Fernando Bittencourt Luciano

In vitro antifungal activity of allyl isothiocyanate (AITC) against Aspergillus parasiticus and Penicillium expansum and evaluation of the AITC estimated daily intake

Isothiocyanates (ITCs) are natural compounds derived from cruciferous vegetables produced by enzymatic conversion of metabolites called glucosinolates. They are potentially useful antimicrobial compounds for food applications have been shown to be promising agents against cancer in human cell culture, animal models, and in epidemiological studies. In this study, the antifungal activity of the allyl isothiocyanate (AITC) was evaluated on two mycotoxigenic fungi as Aspergillus parasiticus and Penicillium expnsum, aflatoxins (AFs) and patulin (PAT) producers, employing an assay on solid medium. Also an approximation of the risk evaluation associated to the intake of food treated with the AITC to reduce the risk of fungi spoilage has been evaluated. On solid medium and after 20 days incubation the strain of Penicillium expansum was inhibited with AITC quantities highest than 50 mg, whereas the strain of A. parasiticus was sensible to AITC doses highest than 5 mg. The analysis of the risk assessment associated to the intake of several food classes treated with the bioactive compound AITC to prevent fungi spoilage evidenced that this product can be considered as safe due that the estimated daily intakes (EDIs) are always lower than the AITC Admissible Daily intake (ADI).

Influence of the antimicrobial compound allyl isothiocyanate against the Aspergillus parasiticus growth and its aflatoxins production in pizza crust

Aflatoxins (AFs) are secondary metabolites produced by different species of Aspergillus, such as Aspergillus flavus and Aspergillus parasiticus, which possess mutagenic, teratogenic and carcinogenic activities in humans. In this study, active packaging devices containing allyl isothiocyanate (AITC) or oriental mustard flour (OMF) + water were tested to inhibit the growth of A. parasiticus and AFs production in fresh pizza crust after 30 d. The antimicrobial and anti-aflatoxin activities were compared to a control group (no antimicrobial treatment) and to a group added with commercial preservatives (sorbic acid + sodium propionate). A. parasiticus growth was only inhibited after 30 d by AITC in filter paper at 5 μL/L and 10 μL/L, AITC sachet at 5 μL/L and 10 μL/L and OMF sachet at 850 mg + 850 μL of water. However, AFs production was inhibited by all antimicrobial treatments in a dose-dependent manner. More importantly, AITC in a filter paper at 10 μL/L, AITC sachet at 10 μL/L, OMF sachet at 850 mg + 850 μL of water and sorbic acid + sodium propionate at 0.5-2.0 g/Kg completely inhibited AFs formation. The use of AITC in active packaging devices could be a natural alternative to avoid the growth of mycotoxinogenic fungi in refrigerated bakery products in substitution of common commercial preservatives.

Occurrence, toxicity, bioaccessibility and mitigation strategies of beauvericin, a minor Fusarium mycotoxin

Emerging Fusarium mycotoxins include the toxic secondary metabolites fusaproliferin, enniatins, beauvericin (BEA), and moniliform. BEA is produced by some entomo- and phytopathogenic Fusarium species and occurs naturally on corn and corn-based foods and feeds infected by Fusarium spp. BEA has shown various biological activities (antibacterial, antifungal, and insecticidal) and possesses toxic activity, including the induction of apoptosis, increase cytoplasmic calcium concentration and lead to DNA fragmentation in mammalian cell lines. Cereals food processing has an important effect on mycotoxin stability, leading to less-contaminated food compared to the raw materials. Different industrial processes have shown to be effective practices to reduce BEA contents due to thermal food processing applied, such as cooking, boiling, baking, frying, roasting and pasteurization. Some studies demonstrated the capacity of lactic acid bacteria to reduce the presence of the BEA in model solution and in food chain through fermentation processes, modifying this mycotoxin in a less toxic derivate. Prebiotic and probiotic ingredient can modulate the bioaccessibility of BEA reducing the risk of intake of this minor Fusarium mycotoxin. This review summarizes the existing data on occurrence, toxicity and especially on BEA reduction strategies in food and feed such as chemical reduction, biocontrol and food processing.

A chemical approach for the reduction of beauvericin in a solution model and in food systems.

