Efficacy of Two Commercially Available Adsorbents to Reduce the Combined Toxic Effects of Dietary Aflatoxins, Fumonisins, and Zearalenone and Their Residues in the Tissues of Weaned Pigs.

Mycotoxins present a significant health concern within the animal-feed industry, with profound implications for the pig-farming sector. The objective of this study was to evaluate the efficacy of two commercial adsorbents, an organically modified clinoptilolite (OMC) and a multicomponent mycotoxin detoxifying agent (MMDA), to ameliorate the combined adverse effects of dietary aflatoxins (AFs: sum of AFB1, AFB2, AFG1, and AFG2), fumonisins (FBs), and zearalenone (ZEN) at levels of nearly 0.5, 1.0, and 1.0 mg/kg, on a cohort of cross-bred female pigs (N = 24). Pigs were randomly allocated into six experimental groups (control, mycotoxins (MTX) alone, MTX + OMC 1.5 kg/ton, MTX + OMC 3.0 kg/ton, MTX + MMDA 1.5 kg/ton, and MTX + MMDA 3.0 kg/ton), each consisting of four individuals, and subjected to a dietary regimen spanning 42 days. The administration of combined AFs, FBs, and ZEN reduced the body-weight gain and increased the relative weight of the liver, while there was no negative influence observed on the serum biochemistry of animals. The supplementation of OMC and MMDA ameliorated the toxic effects, as observed in organ histology, and provided a notable reduction in residual AFs, FBs, and ZEN levels in the liver and kidneys. Moreover, the OMC supplementation was able to reduce the initiation of liver carcinogenesis without any hepatotoxic side effects. These findings demonstrate that the use of OMC and MMDA effectively mitigated the adverse effects of dietary AFs, FBs, and ZEN in piglets. Further studies should explore the long-term protective effects of the studied adsorbent supplementation to optimize mycotoxin management strategies in pig-farming operations.

Incidence and Levels of Aflatoxin M1 in Artisanal and Manufactured Cheese in Pernambuco State, Brazil.

Cheese is one of the most susceptible dairy foods to accumulating aflatoxins due to their high affinity to caseins. The consumption of cheese contaminated with high levels of aflatoxin M1 (AFM1) can be highly harmful to humans. The present work, based on high-performance liquid chromatography (HPLC), highlights the frequency and levels of AFM1 in coalho and mozzarella cheese samples (n = 28) from the main cheese-processing plants in Araripe Sertão and Agreste in the state of Pernambuco, Brazil. Of the evaluated cheeses, 14 samples were artisanal cheeses and the remaining 14 were industrial (manufactured) cheeses. All samples (100%) had detectable levels of AFM1, with concentrations ranging from 0.026 to 0.132 µg/kg. Higher levels (p < 0.05) of AFM1 were observed in artisanal mozzarella cheeses, but none of the cheese samples exceed the maximum permissible limits (MPLs) of 2.5 µg/kg established for AFM1 in cheese in Brazil and 0.25 µg/kg in the European countries by the European Union (EU). The high incidence of low levels of AFM1 found in the evaluated cheeses underscores the need for stringent control measures to prevent this mycotoxin in milk used for cheese production in the study area, with the aim of protecting public health and reducing significant economic losses for producers.

Effect of Lactic Acid Bacteria Strains on the Growth and Aflatoxin Production Potential of Aspergillus parasiticus, and Their Ability to Bind Aflatoxin B1, Ochratoxin A, and Zearalenone in vitro.

