Pyrrolizidine Alkaloids in Food on the Italian Market.

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by over 6000 plant species worldwide. PAs enter the food chain through accidental co-harvesting of PA-containing weeds and through soil transfer from the living plant to surrounding acceptor plants. In animal studies, 1,2-unsaturated PAs have proven to be genotoxic carcinogens. According to the scientific opinion expressed by the 2017 EFSA, the foods with the highest levels of PA contamination were honey, tea, herbal infusions, and food supplements. Following the EFSA's recommendations, data on the presence of PAs in relevant food were monitored and collected. On 1 July 2022, the Commission Regulation (EU) 2020/2040 came into force, repealed by Commission Regulation (EU) 2023/915, setting maximum levels for the sum of pyrrolizidine alkaloids in certain food. A total of 602 food samples were collected from the Italian market between 2019 and 2022 and were classified as honey, pollen, dried tea, dried herbal infusions, dried herbs, and fresh borage leaves. The food samples were analyzed for their PA content via an in-house LC-MS/MS method that can detect PAs according to Regulation 2023/915. Overall, 42% of the analyzed samples were PA-contaminated, 14% exceeded the EU limits, and the items most frequently contaminated included dried herbs and tea. In conclusion, the number of food items containing considerable amounts of PAs may cause concern because they may contribute to human exposure, especially considering vulnerable populations-most importantly, children and pregnant women.

Climate Effects on Ergot and Ergot Alkaloids Occurrence in Italian Wheat.

In recent years, there has been an intensification of weather variability worldwide as a result of climate change. Some regions have been affected by drought, while others have experienced more intense rainfall. The incidence and severity of moldy grain and mycotoxin contamination during the growing and harvesting seasons have increased as a result of these weather conditions. Additionally, torrential rains and wet conditions may cause delays in grain drying, leading to mold growth in the field. In July 2023, a wheat field in Lecco (Lombardy, Italy) was affected by torrential rains that led to the development of the Claviceps fungi. In the field, dark sclerotia were identified on some ears. Wheat ears, kernels, and sclerotia were collected and analyzed by LC-MS/MS at IZSLER, Food Chemical Department, in Bologna. The wheat ears, kernels, and sclerotia were analyzed for 12 ergot alkaloids (EAs) according to (EU) Regulation 2023/915 (ergocornine/ergocorninine; ergocristine/ergocristinine; ergocryptine/ergocryptinine; ergometrine/ergometrinine; ergosine/ergosinine; ergotamine/ergotaminine), after QuEChERS (Z-Sep/C18) purification. The analyzed sclerotia showed significant differences in total alkaloid content that vary between 0.01 and 0.5% (w/w), according to the results of the 2017 EFSA scientific report. EAs detected in sclerotia were up to 4951 mg/kg, in wheat ears up to 33 mg/kg, and in kernels were 1 mg/kg. Additional mycotoxins, including ochratoxin A, deoxynivalenol, zearalenone, fumonisins, T2-HT2 toxins, and aflatoxins, were investigated in wheat kernels after purification with immunoaffinity columns (IAC). The analysis revealed the presence of deoxynivalenol in wheat kernels at a concentration of 2251 µg/kg. It is expected that climate change will increase the frequency of extreme weather events. In order to mitigate the potential risks associated with mycotoxin-producing fungi and to ensure the protection of human health, it is suggested that official controls be implemented in the field.

Overview of Cyanide Poisoning in Cattle from Sorghum halepense and S. bicolor Cultivars in Northwest Italy.

