Bacillus velezensis CL197: a zearalenone detoxifying strain isolated from wheat with potential to be used in animal production.

Zearalenone (ZEA) is a mycotoxin produced by Fusarium species, and cause contamination of food and feed, with impacts in animal production and in food production chain. Effective detoxifying methods, such as biodegradation, are therefore required. This study aimed to isolate microorganisms and screen ZEA detoxifying strains. As a result, 197 microorganisms were isolated, and six were initially selected after colorimetric screening. ZEA (1 µg/mL) was added to culture media, and after 24 h, all six microorganisms were able to degrade ZEA, without the formation of α-ZOL. One isolate eliminated ~ 99% of ZEA and was identified as Bacillus velezensis CL197. ZEA metabolites produced by the bacteria were evaluated, and no metabolites with greater or similar toxicity than ZEA were detected. This strain was applied to swine in vitro digestion, and up to 64% of ZEA was degraded. B. velezensis CL197 significantly degraded ZEA, demonstrating potential to be used as a detoxifying agent in the food production chain as a biocontrol agent.

Characterization of lactic acid bacteria isolated from human breast milk and their bioactive metabolites with potential application as a probiotic food supplement.

The probiotic properties of twenty-five lactic acid bacteria (LAB) isolated from human breast milk were investigated considering their resistance to gastrointestinal conditions and proteolytic activity. Seven LAB were identified and assessed for auto- and co-aggregation capacity, antibiotic resistance, and behavior during in vitro gastrointestinal digestion. Three Lacticaseibacillus strains were further evaluated for antifungal activity, metabolite production (HPLC-Q-TOF-MS/MS and GC-MS/MS) and proteolytic profiles (SDS-PAGE and HPLC-DAD) in fermented milk, whey, and soy beverage. All strains resisted in vitro gastrointestinal digestion with viable counts higher than 7.9 log10 CFU mL-1 after the colonic phase. Remarkable proteolytic activity was observed for 18/25 strains. Bacterial auto- and co-aggregation of 7 selected strains reached values up to 23 and 20%, respectively. L. rhamnosus B5H2, L. rhamnosus B9H2 and L. paracasei B10L2 inhibited P. verrucosum, F. verticillioides and F. graminearum fungal growth, highlighting L. rhamnosus B5H2. Several metabolites were identified, including antifungal compounds such as phenylacetic acid and 3-phenyllactic acid, and volatile organic compounds produced in fermented milk, whey, and soy beverage. SDS-PAGE demonstrated bacterial hydrolysis of the main milk (caseins) and soy (glycines and beta-conglycines) proteins, with no apparent hydrolysis of whey proteins. However, HPLC-DAD revealed alpha-lactoglobulin reduction up to 82% and 54% in milk and whey, respectively, with L. rhamnosus B5H2 showing the highest proteolytic activity. Overall, the three selected Lacticaseibacillus strains demonstrated probiotic capacity highlighting L. rhamnosus B5H2 with remarkable potential for generating bioactive metabolites and peptides which are capable of promoting human health.

Comprehensive Review of Aflatoxin and Ochratoxin A Dynamics: Emergence, Toxicological Impact, and Advanced Control Strategies.

Filamentous fungi exhibit remarkable adaptability to diverse substrates and can synthesize a plethora of secondary metabolites. These metabolites, produced in response to environmental stimuli, not only confer selective advantages but also encompass potentially deleterious mycotoxins. Mycotoxins, exemplified by those originating from Alternaria, Aspergillus, Penicillium, and Fusarium species, represent challenging hazards to both human and animal health, thus warranting stringent regulatory control. Despite regulatory frameworks, mycotoxin contamination remains a pressing global challenge, particularly within cereal-based matrices and their derived by-products, integral components of animal diets. Strategies aimed at mitigating mycotoxin contamination encompass multifaceted approaches, including biological control modalities, detoxification procedures, and innovative interventions like essential oils. However, hurdles persist, underscoring the imperative for innovative interventions. This review elucidated the prevalence, health ramifications, regulatory paradigms, and evolving preventive strategies about two prominent mycotoxins, aflatoxins and ochratoxin A. Furthermore, it explored the emergence of new fungal species, and biocontrol methods using lactic acid bacteria and essential mustard oil, emphasizing their efficacy in mitigating fungal spoilage and mycotoxin production. Through an integrative examination of these facets, this review endeavored to furnish a comprehensive understanding of the multifaceted challenges posed by mycotoxin contamination and the emergent strategies poised to ameliorate its impact on food and feed safety.