The increased consumption of plant-based foods has intensified the concern related to mycotoxin intoxication. This study aimed to investigate the effect of selected lactic acid bacteria (LAB) strains on the growth of Aspergillus parasiticus NRRL 2999 and its production of aflatoxin (AF). The ability of the heat-killed (100°C for 1 h) LAB strains to bind aflatoxin M1 (AFM1) in milk and aflatoxin B1 (AFB1), ochratoxin A (OTA), and zearalenone (ZEN) in potassium phosphate buffer (PPB) was also evaluated in vitro. Ten LAB strains were tested individually, by inoculating them simultaneously with the fungus or after incubation of the fungus for 24 or 48 h at 25°C. Double layer yeast extract sucrose (YES) agar, de Man Rogosa and Sharpe (MRS) agar, and YES broth were incubated for 7 days at 25°C to follow the development of the fungus. Levilactobacillus spp. 3QB398 and Levilactobacillus brevis 2QB422 strains were able to delay the growth of A. parasiticus in YES broth, even when these strains were inoculated 24 h after the fungus. The inhibitory effect of these LAB strains was confirmed by the reduction of fungus colony size, suggesting dominance of LAB by competition (a Lotka-Voltera effect). The production of AFB1 by A. parasiticus was inhibited when the fungus was inoculated simultaneously with Lactiplantibacillus plantarum 3QB361 or L. plantarum 3QB350. No AFB1 was found when Levilactobacillus spp. 2QB383 was present, even when the LAB was inoculated 48 h after the fungus. In binding studies, seven inactivated LAB strains were able to promote a reduction of at least 50% the level of AFB1, OTA, and ZEN. This reduction varied depending on the pH of the PPB. In milk, however, only two inactivated LAB strains were able to reduce AFM1, with a reduction of 33 and 45% for Levilactobacillus spp. 3QB398 (Levilactobacillus spp.) and L. brevis 2QB422, respectively. Nevertheless, these results clearly indicate the potential of using LAB for mycotoxin reduction.

Metataxonomics contributes to unravel the microbiota of a Brazilian dairy.

For this research communication, 90 samples of a Brazilian dairy were combined into four groups (raw material, final product, food-contact and non-food contact surfaces) and analyzed by metataxonomics based on 16S rRNA gene sequencing. The results showed high alpha-diversity indexes for final product and non-food contact surfaces but, overall, beta-diversity indexes were low. The samples were separated in two main clusters, and the core microbiota was composed by Macrococcus, Alkaliphilus, Vagococcus, Lactobacillus, Marinilactibacillus, Streptococcus, Lysinibacillus, Staphylococcus, Clostridium, Halomonas, Lactococcus, Enterococcus, Bacillus and Psychrobacter. These results highlight that rare taxa occur in dairies, and this may aid the development of strategies for food protection.

Aflatoxin M1 absorption by non-viable cells of lactic acid bacteria and Saccharomyces cerevisiae strains in Frescal cheese.

The purpose of this study was to evaluate the ability of heat-killed cells (121 °C, 10 min) from two strains of lactic acid bacteria (LAB) (Lactobacillus rhamnosus and Lactococcus lactis) and one strain of yeast (Saccharomyces cerevisiae), alone or in combination, to reduce the levels of aflatoxin M1 (AFM1) in Frescal cheese during 30 days of storage. The experimental design was totally randomized, in a 2 × 2 × 2 factorial arrangement, corresponding to two levels of LAB (0 and L. rhamnosus at 1010 cells/kg + L. lactis at 1010 cells/kg), two levels of S. cerevisiae in milk (0 and 1010 yeast cells/kg) and two AFM1 levels (0 and 0.5 µg/kg) added to the cheese curd, totaling 8 treatments with three replicates per treatment. AFM1 levels in Frescal cheese were evaluated by using a high-performance liquid chromatography. Cheese fat and protein contents were not affected (P > 0.05) by any of the treatments, and only pH decreased (P < 0.05) in all treatments from days 2 to 30 of storage (usual shelf life of this type of cheese). AFM1 levels detected in contaminated cheeses decreased on day 2 of storage, varying from 0.09 µg/kg (cheese with addition of bacterial cells) to 0.29 µg/kg (no addition of LAB or yeast cells), this may have occurred due to loss of AFM1 in the Frescal cheese whey. The concentrations of detected AFM1 decreased (P < 0.05) in all treatments from days 2 to 10 of storage, and the maximum percentage reduction of the detectable levels (100%) was achieved after 10 and 20 days of storage in cheeses containing LAB and yeast cells, or prepared with yeast cells alone, respectively. The addition of heat-killed LAB (cells of L. rhamnosus and L. lactis) and Saccharomyces cerevisiae alone or in combination, has a potential ability for adsorbing the AFM1 in Frescal cheese during 30 days of storage.

