Effects of a mixed additive based on Saccharomyces cerevisiae and Lactobacillus rhamnosus on broilers exposed to aflatoxin B1 by contaminated feed.

The objective of the present study was to explore the influence of dietary supplementation with a mixed additive (MA) containing a probiotic and anti-mycotoxin (Saccharomyces cerevisiae RC016 and Lactobacillus rhamnosus RC007) and its interaction on the performance and health (biochemistry and liver/intestine histopathology) of broilers fed diets contaminated with aflatoxin B1 (AFB1) at 506000±22.1ng/kg. The MA contained S. cerevisiae RC016 (1×107cells/g) and L. rhamnosus RC007 (1×108cells/g) in relation 1:1. A total of sixty-one-day-old Cobb broilers were randomly allocated into four treatment groups with three replicates of 5 birds each for a five-week-old feeding experiment. The experimental diet for each treatment (T) was formulated as follows: T1, a commercial diet (CD); T2, CD+AFB1; T3, CD+0.1% MA; T4, CD+AFB1+0.1% MA. The MA improved (p<0.01) production parameters (weight gain, conversion rate, and carcass yield) and reduced (p<0.01) the toxic effect of AFB1 on the relative weight of the livers. In addition, the macro and microscopic alterations of livers and the possible intestinal injury related to histological damage in the presence of mycotoxin were reduced. The use of probiotic MA based on S. cerevisiae RC016 and L. rhamnosus RC007 in animal feed provides greater protection against mycotoxin contamination and is safe for use as a supplement in animal feed, providing beneficial effects that improve animal health and productivity. This is of great importance at the economic level for the avian production system.

Chitosan-glucose derivative as effective wall material for probiotic yeasts microencapsulation.

A chitosan-glucose derivative (ChG) with lower antimicrobial activity against whey native probiotic yeast K. marxianus VM004 was synthesized by the Maillard reaction. The ChG derivative was characterized by FT-IR, 1H NMR, and SLS to determine the structure, deacetylation degree (DD), and molecular weight (Mw). In addition, we evaluated the antioxidant, cytotoxic, and antimicrobial activities of ChG. ChG was then used for microencapsulation of K. marxianus VM004 by spray drying. The microcapsules were characterized by evaluating their encapsulation yield, encapsulation efficiency, morphology, tolerance to the gastrointestinal tract, and viability during storage. The results indicated that a non-cytotoxic product with lower MW and DD and higher antioxidant activity than native chitosan was obtained by the Maillard reaction. The yeast ChG microcapsules exhibited an encapsulation efficiency >57 %, improved resistance to gastrointestinal conditions, and enhanced stability during storage. These results demonstrate that ChG may be a promising wall material for the microencapsulation of probiotic yeasts.

In-feed nutritional additive probiotic Saccharomyces boulardii RC009 can substitute for prophylactic antibiotics and improve the production and health of weaning pigs.

Background and Aims: Non-therapeutic antibiotic use is associated with the current decrease in antibiotic therapeutic efficiency and the emergence of a wide range of resistant strains, which constitutes a public health risk. This study aimed to evaluate the use of Saccharomyces cerevisiae var. boulardii RC009 as a nutritional feed additive to substitute the prophylactic use of antibiotics and improve the productive performance and health of post-weaning piglets. Materials and Methods: Four regular nutritional phases were prepared. Post-weaning pigs (21-70 days old) received one of two dietary treatments: T1-basal diet (BD-control group) with in-feed antibiotics as a prophylactic medication (one pulse of Tiamulin in P3 and one pulse of Amoxicillin in P4); and T2-BD without in-feed antibiotics but with Saccharomyces boulardii RC009 (1 × 1012 colony forming unit/T feed). The feed conversion ratio (FCR), total weight gain (TWG-kg), and daily weight gain (DWG-kg) were determined. A post-weaning growth index (GI) was calculated and animals (160 days old) from each treatment were analyzed at the abattoir after sacrifice for carcass weight and respiratory tract lesions. Results: Pigs consuming probiotics had higher TWG and DWG than the control group. The group of animals with low body weight obtained the same results. Saccharomyces boulardii administration decreased diarrhea, and FCR reduction was related to a GI improvement. A significant increase in carcass weight and muscle thickness reduction was observed in animals received the probiotic post-weaning. Conclusion: Saccharomyces boulardii RC009, a probiotic additive, was found to improve the production parameters of pigs post-weaning and enhance their health status, indicating that it may be a promising alternative to prophylactic antibiotics.

