Isolation and characterization of Bacillus amyloliquefaciens ZDS-1: Exploring the degradation of Zearalenone by Bacillus spp.

Zearalenone (ZEN) is a mycotoxin produced mainly by various Fusarium species which occur naturally in many crops worldwide. ZEN causes reproductive disorders and hyperestrogenic syndromes in animals and humans. This study aimed to isolate ZEN-degrading bacteria to develop strategies for detoxifying ZEN contamination in cereal crops. We screened approximately 1000 colonies for degrading ability and found four strains were capable of degrading ZEN. We selected one strain ZDS-1 for further study because it showed the high ZEN-degrading ability. On the basis of morphological, physiological and phylogenetic analysis of its 16SrRNA, gyrA gene sequences, strain ZDS-1 was identified as Bacillus amyloliquefaciens. The optimal conditions for the biodegradation of ZEN by ZDS-1 were temperature; 30 °C, pH; 6.0–7.0, and cell concentration; 5 × 10⁸ cfu/mL. ZDS-1 could degrade ZEN efficiently with the concentration from 1 mg/L to 100 mg/L. ZDS-1 not only could remove ZEN in the culture medium, but also could degrade ZEN in wheat. The ZEN removal by ZDS-1 was not due to binding or absorption, and during the process of ZEN degradation, no ZEN derivatives of ZEN were produced. These results suggested that Bacillus amyloliquefaciens ZDS-1 would be explored further for its ability to degrade ZEN in field trials.     

Biological detoxification of Aflatoxin B1 by Bacillus licheniformis CFR1

Aflatoxin B₁ is the most harmful among the mycotoxins commonly present in food and feed, and it may lead to hepatocellular carcinoma in humans and animals. Therefore, limiting its exposure to humans and livestock is very much essential. The present study aims to isolate and characterize Aflatoxin B₁ detoxifying bacteria from various sources, to develop a safe and environment-friendly strategy for Aflatoxin B₁ management. Fifty-six bacteria were isolated using a media amended with coumarin as a sole carbon source. Seven strains showed more than 70% reduction of AFB₁ in liquid culture media. Among them, isolate CFR1 reduced Aflatoxin B₁ by 94.7%, and it was selected for further studies. CFR1 was identified as Bacillus licheniformis CFR1, by biochemical characterization and 16S rRNA gene sequencing. The cell-free supernatant of B. licheniformis CFR1 was able to degrade AFB₁ efficiently than the cell lysate. The degradation of AFB₁ was examined using High-Performance Thin Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Electron spray ionization-Mass Spectrometry (ESI-MS). The optimal temperature, time, and pH of the medium for the maximum degradation of Aflatoxin B₁ were found to be 37 °C, 24 h and 7, respectively. Furthermore, Ames test for mutagenicity showed that when treated with B. licheniformis CFR1 extracellular fraction coincided with the loss of Aflatoxin B₁ mutagenicity. To the best of our knowledge, this is the first study that shows more than 90% degradation of AFB₁ by B. licheniformis. Thus, B. licheniformis CFR1 might be an excellent candidate for bioremediation and detoxification of Aflatoxin B₁ from both field and food matrices.     

Elimination of aflatoxin B1 in vegetable oil based on immuno-magnetosomes probes from a novel magnetotactic bacterium

Here, a magnetotactic bacterium (MTB) existing in freshwaters was acclimated and mass propagations for producing natural magnetosomes with diameter about 15 nm at ordinary ambient. It was found that the magnetosomes which are mainly composed of iron oxide and iron sulfide hold pretty well magnetic features. Furthermore, the developed magnetosomes-AFB₁ antibody immunomagentic probes through chemical bonding AFB₁ polyclonal antibody onto the natural magnetosomes maintain 28 fold capability on collection of AFB₁ toxin in vegetable oil compared to conventional Fe₃O₄ magnetic nanoparticle-AFB₁ antibody probes. Recovery ratio up to 93.7% from commercial vegetable oil with artificial AFB₁ contamination during 15 min collection were demonstrated. Through circular dichroism (CD) spectral measurements, the mechanism of magnetosomes probe was interpreted for proposing the practical way with high-performance on controlling the aflatoxins contamination in liquid foods basing on immunomagentic separation technique.     

