Evaluation of beauvericin and enniatins in Italian cereal products and multicereal food by liquid chromatography coupled to triple quadrupole mass spectrometry

In this study, 48 multicereal baby foods samples including 25 of pasta and 23 of multicereal baby foods from supermarkets of Campania region (Italy) were analysed for evaluating the presence of beauvericin (BEA) and enniatins (ENs) A, A1, B, B1 and B4. Subsequently to evaluate the risk exposure of Italian population and infant population over the consumption of pasta or multicereal baby food, was, respectively, evaluated.     

Beauvericin and enniatins A,A1, B and B1 in Norwegian grain: a survey

Norwegian grain samples (73 oats, 75 barley, 80 wheat) from the 2000 to 2002 growing seasons were examined for contamination with five different enniatins and the association between the found concentrations and the prevalence or infection level with several common Fusarium species investigated. Enniatin B was the fungal metabolite with the highest prevalence (100%) and the highest maximum concentration (5800 μg/kg, wheat). The maximum concentration of all five enniatins together in a single sample was 7400 μg/kg (wheat). Enniatin concentrations were correlated with several independent variables, among them grain species. Beauvericin was only sporadically detected in barley and wheat and at concentrations just above the limit of detection of 3 μg/kg, while amounts up to 120 μg/kg were found in oats. The likelihood of detecting enniatin A1 as well as the concentrations of enniatins B and B1 could be mainly related to infection with Fusarium avenaceum/arthrosporioides, and the likelihood of detecting beauvericin could be related to infection with Fusarium poae. This survey indicates that the prevalence of enniatins A1, B and B1 in Norwegian grain is high, and that enniatin B concentrations of above 1000 μg/kg are common in barley and wheat.     

Production of enniatins A,A1, B,B1, B4, J1 by Fusarium tricinctum in solid corn culture: Structural analysis and effects on mitochondrial respiration

Enniatins (ENs) are secondary fungal metabolites with hexadepsipeptidic chemical structure and they possess a number of potent biological activities that can contaminate several kind of food and foodstuffs increasing the exposure risk for consumers. ENs are produced by several Fusariun strains including Fusarium subglutinans, Fusarium proliferatum and Fusarium tricinctum. Production of a mixture of ENs was performed by culturing F. tricinctum ITEM 9496 on white corn as substrate. The solid culture components were dried and subsequently extracted with water/methanol (50/50 v/v, 0.5% NaCl), homogenised, filtered, extracted by ethyl acetate and analysed by liquid chromatography with diode array detection (LC-DAD). The crude extract was first separated by low pressure liquid chromatography (LPLC) and then further purified by liquid chromatography (LC), resulting in six compounds with a purity higher than 95% as calculated by ¹H NMR, and with a yield of 30–300 mg per compound. The chemical structures of the ENs were determined by liquid chromatography coupled to mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR). The biological activity of the resulting ENs was determined using a mitochondrial respiration test. We discovered that all the ENs studied induced an increase in the mitochondrial respiration resulting in uncoupling of the oxidative phosphorylation. This effect was most likely due to flux of K⁺ ions into the mitochondrial matrix. The order of potency of the ENs derivatives was: A₁ > B₁ > B > A > B₄ > J₁. These results suggest a correlation between the chemical structures and bioactivity and confirm the severe risks for human associated with consumption of enniatins.     

Effects of feed naturally contaminated with Fusarium mycotoxins on metabolism and immunity of dairy cows

A previous study in dairy cows showed some effect of feed contaminated with Fusarium mycotoxins on metabolism and immunity. A subsequent experiment investigated the effect of feedborne Fusarium mycotoxins on some immune functions in more detail. A total mixed ration (TMR) containing a blend of feedstuffs naturally contaminated with Fusarium mycotoxins was fed for 63 d to 12 mid-lactation Holstein cows with an average milk production of 36 kg/d in a completely randomized design with repeated measures including 1) control TMR and 2) contaminated TMR. Wheat, corn, hay, and corn silage were the contaminated feedstuffs. Deoxynivalenol was the major contaminant and was found in TMR at 3.5 mg/kg of dry matter. The parameters measured were 1) performance: body weight, body condition score, dry matter intake, milk production, composition and somatic cell count; 2) health: blood serum chemistry, hematology, coagulation profile, and rumen fluid ammonia levels; 3) immune function: total serum immunoglobulins (IgA, IgG, IgM), specific antibody response to ovalbumin, and neutrophil phagocytosis. Dry matter intake, body weight, milk production, and milk composition were not affected by diet. Neutrophil phagocytosis was depressed throughout the experiment in cows fed the contaminated diet. Serum sodium concentrations and osmolality were significantly elevated throughout the experiment in cows fed the contaminated diet. Primary antibody response to ovalbumin immunization was higher in cows fed the contaminated diet compared with controls. It was concluded that feed naturally contaminated with Fusarium mycotoxins can affect metabolic parameters and immune function of dairy cows.     

