Extracellular enzyme production and enterotoxigenic gene profiles of Bacillus cereus and Bacillus thuringiensis strains isolated from cheese in Turkey

The aim of the present study was to investigate the biochemical characteristics, extracellular enzyme production and enterotoxigenic genes contents of 6 Bacillus cereus and 22 Bacillus thuringiensis strains, isolated from 100 cheese samples in Turkey. Crystal morphologies of B. thuringiensis strains were found either spherical (n = 12) or spherical and irregular-shaped (n = 10) by phase contrast microscopy. B. cereus and B. thuringiensis strains were found to produce extracellular enzymes, respectively: gelatinase (83% and 91%), DNase (83% and 77%), lecithinase (83% and 95%), protease on skim milk agar (100% and 100%), protease on milk agar (100% and 91%), protease on casein agar (83% and 77%), xylanase (100% and 45%), and cellulase (0% and 41%), and amylase (83% and 27%). All of the strains, except for Bt-D1, hydrolyzed Tween 20 (96%), but not Tween 80 or tributyrin. Pectinolytic activity was obtained to be the least frequent (4%). PCR analysis showed that all strains contained nheA, nheB, nheC and hblD genes. The hblA and hblC genes were present in 2 and 4 of B. thuringiensis strains, respectively. The bceT gene was detected in 1 B. cereus and 9 B. thuringiensis strains. The entFM gene was detected more frequently in B. thuringiensis (82%) than in B. cereus strains (50%). To our knowledge, this is the first report about the isolation and identification of enterotoxigenic B. cereus and B. thuringiensis strains from cheese samples in Turkey.     

Antilisterial activity and stability of nanovesicle-encapsulated antimicrobial peptide P34 in milk

Bacillus sp. P34, a strain isolated from aquatic environments of Brazilian Amazon basin, produces a bacteriocin-like substance (BLS) which was encapsulated in nanovesicles prepared from partially purified soy lecithin. The efficiency of free and encapsulated BLS P34 to control the development of L. monocytogenes and maintenance of antimicrobial activity was assessed over time in milk. The antimicrobial activity of free and encapsulated BLS P34 decreased approximately 50% after 4 days of storage (<4 °C) in skim and whole milk. After this period there was not significant loss of activity up to 21 days. The viable counts of Listeria monocytogenes in skim and whole milk containing 3200 AU/ml of free or encapsulated BLS P34 were always lower than those observed in controls without bacteriocin at both 30 °C and 7 °C. At 1600 AU/ml concentration, free and encapsulated BLS P34 were inhibitory to L. monocytogenes in skim milk, when compared with the control at 7 days. Nanovesicle-encapsulated and free BLS P34 shows potential use as biopreservative for application in milk-derived products.     

Development and application of a centrifugation-plating method to study the biodiversity of Bacillus species in rice products

A centrifugation-plating method was developed, using amylase and Tween 80 pre-treatment, for detection and enumeration of Bacillus spp. in rice products. The high sensitivity of this method improved detection of a variety of Bacillus species in rice products compared to the spread-plate method. Bacillus spp. were detected in 33 out of 35 raw rice samples with the centrifugation-plating method, but only 13 samples using the spread-plating method, even though 1 mL of a 10⁻¹ sample dilution was used, to increase sensitivity of the method. Known toxigenic or potentially toxigenic species (Bacillus cereus/Bacillus thuringiensis, Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus) were those most frequently found in both raw and cooked rice.     

Effectiveness of combined Pulsed Electric Field (PEF) and Manothermosonication (MTS) for the control of Listeria innocua in a smoothie type beverage

The combination of novel, non-thermal technologies for preservation purposes is a recent trend in food processing research. The objectives of the current study were (i) to optimise PEF or MTS treatment conditions which would achieve a maximum reduction of up to 3 log cycles of Listeria innocua in a milk based smoothie, when these technologies were applied individually, and (ii) to investigate possible additive or synergistic effects of the combined technologies. Microbiological analysis was performed by inoculating the smoothie with L. innocua and enumerating populations pre- and post-processing. All technologies applied within combinations significantly reduced L. innocua in the smoothie, when compared to untreated controls (p ≤ 0.0001). The sequence in which the MTS and PEF were applied was found to have a significant impact on the level of microbial reduction achieved (p ≤ 0.05). The sequence of MTS followed by PEF was the most effective in inactivating L. innocua achieving a mean reduction of 5.6 log cfu/ml, thereby exceeding the 5 log cycles minimum requirement specified by the United States Food and Drug Administration (US FDA). Significantly (p ≤ 0.05) lower reductions of 4.2 log cfu/ml were achieved when the PEF + MTS sequence combination was applied. The combination of MTS + PEF achieved inactivation comparable to thermally treated samples (p > 0.05). This study has shown the combination MTS + PEF is a promising hurdle preservation approach to control undesirable microorganisms in milk based smoothie beverages.     

Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR

The increase of foodborne outbreaks associated with fresh vegetables has highlighted the importance of developing rapid and specific methods for the detection and quantification of foodborne pathogens. In this sense, real-time PCR (qPCR) fulfills these requirements although it may detect dead cells. Recently, a potential strategy to specifically detect viable cells has been proposed relying on the use of DNA binding molecules as sample pretreatment previous to the qPCR. In this study propidium monoazide (PMA) and reagent D, combined with qPCR, were evaluated for the detection and quantification of viable Escherichia coli O157:H7, Salmonella and Listeria monocytogenes. Initially, the optimal concentration of both reagents was determined for discrimination between viable and dead bacteria in cell suspensions. Although both reagents showed similar reductions for the three pathogens, reagent D was toxic to L. monocytogenes and Salmonella and therefore only PMA was used to evaluate the applicability of this technique on food samples. A final concentration of 50 μM PMA was assayed in artificially inoculated spinach and mixed salad. PMA-qPCR signal was negative for all dead cell concentrations tested except for mixed salad inoculated with L. monocytogenes at the highest concentration. These results demonstrate that PMA-qPCR is a suitable technique for the detection and quantification of viable pathogens in fresh-cut vegetables at the levels normally found in vegetable samples.     

Quantification and differentiation of Bacillus cereus group species in spices and herbs by real-time PCR

Spores of Bacillus (B.) cereus group species are frequent contaminants in foodstuffs including spices and herbs. However, the distribution of individual B. cereus group species is unknown as standard cultural methods are insufficient for differentiation. Real-time PCR is an alternative method to detect, differentiate and quantify B. cereus group species in food.In our study we applied a combination of previously described real-time PCR assays to detect and quantify the B. cereus group (excluding B. cytotoxicus) with simultaneous discrimination of B. pseudomycoides and cry1-positive B. thuringiensis as well as differentiation of B. weihenstephanensis from B. cereus group species with non-rhizoid colony morphology. For testing food matrices, which can also include PCR inhibiting substances, an internal amplification control was included. In total, five DNA extraction kits were tested on pure spore suspensions to select the one with the best recovery rate when coupled to real-time PCR. The Qiagen DNeasy Blood & Tissue Kit performed best with a limit of detection (LOD) of approximately 100 cfu/ml for spores of each B. cereus, B. weihenstephanensis, B. thuringiensis and B. pseudomycoides strain. However, applied to allspice, paprika, pepper and oregano artificially contaminated with B. cereus spores the LOD was ≥10⁵ cfu/g. In contrast, using the open-formula cetyltrimethylammonium bromide (CTAB) method LODs of 10² to 10³ cfu/g were achieved for paprika, pepper and oregano. For allspice, the LOD was 10⁶ cfu/g.Our quantitative multiplex real-time PCR coupled to DNA extraction by the CTAB method provides a sensitive culture independent technique with the potential to quantitatively detect and differentiate B. cereus group species in several spices and herbs.     

Contamination of milk with Bacillus cereus by post-pasteurization surface exposure as evaluated by automated ribotyping

Over the last decade, increased transportation of refrigerated raw milk from farms to factories has raised concerns over Bacillus cereus contamination in the Brazilian dairy industry. Twelve isolates from pasteurized milk and 30 isolates from the post-pasteurization equipment surfaces of a dairy processing unit were characterized as B. cereus. Seven ribotypes were identified, demonstrating the genetic variability of this microorganism. Most of the isolates belonged to the same ribogroup (RIBO1222-73-S4), and they were found on four surfaces and also in the milk, indicating the role of the equipment surfaces as reservoirs for milk recontamination.     

