Mutual effects on mycotoxin production during co-culture of ochratoxigenic and aflatoxigenic Aspergillus strains.

Mycotoxin co-occurrence compromises the safety of food crops worldwide. Environmental factors, as well as fungal interaction, can substantially influence the infectivity of mycotoxigenic fungi and their subsequent production of multi-mycotoxin. Here, we investigated the mutual effects of the co-culture of ochratoxigenic and aflatoxigenic Aspergillus strains on the co-production of ochratoxin A (OTA) and aflatoxin B1 (AFB1). Single cultures of ochratoxigenic A. carbonarius and A. alliaceus grew optimally at 25 °C, whereas aflatoxigenic A. flavus grew optimally at 35 °C. The maximum levels of OTA and AFB1 were achieved at 25 °C, whereas mycotoxin production decreased at 35 °C. During competitive growth of the ochratoxigenic and aflatoxigenic isolates, inhibition or stimulation of mycotoxin production was dependent on the fungal strain, temperature, and the ratio of the spore concentration. Aspergillus carbonarius and A. alliaceus generally produced OTA, with similar patterns of relative OTA levels at all temperatures. AFB1 production by A. flavus in the presence of ochratoxigenic Aspergillus species was inhibited at 25 °C and stimulated at 35 °C. These results indicated that the temperature, presence of other mycotoxigenic Aspergillus species, and ratio of the initial spore concentration significantly contributed to the co-production of OTA and AFB1.

Optimization and Validation of Dispersive Liquid-Liquid Microextraction for Simultaneous Determination of Aflatoxins B1, B2, G1, and G2 in Senna Leaves and Pods Using HPLC-FLD with Pre-Column Derivatization.

Dispersive liquid-liquid microextraction (DLLME) was optimized for the simultaneous extraction of aflatoxins (AFB1, AFB2, AFG1, and AFG2) from powdered senna leaves and pods. Detection was performed using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and pre-column derivatization. The parameters affecting the DLLME extraction efficiency were evaluated. Chloroform (200 µL) was used as an extraction solvent, 500 µL of distilled water was used as a dispersive solvent, and the extraction was performed at pH 5.6 with no salt added. The optimized method was validated using leaves and pods according to the European Commission guidelines. The linear range for all aflatoxins was 2-50 µg/kg, with values for regression coefficients of determination exceeding 0.995. The recoveries of spiked senna leaves and pods were in the ranges of 91.77-108.71% and 83.50-102.73%, respectively. The RSD values for intra-day and inter-day precisions were in the ranges of 2.30-7.93% and 3.13-10.59%, respectively. The limits of detection and quantification varied in the ranges of 0.70-1.27 µg/kg and 2.13-3.84 µg/kg, respectively. The validated method was successfully applied for the quantification of aflatoxins in 60 real samples of dried senna leaves and pods.

Occurrence of Heat-Resistant Mold Ascospores in Pineapple and Sugarcane Field Soils in Thailand.

Heat-resistant molds (HRMs) are important spoilage fungi of heat-processed fruit products worldwide. Ascospores of HRMs are widely distributed in the soil in which fruits are grown and are often found associated with raw fruit materials. To date, there is little available information on the distribution of HRMs in the soil and on their heat resistance. Thus, this study determined the presence and characterized the heat resistance of HRMs in soil samples from pineapple and sugarcane fields in Thailand. HRMs were detected in all soil samples, and the most dominant species was Aspergillus with 50-99.2% relative abundance. Other isolates, in descending order of frequency, were Penicillium, Talaromyces, Hamigera, and Paecilomyces. Then, 100 representative HRM isolates were identified based on a combination of morphological characteristics and ITS sequences. They were classified into 5 genera and 24 species. The heat resistance of ascospores aged 30 days produced by selected HRMs was qualitatively determined in a glucose-buffered solution. Based on their log reductions after heat shock at 75°C for 30 min, they were classified as less, moderately, or highly heat-resistant ascospores. HRMs belonging to A. chevalieri, A. denticulatus, A. siamensis, A. laciniosus, A. fennelliae, A. spinosus, Paec. niveus, H. pallida, and T. macrosporus produced high heat-resistant ascospores. In addition, soil physicochemical properties significantly influenced the prevalence of HRMs, depending on the fungal genus. The thermal resistance of ascospores was significantly and positively correlated to available phosphorus, whereas it was negatively correlated to soil pH. The results of this study confirmed the presence of HRMs in soils and potential HRM contamination, especially in fruits growing in acidic or high-nutrient soils, or both.