Removal of aflatoxin B1 by roasting with lemon juice and/or citric acid in contaminated pistachio nuts

Nowadays, aflatoxin B₁ (AFB₁) could be considered as one of the most hazardous mycotoxins for humans, and nuts comprise one of the major responsible food categories for human exposure to this mycotoxin. Thus, complete elimination of AFB₁ or reduction of its content in nut foods, such as pistachio attracted lots of attentions. In the current study, the efficacy of roasting process by incorporation of lemon juice and/or citric acid on the reduction of AFB₁ in contaminated pistachio nuts (AFB₁ at two levels of 268 and 383 ng/g) was investigated. Significant degradation of AFB₁ (up 93.1% for AFB₁) was recorded by applied treatment protocols. Although roasting of 50 g pistachio nuts with 30 ml water, 30 ml lemon juice and 6 g of citric acid at 120 °C for 1 h resulted to a significant degradation (93.1 ± 8.2%) of AFB₁, this treatment altered the desired physical properties. Roasting with 30 ml water, 15 ml lemon juice and 2.25 g of citric acid at 120 °C for 1 h reduced the level of AFB₁ in 49.2 ± 3.5% of the initial level without a noticeable change in desired appearance of pistachios. Hence, a synergistic effect between heating and lemon juice/citric acid in order to AFB₁ degradation was observed. It could be concluded that roasting process with lemon juice and citric acid could be applied as a useful and safe degradation method of AFB₁ in naturally contaminated pistachio nuts.     

Degradation and detoxification of aflatoxin B1 using nitrogen gas plasma generated by a static induction thyristor as a pulsed power supply

Toxic fungal metabolite aflatoxin B₁ (AFB1) is a stable carcinogen that is sometimes found in foods, such as peanuts and peppers. In this study, AFB1 was applied to a coverglass and subjected to treatment with nitrogen gas plasma generated by a plasma apparatus using a short high voltage pulse from a static induction thyristor power supply at 1.5 kpps (kilo pulse per second). Enzyme-linked immunosorbent assay showed that a 20 μL aliquot of a 200 ppb solution of AFB1 was efficiently degraded to less than one tenth of the original level within 15 min. High-performance liquid chromatography confirmed the loss of AFB1 after plasma treatment and the generation of small fragments, possibly originating from the degradation process. Moreover, a cell-based assay using HepG2 as an index to measure the cell-growth promoting properties of AFB1 showed that the gas plasma treatment reduced the biological activity of the mycotoxin. Although the amount of heat and ultraviolet light is insufficient to inactivate the toxin, the reactive chemical products appear to contribute to the degradation of AFB1. Controlling and optimizing the conditions for producing these reactive chemical species during plasma generation would lead to efficient inactivation of AFB1.     

Fungal and aflatoxin contamination of marketed spices

Fourteen spice samples were collected from local markets in Doha, Qatar, during 2012, and were surveyed for the presence of potentially harmful mycoflora and for contamination with aflatoxins B₁, B₂, G₁, and G₂ by high-performance liquid chromatography (HPLC). Among the tested spice samples, chili powder showed the highest presence of fungal propagules, while ginger, curry and garlic samples did not present any fungal contamination. A total of 120 isolates, mostly belonging to Aspergillus and Penicillium genera, were collected and 33 representative species were identified by amplification and sequencing of the internal transcribed spacer (ITS) region. Aspergillus flavus, Aspergillus nomius and Aspergillus niger were the most dominant. Thirty-seven Aspergillus strains were screened for their potential to produce aflatoxins using biochemical and molecular tools: only 9 A. flavus strains showed both fluorescence and amplification with all the three primers targeting aflP, aflM and aflR genes. Aflatoxins were detected in five spices (black pepper, chili, tandoori masala. turmeric and garam masala), and with the exception of garam masala, the tested samples of turmeric, black pepper, tandoori masala and chili powder exceeded B₁ and/or total aflatoxin maximum levels. Our results demonstrate the potential for mycotoxin biosynthesis by fungi contaminating imported spice products.     

