Miracle Olaleye Oriowo

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/- Microbial Analysis and Proximate Composition of Boiled and Fried Local Cheese (WARA) Article in International Journal of Scientific and Research Publications · December 2018 DOI:-/IJSRP-.p8491 CITATIONS READS 3 2,091 4 authors, including: Olufunmilola Ajayi Olalekan Balogun Joseph Ayo Babalola University Joseph Ayo Babalola University 11 PUBLICATIONS 57 CITATIONS 26 PUBLICATIONS 147 CITATIONS SEE PROFILE All content following this page was uploaded by Olalekan Balogun on 20 January 2019. The user has requested enhancement of the downloaded file. SEE PROFILE International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018 ISSN- 710 Microbial Analysis and Proximate Composition of Boiled and Fried Local Cheese (WARA) Ajayi, O. O1., Balogun, O. B1., Oriowo-Olaleye, M1. and Faturoti M. O1. Department of Biological Sciences, Joseph Ayo Babalola University, Ikeji Arakeji, Osun State, Nigeria. DOI:-/IJSRP-.p8491 http://dx.doi.org/-/IJSRP-.p8491 Abstract- Fried cheese locally called Wara gets spoiled when attacked by pathogenic bacteria, as a result of poor hygiene practice by the producer and handlers. Wara samples were bought in Ilesha at Atakumosa market square Osun-state. This research was designed to isolate and enumerate microorganisms using standard microbial methods, proximate composition and the nutritional composition of boiled local and fried cheese (wara). Proximate analysis was carried out to determine nutritional composition. Klebsiella species and Esherichia coli Staphylococcus epidermidis, Bacillus species, Escherichia coli, Salmonella species, Streptococcus species, Clostridium species and Lactobacillus species were isolated using standard technique. The highest bacteria count for the fried cheese was 2.1×105cfu/ml the lowest bacterial count was 1.5×105cfu/ml unit highest bacterial count of the boiled local cheese was 4.4×105(cfu/ml), lowest bacterial count was 1.8×105(cfu/ml) while the range of the values was 2.6×10 5. Fungi isolated were Penicillium and Gymnoase. Fat content for the boiled cheese has the highest mean value of 44.78 ±0.028cfu/ml and Ash had lowest mean value of 2.46±0.014cfu/ml. The moisture content of fried cheese had the highest mean value 55.32±0.014 while the carbohydrate had the lowest mean value 0.63±0.02 for fried chesse. Fibre content was not detected in both samples. The microbial contamination might generally occur based on lack of standardization, fecal contamination and poor personal hygiene in the production, handling and storage. Therefore, all cheese producers and consumers should take care during processing and storage of the cheese to prevent contamination. Index Terms- Pathogenic bacteria, Microorganisms, Proxinate composition and Microbial methods are not standardized (Adetunji, 2011). It could also be coagulated with the juice extract of leaf of sochom apple (Colotropis procera) or pawpaw ). (Augusti, 1996; Adetunji and Alonge, 2009; FAO, 2009). Cheese is usually stored in its whey and consumed fresh, but this can only last for 3-5 days after which spoilage occurs. It is sometimes fried and used as a meat-substitute in stews and soups, or smoke-dried to enhance its keeping qualities. However, all these increase its shelf life by only a few extra days or few weeks at best (Belewu et al., 2005). Cheese is made mostly from the milk of cows, buffaloes, sheeps or goats, and it is an important food component in the healthy diet of human’s highly nutritious rich food and source of protein, peptides, aminoacids, vitamins, salt, and essential minerals including calcium (Augusti, 1996; Belewu and Aina, 2000). Cheese however, has been shown to have antimicrobial properties that prevent disease, it has been used as drugs for certain infection when common antimicrobial agents failed it had also been found that soft cheese has growth inhibitory activity against common bacteria that caused diarrhoea in South West Nigeria (Ibrahim and Falegan, 2013). Microorganisms such as Streptococci, Lactobacilli, Coliform bacteria and some fungi are associated with milk and milk products (Baba et al., 2004; Raheem and Saris, 2009). The consumption of cheese is of utmost public health importance and high consumption rate in Nigeria calls for the periodical examination