Inhibitory Effect of GRAS Essential Oils and Plant Extracts on the Growth of Aspergillus westerdijkiae and Aspergillus carbonarius Strains.

The effect of essential oils (obtained using hydrodistillation) and plant extracts (ethanolic, aqueous, and hexanic extractions) of 10 different plants cultivated in Brazil were tested using the diffusion agar method, with the objective of evaluating the inhibitory effect of the oils and extracts on the mycelial growth of Aspergillus westerdijkiae NRRL 3174 and A. carbonarius RC 2054 (UNRC). Of the 40 essential oils and plant extracts analyzed, oregano essential oil and plant extract, rosemary essential oil, and the clove ethanolic extract were the best choice to obtain the growth parameters (radial growth rates (mm day-1) and lag phase (h)) due the good results presented and the volume of oil/extract obtained. Comparing all the essential oils and plant extracts that were tested for growth parameters, the best results were obtained for the clove ethanolic extract for both strains assayed. These results demonstrated an outstanding potential use of some of these products in prevention of fungal contamination in food. However, further studies need to be conducted to determine the ability of these oils and extracts to inhibit or reduce ochratoxin A production.

Fist report on fumagillin production by Aspergillus fumigatus sensu stricto gliotoxigenic strains recovered from raw cow milk and clinical samples in Argentina / Primer reporte en Argentina sobre la producción de fumagilina por cepas deAspergillus fumigatus sensu stricto productoras de gliotoxina, aisladas de lechecruda y de casos clínicos

Abstract In Argentina there are no reports on Aspergillus fumigatus fumagillin-producingstrains. In this study we describe the isolation and mycotoxin production capacity of ten A.fumigatus strains isolated from farm and clinical samples. Farm strains were isolated frommilk samples taken from dairy cows in Córdoba province, some of which were associated withsubclinical mastitis. A culture medium was defined to optimize fumagillin production and adetection method was developed by HPLC chromatography. It is known that in addition to thehost immune status, strain virulence is a fundamental characteristic that will determine itspathogenicity and, in this sense, fumagillin is considered to be among the virulence factors. Inthe present work, all the strains tested for the production of fumagillin were able to synthesizeit, highlighting that the strain A. fumigatus RC2243, from a milk sample from a cow with clinicalmastitis, was the most productive. The existence of fumagillin-producing strains represents apotential risk of mycotoxins being transferred to raw milk, constituting a public health risk.

Resumen En Argentina no existen reportes sobre cepas de Aspergillus fumigatus productoras de fumagilina. En este trabajo se describe el aislamiento y la producción de dicha micotoxina clínicaspor 10 cepas, provenientes del medioambiente rural y aisladas de muestras clínicas. Las cepasde origen rural fueron aisladas de vacas lecheras en tambos de la provincia de Córdoba, yalgunas de esas cepas se asociaron a casos de mastitis subclínica. Se definió la composición deun medio de cultivo para optimizar la producción de fumagilina y se desarrolló un método decromatografía HPLC para su determinación. Es conocido que, además del estado inmunitario delhuésped, la virulencia de la cepa es una de las características fundamentales que determinansu potencial patogénico y, en este sentido, la fumagilina es considerada un factor de virulencia. En el presente trabajo todas las cepas estudiadas fueron capaces de sintetizarla y la cepa A.fumigatus RC2243, proveniente de leche de una vaca con mastitis subclínica, se destacó comola cepa más productora. La existencia de cepas productoras de fumagillina representa un riesgopotencial por el pasaje de dicha micotoxina a la leche, lo cual constituye un problema para lasalud pública.

Fist report on fumagillin production by Aspergillus fumigatus sensu stricto gliotoxigenic strains recovered from raw cow milk and clinical samples in Argentina.

