Fungal diversity of “solom” a Ghanaian traditional beverage of millet (Pennisetum glaucum)

Abstract 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.

"Solom" a relatively lesser-known cereal beverage obtained from millet is consumed in West Africa particularly in Ghana, Togo, and some parts of Benin. It is an opaque (Plate 1) reddish-brown drink usually served chilled (with ice blocks). It is used for refreshment in both the rural and urban settlements. Culturally, the serving of solom like many other cereal-based beverages in Africa to visitors is considered a gesture of welcome and hospitality, and the beverage is served as part of the traditional initiation of young girls into womanhood (Misihairabgwi & Cheikhyoussef, 2017). "Solom," according to oral tradition, is served at traditional weddings and other important ceremonies as well as at daily social interactions (Embashu et al., 2013). The main ingredients for "solom" production are pearl millet meal, sorghum, or maize malt and water. The bran of millet is optionally added. Fermentation is achieved via chance inoculation, under uncontrolled environmental conditions, and the peculiar nutritional and sensory properties of "solom" derived mainly from the ingredients, with variable qualities which depend largely on the skills of the households. In some cases, back slopping is conducted by the local producers to enhance fermentation and product quality.
Fungal contamination of seeds, grains, and feedstuff is a chronic problem in the developing countries because most of the tropical hot and humid climates support the growth of these fungi in the field and storage systems. The presence and growth of fungi mostly cause spoilage of food quality and quantity-wise. These agricultural raw ingredients have been implicated in the transfer of fungal contaminants to processed foods (Candlish et al., 2001;Rasooli & Abyaneh, 2004).
The occurrence of some genera of fungi in our body system (mycosis) may be detrimental subsequently, certain mycotoxins (mycotoxicosis) produced have carcinogenic, mutagenic, teratogenic, and immunosuppressive implications on humans and animals alike. This is of major concern in evaluating the safety of the final product solom (Mastanjević et al., 2018). Despite the numerous reports on the spectrum and quantities of fungi and their metabolites in cereals used in the preparation and processing of traditional beverages, few studies have analyzed the transfer rate of fungal spores from ingredients to beverages, their concentrations in the beverages, and exposure rates for consumers (Ezekiel et al., 2019).
On another hand, filamentous molds are also responsible for the quality of alcoholic beverages including nutritional values and organoleptic properties such as flavor, taste, and color (Tamang et al., 2016).
It is noteworthy that no strict process control is imposed on traditional brewing, fermentation, or storage of raw cereal grains in Ghana. Poorly enforced regulations targeted at ensuring the microbiological safety levels in African traditional fermented beverages exist, despite the high fungal contamination levels in their raw materials and the high consumption levels of the beverages (Ezekiel et al., 2019). Fungal detection, quantification, and determination of fate during processing are therefore imperious in the cereals and traditionally fermented beverages.
Although scanty data exist (Aboagye et al., 2020;Minamor, Mensah, et al., 2017) on the fungal diversity of cereal-based drinks in Ghana, the ability of fungi to grow on some cereals has been demonstrated by some researchers Kpodo et al., 1996Kpodo et al., , 2000Narh et al., 2011) which points to the likelihood of transfer of fungal contaminants from these grains into these local cereal beverages and so good manufacturing and hygiene practices must be observed for the safety of these beverages. The aim of this study, therefore, was to investigate the mycofloral (fungal) and chemical (pH) qualities of the "solom" beverage available for consumption from their respective sales points.

| Study design and study area
This study was quantitative and purely experimental. It was conducted at the Microbiology Laboratory, School of Allied Health Sciences, University of Health and Allied Sciences, Ho, Volta Region, Ghana.

| Sampling
A total of 30 "solom" samples were purchased from ten (10) different locations in Ho, Volta Region, Ghana (Figure 1). From each location, P L AT E 1 Solom fetched in a calabash ready for drinking as sold on the markets three (3) different samples were purchased. They were stored in sterile plastic bottles and kept in an ice chest immediately after purchase and were transported to the laboratory for analysis.

| Equipment and materials sterilization
Sterilization of equipment was carried out before and after the analysis to prevent cross-contamination of samples, and equipment Petri dishes and universal bottles were washed and rinsed very well with soap and under running tap water and kept in a hot air oven for 1 hr at 180°C to ensure sterility. The media were prepared according to manufacturer's guidelines under safety conditions. A naked flame was turned on to prevent microbial contamination during processing sterilization, and 70% alcohol was also used to clean the working bench before and after use.

