Research Article - Journal of Food Microbiology (2022) Volume 6, Issue 5

IDENTIFICATION OF SPOILAGE BACTERIA FROM FERMENTED FOOD PRODUCTS IN EDO STATE, NIGERIA

Olatunji EO¹, Isola OB^2*, Okanlawon TS¹

¹Department of Biological Sciences, Samuel Adegboyega University, Ogwa, Edo State, Nigeria

²Department of Chemical Sciences, Samuel Adegboyega University, Ogwa, Edo State, Nigeria

Corresponding Author:

Isola OB
Department of Chemical Sciences,
Samuel Adegboyega University,
Ogwa,
Edo State,
Nigeria, Email: dotmanchope@gmail.com

Received: 30-Jun-2022, Manuscript No. AAFMY-22-68214; Editor assigned: 04-Jul-2022, AAFMY-22-68214 (PQ); Reviewed: 18-Jul-2022, QC No. AAFMY-22-68214; Revised: 29-Aug-2022, Manuscript No. AAFMY-22-68214 (R); Published: 05-Sep-2022, DOI: 10.35841/AAFMY-6.5.121.

Citation: Olatunji EO, Isola OB, Okanlawon TS. Identification of spoilage bacteria from fermented food products in Edo state, Nigeria. J Food Microbiol. 2022;6(5):1-10.

Abstract

This study assessed the bacteriological quality of fermented food products by the enumeration of the bacteria that cause their spoilage. Three different samples of fermented food (“Palm wine”, Ogi” and “Fufu”) were purchased from two selling points in Ogwa and Ebelle, Esan-West and Igueben local government areas of Edo state, Nigeria respectively. de Man Rogosa Sharpe, Maconkey and nutrient agars were used for the isolation and determination of bacterial load. Standard morphological and biochemical tests were carried out for the identification and characterization of isolates. A total of twelve (12) bacteria species were isolated. The Total Heterotrophic Bacterial Count (THBC) and Lactic Acid Bacterial (LAB) count which ranged from 1.57 × 10⁴ cfu/ml to 2.47 × 10⁴ cfu/ml and 0.79 × 10⁴ cfu/ ml to 2.38 × 10⁴ cfu/ml before spoilage respectively while total heterotrophic bacterial count and lactic acid bacterial count ranged from 3.47 × 10⁴ cfu/ml to 6.15 × 10⁴ cfu/ml and 1.91 × 10⁴ cfu/ml to 4.05 × 10⁴ cfu/ml after spoilage respectively. The bacteria identified include; Salmonella enteritica, Pseudomonas aeruginosa, Bacillus subtilis, Citrobacter freundii, Klebsiella oxytoca, Staphylococcus aureus and Esherichia coli while lactic acid bacteria were Lactobacillus, Pedococcus, Leuconostoc, Lactococcus and Lactobacillis species. Locally fermented food should be given proper handling so as to prevent food spoilage and food borne infections.