The Probiotic Potential and Metabolite Characterization of Bioprotective Bacillus and Streptomyces for Applications in Animal Production.

Probiotics are increasingly recognized for their potential in managing bacterial challenges in animal production. This study aimed to evaluate the probiotic potential of Bacillus and Streptomyces strains, specifically their bioprotective ability against Salmonella. In agar inhibition assays, these bacteria supported Salmonella-inhibition zones, ranging from 2.5 ± 0.5 to 6.3 ± 2.0 mm. Analyses of antimicrobial metabolites revealed their capacity to produce compounds with anti-Salmonella properties, except for Bacillus subtilis MLB2. When Salmonella was exposed to lyophilized metabolites, inhibition occurred in both liquid (at concentrations between 250 and 500 g/L) and solid cultures (at 500 g/L). To confirm their probiotic potential, the S. griseus and Bacillus strains underwent evaluations for antimicrobial resistance, bile salt tolerance, auto- and co-aggregation, pH resistance, and their ability to adhere to and inhibit Salmonella in Caco-2 cells. These assessments confirmed their probiotic potential. The probiotic strains were further encapsulated and subjected to simulated swine and poultry digestion. They demonstrated survival potential through the gastrointestinal tract and significantly reduced the Salmonella population. Thus, these strains exhibit considerable promise for producing biotechnological products aimed at controlling Salmonella in animal production. This approach ensures the health and hygiene of farming facilities, mitigates the spread of zoonotic bacteria, and contributes positively to public health.

Manufacture of a Potential Antifungal Ingredient Using Lactic Acid Bacteria from Dry-Cured Sausages.

The growing interest in functional foods has fueled the hunt for novel lactic acid bacteria (LAB) found in natural sources such as fermented foods. Thus, the aims of this study were to isolate, identify, characterize, and quantify LAB's antifungal activity and formulate an ingredient for meat product applications. The overlay method performed a logical initial screening by assessing isolated bacteria's antifungal activity in vitro. Next, the antifungal activity of the fermented bacteria-free supernatants (BFS) was evaluated by agar diffusion assay against six toxigenic fungi. Subsequently, the antifungal activity of the most antifungal BFS was quantified using the microdilution method in 96-well microplates. The meat broth that showed higher antifungal activity was selected to elaborate on an ingredient to be applied to meat products. Finally, antifungal compounds such as organic acids, phenolic acids, and volatile organic compounds were identified in the chosen-fermented meat broth. The most promising biological candidates belonged to the Lactiplantibacillus plantarum and Pediococcus pentosaceus. P. pentosaceus C15 distinguished from other bacteria by the production of antifungal compounds such as nonanoic acid and phenyl ethyl alcohol, as well as the higher production of lactic and acetic acid.

Bioactive Antimicrobial Peptides from Food Proteins: Perspectives and Challenges for Controlling Foodborne Pathogens.

Bioactive peptides (BAPs) derived from food proteins have been extensively studied for their health benefits, majorly exploring their potential use as nutraceuticals and functional food components. These peptides possess a range of beneficial properties, including antihypertensive, antioxidant, immunomodulatory, and antibacterial activities, and are naturally present within dietary protein sequences. To release food-grade antimicrobial peptides (AMPs), enzymatic protein hydrolysis or microbial fermentation, such as with lactic acid bacteria (LAB), can be employed. The activity of AMPs is influenced by various structural characteristics, including the amino acid composition, three-dimensional conformation, liquid charge, putative domains, and resulting hydrophobicity. This review discusses the synthesis of BAPs and AMPs, their potential for controlling foodborne pathogens, their mechanisms of action, and the challenges and prospects faced by the food industry. BAPs can regulate gut microbiota by promoting the growth of beneficial bacteria or by directly inhibiting pathogenic microorganisms. LAB-promoted hydrolysis of dietary proteins occurs naturally in both the matrix and the gastrointestinal tract. However, several obstacles must be overcome before BAPs can replace antimicrobials in food production. These include the high manufacturing costs of current technologies, limited in vivo and matrix data, and the difficulties associated with standardization and commercial-scale production.