Characterization of a novel bacteriocin produced by Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish

A novel bacteriocin, sakacin LSJ618, produced by the strain Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish, was studied. L. sakei LSJ618 was identified by both phenotypical and physiological tests combined with 16S rDNA sequence analysis. Sakacin LSJ618 is sensitive to hydrolytic enzymes including lipase, is stable between pH 2-8, and is heat resistant (30 min at 121 °C). Sakacin LSJ618 exhibits inhibitory activity against food-spoiling bacteria and food-borne pathogens, including the Gram-positive Listeria monocytogenes, Staphylococcus aureus, Sarcina sp., Micrococcus luteus, and the Gram-negative Proteus sp. and Escherichia coli, but not against most of the lactic acid bacteria tested. Maximal production of bacteriocin was reached in the late stationary phase, and inhibitory activity declined within 26 h. The mode of action of sakacin LSJ618 was determined to be bactericidal, as evidenced by its action upon Micrococcus tetragenus. After partial purification by ammonium sulfate precipitation and Sephadex G-25 chromatography, the molecular weight of sakacin LSJ618 was determined to be 5.2 kDa by Tricine-SDS-PAGE. The identified properties of sakacin LSJ618 indicate that it is a novel bacteriocin with potential application as a biopreservative in the food industry.     

Application of MPN-PCR in biosafety of Bacillus cereus s.l. for ready-to-eat cereals

Since Bacillus cereus is one of the important foodborne pathogens, it is interesting to investigate the biosafety of Bacillus spp. and B. cereus in ready-to-eat cereals marketed local supermarkets. For this investigation, the prevalence and enumeration of Bacillus spp. and B. cereus were assayed using MPN-PCR method. Results showed that 78% of the processed cereal products intended for direct consumption were positive for the presence of B. cereus with concentrations ranging from as low as 30 MPN/g to more than 24,000 MPN/g. The concentration obtained from this study also reflects on the differences in the contamination level between the infant food, raw cereals, cereal bars, ready-to-eat breakfast cereals and pre-mixed drinks examined. Hence, application of the MPN-PCR method was found to be useful to address the biosafety concerns of B. cereus in ready-to-eat cereals.     

Removal of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 biofilms on stainless steel using scallop shell powder

Biofilms on steel surfaces containing Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 continue to threaten dairy and meat processors. In this study, the ability of scallop shell powder (SSP) to remove biofilms formed by these three pathogens on stainless steel plates was examined. Whey powder solution (WPS) and bench wash water (BWW) provided by dairy and meat factories, respectively, were inoculated with L. monocytogenes, S. aureus or E. coli O157:H7 (9 log₁₀ CFU/ml). Stainless steel plates (10 cm²) were placed in the inoculated fluids and incubated at 20 °C at 48 h to form biofilms. After drying and washing in sterile water, the plates were treated with 0.0, 0.25, or 0.50% (w/v) SSP slurries for 1, 5, or 10 min and then quantitatively examined for the three pathogens. Both 0.25 and 0.50% SSP reduced L. monocytogenes on the plates by 4 log CFU/cm² with a 1 min exposure to 0.50% SSP decreasing S. aureus by 5 logs CFU/cm². After 1 min in 0.25 and 0.50% SSP, E. coli O157:H7 populations in WPS and BWW biofilms decreased 4 and 6 log CFU/cm² and 3 and 5 log CFU/cm², respectively. Increasing the concentration of SSP led to significantly increased efficacy against the tested pathogens (P < 0.05). In conclusion, this study showed that SSP slurries could significantly reduce the numbers of L. monocytogenes, S. aureus and E. coli O157:H7 in biofilms on stainless steel surfaces.     

Control of milk pH reduces biofilm formation of Bacillus licheniformis and Lactobacillus paracasei on stainless steel

Microbial biofilms present in dairy farms may contaminate milk during milk harvest and transfer diseases from the environment to cows. In order to reduce biofilm formation with respect to the role of pH, a study involving the control of milk pH during long-term biofilm formation of Bacillus licheniformis NBRC 12195 and Lactobacillus paracasei subsp. paracasei NBRC 15889 on stainless steel coupons in different dilutions of skim milk (0.1%, 1.0% and 5.0%) was conducted. During long incubation at 30 °C, pH decreased due to bacterial development in unadjusted samples. In pH-adjusted samples, pH was kept at around 7.0 by the addition of sterile sodium hydroxide. Biofilms formed on stainless steel coupons were daily stained by 0.1% Crystal Violet solution and assessed by the evaluation of optical density. The bacterial count of the suspensions showed that the control of pH enhanced the growth of bacteria in free-floating form. In contrast, optical densities of biofilms formed in the pH-adjusted samples were significantly lower than in the pH-unadjusted samples in all of three skim milk dilutions. Comparison of maximum OD values of adhered cells at different nutrient levels also implicated that for both tested strains, thicker biofilms were formed in milk dilutions at higher nutrient levels. These results suggested that, control of milk pH and milk residue level could significantly reduce biofilm formation of the tested bacteria.     

Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B. cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B. cereus and 16S rRNA for non-emetic B. cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B. cereus, 77 non-emetic B. cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B. cereus without PMA treatment in pure bacteria culture was 4.0 × 10² CFU/mL, as low as 7.5 × 10⁰ CFU/mL for viable B. cereus without PMA treatment, and 7.5 × 10¹ CFU/mL for viable B. cereus with PMA treatment. B. cereus in spiked food produce was detected specifically and sensitively at 1.0 × 10³ CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples.     

Antioxidant and anti food-borne bacterial activities of extracts from leaf and different fruit parts of Myristica fragrans Houtt

The main products of Myristica fragrans are the dried seed (nutmeg) and aril (mace), which are used as spices or condiments to flavor foods. In this study, its leaf and different parts of fruit (pericarp, aril, seed-kernel and shell) were compared for their total phenolic content, antioxidant and anti food-borne bacterial capacities. The 80% methanol extracts of aril, seed-kernel and shell shared the highest total phenolic content with shell extract acted as the greatest primary antioxidant, by having the highest ferric reducing antioxidant power (FRAP) activity (EC₅₀ 9.7 ± 0.1 μg/mL), β-carotene-bleaching activity (EC₅₀ 21.5 ± 2.7 μg/mL) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity (EC₅₀ 160.9 ± 13.9 μg/mL), whereas the pericarp extract exhibited highest secondary antioxidant activity as a metal chelator (EC₅₀ 75.6 ± 14.4 μg/mL). Only the aril and seed-kernel extracts were found to inhibit the food-borne bacteria with the lowest minimum inhibition concentration of 50 μg/mL, against Staphylococcus aureus (ATCC12600) and Bacillus cereus (ATCC10876). Our findings suggest the possibility of using the aril and seed-kernel extracts as natural food preservative and other parts as a new source of natural antioxidant for food and pharmaceutical industries.     

Aflatoxin M1 contamination in dairy products marketed in Iran during winter and summer

In the present study, 298 dairy product samples consisting of pasteurized milk (91 samples), yoghurt (68 samples), white cheese (72 samples), butter (31 samples) and ice cream (36 samples) collected from popular markets in four large Iranian cities were examined for aflatoxin M₁ (AFM₁) by thin layer chromatography (TLC) technique. The toxin was detected in 66 (72.5%) pasteurized milk samples (mean: 0.052 μg/l; range: 0.013–0.250 μg/l), 45 (66.1%) yoghurt samples (mean: 0.032 μg/kg; range: 0.015–0.119 μg/kg), 59 (81.9%) white cheese samples (mean: 0.297 μg/kg; range: 0.030–1.200 μg/kg), 8 (25.8%) butter samples (mean: 0.005 μg/kg; range: 0.013–0.026 μg/kg) and 25 (69.4%) ice cream samples (mean: 0.041 μg/kg; range: 0.015–0.132 μg/kg). The concentration of AFM₁ in 36.2%, 20.6%, 30.5%, 9.6% and 27.7% of pasteurized milk, yoghurt, white cheese, butter and ice cream samples, respectively, were higher than Iranian national standard limits. Levels of AFM₁ in samples of pasteurized milk, yoghurt, butter and ice cream collected in winter were significantly higher (P < 0.05) than those collected in summer. In the case of white cheese, level of AFM₁ was higher in winter than in summer, but the difference was not statistically significant (P > 0.05). The results indicated that the contamination of the dairy products in such a level could be a serious public health problem at the moment.     