Degradation of fumonisin B1 by cinnamon essential oil

Fumonisins are group of mycotoxins produced mainly by Fusarium verticillioides and Fusarium proliferatum. They frequently contaminate corn and corn based products, and cause several diseases in humans and animals. Fumonisin B₁ (FB₁) is the most prevalent fumonisin and is highly toxic to human and animal. The essential oils from plants offer a hope in the prevention and detoxification of these mycotoxins. The present study investigates the degradation effect of cinnamon, citral, Litsea cubeba, clove, eucalyptus, anise, spearmint and camphor oils on FB₁. The degradation level of FB₁ was determined by ELISA. Cinnamon oil proves to be effective essential oil in reducing FB₁, followed by citral, eugenol oil, eucalyptus oil, anise oil and camphor oil. The effects of incubation time, and temperature with respect to the concentration of cinnamon oil on their degradation effect on FB₁ by cinnamon oil were investigated. Results showed that at 120 h time with the 280 μg/ml concentration of cinnamon oil, under 30 °C is optimal for FB₁ reduction. Under optimal condition, FB₁ was reduced from 15.03 to 0.89 μg/ml (94.06%). Cinnamon oil could be a promising candidate in the detoxification and control of FB₁ in corn based products.     

Esterase activity inspired selection and characterization of zearalenone degrading bacteria Bacillus pumilus ES-21

Zearalenone (ZEN), mainly produced by Fusarium species, is an estrogenic mycotoxin which causes reproductive disorders in livestock. In this study, we described a simple and rapid method for screening of ZEN-degrading bacteria by esterase activity assay. Soil bacteria strains were first tested for their esterase activities, then active strains were further evaluated for their ZEN-degrading potentials. A bacterial strain named Bacillus pumilus ES-21 was detected to be able to eliminate ZEN in the culture medium. ZEN degradation conditions were optimized through response surface methodology and the result showed that the degradation rate of ZEN by Bacillus pumilus ES-21 was up to 95.7% at the ZEN concentration of 17.9 μg/ml within 24 h. One of the degradation product was proposed to be 1-(3,5-dihydroxyphenyl)-6′-hydroxy-l′-undecen-l0′-one according to LC-TOF-MS/MS analysis. This study provided a strategy for the isolation of ZEN degrading microbes and a promising degrading strain.     

Isolation and characterization of Pseudomonas otitidis TH-N1 capable of degrading Zearalenone

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced by various Fusarium species and causes hyperestrogenism and related toxicosis of farm animals and humans. The present study aimed to isolate and identify ZEA-resistant bacteria from rumen in order to develop some strategies for detoxifying ZEA-contaminated food and feed. A bacterial strain was isolated from the rumen contents for its ability to utilize ZEA as the sole carbon and energy source. The isolate was an aerobic, Gram-negative, rod-shaped bacterium with single polar flagellum and was named Pseudomonas otitidis TH-N1 based on the morphology and 16S rRNA gene sequence. Meanwhile, the present study investigated that how various influence factors of P. otitidis TH-N1 could remove ZEA from a liquid medium. The optimal temperature, pH value, and concentrations of bacteria for the biodegradation of ZEA were 37 °C, 4.5, and 10⁹ cfu/ml, respectively. These results suggest that P. otitidis TH-N1 is a new bacterium found from the rumen and exhibited remarkable degradation activity of ZEA. It is probably a new bacterial resource to detoxify ZEA from ZEA-contaminated food and feed.     

Aflatoxin B1 degradation by culture and lysate of a Pontibacter specie

The presence of aflatoxin B₁ (AFB₁) along the food chain poses a significant threat, thus propelling the need for an effective approach to control it. This study was therefore, aimed at investigating AFB₁ degradation of liquid cultures and lysates of an isolated Pontibacter sp. (VGF1). Liquid cultures, lysed bacterial cells in the absence (uninhibited lysates) and presence of protease inhibitors (protease inhibited lysates) were respectively incubated with AFB₁ for 3, 6, 12, 24 and 48 h. AFB₁ degradation was monitored during this period on high performance liquid chromatography (HPLC) and results obtained revealed that after 6 h of incubation, the protease inhibited (PI) lysates yielded a 65% AFB₁ degradation, whereas after 12 h, no residual AFB₁ was detected. Conversely, after 48 h of incubation, a significantly (p≤0.05) lower AFB₁ degradation of 50 and 36% by the liquid culture and uninhibited lysate, respectively, were noted. It was further confirmed that the degradation mechanism was enzymatic. Data from cytotoxicity studies against human lymphocytes further demonstrated that extracts of biotransformed AFB₁ were less toxic when compared to that of AFB₁. Findings from this study have demonstrated an alternative approach for the decontamination and biocontrol of AFB₁ in various agricultural commodities.     