Keywords

Fermented foods, Spoilage bacteria, Lactic acid bacteria, Fermentation

Introduction

The deliberate fermentation of foods by man predates written history and is possibly the ancient method of preserving consumable foods. Evidence suggests that fermented foods were used up 7,000 years ago in Babylon. Scientist speculates that our ancestors possibly discovered fermentation by accident and continued to use the process out of first choice or necessity. Preservation through fermentation did not only make foods available for future use, but also add flavour to the food making it more nutritious [1]. According to Chelule, foods are fermented to increase shelve life or for preservation, to improve flavour, texture, add aroma, increased solubility and digestibility. Fermented food products are more palatable and do not easily get spoilt because the by-products of fermentation which also include organic acids, hydrogen peroxide, diacetyl and bacteriocin serve as preservatives against spoilage organisms apart from imparting their desired flavours and aroma. Spoilage of food may be seen as any change that occurs in a food, making it undesirable for human consumption. Food spoilage is caused by microorganisms and this is attained with the help of extracellular enzymes they manufactured, which convert the food product into new substance with the production of gas and change in organoleptic properties. Most food spoilage are due to the activities of bacteria, molds and yeasts, which alter the smell, taste, colour, texture or chemical composition of the food significantly and render it inedible. The chemical properties of the food determine how satisfactory it will be as a culture medium for microbial growth. Carbohydrate rich food is often spoilt by fungi, especially species of Rhizopus, Mucor and Penicillium which grow on starchy foods if kept in humid condition. Fermented foods are always from plant or animal origins which formed great part of an average Nigerian diet contributing main nutrients such as proteins, calories, minerals and some vitamins, and they include indigenous foods like “Nunu,” “Cheese,” “Youghurt,” “Dawadawa,” “Palm wine,” “Ogi” “Fufu” etc. Local fermentation of food is a method of food refinement, where microbes, for example, Lactic Acid Bacteria (LAB) are applied in food production through the method acknowledged as fermentation. Fermentation in food processing involves the conversion of carbohydrates to alcohol and carbon dioxide or organic acids using yeast and/or bacteria, under anaerobic conditions. Fermentation is mainly one of the standard methods of food preservation with Lactic Acid Bacteria (LAB) and yeasts playing the major role in this fermentation [2]. The load of the microorganisms in the food is commonly small, but their consequence on the nature of the food, mainly in terms of flavour, and other organoleptic properties, is profound. Fermented food products are more palatable and do not easily get spoilt because the by-products of fermentation which also include organic acids, hydrogen peroxide, diacetyl and bacteriocin serve as preservatives against spoilage organisms apart from imparting their desired flavours and aroma. Spoilage of food may be seen as any change that occurs in a food, making it undesirable for human consumption. Most food spoilage are due to the activities of bacteria, molds and yeasts, which alter the smell, taste, colour, texture or chemical composition of the food significantly and render it inedible [3]. Lactic acid bacteria like Lactobacillus, Pediococcus, Streptococcus, Oenococcus, etc. are the most essential bacteria in fermented foods, followed by Acetobacter species, which oxidize alcohol to acetic acid. The acetic acid fermentation has been used broadly to produce fruit vinegars including cider vinegar. Lactic Acid Bacteria (LAB) isolated from fermented foods demonstrated probiotic properties such as hypo-lipid emic, hepato-protective and antibacterial and had been set up to be effective in treating gastroenteritis in man and animals. Moulds are as well important organisms in food processing both as spoilers and preservers of foods [4]. Many moulds have ability to produce enzymes of economic importance such as pectinase by Aspergillus niger. Species of Aspergillus are intricate in the production of citric acid from excess like apple pomace. The Aspergillus species are often responsible for unwanted changes in foods triggering spoilage. In the other way, Penicillium species are associated with the ripening and flavour development in cheeses. While the species of ceratocystis are involved in fruit flavour production, at the same time, Penicillium is the causative agent for manufacture of toxin like patulin. Like bacteria and molds [5], yeasts have beneficial and non-beneficial effects in food fermentations. The most beneficial yeast in terms of desirable food fermentations are from the Saccharomyces family, especially Saccharomyces cerevisiae involved in bread production and alcohol in wine fermentations. Saccharomyces cerevisiae var. ellipsoideus is employed extensively in wine production. Schizosaccharomyces pombe and S. boulderi are the dominant yeasts in the production of traditional fermented beverages, mainly those obtained from maize and millet. Saccharomyces cerevisiae var. carlbergenisis is the yeast intricate in beer production. Schizosaccharomyces pombe has [6] capacity to degrade malic acid into ethanol and carbon dioxide and has been used positively to reduce the acidity in the grape and plum musts. A number of yeasts like Rhodotorula and Cryptococcus have ability to produce pigment to be used as biocolur [7].

The chemical properties of the food determine how satisfactory it will be as a culture medium for microbial growth. Carbohydrate rich food is often spoilt by fungi, especially species of Rhizopus, Mucor and Penicillium which [8] grow on starchy foods if kept in humid condition. The fermentation procedures for these foods are a local knowledge picked up by observations and practice, and move on from age to age [9].