Sorghum plants naturally produce dhurrin, a cyanogenic glycoside that may be hydrolysed to cyanide, resulting in often-lethal toxicoses. Ruminants are particularly sensitive to cyanogenic glycosides due to the active role of rumen microbiota in dhurrin hydrolysis. This work provides an overview of a poisoning outbreak that occurred in 5 farms in Northwest Italy in August 2022; a total of 66 cows died, and many others developed acute toxicosis after being fed on either cultivated (Sorghum bicolor) or wild Sorghum (Sorghum halepense). Clinical signs were recorded, and all cows received antidotal/supportive therapy. Dead animals were subjected to necropsy, and dhurrin content was determined in Sorghum specimens using an LC-MS/MS method. Rapid onset, severe respiratory distress, recumbency and convulsions were the main clinical features; bright red blood, a bitter almond smell and lung emphysema were consistently observed on necropsy. The combined i.v. and oral administration of sodium thiosulphate resulted in a rapid improvement of clinical signs. Dhurrin concentrations corresponding to cyanide levels higher than the tolerated threshold of 200 mg/kg were detected in sorghum specimens from 4 out of 5 involved farms; thereafter, such levels declined, reaching tolerable concentrations in September-October. Feeding cattle with wild or cultivated Sorghum as green fodder is a common practice in Northern Italy, especially in summer. However, care should be taken in case of adverse climatic conditions, such as severe drought and tropical temperatures (characterising summer 2022), which are reported to increase dhurrin synthesis and storage.

Content of Toxic Elements (Arsenic, Cadmium, Mercury, Lead) in Eggs from an Ethically Managed Laying Hen Farm.

Domestic chicken farming has been promoted and spread in several Italian municipalities and worldwide as an aid to the self-consumption of domestically produced food. This study investigated the levels of four toxic elements (As, Cd, Hg, and Pb) in eggs from an ethical laying hen farm, comparing the element concentrations with those possibly present in supermarket eggs. A total of 201 eggs, 141 from the farm and produced by different hen genotypes, and 60 from the supermarket, were collected. The levels of the toxic elements were evaluated in the yolk, albumen, and eggshells of all eggs. The results show that the supermarket eggs' yolk and albumen were more contaminated with lead, compared to the rural eggs. Contrarily, the mean content of arsenic was higher in the albumen and eggshells of the rural eggs, compared to the supermarket eggs. The cadmium content was below the LOQ (0.005 mg/kg) in all samples. The mercury content was below or around the LOQ in all rural eggs. Overall, the supermarket egg albumens were significantly more contaminated than the rural ones. No significant differences were found in quality parameters for both types of eggs. The toxic element values that were detected were in line with other studies in the literature. However, despite the concentrations found not representing a risk to the consumers' health, the results of this study raise a potential food safety issue, and it would be desirable to set specific MRLs for eggs for consumers' protection.

Pyrrolizidine Alkaloids from Monofloral and Multifloral Italian Honey.

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by plants as a self-defense against insects. After bioactivation in the liver, some PAs can cause acute or chronic toxicity in humans. The aim of this study was to determine the presence of PAs in 121 samples of monofloral and multifloral honey from three different Italian regions (Friuli-Venezia Giulia, Marche and Calabria) to meet the European Food Safety Authority (EFSA) suggestion. An in-house liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was validated according to European Union Reference Laboratory (EURL) performance criteria. This method allowed the detection and quantification of 35 PAs. Of the 121 honey samples, 38 (31%), mostly from Calabria, contained PAs. The total content of the PAs ranged from 0.9 µg/kg to 33.1 µg/kg. In particular, echimidine was the most prevalent PA. A rapid human exposure assessment to PAs in honey and a risk characterization was performed using the EFSA RACE tool. The assessment highlighted a potential health concern only for toddlers who frequently consume elevated quantities of honey. This study showed a low presence of PAs in Italian honey; however, the importance of continuously monitoring these compounds is stressed, along with the suggestion that the relevant authorities establish maximum limits to guarantee support for producers and consumer safety.

Development of Histamine in Fresh and Canned Tuna Steaks Stored under Different Experimental Temperature Conditions.

