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International Journal of Research in Pure and Applied Microbiology Universal Research Publications. All rights reserved
ISSN 2277–3843
Original Article Isolation and characterization of microorganisms in dakuwa (a Nigerian cereal/groundnut snack). 1*
Ocheme, O.B. 2Igyor, M.A. and 1Hassan, H. 1 Food Science and Technology Department, Federal University of Technology, Minna, Nigeria 2 Food Science and Technology Department, University of Agriculture, Makurdi, Nigeria *Corresponding author. E-mail:
[email protected] and
[email protected]; Tel.: +2348037885078 Received 02 January 2014; accepted 24 January 2014 Abstract The micro flora of dakuwa (a Nigerian cereal/legume snack) under ambient (32±4 0C) storage was investigated. Dakuwa balls were produced and packaged in aluminium foil, low density (LDP) and high density (HDP) polyethylene bags and stored at 32±40C for four months. The microorganisms present in dakuwa were isolated and identified on a monthly basis. Staphylococcus aureus, Streptococcus pyogene and Bacillus subtilis were the bacteria found in dakuwa while the moulds were Mucor spp, Aspergillus niger Aspergillus flavus. Only Bacillus subtilis and Mucor spp survived for up to four months in the packaged samples. Although packaging limited the growth and survival of some types of microorganisms, type of packaging had no effect on the type of microorganisms that survived in packaged dakuwa. © 2014 Universal Research Publications. All rights reserved Keywords: Dakuwa, ambient, packaged, Bacillus subtilis, Mucor Spp, Aspergillus, Streptococcus, Staphylococcus Introduction Snacks are sweet or savoury foods eaten to provide light sustenance in a quick and convenient format. They are eaten or as an alternative to main meals (IFIS, 2005). Snack foods are essential vehicles for delivery of essential nutrients because of the growing change in eating habits (Henshaw and Agunbiade, 2004). Dakuwa is a cereal and groundnut based snack prepared at home by vendors and consumed mainly in the northern parts of Nigeria. Dakuwa is prepared from mixtures of cereals (maize, millet and sorghum), tigernuts, groundnut, ground pepper, ginger, sugar and salt (Nkama and Gbenyi, 2001). The ingredients are thoroughly mixed, pounded and moulded into balls that can be eaten without further processing (Abdulrahman and Kolawole, 2003). In Nigeria, dakuwa, like other snack foods, is mainly consumed in its areas of production. Its production varies with people, culture and geographical locations: these leads to possession of variable characteristics (Ingbian and Akpapunan, 2005). Some of the problems associated with the local production of snacks such as dakuwa include non standardization of equipment, process and raw material, inadequate hygiene during and after production, and little or no packaging which results in poor preservation techniques and high levels of contaminants in the food resulting in food borne illnesses (Ingbian and Akpapunam, 2005). According to Nkama and Gbenyi (2001) the local production of dakuwa does not take into consideration the
15
presence of anti-nutrients such as phytate and tannins as a cereal/legume based product. This stimulated a survey of the traditional methods of processing dakuwa and an assessment of traditionally produced dakuwa in Niger State, north central Nigeria (Ocheme et al. 2011a, 2011b). Furthermore, the effect of maize/groundnut ratio on the quality and acceptability of dakuwa as well as the moisture sorption characteristics of dakuwa have been studied and documented (Ocheme et al. 2012, 2013). The objective of this study was to identify the microorganisms associated with dakuwa. Knowledge of the type of microorganisms associated with dakuwa will be useful in selecting appropriate processing, packaging and storage conditions which will maximize the product’s stability and wholesomeness. Materials and Methods Source of materials Maize (yellow variety) and groundnut (redskin) used for preparing dakuwa were obtained from local farmers at Gidan Kwano, Chanchaga Local Government area while sugar and granulated red pepper were obtained from Minna Central Market, Niger State, Nigeria. Preparation of raw materials Maize and groundnut seeds were manually cleaned and washed in cold tap water. 400g each of maize grains and groundnut were soaked in 2 litres of water for 12 hours after which they were germinated for 72 hours. At the end of germination, the maize grains and groundnuts were oven