In Argentina there are no reports on Aspergillus fumigatus fumagillin-producing strains. In this study we describe the isolation and mycotoxin production capacity of ten A. fumigatus strains isolated from farm and clinical samples. Farm strains were isolated from milk samples taken from dairy cows in Córdoba province, some of which were associated with subclinical mastitis. A culture medium was defined to optimize fumagillin production and a detection method was developed by HPLC chromatography. It is known that in addition to the host immune status, strain virulence is a fundamental characteristic that will determine its pathogenicity and, in this sense, fumagillin is considered to be among the virulence factors. In the present work, all the strains tested for the production of fumagillin were able to synthesize it, highlighting that the strain A. fumigatus RC2243, from a milk sample from a cow with clinical mastitis, was the most productive. The existence of fumagillin-producing strains represents a potential risk of mycotoxins being transferred to raw milk, constituting a public health risk.

Respiratory tract clinometry, fat thickness, haematology and productive parameters associated with direct-fed microbials used as growth promoter antibiotic alternative in weaned piglets.

The objective was to evaluate the effect of two probiotic yeast strains (Saccharomyces. cerevisiae RC016 and Kluyveromyces marxianus VM004) as a substitute of growth promoter antibiotics on health status and productive parameters in weaned piglets. Commercial line hybrid piglets (Choice n=200), weaned at 21 d age were allotted by sex, and assigned in 4 pens per treatment (2 pens males and 2 pens females), 10 pigs per pen divided into 2 blocks (with or without antibiotics). Dietary treatments included a basal diet (BD) supplemented with probiotic Saccharomyces cerevisiae RC016 and Kluyveromyces marxianus VM004 (100 g, 1 × 1010 CFU/g, respectively), with or without antibiotics, mixed per ton of growth phases diets. Pigs were fed ad libitum with treatments T1) BD with antibiotics (BD); T2) BD with antibiotics + Saccharomyces cerevisiae; T3) BD without antibiotics + Saccharomyces cerevisiae; T4) BD with antibiotics + Kluyveromyces marxianus; T5) BD without antibiotics + Kluyveromyces marxianus. The effects on respiratory tract clinometry, carcass quality, organs weight, blood haematology and productive parameters were evaluated. When clinical signs occurred (diarrhoea, stomach ulcers, respiratory signs), they decreased with both probiotics addition, mainly Saccharomyces cerevisiae. The productive parameters promotion by both probiotics was similar than that using antibiotics. The probiotics inclusion increased the carcass weight and significantly reduced the lumbar fat thickness (P ≤ 0.05). Supplementation with both probiotics demonstrated their ability to substitute the antibiotics use on clinometry, carcass quality and on the productive parameters promotion of weaned piglets.

Influence of technological procedures on viability, probiotic and anti-mycotoxin properties of Saccharomyces boulardii RC009, and biological safety studies.

The objective was to evaluate the technological processing (protection strategies and storage conditions) influence on viability, on probiotic properties and adsorbent aflatoxin B1 capacity of S. boulardii RC009. Also, the yeast biological safety was evaluated. Lyophilisation (DL) and encapsulation â€‹+ â€‹lyophilisation (EL) were conducted. Yeast protected with maltodextrin (M) or WPC stored at 4 â€‹°C reduced 1 and 2 log the viability, respectively. Yeast protected with M stored at 25 â€‹°C reduced 1 log after 70 â€‹d; with WPC the viability significantly reduced 3 log after 30 â€‹d. Technological processing improved the coaggregation's capacity with pathogens and DL process allowed the greatest AFB1 adsorption. S. boulardii 106 â€‹cells/mL were no toxic to Vero cells (p˂0.05). Saccharomyces boulardii RC009 protected with M or WPC maintained viability after technological processing. It possesses a great capacity for AFB1 adsorption and probiotic properties and could be considered a candidate with proven safety for functional food products development.

Lithothamnium calcareum (Pallas) Areschoug seaweed adsorbs aflatoxin B1 in vitro and improves broiler chicken's performance.