Association studies on the porcine RETN, UCP1, UCP3 and ADRB3 genes polymorphism with fatness traits

Searching for effects of candidate gene polymorphisms on fatness traits is an important goal for pig industry. In this study we evaluated polymorphism of four porcine genes involved in energy metabolism (RETN, UCP1, UCP3 and ADRB3). Moreover, their association with fat deposition traits was analyzed in two breeds (Polish Landrace, Polish Large White) and a Polish synthetic line (L990). Altogether, five SNPs were identified, including two novel ones in the 5′-flanking region of the RETN gene and a novel missense substitution in the UCP3. Distribution of these polymorphisms in the studied five breeds and the synthetic line was not uniform. Two of the analyzed SNPs: g.−178G > A in the RETN and g.946C > T in the UCP3 gene revealed a significant association with abdominal fat weight or backfat thickness. Such associations were not observed for the UCP1 or ADRB3 gene polymorphisms. Our study showed that polymorphisms of the UCP3 and RETN genes are potentially associated with porcine fatness traits.     

Multiplex conventional and real-time PCR for fish species identification of Bianchetto (juvenile form of Sardina pilchardus), Rossetto (Aphia minuta), and Icefish in fresh,marinated and cooked products

A conventional and a realtime multiplex PCR were developed to detect fraudulent substitutions of Bianchetto (juvenile form of Sardina philcardus) and Rossetto (Aphia minuta) with Icefish (Neosalanx spp.), which show similar morphological characteristics. Since it is the major by-catch species, Engraulis encrasicolus was also included in the analytical procedure. A common reverse primer and forward species-specific primer were designed on the mitochondrial cytochrome b gene to amplify sequences of different lengths by conventional PCR. Specific peaks were therefore also provided after melting temperature analysis in real-time PCR, thus enabling each species to be clearly differentiated. The two PCR methods were validated on fresh and processed products after preparing four typical dishes: two marinades (from raw or lightly boiled fish), a pasta sauce, and batter-fried fish cakes. All samples were correctly identified, although there was some DNA degradation after processing.     

Effect of suboptimal temperatures and sublethal CO2 levels on multiplication of Tribolium castaneum (Coleoptera: Tenebrionidae), alone or competing with Cryptolestes ferrugineus (Coleoptera: Cucujidae)

Groups of adults of Tribolium castaneum alone, or in combination with adults of Cryptolestes ferrugineus, were exposed to suboptimal temperatures (15°C, 20°C, and 25°C) and sublethal CO₂ levels (2%, 5%, and 10%) in dry (12%, wet mass basis) and damp (15%, wet mass basis) stored wheat in the laboratory, to investigate effects on population size. The mean adult numbers in single- and mixed-species tests were positively correlated with higher temperature and moisture content and negatively correlated with higher CO₂ levels. Adult numbers in single- and mixed-species tests were lower at sublethal CO₂ levels compared to ambient CO₂ levels at all the test temperatures and decreased in dry grain compared to damp grain. Although, a specific trend was not observed in population inhibition between mixed-species and single-species tests, overall the adult populations of T. castaneum were reduced in the presence of C. ferrugineus. A mathematical model was derived to predict the size of adult populations of T. castaneum alone, or in the presence of C. ferrugineus considering all the variables in this study. The model had an R² value of 0.72 but needs to be validated and refined with field data.     

Inhibition of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) development with essential oil extracted from Cymbopogon schoenanthus L. Spreng. (Poaceae), and the wasp Dinarmus basalis (Rondani) (Hymenoptera: Pteromalidae)

The control of the development of Callosobruchus maculatus was studied using a method that combined exposure to essential oil extracted from Cymbopogon schoenanthus and the introduction of a pteromalid natural enemy of the bruchid, Dinarmus basalis. The effect of the essential oil used was evaluated on all developmental stages of C. maculatus and on adults of D. basalis. At the highest concentration tested (33.3 μl/l) all adults of C. maculatus were killed within 24 h of exposure to the oil and the development of newly laid eggs and neonate larvae was also inhibited. However, the oil had variable efficacy against the bruchid instars developing inside the seeds: 5-day-old larvae (63% LI and 37% LII) of C. maculatus developing inside the seeds proving to be highly susceptible while 15-day-old insects (84% of pupae and 16% of larvae) were tolerant. Under the same conditions (33.3 μl/l), adults of D. basalis were very susceptible to oil vapours and to the residual activity of the oil after 3 or 6 days. However, the introduction of 10 pairs of adult D. basalis into a jar containing 100 hosts aged 10 days, 3 or 6 days before the oil application, gave respectively an emergence of 26 or 18 adults of the parasitoid compared to 28 in the control and there was no adult emergence of the host. The possibility of an integrated pest management strategy by using allelochemicals such as essential oils and indigenous natural enemies to control C. maculatus development in cowpea stocks is discussed.     