International Journal of Research in Pure and Applied Microbiology 2014; 4(1): 15-19
dried at 600C for 1 hour. The groundnuts and maize grains were roasted at 1400C for 30 and 60 minutes respectively. The groundnut was decoated after which both maize and groundnut were milled separately using a local attrition mill. After milling, the maize flour was sieved to obtain a particle size of 0.05mm. The maize flour and groundnut paste were then mixed together in a 1:1 ratio. To 100g of this mixture 10%, 5% and respectively of table sugar and granulated red pepper were added. The mixture was then passed through the attrition mill once and moulded into balls. The balls were then packaged in aluminium foil, low density polyethylene (LDP) and high density polyethylene (HDP). The packaged samples were kept at ambient temperature (32±40C) for four months and microbiological analyses were carried out monthly. Microbiological analyses Bacteriological analysis was carried out by selective media method as described by Prescott (2002) and Sherman (2005). Morphological characterization of the isolated microbes were done on the basis of simple staining and gram staining as described by Holt et al. (1994) and Sherman (2005). Biochemical characterization of the isolates were accomplished by indole test, methyl red test, Voges Proskauer test and citrate utilization test, nitrate reduction test, lactose, sucrose, dextrose fermentation reaction test as described by Sherman (2005) and Holt et al. (1994). Results and Discussion Tables 1 - 4 shows the bacteria present in dakuwa in different packaging materials. The bacteria that were isolated and identified were Staphylococcus aureus,
Streptococcus pyogene and Bacillus subtilis. Bacillus subtilis and Staphylococcus aureus were present in the fresh sample. For the unpackaged sample, the Bacillus subtilis disappeared after one month of storage while the Staphylococcus was present throughout the four months of storage. Furthermore, Streptococcus pyogene was found in the unpackaged sample in the second and fourth month alongside the Staphylococcus aureus. With respect to the packaged samples, the Staphylococcus aureus was not seen again after one month. Only bacillus subtilis was found in them all through the period of storage. Staphylococcus aureus and Bacillus subtilis are common contaminants of the human skin and soil, hence their presence in dakuwa is most likely due to poor handling in the course of production. The presence of Staphylococcus aureus and Streptococcus pyogene in dakuwa is of public health significance given that they can cause food poisoning. The absence of Staphylococcus aureus and Staphylococcus pyogene in the packaged samples indicates that they thrive better in unpackaged dakuwa. This may be due to their inability to tolerate limited oxygen (Adam and Moss, 2008) which, as a result of the barrier properties of the packaging materials, will be higher around the unpackaged sample than in the packaged sample. Antagonism from the Bacillus subtilis may also be responsible for the absence of the Staphylococcus in the packaged sample. Under adverse conditions Bacillus subtilis is able to form spores (Adams and Moss, 2008) and this may be why it was present in the packaged samples all through the period of storage.
Table 1: Characterization and identification of bacteria isolated from unpackaged dakuwa Month of storage 0 (Fresh sample)
S
GS
CT
CG T
SH
IT
MT Y
CU
HSP
GT
Cocci
+
+
Rods
+
+
1
Cocci
+
2
Cocci
FT
ST
MN T
LC T
Microorganism
+
-
+
+
+
+
-
+
-
+
+
-
+
-
+
+
+
-
+
-
Staphylococcus aureus
-
+
+
+
-
+
+
+
+
+
-
Bacillus subtilis
+
+
-
Staphylococcus aureus
+
+
+
-
+
+
+
+
+
-
+
+
-
Staphylococcus aureus
Cocci
+
+
-
-
+
+
+
-
+
-
+
+
+
Staphylococcus pyogene
3
Cocci
+
+
+
-
+
+
+
+
+
-
+
+
-
Staphylococcus aureus
4
Cocci
+
+
+
-
+
+
+
+
+
-
+
+
-
Staphylococcus aureus
Cocci
+
+
-
-
+
+
+
-
+
-
+
+
+
Staphylococcus pyogene