This study aimed to evaluate the aflatoxin B1 (AFB1) adsorption capacity of the seaweed Lithothamnium calcareum in vitro and to prevent aflatoxicosis in broiler chickens. In vitro adsorption assays were performed at a single AFB1 concentration (1 µg/mL) and four seaweed concentrations (0.50, 1, 1.5 and 2 mg/mL) at pH 3 and pH 6. The maximum adsorption was obtained at the lowest seaweed content (0.62 and 0.78 µg/mg). Male broiler chickens (256) were housed in metallic cages. Experimental diets were T1 (control), 18 µg/kg AFB1; T2, 18 µg/kg AFB1 and 0.2% L. calcareum (2.0 kg/ton); T3, 1018 µg/kg AFB1; and T4, 1018 µg/kg AFB1 and 0.2% L. calcareum. Performance parameters (live weight, weight gain and feed conversion rate) improved when seaweed was applied. The aspartate-aminotransferase and alanine-aminotransferase levels tend to decrease in birds receiving only seaweed, also the uric acid levels reduced significantly (P Ë‚ 0.05), while birds receiving only AFB1 increased the biochemical parameter levels. The livers from animals fed with AFB1 showed histopathological alterations with disorganization of periportal hepatocytes, necrosis with multifocal coagulation and mild fat degeneration; the livers from T4 had normal appearance. Lithothamnium calcareum was able to prevent aflatoxicosis in broiler chickens and also improved their zootechnical performance.

A Saccharomyces cerevisiae RC016-based feed additive reduces liver toxicity, residual aflatoxin B1 levels and positively influences intestinal morphology in broiler chickens fed chronic aflatoxin B1-contaminated diets.

The present study was conducted to investigate the ability of Saccharomyces cerevisiae RC016 (Sc)-based feed additive to reduce liver toxicity, residual aflatoxin B1 (AFB1) levels and influence intestinal structure in broiler chickens fed chronic aflatoxin B1-contaminated diets. A total of 100 one-day-old male commercial line (Ross) broiler chickens were divided into 4 treatments, with 5 pens per treatment and 5 broiler chickens per pen. Birds were randomly assigned to 4 treatments, which were namely treatment 1 (T1), control diet (CD); T2, CD + Sc at 1 g/kg; T3, CD + AFB1 at 100 µg/kg; T4, CD + Sc at 1 g/kg + AFB1 at 100 µg/kg. The liver histopathology of broiler chickens fed diets with AFB1 showed diffused microvacuolar fatty degeneration. The addition of Sc showed normal hepatocytes similar to the control. The small intestine villi from AFB1 group showed atrophy, hyperplasia of goblet cells, prominent inflammatory infiltrate and oedema. In contrast, the small intestine villi from birds that received the yeast plus AFB1 showed an absence of inflammatory infiltrate, and atrophy; moreover, a lower number of goblet cells compared to the groups with AFB1 was observed. The morphometric intestine studies showed that a significant decrease (P < 0.05) in the crypt depth values when Sc was applied to AFB1-contaminated diets. Although the intestinal villus height and apparent adsorption area did not show significant differences (P > 0.05), there was a tendency to improve these parameters. The residual levels of AFB1 in livers were significantly reduced (P < 0.05) in the presence of the yeast. The present work demonstrated that the addition of Sc alone or in combination with AFB1 in the broiler chicken diets had a beneficial effect in counteracting the toxic effects of AFB1 in livers besides improving the histomorphometric parameters and modulating the toxic effect of AFB1 in the intestine.

Antioxidant and cytoprotective effect of peptides produced by hydrolysis of whey protein concentrate with trypsin.

Whey protein is one of the most relevant co-products manufactured by the dairy industry and it is a powerful environmental pollutant. Therefore, the enzymatic hydrolysis of whey protein concentrate (WPC 35) to produce antioxidant peptides is an innovative approach which can provide added value to whey. The WPC 35 hydrolysis with trypsin was carried out for 4.31 h at 41.1 °C with an enzyme/substrate ratio of 0.017. Under such hydrolysis conditions, the peptides produced have the highest radical scavenging activity and cytoprotector effect. The WPC hydrolysate and a permeate ≤3 kDa were characterized by SDS-page, RP-HPLC and MALDI-TOF-MS. Furthermore, O2•- and HO• scavenging activity and the cytoprotective effect against a stress agent in epithelial cells of the rat ileum (IEC-18) were determined. In this study, strong antioxidant and cytoprotective peptides were obtained from a low-cost dairy industry product, which could improve consumers' health when used as functional ingredients.

Effect of fermented whey with a probiotic bacterium on gut immune system.

