (PP) (from 19% to 62%). A strong correlation was found between the inhibition of increase of putrescine, cadaverine, histamine and their bacteria producers such as Enterobacteriaceae, Pseudomonas spp. and Brocothrix thermospacta. By exploiting antimicrobial and antioxidant action of essential oil of R. officinalis, the new APs contribute to increase the shelf life of fresh meat and to preserve its important nutrients

Development a new active packaging system for fresh food through the studies of chemical and biological markers

Packaging and food should not be considered two separate entities, but as elements that can interact, always with the aim to improve the acceptability of the packaged product. In the last two decades many researchers have placed their attention on the study of new packaging systems that improves food preservation. The most investigated new preservation technologies for fresh meat are new packaging systems such as active packaging (AP) and modified atmosphere packaging (MAP) which use natural antimicrobials for bio preservation. AP is the incorporation of specific compounds into packaging systems that interact with the contents environment to maintain or increase product quality and shelf life. AP functions and technologies include moisture control, O2 scavengers or absorbers, CO2 controllers, odours controllers, antimicrobial and antioxidant agents, either natural or synthetic. By preserving the important nutrients of fresh meat such as proteins, essential amino acids, vitamins of group B and others, AP technology can be useful for enhancing the intake of these nutrients too. Natural extracts and essential oils of plants are extraordinary sources of bioactive molecules mixtures, having important antioxidant and antimicrobial actions. The use of natural extracts categorized as flavourings by the European Union (EU) and generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA), represents an interesting approach in AP technology. Considerable results, especially from a biological point of view, have been carried out on the assessment of the antioxidant activity of many herbs such as rosemary (Rosmarinus officinalis), oregano (Origanum vulgare) and others incorporated in an active packaging film. The Rosemary (Rosmarinus officinalis L.) essential oil (REO) contains bioactive compounds having important antioxidant and antimicrobial activities. In this research work the shelf life of different types of fresh food (chicken and beef meat, raw and cooked ham) was studied by monitoring: biogenic amines (BAs), hexanal, microbial growth (such as psychrotrophics, Brochothrix thermosphacta, Pseudomonas spp., and Enterobacteriaceae), pH, colorimetric measurement (L*, a*, b* values) and sensory analyses. The packaging used were Ideabrill® enriched with REO (called ''AP''), Ideabrill® (NAP) and the common packaging (PP). Fresh food wrapped in different systems, were stored at 4°C in different condition: air, under-vacuum and with high-O2 concentration (50:30:20; O2:CO2:N2 %). First of all, more attention was focused on the studies of BAs, that are basic nitrogenous compounds considered important indicator of freshness and quality of food. The active packaging system (AP) that incorporating essential oil of Rosmarinus officinalis at 4% (w/w), inhibits the increase of BAs and the bacteria involved in to their production was developed. BAs were analyzed by a SPE-HPLC-DAD method during the storage time of meat (0-7 days, 4°C). Results showed that, in each monitored day, Biogenic Amine Index (BAI) expressed in mg kg-1 is lower in meat wrapped in AP with respect to that packed in polycoupled packaging (PP) (from 19% to 62%). A strong correlation was found between the inhibition of increase of putrescine, cadaverine, histamine and their bacteria producers such as Enterobacteriaceae, Pseudomonas spp., Brocothrix thermospacta. By exploiting antimicrobial and antioxidant action of essential oil of Rosmarinus officinalis, the new APs contributes to increase the shelf life of fresh meat and to preserve its important nutrients. Moreover, the antioxidant action of new AP system was tested by monitoring hexanal as marker of lipidic oxidation of fresh meat. Preliminary results showed good results in favour of food wrapped in AP system in comparison with PP. In collaboration with Ghent University, the shelf life of beef meat wrapped in AP and in NAP under different air conditions were studied. Microbial growth, sensory characteristics, color parameters, and pH were measured. The use of REO has proved to be efficacious in every storage conditions, because the counts of psychrotrophics, Brochothrix thermosphacta, Pseudomonas spp., and Enterobacteriaceae were lower in AP meat compared to NAP meat. Sensorial and colorimetric analysis showed that the best packaging conditions were high-O2 atmosphere in combination with REO. Based on microbiological data, shelf life of beef meat was 5 days for air packaged samples (AP and NAP), 6-7 days for NAP under vacuum condition samples, 8-9 days for AP under vacuum condition samples, 9-10 for NAP in high-O2 condition samples, and until 15 days for AP in high-O2 condition samples. During the last part of the work, a new analytical method for analyzing ten underivatized BAs in meat by HPLC-MS/MS has been developed. The comparison between ion trap and triple quadrupole as mass analyzers elected triple quadrupole for its higher sensitivity and selectivity. The correlation coefficients of calibration curves of the analyzed compounds were in a range 0.987-0.999, and the Limits of Detection (LODs) and Limits of Quantification (LOQs) were in the range 0.002-0.1 mg l-1 and 0.008-0.5 mg l-1, respectively. The validated method was applied to the analysis of sixteen commercial meat samples for evaluating the freshness of food through the study of BA indices, i.e. Biogenic Amine Index (BAI) and the ratio spermidine/spermine (SPD/SPE). Results showed that all analyzed samples presented good freshness quality with a BAI lower than 1.49 mg kg-1 and a SPD/SPE ratio lower than 0.41 in each case. This methodology to test the freshness of meat could have a great potentiality of application along the entire production chain of meat products