Key : S = shape; GS = Gram staining; CT = catalase test; CGT = coagulase test; SH = starch hydrolysis; IT = indole test; MTY = motility; CU = citrate utilization; HSP = hydrogen sulphide production; GT = glucose test; FT = fructose test; ST = sucrose test; MNT = mannitol test; LCT = lactose test
Table 2: Characterization and identification of bacteria isolated from dakuwa packaged in LDP Month of storage
S
GS
CT
CGT
SH
IT
MTY
CU
HSP
GT
FT
ST
MNT
LCT
0 (Fresh sample)
Cocci
+
+
+
-
+
+
+
+
+
-
+
+
-
Rods Rods Rods Rods Rods
+ + + + +
+ + + + +
-
+ + + + +
-
+ + + + +
+ + + + +
-
+ + + + +
+ + + + +
-
-
+ + + + +
1 2 3 4
Microorganism
Staphylococcus aureus Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis
Key : S = shape; GS = Gram staining; CT = catalase test; CGT = coagulase test; SH = starch hydrolysis; IT = indole test; MTY = motility; CU = citrate utilization; HSP = hydrogen sulphide production; GT = glucose test; FT = fructose test; ST = sucrose test; MNT = mannitol test; LCT = lactose test
16
International Journal of Research in Pure and Applied Microbiology 2014; 4(1): 15-19
Table 3: Characterization and identification of bacteria isolated from dakuwa packaged in HDP Month of storage 0 (Fresh sample) 1 2 3 4
S
GS
CT
CGT
SH
IT
MTY
CU
HSP
GT
FT
ST
MNT
LCT
Cocci
+
+
+
-
+
+
+
+
+
-
+
+
-
Rods Rods Rods Rods Rods
+ + + + +
+ + + + +
-
+ + + + +
-
+ + + + +
+ + + + +
-
+ + + + +
+ + + + +
-
-
+ + + + +
Microorganism Staphylococcus aureus Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis
Key : S = shape; GS = Gram staining; CT = catalase test; CGT = coagulase test; SH = starch hydrolysis; IT = indole test; MTY = motility; CU = citrate utilization; HSP = hydrogen sulphide production; GT = glucose test; FT = fructose test; ST = sucrose test; MNT = mannitol test; LCT = lactose test Table 4: Characterization and identification of bacteria isolated from dakuwa packaged in aluminium foil Month of storage 0 (Fresh sample) 1 2 3 4
S
GS
CT
CGT
SH
IT
MTY
CU
HSP
GT
FT
ST
MNT
LCT
Cocci
+
+
+
-
+
+
+
+
+
-
+
+
-
Rods Rods Rods Rods Rods
+ + + + +
+ + + + +
-
+ + + + +
-
+ + + + +
+ + + + +
-
+ + + + +
+ + + + +
-
-
+ + + + +
Microorganism Staphylococcus aureus Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis
Key :S = shape; GS = Gram staining; CT = catalase test; CGT = coagulase test; SH = starch hydrolysis; IT = indole test; MTY = motility; CU = citrate utilization; HSP = hydrogen sulphide production; GT = glucose test; FT = fructose test; ST = sucrose test; MNT = mannitol test; LCT = lactose test Table 5 : Morphological characteristics of moulds isolated from unpackaged dakuwa Month of storage
Colour of aerial hyphae
Colour of substrate hyphae
Nature of hyphae
Shape and asexual structure
0 (Fresh sample)
Grey
Brown
Nonseptate
Oval, black
Whitish black
Brown
Septate
Oval, green
Yellow
Brown
Septate
Oval, green
Whitish black
Brown
Septate
Oval, green
2
Yellow
Brown
Septate
Oval, green
3
Yellow
Brown
Septate
Oval, green
4
Yellow
Brown
Septate
Oval, green
1
Appearance of sporangiosphore Long, erect and non-septate conidia Long, erect and septate conidia Long, erect and septate conidia Long, erect and septate conidia Long, erect and septate conidia Long, erect and septate conidia Long, erect and septate conidia
Characteristic spore head
Microorganism
Elliptical
Mucor Spp
Multi-nucleated
Aspergillus niger
Multi-nucleated
Aspergillus flavus
Multi-nucleated
Aspergillus niger
Multi nucleated Multi nucleated Multi nucleated
Aspergillus flavus Aspergillus flavus Aspergillus flavus
Table 6 : Morphological characteristics of moulds isolated from dakuwa packaged in LDP Month of storage
Colour of aerial hyphae
Colour of substrate hyphae
Nature of hyphae
Shape and asexual structure
0 (Fresh sample)
Grey
Brown
Nonseptate
Oval, black
Whitish black
Brown
Septate
Oval, green
1
Grey
Brown
Nonseptate
Oval, black
2
Grey
Brown
Nonseptate
Oval, black
3
Grey
Brown
Non-
Oval, black
17
Appearance of sporangiosphore Long, erect and non-septate conidia Long, erect and septate conidia Long, erect and non-septate conidia Long, erect and non-septate conidia Long, erect and
Characteristic spore head
Microorganism
Elliptical
Mucor Spp
Multi-nucleated
Aspergillus niger
Elliptical
Mucor Spp
Elliptical
Mucor Spp
Elliptical
Mucor Spp
International Journal of Research in Pure and Applied Microbiology 2014; 4(1): 15-19