Antimicrobial Mechanism of Salt/Acid Solution on Microorganisms Isolated from Trimmed Young Coconut.

This study investigated the inhibitory activity of organic solutions containing 5, 10, 15, 20 and 30% (w/v) sodium chloride and citric acid solution and 15:10, 15:15, 15:20 and 15:30% (w/v) sodium chloride (NaCl) combined with citric acid (CA) solution (salt/acid solution) for 10 min against microorganisms isolated from trimmed young coconut: Bacillus cereus, B. subtilis, Staphylococcus aureus, S. epidermidis, Enterobacter aerogenes, Serratia marcescens, Candida tropicalis, Lodderromyces elongisporus, Aspergillus aculeatus and Penicillium citrinum. Commercial antimicrobial agents such as potassium metabisulfite and sodium hypochlorite (NaOCl) were used as the controls. Results showed that 30% (w/v) NaCl solution displayed antimicrobial properties against all microorganisms, with s reduction range of 0.00-1.49 log CFU/mL. Treatment of 30% (w/v) CA solution inhibited all microorganisms in the reduction range of 1.50-8.43 log CFU/mL, while 15:20% (w/v) salt/acid solution was the minimum concentration that showed a similar antimicrobial effect with NaOCl and strong antimicrobial effect against Gram-negative bacteria. The mode of action of this solution against selected strains including B. cereus, E. aerogenes and C. tropicalis was also determined by scanning electron microscopy and transmission electron microscopy. B. cereus and E. aerogenes revealed degradation and detachment of the outer layer of the cell wall and cytoplasm membrane, while cytoplasmic inclusion in treated C. tropicalis cells changed to larger vacuoles and rough cell walls. The results suggested that a 15:20% (w/v) salt/acid solution could be used as an alternative antimicrobial agent to eliminate microorganisms on fresh produce.

The Occurrence and Co-Occurrence of Regulated, Emerging, and Masked Mycotoxins in Rice Bran and Maize from Southeast Asia.

Raw feed materials are often contaminated with mycotoxins, and co-occurrence of mycotoxins occurs frequently. A total of 250 samples i.e., rice bran and maize from Cambodia, Laos, Myanmar, and Thailand were analysed using state-of-the-art liquid chromatography-mass spectrometry (LC-MS/MS) for monitoring the occurrence of regulated, emerging, and masked mycotoxins. Seven regulated mycotoxins - aflatoxins, ochratoxin A, fumonisin B1, deoxynivalenol, zearalenone, HT-2, and T-2 toxin were detected as well as some emerging mycotoxins, such as beauvericin, enniatin type B, stachybotrylactam, sterigmatocystin, and masked mycotoxins, specifically zearalenone-14-glucoside, and zearalenone-16-glucoside. Aspergillus and Fusarium mycotoxins were the most prevalent compounds identified, especially aflatoxins and fumonisin B1 in 100% and 95% of samples, respectively. Of the emerging toxins, beauvericin and enniatin type B showed high occurrences, with more than 90% of rice bran and maize contaminated, whereas zearalenone-14-glucoside and zearalenone-16-glucoside were found in rice bran in the range of 56-60%. Regulated mycotoxins (DON and ZEN) were the most frequent mycotoxin combination with emerging mycotoxins (BEA and ENN type B) in rice bran and maize. This study indicates that mycotoxin occurrence and co-occurrence are common in raw feed materials, and it is critical to monitor mycotoxin levels in ASEAN's feedstuffs so that mitigation strategies can be developed and implemented.

Detection of Profenofos in Chinese Kale, Cabbage, and Chili Spur Pepper Using Fourier Transform Near-Infrared and Fourier Transform Mid-Infrared Spectroscopies.

