Isolation of Fungal Species from Fermentating Pearl Millet Gruel and Determination of their Antagonistic Activities against Indicator Bacterial Species

Nigerian Food Journal Official Journal of Nigerian Institute of Food Science and Techonology

www.nifst.org

NIFOJ Vol. 30 No. 1, pages 35 – 42, 2012

Isolation of Fungal Species from Fermentating Pearl Millet Gruel and Determination of their Antagonistic Activities against Indicator Bacterial Species * Osamwonyi, U.O. 1 and Wakil, S.M.2

ABSTRACT The mycological and physicochemical qualities (pH and titratable acidity) of fermenting pearl millet gruel were evaluated using routine methods. Several species of yeasts and moulds were isolated and the mould species identified based on their observable macroscopic and microscopic characteristics. The moulds identified included: Penicillium sp (FM1), Rhizopus spp (FM2, FM3 and FM6), Aspergillus flavus (FM7) and Aspergillus niger (FM8). These isolates were screened for production of antimicrobial compounds using agar well diffusion method. Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus lichieniformis, Salmonella spp., Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas syringae, Proteus sp. and Serratia sp. were utilized as indicator organisms. Secondary metabolites were also extracted from the respective moulds and the antimicrobial properties of these metabolites were tested against pure cultures of E. coli, S. aureus, P. flourescens and B. lichieniformis. All the moulds exhibited antimicrobial activity against P. flourescens while the metabolic extract of Aspergillus flavus (FM7) displayed the highest zone of inhibition (24 mm) against an overnight culture of P. flourescens.

Keywords: Fungi, fermentation, millet. Introduction Cereal grains are the major source of calories and proteins for the people of Nigeria (Nkama and Gbenyi, 2001). The major cereals cultivated in Nigeria are sorghum, millet, rice and maize. The major states that produce pearl millet in Nigeria are Borno, Yobe, Jigawa, Kano, Katsina, Zamfara, Sokoto and Kebbi States. Cereal grains constitute an important group of substrates for fermented foods in tropical Africa. The common cereals which are used include maize, sorghum and millet. Fermented foods from cereals include: Ogi produced from millet, sorghum or maize; Kenkey, a staple food in Ghana produced Faculty of Basic and Applied Science, Department of Microbiology, Wellspring University, Benin City, Nigeria. 2 Faculty of Science, Department of Botany and Microbiology, University of Ibadan, Ibadan, Nigeria. * corresponding author: [email protected] 1

from maize. Millets are usually cultivated under extremely harsh conditions of high temperature, low and erratic precipitation, short growing seasons and acidic and infertile soils with poor water holding capacity (Nkama and Ikwelle, 1998). Millets are rich in B vitamins, especially niacin, B17, B6 and folic acid, calcium, iron, potassium, magnesium and zinc (Seenappa, 1987). Pearl millet (Pennisetum glaucum) is known to have a higher protein content and better amino acid balance than Sorghum (Nkama and Ikwelle, 1998). The higher ratio of germ to endosperm is responsible for the higher protein content (Dendy, 1995). Pearl millet is no doubt superior to other cereals with respect to some of the nutrients especially average protein, mineral and fat (Usha et al., 1996). However, the presence of various antinutrients, poor digestibility of the protein and carbohydrates and low palatability greatly affect its utilization as food. Fermentation (Khetarpaul and Chauhan, 1990; Usha et al., 1996)

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Nigerian Food Journal Vol. 30 No. 1, 2012 ... Research Note

and sprouting (Chavan and Kadam, 1989) have been reported to increase the protein digestibility of millet. Traditional technologies available for processing of millet include threshing, cleaning, washing, dehulling, soaking, germination, wet and dry milling and fermentation (Makuru, 1992). Pearl millet has a wide array of uses. In its traditional growing areas in India and many African countries, pearl millet is consumed in the form of leavened or unleavened bread, porridges, boiled or steamed foods, and (alcoholic) beverages. The stalks are a valued building material, fuel and livestock feed (Wilson et al., 2006). Fermentation can be defined as a process in which biochemical changes are brought about in organic substances (carbohydrates, proteins and fats) through the action of enzymes which are produced by microorganisms (Fields et al., 1981). Fermentation is indigenous to African culture and meets the challenges of food spoilage and food- borne diseases which are experienced in Africa (Oyewole, 1997). Most traditional fermentations employ a whole array of natural microflora that could function under the varied environmental and non-sterile conditions presented by the different processes. Such fermentations are characterized by numerous microorganisms of varying functions that could be beneficial or detrimental to the fermentation processes; mixed cultures that produce the blend of rich flavours and aromas of the product; lactic acid bacteria creating unfavourable environment for pathogenic Bacillaceae and Enterobacteriaceae; and some microorganisms that could accelerate spoilage, particularly in the finished products (Steinkraus, 1991). Fungi are a diverse and valuable resource for the discovery of novel beneficial antimicrobial substances. The chemical potential of fungi is enormous and new approaches need to be devised to efficiently access this genetic and chemical diversity for the development of new bio preservatives (El-Banna et al., 1987). Many fungal species grow on food products, often contributing to their spoilage, as in fruits, vegetables and other fresh or prepared products. However, in many