Preparation of Sourdoughs Fermented with Isolated Lactic Acid Bacteria and Characterization of Their Antifungal Properties.

Traditional sourdough is obtained using a mixture of flour and water stored at room temperature until acidification. Therefore, adding lactic acid bacteria (LAB) can improve the quality and safety of sourdough bread. Faced with this problem, four drying techniques-freeze-drying, spray-drying, low-temperature drying, and drying at low humidity-have been applied. Our goals were to isolate LAB strains with antifungal potential against Aspergillus and Penicillium fungi. The antifungal capacity was evaluated with agar diffusion, co-culture in overlay agar, and a microdilution susceptibility assay. In addition, the antifungal compounds generated in sourdough were analyzed. As a result, dried sourdoughs were prepared with Lactiplantibacillus plantarum TN10, Lactiplantibacillus plantarum TF2, Pediococcus pentosaceus TF8, Pediococcus acidilactici TE4, and Pediococcus pentosaceus TI6. The minimum fungicidal concentrations ranged from 25 g/L versus P. verrucosum and 100 g/L against A. flavus. A total of 27 volatile organic compounds were produced. Moreover, the lactic acid content reached 26 g/kg of dry product, and the phenyllactic concentration was significantly higher than the control. The P. pentosaceus TI6 exhibited a higher antifungal capacity in vitro and demonstrated a higher production of antifungal compounds compared to the other strains; therefore, further studies will evaluate the impact of this sourdough in bread manufacture.

Bread Biopreservation through the Addition of Lactic Acid Bacteria in Sourdough.

Nowadays, the consumer seeks to replace synthetic preservatives with biopreservation methods, such as sourdough in bread. Lactic acid bacteria (LAB) are used as starter cultures in many food products. In this work, commercial yeast bread and sourdough breads were prepared as controls, as well as sourdough breads with L. plantarum 5L1 lyophilized. The impact of L. plantarum 5L1 on the properties of bread was studied. Antifungal compounds and the impact on the protein fraction by the different treatments in doughs and breads were also analyzed. In addition, the biopreservation capacity of the treatments in breads contaminated with fungi was studied and the mycotoxin content was analyzed. The results showed significant differences with respect to the controls in the properties of the bread and a higher total phenolic and lactic acid content in breads with higher amounts of L. plantarum 5L1. In addition, there was a higher content of alcohol and esters. Furthermore, adding this starter culture produced hydrolysis of the 50 kDa band proteins. Finally, the higher concentration of L. plantarum 5L1 delayed fungal growth and reduced the content of AFB1 and AFB2 compared to the control.

Use of Mustard Extracts Fermented by Lactic Acid Bacteria to Mitigate the Production of Fumonisin B1 and B2 by Fusarium verticillioides in Corn Ears.

Corn (Zea mays) is a worldwide crop subjected to infection by toxigenic fungi such as Fusarium verticillioides during the pre-harvest stage. Fusarium contamination can lead to the synthesis of highly toxic mycotoxins, such as Fumonisin B1 (FB1) and Fumonisin B2 (FB2), which compromises human and animal health. The work aimed to study the antifungal properties of fermented yellow and oriental mustard extracts using nine lactic acid bacteria (LAB) in vitro. Moreover, a chemical characterization of the main phenolic compounds and organic acids were carried out in the extracts. The results highlighted that the yellow mustard, fermented by Lactiplantibacillus plantarum strains, avoided the growth of Fusarium spp. in vitro, showing Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) values, ranging from 7.8 to 15.6 g/L and 15.6 to 31.3 g/L, respectively. Then, the lyophilized yellow mustard fermented extract by L. plantarum TR71 was applied through spray-on corn ears contaminated with F. verticillioides to study the antimycotoxigenic activity. After 14 days of incubation, the control contained 14.71 mg/kg of FB1, while the treatment reduced the content to 1.09 mg/kg (92.6% reduction). Moreover, no FB2 was observed in the treated samples. The chemical characterization showed that lactic acid, 3-phenyllactic acid, and benzoic acid were the antifungal metabolites quantified in higher concentrations in the yellow mustard fermented extract with L. plantarum TR71. The results obtained confirmed the potential application of fermented mustard extracts as a solution to reduce the incidence of mycotoxins in corn ears.