The aim of the work presented in this Research Communication was to evaluate the effect of fermented whey (FW) with Lactobacillus rhamnosus RC007 in a mice model. BALB/c mice were divided into three groups: control group: animals received orally 0.1 ml of phosphate buffered saline (PBS); FW group: animals received orally 0.1 ml of FW; whey (W) group: animals received orally 0.1 ml of W without fermentation with probiotic bacterium. After 10 d mice were sacrificed. Small intestines were collected for determination of IL-10; IL-6, TNFα, goblet cells and intraepithelial lymphocytes. Increases of all the cytokines assayed were observed in mice that received FW compared to control and W group. The ratio between the anti and pro-inflammatory cytokines (IL-10/TNFα) increased in the group of mice that received FW. The number of goblet cells and intraepithelial lymphocytes were also increased in animals that received FW. The results showed that FW with L. rhamnosus RC007 was able to stimulate and to modulate mouse immune system. Whey fermented by this probiotic bacterium is an interesting alternative for development of a new food additive for pig production, taking advantage of the beneficial properties of probiotic bacterium and the nutritional properties of whey.

In Vitro Activity of Neem (Azadirachta indica) Oil on Growth and Ochratoxin A Production by Aspergillus carbonarius Isolates.

Aspergillus carbonarius is a saprobic filamentous fungus, food spoiling fungus and a producer of ochratoxin A (OTA) mycotoxin. In this study, the in vitro antifungal activity of neem oil (0.12% p/p of azadirachtin) was evaluated against the growth of six strains of A. carbonarius and the production of OTA. Four different concentrations of neem oil were tested in addition to three incubation times. Only the concentration of 0.3% of neem oil inhibited more than 95% of the strain's growth (97.6% ± 0.5%), while the use of 0.5% and 1.0% of neem oil showed lower antifungal activity, 40.2% ± 3.1 and 64.7% ± 1.1, respectively. There was a complete inhibition of OTA production with 0.1% and 0.3% neem oil in the four strains isolated in the laboratory from grapes. The present study shows that neem essential oil can be further evaluated as an auxiliary method for the reduction of mycelial growth and OTA production.

Effect of prepubertal nutrition on cellular apoptosis and proliferation in at term placenta of Anglo-Nubian goats.

The aim of this study was to evaluate the effect of feed restriction with or without monensin supplementation, followed by a re-feeding period on cellular apoptosis and proliferation in at term placenta of Anglo-Nubian goats. To evaluate the induction of apoptosis through the intrinsic pathway, proteins Bax and Bcl-2 were determinated. The apoptosis was related with the cell proliferation indices through Ki67 determination. The treatments were applied for 250 days and were (a) ad libitum feeding (control; n = 5); (b) restricted feeding at 70% of control (restricted; n = 7); and (c) restricted with monensin supplementation (monensin; n = 7). After treatments, all the animals were fed to support their requirements. After parturition, 27 placentas were gathered. The placental cellular structure was studied by high-resolution light microscopy and transmission electron microscopy; the cellular proliferation was determined by Ki67 index, and Bax and Bcl-2 proteins were localized by immunohistochemical analysis. Differences in cell proliferation through the Ki67 index were found in monensin group placentas. Monensin supplementation stimulated the placental cell proliferation reversing the effect of feed restriction during the peripuberal period. A significant increase of Bcl-2 in placentas of restricted group was found, and it would provide a protective effect on the placental structure. A lack of the Bcl-2 protective effect was observed in control and monensin group placentas, probably meaning that the observed apoptosis would be induced through the intrinsic signalling pathway. A balance between apoptosis and cell proliferation is necessary to maintain tissue homoeostasis during caprine placental development.

Fusarium species and moniliformin occurrence in sorghum grains used as ingredient for animal feed in Argentina.

