Oxytetracycline Glucose Yeast Extract Research Articles

Fungal profile, levels of aflatoxin M1, exposure, and the risk characterization of local cheese ‘wagashi’ consumed in the Ho Municipality, Volta Region, Ghana

Wagashi is a West African type cottage cheese locally prepared from cow milk. Wagashi like other milk products, is prone to microbial contamination, particularly by fungi. Many of these fungal species produce mycotoxins which are of serious public health concern. This work aimed to update the mycoflora profile and determine the concentrations of aflatoxin M1 and its health risk characterization due to the consumption of wagashi. Culturing the wagashi on mycological media (Oxytetracycline Glucose Yeast Extract OGYE, Dichloran Rose Bengal Chloramphenicol DRBC) caused a de-novo growth of the quiescent spores at 28–30 °C for 5–7 days. The analysis of AFM1 levels in the samples was done using High-Performance Liquid Chromatography connected to a Fluorescence detector (HPLC-FLD). The exposure and risk assessment to the AFMI levels were determined using deterministic models prescribed by the European Food Safety Authority (EFSA). The fungal counts ranged between 2.36–4.30 log10 CFU/g. In total, thirteen (13) fungal species from eight (8) genera were isolated from all wagashi samples. They are; Fusarium oxysporum, Aspergillus flavus, Aspergillus niger, Fusarium verticillioides, Penicillium digitatum, Trichoderma harzianum, Aspergillus terreus, Rhodotorula mucilaginosa, Rhizopus stolonifer, Aspergillus fumigatus, Yeast sp., Mucor racemosus and Fusarium oligosporum belonging to the genera Fusarium, Aspergillus, Penicillium, Trichoderma, Rhodotorula, Rhizopus, Yeast, and Mucor. The AFM1 observed in the wagashi samples' analysis was low, ranging from 0.00 (Not Detected) ± 0.00 − 0.06 ± 0.002 µg/Kg. Risk assessments of AFM1 using deterministic models produced outcomes that ranged between 5.92 × 10−3- 0.14 ng/kg bw/day, 1.42 –44.35, 0–0.0323 ng aflatoxins/kg bw/day, and 1.51 × 10−3 − 9.69 × 10−4 cases/100,000 person/yr for estimated daily intake (EDI), margin of exposure (MOE), average potency, and cancer risks, respectively, for the age categories investigated. Fungal counts were interpreted as medium to high. It was also established that the consumption of wagashi may pose adverse health effects on all age categories in the selected zones of the study since all calculated MOE values were less than 100,000.

Toxicogenic fungal profile, Ochratoxin A exposure and cancer risk characterization through maize (Zea mays) consumed by different age populations in the Volta region of Ghana

Maize (Zea mays) is an important staple food crop for the majority of Ghanaians. Maize is mostly contaminated by fungal species and particularly mycotoxins. This work aimed to identify and quantify the incidence of fungal infection and exposure to Ochratoxin A (OTA) as well as the health risk characterization in different age populations due to maize consumption in the Volta region. Maize samples were plated on Dichloran Rose Bengal Chloramphenicol (DRBC) agar, and Oxytetracycline Glucose Yeast Extract (OGYE) agar. All media were prepared in accordance with the manufacturers' instructions. The plates were incubated at 28±2°C for 5-7 days. High-Performance Liquid Chromatography connected to a fluorescence detector (HPLC-FLD) was used to analyze the ochratoxin A (OTA) levels in maize. Cancer risk assessments were also conducted using models prescribed by the Joint FAO/WHO Expert Committee on Additives (JECFA). The maize samples collected from the Volta region contained fungal population between the range of 3.08-4.58 log10CFU/g. Eight (8) genera were recorded belonging to Aspergillus, Trichoderma, Penicillium, Fusarium, Saccharomyces, Mucor, Rhodotorula and Rhizopus. The species diversity includes A. flavus, A. niger, T. harzianum, P. verrucosum, F. oxysporum, Yeast, F. verticillioides, Rhodotorulla sp, A. fumigatus, R. stolonifer, M. racemosus species. Additionally, the ochratoxins level contained in the samples were very noteworthy and ranged from 1.22 to 28.17 μg/kg. Cancer risk assessments of OTA produced outcomes also ranged between 2.15 and 524.54ng/kgbw/day, 0.03-8.31, 0.0323, and 0.07-16.94 for cases/100,000 person/yr for Estimated Daily Intake (EDI), Margin of Exposure (MOE), Average Potency, and Cancer Risks respectively for all age categories investigated. There was very high mycoflora load on the maize sampled from the Volta region, likewise the range of mycotoxins present in the maize grains, suggesting the potential to pose some adverse health effects with the populace of the Volta region.