of cheese produced in different locality.. The study was designed to isolate and identify microorganisms found in both fried and boiled cheese; to determine the proximate composition and assess the best form of cheese for humans consumption. I. INTRODUCTION C heese (wara) it is a Nigerian soft, white, unsalted and unripened cheese usually processed from cow milk by the Fulani tribes who are mainly cattle rearers in Nigeria (Raheem, 2006; Ogunbanwo et al., 2011). Wara making started with the Fulani’s but as a result of their nomadic lifestyle, it spread to other parts of the country including Kwara, Oyo, Ogun, Ondo and the Benin Republic (Raheem, 2006). It is obtained by coagulating the casein with rennette or similar enzymes in the presence of lactic acid produced by adding microorganisms (Adegoke et al., 1992). However in many cases starter culture are not used as processing condition http://dx.doi.org/-/IJSRP-.p84XX II. MATERIALS AND METHOD 2.1 Study Area The cheese sample (Wara) used for this study was purchased from Ilesa West Local Government at Atakumosa market square in Osun state, Nigeria. The market was sited close to the place of the king of the town (Ereja square) on the outskirt of the city of Ilesha. The total population according to 2006 census was 103,555 having the total area as 63km2 (24 sq mi) and the coordinate of 7°39N 4°43’E. 2.2 Techniques for preparation of media 2.2.1 Sterilization Technique www.ijsrp.org International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018 ISSN- The glassware used was thoroughly washed with detergent rinsed with distilled water and air dried. The glassware were wrapped with aluminum foil paper and sterilized in an autoclave at 121°C for 15 minutes. 2.2.2 Collection of Samples Wara sample processed traditionally by Fulani woman were bought in highly populated location in Ilesha at Atakumosa Market Ilesa Osun State, Nigeria. The Fried cheese was bought into a sterile plastic plate and transported to Microbiology laboratory for analysis within 2 hours of purchase. III. PREPARATION OF MEDIA 3.1 Nutrient agar: This was prepared according to manufacturer’s description. Seven (7g) of nutrient agar powder was weighed and dissolved into 250 ml of distilled water. It was heated to get it homogenized and allowed to change to golden yellow before it was autoclave at 121º C for 15 minutes. 3.2 MacConkeyAgar: This was prepared according to manufacturer’s description. 14g of MacConkey agar powder was weighed and dissolved in 250ml of distilled water and homogenized using heat to boil. This was then sterilized by autoclaving at 121ºC for 15minutes. 3.3 Agar Slant Agar slant were prepared by dispensing 15ml of nutrient agar into different MacCartney bottles. The bottles were placed in a slanting position and allowed medium to gel. 3.4 Serial dilution of Samples Five grams (5g) of the cheese was meshed using the crucible and pestle, serial dilution was done by measuring 9ml of sterile water into first test tube till the tenth test tube. 1ml of the sample was then added into each labeled and properly covered test tube using a sterile syringe. 1ml was then taken from 10 5 or the fifth test tube into a petridish and gently swirled and allow to set. The petridishes were then incubated at 37°C for 24 hours. After the incubation, the colonies that developed on nutrient agar plate were counted and used to determine the total bacteria count of the sample (cfu/ml). The representative colonies on the plates were subcultured on a fresh nutrient agar to obtain a pure culture of isolates. The pure culture was then transformed into nutrient agar slant for biochemical test. 3.5 Isolation and identification of the bacteria Distinct and well isolated colonies were sub-cultured and examined for various sizes, shapes, colours and texture, a series of tests such as Catalase, Oxidase, Indole, Nitrate reduction, and sugar fermentation were carried out to identify the bacteria according to (Cheesbrough, 2006). 3.6 Isolation of Fungi Nine point eight gram (9.8g) of Potato Dextrose Agar (PDA) powder added with antibiotics (to inhibit the growth of http://dx.doi.org/-/IJSRP-.p84XX 711 bacteria) was dissolved into 250ml of sterile water and autoclaved at 121°C for 15 minutes and allowed to cool before pouring aseptically into the petridishes. The prepared potato dextrose agar (PDA) was aseptically dispensed into the Petri dish containing the sample and swirled gently before cooling. The plate was then incubated in an incubator at 26°C for five (5) days before observation (Onyeagba, 2004). 