Inhibitory effect of the essential oil of Curcuma longa L. and curcumin on aflatoxin production by Aspergillus flavus Link

Aflatoxins are highly toxic, mutagenic, teratogenic and carcinogenic mycotoxins. Consumption of aflatoxin-contaminated food and commodities poses serious hazards to the health of humans and animals. Turmeric, Curcuma longa L., is a native plant of Southeast Asia and has antimicrobial, antioxidant and antifungal properties. This paper reports the antiaflatoxigenic activities of the essential oil of C. longa and curcumin. The medium tests were prepared with the oil of C. longa, and the curcumin standard at concentrations varied from 0.01% to 5.0%. All doses of the essential oil of the plant and the curcumin standard interfered with mycotoxin production. Both the essential oil and curcumin significantly inhibited the production of aflatoxins; the 0.5% level had a greater than 96% inhibitory effect. The levels of aflatoxin B₁ (AFB₁) production were 1.0 and 42.7 μg/mL, respectively, for the samples treated with the essential oil of C. longa L. and curcumin at a concentration of 0.5%.     

Resistance in maize to the Larger Grain Borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae)

Host plant resistance in maize can be a useful component of Integrated Pest Management (IPM) of the Larger Grain Borer (LGB), Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae). The germplasm bank of the International Maize and Wheat Improvement Centre (CIMMYT) has more than 14,000 maize accessions from all over the world. In 1993, from the seed regeneration nursery of Caribbean land races, 105 land races were selected using good plant type as the criterion. When grains of these land races were infested with LGB in the laboratory, 19 showed resistance as evidenced by the low amount of powder (chewed uneaten maize plus feces) formed 140 days after the infestation. Through a series of infestation, selection and inbreeding (self-pollination of land races) over four generations, S₃ maize ears were generated. These ears showed a high level of resistance as indicated by a low powder production relative to the susceptible control. The LGB reproduction was adversely affected on resistant ears as indicated by the small size of adult populations. Thus, antibiosis could be the mechanism of resistance operating within the S₃ progenies of selected land races. Such sources of resistance can be very useful in developing maize populations/hybrids for use in IPM of LGB.     

Essential oils of the aerial parts of three Salvia species from Jordan: Salvia lanigera, S. spinosa and S. syriaca

The compositions of the essential oils of three Jordanian Salvia species, S. lanigera and S. spinosa from a desert climate, and S. syriaca from a Mediterranean habitat, were studied. All three oils are rich in monoterpene derivatives (68–73%).     

Development and survival of the cheese mites, Acarus farris and Tyrophagus neiswanderi (Acari: Acaridae), at constant temperatures and 90% relative humidity

Two species of acarid mites, Acarus farris and Tyrophagus neiswanderi, have been identified infesting Cabrales cheese in an Asturian maturing cave, the former being the prevalent species. The developmental rate and survival of immature stages of these mites were examined at constant temperatures, ranging from 7 to 29.7 °C for A. farris, and 10 to 31 °C for T. neiswanderi, and a relative humidity (r.h.) of 90±5%. The larval stage of A. farris was particularly susceptible to low and high temperatures with 81.7% and 95.2% mortality at 7 and 29.7 °C, respectively. Tyrophagus neiswanderi larvae also showed the greatest mortality at extreme temperatures among immature stages, though at a lower level than for A. farris (8.6% and 25.6% at 10 and 31 °C, respectively). The optimal temperature for development appeared to be 27-28 °C for both species and the developmental rates were higher for A. farris than T. neiswanderi within the range of the cooler temperatures prevalent in the cheese-maturing caves. The nonlinear Logan type-III model provided the best fit for the relationship between developmental rates and temperature (R_a²>0.99) for all immature stages of A. farris, whereas the development of T. neiswanderi was better described by the Lactin model (R_a²>0.97). The lower and upper developmental threshold temperatures predicted for each stage of A. farris were 3-4 °C lower than those predicted for T. neiswanderi. The differential temperature-development rate for each species might explain the greater abundance of A. farris compared to T. neiswanderi. Furthermore, manipulation of temperature based on modeling predictions may well be used to control mite populations during the cheese maturing process.     