Beauvericin (BEA) is a bioactive compound produced by the secondary metabolism of several Fusarium strains with a strong antibacterial, antifungal, and insecticidal activities. This study evaluated the reduction of BEA added at 25 mg/kg in phosphate buffer saline (PBS) solutions at pH of 4, 7 and 10, or to different cereal products (kernels and flours) by the bioactive compounds phenyl isothiocyanate (PITC) and benzyl isothiocyanate (BITC). The concentration of the mycotoxin was evaluated using liquid chromatography coupled to the diode array detector (LC-DAD). In solution, BEA reduction ranged from 9% to 94% on a time-dependent fashion and lower pH levels resulted in higher BEA reduction. Cereal kernels and flours treated with gaseous PITC and BITC (50, 100 and 500 μL/L) presented BEA reduction from 9% to 97% and was dose-dependent. Among the crops, corn was the vehicle where BEA was mostly affected by the action of the ITCs, followed by wheat and rice, and lastly barley. Overall, PITC caused higher reduction of BEA and should be chosen over BITC as a fumigant to decrease the levels of this mycotoxin in grains and flours.

Antimicrobial packaging based on ɛ-polylysine bioactive film for the control of mycotoxigenic fungi in vitro and in bread

Abstract ɛ‐Poly‐l‐lysine (ɛ‐PL) is a cationic peptide with a broad‐spectrum antimicrobial activity. This study investigates the use of ɛ‐PL as natural antimicrobial to inhibit fungal growth and to reduce aflatoxins (AFs) production. Antifungal activity of starch biofilms with different concentrations of ɛ‐Poly‐l‐lysine (ɛ‐PL) was determined in solid medium against Aspergillus parasiticus (AFs producer) and Penicillium expansum. Then, biofilms were tested as antimicrobial devices for the preservation of bread loaf inoculated with A. parasiticus CECT 2681 and P. expansum CECT 2278. Shelf life and AFs content were examined. Biofilms with concentrations of ɛ‐PL less than 1.6 mg/cm2 showed no fungal growth inhibition in solid medium, while the antifungal activity of the films with greater than 1.6 mg/cm2 of ɛ‐PL was dose dependent. The shelf life of bread inoculated with A. parasiticus was increased by 1 day with the use of films containing 1.6–6.5 mg ɛ‐PL/cm2, while shelf life of bread tainted with P. expansum was increased by 3 day with 6.5 mg ɛ‐PL/cm2. AFs production was greatly inhibited by ɛ‐PL biofilms (93–100%). Thus, ɛ‐PL biofilms could be potentially used as antimicrobial device during bread storage as a natural alternative to the synthetic preservatives. Practical applications Ɛ‐Polylysin is a natural substance from microbial metabolism. Polylysine has a function to prevent a microbe from proliferating by ionic adsorption in the microbe. ɛ‐polylysine has a wide antibacterial spectrum and has an obvious lethal effect on Gram‐positive and Gram‐negative bacteria, yeast, mold, viruses, etc. It has a good antibacterial effect on the Gram‐negative bacteria E. coli and Salmonellae, which are difficult to control with other natural preservatives. ɛ‐Polylysine has already been used generally as a food additive in Japan, Korea and other part of world. In the United States, FDA has recognized the polylysine as a GRAS material. Considered the positive results obtained in the study, this compound could be used for the production of antimicrobial biofilms, applied as separator slices in the loaf bread production, to prevent the growth of the mycotoxigenic fungi A. parasiticus and P. expansum, contributing to reduce the use of the synthetically preservatives in bakery industry and also of the negative impact that these compounds could generate on the health of the end users.

Bioaccessibility of glucoraphanin from broccoli using an in vitro gastrointestinal digestion model

This study investigated the effect of cooking methods on the degradation of glucoraphanin (GR) in broccoli and the bioaccessibility of this compound through simulated gastrointestinal digestion. Broccoli was cooked using three different techniques: boiling, steaming and microwave cooking. Then, GR was extracted and quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In addition, the cooked samples were added to a system that simulates the digestion characteristics of the mouth, stomach and duodenum. Samples were drawn before and after the digestion, and GR bioaccessibility was calculated. GR losses were higher when broccoli was boiled (47.03%), followed by steaming (31.96%) and microwaving (11.87%). However, steamed broccoli presented the highest bioaccessibility (94.59%), followed by boiled (89.69%) and microwaved (60.88%). Taking into consideration the levels of remaining GR after cooking and its bioaccessibility after simulated digestion, steaming can be considered the best method and may result in the highest levels of this bioactive glucosinolate after broccoli intake.