| Fungal plating and incubation
This was carried out according to the procedure outlined for plating by Kortei et al. (2018) with media and process modifications as follows: aliquots of one milliliter (1 ml) of each test sample were added to 9 ml of sterile distilled water and agitated vigorously and was used as the stock solution. The samples were serially diluted 10 -2 up to 10 -4 and then plated on Oxytetracycline Glucose Yeast Extract (OGYE) and Dichloran Rose Bengal Chloramphenicol (DRBC) agars. All media were prepared according to the manufacturer's specifications.
It was followed by incubation at 37°C for 7 days. After 1 week, observable molds and yeast appeared for counting and identification.

| Lacto phenol cotton blue teased mount procedure for identification
A drop of lactophenol cotton blue dye was placed on the slide, and a sterile iron needle was used to transfer a tiny piece of a colony into Lacto Phenol Cotton Blue Dye on the slide. The colony was then teased into very tiny pieces using an iron needle. The slide was covered with a coverslip with a magnification ×400 used. The identification of the fungi was done macroscopically (texture and color in the plate) and microscopically by observation of their cultural and morphological features (Table 1) under the microscope.
Molds and yeast that appeared were identified by their cultural and morphological characteristics using standard identification manuals (Moss, 1989;Samson et al., 2000). Fungal counts were recorded in standard form and then transformed into the logarithmic form as described by Kortei et al. (2018) and Odamtten et al. (2018).
Percentage occurrence of fungal species was calculated using the formula,

| Determination of pH
The pH of samples was determined directly with a bench pH meter (Jenway 3510) after calibration using standard buffers 4.0 and 7.0 pH.

| Data analysis
Procedures of fungal counts and pH were carried out in triplicates, and data collected were subjected to a single-factor analysis of variance (ANOVA). Differences among means were separated using Duncan's multiple range test (DMRT) and significances were accepted at a 5% level (p < .05) using Statistical Package for the Social Sciences (SPSS) software version 22. The analysis was done using the mean counts expressed in the standard forms which were transformed into logarithmic values and results reported as means + standard deviation.

| RE SULTS
Results of the different fungal counts of solom from the different locations are represented in Table 2. For OGYE, the fungal counts ranged between 1.68 ± 0.8 and 4.11 ± 0.9 log 10 CFU/ml for locations H1 and H4, respectively. Counts of solom from the different locations were not statistically significant (p > .05). However, H1 and H4 were not comparable (p < .05).
For DRBC, the counts were in the range of 1.71 ± 0.8-3.52 ± 0.8 log 10 CFU/ml for locations H6 and H3, respectively.
Statistically, the counts were all comparable (p > .05). There was an observed low fungal count generally. The range of counts was within the acceptable to borderline range of microbiological counts for ready-to-eat foods as prescribed by the International Commission for Microbiological Specification of Foods (ICMSF, 1998) (Table 3).
(2) The pH values were in the range of 3.03 ± 0.09-4.03 ± 0.23, respectively, for HS6 and HS10. Statistically, there were no significant differences (p > .05) observed among the pH values of the solom from the respective locations. The trend of pH of the samples collected from the Ho municipality showed no particular trend (Table 4).

| pH
Cereals are susceptible to infections by a broad continuum of plant pathogens. The globalization of cereal commerce is largely linked to increased fungal infection and cross-contamination hazards (Waage et al., 2006).  as appropriate for fungal growth in samples of "akamu" and "kunun- Presumably, the drinks of lower pH had been stored over a longer period of time which is likely to eliminate most fungi thus safer for consumption.
It is noteworthy the range of pH values recorded in this work were lower than pH ranges of 4.6-6.8 and 4.5-7.0 described as optimum for the growth of fungi by Jackson et al. (1991) and Weyman-Kaczmarkowa and Pędziwilk (2000), respectively. Likewise, Anupma and Tamang (2020) recorded an average of pH 5.3. Nonetheless, a much higher pH range was reported by Yamanaka (2003) as 7-9 for optimum growth of fungi. The acidity of this beverage tended to be directly proportional to the storage period (increase with an increase in the fermentation period) resulting in spoilage. Consequently, the low pH values may have inhibited the growth of some fungal species isolated.

