Isolation and Identification of Bacteria Associated with Pre and Post Processing of Groundnut Cake in Sokoto State, North-Western Nigeria

This study was conducted to assess the bacterial contamination between the groundnut cakes and paste obtained from Rural areas (Bodinga, kware) and that obtained within the metropolis at Urban areas (Arkilla and ‘Yargoriba) of Sokoto metropolis. A total of 8 samples of ready to eat groundnut cake and pastes were purchased, 4 of which were cake packaged in polythene and 4 paste packaged in plastic rubbers. The samples were analyzed microbiologically using standard procedures. Bacteria species associated with the samples collected from the rural areas were identified as Bacillus lentus, B. megaterium, B. licheniformis, Listeria monocytogene, Staphylococcus aureus while those associated with samples collected from the urban areas were identified as Bacillus lentus, B. megaterium, B. licheniformis, Listeria monocytogene, Staphylococcus aureus and S. hominis. There was higher bacteria count on samples from rural areas compared to those from urban areas. This might be as a result of unhygienic practices during the processing of the product which may range from methods of obtaining and quality of raw materials including sources of water, packaging, handling and distribution of the finished product to the final consumers. Based on these, it is recommended that Producers of groundnut cake should use good hygienic places, raw material and equipment to reduce the risk of microbial contamination. =Groundnut paste collected from ‘Yar goriba area. CA= Catalase test, SH= Starch hydrolysis, CI= Citrate, IN= Indole, MR= Methyl Red, VP=Voges ProskaurMO= Motility, GL= glucose, LA= Lactose, SU= Sucrose, H2S= Hydrogen Sulphite, UR= Urease, Trt= Gram Stain


INTRODUCTION
Groundnut (Arachis hypogaea L.) cake, popularly called "Kulikuli", is produced by extracting oil from the groundnut kernels (Atanda and Akano, 1990). It is a popular snack in Nigeria that is processed locally which exposes it to microbial contamination, as a result of improper processing associated with handling, water impurities during oil extraction, the use of contaminated equipment, air, packaging materials and storage conditions. Groundnut paste and cake have been frequently associated with food illness in which the initial contamination is traceable to food handlers. Numerous epidemiological reports and studies have implicated food of ready to eat origin as the major vehicle associated with illness caused by food born pathogen (Sokari, 1991). The microbial quality of groundnut cake primarily depends on the quality of raw materials, production process, processing environment, hygiene of the personnel involved in processing and the storage condition. Since the production of groundnut cake and paste is no more limited to oil mills but to local processors, there is possibility of microbial contamination due to the unhygienic practices during the processing, groundnut cakes and pastes can be contaminated with microorganisms. Hence, Microbial and Aflatoxin contamination is a major concern in food safety as they affect humans, animals and the economic growth of any nation (Hwang et al., 2004). Groundnut cake and paste may be contaminated by bacteria such as Staphylococcus spp, Escherichia coli; an indicator of fecal contamination resulting from water supply. The presence of these contaminating organisms in food consumed by large population is of public health concern (Oko et al., 2015). Therefore, this study was undertaken to assess the bacterial contamination between the groundnut cakes and paste obtained from rural area and that obtained within the Sokoto metropolis of Sokoto state, Nigeria.

Study Area
The study area is within Sokoto State; Bodinga, Kware and within Sokoto metropolis; Arkilla and 'Yargoriba. Sokoto lies on 13⁰01'N, 15⁰05'E; at an altitude of 350m above sea level (SERC, 2015). The main occupation of Sokoto inhabitants includes; Farming, Fishing and leather work (MOI, 2008).

Materials and Reagents
The materials and reagents used in the laboratory for these research work are; Nutrient agar, Ethanol, Nutrient broth, Urea agar base, Triple Sugar Iron (TSI) medium, Gram stain reagents, Streptomycin, Petri dishes, Test tubes, Bend glass rod, Glass slide and Cover slip, Conical flask, Distilled water, Aluminum foil, Syringes, Slant bottles, Wire Lupe, Masking tape, Cotton wool, Face mask and Hand gloves. Samples Collection Groundnut cake (kulikuli) both pre and post processed where collected at random from four (4) processing units of Sokoto State; Two from Rural areas (Bodinga and Kware) and two from Urban area, that is within Sokoto metropolis (Arkilla and 'Yargoriba). A sample each, of the groundnut paste and cake was collected from each of the four processing units making a total number of eight samples. The Groundnut cake samples were collected in a polythene bag while the Groundnut paste samples in a plastic rubber.