PD-1菌株的原油降解能力研究

应用微生物法,对中国石油抚顺石化公司石油三厂隔油池附近被石油污染的发黑土壤试样进行了筛选处理,培养出了PD-1菌株,并考察了其对大庆原油的降解能力。结果表明,PD-1活细胞对质量浓度为2.0 g/L的原油显示出了较强的降解能力,随着培养时间的延长,OD610(培养液光密度值)先增大后减小,η(原油降解率)逐渐增大并趋于平缓,OD610值在培养第6天达到最大值8.383,η在第9天接近40.1%;PD-1休止细胞提高了原油降解酶的浓度,并减小了原油代谢产物对降解酶的抑制作用,从而具有更高效的原油降解能力,正交实验中的η高达95.5%。结合生产实际,最终选择的PD-1休止细胞降解原油的最佳条件为:反应时间为24 h,培养基中的原油质量浓度为4.0 g/L,OD610为50,反应温度为20~30℃。PD-1菌株能在常温下及较短时间内高效地降解原油,推广应用的潜力较高。     

Bioaccessibility and bioavailability of fumonisin B2 and its reaction products with isothiocyanates through a simulated gastrointestinal digestion system

Fumonisins (FBs) are toxins produced mainly by the molds Fusarium verticillioides (also known as Fusarium moniliforme) and Fusarium proliferatum. These mycotoxins are contaminants of wheat, maize, maize-based foods and other grains worldwide. Isothiocyanates (ITCs) are natural compounds produced by the enzymatic hydrolysis of glucosinolates, which are found in plants of the Brassicaceae family. The use of ITCs as food preservatives has been extensively researched. In this study, allyl (AITC), phenyl (PITC) and benzyl isothiocyanates (BITC) fumigation systems (500 μL/L) were employed to reduce FB₂ levels naturally produced in bread by Gibberella moniliformis CECT 2987. Reaction products formed between FB₂ and ITCs, their bioaccessibility and bioavailability were examined. Reduction of FB₂ in bread ranged from 90 to 99%, whereas its bioaccessibility ranged from 57 to 65%. The bioaccessibility found for the FB₂-ITCs conjugates ranged from 33 to 71%, whereas the bioavailability of the FB₂ and of the FB₂-ITCs adducts ranged from 0.6 to 3.0%.     

An initial characterization of aflatoxin B1 contamination of maize sold in the principal retail markets of Kigali,Rwanda

Food security considerations have shifted in recent years, with the recognition that available food should also be nutritious and safe. There is a growing evidence base for contamination of maize and other crops by fungal toxins in the tropics and sub-tropics. As an initial snapshot of contamination by one of these toxins in Rwanda, Aflatoxin B1 (AFB1) was analyzed in 684 samples of maize flour collected in seven principal retail markets of Kigali and in 21 samples of animal feed from seven feed vendors. Two rounds of sample collections were carried out, the first in September 2014 and the second in January 2015. A questionnaire given to vendors was used to determine if gender and education level of vendors, origin of maize and awareness of aflatoxins had any significant effect on AFB1 level in collected samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Immuno-affinity fluorimetery were used to analyze samples. Only markets had a significant effect on AFB1 level; for the two collections, differences were inconsistent among markets. In the first round, market means of AFB1 varied between 8.0 ± 5.57 μg/kg and 24.7 ± 23.74 μg/kg and for the second round, between 10.4 ± 8.4 μg/kg and 25.7 ± 25.85 μg/kg. In most animal feed samples AFB1 was >100 μg/kg. None of the vendors interviewed was aware of the risk of mycotoxin contamination in their maize-based flours and feed. Limits set by the United States Food and Drug Administration (20 μg/kg) for total aflatoxins and European Commission (2 μg/kg) for AFB1 for maize flour imports, were varied between 2–35% and 66–100% of samples, respectively. The implications of this study for human and animal health in Rwanda suggest that expanded surveys are needed to understand the scope of contamination, given the influence of environment and other factors on aflatoxin accumulation. Available options to mitigate and monitor aflatoxin contamination can be further deployed to reduce contamination.     