3.8 Proximate Analysis of Nutritional Composition of Fried Cheese This refers to the determination of the major constituents of food sample and it is used to assess if a sample is within it’s normal compositional parameters or somehow been adulterated. This method partitioned nutrient in food sample into six components: ash, crude protein, fat, crude fiber, moisture content, and carbohydrate (FAO, 2008). IV. RESULT Table 1: It shows the total bacterial count in fried cheese calculated in colony forming units per ml, the sample four had no bacterial population . Table 2: Total bacterial load of boiled local cheese (wara) samples calculated in colony forming units per ml, all the sample had bacterial population which varies from sample one till sample five Table 3: It shows the morphological characteristics and biochemical test of bacteria isolate from boiled local cheese (wara) based on size, texture, colour, opacity, surface, elevation and margin. Table 4: It shows the morphological characteristics and biochemical test of bacteria isolate from fried local cheese (wara) is based on size, texture, colour, opacity, surface, elevation and margin. Table 5: It shows the count for fungi isolated in the fried cheese sample. The fungi isolate were: penicillium spp and Gymnoaseus sp. Table 6: Proximate analysis showing the nutrition evaluation of cheese, which is based on Moisture content, Fat, Fibre, Crude protein and Carbohydrate. Table 7: Statistical analysis of proximate composition shows mean Standard deviation, Variance and Coefficient of variation. Figure 1: Bacterial count in fried cheese which is calculated in colony forming units per ml. . Figure 2: Bacterial load of local cheese (wara) samples. Figure 3: The boxplot of the statistical analysis of proximate composition www.ijsrp.org International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018 ISSN- 712 TABLE 1: Total bacterial load of Fried Cheese (wara) sample Replicate Sample 1(Cfu/ml) Sample 2(Cfu/ml) Sample 3(Cfu/ml) Sample 4 (Cfu/ml) RI 1×105 R2 2×105 Mean 1.5×105 SD 0.70710 CV 47.40 VAR 0.5 Keys: SD: Standard deviation VAR: Variance CV: Coefficient of variance 2×105 1×105 1.5×- 2×105 2×105 2 ×105 0 0 0 0 2×105 1×- Table 2: Total bacterial load of boiled local cheese (wara) samples. Replicate Sample (Cfu/ml) 1 Sample (Cfu/ml) 2 Sample (Cfu/ml) 3 Sample (Cfu/ml) 4 Sample (Cfu/ml) 5 Sample 6 (Cfu/ml) R1 4.5×105 3.3×105 2.7×105 1.8×105 2.1×105 1.7×105 R2 4.3×105 3.7×105 3.0×105 2.3×105 1.5×105 2.6×105 Mean 4.4×105 3.5×105 2.85×105 2.05×105 1.8×105 2.15×105 SD - - - - - - CV 3.2% 8% 7.4% 17% 24%% 29% VAR 2 8 4.5 12.5 18 40.5 KEYS: SD: Standard deviation CV: Coefficient of variation VAR: Variance R1:Replicate 1 http://dx.doi.org/-/IJSRP-.p84XX R2: Replicate 2 www.ijsrp.org International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018 ISSN- 713 Sucrose Probabale organism G Escherichia coli - - G G Salmonella spp Round Cream Smooth Small Transluscent - - + + - + + d Entire Cream Smooth Small Opaque - + + + + + d Entire Cream Dry smooth Big Opaque - - + - + d Round cream Dry smooth Small Translucent - - + - + Motility d Hydrogen sulphide G Indole - VP - Methyl red Lactobacillus spp Gram staining G Spore staining G Optical - Size Klebsiellia spp Surface G Colour G Edge Gluose TABLE 3: Morphological characteristics and biochemical test of Bacterial isolates from fried local cheese (wara) Shape Size Texture Color Opacity Edge Gram stain Spore stain Methyl red catalase Indole Motility VP H2S Lactose Glucose Sucrose Small rough Cream opaque entire + + + + + - A A A Small rough Cream opaque entire + Small smooth Yellow opaque center + Small smooth Cream opaque entire Lactobacillus spp Rod Clostridium spp + Cocci Streptococcus spp + Rod Bacillus spp Cocci Probable Organisms Table 4: Morphological characteristics and biochemical test of bacterial isolates from boiled local cheese (wara) + http://dx.doi.org/-/IJSRP-.p84XX - + + + - - - - A A + + + + - + + - AG AG - - + - - - + G AG AG www.ijsrp.org Cream opaque center opaque center smooth rough + Pink Small Small