Rhamnogalacturonan, a chemically-defined polysaccharide, improves intestinal barrier function in DSS-induced colitis in mice and human Caco-2 cells

Natural polysaccharides have emerged as an important class of bioactive compounds due their beneficial biological effects. Here we investigated the protective and healing effects of rhamnogalacturonan (RGal) isolated from Acmella oleracea (L.) R.K. Jansen leaves in an experimental model of intestinal inflammation in mice and in heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2). The findings demonstrated that RGal treatment for 7 days reduced the severity of DSS-induced colitis by protecting mice from weight loss, macroscopic damage and reduction of colon length. When compared to the DSS group, RGal also protected the colon epithelium and promoted the maintenance of mucosal enterocytes and mucus secreting goblet cells, in addition to conserving collagen homeostasis and increasing cell proliferation. In an in vitro barrier function assay, RGal reduced the cellular permeability after exposure to IL-1β, while decreasing IL-8 secretion and claudin-1 expression and preserving the distribution of occludin. Furthermore, we also observed that RGal accelerated the wound healing in Caco-2 epithelial cell line. In conclusion, RGal ameliorates intestinal barrier function in vivo and in vitro and may represent an attractive and promising molecule for the therapeutic management of ulcerative colitis.

Avaliação in vitro de atividade antimicrobiana de peptídeos anfipáticos derivados de proteínas do soro de leite

O objetivo deste estudo foi avaliar, in vitro, a atividade antimicrobiana de peptideos anfipaticos derivados de proteinas do soro de leite contra importantes microrganismos patogenicos e toxigenicos. Para isto, foram testados dois peptideos de 16 e 6 aminoacidos, de sequencias HQPHQPLPPTVMFPPQ e KIPAVF respectivamente, frente as bacterias Salmonella Typhimurium, Staphylococcus aureus , Listeria monocytogenes e Escherichia coli , e frente aos fungos Aspergillus parasiticus , Fusarium verticillioides e Fusarium graminearum . Buscou-se determinar as concentracoes inibitorias minimas (CIMs) dos dois peptideos contra os microrganismos utilizando o metodo de microdiluicao em placas de 96 pocos. Foram testadas doses de ate 5000 mg.L-1 de cada peptideo, contudo, nao foi possivel determinar suas CIMs frente aos patogenos e contaminantes testados. Analisando trabalhos similares com peptideos e/ou lisados proteicos de soro de leite, percebe-se uma variacao muito grande entre os resultados. Isso demonstra a divergencia de bioatividade de peptideos com diferentes sequencias, alem das possiveis diferentes respostas celulares a estas moleculas, tais como metabolizacao e alteracoes nao-letais. Assim, conclui-se que os peptideos aqui testados nao sao bons candidatos para aplicacao na industria de alimentos. Doses mais elevadas dos peptideos nao foram testadas, pois inviabilizaria sua aplicacao em alimentos devido ao alto custo de obtencao.

Aflatoxins and A. flavus Reduction in Loaf Bread through the Use of Natural Ingredients

In this study, the antifungal activity of yellow mustard (YMF) and oriental mustard (OMF) meal extracts against 14 strains of fungi was tested on a solid medium. The results obtained with the YMF were next confirmed in liquid medium determining the minimum inhibitory concentration (MIC) and the minimum fungicide concentration (MFC). Finally, the use of YMF as a natural preservative to extend the useful life of bread was evaluated. Breads with different concentrations of YMF (2, 4, 6 and 8 g/kg) were prepared and contaminated with Aspergillus flavus ISPA 8111 and Penicillium nordicum CECT 2320. For 10 days the formation of mycelium was observed, and after that the fungal growth and the mycotoxins production was determined. The results obtained with the YMF were compared with breads treated with the commercial additive sodium propionate (E-281). The results showed a significant reduction of the fungal population using 6 g/kg and 8 g/kg of YMF in bread contaminated with A. flavus and with P. nordicum and an extensions of the breads shelf life of 7 and 5 days, respectively, in comparison with the control experiment. A reduction of 78% of AFB1 was observed using 6 g/kg of YMF while no AFB1 production was detected employing 8 g/kg of YMF in bread preparation.

Combination of garlic essential oil, allyl isothiocyanate, and nisin Z as bio-preservatives in fresh sausage

The effects of natural antimicrobial compounds (garlic essential oil [GO], allyl isothiocyanate [AITC], and nisin Z [NI]) on microbiological, physicochemical and sensory characteristics of fresh sausage were assessed. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) towards Escherichia coli O157:H7 and Lactobacillus plantarum were determined in vitro. Sausages inoculated with E. coli O157:H7, were treated with different combinations of antimicrobials and assessed for microbiological and physicochemical parameters during storage (6C for 20 d). Treatments that presented the greatest antimicrobial effects were subjected to sensory evaluation. Combinations of 20 mg/kg NI + 125 μL/kg GO + 62.5 μL/kg AITC or 20 mg/kg NI + 62.5 μL/kg GO + 125 μL/kg AITC were effective in reducing E. coli O157H7 and spoilage lactic acid bacteria, and maintained the physicochemical characteristics of fresh sausage. Combinations of NI, GO and AITC were effective to improve the safety and the shelf life of fresh sausage, with no impact on its sensory acceptance.