Molecular characterization of biosurfactant producing Bacillus cereus strain DRDU1 for its potential application in bioremediation and further EOR studies

Abstract

Environmental pollution by crude oil during petroleum exploration is a global issue of concern. It largely alters the physicochemical property of soil and water which makes them toxic for crops and aquatic organisms to survive. Bacillus cereus strain DRDU1 (Genbank accession no. KF273330.1) is an endospore forming potential hydrocarbon degrader which was isolated from an automobile engine. The isolate could achieve up to 96 and 84% kerosene and crude oil degradation with respective cfu of (6.3?±?2.31)?×?10⁹ and (13.67?±?2.52)?×?10⁸ cells/mL. On the other hand it also showed its potential to degrade 74 and 67% of kerosene and crude oil in nitrate (N) and phosphate (P) deficient media with corresponding cfu (10?±?1.73)?×?10⁸ and (6?±?2.65)?×?10⁶ cells/mL. This indicates its potential to survive under nutrient stress condition. The strain was reported to be capable of producing biosurfactant which may play role in petroleum biodegradation and may be a potential tool for Microbial Enhanced Oil Recovery (MEOR) studies in future. Herein the presence of sfp gene in the isolate was confirmed which may be responsible for its ability to synthesize biosurfactant for hydrocarbon degradation and also for its stress tolerant potential. It was further characterized and the hypothetical protein sequence encoded by this gene is also proposed.     

Survey of aflatoxin B1 in helva,a traditional Turkish food,by TLC

A total of 102 helva samples consisting of 34 plain helva, 34 helva containing cacao, and 34 helva containing pistachio nuts purchased from helva-factories and supermarkets in Adana of Turkey were analysed for aflatoxin B₁ (AFB₁) by thin-layer chromatography. The detection limit of AFB₁ was 1 μg kg⁻¹. Recovery experiments were carried out with spiked samples in the range 2–10 μg kg⁻¹ of AFB₁. No AFB₁ was found in any plain helva and helva containing cacao samples. On the other hand, of 34 helva containing pistachio nuts AFB₁ was determined in eight samples. AFB₁ was found in excess of Turkish legal limit of 5 μg kg⁻¹ in 4 of 102 helva samples. This paper reports the data of the first survey for the presence of AFB₁ in helva in Turkey.     

Effect of ozone treatment on aflatoxin B1 and safety evaluation of ozonized corn

This paper studies the ozone treatment effect on degradation of aflatoxin B₁ (AFB₁) in corn with different moisture content (MC). The toxicity of the degradation products (DPs) of the ozone-treated AFB₁-Contaminated Corn (ACC) was also evaluated using the human hepatocellular carcinoma cell line (HepG2) as model cells. The degradation rate of AFB₁ in corn increases with ozone concentration and treatment time. The results showed that ACC with 13.47% MC was easier to be degraded by ozone than with 20.37% MC. Treated with 90 mg L⁻¹ ozone for 20 min and 40 min, AFB₁ in corn with 13.47% MC decreased from 83 μg kg⁻¹ to 18.12 μg kg⁻¹ and 9.9 μg kg⁻¹, respectively, well meeting the China National Standard of AFB₁ in corn (20 μg kg⁻¹). In order to evaluate the safety of ozone used on ACC, the impacts of AFB₁ as well as untreated and ozone-treated ACC with the same level of AFB₁ content on HepG2's survival rate, morphology, and apoptosis were studied. The results showed that ACC had high cell toxicity while the toxicity of ozone-treated ACC had no significant difference with that of the AFB₁-free culture solution. It is concluded that ozonation can quickly and effectively degrade AFB₁ in corn and diminish ACC's toxicity, and therefore, ozonation is expected to be an effective, fast, and safe method for AFB₁ degradation in ACC.     