Nigeria is a multilingual and multicultural nation which most likely reflects in their dressings, cultural practices and modes of nutrition. Each settlements and tribes has its own indigenous preferred food that is defined by customs, tradition and religion [10]. The fermentation practices are often in a small scale on domestic basis, characterized by the usage of simple nonsterile equipment, chance or natural inoculums, uncontrollable conditions, sensory variations, poor stability and unattractive package of the refined products resulting in food of unstable quality. With increasing industrialization and urbanization, efforts are presently geared toward the direction of developing a large-scale factory processing facilities [11] for these foods where the quality of the refined product will be guaranteed.

Today, there is a high consumption rate of fermented food products such as palm wine, ogi and fufu by residents in Ogwa. The consumption of these spoilt fermented food products results in illness such as diarrhea etc. The probable cause of these problems sometimes lies in the unhygienic and poor preservation methods employed by fermented foods dealers as a result of scanty information on the storage status and the organisms involved in the decay of fermented foods commonly used up. This research was carried out to study the quality of palm wine, ogi and fufu sold in Ogwa market and enumerate bacterial associated with them [12].

Materials and Methods

Collection of sample

Three different samples of fermented food (“Palm wine,” Ogi” and “Fufu”) were purchased from two selling points in Ogwa and Ebelle and were collected in sterile container. They were left at room temperature for 2-3 days to undergo spoilage [13].

Chemical analysis

pH determination: pH of samples was determined using a calibrated pH meter Jenway 3510, three steps calibration was done using automatic buffer of pH 4, 7 and 10 before taking the pH of sample solutions.

Microbiological analysis

Media used and their preparations: Nutrient Agar (NA) and MacConkey Agar (MCA) were used for the enumeration of total heterotrophic bacteria and coliform bacteria respectively using pour plate method. Heterotrophic bacteria and coliform were determined by incubation at 37°C for 24 hrs. Incubation was at 28°C for 72 hrs. Each medium was prepared according to the manufacturer’s instructions.

Isolation of the associated bacteria

Serial dilution of sample: Ten millilitres of each sample was homogenized into 90 ml distilled water. Tenfold serial dilution (10^-1 to 10^-9) was made using sterile test tubes. Nine millimetres of normal saline was pipette into the test tube and 1 ml from the initial mixture was aseptically transferred to the first test tube, 1 ml of same diluents was transferred to the second test tube [14]. This procedure was repeated until the ten-fold dilution (10^-10) was achieved. 1 ml from dilution was pour plated in duplicates on various media for enumeration of isolates. Bacteria were enumerated on pour plates of MacConkey Agar (MCA), Nutrient Agar (NA). The pour plate method of inoculation after serial dilution of the samples by 10^-4 dilution factor was conducted for viable count, after sterilizing media at 121°C for 15 mins using autoclave. All plates were incubated for 48 hours at 37°C as described by Manga and Oyeleke [15].

Inoculation and enumeration: Nutrient agar (NA) and MacConkey Agar (MCA) was used for the enumeration of bacteria coliform respectively [16]. The inoculated plates were incubated at 37°C 24 hrs. for the enumeration of total heterotrophic bacteria and total coliform bacteria. The agars were prepared according to the manufacturer’s procedure. Aliquot 1 ml of appropriate ten-fold serial dilution (10^-3, 10^-6, 10^-9) of the food sample was inoculated into the nutrient agar plates 1 ml from dilution was pour pated in duplicates on various media for enumeration of isolates. Bacteria were on pour plates of nutrient agar. The pour plate method of inoculation after serial dilution of the sample by 10^-4 dilution factor was conducted for viable count, after sterilizing media at 121°C for 15 minutes using autoclave. All plates were incubated for 48 hours at 37°C as described by Manga and Oyeleke, [17].