Different types of quantitative technology based on infrared spectroscopy to detect profenofos were compared based on Fourier transform near-infrared (FT-NIR; 12,500-4000 cm-1) and Fourier transform mid-infrared (FT-MIR; 4000-400 cm-1) spectroscopies. Standard solutions in the range of 0.1-100 mg/L combined with the dry-extract system for infrared (DESIR) technique were analyzed. Based on partial least-squares regression (PLSR) to develop a calibration equation, FT-NIR-PLSR produced the best prediction of profenofos residues based on the values for R 2 (0.87), standard error of prediction or SEP (11.68 mg/L), root-mean-square error of prediction or RMSEP (11.50 mg/L), bias (-0.81 mg/L), and ratio performance to deviation or RPD (2.81). In addition, FT-MIR-PLSR produced the best prediction of profenofos residues based on the values for R 2 (0.83), SEP (13.10 mg/L), RMSEP (13.00 mg/L), bias (1.46 mg/L), and RPD (2.49). Based on the ease of use and appropriate sample preparation, FT-NIR-PLSR combined with DESIR was chosen to detect profenofos in Chinese kale, cabbage, and chili spur pepper at concentrations of 0.53-106.28 mg/kg. The quick, easy, cheap, effective, rugged, and safe technique coupled with gas chromatography-mass spectrometry was used to obtain the actual values. The best FT-NIR-PLSR equation provided good profenofos detection in all vegetables based on values for R 2 (0.88-0.97), SEP (5.27-11.07 mg/kg), RMSEP (5.25-11.00 mg/kg), bias (-1.39 to 1.30 mg/kg), and RPD (2.91-5.22). These statistics revealed no significant differences between the FT-NIR predicted values and actual values at a confidence interval of 95%, with agreeable results presented at pesticide residue levels over 30 mg/kg. FT-NIR spectroscopy combined with DESIR and PLSR should be considered as a promising screening method for pesticide detection in vegetables.

Isolation, identification and toxigenic potential of ochratoxin A-producing Aspergillus species from coffee beans grown in two regions of Thailand.

In 2006 and 2007, 32 Thai dried coffee bean samples (Coffea arabica) from two growing sites of Chiang Mai Province, and 32 Thai dried coffee bean samples (Coffea canephora var. robusta) from two growing sites of Chumphon Province, Thailand, were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The overall percentage of fungal contamination in coffee was 98% and reduced to 60% after surface disinfection. There were remarkable ecological differences in the composition of ochratoxigenic species present in these two regions. Arabica coffee bean samples from the North had an average of 78% incidence of colonization with Aspergillus of section Circumdati with Aspergillus westerdijkiae and A. melleus as the predominant species. Aspergillus spp. of section Nigri were found in 75% of the samples whereas A. ochraceus was not detected. Robusta coffee beans from the South were 98-100% contaminated with predominantly A. carbonarius and A. niger. A. westerdijkiae was only found in one sample. The diversity of the fungal population was probably correlated with the geographical origin of the coffee, coffee cultivar, and processing method. Representative isolates of section Circumdati (52) and Nigri (82) were examined for their OTA production using HPLC with fluorescence detection. Aspergillus westerdijkiae (42 isolates out of 42), A. steynii (13/13), and A. carbonarius (35/35) in general produced large amounts of OTA, while one isolate of A. sclerotiorum produced intermediate amounts of OTA. 13% of the A. niger isolates produced OTA in intermediate amounts. OTA levels in coffee bean samples were analyzed using the Ridascreen OTA ELISA kits. Of the 64 coffee bean samples analyzed, 98% were contaminated with OTA in levels of <0.6-5.5 microg/kg (Arabica) and 1-27 microg/kg (Robusta). Presence of OTA in representative coffee samples was also confirmed by LC-MS/MS after ion-exchange purification.

Prospects for Food Fermentation in South-East Asia, Topics From the Tropical Fermentation and Biotechnology Network at the End of the AsiFood Erasmus+Project.

Fermentation has been used for centuries to produce food in South-East Asia and some foods of this region are famous in the whole world. However, in the twenty first century, issues like food safety and quality must be addressed in a world changing from local business to globalization. In Western countries, the answer to these questions has been made through hygienisation, generalization of the use of starters, specialization of agriculture and use of long-distance transportation. This may have resulted in a loss in the taste and typicity of the products, in an extensive use of antibiotics and other chemicals and eventually, in a loss in the confidence of consumers to the products. The challenges awaiting fermentation in South-East Asia are thus to improve safety and quality in a sustainable system producing tasty and typical fermented products and valorising by-products. At the end of the "AsiFood Erasmus+ project" (www.asifood.org), the goal of this paper is to present and discuss these challenges as addressed by the Tropical Fermentation Network, a group of researchers from universities, research centers and companies in Asia and Europe. This paper presents current actions and prospects on hygienic, environmental, sensorial and nutritional qualities of traditional fermented food including screening of functional bacteria and starters, food safety strategies, research for new antimicrobial compounds, development of more sustainable fermentations and valorisation of by-products. A specificity of this network is also the multidisciplinary approach dealing with microbiology, food, chemical, sensorial, and genetic analyses, biotechnology, food supply chain, consumers and ethnology.