Aflatoxins and fumonisins in feed from a broiler operation system from São Paulo state, Brazil / Aflatoxinas e fumonisinas em rações de um sistema de produção de frangos de corte no estado de São Paulo, Brasil

ABSTRACT: The aim of the present study was to assess the occurrence of aflatoxins (AFs) and fumonisins (FBs) in feed ingredients (corn and soybean meal) and finishing feed in a broiler operation system, as well was to evaluate their effect on the productivity of 20 batches of broilers produced and the histology status of broilers' liver after slaughter. Corn samples presented the highest frequencies of AFs and FBs, at mean levels of 29.1 and 2,100µg/kg, respectively. Soybean samples presented mean levels of 1.5 and 70µg/kg for AFs and FBs, respectively. Batches of broilers receiving feed containing FB levels higher than 1,000µg/kg had lower weight gain and higher mortality rates, while those fed rations with AFs equal or above the limit of quantification (LOQ) of the analytical method presented higher scores of histological changes in the liver. A dilution effect was observed for AFs and FBs from ingredients, especially corn, to feed during manufacture, whilst not enough to prevent losses in productivity. Results of this trial highlighted the need for strict control of mycotoxins in corn intended for broilers.

RESUMO: O objetivo do presente trabalho foi verificar a ocorrência de aflatoxinas (AFs) e fumonisinas (FBs) em ingredientes (milho e farelo de soja) e na ração de abate sobre a produtividade de uma empresa integradora de frangos de corte, bem como avaliar seus efeitos sobre produtividade de 20 lotes de frangos produzidos pela empresa e a histologia dos fígados dos frangos após o abate. As amostras de milho apresentaram as maiores frequências de AFs e FBs, em concentrações médias de 29,1 e 2.100µg/kg, respectivamente. As amostras de farelo de soja apresentaram níveis médios de 1,5 e 70µg/kg para AFs e FBs, respectivamente. Os lotes de aves que receberam ração contendo níveis de FBs maiores que 1,000µg/kg apresentaram menor ganho de peso e maior percentual de mortalidade, enquanto que as que receberam ração com AFs iguais ou superiores ao limite de quantificação (LQ) do método analítico apresentaram maior grau de alteração histopatológica no fígado. Houve efeito de diluição de AFs e FBs dos ingredientes, especialmente o milho, à ração no processo de fabricação, porém não suficiente para evitar perdas na produtividade. Os resultados do estudo reforçam a necessidade do controle estrito de micotoxinas no milho destinado à alimentação de frangos de corte.

Behavior of Foodborne Pathogens Listeria monocytogenes and Staphylococcus aureus in Mixed-Species Biofilms Exposed to Biocides.

In nature and man-made environments, microorganisms reside in mixed-species biofilms, in which the growth and metabolism of an organism are different from these behaviors in single-species biofilms. Pathogenic microorganisms may be protected against adverse treatments in mixed-species biofilms, leading to health risk for humans. Here, we developed two mixed five-species biofilms that included one or the other of the foodborne pathogens Listeria monocytogenes and Staphylococcus aureus The five species, including the pathogen, were isolated from a single food-processing environmental sample, thus mimicking the environmental community. In mature mixed five-species biofilms on stainless steel, the two pathogens remained at a constant level of ∼105 CFU/cm2 The mixed five-species biofilms as well as the pathogens in monospecies biofilms were exposed to biocides to determine any pathogen-protective effect of the mixed biofilm. Both pathogens and their associate microbial communities were reduced by peracetic acid treatments. S. aureus decreased by 4.6 log cycles in monospecies biofilms, but the pathogen was protected in the five-species biofilm and decreased by only 1.1 log cycles. Sessile cells of L. monocytogenes were affected to the same extent when in a monobiofilm or as a member of the mixed-species biofilm, decreasing by 3 log cycles when exposed to 0.0375% peracetic acid. When the pathogen was exchanged in each associated microbial community, S. aureus was eradicated, while there was no significant effect of the biocide on L. monocytogenes or the mixed community. This indicates that particular members or associations in the community offered the protective effect. Further studies are needed to clarify the mechanisms of biocide protection and to identify the species playing the protective role in microbial communities of biofilms.IMPORTANCE This study demonstrates that foodborne pathogens can be established in mixed-species biofilms and that this can protect them from biocide action. The protection is not due to specific characteristics of the pathogen, here S. aureus and L. monocytogenes, but likely caused by specific members or associations in the mixed-species biofilm. Biocide treatment and resistance are a challenge for many industries, and biocide efficacy should be tested on microorganisms growing in biofilms, preferably mixed systems, mimicking the application environment.