Mycobiota profile, phenology, and potential toxicogenic and pathogenic species associated with stored groundnuts (Arachis hypogaea L.) from the Volta Region, Ghana

This study updates the mycobiota resident in groundnut seeds, their phenology during storage with the view to ascertain their occurrence, potential toxigenic species, and pathologically important species in the stored samples. The moisture content of the seeds ranged from 5.7% to 6.5% within the stipulated safe moisture content of 8% for extension of shelf life. Culturing the seeds on mycological media (Sabouraud's Dextrose Agar SDA; Oxytetracycline Glucose Yeast Extract OGYE, Potato Dextrose Agar, PDA) caused a de novo growth of the quiescent spores at 28–30°C for 7–14 days. Fungal population counts on the three media ranged from 2.01 to 2.16 log10 CFU/g samples to a final 6‐month count of 1.67–2.60 log10 CFU/g. Eighteen different fungal species belonging to ten genera were encountered on the media, namely Aspergillus, Cladosporium, Curvularia, Fusarium, Penicillium, Trichoderma, Rhizopus, Rhodotorula, Sporendonema, and Paecilomyces. Aspergillus spp. (A. niger, A. flavus, A. fumigatus, and A. terreus) were the most frequently isolated, followed by Fusarium species (F. oxysporum, F. solani, and F. verticillioides), Trichoderma (T. harzianum and T. viride), Rhizopus spp (R. oligosporus and R. stolonifer), and Penicillium verrucosum. The species which were seed borne (A. niger, A. flavus, A. terreus, A. fumigatus, F. solani, F. verticillioides, T. viride, C. herbarum, and Curvularia lunata) were isolated on both surface sterilized and non‐surface sterilized seeds. The phenology of the encountered fungal species generally followed five patterns. The most frequently isolated Aspergillus niger, A. flavus, and A. fumigatus predominated throughout the 6 months sampling period, while A. ustus and A. terreus appeared sporadically and disappeared. The early colonizers (R. oligosporus, R. stolonifer, and Paecilomyces) could not be isolated after 2–3 months owing presumably to stronger antibiosis competition from the Aspergillus species. The most predominant Aspergillus species initially constituted 36%–48% of the total population but declined to 10%–36% in 6 months. Mycobiota encountered with mycotoxigenic potential and human health importance were A. niger, A. flavus, A. fumigatus, F. verticillioides, and Penicillium verrucosum. Other species of pathological importance to plants were Curvularia lunata and Fusarium oxysporum. The practical implications of these findings are discussed.

Fungal diversity of "solom" a Ghanaian traditional beverage of millet (Pennisetum glaucum).

The association of cereals with fungi cannot be disregarded as their manifestation in our foods poses serious health risks. The aim of this study was to investigate the mycofloral (fungal) and chemical (pH) qualities of the “solom” (beverage of millet) available for consumption from their respective sales points in Ho. “Solom” a cereal beverage of millet was sampled from ten (10) different locations in the Ho Municipality of Ghana and evaluated for their pH, fungal counts, and species diversity. Mycological analyses were done on Oxytetracycline Glucose Yeast Extract (OGYE) and Dichloran Rose Bengal Chloramphenicol (DRBC) media from three (3) points per location using serial dilution. A total of fourteen (14) fungal species belonging to eight (8) genera were isolated on both media; Aspergillus (A. niger, A. flavus, A. fumigatus, A. parasiticus, A. alutaceaus, A. terreus), Rhizopus (R. stolonifer), Mucor (M. racemosus), Fusarium (F. oxysporum), Penicillium (P. digitatum, P. verucosum), Cladosporium (C. cladosporoides), Curvularia (C. lunata), and Rhodotorula sp. were recorded. Fungal counts on both media ranged between 1.68 ± 0.8 and 4.11 ± 0.9 log10 CFU/ml. There were statistically significant (p < .05) differences observed in the samples from different locations. The values of pH recorded were in the range of 3.03 ± 0.09–4.03 ± 0.23 and showed no significant differences (p > .05) among them. All samples were found to be in the acceptable range of values prescribed by the International Commission for Microbiological Specification of Foods (ICMSF, 1998). Good Manufacturing Practices (GMP) and Good Hygiene Practices (GHP) should be employed to enhance food safety.