Characterization and identification of bacterial isolates: Bacteria cultures were characterized and identified using various morphological and biochemical tests including: Gram stain, motility, catalase, coagulase, indole, citrate, oxidase and sugar utilization test. The motility test was done according to the technique described by Cheesbrough to distinguish motile bacteria from the non-motile one. Pure cultures of each isolate were [18] obtained by streaking the specific colonies on nutrient agar slants and incubated overnight theses were maintained in an agar slant in bijoux bottles. The identification of the microbial isolates was established on classification scheme offered by Harrigan and McCance, Buchanan and Gibbson and Collin and Lyne. The identification was based essentially on morphological and biochemical reactions [19].

Results and Discussion

The results were presented as mean of replicate readings (Tables 1-5).

Table 1. The averages of THBC (x 10⁴ cfu/ml) counts and LAB (x 10⁴ cfu/ml) counts are summarized in the table below.

Table 2. Bacteria isolate from nutrient agar.

Table 3. Lactic acid bacteria from De Man Rogosa Sharpe (Mrs) agar.

Table 4. Distribution of Isolate before spoilage.

Table 5. Distribution of isolates after spoilage.

It was recorded in the (Table 1) that among Ebelle fufu and Ogwa fufu, Ebelle fufu 1 had the highest Total Heterotrophic Bacterial Count (THBC) of 2.47 × 10⁴ cfu/ml before spoilage and also had the highest count of 5.54 × 10⁴ cfu/ml after spoilage [20]. For Lactic Acid Bacterial count (LAB), Ebelle fufu 1 had the highest count of 1.36 × 10⁴ cfu/ml before spoilage and 3.05 × 10⁴ cfu/ml after spoilage. The least among the Ebelle fufu in THBC is Ebelle Fufu 2 which had 1.67 × 10⁴ cfu/ml before spoilage and the least after spoilage is Ebelle fufu 2 had 3.75 × 10⁴ cfu/ml. for LAB count Ebelle fufu 2 had the least of 0.92 × 10⁴ cfu/ml before spoilage and 2.06 × 10⁴ cfu/ml after spoilage [21].

The results in Table 1 also recorded that among Ebelle ogi and Ogwa ogi, the highest THBC Ogwa ogi 2 had 2.44 × 10⁴ cfu/ml before spoilage and 6.15 × 10⁴ cfu/ml after spoilage. For LAB count before spoilage, Ogwa ogi 2 recorded as the highest count of 1.42 × 10⁴ cfu/ml and 4.05 × 10⁴ cfu/ml after spoilage. The least among Ogwa ogi and Ebelle ogi before spoilage for THBC recorded Ebelle ogi 1, had 1.58 × 10⁴ cfu/ml and 3.58 × 10⁴ cfu/ml after spoilage [22]. For LAB count Ebelle ogi 1 had the least of 0.86 × 10⁴ cfu/ml before spoilage and 1.97 × 10⁴ cfu/ml after spoilage [23].

The results in the table recorded that among Ebelle palm wine and Ogwa Palm wine, the highest THBC before spoilage was Ogwa palm wine 2, with 2.15 × 10⁴ cfu/ml and Ebelle palm wine 2 with 4.19 ×10⁴ cfu/ml after spoilage [24]. For LAB count the highest count is Ebelle palm wine 2 gave 2.38 × 10⁴ cfu/ml before spoilage and Ogwa palm wine 2 with 2.69 × 10⁴ cfu/ml after spoilage. The least among Ebelle palm wine and Ogwa palm wine for THBC recorded that, [25] Ebelle palm wine 2 had the least of 1.57 × 10⁴ cfu/ml before spoilage and Ebelle palm wine 1 had the least of 3.47 × 10⁴ cfu/ml after spoilage. For LAB count, Ogwa palm wine 2 had the least of 0.79 × 10⁴ cfu/ml before spoilage while Ebelle palm wine 1 had the least of 1.91 × 10⁴ cfu/ml after spoilage [26].

