The Journal of Food Technology in Africa Innovative Institutional Communications ISSN: 1028-6098 Vol. 6, Num. 1, 2001, pp. 21-24

The Journal of Food Technology in Africa, Vol. 6, No. 1, Jan-Mar, 2001, 21-24

Microbial load and microflora of cassava (Manihot esculenta Crantz) and effect of cassava juice on some food borne pathogens

Gulelat Desse, Mulugeta Taye

Debub University, Awassa College of Agriculture P. O. Box 5 Awassa, Ethiopia

Code Number: ft01007

Abstract

The microbial load of cassava tubers that were grown at Awassa, Ethiopia, was analysed. The total viable microbial count of fresh cassava, before cleaning, ranged from 8.7x10⁴ to 2.1x10⁹
c.f.u/g whereas, in thoroughly cleaned product it was reduced to 10⁶ c.f.u/g (% C.V., 19.5). Enterobacteriacae and spore former bacteria had mean counts of 10⁴ and 10³ c.f.u/g respectively. The dominant bacteria groups within the aerobic mesophillic flora were Acinetobactor spp. (29.1%), Micrococcus spp. (17.4%) and Enterbacteriacae (16%). Bacterial spores, Pseudomonas, Moraxella and Aeromonas spp. Were detected in a small proportions. Fate of Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes in cassava juice was also evaluated. Except in B. cereus the growth of the bacterial strains was retarded only at higher concentrations.

Introduction

Cassava is a major source of carbohydrate particularly for low-income consumers. It grows in a wide range of climatic conditions and yields better than most other crops under drought conditions (Cock, 1985). At present, cassava is a major source of food for 800 million people (Nweke, 1996). It has also been used as a food in some parts of Ethiopia, particularly in the south and southwestern regions for a century (Taye, 1994).

Information on the microbiology or nutritional quality of food items in Ethiopia are limited (Gashe et al. 1982; Gifawosen nad Bisrat, 1982; Gashe; 1985, 1987; Ashenafi, 1994). There are very few reports on the microbial quality and fate of the various food borne pathogens during the storage of cassava cultivars in Ethiopia.

The level of micro organisms in food items is essential as some of them are hazardous to health. For instance, if the number of Bacillus cereus and Staphylococcus aureus exceeds 106 cfu/ml, it is suspected of causing food poisoning (Geopfert, et al. 1972; Tatini, et al. 1973) and Listeria monocytogenes causes menengitism abortion and prenatal septicaemia mainly in new born, infants and immuno suppressed adults (Gray and Killinger, 1966).

The purpose of this study was, therefore, to evaluate the microbial load of fresh cassava and the behaviour of B. cereus, S. aureus and L. monocytogenes in cassava juice.

Materials and Methods

Sampling

A total of 120 matured fresh tuber roots of Amarokello Red and Umbure cultivars were collected from the experimental site of Awassa College of Agriculture, Awassa. Samples were put in sterile containers, immediately brought to the laboratory and processed for the following microbiological parameters.

Microbiological analysis: 25 gm of sample was aseptically removed and homogenised in 225 ml sterile 0.1% peptone water using lab blender for ten minutes.

Aerobic mesophillic bacteria: samples were further diluted in sterile tap water and volumes of 0.1 ml of appropriate dilutions were spread-plated in duplicates on pre-dried surfaces of plate count agar (PC; Merck). Colonies were counted after incubation at 30 to 32°C for 48 h.

Staphlococci: To identify Staphylococcus aureus appropriate dilutions were spread-plated in duplicate plates of Manitol Salt Agar (Oxoid) and incubated at 30 to 32° C for 48-72 h. Ten colonies from countable plates were picked and slide and tube coagulase test was done.

Enterbactriaceae: From the appropriate dilutions 0.1 ml volumes were taken, spread-plated in duplicates onto Violet Red Glucose agar (Oxoid) plates and incubated at 30 to 32° C for 24 h.

Bacterial spores: 0.1 ml of appropriate dilutions (after heating the sample for fifteen minutes) in a water bath of 80° C) were spread-plated in duplicates on the surface of PC (Merck) and colonies were counted after incubation at 30 to 32° C for 24 h.

Yeasts and Molds: Volumes of 0.1 ml appropriate dilutions were spread-plated in duplicates in pre-dried surfaces of Chloramphenicol-Bromophenol-Blue agar (CBB), (consisting of g/l in distilled water: yeast extract, 5.0; glucose, 20.0; chloramphenicol, 0.1; Bromophenol Blue, 0.01; agar, 15). The pH was adjusted to 6.0 to 6.4 yeast colonies were counted after incubating the plates at 25-27° C for 5 days.

Flora assessment: After colony counting, 10-15 colonies were selected at random from countable PC agar plates. Sub-cultures were further purified by repeated plating and a total of 700 strains were isolated and tentatively differentiated into various bacterial groups by the following characteristics: Phase contrast microscopy was used to examine cell shape and grouping, presence or absence of endospores and motility; Gram reaction was determined using the KOH test of Gregerson (Gregerson, 1978); cytochrome oxidase was tested by the method of Kovacs (Kovacs, 1956); catalase test was made with 3% (v/v) H₂O₂ solution; and glucose metabolism was investigated by the O/F test of Hugh and Leifson (Hugh and Leifson, 1953).