Evaluating the Effect of Gamma Irradiation and Steam Sterilization on the Survival and Growth of Composted Sawdust Fungi in Ghana

The growth and survival of some fungi associated with composted and pretreated sawdust particularly for mushroom cultivation were investigated on two growth media; Cooke’s and Oxytetracycline Glucose Yeast Extract (OGYE). Some fungi were isolated during the composting of sawdust over a period of 28 days as well as after pretreatment with gamma irradiation doses of 5, 10, 15, 20, 25 and 32 kGy and moist heat of 100±2oC for 2.5 hours. Fungal counts ranged 4.725.77 log10 CFU/g and 3.44.1 log10 CFU/g respectively for both media. Both pretreatment methods effectively reduced (p<0.05) fungal counts by an average of 1.48 (irradiation) and 2.22 (steam) log-cycle reductions on OGYE while there was an average of 3.13 (irradiation) and 1.10 (steam) Original Research Article Kortei et al.; BMRJ, 7(4): 180-192, 2015; Article no.BMRJ.2015.110 181 log-cycle reduction on Cooke’s. Corresponding radiation sensitivities (D10 values) of 5.94±2.06 kGy and 5.64±1.12 kGy were recorded for fungi on both media respectively. Five species belonging to three genera were isolated on OGYE and among the fungi were Aspergillus niger, Aspergillus fumigatus, Aspergillus ustus, Mucor racemosus and Rhizopus stolonifer. Ten species belonging to four genera were also isolated on Cooke’s medium included Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Aspergillus ustus, Aspergillus terreus, Aspergillus parasiticus, Aspergillus alutaceus, Rhizopus stolonifer, Fusarium oxysporum and Mucor racemosus. There was an observed reduction (p<0.05) in species diversity after pretreatment. The presence of these microorganisms on sawdust also has serious repercussions on animal and human health.

Comparison of selective media for the recovery and enumeration of probiotic yeasts from animal feed

Six selective media (acidified malt extract agar, CHROMagar Candida, dichloran rose bengal chloramphenicol, molybdate, oxytetracycline glucose yeast extract and Petrifilm Yeast and Mould agar) were examined for the recovery of three yeasts commonly used in animal feeds as probiotic additives ( Candida pintolopesii, C. saitoana and Saccharomyces cerevisiae). The highest recovery was obtained on oxytetracycline glucose yeast extract agar, although this was susceptible to overgrowth by moulds. CHROMagar Candida also gave good recovery and species were easily discriminated by the differential colour formation of colonies.

Penicillium species during the manufacturing and ripening of Armada raw goat's milk cheese: identification, characteristics and in vitro potential toxins production

The species and characteristics of the moulds of the Penicillium genus were studied throughout the manufacturing and ripening of Armada cheese. Along the ripening of four batches of cheese, samples of milk, curd, and 1-,2-,4-, 8-, and 16-week-old chee se were studied. Of the 274 iso- lates obtained from the Oxytetracycline Glucose Yeast-Extract Agar medium throughout the manu- facturing and ripening, 28 were considered moulds belonging to the Penicillium genus and were identified using both morphological and physiological tests as following: Penicillium roqueforti (10 strains), P. crustosum (6 strains), P. commune (4 strains), P. aurantiogriseum (3 strains), P. ver- rucosum chemotype 1(2 strains), and P. chrysogenum (3 strains). The presence of the species of the Penicillium genus was more important in the last stages of ripening. This fact could be related to the marked increase in the content of total free fatty acids, and of short-chain fatty acids in particular, which is observed after the second mon th of ripening in this cheese. The mycotoxin production was inves- tigated by thin-layer chromatography techniques. Ali the strains were toxigenic in vitro. © InralElse- vier, Paris. Armada goat cheese 1 mou Id 1Penicillium 1 mycotoxin