Among biogenic amines, histamine is most frequently involved in foodborne intoxication. To evaluate histamine formation in tuna, several storage conditions were reproduced. An LC-MS/MS method was used for analytical determinations. Fresh tuna samples (not contaminated and grafted with tuna muscle naturally incurred with histamine at 6000 mg/kg) were stored at 4, 12, and 20 °C, and daily samples were collected for 6 days. The development of histamine was observed only in grafted tuna samples. At 4 °C, histamine formation progressed from 12.8 mg/kg (day 1) up to 68.2 mg/kg (day 6). At 12 °C, higher concentrations developed (23.9 mg/kg on day 1 up to 2721.3 mg/kg on day 6) relative to 20 °C (from 12.0 to 1681.0 mg/kg). It was found that at 4 °C, if grafted tuna was submerged in oil, histamine formation progressed more slowly. In a naturally contaminated sample, it was observed that the histamine distribution was uniform, while the normal cooking process did not affect the histamine level. Furthermore, it was found that the use of histamine-contaminated equipment for food handling may result in histamine formation in food. These results confirm the importance of implementing good hygiene practices and respecting the cold chain.

Occurrence of Histamine in Commercial Cat Foods under Different Storage Conditions.

In fish-based foods, one of the effects of inappropriate storage can be the formation of biogenic amines. Among these, histamine is considered one of the most toxic. The purpose of the present study is to assess the occurrence of histamine in fish-based pet foods, and to evaluate the changes in histamine content during storage at different temperatures. For the analysis, an LC-MS/MS method was used. Fifty-eight pet foods were purchased, and an aliquot of them was analyzed just after opening the packages. Histamine was detected in 12 samples and concentrations ranged from 1.5 to 30.1 mg/kg. The remaining of each positive sample was divided into seven subsamples. One of them was used as a control sample and kept at -20 °C, while the other six were exposed to different environmental conditions. Samples exposed to room temperature showed no significant changes in histamine levels, while samples exposed to high temperatures showed significant increases in histamine content. Finally, samples exposed to refrigerator temperature showed a slight decrease in histamine levels. Under the experimental conditions, the EU limit of 100 mg/kg established for fishery products was never exceeded. These results seem to indicate a low risk of histamine intoxication in cats fed fish-based pet food.

Occurrence of Ochratoxin A in Different Types of Cheese Offered for Sale in Italy.

The detection of Ochratoxin A (OTA) in the milk of ruminants occurs infrequently and at low levels, but its occurrence may be higher in dairy products such as cheese. The aim of this study was to investigate the presence of OTA in cheeses purchased in the metropolitan city of Bologna (Italy) and the surrounding area. For the analysis, a LC-MS/MS method with a limit of quantification (LOQ) of 1 µg/kg was used. OTA was detected in seven out of 51 samples of grated hard cheese (concentration range 1.3-22.4 µg/kg), while it was not found in the 33 cheeses of other types which were also analysed. These data show a low risk of OTA contamination for almost all types of cheese analysed. To improve the safety of cheese marketed in grated form, more regulations on cheese rind, which is the part most susceptible to OTA-producing moulds, should be implemented or, alternatively, producers should consider not using the rind as row material for grated cheese. It would be interesting to continue these investigations particularly on grated hard cheeses to have more data to update the risk assessment of OTA in cheese, as also suggested by EFSA in its 2020 scientific opinion on OTA.

Investigation of Lepechinia graveolens for its antioxidant activity and phenolic composition.

A bioguided separation of Lepechinia graveolens (Reg.) Epling. (Lamiaceae) for antioxidant activity was carried out. The ethyl acetate fraction (EAF) from the methanolic extract was chromatographed on Sephadex LH-20 affording five active fractions. The radical scavenging activity of each fraction, as well as that of the isolated compounds, was tested using three different methods. The major isolated antioxidant compounds were identified as luteolin-7-O-glucuronide, rosmarinic acid, and rosmarinic acid methyl ester by means of 1H and 13C NMR and mass spectrometry. Twenty other minor phenolic compounds were determined by liquid chromatography-negative electrospray ionization tandem mass spectrometry. They included hydroxycinnamic acid derivatives, flavonoids and phenolic diterpenes. The major phenolic compound was found to be rosmarinic acid, which was quantitatively determined by high-performance liquid chromatography (HPLC)-diode array detector (DAD). The content of rosmarinic acid was 2.8% (referred to dry weight), this amount being around 30% of the total phenolic content (TPH) of the crude extract. It can be concluded that the antioxidant activity of Lepechinia graveolens was mainly due to rosmarinic acid, which allows this plant species to be considered as a potential new source of this well known natural antioxidant.