Sample Preparation
The collected groundnut cake samples were reduced to fine particles in the laboratory through the use of sterile porcelain pestle and mortar. Media Preparation All glass wares were washed with detergent solution, thoroughly rinsed in several changes of tap water and then air dried. The glass wares were put into a hot air oven and sterilized at temperature of 121 for 15minutes then allowed to cool down to at room temperature before usage. All media were prepared according to the manufacturer's instructions (labels). Preparation of Nutrient agar 28g of nutrient agar (NA) powder was weighed on an electrical weighing balance and transferred into a conical flask containing 1000ml distilled water. The mouth of the conical flask was plugged with a non-absorbent cotton wool, and then wrapped with aluminum foil. The mixture was swirled and heated to obtain a homogenized solution. The solution was autoclaved at 120⁰C for 15 minute with pressure of 15psi, after which the prepared medium was allowed to cool down to about 45˚C, and then poured into Petri dishes and allowed to solidify. Serial Dilution Five (5) test-tubes each containing 9ml distilled water were used for the dilution; 1g of each sample was taken and transferred into the first test tube and allowed to dissolve, then mixed; to the second tube, 1ml of the content from the first test-tube was aseptically taken using sterile syringe and poured into the second (2) test tube. This procedure was repeated up to the last test tube (5 test tubes). Thus, making a dilution factor of 10ˉ1, 10ˉ2, 10ˉ3, 10ˉ4, and 10ˉ5 respectively. Bacterial Plates Samples collected were inoculated into a prepared petri dish containing Nutrient agar (medium for isolation of bacteria). The inoculated Ager media was incubated in an oven at 37°c for 24hours, after which colony growth was observed.
Identification of the isolated bacteria was carried out by first observing the appearance of the colonies produced which is followed by Gram's staining and subsequent sub-culture onto slant bottles containing nutrient Ager, followed by biochemical test. Identification and Characterization of the Bacteria Isolates Identifiable colonies were carefully examined microscopically for characteristics such as the shape, color, size and consistency. Bacteria isolates were characterized based on colony morphology, microscopic appearance and gram staining reactions as well as biochemical tests. Microscopy The isolates were identified by picking a colony and placing it on a sterile glass slide, gram stained and viewed under the microscope. Procedure Gram staining was conducted by making a smear of the isolates on a glass slide using fixed heat. To the smear on the slides, Crystal violet was poured in drops and allowed for 30seconds then rinsed with water, the films were again covered with Iodine and allowed for another 30seconds then rinsed with water and decolorized with 90% alcohol. They were again rinsed with water and color stained with safranin for about 20-30 seconds and finally rose with water and dried. Slides were then examined under the oil immersion objective (100 ⁎ ) Biochemical tests The pure bacteria isolates were subjected to biochemical characterization. the following biochemical test performed are catalase test, coagulase test, urease test, indole test, citrate utilization test starch hydrolysis and Triple sugar iron(T.S.I) test, which includes hydrogen sulphite, gas, lactose, sucrose, glucose and motility. Lactose fermentation growth at 45°C and haemolysis. The tests were performed in accordance with standard procedures described by (Barrow and Feltham 1993). Catalase Test This test was used to differentiate those bacteria that produce the enzyme catalase from non-catalase producing Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.10, No.12, 2020 15 bacteria.
A drop of 3% hydrogen peroxide was placed on a glass slide, using a wire loop, the 24-hour colony of each isolate was emulsified into the hydrogen sulphide and observed. A positive test will be indicated by bubbling and frothing. Whereas in a negative test, there will be no bubbling or frothing. (Oyeleke and Manga, 2008) Indole Test This is used in determining an organism's ability to produce indole from tryptophan. The organism was grown in tryptophan broth for 24hours, kovacs iodole reagent was then added. A positive reaction will be indicated by the development of a red color in the reagent layer above the broth within 1minute.whereas a negative reaction is indicated by yellow color on the medium (Oyeleke and Manga, 2008). Urease Test This test is used to detect the ability of an organism to produce the enzyme urease that hydrolysis urea into ammonia and carbon dioxide. A culture was incubated in urease agar for 48hours. A positive reaction is indicated by the development of a bright pink or red coloring agar (Beishir, 1991). Citrate Utilization Test A Simon citrated agar was used. The development of a deep blue color indicates a positive reaction. Methyl Red (MR) and Voges Proskaur (VP) Test. These considered together since they are physiologically related and are incubated into the same medium MR-VP broth and the results obtained are usually opposite (i.e. MR+, VP-or MR-, VP+) (Beishir,1991) A wire loop full of each isolate was inoculated into glucose phosphate peptone water and then incubated at 37 o c for 48 hours. Few drops of methyl red were added to the culture (Cheesbrough, 2006). Vogesproskaur test (VP). 0.5ml of 40% potassium hydroxide was added followed by 1ml Naphtol. This was shaken vigorously and placed in a sloppy position and was examined for 30-60 minute (Cheesbrough, 2006). Coagulase Test Slide Method Two drops of water were dropped on a clean glass slide. One colony was dropped on each drop of water and emulsified. A flame wire loop was dipped in human plasma and used to stir the bacteria suspension. Visible clumping in 10 seconds shows a positive result. Starch hydrolysis Nutrient agar along with starch was prepared and poured into petri dishes. Sub-culture was made from the stock then incubated for 24hours. Iodine was dropped on the sub-cultures and allowed for 1min then poured out. The organism that appears visible is said to be able to utilize starch while those that appear black are not able to utilize starch. Triple Sugar Iron (T.S.I) Inoculum from sub-cultured plates were picked with a sterile straight wire loop and stabbed on the butt and then streak on the surface of slant. The inoculated test tubes were then incubated at 37°C for 24hours. Since TSI is a composite media, several reactions were read from it after the 24hours of incubation. Gas production was determined by the presence of bubbles or cracks on the agar or complete disruption of medium, hydrogen sulphide production can be determined by the blackening of the whole butt or a streak or ring of blackening at the slant butt junction, Motility can be determined by the presence of growth along the area been stabbed by the straight wire loop. If the butt becomes yellow, it is an indication of glucose fermentation, formation of a pink/red color at the slant indicates the presence of lactose and sucrose fermentation (CheeseBrough, 2006).