Table 2 revealed the presence of Salmonella enteritica, Pseudomonas aeruginosa, Bacillus ssubtitles, Citrobacter freundii, Klebsiella oxytoca, Staphylococcus aureus and Escheichia coli from Nutrient agar. There was dominance of gram negative isolates over the gram positive [27].

Table 3 recorded that Lactobacillus sp, Pedococcus sp, Leuconostoc sp, Lactococcus sp and Lactobacillus sp, were isolated from MRS agar from [28] Ogi, fufu, and Palm wine samples from Ogwa and Ebelle communities [29].

Table 4 results show that, in Citrobacter freundii, Klebsiella oxytoca, Staphylococcus aureus and Lactobacillus sp, the organisms [30] were present in all the samples and batches while Salmonella enteritica, Pedococcus sp, Leuconostoc sp and Lactococcus sp were not present in all samples and batches [31].

The results show that in Citrobacter freundii, Klebsiella oxytoca, Staphylococcus aureus, Lactobacillus sp and Lactobacillis sp, the organisms were present in all samples and batches (Table 5) [32].

In this study, the results of the experiment indicated that the fermented foods (Palm wine, Ogi and Fufu) [33] had a Total Heterotrophic Bacterial Count (THBC) and Lactic acid Bacterial (LAB) count which ranged from 1.57 × 10⁴ cfu/ ml-2.47 × 10⁴ cfu/ml and 0.79 × 10⁴ cfu/ml-2.38 × 10⁴ cfu/ml before spoilage respectively while Total heterotrophic bacterial count and Lactic acid bacterial count ranged from 3.47 × 10⁴ cfu/ml-6.15 × 10⁴ cfu/ml and 1.91 × 10⁴ cfu/ml-4.05 × 10⁴ cfu/ml after spoilage respectively [34]. This may be due to the known fact that fermentation which usually occurs during fermented food processing usually involves many bacteria species especially the lactic acid bacteria [35].

The bacteria associated with spoilage of fermented food [36] (palm wine, Ogi and Fufu) included Salmonella enteritica, Pseudomonas aeruginosa, Bacillus subtilis, Citrobacter freundii, Klebsiella oxytoca, Staphylococcus aureus and Esherichia coli while lactic acid bacteria were Lactobacillus, Pedococcus, Leuconostoc, Lactococcus and Lactobacillis species [37].

The presence of some microorganisms such as Lactobacillis, Leuconostoc, Pedococcus, Lactobacillus, Lactococcus species may be beneficial. Several of these have been found to have probiotic properties and immunomodulatory functions [38]. Several Lactobacillus sp. [39] have been reported to display stimulatory properties on cells of innate immunity in vitro and in vivo in both animal models and in humans given fermented food products containing probiotics. In humans, Lactobacilli are normally present in the vagina [39], gastrointestinal tract and are together with Bifidobacterium one of the first bacteria to colonize the infant gut after delivery. The spoilage fermented drinks has been associated with the presence of both Bacillus subtilis and Pseudomonas aeruginosa, the findings of the work agrees with the submissions of Nwachukwu [40].

Most of the bacteria identified in the study have been implicated in spoilage of ferment foods and other related infections [40]. When fermented foods are left in ambient temperature the contaminating organisms proliferates and this results in the spoilage of the fermented foods. The results were similar to the Citrobacter freundii, Klebsiella oxytoca, Staphylococcus aureus and Lactobacillus sp, were present in all the samples and batches due to their ubiquitous nature in the environment.

Conclusion

In conclusion, the study revealed that, the total Heterotrophic bacteria count and lactic acid bacteria counts in Ebele fufu 1 was very. In the three different cases reported. The after counts were more than twice the before counts in both total Heterotrophic bacteria count and lactic acid bacteria count after spoilage. This reveals high concentration of bacteria in ebele fufu 1 after spoilage. Similar findings were reported of Ebele fufu 2. Over 100% of the previous counts of bacteria were reported after food spoilage. in other food type, the results obtained were not different as the counts for bacteria increased by more than 100% after food spoilage. Ogwa Fufu is recommended for eating because it has the least total heterotrophic count and lactic acid bacterial count.