Study of the yeasts during the ripening process of Armada cheeses from raw goat's milk

The counts, species and characteristics of yeasts were studied throughout the manu- facturing and ripening of four batches of Armada chee se, a craft variety made in the north of Spain from raw goat's milk. Counts in Oxytetracycline Glucose Yeast-Extract Agar (1.82 x 104 - 1.15 x lOS cfu·g-1 in milk) increased one log. unit in curd and another log. unit during the first week ofripening, remaining stable in two- and four-week-old cheeses and decreasing from this point to the end of the ripening process. The yeast percentage of the isolates carried out remained practically constant at around 75 % throughout the ripening process, dropping to 27 % at the last sampling point (16-week-old cheese). Of the 181 isolated yeast strains, 60 were identified as Geotrichum candidum, 58 as Candida lambica, II as Candida krusei, 4 as Candida lipolytica, 2 as Candida ingens, 1 as Candida catenu- lata, 1 as Candida pintolopesii, 1 as Candida colliculosa, 19 as Kluyveromyces lactis, II as Kluyveromyces marxianus, 10 as Saccharomyces unisporus and 3 as Trichosporon beigelii. G. can- didum predominated in 1- and 2-week-old cheeses and C. lambica was the most abundant species in milk and in 4- and 8-week-old cheese while C. krusei dominated in 16-week-old cheese. Judging from their metabolic characteristics, the yeasts in Armada cheese seem to participate in the initial acidifi- cation of the curd and in the consumption of lactic acid which takes place throughout the ripening pro- cess, though, with the exception of G. candidum, they do not seem to play a relevant role in the lipolysis, either way its resistance to NaCI is not very high. However, they seem to play an important role in the degradation of the biogenic amines; of the 181 strains identified, 136 assimilated the four biogenic amines tested (cadaverine, histamine, putrescine and tyramine) and ail the strains assimilated at least one ofthese amines. © Inra/Elsevier, Paris.

Mycoflora of paddy and milled rice produced in the region of Northeastern Argentina and Southern Paraguay

Thirty samples of paddy rice and twenty-five of milled rice were obtained from processing centers located in two northern Provinces of Argentina and one southern Province of Paraguay. Contaminating fungi were enumerated by direct plating on dichloran rose bengal chloramphenicol agar and oxytetracycline glucose yeast extract agar before and after surface disinfection. All fungi were isolated and identified to the genus level and percentage infection of samples calculated. Those belonging to the genera Penicillium, Aspergillus and Fusarium were identified to species level. The surface mycoflora was dominated by storage fungi, notably Penicillium citrinum (73% of samples), P. islandicum (60% of samples), Aspergillus niger, A. flavus and Fusarium semitectum. The major fungi found as internal contaminants of paddy rice were, again, Penicillium citrinum (66% of samples) and P. islandicum (50% of samples). Milled rice showed a lower level of contamination, but with a similar species distribution, Penicillium citrinum and P. islandicum again being the main contaminants. The presence of these species suggests a potential for mycotoxin production. Further studies are needed to establish the mycotoxin quality of rice from this region.