Results
Fourteen (14) Bacteria Species were isolated, the frequency of occurrence of these organisms showed that Bacillus megaterium has the highest frequency with 35.7% followed by Bacillus lentus, Staphylococcus hominis and Listeria monocytogene with (21.2%, 14.3% and 14.3%). The least frequency with 7.1 was from Bacillus licheniformis and Staphylococcus aureus ( Table 1).
The results of biochemical tests on groundnut cake and paste collected from both rural and Urban areas of Sokoto are presented in Table 3 and Table 4. They showed the presence of Bacillus lentus, B. megaterium, B. licheniformis, Listeria monocytogene, Staphylococcus aureus and S. hominis with the exception of Staphylococcus hominis which only appeared on samples collected from the Urban area.
Staphylococcus hominis is a coagulase-negative member of the bacteria genus Staphylococcus, consisting of Gram-positive, spherical cells in clusters. S. hominis is normally found on human skin of usually harmless people, but it can sometimes cause infection in people with abnormally weak immune systems, their presence in food are indication of excessive human handling (Abdalla et al., 2012).
Bacillus lentus, B. megaterium, Listeria monocytogene, Staphylococcus aureus and S. hominis were found to appear on the Groundnut cake samples while Bacillus lentus, B. megaterium, B. licheniformis and Listeria monocytogene, appeared on the Groundnut paste samples.
The presence of Bacillus specie might be due to the fact that it is an abundant spore former in soil, air and water, hence can be present in the food sample as a result of exposure of the product on the road side. This report is in agreement to the studies of (Clarence et al., 2009;Hassan et al., 2014;Okonko et al., 2009;Oluwafemi and Simisaye, 2005). They isolated similar organisms from sausages, meat pie, sea foods and puff, respectively. Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.10, No.12, 2020

DISCUSSION
In this study, a total of eleven (11) organisms were recorded from the microbial analysis on Groundnut cake and paste collected from Urban and rural areas of Sokoto State respectively. Six (6) of which were bacteria and five (5) fungi. The bacteria isolates were identified on the bases of their reactions to biochemical reagents. These organisms include; Bacillus lentus, B. megaterium, B. licheniformis, Listeria monocytogene, Staphylococcus aureus and S. hominis. Morphological characteristics and microscopic examination were used for the identification of fungi isolates. This research has revealed a number of bacteria associated with the contamination of groundnut cake and paste which is in agreement with Okwu et al. (2017) who isolated bacteria species of Bacillus spp., Staphylococcus spp., and fungi specie Aspergillus spp. from groundnut in their study. Staphylococcus spp in the groundnut pastes will result if the grinding machine and other utensils are contaminated. According to Doyle (2007), Bacillus spp. and Pseudomonas spp. are normal inhabitants of the soil and are capable of causing disease in humans and animals. These organisms are capable of surviving harsh conditions such as roasting and blending processes. Table 3 shows the statistical analysis using mean and standard error mean (SEM) from the average bacteria count. Groundnut cake from Rural area when compared with Groundnut paste from Rural area showed that between sample A 67.33±7.58 and C 35.50±1.00 there is significant difference between the mean, and comparing sample B 50.0±6.25 Groundnut cake from Urban area with D 26.33±4.79 Groundnut paste from Urban area, there was also a significant difference. Therefore, among the whole samples, sample D is recommended (more acceptable) since it has the least mean ± SEM value hence, it has less contamination.

CONCLUSION
The results obtained showed higher bacteria count in samples from Rural areas compared to those from Urban areas, this might be as a result of unhygienic practices during the processing of the product. This may range from methods of obtaining and quality of raw materials including sources of water, to the packaging, handling and distribution of the finished product to the final consumers. Considering the nutritional importance of groundnut cake and its suitability for incorporation into traditional and conventional products, it is hereby suggested that a small to medium scale production of groundnut cake be carried out under standard hygienic operation procedures. This will drastically reduce contamination and epidemics that may result in the consumption of improperly produced food and food products as well as popularize the nutritional benefits of the product.

RECOMMENDATIONS
Based on the results obtained above, it is recommended that: -The National Agency for Food Drug Administration Control (NAFDAC) should pay attention to small scale businesses so as to ensure a wholesome food for the consumers.
-The producers of groundnut cake should use hygienic area, raw materials and equipment to reduce the high risk of microbial contamination -The public should be enlightened, especially the producers, hawkers and consumers of this food product, on the resulting negative implication of unhygienic practices on human health.