Listeria monocytogenes incidence changes and diversity in some Brazilian dairy industries and retail products.

Listeria monocytogenes can cause listeriosis, a severe foodborne disease. In Brazil, despite very few reported cases of listeriosis, the pathogen has been repeatedly isolated from dairies. This has led the government to implement specific legislation to reduce the hazard. Here, we determined the incidence of L. monocytogenes in five dairies and retail products in the Southeast and Midwest regions of Brazil over eight months. Of 437 samples, three samples (0.7%) from retail and only one sample (0.2%) from the dairies were positive for L. monocytogenes. Thus, the contamination rate was significantly reduced as compared to previous studies. MultiLocus Sequence Typing (MLST) was used to determine if contamination was caused by new or persistent clones leading to the first MLST profile of L. monocytogenes from the Brazilian dairy industry. The processing environment isolate is of concern being a sequence-type (ST) 2, belonging to the lineage I responsible for the majority of listeriosis outbreaks. Also, ST3 and ST8 found in commercialized cheese have previously been reported in outbreaks. Despite the lower incidence, dairy products still pose a potential health risk and the occurrence of L. monocytogenes in dairies and retail products emphasize the need for continuous surveillance of this pathogen in the Brazilian dairy industry.

Biofilm-producing ability of Listeria monocytogenes isolates from Brazilian cheese processing plants.

The persistence of Listeria monocytogenes in food industry environments has been associated to the ability of specific isolates to produce biofilms. This study aimed to evaluate the biofilm production of 85 L. monocytogenes strains previously isolated from samples of cheese, brine and the environment of two cheese processing plants located in São Paulo, Brazil. The L. monocytogenes isolates belonged to serotypes 4b, 1/2b and 1/2c, yielded 30 different pulsotypes by pulsed-field gel electrophoresis (PFGE), and were submitted to biofilm-formation assays on polystyrene microplates and stainless steel coupons incubated statically at 35±0.5°C for 48h. All isolates from different sources showed ability to produce biofilms on polystyrene microplates, from which 21 (24.7%) also produced biofilms on stainless steel. Four isolates (4.7%) belonging to four different pulsotypes were classified as strong biofilms-producers on polystyrene microplates, while isolates belonging to four pulsotypes previously evaluated as persistent had weak or moderate ability to produce biofilms on polystyrene microplates. No relationship between the serotypes or pulsotypes and their biofilm-forming ability was observed. This study highlights the high variability in the biofilm production among L. monocytogenes strains collected from cheese and cheese-production environment, also indicating that strong biofilm-formation ability is not a key factor for persistence of specific isolates in cheese processing plants.

Determination of serum aflatoxin B1-lysine to evaluate the efficacy of an aflatoxin-adsorbing feed additive in pigs fed an aflatoxin B1-contaminated diet.

In this study, serum aflatoxin B1 (AFB1)-lysine was determined in order to evaluate the in vivo efficacy of a hydrated sodium calcium aluminosilicate (HSCAS) in pigs fed AFB1. Twenty-four 49-day-old crossbred barrows were maintained in individual cages and allowed ad libitum access to feed and water. A completely randomized design was used with six animals assigned to each of four dietary treatments for 21 days as follows: (A) basal diet (BD), (B) BD supplemented with 0.5 % HSCAS, (C) BD supplemented with 1.1 mg/kg AFB1, and (D) BD supplemented with 0.5 % HSCAS and 1.1 mg/kg AFB1. HSCAS was able to alleviate the toxic effects of AFB1 on pigs and reduce (P < 0.05) the levels of serum AFB1-lysine. Cumulative reductions of adduct yield values, calculated through the equation [(pg AFB1-lysine/mg albumin) / (µg AFB1/kg body weight)], were 53.0, 62.8, and 72.1 after 7, 14, and 21 days of oral exposure, respectively. AFB1-lysine has potential as an AFB1-specific biomarker for diagnostic purposes and for evaluating the efficacy of chemoprotective interventions in pigs.