Effect of water activity, modified atmosphere packaging and storage temperature on spore germination of moulds isolated from prunes

The combined effect of medium water activity ( a w) modified atmosphere packaging (MAP) on the conidial germination of Aspergillus niger, Eurotium amstelodami, Penicillium chrysogenum and Fusarium oxysporum was studied by applying response surface methodology (RSM) for 45 days incubation at 20, 30 and 40 °C. Oxytetracycline glucose yeast extract (OGYE) agar media were prepared over a wide range of a w (0.80–0.95), controlled by glucose. The headspace atmosphere composition varied from 0–20% for O 2 and from 0–80% for CO 2 with N 2 as balance in the ratio medium gas mixture of 1 3 ( v v ). The results of this experiment were confirmed by repeating some combinations ( a w-MAP) in the zone where the inhibition was effective. In contrast to A. niger which germinated and grew better at 30 and 40 °C, P. chrysogenum and F. oxysporum were capable of germinating and growing at 20 °C but not at 40 °C. E. amstelodami was less influenced by the incubation temperature than were the other species. Under anaerobic atmospheres, germination and growth of all tested species were completely inhibited at low a w, while at high a w because of residual O 2, conidia germinated but failed to grow. At any a w value between 0.88 and 0.92, conidia failed to germinate when 100% CO 2 was applied. Similar results were obtained for E. amstelodami and F. oxysporum in 80% CO 2-20% N 2 and 60% CO 2-40% N 2. Under aerobic conditions, at 5% O 2 germination and growth occurred only at high a w, while 10 or 20% O 2 combined with either 80 or 60% CO 2 conidial germination and mould growth were only delayed comparing to the control (air).

Enumeration of xerophilic yeasts in the presence of xerophilic moulds: a collaborative study

A collaborative study was undertaken to compare the performance of Dichloran 18% Glycerol agar (DG18) with three other media widely used in food mycology, for the enumeration of xerophilic yeasts in the presence of xerophilic moulds. Oxytetracycline Glucose Yeast extract agar, (OGY), Dichloran Rose Bengal Chloramphenicol agar (DRBC) and Malt extract Yeast extract 50% Glucose agar (MY50G) were evaluated. Three reference samples (A, B, C) were prepared using skimmed milk powder inoculated with mixed lyophilized cultures of selected xerophilic yeasts and moulds, at levels around 10 4 to 10 5 CFU/g. Yeast species used were Candida glucosophila, C. versatilis, Zygosaccharomyces ballii and Z. rouxii, together with Eurotium spp. and some other moulds. Collaborators were asked to examine each sample twice, by dilution plating on the four media. Colonies were counted after 3, 5 and 7 days incubation at 25 °C. Fourteen participants from seven laboratories in six countries collaborated in this study. The ability of collaborators to detect and count yeasts after 5 and 7 days on DG18, DRBC and OGY varied depending on the sample. Few participants were able to count yeasts even after 7 days on MY50G. The overall results demonstrated that DG18 incubated 5 days at 25 °C was superior to the other three media tested for the enumeration of xerophilic yeasts in the presence of xerophilic moulds.

A 2‐year survey of the airborne mycoflora in a hospital environment

Studies on levels of airborne fungal spores in hospital wards and a diet kitchen were carried out for a period of 24 months using an air sampler. In a preliminary study using Sabouraud dextrose agar (SDA), SDA with chloramphenicol, acidified malt extract agar, oxytetracycline glucose yeast extract and Rose‐Bengal chloamphenicol varying levels of fungi were recovered on each of these media. Sabouraud medium, which is used to recover medically important fungi, showed relatively lower fungal counts with an average of 89 cfu m‐3 as compared with counts > 210 cfu m‐3 on all the other media. Fungi accounted for about 68% of the total viable counts and the predominant genera were Aspergillus, Fusarium, Monilia, Penicillium, Rhizopus, Monilia, Candida, Rhodotorula and Pichia. Spore counts were higher in the wards (303 cfu m‐3) than in the diet kitchen (208 cfu m‐3) The aerial fungal spore counts were 51% higher in all the areas sampled during the warmer months than the colder months but there was no major change in the types of fungi except that Chrysonilia species were only isolated between October and March when they accounted for < 5% of the total fungal isolates.