Biogenic Amines: an important marker for evaluating the quality of fresh food.

Biogenic amines (BAs) are organic bases found in food. The presence of high levels of BAs constitutes a potential public concern due to their physiological and toxicological effects. BAs are mostly analysed by liquid chromatography (LC) with pre and post column derivatization. LC coupled to mass spectrometry (LC-MS) offers a great potential because it has the advantage that not derivatization process is needed. A LC-MS/MS method was developed and applied to the analysis of BAs in fresh food. BAs were used as a freshness index of meat to attest the performance of a new type of active packaging

Analysis of ten underivatized biogenic amines in meat by liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Biogenic Amines (BAs) are basic nitrogenous compounds considered to be important indicators of freshness and quality in food (1). Over the years, various methods have been proposed for analyzing BAs in food, with the use of high performance liquid chromatography (HPLC) coupled to a number of detectors. The most frequently used methods are based on derivatization reactions and separation processes followed by ultraviolet (UV) or fluorescence (FL) detection (2). In this work, a new analytical method for analyzing ten underivatized BAs in meat by HPLC-MS/MS has been developed. Comparison between ion trap and triple quadrupole as mass analyzers indicated that the latter provides greater sensitivity and selectivity. The range of calibration curves correlation coefficients of the analyzed compounds was 0.987-0.999, and the Limits of Detection (LODs) and Limits of Quantification (LOQs) were in the range of 0.002-0.1 mg l-1 and 0.008-0.5 mg l-1, respectively. Once validated, the method was used to analyze the concentrations of BAs in sixteen commercial meat samples, for evaluating the freshness of food through the study of BA indices, i.e. Biogenic Amine Index (BAI) and the spermidine/spermine (SPD/SPE) ratio. The results indicated that all samples were fresh, with a BAI lower than 1.49 mg kg-1 and a SPD/SPE ratio lower than 0.41, in each case. This methodology for testing the freshness of meat has potential for quality control applications along the entire production chain of meat products

Effect of the fruit ripening on the antioxidant capacity and total phenolics content on walnuts and almonds.