instances, the presence of fungi on some products does not have a deleterious effect and may even contribute to the ripening and development of important organoleptic characteristics typical of a particular food (Grazia et al., 1986; Huerta et al., 1987). Fungi offer an enormous potential for new products. Most fungi studied to date have been isolated from soil and were proven to have a high creative index, i.e. new and interesting secondary metabolites could be isolated. Genera such as Aspergillus, Penicillium, Acremonium and Fusarium, all typical soil isolates, are known for their ability to synthesize diverse chemical structures. Dreyfuss (1994), however, described a problem which is often encountered during microbiological screening of fungal isolates for their content of secondary metabolites. Increasingly, known metabolites are rediscovered making screening programmes less efficient. This may be due to the use of the same well-established isolation methods for fungi. Thus, often, the same fungal strains are re-isolated and this could also result in the rediscovery of known compounds as the same taxa produce the same metabolites with high coincidence (Skujins, 1984). Therefore, the objectives of this study were to to evaluate the antimicrobial activities of fungal species present in fermenting pearl millet gruel. Materials and Methods Sample collection Samples of pearl millet (Pennisetum glaucum) used for this study were obtained from Bodija market, Ibadan, Oyo State, Nigeria. The millet was purchased in sterile polythene bags and brought to the microbiology laboratory of the Department of Botany and Microbiology, University of Ibadan, Ibadan and kept in the refrigerator for further analysis. Sample preparation and isolation of fungi The millet grains were cleaned and washed using distilled water and then sun-dried. Two hundred grams of pearl millet was weighed and dry milled using a clean surface sterilized domestic blender.

Isolation of Fungal Species from Fermentating Pearl Millet Gruel and their Antagonistic Activities ... Osamwonyi and Wakil

The resultant 230 g of blended millet powder was mixed with I litre of sterile distilled water and allowed to ferment spontaneously for 72 h. The fermenting gruel was stirred to ensure mixing of the steep water and fermenting millet. One (1) ml of the mixture was aseptically removed from the fermenting vessel after stirring for serial dilution at 0 h, 24 h, 48 h and 72 h. Five (5) fold serial dilution of the sample collected was plated out on the three different media and pure cultures of organisms obtained by streaking were stored on slants using medium of isolation for further analysis. Malt extract agar (MEA) was used for isolation of moulds and yeasts from the fermenting gruel using the pour plate technique and the medium was prepared according to manufacturer’s specification and sterilized at 121ºC for 15 min. Characterization of fungal isolates Pure cultures of the fungal isolates were aseptically inoculated into sterile potato dextrose agar by the use of a sterile inoculating needle. The plates were incubated at room temperature (28 ± 2oC) and isolates were examined after 72 h for their growth pattern, spores, mycelia colouration and distribution of spores. A pin head size of mycelia from 72 h culture was put onto a drop of cotton blue in lactophenol on a clean grease free slide. The mycelia were properly teased apart using sterile inoculating needle and covered with a cover slip. The slide was then observed under x10 and x40 objective lenses. The shape and arrangement of the fruiting bodies were observed. Identification of the isolates were done using compendium of soil fungi (Domsch et al., 1980). Screening for antimicrobial producing microorganisms Indicator organisms used were Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus lichieniformis, Salmonella species, Pseudomonas flourescens, Pseudomonas aeruginosa, Proteus species, Serratia species and Pseudomonas syringae collected from the postgraduate laboratory of the Department of Botany and Microbiology, University of Ibadan. The organisms were stored in nutrient broth and day-old cultures