Effect of peracetic acid on biofilms formed by Listeria monocytogenes strains isolated from a Brazilian cheese processing plant

ABSTRACT This study aimed to investigate the effect of peracetic acid (PAA, 0.5%) on adherent cells of three strains of Listeria monocytogenes strains belonging to serotypes 4b and 1/2b that had been previously isolated from the environment of a Brazilian cheese plant. The assays were conducted using polystyrene microplates and stainless steel coupons and the adhered cells were treated with PAA for 60, 120 and 180 s. On stainless steel, biofilms were partially inactivated by PAA after 60 s and almost 100% of the cells were damaged within 180 s using epifluorescence microscopy with LIVE/DEAD® staining. On polystyrene microplates, PAA decreased (P<0.05) biofilm biomass produced by the three L. monocytogenes isolates at 60 s, when compared with controls (no PAA treatment). However, PAA did not completely eliminate L. monocytogenes cells on polystyrene microplates (decreasing 1.8-2.5 log cycles after treatment with PAA for 180 s). The correct concentration and contact time of PAA is critical for eliminating biofilms formed by L. monocytogenes on stainless steel surfaces, although further studies are needed for defining efficient PAA treatments to remove adherent cells of this pathogen on plastic polymers

Relação entre níveis de fumonisina B1 e ácido fólico em farinha de milho e a concentração de ácido fólico no soro humano / Relationship between fumonisin B1 and folic acid levels in foods and the folic acid concentration in human serum

Neste trabalho foram determinados os níveis de ácido fólico e de fumonisina B1 (FB1) em farinha de milho consumida por 24 voluntários residentes em um campus universitário no estado de São Paulo, bem como sua relação com as concentrações de ácido fólico sérico nos indivíduos. As análises de ácido fólico e de FB1 em farinha de milho foram realizadas por cromatografia líquida de alta eficiência (CLAE), enquanto a determinação de ácido fólico sérico foi feita por kit de imunoensaio. Detectou-se a FB1 em 100% das amostras de farinha de milho, em níveis que variaram de 142 a 3.037 µg kg-1 (média: 738 ± 591 µg kg-1). As concentrações de ácido fólico nas amostras de farinha de milho ficaram entre < 0,3 µg kg-1 (limite de quantificação) e 1.705 µg kg-1, com média de 713 ± 435 µg kg-1, o que representa 47% do limite mínimo exigido pela Agência Nacional de Vigilância Sanitária (ANVISA) para farinhas de milho comercialmente disponíveis. Nas amostras de soro humano, os níveis de ácido fólico variaram de 6,7 a 24,0 ng mL-1 (média: 13,4 ± 5,4 ng mL-1). Não houve correlação (p < 0,05) entre os níveis de ácido fólico no soro dos indivíduos e as concentrações de FB1 ou ácido fólico nas amostras de farinha de milho. Outros estudos são necessários para estimar a ingestão total de FB1 por meio da dieta para averiguar os efeitos das fumonisinas sobre a absorção de ácido fólico nos indivíduos avaliados.(AU)

In the present study, folic acid and fumonisin B1 (FB1) levels were determined in corn flour consumed by 24 volunteers, residents in a university campus in São Paulo State, as well as its relationship with folic acid in serum of individuals. Analyses of folic acid and FB1 in corn flour were performed by high-performance liquid chromatography (HPLC), while the determination of folic acid in serum was accomplished using an immunoassay kit. FB1 was detected in 100% of corn samples, at levels ranging from 142 to 3,037 µg kg-1 (which means: 738 ± 591 µg kg-1). The concentrations of folic acid in corn flour samples ranged from < 0.3 µg kg-1 (limit of quantification) to 1,705 µg kg-1, with a mean of 713 ± 435 µg kg-1, which represents 47% of the minimum required by National Agency of Health Surveillance (ANVISA) for corn flour commercially available. The levels of folic acid in human serum samples ranged from 6.7 to 24.0 ng mL-1 (meaning: 13.4 ± 5.4 ng mL-1). No correlations were observed (p < 0.05) between the folic acid levels in serum of individuals and the concentrations of FB1 or folic acid in corn flour samples. Further studies are needed to estimate the total intake of FB1 in the diet to assess the effects fumonisins on the absorption of folic acid in the individuals evaluated.(AU)