Walnuts (Juglans regia L.) and almonds (Prunus amygdalus) are a valuable crop traditionally cultivated for their wood and their largely consumed fruits. Not only dry fruits (nuts and almonds) are used, but also green fruits, shells, kernels, bark, green husks (epicarp) and leaves have been used in both cosmetic and pharmaceutical industry [1]. In recent decades an increasing tendency towards the use of natural substances in place of the synthetic ones has been observed in order to reduce environmental pollution, and natural antioxidants, such as phenolic compounds, are gaining importance as a good alternative to chemical preservatives [2]. Polyphenols are a large and diverse group of phytochemicals in plants, known for their antioxidant activity; several studies demonstrated polyphenols benefits for human health, decreasing the risk of degenerative diseases reducing the oxidative stress, inhibiting macromolecular oxidation and having antimicrobial activity [3]. Different works assessed the total polyphenolic content and demonstrated the potential antioxidant of walnut and almond products, especially fruits but also leaves and liqueurs produced by green fruits, bark and specific compounds like juglone. However, despite the abundant literature about this topic, very little is known about the effects of the ripening stage on the changes in the phenolic content and antioxidant activity in husk, kernel and seed. In the present work, almonds and walnuts were collected manually from no pesticides treated trees located in San Venanzo di Castignano, AP, Italy, on the geographic rid reference longitude 42°57'20.4"N and latitude 13°38'39.2"E. The fruits were harvested periodically from May to October 2015 and polyphenols were immediately extracted with acidified ethanol from the husk, kernel and seed separately. The total phenolics were assayed colorimetrically by means of the Folin-Ciocalteu method and their content was expressed as gallic acid equivalents. The antioxidant power of extracts was evaluate by their ability to scavenge the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical; it was calculated from the decrease in absorbance of the DPPH radical caused by antioxidants, due to the scavenging of the radical by hydrogen donation. The degree of discoloration indicates the scavenging potential of the antioxidant extract and ascorbic acid was used as reference compound. Both fruits extracts were found to be important sources of phenolic antioxidants. The average polyphenol content ranged from 322.5 mg/GAE to 1687.3 mg/GAE in seeds and kernels respectively for walnuts and from 175.1 mg/GAE to 1569.6 mg/GAE in the seeds and green husk respectively for almonds. Generally the highest polyphenol content was observed in any case between the 40th and the 50th day of harvesting. Highest radical scavenging capacity (RSC) was observed in walnuts at the late ripening stages in husks, kernels and seeds: after 75 days from the first harvesting, the seeds showed the greatest RSC inhibiting the 87.7% of the DPPH. In almonds, the same radical scavenging behaviour was also observed at the late stages of fruits ripening and almond kernels displayed the highest antiradical activity being able to scavenge the 90.8% of DPPH. The results showed in this study may be valuable with regard to the utilization of by-products of walnuts and almonds production as a functional food material

ISOLAMENTO E PURIFICAZIONE DELLE SOIASAPONINE I E βg DAI SEMI DI SOIA MEDIANTE HPLC-DAD-MS

I legumi sono costituenti molto importanti nella Dieta Mediterranea, sono semi ad alto valore nutritivo (fibra, proteine, carboidrati, grassi vegetali di tipo insaturo, fosforo, ferro, calcio, vitamine del gruppo B). Di particolare importanza tra i composti di interesse nutraceutico troviamo le saponine che sono glicosidi di origine vegetale. Queste molecole hanno dimostrato di possedere diverse proprietà altamente salutari: abbassano i livelli di colesterolo e inibiscono il suo assorbimento, promuovono una azione anti-epatotossica, esplicano un’ azione anti-carcinogenica e promuovono un’azione anti-antinfettiva nei confronti del virus dell’HIV. Le lenticchie e la soia in particolare, ma anche altri legumi come piselli, fagioli e ceci, contengono un alto tenore di queste saponine, in particolare delle soiasaponine (glicosidi triterpenici) I (C48H78018) e βg (C54H84021). Il nostro obiettivo è stato quello di estrarre e purificare entrambe le soiasaponine a partire dai semi di soia. Le soiasaponine I e βg, una volta estratte e purificate dalla soia, verranno successivamente usate come standard per la quantificazione delle stesse in varie cultivar di lenticchie italiane, in particolare in quelle endemiche dell’Appennino Umbro-Marchigiano ad esempio quelle dell’altopiano di Castellucio di Norcia .Per effettuare l’isolamento di queste due sostanze bioattive dai semi di soia, sono necessari diversi step. Inizialmente vengono macinati 120 gr di soia cruda, ottenendo una farina di soia, la quale viene posta in una beuta con 1 L di EtOh aquoso al 70 %, sotto agitazione a temperatura ambiente. Dopo la fase di estrazione, si filtra e si scarta il precipitato. Il liquido poi viene successivamente evaporato mediante rotavapor ottenendo un estratto di soia. Una volta evaporato, viene effettuato il primo step di purificazione mediante cromatografia per gravità, dove 5 g di questo estratto vengono caricati in una colonna cromatografica con fase stazionaria costituita da silice C-18 (fase inversa) e fase mobile costituita da una miscela di H20/CH3CN in rapporto 70/30. Le frazioni eluite vengono raccolte in base alla presenza di soiasaponina I e βg, confermata dalla successiva analisi in TLC e HPLC-MS. Un’ ulteriore purificazione viene effettuata con HPLC SEMIPREPARATIVO accoppiato a rivelatore DAD utilizzando 206 e 292 nm come lunghezze di onda per la soiasaponina I e per la soiasaponina VI rispettivamente. Infine l’ analisi viene conclusa con la caratterizzazione e la quantificazione della soiasaponina I e VI tramite esperimenti in spettrometria di massa-massa (MS/MS) e HPLC-MS ( Fig.1 ). In queste operazioni è necessaria molta accuratezza vista la difficoltà nel reperire la soiasaponina βg, altamente instabile e termolabile. Quest’ultima, infatti, si trasforma facilmente in soiasaponina I a temperature maggiori di 25 gradi