Application of White Mustard Bran and Flour on Bread as Natural Preservative Agents.

In this study, the antifungal activity of white mustard bran (MB), a by-product of mustard (Sinapis alba) milling, and white mustard seed flour (MF) was tested against mycotoxigenic fungi in the agar diffusion method. The results obtained were posteriorly confirmed in a quantitative test, determining the minimum concentration of extract that inhibits the fungal growth (MIC) and the minimum concentration with fungicidal activity (MFC). Since MF demonstrated no antifungal activity, the MB was stored under different temperature conditions and storage time to determine its antifungal stability. Finally, an in situ assay was carried out, applying the MB as a natural ingredient into the dough to avoid P. commune CECT 20767 growth and increase the bread shelf life. The results demonstrated that the antifungal activity of MB was dose-dependent. The higher assayed dose of MB (10 g/kg) reduced the fungal population in 4.20 Log CFU/g regarding the control group. Moreover, the shelf life was extended four days compared to the control, equaling its effectiveness with the synthetic preservative sodium propionate (E-281). Therefore, MB could be an alternative to chemical additives in bread formulations since it satisfies consumer requirements. Also, the formulation of bread with MB valorizes this by-product generated during mustard seed milling, thereby helping the industry move forward sustainably by reducing environmental impact.

Development of an Antifungal Device Based on Oriental Mustard Flour to Prevent Fungal Growth and Aflatoxin B1 Production in Almonds.

The present study describes the manufacture of an antifungal device composed of oriental mustard flour and hydroxyethyl-cellulose (H-OMF) and evaluates its efficacity in inhibiting Aspergillus flavus growth and aflatoxin B1 (AFB1) production in almonds. Additionally, it compares the H-OMF with allyl isothiocyanate (AITC) and a freeze-dried extract of yellow mustard flour (YMF-E); such substances were previously described as antifungal. Minimum inhibitory concentration (MIC), Minimum fungicidal concentration (MFC), the H-OMF in vitro antifungal activity, and the residual fungal population, as well as the production of AFB1 in almonds were determined. AITC and YMF-E showed significant antifungal activity in vitro. Additionally, the in vitro activity of H-OMF avoided mycelial growth by applying 30 mg/L. Almonds treated with AITC (5.07, 10.13, and 20.26 mg/L) and H-OMF (2000 and 4000 mg/L) showed a reduction in the population of A. flavus and the production of AFB1 to values below the limit of detection. YMF-E showed effectiveness by in vitro methodologies (MIC and MFC) but did not show efficacy when applied in almonds. Our findings indicated that the hydroxyethyl-cellulose-based device containing oriental mustard flour might be utilised as a fumigant to increase the safety of almonds and could be extended to other cereals or dry fruits.

Effect of allyl isothiocyanate on transcriptional profile, aflatoxin synthesis, and Aspergillus flavus growth.

The goals of this study were to determine the efficacy of allyl isothiocyanate (AITC) against the growth of A. flavus and Aflatoxin B1 (AFB1) production as well as to evaluate changes in the transcriptome profile when colonizing maize. A. flavus was inoculated in potato dextrose agar (PDA), the plates were placed inside glass jars and the mycelial growth (MG) was monitored for 7 d. Likewise, maize grains were contaminated with A. flavus in glass jars of 1 L and treated with 0.125, 0.25, 0.5, 1 and 5 µL of AITC. The moisture content (MC) of grains was 15 and 21%. After 7 days of storage, the MG was significantly reduced in doses higher than 0.125 µL/L of AITC. All doses of AITC reduced significantly the fungal growth and AFB1 production in maize after 30 d, regardless of MC. The transcriptional changes caused by AITC treatment showed significant overexpression for environmental and global transcription factors. These results suggest that AITC could be used as a fumigant to avoid the growth of A. flavus and the production of AFB1, moreover, confirm transcriptional alteration of genes involved in AFB1 and other processes key for normal fungal growth and development.