Identification of aflatoxin B1 on maize kernel surfaces using hyperspectral imaging

A shortwave infrared (SWIR) hyperspectral imaging system with wavelength range between 1000 and 2500 nm was used to assess the potential to detect aflatoxin B₁ (AFB₁) contaminants on the surface of healthy maize kernels. Four different AFB₁ solutions were prepared and deposited on kernels surface to achieve 10, 20, 100, and 500 ppb, respectively. A drop of 20% methanol was dipped on the surface of 30 healthy kernels in the same way to generate the control samples. Based on the standard normal variate (SNV) transformation spectra, principal components analysis (PCA) was used to reduce the dimensionality of the spectral data, and then stepwise factorial discriminant analysis (FDA) was performed on latent variables provided by the PCA's. Furthermore, beta coefficients of the first three of four discriminant factors were analyzed and key wavelengths, which can represent AFB₁ and be used to differentiate different level of AFB₁ were indentified. Furthermore, 150 independent samples were used as verification set to test the reproducibility of the proposed method. A minimum classification accuracy of 88% was achieved for the validation set and verification set. Results indicated that hyperspectral imaging technology, accompanied by the PCA-FDA method, can be used to detect AFB₁ at concentrations as low as 10 ppb when applied directly on the maize surface.     

Fungal contamination and aflatoxin B1 of ‘egusi’ melon seeds in Nigeria

One hundred and thirty seven samples of melon seeds (Colocynthis citrullus L.) from randomly selected farmers’ stores in the humid forest and Northern Guinea savanna of Nigeria were analysed for the incidence of diseased seeds, moisture content, associated moulds and levels of aflatoxin B₁ contamination. The proportion of diseased seeds ranged from 2.5 to 37.3% in the forest and 2.1 to 17.9% in the savanna, while the seed moisture content varied from 5.3 to 10.4%, and 4.6 to 9.5% respectively. All the samples contained moulds, with the two genera, Aspergillus and Penicillium predominating, while A. flavus had the highest species count. The other common fungal isolates in order of decreasing incidence were A. niger, P. citrinum, Botryodiplodia theobromae, Cladosporium sp and A. clavatus. Thin layer chromatography analysis showed that 32% in the forest and 21% samples in the savanna contained aflatoxin B₁ with mean levels of 14.8 μg/kg in the forest and 11.3 μg/kg in the savanna respectively. Significant positive correlations were found between number of aflatoxin B₁ positive samples and the percentage of A. flavus infected samples and between the levels of diseased seeds and the levels of aflatoxin B₁ contamination.     

Detection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilization platform

We report an aptamer-based biosensor for detection of aflatoxin B₁ (AFB₁), a mycotoxin identified as contaminant in food. The sensor is assembled in a multilayer framework that utilizes cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) for acquiring the signal response by means of redox indicators: K[Fe(CN)₆]^−3/−4. Poly (amidoamine) dendrimers of fourth generation (PAMAM G4) immobilized on gold electrode covered by cystamine, were employed for attachment of single stranded amino-modified DNA aptamers specific to AFB₁. The cystamine-dendrimers (Cys-PAMAM) layers were compared with other immobilization platforms such as cystamine (Cys), 11-mercaptoundecanoic acid (MUA) and 11-mercaptoundecanoic acid-dendrimers (MUA-PAMAM), being the first approach the most appropriate for producing sensitive and reproducible signal in the range of concentrations 0.1–10 nM AFB₁. The sensor was validated in certified contaminated peanuts extract as well as in spiked samples of peanuts-corn snacks and the sensing response was evaluated and compared in terms of the matrix effect. The aptamer specificity was analyzed by testing the sensor in other mycotoxins such as aflatoxin B₂ (AFB₂) and ochratoxin A (OTA). The limit of detection achieved by this sensor was LOD = 0.40 ± 0.03 nM, it was regenerable in 0.2 M glycine-HCl and it did not lose its stability up to 60 h storing at 4 °C. Atomic Force Microscopy (AFM) studies were also performed for illustrating individual steps of biosensor assembly.