Effect of Rosmarinus officinalis L. essential oil combined with different packaging conditions to extend the shelf life of refrigerated beef meat

Rosemary essential oil (REO) contains bioactives having antioxidant and antimicrobial properties. This work investigated the effect of REO combined with modified atmosphere packaging conditions (MAP), in our case, aerobic, vacuum or high O-2, to extend the shelf life of beef. Beef slices were wrapped in special three-layer sheets of packaging material, some with a coating of REO (active packaging, AP), and some without REO (non active packaging, NAP), and stored at 4 degrees C for 20 days. The use of REO proved efficacious in every storage condition, as seen in the lower counts of psychrotrophics, Brochothrix thermosphacta, Pseudomonas spp., and Enterobacteriaceae in AP meat compared to NAP meat. Sensory and colourimetric analyses showed that the best packaging conditions were high-O-2 atmosphere in combination with REO. Based on microbiological data, shelf life of beef was 5-6 days for AP samples packaged under aerobic conditions and 14-15 days for AP samples in high-O-2 conditions

Sviluppo di una metodica analitica per l’analisi di soiasaponine I e βg nelle lenticchie mediante HPLC-MS/MS triplo quadrupolo.

Il modello di dieta mediterranea consiglia l’assunzione di legumi, in quanto presentano molteplici effetti benefici sulla salute dell’uomo. La lenticchia (Lens culinaris), legume tipico e endemico dell’Appennino Umbro-Marchigiano (centro Italia), è un alimento ad alto valore nutritivo; infatti è ricco in fibra, aminoacidi essenziali, carboidrati, grassi vegetali di tipo insaturo, acido folico, fosforo, ferro, calcio, magnesio, potassio, vitamina A, C, e vitamine del gruppo B e inoltre manifesta un basso indice glicemico. Le lenticchie contengono un’ampia varietà di composti nutraceutici come le saponine (glicosidi triterpenici), in particolare soiasaponine del gruppo B quali la soiasaponina I (C48H78018) e la soiasaponina βg (C54H84021). Queste molecole bioattive hanno dimostrato di possedere diverse proprietà altamente salutari: abbassano i livelli di colesterolo e inibiscono il suo assorbimento, promuovono un’azione anti-epatotossica, esplicano un’attività anti-carcinogenica e manifestano un’azione anti-replicativa nei confronti del virus dell’HIV1. L’obiettivo di questo lavoro è stato lo sviluppo di un metodo molto rapido e specifico per l’estrazione e la quantificazione della soiasaponina I e βg in diversi cultivar di lenticchie mediante HPLC-MS/MS (triplo quadrupolo). In tutti i lavori riportati in letteratura, tra i quali due sviluppati nel nostro laboratorio,2,3 la determinazione della soiasaponina I e βg è stata sempre effettuata mediante HPLC-DAD o HPLC-MS (singolo quadrupolo). Lo sviluppo di questo nuovo metodo in massa/massa ci permette di risparmiare tempo, evitando lo step di purificazione mediante SPE e al tempo stesso di aumentare la specificità e la selettività. Questo nuovo metodo HPLC-MS/MS triplo quadrupolo è stato successivamente applicato all’analisi di 44 campioni di lenticchie prelevate da località del centro Italia, situate a diverse altitudini. I risultati ottenuti dimostrano che le lenticchie sono una fonte molto importante di soiasaponine. La soiasaponina I è presente in concentrazioni che vanno da 55 a 248 mg kg-1, mentre la soiasaponina βg in concentrazioni che vanno da 422 a 1272 mg kg-1. Per dimostrare l’effetto ipocolesterolemizzante di queste molecole, stiamo svolgendo uno studio in vivo su ratti sottoposti a dieta ipercolesterolemica e successivamente trattati con estratti di lenticchie. I risultati finora ottenuti sono molto positivi