In vitro control of multiplication of some food-associated bacteria by thyme, rosemary and sage isolates

Antibacterial activity of thyme, rosemary and sage isolates obtained by supercritical fluid extraction and hydrodistillation was investigated on Geobacillus stearotermophillus, Bacillus cereus, Bacillus subtilis var. niger, Enterococcus faecium, Salmonella enteritidis and Escherichia coli strains. Bacillus species were the most susceptible to all tested isolates. The thyme isolates showed the strongest antibacterial activity against tested bacteria with MIC values of 40-640 μg/ml, followed by rosemary (MIC = 320-1280 μg/ml) and sage (MIC = 160-2560 μg/ml) isolates. Therefore, the antibacterial activity of the most abundant components found in the thyme isolates, thymol, p-cymene and their mixture was investigated as well. The thyme isolates, especially supercritical extract, showed stronger antibacterial activity against Bacillus strains compared to the single components and their mixture, which indicated synergetic effect of the other components. Results of this study indicated thyme as a valuable source of natural antibacterial agents and supercritical fluid extraction as an efficient isolation method.     

Effect of Hibiscus sabdariffa extract and Nigella sativa oil on the growth and aflatoxin B1 production of Aspergillus flavus and Aspergillus parasiticus strains

This study was undertaken to evaluate the inhibitory effect of Hibiscus sabdariffa calyx extract at concentrations of 5, 7.5, 10 and 12.5 g/100 ml and Nigella sativa oil at concentrations of 1, 2 and 3 ml/100 ml on the growth and aflatoxin B₁ production by Aspergillus parasiticus (CBS 921.7) and Aspergillus flavus (SQU 21) strains. The inhibition of aflatoxin B₁ production by the different concentrations of H. sabdariffa calyx ranged between 91.5-97.9% and 87.1-93.3% for A. flavus and A. parasiticus strains, respectively, whereas the inhibition by different concentrations of N. sativa oil ranged between 47.9 and 58.3% for A. flavus and 32-48% for A. parasiticus strains. The different concentrations of H. sabdariffa calyx and N. sativa oil had no significant effect on the growth of either Aspergillus species. Neither H. sabdariffa calyx nor N. sativa oil detoxified pure aqueous aflatoxin. Our results suggest that H. sabdariffa calyx and N. sativa oil extracted from seeds had metabolic effects on aflatoxin biosynthesis pathway of both Aspergillus species and can be used as an effective biocontrol and non-toxic biopreservatives in food industry against aflatoxin contamination.     

Quantitative risk assessment of Mycobacterium avium subsp. paratuberculosis survival in pasteurized milk in three dairy plants in Italy

The objective of this study was to carry out a quantitative risk assessment (QRA) of Mycobacterium avium subsp. paratuberculosis (MAP) survival in pasteurized milk produced by industrial dairy plants. Data were collected in three dairy plants (A, B and C) located in three different Italian regions and processing 38.75 (plant A), 89.29 (plant B) and 190.56 million litres (Plant C) of milk yearly. Plants A and plant C produce pasteurized milk, soft and hard industrial cheeses and yogurt; plant B produces only pasteurized milk. In-line milk filter (ILMF) samples and/or bulk milk samples were collected from all 569 herds delivering milk to the three dairy plants. Samples were analysed by quantitative real-time PCR (qPCR). The QRA considered the presence of MAP in ILMF and in bulk milk of all the dairy herds delivering milk to the three investigated dairy plants, estimating MAP concentration in raw milk on the basis of these data, the dilution effect due to mixing milk in collecting trucks and in plant silos, and the effect of pasteurization in reducing the MAP load. The expected fraction of litres of pasteurized milk with 0 MAP would be 99.02%, 99.45% and 99.12%, in plants A, B and C respectively, and an overall percentage 0.55% to 0.98% of pasteurized milk having a MAP contamination >0 colony forming units (CFU)/l and 0.04%–0.11% of pasteurized litres with a MAP contamination > 100 CFU/l was predicted. A daily variation was observed in the proportion of MAP-contaminated litres of milk. The study demonstrated that milk in the dairy plants investigated may be a source of MAP exposure for humans. The between-herd and within-herd MAP apparent prevalence in the investigated areas are likely comparable to those in other areas in Italy, Europe and North America, and the results are applicable to other geographical areas.     

