Natural antimicrobials for control of Salmonella Enteritidis in feed and in vitro model of the chicken digestive process.

This study evaluated the antimicrobial effect of essential oils (EO) and organic acids (OA) against Salmonella Enteritidis in chicken feed and during an in vitro model that mimics the chicken digestive process. The minimal inhibitory concentration (MIC) of allyl isothiocyanate (AITC), carvacrol (CV), propionic acid (PROP) and caproic acid (CAP) were individually determined. Then, based on the MICs of each compound, combinations of EOs and/or OAs were tested to evaluate their synergic antimicrobial effect. The synergic effect of AITC and CAP was the most efficient against the bacterial strain tested. Commercial feed was inoculated with a 5-strain cocktail of S. Enteritidis and treated with different doses of AITC + CAP to evaluate their effect on the growth/survival of the pathogen. In addition, the simulated digestion model was used to access the antimicrobial effect of AITC + CAP added to the feed towards S. Enteritidis and Lactobacillus plantarum. Synergistic effect was found between AITC (0.065 mM) and CAP (17.5 mM) against S. Enteritidis in chicken feed, where S. Enteritidis was reduced to undetectable levels (<1.00 log CFU/g). AITC (1.95 mM) + CAP (45 mM) also decreased (p < 0.05) the population of S. Enteritidis in the simulated digestion, while the growth of L. plantarum was not affected. Therefore, the addition of AITC + CAP in feed might be a potential natural antimicrobial able to prevent economic losses caused for Salmonella in chicken.

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 Penicilliumnordicum 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.

Evaluation of gaseous allyl isothiocyanate against the growth of mycotoxigenic fungi and mycotoxin production in corn stored for 6 months.

BACKGROUND: Brazil produces approximately 63 million tons of corn kernels annually, which is commonly contaminated with fungi and mycotoxins. The objective of this study was to evaluate the efficacy of gaseous allyl isothiocyanate (AITC) to inhibit the growth of Aspergillus parasiticus and Fusarium verticillioides, and mycotoxin production (aflatoxins B1 , B2 , G1 and G2 , fumonisins B1 and B2 ) in corn during 180 days of storage. RESULTS: AITC at 50 µL L-1 resulted in a significant reduction of the fungal population (P < 0.05) after 180 days, decreasing 3.17 log(CFU g-1 ) and 3.9 log(CFU g-1 ) of A. parasiticus and F. verticillioides respectively in comparison with the control. In addition, 10 and 50 µL L-1 treatments prevented the production of fumonisin B1 for the whole period. Aflatoxins were not detected in either control or treated groups. Residual levels of AITC in corn treated with 10 µL L-1 and 50 µL L-1 were detected up to 14 days and 30 days respectively. CONCLUSION: Prophylactic treatment with AITC reduced the fungal population and inhibited fumonisin B1 production in stored corn, exhibiting great potential to be applied in corn silos to prevent fungi contamination and minimize mycotoxin levels. © 2018 Society of Chemical Industry.

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

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.

Fumigation of Brazil nuts with allyl isothiocyanate to inhibit the growth of Aspergillus parasiticus and aflatoxin production.

BACKGROUND: Brazil produces approximately 40 000 tons of Brazil nuts annually, which is commonly contaminated with fungi and mycotoxins. Gaseous allyl isothiocyanate (AITC) was used to inhibit the growth of Aspergillus parasiticus and its production of aflatoxins (AFs) in Brazil nuts. RESULTS: Nuts were inoculated with 104 spores g-1 of A. parasiticus and placed in airtight glass jars with controlled relative humidity (RH = 95 or 85%). Samples were treated with 0, 0.5, 1.0 or 2.5 µL L-1 of gaseous AITC and analyzed after 30 days to determine the fungal population and AFs content. Samples were also submitted to sensory evaluation. AITC at 2.5 µL L-1 could completely inhibit the fungal growth and AFs production in both the RH tested. AITC at 0.5 and 1 µL L-1 did not affect the microbial growth at RH = 95%, but 1 µL L-1 reduced the production of AFs by ∼50%. All AITC treatments reduced the fungal population and AFs to undetectable levels at RH = 85%. None of the concentrations altered sensory characteristics of Brazil nuts. CONCLUSION: Gaseous AITC could be used as an alternative to inhibit the growth of A. parasiticus during storage and transport of Brazil nuts. © 2017 Society of Chemical Industry.

Combination of essential oil compounds and phenolic acids against Escherichia coli O157:H7 in vitro and in dry-fermented sausage production.