Escherichia coli Cocci Staphylococcus epidermidis Rod International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018 ISSN- - 714 - - + - - + + - AG AG - + + + + - - AG AG AG Error bar ±1 SE Figure 1: Bacteria count in fried cheese. http://dx.doi.org/-/IJSRP-.p84XX www.ijsrp.org International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018 ISSN- 715 Error bar ±1 SE Figure 2: Bacterial load of local boiled cheese (wara) samples. Table 5: Total fungi count in fried cheese (Wara) Sample- Cfu/ml 6×103 4×105 Fungi organism Penicillium spp Gymnoase spp Table 6: Proximate analysis result showing nutritional evaluation in fried cheese S/N %Ash % MC %CP %Fat %Fibre %CHO R1 2.45 10.33 21.11 44.8 0 21.31 R2 2.47 10.31 21.1 44.76 0 21.36 Mean 2.46 10.32 21.105 44.78 0 21.335 SD - - - - 0 - VAR 0.002 0.002 5E-05 0.008 0 0.0125 CV 0.05749 0.0137 0.335 0.0632 0 0.01657 Sample 2.46±0.014 Keys: MC: Moisture Content SD: standard deviation VAR: variance CV: coefficient of variance 10.31±0.014 21.1±0.007 44.76±0.02 0 21.36±0.03 CP: Crude protein CHO: Carbohydrate Table 7: Statistical analysis of proximate composition in boiled cheese Replicate ASH MC CP FAT R1 1.16 55.31 18.11 24.78 0 0.64 R2 1.18 55.33 18.12 24.76 0 0.61 Mean 1.17 55.32 18.115 24.77 0 0.625 SD - - - - 0 - CV 1.41% 0.03% 0.04% 0.1% 0 3.4% VAR 0.0002 0.0002 5E-05 0.0002 0 0.00045 http://dx.doi.org/-/IJSRP-.p84XX FIBRE CHO www.ijsrp.org International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018 ISSN- 1.17±0.014a Sample 55.32±0.014b 18.2±0.007c 716 24.8±0.014d 0e 0.63±0.021f Data are presented as Mean±SD (n=2) from duplicate determination. Different superscripts in the same column are significantly different (P<0.05). Keys: MC: Moisture content CP: Crude protein CHO: Carbohydrate ND: Not detected Boxplot of ASH, MC, ... 60 50 Data 40 % 30 20 10 0 ASH MC CP FAT FIBRE CHO Figure 3: The boxplot of the statistical analysis of proximate composition V. DISCUSSION AND CONCLUSION The results obtained from the microbial analysis of fried cheese (wara) show that the products were contaminated with microorganisms of public health concern. The bacterial count in the sample may be a consequence of low level of hygiene maintained during the processing and sale of the products. This includes the handlers, quality of water used and the utensils. During the sale of fried cheese (wara), dirty hands and spoons are dipped into the bowl for product selection by both hawkers and consumers. The exposure of wara and while they are displayed for sale in bowls can serve as source of contamination. The detection Klebsiella spp and E.coli in wara may indicates possible faecal contamination because the fulani’s do not disinfect the teats and udders prior to milking despite the fact that the cow lies in a muddy barnyard and dirty environment which inevitably contaminate the milk and could increase the microbial load. The http://dx.doi.org/-/IJSRP-.p84XX presence of Staphyloccus epidermidis, as supported by the study of Ajayi et al. (2016) may lead to contamination of food and eventually affects the health of the consumers. The presence of Bacillus species which produces several toxins, being isolated in this study probably is an indication of poor hygienic habit of the milker milking the cow. Clostridium species isolated which are important cause of diarrhea inhabits the soil and intestinal tract of animals including humans and can cause food intoxication which is congruent to the findings of Ajayi et al. (2016). Being enteric bacteria, their presence indicates poor hygienic practices among handlers of wara. Due to the significance of the faecal-oral route transmission for many bacterial food-borne diseases, basic hygienic measures assume a decisive importance in food safety management (Alalade and Adeneye, 2006). The detection of Lactobacillus species isolated from sample show that they are organism used for the production of cheese. Lactobacillus species allows the production of gas in lactose, sucrose and glucose Aworh and Egounlety (1985). They are motile, gram positive bacteria which tests positive for www.ijsrp.org International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018 ISSN- catalase test. The fungal isolates: Gymnoase and Penicillium, species which were isolated are known as spore formers, which