Comparing results obtained for Ebele and Ogwa, it can be seen that there was no much difference between bacteria counts before and after food spoilage.

Recommendations

Locally fermented food should be given proper handling so as to prevent food borne infections. Advanced techniques for the production of locally fermented food should be encouraged.

References

1. Aderiye BI, Laleye SA, Odeyemi AT, et al. Hypolipidemic effect of Lactobacillus and Streptococcus species from some Nigerian fermented foods. Res J Microbiol. 2007;2(6):538-44.
2. Abdel AA, Dardir HA. Hygienic Quality of Local Traditional Fermented Skimmed Milk (Laban Rayb) Sold in Egypt. World J Dairy Food Sci. 2009;4(2):205-9.
3. Aworh OC. The Role of Traditional Food Processing Technologies in National Development: The West African Experience. Int Union Food Sci Technol. 2008;1:1-18.
4. Ogunshe AA, Omotosho MO, Ayansina AD, et al. Microbial studies and biochemical characteristics of controlled fermented afiyo-a Nigerian fermented food condiment from Prosopis africana (Guill and Perr.) Taub. Pak J Nutr. 2007;6(6):620-7.

   [Googlescholar]

5. Adeleke RO, Abiodun OA. Physicochemical properties of commercial local beverages in Osun State, Nigeria. Pak J Nutr. 2010;9(9):853-5.

   [Googlescholar]

6. Adebayo GB, Otunola GA, Ajao TA, et al. Physicochemical, microbiological and sensory characteristics of kunu prepared from millet, maize and guinea corn and stored at selected temperatures. Adv J Food Sci Technol. 2010;2(1):41-6.

   [Googlescholar]

7. Agarry OO, Nkama I, Akoma O, et al. Production of Kunun-zaki (A Nigerian fermented cereal beverage) using starter culture. Int Res J Microbiol. 2010;1(2):18-25.

   [Googlescholar]

8. Battcock M. Fermented fruits and vegetables: a global perspective. Food Agric Org. 1998.

   [Googlescholar]

9. Benito C, Palomero S, Calderon P, et al. Schizosaccharomyces. Encyclopedia of Food Microbiology,2nd edition, Netherlands. 2014;370.
10. Benito Á, Calderón F, Benito S, et al. The combined use of Schizosaccharomyces pombe and Lachancea thermotolerans-Effect on the anthocyanin wine composition. Molecules. 2017;22(5):739.

    [Crossref][Googlescholar][Indexed]

11. Chelule PK, Mbongwa HP, Carries S, et al. Lactic acid fermentation improves the quality of amahewu, a traditional South African maize-based porridge. Food Chem. 2010;122(3):656-61.

    [Crossref][Googlescholar]

12. Klaenhammer TR. Bacteriocins of lactic acid bacteria. Biochimie. 1988;70(3):337-49.

    [Crossref][Googlescholar][Indexed]

13. Joshi VK, Parmar M, Rana NS, et al. Pectin esterase production from apple pomace in solid-state and submerged fermentations. Food Technol Biotechnol. 2006;44(2).

    [Googlescholar]

14. Joshi VK, Thakur NS. Vinegar: Composition and production. Indus Publishing. New Delhi. India, 2000;1222.
15. Joshi VK, Sharma S. Cider Vinegar: Microbiology, technology and quality. J Food Sci Technol 2009;25:306–7.

    [Googlescholar]

16. Joshi VK, Attri D, Bala A, et al. Microbial pigment. Indian J Biotechnol. 2003;2:362–9.

    [Googlescholar]

17. Joshi VK. Special Issue on Production of Wine from Non-Grape Fruits. Nat Prod Radiance. 2009;8(4):311-472.
18. Joshi VK, Attri D. Optimization of apple pomace based medium and fermentation conditions for pigment production by Rhodotorula species. Proceed National Acad Sci. 2006; 76:171-6.
19. Joshi, VK, Lakhanpal P, Chopra V, et al. Occurrence of patulin its dietary intake through consumption of apple and apple products and methods of its removal. Int J Food Ferment Technol. 2013;3(1):15-32.