Selective media for detecting and enumerating foodborne yeasts

No one medium is satisfactory for detecting, isolating and enumerating all yeasts in all foods. Antibiotic-supplemented media such as dichloran rose Bengal chloramphenicol agar, tryptone glucose yeasts extract chloramphenicol agar, oxytetracycline glucose yeast extract agar and rose Bengal chloramphenicol agar are superior to acidified potato dextrose agar and other acidified media for enumeration of the vast majority of spoilage yeasts. Dichloran glycerol (18%) agar performs well for enumerating moderately xerotolerant yeasts. Malt extract yeast extract glucose (up to 60%) can be used for detecting and enumerating moderate and extreme xerophiles. These media also support the growth of moulds. Lysine agar, Schwarz differential agar and Lin's wild yeast differential agar are used by the brewing industry to differentiate wild yeasts from brewer's strains. Lysine agar is selective for apiculate yeasts and ethanol sulfite yeast extract agar is selective for Saccharomyces. Both have application in wineries. Modified molybdate agar can be used to selectively isolate yeasts from tropical fruits. Preservative-resistant yeasts can be detected on malt acetic agar. The recommended incubation temperature is 25°C, but incubation time between plating and counting colonies ranges from 5 days for determination of general populations of yeasts to 10 days for more for xerotolerant yeasts. There is need for new and improved media for selectively isolating various groups, genera, species and strains of yeasts capable of growing only under specific environmental conditions in specific types of foods and beverages.

Media for detecting and enumerating yeasts and moulds

Dilution plating techniques are designed to determine populations of viable fungal propagules per unit weight or volume of food. Direct plating techniques, on the other hand, are designed to assess the internal mycoflora of individual pieces of foods, e.g., seeds or dried fruits, and results are expressed as a percentage of infected pieces. Both techniques are used by industry and regulatory agencies to monitor levels of fungal contamination at various stages of food handling, storing, processing and marketing. Peptone (0.1%) water is commonly used as a diluent for samples to be homogenized or blended. Buffered diluents containing up to 30% glucose are sucrose are recommended for enumerating xerophiles. No one medium is satisfactory for detection or enumeration of yeasts and moulds in all foods. Dichloran rose bengal chloramphenicol agar, oxytetracycline glucose yeast extract agar and rose bengal chloramphenicol agar are superior to acidefied potate dextrose agar for enumeration of yeasts and moulds. Dichloran 18% glycerol agar performs well for enumerating moderating moderately xerophilic yeasts and moulds. Fastidous xerophiles require media containing high concentrations of sugars and/or sodium chloride. Media have been formulated to detect potentially aflatoxigenic aspergilli and mycotoxigenic strains of penicillia and fusaria, but increased selectivity and specificity of media for detecting mycotoxigenic moulds are needed. Heat-resistant mould ascospores often require heat treatment prior to plating in order to activate the fermination process. The spread-plate technique is strongly preffered over the pour-plate technique for enumerating yeasts and moulds. The recommended incubation temperature is 25°C, but incubation time between plating and counting colonies ranges from 5 days for determination of general populations of mycoflora to 4 weeks or more for fastidious xerophiles. There is a need for new and improved media for selectively isolating various groups, genera, species and/or strains of fungi capable of growing only under specific environmental conditions, e.g., low a w or, in the case of sublethally injured cells, under conditions which facilitate resuscitation. Improved media are needed which accurately detect moulds producing specific mycotoxins in a wide range of food types.

Enumeration and identification of airborne viable mould propagules in houses

A number of techniques for the enumeration and identification of viable mould propagules in the indoor air of houses were evaluated in order to document to what extent different results are obtained when different methods are used. A comparison was made between the results obtained with five commercially available air sampling devices (Slit-to agar sampler, N6-Andersen sampler, Surface Air System sampler, Reuter Centrifugal Air sampler, Gelatine Filter sampler) and a non-volumetric sampler (the Open Petri Dish), in combination with four culture media (malt extract agar, dichloran glycerol-18 agar, oxytetracycline glucose yeast extract agar and dichloran rose bengal chloramphenicol agar). The coefficients of variation were high (generally greater than 20%) for all combinations. Statistical analysis showed that the Slit sampler and the N6-Andersen sampler in combination with DG18 and MEA gave the best precision and the highest yield in terms of colony forming units per square cubic meter of air (CFU/m3) and number of species isolated.