septate
4
Grey
Brown
Nonseptate
Oval, black
non-septate conidia Long, erect and non-septate conidia
Elliptical
Mucor Spp
Characteristic spore head
Microorganism
Elliptical
Mucor Spp
Multi-nucleated
Aspergillus niger
Elliptical
Mucor Spp
Elliptical
Mucor Spp
Elliptical
Mucor Spp
Elliptical
Mucor Spp
Table 7 : Morphological characteristics of moulds isolated from dakuwa packaged in HDP Month of storage
Colour of aerial hyphae
Colour of substrate hyphae
Nature of hyphae
Shape and asexual structure
0 (Fresh sample)
Grey
Brown
Nonseptate
Oval, black
Whitish black
Brown
Septate
Oval, green
1
Grey
Brown
Nonseptate
Oval, black
2
Grey
Brown
Nonseptate
Oval, black
3
Grey
Brown
Nonseptate
Oval, black
4
Grey
Brown
Nonseptate
Oval, black
Appearance of sporangiosphore Long, erect and non-septate conidia Long, erect and septate conidia Long, erect and non-septate conidia Long, erect and non-septate conidia Long, erect and non-septate conidia Long, erect and non-septate conidia
Table 8 : Morphological characteristics of moulds isolated from dakuwa packaged in aluminium foil Month of storage
Colour of aerial hyphae
Colour of substrate hyphae
Nature of hyphae
Shape and asexual structure
Appearance of sporangiosphore
Characteristic spore head
Microorganism
0 (Fresh sample)
Grey
Brown
Nonseptate
Oval, black
Long, erect and non-septate conidia
Elliptical
Mucor Spp
Whitish black
Brown
Septate
Oval, green
Multi-nucleated
Aspergillus niger
1
Grey
Brown
Elliptical
Mucor Spp
2
Grey
Brown
Elliptical
Mucor Spp
3
Grey
Brown
Elliptical
Mucor Spp
4
Grey
Brown
Elliptical
Mucor Spp
Nonseptate Nonseptate Nonseptate Nonseptate
Oval, black Oval, black Oval, black Oval, black
Long, erect and septate conidia Long, erect and non-septate conidia Long, erect and non-septate conidia Long, erect and non-septate conidia Long, erect and non-septate conidia
The results of the characterization and identification of the As shown in tables 2, 3, 4, 6, 7 and 8, type of packaging mould present in dakuwa in different packaging materials material had no effect on the type of microbe present in are shown in tables 5-8. The moulds that were isolated and dakuwa. From the tables mentioned above, it was observed identified were Mucor spp, Aspergillus niger and that the same types of microbes were present in the Aspergillus flavus. Only Mucor spp and Aspergillus niger different packaging materials during the period under were present in the fresh sample. After one month of observation. storage, Mucor spp was not found in the unpackaged Conclusion sample but Aspergillus flavus was present alongside Staphylococcus aureus, Streptococcus pyogene and Aspergillus niger. In the third and fourth month of storage, Bacillus subtilis were the bacteria found in dakuwa while only Aspergillus flavus was present in the unpackaged the moulds were Mucor spp, Aspergillus niger Aspergillus sample. For the packaged samples, the Aspergillus niger flavus. However, only Bacillus subtilis and Mucor spp can that was present in the fresh sample disappeared after one survive for extended period of time if the product is month of storage leaving only Mucor spp which was packaged in either aluminium foil or polyethylene. Apart present all through the period of storage. The presence of from packaging, the incidence of these microorganisms in Aspergillus flavus may be due to contamination of the dakuwa can be highly reduced through proper handling of grains due to poor post harvest handling. The presence of grains from the point of harvest to the point of processing. Aspergillus flavus exposes dakuwa consumers to the risk of Furthermore, observation of good manufacturing practices aflatoxin food poisoning especially if aflatoxigenic strains during processing is also very important in order to prevent are present since not all strains of Aspergillus flavus contamination by microorganisms of public health produce the toxins. significance. International Journal of Research in Pure and Applied Microbiology 2014; 4(1): 15-19
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Source of support: Nil; Conflict of interest: None declared
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International Journal of Research in Pure and Applied Microbiology 2014; 4(1): 15-19