Preparation of cassava juice: juice from cassava was extracted by fruit grinding centrifugal machine and steam-sterilized. The juice was diluted in Brain Heart Infusion (BHI) Broth (Merck) to give final concentrations of 25%, 50% and 75% Cassava juice. 100% was considered as undiluted juice and BHI broth served as a control. An electrode of a pH meter was dipped into the juice to measure its pH.

Test Organisms: Baccilus cereus (WS1537), Staphylococcus aureus (WS 1759) and Listeria monocytogenes (WS 2300) were obtained from the culture collections of Bacterologosches Institu, SVA, Weihenstephan, Germany.

Inoculation of cassava juice with test strains: For the purpose, cultures (overnight) of the test strains were separately inoculated in the various concentrations of the juice and the control tubes having a final innoculum level of 10² - 10³ cfu/ml. After mixing thoroughly, the tubes were incubated at 30 - 32° C for 8 hours.

Analysis of samples: Appropriate dilutions from the freshly inoculated tubes were surface plated on Brian Heart Infusion Agar (MERCK), in duplicate to determine the initial innoculum level. All the tubes were then sampled aseptically after four and eight hours. The media used were: Manitol Slat agar for S. aureus, Modified McBride agar for L. monocytogenes and Baccilus cereus agara for B. cereus. All these media are from Oxoid and inoculated plates were then incubated at 32°C for 24-48h after which colonies were counted.

Results

The total viable microbial count of un-cleaned cassava ranges from 8.7x10⁴ to 2.1x10⁹ cfu/g. After a thorough cleaning with a chlorinated tap water, however, the count reduced to 10⁶ cfu/g (table 1). From the various bacterial groups isolated from well-cleaned cassava, Staphylococcus spp., yeasts and molds were detected in a very low proportion (<10³ and 10² cfu/g respectively), whereas, plate count agar and Violet Red Bile Glucose agar yielded relatively higher microbial counts. The mean counts of AMB were around 10⁶ cfu/g with variation among samples (C.V., 19.6%). Enterobacteriacae and Bacterial spores had mean counts of 10⁴ and 10³ cfu/g respectively. Marked variations, however, were noted in the counts of Bacterial spores (C.V., 29.5%). Isolates from the plate count agar yielded different bacterial genera of epidemiological significance at varying frequencies (Table 2). The dominant ones were Acinetobacter spp., 29.1%; Micrococcus spp., 17.4% and Enterobacteriacae, 16%. Acinetobacter, Micrococcus, and Enterobacteiacae were isolated from 65.8%, 46.7% and 34.1% of the samples respectively.

Fig.1 Fig. 2 Fig 3

The pH value of cassava juice was found to be 6.3. In the media containing no cassava juice all the test strains grew without any retardation. At 50% concentration the growth of L. monocytogenes was affected and complete inhibition was observed at 100% concentration. Concerning B. cereus, its growth was completely inhibited at concentrations of 25%, whereas, with increasing concentrations counts for S. aureus decreased.

Discussion

Coryneforms, lactic acid bacteria, Sporeformers, coliforms, micrococci and pseudomonas are saprophytes that were observed in most vegetables and tubers (Geopfert, 1980). The microflora of cassava growing in Ethiopia may, thus, be considered saprophytes, which usually are found in fresh vegetables. As cassava tubers are not properly stored here in this country, the bacteria load and its multiplication should not be considered acceptable. The cassava samples are found to consist staphylococcus, that may cause staphycoccal food poisoning and it was reported that production of enterotoxin occurs at Staph. Aureus count of 106 cfu/g (Tatini, et al. 1970). If the level of contamination with B. cereus exceeds 10⁶ cfu/g, it can play a role in the transmission of B. creus food poisoning (protnoy, et al., 1976).

L. monocytogenes is ubiquitous in nature (Farber, et al. 1987). There is a possibility of contamination of cassava with L. monocytogenes and this results in abortion in pregnant women, menengitis and encephalitis in immunocompromised hosts (McLaughlin, 1987).

The high bacterial load of cassava observed in this study is, therefore, due to its unhygienic handling, which as a result, may initiate spoilage bacteria to multiply. As is shown in table 1, counts of the various bacterial groups from well cleaned cassava is low. However, the potential of their multiplication makes it hazardous for consumption. Among the AMB, Acinetobacter is found to be the dominant one. Although, the growth failed to continue during storage trials, an insignificant amount of Salmonella, only in some of the fresh samples, was detected in this study. However, some reports (Yoo, et al. 1983; Frank and Bayer, 1984) have shown that there is an incidence of salmonella in many food items like fish and egg. It is, therefore, important that everything likely to come into contact with the cassava is clean. Moreover, the natural process is irreversible ad preservation principle should be employed to slow down the deterioration, hence increasing the overall quality and storage shelf life of the product. The sooner any preventive measures are taken after harvest, the greater the chance of reducing post-harvest losses.

Wastage of food due to poor handling practices of food products in Ethiopia is extreme. On the other hand, low income and the low level of technology in the country has given no chance to the people to use suitable ways of packing and storage. Hence famine, nutritional deficiencies, rural-urban migration and mortality rate may be minimised if proper health education is given and improved traditional food preservation techniques are widely introduced. These foods should be handled hygienically to minimise contamination and avoid unnecessary loss. This study, thus, indicates that the shelf life of cassava product can be improved from microbiological point of view and that cassava juice may not have an inhibitory effect on Pathogens, which may contaminate the tubers.

Acknowledgement

The technical assistance of Amsalu Gobena and Wondosen Tadesse is highly acknowledged.

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