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were used to test all isolates for antimicrobial activity and further study was done on organisms which showed positive antimicrobial activity. Antimicrobial activity of the fungal isolates was determined using the agar-well diffusion method (Tagg and McGiven, 1971). Twenty (20) ml of Mueller Hinton agar (Becton Dickinson, USA) was inoculated with overnight culture (106) of an indicator strain and overlaid on an agar plate after cooling a 10 mm diameter of the fungi isolate obtained from 72 h old plates using a cork borer placed in the plate. After incubation overnight, the antimicrobial activity was expressed as the diameter of the inhibition zones around the fungi isolates (Atalla et al., 2008). Determination of secondary metabolite production in fungi Extracts were prepared using a modification of the method of Belofsky et al. (1998). Twenty-five ml of Malt Extract Broth dispensed into bottles and sterilized was inoculated with 10 mm disc of the test isolate and incubated for seven (7) days at room temperature (28ºC ± 2°C), then the mycelia was harvested by filtering through Whatman filter paper. The liquid was centrifuged at 4000 revs/min for 20 min, then filtered using Whatman filter paper. The mycelia were crushed using silica gel 60 in a mortar and then the mycelia were extracted using ethyl acetate to obtain intracellular metabolites. It was left in a separating funnel for 15 min to precipitate. The crude ethyl acetate was collected. The filtrate of each fungus was also extracted several times using ethyl acetate (v/v) in a separating funnel and the extract added to the extract from the mycelia. The mixture obtained was evaporated to dryness using a water bath at 50ºC. The extract was dissolved in ethyl acetate and tested for antimicrobial activity against selected indicator organisms using the agar well diffusion method. Mueller Hinton agar was seeded with the indicator organisms (E. coli, S. aureus, P. flourescens and B. licheniformis) inhibition zones around each well were measured in mm after incubation at 28ºC for 24 h.

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Determination of pH The pH change of the fermenting gruel and steeping water was monitored daily using a pH meter. Ten millilitres of steep water and the fermenting gruel was aseptically removed and its pH was determined using a pH meter equipped with a glass electrode. Titratable acidity The titratable acidity (expressed as percentage lactic acid) was determined by shaking the fermenting vessel to enable mixing of steep water and fermenting gruel and pipetting ten millilitres of steep water and fermenting gruel which was titrated against 0.1M NaOH and phenolphthalein served as indicator. The percentage acidity was calculated as described by AOAC (1990). % acidity = ml NaOH x MNaOH x ME x 100 volume of sample Where ml NaOH = volume of NaOH used M NaOH = Molarity of NaOH ME = equivalence factor (90.08 mg). Results Table 1 shows the plate count obtained on the Malt Extract Agar for total fungal counts. Counts were recorded in colony forming units per ml (cfu/ml). Presence of various mould species was detected only between 0 and 24 h with counts increasing from 4 x 103 – 6 x 104 cfu/ml while the moulds were replaced with yeast which had counts of 4 x 103 cfu/ml at 48 h and increased very rapidly to 8.5 x 107 cfu/ml at 72 h. The table also shows the pH and Total Titratable Acidity (TTA) of the fermenting gruel with respect to fermentation time. The pH decreased progressively from 0 – 72 h from 6.1 – 3.7 while TTA increased from 0.54 at 0 h to 4.05 at 72 h. A total of fifteen (15) yeast isolates and eight (8) fungi were identified from MEA. Table 2 shows the morphological characterization of the respective moulds. The macroscopic examination included checking for mycelia colour (which ranged from hyaline to various shades of

green, brown and black), mycelia where either ariel or spreading and pigmentation of conidiophores was also observed. Microscopic examination involved viewing teased portions of mycelia for conidia shape and wall (either smooth or rough), differentiation of mycelium, presence or absence of foot cells for conidiophores and branching of conidiophores. Table 1: Plate count of microorganism and acidity obtained during fermentation of pearl millet Fermentation Microbial Count Time (h) (cfu/ml)

pH

TTA

6.1 4.4 3.9 3.7

0.54 2.9 3.42 4.05

MOULDS YEASTS

0 24 48 72

4 x 103 6 x 104 ND ND

ND ND 4 x 103 8.5 x 107

MEA – Malt Extract Agar TTA – Total Titratable Acidity ND – Not Detected

All the eight fungi isolates showed antimicrobial activity against the indicator organisms (Table 3). Isolates FM 1 and FM 5 showed inhibition against the highest number of organisms (5) while isolate FM 3 showed the least inhibition by inhibiting only 2 of the indicator organisms. The largest zone of inhibition (20 mm) was observed in isolates FM 5 and FM 7 both against E. coli. P. fluorescens showed the highest susceptibility and was inhibited by all the fungi isolates. Figure 1 shows the zones of inhibition of E. coli by Penicillium spp. Table 4 shows the zone of inhibition on the indicator organisms by the various metabolites produced by the moulds. The highest zone of inhibition was exhibited by metabolites of Aspergillus niger (FM 7) against P. fluorescens (27mm) while Figures 1 – 2 show plates of zones of inhibitions on test organisms by mould isolates and their metabolites.