Escherichia coli O157:H7 is a foodborne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. The low dose of infection and severity of the disease represent a concern to public health. Natural compounds have been widely applied as food additives to replace synthetic preservatives. The aim of this study was to determine the efficiency of essential oil compounds (EOCs) in combination with phenolic acids (PA) in vitro and in dry-fermented sausage production. Minimum Inhibitory Concentration (MIC) and Fractional Inhibitory Concentration index (FICindex) were determined for a 5-strain mixture of E. coli O157:H7. Batches of sausage tainted with E. coli O157:H7 were produced using Pediococcus pentosaceus UM 116P and Staphylococcus carnosus UM 123M as starter cultures. The best combination of EOCs and PAs found in vitro was used as an additive. Chemical-physical and microbiological analyses were evaluated weekly from day 0 to 35 after production. Sensory evaluation (texture, odor, flavor, appearance and overall evaluation) of E. coli-free sausages was conducted using a 9-point hedonic scale with 56 untrained volunteers. The MIC values of allyl isothiocyanate (AITC), carvacrol (CAR), ferulic acid (FA), o-coumaric acid (CA) and p-hydroxybenzoic acid (AHB) were, respectively, 0.25; 1.3; 5.12; 18.27; and 37mM. AITC combined with CA had a synergistic effect (FICindex=0.25) and together they were applied in the production of dry fermented sausage at concentrations of 10× FIC and 20× FIC. Aw had no significant difference among treatments, whereas the pH of 10× FIC and 20× FIC were higher than the control. E. coli O157:H7 was reduced by >5logCFU/g with 20× FIC after 21d, and by 2.8logCFU/g with 10× FIC after 35d. Sensory analysis showed that the combination of AITC and ο-coumaric acid in both treatments presented lower scores in the 5 categories when compared to the control, but none of the parameters received a negative score. This study demonstrated that the combination of AITC and ο-coumaric acid at 20× FIC reduced E. coli O157:H7 in compliance with the North American legislation, but adjustments in the dose are necessary to improve the sensory characteristics of the final product.

Microbial and chemical origins of the bactericidal activity of thermally treated yellow mustard powder toward Escherichia coli O157:H7 during dry sausage ripening.

Work examines the origin of bactericidal activity in mustard flour and explores the relative contribution from starter cultures, E. coli O157:H7 itself and other sources. Bacteria can degrade naturally occurring glucosinolates in mustard and form isothiocyanates with antimicrobial activity. In the present work, 24 starter cultures (mostly from commercial mixtures) were screened for their capacity to decompose the glucosinolate, sinalbin. The most active pair, Pediococcus pentosaceus UM 121P and Staphylococcus carnosus UM 123 M, were used together for the production of dry fermented sausage contaminated with E. coli O157:H7 (~6.5 log CFU/g). They were compared to industrial starters used previously (P. pentosaceus UM 116P and S. carnosus UM 109 M) for their reduction of E. coli O157:H7 viability. Sausage batches containing hot mustard powder (active myrosinase), cold mustard powder (inactivated myrosinase), autoclaved mustard powder (inactivated myrosinase) and no mustard flour (control) were prepared. Interestingly, both pairs of starter cultures yielded similar results. Elimination of E. coli O157:H7 (>5 log CFU/g) occurred after 31 days in the presence of hot flour and in 38 days when the cold flour was added. Reductions >5 log CFU/g of the pathogen did not occur (up to 38 days) in the control group. It was found that E. coli O157:H7 itself had a greater effect on sinalbin conversion than either pair of starter cultures, and glucosinolate degradation by the starter cultures was less important in determining E. coli survival. The autoclaved powder caused more rapid bactericidal action against E. coli O157:H7, yielding a >5 log CFU/g reduction in 18 days. This may have been a result of the formation and/or release of antimicrobial substances by the autoclave treatment. Autoclaved mustard powder could potentially solve an important challenge facing the meat industry as it strives to manufacture safe dry fermented sausages.

Enzymatic inhibition by allyl isothiocyanate and factors affecting its antimicrobial action against Escherichia coli O157:H7.

Allyl isothiocyanate (AIT) is derived from the glucosinolate sinigrin found in plants of the family Brassicaceae. It is a well-recognized antimicrobial agent against a variety of organisms, including foodborne pathogens such as Escherichia coli O157:H7. The efficiency of this natural agent in reducing E. coli O157:H7 numbers in food products have been reported. However, few have examined the mechanism by which AIT, and perhaps most of the isothiocyanates, kill E. coli O157:H7. In the present report, AIT showed greater antimicrobial activity at low pH values. For example, at pH 4.5 and 5.5 the MIC was 25 microL/L, while at pH 8.5, 500 microL/L was required to inhibit bacterial growth. This mustard-derived compound exhibited a high decomposition rate in water at 37 degrees C. Its degradation profile contained 3 major products and of these, diallylthiourea represented the largest ( approximately 80%) component. The decomposition products did not show antimicrobial activity towards E. coli O157:H7, even when combined with a sub-lethal dose of AIT (10 microL/L). AIT may only be antimicrobial in its original form and any further degradation in water is undesirable. AIT interactions with thioredoxin reductase and acetate kinase were also subjects of this study. AIT at 10 to 100 microL/L was able to significantly inhibit both enzymes, but only 1 microL/L was needed to decrease the activity of thioredoxin reductase. From these results, it can be postulated that: 1) AIT is a more effective antimicrobial at low pH values and its degradation reduces this activity; 2) decomposition products in water might not participate in the antimicrobial action of AIT; and 3) AIT seems to have a multi-targeted mechanism of action, perhaps inhibiting several metabolic pathways and damaging cellular structures.