Schisarisanlactones A and B: A new class of nortriterpenoids from the fruits of Schisandra arisanensis

Two novel highly oxygenated nortriterpenoids, schisarisanlactones A (1) and B (2), have been isolated from the fruits of Schisandra arisanensis, an endemic plant of Taiwan. Compounds 1 and 2 possess an unprecedented 5/5/7/5/5-fused pentacyclic ring system. The structures of both compounds were determined on the basis of spectroscopic analyses, especially 2D NMR and MS. A plausible biogenetic pathway of 1 was proposed. Schisarisanlactone A (1) showed significant anti-HIV activity.     

The inhibitory effect of Plectranthus barbatus and Plectranthus ecklonii leaves on the viability,glucosyltransferase activity and biofilm formation of Streptococcus sobrinus and Streptococcus mutans

Aqueous extracts of Plectranthus barbatus and Plectranthus ecklonii are traditionally used as anti-inflammatory and anti-fungal agents. The effect of these extracts and of its main component, rosmarinic acid, on the viability of the cariogenic bacteria, Streptococcus sobrinus and Streptococcus mutans, was determined by MIC and MBC. The influence of these extracts on the biofilm formation as well as on the inhibition of glucosyltransferase enzyme, produced by these species, was also analysed. Aqueous extracts of P. barbatus and P. ecklonii were stronger inhibitors than rosmarinic acid. MIC values of 3.8 and 4.7 mg/ml for S. sobrinus and 2.9 and 5.0 for S. mutans were obtained, while rosmarinic acid presented MIC values of 8.4 and 7.3 mg/ml. P. barbartus, P. ecklonii and rosmarinic acid presented MBC values of 9.5, 9.0 and 12.0 mg/ml for S. sobrinus, and 9.5, 10.0 and 12.5 mg/ml for S. mutans. The inhibition of biofilm formation by P. barbatus, P. ecklonii and rosmarinic acid presented IC₅₀ values of, respectively, 0.6, 1.0 and 3.1 mg/ml for S. sobrinus and 1.4 and 2.7 and 1.3 mg/ml for S. mutans. The glucosyltransferase inhibition activity by theses extracts and rosmarinic acid was calculated and IC₅₀ values presented were, respectively, 1.1, ca 1.2 and 2.1 mg/ml for S. sobrinus and 3.1, 1.6 and 3.9 mg/ml for S. mutans were obtained. These extracts may be useful in the prevention of dental carie.     

Insecticidal activities of constituents identified in the essential oil from leaves of Chamaecyparis obtusa against Callosobruchus chinensis (L.) and Sitophilus oryzae (L.)

Analysis by gas chromatography-mass spectrometry led to the identification of 20 volatiles from the steam distilled oil of the leaves from Chamaecyparis obtusa Siebold et Zuccarni. Seven constituents of the oil were tested for contact and fumigant activity against adults of Callosobruchus chinensis (L.) and Sitophilus oryzae (L.). Responses varied with compound and dose rather than increasing exposure time beyond 1 or 2 days. In the impregnated-paper test with the constituents [bornyl acetate, (+)-limonene, myrcene, α-phellandrene, α-pinene, sabinene and terpinolene], at 0.1 mg/cm², bornyl acetate, α-phellandrene and terpinolene caused 97%, 97% and 87% mortality, respectively, against C. chinensis adults 1 day after treatment. At 0.05 mg/cm², moderate activity was achieved with bornyl acetate (56% mortality), α-phellandrene (75%) and terpinolene (55%). Against S. oryzae adults, at 0.26 mg/cm², terpinolene caused 93% mortality 2 days after treatment, whereas 80% mortality 4 days after treatment was observed with bornyl acetate and α-phellandrene. The toxicity of these compounds was significantly decreased at 0.18 mg/cm². In a fumigation test with S. oryzae adults, bornyl acetate and terpinolene were much more effective in sealed containers than in open ones, indicating that the insecticidal activity of these compounds was largely attributable to fumigant action. These naturally occurring materials derived from leaves of Chamaecyparis could be useful for managing populations of C. chinensis and S. oryzae.     