Isolation of Fungal Species from Fermentating Pearl Millet Gruel and their Antagonistic Activities ... Osamwonyi and Wakil

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Table 2: Morphological characteristics of the fungi isolates Isolates

Macroscopic

Microscopic

FM 1

Thin spreading colonies velvety, Conidia smooth walled, ellipsoidal bluish green with pale yellow reverse species pigmentation

Penicillium

FM 2

Fast growing colonies forming hyaline ariel hyphae, rhizoids are pigmented with brownish sporangiosphores

Rhizopus species

Differentiated into stolons and nodes with rhizoids and sporangiophores, sporangiophores are short ellipsoidal with almost pointed ends

Probable

FM 3 Yellowish green colonies Conidiophore hyaline and rough-walled

Aspergillus flavus

FM 4 Fast growing pale colonies with fluffy ariel mycelium

Conidiophores are blastonously branched. Terminal branches are slender slightly tapering fusiform are sickle-shaped and septate

Fusarium species

FM 5

Fast growing colonies forming hyaline ariel hyphae, rhizoids are pigmented with brownish sporangiophores

Differentiated into stolons and nodes with rhizoids and sporangiophores, sporangiophores are short ellipsoidal with almost pointed ends

Rhizopus species

FM 6

Fast growing colonies forming hyaline ariel hyphae, rhizoids are pigmented with brownish sporangiophores

Differentiated into stolons and nodes with rhizoids and sporangiophores, sporangiophores are short ellipsoidal with almost pointed ends

Rhizopus species

FM 7 Black powdery conidiophores

Conidiophores arising from long branched foot calls, irregularly roughened

Aspergillus niger

FM 8 Black powdery conidiophores

Conidiophores arising from long branched foot calls, irregularly roughened

Aspergillus niger

Table 3: Diameter of zones of inhibition (MM) of indicator organisms by fungi isolates

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Table 4: Diameter of zones of inhibition (MM) of indicator organisms by metabolites obtained from fungi isolates

Discussion Various species of mould and yeast were isolated from the fermenting gruel. The surface microflora of millets, including Aspergillus species, Rhizopus species, Penicillum species and Fusarium species, which were quickly eliminated during the fermentation period was in accordance with the finding of Akinrele (1970). This may probably be as a result of increased anaerobiosis which prevailed during the fermentation process. All the moulds isolated were able to inhibit the growth of indicator organisms to varying degrees, but all yeasts isolated in the course of this study were unable to inhibit the growth of the respective indicator organisms. The highest zone of inhibition (20 mm) was observed for both Aspergillus niger and Rhizopus spp. against E. coli. This might not be surprising given the fact that these fungi can survive in a variety of environments and, as a consequence, produce a variety of extracellular metabolites which can be toxic to other competing flora. Aspergillus flavus elaborated zones of inhibition against the least number of isolates (2), while Penicillium spp. and Rhizopus spp. produced zones of inhibition against the highest number of test isolates (5). This was collaborated by the antibacterial activities of their metabolites which produced zones of inhibition against all the indicator organisms. This could be

indicative of the potential antibiotic producing abilities of these fungal isolates. The ability of the secondary metabolites such as proteins extracted from the fungi species to inhibit the indicator organisms used is in line with the findings of Atalla et al. (2008) who reported obtaining metabolites from fungi associated with marine algae considered as antibacterial and antifungal compounds, as they were active against gram positive, gram negative bacteria and a fungus. Some of the fungi species isolated are the world highest sources of antibiotics, i.e. Penicillium species and Fusarium species. Conclusion In view of the antibiotic producing capabilities of some of these fungi, i.e. Penicillium spp. and Rhizopus spp., these fungi or their purified antibacterial metabolites can be used to improve preservative effect and add flavour and taste to several fermented or processed foods. Also, these isolated fungal isolates can have a positive impact when incorporated in starter cultures used for traditional fermented foods, as these fungi can improve the shelf life and safety of the resultant fermented food so produced. Further work is needed to standardize the methods of application and quantity of the antimicrobial products to be applied to foods so that they can be used industrially for food preservation.

Isolation of Fungal Species from Fermentating Pearl Millet Gruel and their Antagonistic Activities ... Osamwonyi and Wakil

Fig. 1:

The zones of inhibition of Escherichia coli by metabolites from isolates FM 5

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Zones of inhibition of Pseudomonas aeruginosa by secondary metabolites from isolate FM 1

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