| Fungal counts
Fungal counts observed in this work were in the same range of values of 2.098-4.23 log 10 CFU/ml reported by Aboagye et al. (2020) as counts of "asaana" a beverage of maize from Ghana. Minamor, Mensah, et al. (2017) recorded fungal counts of <10 4 CFU/ml in "pito" a cereal beverage of sorghum were found to be within the permissible limits.
Contrarily, Oriola et al. (2017) reported higher fungal counts of range 3.4 × 10 5 ± 0.10-4.5 × 10 6 ± 0.10 (5.53-6.65 log 10 ) CFU/ml from "Otika" a Nigerian cereal beverage of sorghum. In a related study, Anupma and Tamang (2020) recorded an average fungal population of 4.9 × 10 5 (5.69 log 10 ) CFU/g from amylase and alcohol-producing starters in India. From Nigeria, Popoola (2019) reported a range between 1.30 × 10 5 CFU/ml and 1.74 × 10 5 (5.11-5.23 log 10 ) CFU/ml in "akamu" samples obtained from different cereals. High colony counts >10 4 are an indication of spoilage as a result of either poor hygiene or poor quality of cereals and water used in the preparation of the beverage. Mossel et al. (1986) highlighted that variation in fungal counts could be attributed to differences in compliance with Good Manufacturing Practices (GMP) conditions during the growing, processing, or storage of the raw material of the food. Furthermore, the effect of storage on the viability of the fungal propagules could also be worth considering. Lastly, the high incidence of a particular fungal specie may indicate the presence of mycotoxins. A consumer must be made aware the consequences of drinking "solom" not hygienically prepared and also stored for longer periods as may contain greater fungal counts; hence, its safety is doubtful.
The range of counts recorded in this work were, however, within the acceptable range of microbiological counts for ready-to-eat foods as prescribed by the International Commission for Microbiological Specification of Foods (ICMSF, 1998). Although mycotoxins were not investigated for in this study, several reports exist on the occurrence of some kinds of mycotoxins of public health interests viz-viz their toxicogenic fungi.

| Fungal species
In a related study, Aboagye et al. (2020) recorded a total of 11 fungal species belonging to 4 genera were isolated, and namely Aspergillus, Fusarium, Penicillium, and Rhodotorulla spp from "asaana" (corn malted drink) from Ghana. From Nigeria and some parts of Benin, fungal species belonging to the genera Aspergillus and Penicillium have been reported to be associated with "Ogi" (Phiri et al., 2020).
The incidence of these fungal species has been linked to the spoilage of beverages as explained by Kolawole et al. (2007)   also isolated Saccharomyces cerevisiae, as the only fungus associated with fermentation of "pito" in Accra, Ghana. Lee and Lee (2002) explained fungi and lactic acid bacteria grow simultaneously in cereal beverages and play important roles in the later stage of alcoholic fermentation, and the mixed culture also contributes to some sensorial properties of the beverage such as the more intense flavor of some beverages. Molds and other microorganisms convert unpalatable carbohydrates of low digestibility and proteins into palatable sugars and amino acids with a high conversion efficiency.
The occurrence of toxigenic fungi in these cereals and beverages is suggestive of the potential presence of their mycotoxins. Mycotoxins are secondary metabolites of filamentous fungi which are mostly toxic, carcinogenic, mutagenic, and teratogenic even at the minutest of concentrations (Ezekiel et al., 2018;Omotayo et al., 2019).
Although not investigated in this present study, incidences of different mycotoxins in traditional as well as nontraditional cereal beverages have been reported in the literature. Of particular concern to food safety and public health are those of the toxicogenic fungi belonging to the genera; Fusarium (zearalenone, fumonisins, deoxyni- Kwashiorkor in children is aggravated by long-term exposure to aflatoxin. Commonly, aflatoxins cause jaundice, hepatomegaly, splenomegaly, tachycardia, and anorexia beside others. Ochratoxins are also well known for causing damage to the liver (Nyanzi & Jooste, 2012).
It is noteworthy in many illustrations, assigning a favored basis of contamination can be thought-provoking. Moreover, the environment of a specific manufacturing plant can be highly variable and unique, such that it may be necessary to clarify the specific sources of contamination in each particular situation (Garijo et al., 2015;Hernández et al., 2018). Microbial contamination throughout the market chain is an inevitable occurrence.
The presence of pathogenic microorganisms and mycotoxins in beverages constitutes a Public Health hazard for consumers and economic loss for the producers (Granados-Chinchilla et al., 2018) as many of these fungi are in the spoilage category and the presence of toxigenic fungi is indicators of a possible mycotoxin contamination risk (Anjorin et al., 2013). It is of paramount importance that these fermented products be prepared under good sanitary and hygienic conditions including application. To mitigate the effects of the natural presence of microbes in the food chain, the application of Good Hygiene Practices (GHP) and Good Manufacturing Practices (GMP) allows a minimization in the contamination to satisfactory levels.

| CON CLUS ION
Our findings on fungal diversity in Solom from the Ho Municipality of Ghana may supplement the microbial miscellany in ecosystems of Ghana, which is one of the potential biodiversity hot spots of the world. We identified eight (8)  Fungal species present in this traditional cereal-based beverage ("solom") were morphologically, ecologically, and showed high diversity within the community. The pH of the cereal beverage (solom) was found to be between the ranges of 3.03 ± 0.09-4.03 ± 0.23 which suggested acidity.

ACK N OWLED G M ENTS
The authors wish to acknowledge the contribution of the techni-

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.