Atividade de plasmina e plasminogênio no leite longa vida com alta e baixa contagem de células somáticas durante o armazenamento / Activity of plasmin and plasminogen in ultra high temperature milk with high and low somatic cell counts during storage

O objetivo deste estudo foi avaliar o efeito da contagem de células somáticas (CCS) do leite na atividade de plasmina e plasminogênio durante o período de armazenamento do leite longa vida integral. Os leites crus foram categorizados em grupos de CCS de baixa (342.000-487.000 células mL-1) e alta contagem (603.000-808.000 células mL-1). Dois lotes de leite longa vida em cada categoria de CCS foram analisados para determinação de plasmina e plasminogênio após 10, 30, 60, 90 e 120 dias de armazenamento em temperatura ambiente. Para a fabricação do leite longa vida, o leite cru foi submetido à pasteurização rápida seguida da esterilização industrial do leite por injeção de vapor pelo método direto e embalagem asséptica do produto. A CCS não apresentou efeitos sobre as características físico-químicas do leite cru, e nem sobre a atividade de plasmina e plasminogênio nos leites cru e longa vida, armazenados por 120 dias. Entretanto, independentemente da CCS, a atividade de plasmina e plasminogênio aumentou no leite longa vida ao longo do armazenamento, indicando a possibilidade de aumento da proteólise no produto durante sua vida de prateleira.

This study aimed to evaluate the effect of somatic cell counts (SCC) in milk on plasmin and plasminogen activities of ultra high temperature (UHT) milk during storage. Raw milks were categorized in SCC groups of low (342,000-487,000 cells mL-1) and high cells (603,000-808,000 cells mL-1). Two replicates of UHT milks within each SCC category were analyzed for plasmin and plasminogen activities after 10, 30, 60, 90 and 120 days of storage at room temperature. For manufacture of UHT milk, raw milk was pasteurized and sterilized by direct vapor injection process, followed by aseptic packaging. SCC had no effect on physical-chemical characteristics of raw milk, and on plasmin or plasminogen activities in raw and UHT milks during 120 days of storage. However, independently of the SCC in raw milk, the activity of plasmin and plasminogen increased in UHT milk during storage, hence indicating a possible increase in proteolysis in the product during its shelf-life.

Determinaçäo do número mínimo de amostragens mensais de leite, para pagamento por qualidade / Determination of minimum samples of milk to the payment by quality

Devido à total ausência de trabalhos relativos ao número de amostragens necessárias, o objetivo foi determinar o número mínimo para que o pagamento de leite aos produtores no Brasil seja adequado. Para isto, foram coletadas semanalmente amostras de leite do tanque, proveniente do rebanho de vacas leiteiras do Departamento de Produçäo Animal, da ESALQ/USP, por 27 meses, totalizando 134 amostras, que foram analisadas, no mesmo dia da coleta, pela Clínica do Leite, para verificar as concentraçöes de gordura, proteína e sólidos totais e a contagem de células somáticas (CCS). O desvio na média dos componentes do leite, em funçäo do tamanho da amostra, foi calculado a partir da variância amostral. O maior ganho na precisäo ocorre quando da coleta de 4-5 amostras mensais, considerando o cálculo da média por mês, bimestral ou trimestral. Entretanto, com base nas classes utilizadas pela indústria, seriam necessárias 13 amostras mensais para uma estimativa precisa da média mensal, 9 para a média bimestral e 7 para a média trimestral, considerando que a porcentagem de gordura é a maior limitante para o pagamento do leite.