Effects of spray volume, type of surface tissue and inoculum level on the survival of Escherichia coli on beef sprayed with 5% lactic acid

Membrane, fat and cut muscle surfaces of beef were inoculated with Escherichia coli at numbers about 4, 1 or −1 log cfu/cm². The inoculated meat was sprayed with water or 5% lactic acid at volumes of 0.5, 0.1 or 0.02 ml/cm². Spraying with water reduced the numbers of E. coli on membrane surfaces by up to 1 log unit, but had little effect on the numbers of E. coli on fat or cut muscle surfaces. Spraying with 5% lactic acid reduced the highest numbers of E. coli on membrane surfaces by up to 4 log units; but those numbers on fat or cut muscle surfaces were reduced by ≤1.5 log unit, and the reductions declined with decreasing volumes of 5% lactic acid. With inocula of 1 log cfu/cm², spraying lactic acid in any volume reduced the numbers of E. coli on membrane or fat surfaces by about 1 log unit, and the numbers on cut muscle surfaces by between 0.8 and 0.2 log unit. E. coli were detected in enrichment cultures of samples from all surfaces inoculated with E. coli at −1 log cfu/cm² and sprayed with 5% lactic acid at 0.5 ml/cm². The findings indicate that spraying relatively heavily contaminated cuts or trimmings with 5% lactic acid at ≥0.1 ml/cm² can be expected to reduce numbers of E. coli and, presumably, associated pathogens by between 0.5 and 1 log unit. However, such a treatment is likely to be at best marginally effective for reduce the numbers of these organisms on lightly contaminated product.     

Study of physicochemical parameters and spontaneous fermentation during traditional production of munkoyo, an indigenous beverage produced in Democratic Republic of Congo

The evolution of physicochemical parameters and spontaneous fermentation were investigated during traditional production of munkoyo. During munkoyo preparation, starch hydrolysis by amylolytic enzymes contained in munkoyo roots was performed at temperatures above 43 °C. After 90 h, pH decreased from 6.4-6.2 to 3.7-3.6; lactic acid concentration and acidity increased respectively from 4.4-15.0 mmol l⁻¹ to 42.0-44.0 mmol l⁻¹ and 0.05% to 0.45-0.6%; alcohol content did not exceed 394 mmol l⁻¹. When the temperature dropped from 43-42 °C to 29-25 °C, in the first 15 h of fermentation, the rate of pH decrease was significant, from 6.4-6.2 to 4.1-4.0. The acidification of munkoyo was due to lactic acid, largely in the form of the d (−) lactate isomer. At temperatures around 20 °C, secondary products also contributed to the acidification of the beverage. Lactic fermentation, observed from 43 °C in the wort, was promoted by thermophilic lactic acid bacteria. Lactobacillus delbrueckii lactis was identified as the representative lactic acid bacteria. Alcohol production in munkoyo, beginning only after 15 h of fermentation, is due mainly to Saccharomyces cerevisiae.     

Aflatoxin and fumonisin contamination of yam flour from markets in Nigeria

The natural occurrence of aflatoxins (AFs) and fumonisins (FBs) in yam flour samples (n = 100) obtained in south-western Nigeria was evaluated. AFs were determined by HPLC with fluorescence detection and FBs by HPLC coupled with mass spectrometry. Aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁) were found in 57% and 21% of flours from white yam with concentrations ranging from <0.02 (limit of detection, LOD) to 3.2 μg kg⁻¹ (mean = 0.4 μg kg⁻¹) and from <0.05 to 3.5 μg kg⁻¹, respectively. AFB₁ was the only aflatoxin detected in samples from water yam, contaminating 32% of the samples with values ranging from <LOD to 0.6 μg kg⁻¹ (mean = 0.1 μg kg⁻¹). Fumonisin B₁ was found in 32% of the white yam samples (<0.5 (LOD) to 91 μg kg⁻¹; mean = 5 μg kg⁻¹) and in 5% of water yam samples (<LOD to 2 μg kg⁻¹). AFs and FBs were significantly higher (P < 0.05) in white yam flours compared to water yam flours. Preparation of amala from naturally-contaminated yam flour resulted in reduction of AFB₁ and AFG₁ by 44% and 51% respectively. From this study, only 7% of the samples contained AFs above the European standard limits for cereals intended for direct human consumption, while all the FBs-positive samples were well below the limits. The occurrence of ochratoxin A, zearalenone and deoxynivalenol was also evaluated in 20 samples; these mycotoxins were never detected.