BACKGROUND: A survey on Fusarium species and moniliformin (MON) occurrence in sorghum grains collected from one of the main sorghum-producing areas of Argentina was conducted. Also, growth of F. thapsinum, one of the main sorghum pathogens, and MON production under different water activity (aw ) conditions on a sorghum-based medium were determined. RESULTS: Infection of sorghum grains by Fusarium species ranged from 82.5 to 99%; closely related species F. verticillioides, F. thapsinum and F. andiyazi were the most frequently recovered, followed by F. proliferatum and F. subglutinans. By sequencing a portion of the translation elongation factor-1α (TEF-1α) gene and by maximum parsimony analysis, F. verticillioides and closely related species were identified as F. thapsinum, F. andiyazi and F. verticillioides. Species within the F. graminearum species complex (FGSC) were isolated in high frequency. Maximum growth rates of 12 F. thapsinum strains were obtained at 0.995 aw . All evaluated strains were able to produce MON at all aw values tested, but MON production was higher at 0.995-0.982 aw . MON was detected in 41% of the samples at levels ranging from 363.2 to 914.2 µg kg-1 . CONCLUSION: This study provides new data on the occurrence of Fusarium species in sorghum grains destined for animal consumption in Argentina. The production of MON at different aw values showed that the toxin can be produced under field conditions. The risk to livestock exposed to daily low levels of MON associated with the toxin occurrence in the sorghum grains analyzed is unknown. © 2018 Society of Chemical Industry.

Molecular characterization of Aspergillus fumigatus isolated from raw cow milk in Argentina: Molecular typing of A. fumigatus from raw cow milk.

Aspergillus fumigatus, the major etiological agent of human and animal aspergillosis, is a gliotoxinogenic species into section Fumigati commonly found in contaminated animal environments. In dairy herds, exposed areas of lactating cows, as mammalian glandule, can be easily contaminated by them. This study was aimed to identify A. fumigatus sensu lato strains (identified based on morphology) isolated from raw cow milk at species level, by morphological and molecular techniques, and to estimate their genetic variability. Forty-five A. fumigatus strains showed similar RAPD profiles (generated with PELF and URP1F primers) to each other and to A. fumigatus sensu stricto reference strains; also, they were almost identical to clinical human and feed-borne A. fumigatus strains included in the assay, since their similarity coefficient ranged from 0.7 to 1.00. Therefore, all strains were characterized as belonging to A. fumigatus sensu stricto species. This result was supported by sequencing the benA gene of selected strains and by maximum parsimony analysis. In addition, RAPD fingerprinting demonstrated intra-specific genetic variability into the A. fumigatus sensu stricto cluster. The results found in this study strengthen the fact that A. fumigatus sensu stricto is the predominant species in the Aspergillus section Fumigati found in animal environments such as dairy herd environments, while other species such as A. novofumigatus, A. fumigatiaffinis, A. udagawae and A. lentulus may be rarely isolated. Since no differences between animal and human strains were observed they can become pathogenic also for farm handlers'. Moreover, the presence of A. fumigatus sensu stricto in raw cow milk is probably a very important risk factor since milk and its by-products are generally indented for human consumption, then gliotoxin could be transferred to them.

Intestinal toxicity of deoxynivalenol is limited by Lactobacillus rhamnosus RC007 in pig jejunum explants.

Probiotics have been explored to stimulate gut health in weaned pigs, when they started to consume solid diet where mycotoxins could be present. The aim of this study was to evaluate the effect of Lactobacillus rhamnosus RC007 on the intestinal toxicity of deoxynivalenol (DON) in an ex vivo model. Jejunal explants, obtained from 5-week-old crossbred castrated male piglets, were kept as control, exposed for 3 h to 10 µM DON, incubated for 4 h with 109 CFU/mL L. rhamnosus, or pre-incubated 1 h with 109 L. rhamnosus and exposed to DON. Histological lesions were observed, para- and transcellular intestinal permeability was measured in Ussing chambers. The expression levels of mRNA encoding six inflammatory cytokines (CCL20, IL-10, IL-1ß, TNFα, IL-8 and IL-22) were determined by RT-PCR. The expressions of the phosphorylated MAP kinases p42/p44 and p38 were assessed by immunoblotting. Exposure to DON induced histological changes, significantly increased the expression of CCL20, IL-1ß, TNFα, IL-8, IL-22 and IL-10, increased the intestinal paracellular permeability and activated MAP kinases. Incubation with L. rhamnosus alone did not have any significant effect. By contrast, the pre-incubation with L. rhamnosus reduced all the effects of DON: the histological alterations, the pro-inflammatory response, the paracellular permeability and the phosphorylation of MAP kinases. Of note, L. rhamnosus did not adsorb DON and only slightly degrade the toxin. In conclusion, L. rhamnosus RC007 is a promising probiotic which, included as feed additive, can decrease the intestinal toxicity of DON.