therefore means that they can easily contaminate the dairy products which are usually exposed during processing, storage, and hawking. They are major spoilage organisms of carbohydrate foods (Rhodes and Fletcher, 1966). However, their growth can result in the production and accumulation of mycotoxins which are of public health and economic importance (Burnett and Beuchat, 2001). The nutritional analysis of fried cheese shows that they are of appreciable nutritional status especially in the protein and fat content. The dairy products particularly cheese are good sources of protein. Higher fat content was observed in fried cheese wara. This could be due to vegetable oil used in frying of the cheese. [9] [10] [11] [12] [13] [14] [15] REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] Alalade, O.A. and Adeneye, J.A. (2006). The effects of short term frozen storage on chemical composition and coliform and coliform microflora of Wara cheese “Wara cheese under frozen storage. Advance Journal of Food Technology. 2(1): 44-44 Augusti, K.T. (1996). Therapeutic values of onion and garlic. Indian J ExpBiol 64: 334- 336. Aworh, O.C. and Egounlety, M. (1985). Preservation of West African soft cheese by chemical treatment. Journal of Dairy production Research. 52: 189-195. Baba, K.M., Orire, A.O. and Suleiman, H W. (2004). Profitability of cheese production and marketing among rural women in Moro local Government area of Kwara state. Proc. American Journal of Food and Nutrition., 3(1): 13-21 Belewu M.A and Aina (2000). Microbial evaluation of indigenous milk products with special reference to the bacterial flora of public health importance in Nigeria. African Journal of Experimental Microbial analysis. 1 (1): 13-19. Belewu, M.A., Belewu, K.Y and Nkwunonwo C.C. (2005). Effect of biological and chemical preservatives on the shelflife of West African soft cheese. African Journal of Biotechnology. 4:-. Belewu, M.A., Belewu, K.Y. and Ganiyu, R.O. (2011). Evaluation of lemon Grass Cymbopogon citrates) oil and pure natural honey on the shelf life of cheese. Electronic Journal of Environmental, Agricultural and Food Chemistry (EJEAFChe), 10(4),-. Burnett and Beuchat, (2001). Emerging infectious Diseases. Produce Handling and Processing Practices; 5:6 http://dx.doi.org/-/IJSRP-.p84XX View publication stats [16] 717 Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries. Part 2 Press Syndicate of the university of Cambridge, United Kingdom pp. 63-70. FAO: Food and Agricultural organization.(2008). Milk for health and wealth .FAO, Rome, Italy. Journal of agricultural, 19:34-12. Ibrahim, T.A. and Falegan C.R. (2013). Anti-bacterial activities of crude cell free supernatants of Lactic acid bacteria from wara (Nigeria soft cheese). Research and Reviews..Journal of food and Dairy Technology, 1 (2):1-4. Ogunbanwo, S. T., Sanni, A. I. and Onilude, A. A. (2011). Effect of bacteriocinogenic Lactobacillus spp. On the shelf life of fufu, a traditional fermented cassava product. World Journal of Microbiology and Biotechnology, 20:57-63. Oladipo I.C., Onyeruke I.C. and Adebiyi, A.O. (2009); Microbiological analysis of some Vended cheese in Osun, Nigeria. Afrrican Journal of Food Science. 3 (12) pp: 406-412. Onyeagba , A. (2004) Laboratory guide for microbiology. Crystal Publishers, Okigwe, Imo State. pp 95-97. Raheem, V.D. and Saris, D.E.J. (2009). Characterization and Application of Calotropisprocera, a coagulant in Nigeria wara cheese. International Journal of Food Science and Technology. 20:23-12. Sanni A.I., Onilude A.A. and Momoh M.O. (2011). Selection of starters and a Starter-mediated novel procedure for production of wara, a West African soft cheese. International Journal of Food Science Technology, 34: 325-333. AUTHORS First Author – Ajayi, O. O, Department of Biological Sciences, Joseph Ayo Babalola University, Ikeji Arakeji, Osun State, Nigeria. Second Author – Balogun, O. B, Department of Biological Sciences, Joseph Ayo Babalola University, Ikeji Arakeji, Osun State, Nigeria. Third Author – Oriowo-Olaleye, M, Department of Biological Sciences, Joseph Ayo Babalola University, Ikeji Arakeji, Osun State, Nigeria., Corresponding Author e-mail:- Fourth Author – Faturoti M. O, Department of Biological Sciences, Joseph Ayo Babalola University, Ikeji Arakeji, Osun State, Nigeria. www.ijsrp.org