    [Googlescholar]

20. Malhotra B, Keshwani A, Kharkwal H, et al. Antimicrobial food packaging: Potential and pitfalls. Front Microbiol. 2015;6:611.

    [Crossref][Googlescholar][Indexed]

21. Nwachukwu IN, Ibekwe VI, Nwabueze RN, et al. Characterization of Palm wine Yeasts Isolates from Industrial Utilization. Afr J Biotechnol. 2006;5(19):1725-8.
22. Okafor N. Palm wine yeasts from parts of Nigeria. J Sci Agric. 1972;23(12):1399-1407.

    [Crossref]

23. Okafor N. Fermented foods and their processing. Biotechnol. 2009;8:1-10.
24. Olotu OO, Oyetayo VO, Titilayo TA, et al. Safety of Small-Scale Food Fermentations in Developing Countries. Int J Food Safety. 2009;11:29-34.

    [Googlescholar]

25. Klaenhammer TR, Russell WM. Species of the Lactobacillus acidophilus complex. J Food Microbiol. 2000;2,1151-7.
26. Walter J. Ecological role of Lactobacilli in the gastrointestinal tract: implications for fundamental and biomedical research. Appl Environ Microbiol. 2008;74(16):4985-96.

    [Crossref][Googlescholar][Indexed]

27. Walker WA. Initial intestinal colonization in the human infant and immune homeostasis. Ann Nutr Metab. 2013;63(2):8-15.

    [Crossref][Googlescholar][Indexed]

28. Nwachukwu E, Onovo OM, Ezeama CF, et al. Effect of Lime Juice on the Bacterial Quality of Zobo Drinks Locally Produced in Nigeria. Res J Microbiol. 2007;2(10):787-91.
29. Donkor ES, Aning KG, Quaye J, et al. Bacterial contamination of informally marketed raw milk in Ghana. Ghana Med J. 2000;41(2):200.

    [Crossref][Googlescholar][Indexed]

30. Rawat S. Food Spoilage: Microorganisms and their prevention. Asian J Plant Sci Res. 2015;5(4):47-56.

    [Googlescholar] [Indexed]

31. Amusa NA, Ashaye OA. Effect of Processing on Nutritional, Microbiological and Sensory Properties of Kunun-Zaki (A Sorghum Based Non-Alcoholic Beverage) Widely Consumed in Nigeria. Pak J Nutr. 2009;8(3):288-92.

    [Googlescholar]

32. Saranraj P, Geetha M. Microbial Spoilage of Bakery Products and Its Control by Preservatives. Int J Pharm Biol Arch. 2012;3(1):38-48.

    [Googlescholar]

33. William CF, Dennis CW. Food Microbiology. 4th edition. McGraw Hill, India. 2011;330.
34. Salasiah K, Gulam R, Son R, et al. Bacteriocin-Producing Lactic Acid Bacteria Isolated from Traditionally Fermented Food. Malays J Med Sci. 2001;8(1):63-8.
35. Dhawale and La Master. Microbiology Laboratory Mannual. The McHill Company, USA, 2003;226.
36. Oyeleke SB, Manga BS. Essential of Laboratory Practical in Microbiology, 1st edition, Tobest Publisher, Niger, 2008;36-58.
37. Cheesbrough M. District Laboratory Practice in Tropical countries. 2000;182-4.
38. Harrigan WF, McCance ME. Laboratory Methods in Food and Dairy Microbiology. 1976.

    [Googlescholar]

39. Buchanan RE, Gibson NE. Bergey's Manual of Determinative Bacteriology. 8th edition. Williams and Wilkins, Baltimore, USA, 1974.
40. Collins CH, Lyne PM. Microbiological Methods. 5th Edition, Butterworth and Co. Publisher, London 1984;331-45.