Validation of a liquid chromatography/tandem mass spectrometry method for the detection of aflatoxin B1 residues in broiler liver.

Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin produced mainly by Aspergillus flavus and Aspergillus parasiticus. It is the predominant mycotoxin found in raw materials used for the manufacture of broiler feeds. The aim of the present study was to develop a new and optimized method for the detection and quantification of aflatoxin B1 (AFB1) residues in broiler liver using solid phase extraction (SPE) clean-up and liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS) detection. The method was validated for linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ). The validation parameters indicated satisfactory linearity (r2>0.99), accuracy and precision (4.57% intra-day RSD; 14.65% inter-day RSD) a very high recovery (99±13%) and high sensitivity achieved for AFB1 in animal samples (LOD=0.017 and LOQ=0.050ng/g). The method was effective for the detection and quantification of AFB1 residues in broiler liver and could also be potentially used for detecting AFB1 in other edible animal tissues after natural or experimental AFB1 exposure with high sensitivity and precision.

Pediococcus acidolactici and Pediococcus pentosaceus isolated from a rainbow trout ecosystem have probiotic and ABF1 adsorbing/degrading abilities in vitro.

Probiotics are being used in biological control of bacterial pathogens, as an alternative to antibiotics, to improve health and production parameters in fish farming. Fish farming production is severely affected by aflatoxins (AFs), which are a significant problem in aquaculture systems. Aflatoxins exert substantial impact on production, causing disease with high mortality and a gradual decline of reared fish stock quality. Some aspects of aflatoxicosis in fish, particularly its effects on the gastrointestinal tract, have not been well documented. The aim of the present study was to evaluate probiotic properties of lactic acid bacterial (LAB) strains isolated from rainbow trout intestine and feed. Moreover, AFB1-binding and/or degrading abilities were also evaluated to assess their use in the formulation of feed additives. Growth at pH 2, the ability to co-aggregate with bacterial pathogens, inhibition of bacterial pathogens, and determination of the inhibitory mechanism were tested. Aflatoxin B1 (AFB1) adsorption and degradation ability were also tested. All strains were able to maintain viable (107 cells ml-1) at pH 2. Pediococcus acidilactici RC001 and RC008 showed the strongest antimicrobial activity, inhibiting all the pathogens tested. The strains produced antimicrobial compounds of different nature, being affected by different treatments (catalase, NaOH and heating), which indicated that they could be H2O2, organic acids or proteins. All LAB strains tested showed the ability to coaggregate pathogenic bacteria, showing inhibition percentages above 40%. Pediococcus acidilactici RC003 was the one with the highest adsorption capacity and all LAB strains were able to degrade AFB1 with percentages higher than 15%, showing significant differences with respect to the control. The ability of some of the LAB strains isolated in the present work to compete with pathogens, together with stability against bile and gastric pH, reduction of bioavailability and degradation of AFB1, may indicate the potential of LAB for use in rainbow trout culture.

Encapsulated whey-native yeast Kluyveromyces marxianus as a feed additive for animal production.

Whey is the main byproduct of the cheese industry. While the composition is variable, it retains up to 55% of milk nutrients. The beneficial features of whey indicates a promising source of new potentially probiotic strains for the development of food additives destined for animal production. The aim of this study was to identify Kluyveromyces spp. isolated from whey, to study some probiotic properties and to select the best strain to be encapsulated using derivatised chitosan. Kluyveromyces marxianus strains (VM003, VM004 and VM005) were isolated from whey and identified by phenotypic and molecular techniques. These three yeast strains were able to survive under gastrointestinal conditions. Moreover, they exhibited weak auto-aggregation and co-aggregation with pathogenic bacteria (Salmonella sp., Serratia sp., Escherichia coli and Salmonella typhimurium). In general the K. marxianus strains had a strong antimicrobial activity against pathogenic bacteria. The potential probiotic K. marxianus VM004 strain was selected for derivatised-chitosan encapsulation. Material treated with native chitosan exhibited a strong antimicrobial activity of K. marxianus, showing a total growth inhibition at 10 min exposure. However, derivatised-chitosan encapsulation showed a reduced antimicrobial activity. This is the first study to show some probiotic properties of whey-native K. marxianus, in vitro. An encapsulation strategy was applied using derivatised chitosan.