Effects of milk fat composition, DGAT1, and SCD1 on fertility traits in Dutch Holstein cattle

Recently, selective breeding was proposed as a means of changing the fatty acid composition of milk to improve its nutritional quality. Before implementing such breeding objectives, effects on other economically important traits should be investigated. The objectives of this study were to examine 1) the effect of milk fat composition, and 2) the effect of polymorphisms of DGAT1 and SCD1 genes on female fertility in commercial Dutch Holstein-Friesian cattle. Data on 1,745 first-lactation cows were analyzed by fitting linear mixed models. We found that higher concentrations of trans fatty acids within total milk fat negatively affected reproductive performance. Furthermore, results suggested a potential effect of the DGAT1 polymorphism on nonreturn rates for insemination 28 and 56 d after the first service. Our results can be used to assess the correlated effects of breeding for improved milk fat composition on reproduction, thereby allowing for better evaluation of breeding programs before implementation.     

A new variant of antimetabolic protein,arcelin from an Indian bean, Lablab purpureus (Linn.) and its effect on the stored product pest, Callosobruchus maculatus

The anti-metabolic or insecticidal gene, arcelin (Arl) was isolated, cloned and sequenced using sequence specific degenerate primers from the seeds of Lablab purpureus collected from the Western Ghats, Tamil Nadu, India. The L. purpureus arcelin nucleotide sequence was homologous to Arl-3 and Arl-4 alleles from Phaseolus spp. The protein it encodes has 70% amino acid identity with the amino acid sequences of Arl-3I, Arl-3III, Arl-4 precursor, Arl-4 and Arl-4I. The partially purified arcelin from the seeds of L. purpureus using an artificial diet confirmed the complete retardation of development of the stored product pest Callosobruchus maculatus at 0.2% w/w arcelin-incorporated artificial seeds.     

Comparative study of field and laboratory evaluations of the ethnobotanical Cassia sophera L. (Leguminosae) for bioactivity against the storage pests Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) and Sitophilus oryzae (L.) (Coleoptera: Curculionidae)

The powdered leaves of Cassia sophera along with hot- and cold-water leaf extracts of this plant were tested in laboratory experiments in the UK and in field trials in Tamale, Northern Ghana, using traditional storage containers, to determine their inhibitory and toxic effects against Sitophilus oryzae and Callosobruchus maculatus infestation of stored rice and cowpea, respectively. Laboratory and field experiments with cowpea showed that the use of C. sophera hot-water extracts was more effective at reducing C. maculatus infestation and adult emergence on cowpea than the traditional leaf-powder application (1% and 5% w/w) or the use of a cold-water extract of C. sophera. Hot-water extracts of C. sophera might be a more effective technique of applying the plant material on to stored cowpea than using powdered C. sophera leaves, the currently used application by small-scale farmers. In contrast, experiments with S. oryzae on rice showed that C. sophera leaf powder (5% w/w) effectively reduced adult emergence in the laboratory, but this could not be confirmed under field conditions. The hot and dry climatic conditions in the field might impart a natural protection against rice infestation by S. oryzae, making the use of protectants and pesticides less necessary for farmers. This was supported by the negligible rice grain damage after 6 months of field storage and by the failure of the S. oryzae population to establish itself under field conditions. The implications of using botanicals in pest control are discussed.     

A laboratory assessment of the potential of different strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpea

Twelve indigenous and exotic isolates of Beauveria bassiana and Metarhizium anisopliae were evaluated for their virulence and their ability to suppress populations of Callosobruchus maculatus in stored cowpea. LT₅₀ values ranged from 3.11 to 6.12 days following immersion in aqueous suspensions containing 1×10⁸ conidia ml⁻¹. Indigenous isolates that had been recovered from C. maculatus were more virulent in laboratory bioassays than exotic isolates from other insects. The two isolates with the shortest LT₅₀ values were compared in dose-response assays by immersion and by exposure to cowpea grains treated with dry conidia. In both assays B. bassiana 0362 was consistently more virulent than M. anisopliae 0351. By immersion, LC₅₀ values on day 6 post-treatment were 9.10×10⁴ and 7.10×10⁵ conidia ml⁻¹ for B. bassiana 0362 and M. anisopliae 0351, respectively. Exposure to treated grains gave LC₅₀ values of 1.15×10⁷ and 4.44×10⁷ conidia g⁻¹ grain for B. bassiana 0362 and M. anisopliae 0351, respectively. In 1 kg batches of cowpea stocked with 50 adult C. maculatus, B. bassiana 0362 at both 1×10⁷ and 1×10⁸ conidia g⁻¹ grain led to significant adult mortality and reduced F₁ emergence relative to untreated populations. At 1×10⁸ conidia g⁻¹ the effect of the fungus persisted into the F₁ generation. The net reproductive rates, R₀, measured 26 days after insects were released were 5.16 and 7.32 for the high and low doses compared to 9.52 for the untreated control.