Antimicrobial effect of Thai spices against Listeria monocytogenes and Salmonella typhimurium DT104.

The objective of this study was to determine the potential antimicrobial activity of extracts and essential oils of spices from Thailand against foodborne pathogenic bacteria. The antimicrobial efficacy of ginger (Zingiber officinale), fingerroot (Boesenbergia pandurata), and turmeric (Curcuma longa) was evaluated against five strains of Listeria monocytogenes and four strains of Salmonella enterica ssp. enterica serovar Typhimurium DT104. Antimicrobial activity was investigated in microbiological media by using an agar dilution assay and enumeration over time and a model food system, apple juice, by monitoring growth over time. In the agar dilution assay, water extracts of the three spices had no effect on L. monocytogenes. Similarly, 50% ethanol extracts of ginger or turmeric had no effect. In contrast, ethanolic fingerroot extracts at 5 to 10% (vol/ vol) inhibited most L. monocytogenes strains for 24 h in the agar dilution assay. Commercial essential oils (EO) of ginger or turmeric inhibited all L. monocytogenes at < or = 0.6 or < or = 10%, respectively. Fingerroot EO inhibited all strains at < or = 0.4%. In the enumeration-over-time assay, a 5% fingerroot ethanol extract reduced ca. 4 log CFU/ml Listeria by around 2 log in 24 h while 10% inactivated the microorganism in 9 h. Fingerroot EO at 0.2% inactivated 4 log CFU/ml L. monocytogenes in 6 to 9 h. Neither extracts nor commercial EO had any effect on Salmonella Typhimurium DT 104 with the exception of fingerroot EO, which inhibited all strains at < or = 0.7%. Addition of 0.2% fingerroot EO to apple juice reduced 4 log of L. monocytogenes Scott A and both strains of Salmonella Typhimurium to an undetectable level within 1 to 2 days. It was concluded that fingerroot EO and extract have potential for inhibiting pathogens in food systems.

Fumonisin and T-2 toxin production of Fusarium spp. isolated from complete feed and individual agricultural commodities used in shrimp farming.

Fusarium spp. are plant pathogens producing fumonisins and trichothecenes that both affect human and animal health. In the present study, 40 fungal strains were isolated and species identified from 35 shrimp feed samples and from 61 agricultural raw materials. F. verticillioides was the predominant species (85 %) mostly found in corn and soybean meal, while no Fusarium contamination was detected in shrimp feed. Levels of 10 % of F. oxysporum were isolated from peanut and 5 % of F. equiseti contamination in corn and peanut. To determine the ability of toxin production, enzyme-linked immunosorbent assay, polymerase chain reaction, and ultra-pressure liquid chromatography-tandem mass spectrometry were performed. All but four of the fumonisin-producing strains contained the FUM1 gene. No Fusarium synthesized T-2 toxin nor contained the Tri5 gene. This survey brings more data on mycotoxin contamination in the food chain of animal feed production, and leads to the awareness of the use of contaminated raw materials in shrimp farming.

Two novel species of Aspergillus section Nigri from Thai coffee beans.

Two novel species of Aspergillus section Nigri from Thai coffee beans are described as Aspergillus aculeatinus sp. nov. and Aspergillus sclerotiicarbonarius sp. nov. Their taxonomic status was determined using a polyphasic taxonomic approach with phenotypic (morphology and extrolite profiles) and molecular (beta-tubulin, internal transcribed spacer and calmodulin gene sequences) characteristics. A. aculeatinus sp. nov. is a uniseriate species with a similar morphology to Aspergillus aculeatus and Aspergillus japonicus, but producing smaller conidia (2-5 microm). A. aculeatinus sp. nov. produced neoxaline, secalonic acid D and F, and aculeacins. A. sclerotiicarbonarius sp. nov. is a biseriate species similar to Aspergillus carbonarius and Aspergillus ibericus, but produces abundant sclerotia and some unique indol-alkaloids. The type strain of Aspergillus sclerotiicarbonarius sp. nov. is CBS 121057(T) (=IBT 28362(T)) and the type strain of Aspergillus aculeatinus sp. nov. is CBS 121060(T) (=IBT 29077(T)).