Development of an Antifungal and Antimycotoxigenic Device Containing Allyl Isothiocyanate for Silo Fumigation.

The aims of this study were to evaluate the antifungal activity of the bioactive compound allyl isothiocyanate (AITC) against Aspergillus flavus (8111 ISPA) aflatoxins (AFs) producer and Penicillium verrucosum (D-01847 VTT) ochratoxin A (OTA) producer on corn, barley, and wheat. The experiments were carried out initially in a simulated silo system for laboratory scale composed of glass jars (1 L). Barley and wheat were contaminated with P. verrucosum and corn with A. flavus. The cereals were treated with a hydroxyethylcellulose gel disk to which 500 µL/L of AITC were added; the silo system was closed and incubated for 30 days at 21 °C. After that, simulated silos of 100 L capacity were used. Barley, wheat, and corn were contaminated under the same conditions as the previous trial and treated with disks with 5 mL of AITC, closed and incubated for 90 days at 21 °C. In both cases, the control test did not receive any antifungal treatment. The growth of the inoculated fungi and the reduction in the formation of AFs and OTA were determined. In the lab scale silo system, complete inhibition of fungal growth at 30 days has been observed. In corn, the reduction of aflatoxin B1 (AFB1) was 98.5%. In the 100 L plastic drums, a significant reduction in the growth of A. flavus was observed, as well as the OTA formation in wheat (99.5%) and barley (92.0%).

Development of a Bioactive Sauce Based on Oriental Mustard Flour with Antifungal Properties for Pita Bread Shelf Life Improvement.

Ochratoxin A (OTA) is a mycotoxin produced in the secondary metabolism of fungus belonging to the genus Aspergillus and Penicillium. In this study, the employment of oriental mustard flour (OMF) as an ingredient in a packaged sauce was evaluated for the generation in situ of the antimicrobial compound allyl isothiocyanate (AITC) in order to preserve pita bread contaminated with Penicillium verrucosum VTT D-01847, an OTA producer, in an active packaging system. Four different concentrations (8, 16, 33 and 50 mg/g) were tested. Mycelium formation, mycotoxin production, AITC absorbed by the food matrix, and volatilization kinetics were studied for each concentration. The results obtained were compared with bread treated with the commercial additive calcium propionate (E-282). The results showed a shelf life increase of two and three days with the employment of 33 and 50 mg/g of OMF, with a significant reduction of the fungal population (3.1 and 5.7 logs, respectively) in comparison with the control experiment. The use of 16 and 33 mg/g of OMF in the sauce formulation decreased the concentration of OTA in the bread samples while no OTA production was detected employing 50 mg/g of OMF.

Potential Application of Lactic Acid Bacteria to Reduce Aflatoxin B1 and Fumonisin B1 Occurrence on Corn Kernels and Corn Ears.

Fungal spoilage is an important issue for the food industry, leading to food sensory defects, food waste, economic losses and public health concern through the production of mycotoxins. Concomitantly, the search for safer natural products has gained importance since consumers began to look for less processed and chemically treated foods. In this context, the aim of this study was to evaluate the antifungal and antimycotoxigenic effect of seven strains of Lactobacillus plantarum. Lactic acid bacteria (LAB) were grown on Man Rogosa Sharpe (MRS) broth at 37 ºC in anaerobic conditions. After that, the cell-free supernatant (CFS) were recovered to determine its antifungal activity by halo diffusion agar test. In addition, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) was determined for each L. plantarum CFS by 96-well microplates method. Additionally, CFS was used as a natural biocontrol agent on corn kernels and corn ears contaminated with Aspergillus flavus and Fusarium verticillioides, respectively. The L. plantarum CECT 749 CFS showed the highest antifungal effect against all essayed strains. Moreover, the employment of this CFS in food reduced the mycotoxin production at a percentage ranging from 73.7 to 99.7%. These results suggest that the L. plantarum CECT 749 CFS could be promising for the biocontrol of corn.

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.