Abstract

The bacteriological profiles of the different cassava products were investigated. Ground cassava samples, serially diluted with sterile normal saline were cultured in different sterile bacteriological media. Hydrogen cyanide content was determined by spectrometric method. Colonies isolated from the different locations were statistically the same for total viable bacterial counts (TVBC). Cassava peels had the highest values of TVBC (4.456 log10CFU/g) TCC (3.025 log10CFU/g) EC (1.663) and SA counts. Gari had the lowest values of TVBC (3.193) TCC (2.580) EC (not isolated) and SA counts. The traditionally processed cassava products showed no statistically significant differences (p<0.05) with the mechanically processed products except in fufu. The total viable bacterial count was highest in cassava products dried along the roadside (6.980 log10CFU/g) and lowest oven-dried cassava products (2.763 log10CFU/g). No Salmonella and Shigella spp. were isolated in most of the assayed cassava products. The identified storage places of the cassava products did not significantly impact any difference on the bacterial load. There was a progressive decrease in the cyanide contents from cassava peels to cassava chips to fufu and least in gari. The highest hydrogen cyanide concentration of 14.50 mg/kg was recorded in cassava peels and was higher than 10 mg of HCN/kg body weight recommended by WHO. Gari had the least content of 2.25 mg/kg. The study provides information on potential infections and toxicities due to the microorganisms and cyanide level.

Keywords: Bacteriological quality, Cassava products, Cyanide, Total viable bacterial count, Total coliform count.

Received: 10 December 2019 / Revised: 30 December 2020 / Accepted: 26 March 2021/ Published: 13 April 2021

Contribution/ Originality

This study is one of the very few studies which have investigated bacteriological quality of different cassava products processed in Benue State for use as food for man or feedstock for animals.

1. INTRODUCTION

Cassava (Manihot esculenta Crantz) is a basic root crop that is utilized globally as food for humans and feed for animals. It serves as a major source of carbohydrates for millions of people especially the low income earners in the tropics and sub-tropical countries [1]. In terms of agricultural food security, it is the most robust and important food crop in Nigeria [2] and Nigeria is the world’s largest producer for over a decade [3].
In Nigeria, cassava tubers are processed and consumed in different forms, namely: cassava chips which is processed for flour, gari and cassava peels used for feedstock for animals. Other economic uses of cassava include making fufu, confectionaries, glues, monosodium glutamate, sweeteners, pharmaceuticals and textiles [2]. However, most indigenous low-income earners of the society use the cassava in the form of gari, fufu, chips and the peels are consciously or unconsciously served to ruminant animals as feedstock.

Microorganisms are ubiquitous and are found virtually everywhere. They have a wide range of carbon substrates for growth and cellular metabolism. In cassava production systems, many microorganisms have been associated with its contamination including fungi [4, 5] and bacteria [2, 5] rendering it a public health issue. Microbial quality/ public health issues relating to consumption of cassava products include the following.
Cassava contains cyanogenic glucoside which is made up of linamarin and lotaustrain [6]. The quantity of cyanogenic glucoside is dependent on the cassava variety [7]. Diseases arising from excessive consumption of cassava with high cyanide contents include but not limited to cretinism, goiter, neuropathy and tropical diabetics Nhassico, et al. [8]. WHO [9] stipulated the limits of cyanogenic glucoside in consumed food to which if exceeded, becomes adverse. The implication of consumption of cassava product with high cyanide content is well known. The present work investigated the bacteriological quality cyanide compositions of different cassava products sold in Benue State for use as food for man or feedstock for animals.

2. MATERIALS AND METHODS

2.1. Collection of Cassava Product Samples

Survey was carried out in Benue State in November 2018 in three local government areas of the state to assess the techniques deployed in cassava processing, drying and storage in the various places visited and in addition determine the microbial load. Multistage sampling involving purposive and random methods were used. Cassava producing communities in Benue State were selected and then random samples were collected from livelihoods in the selected communities.

2.2. Bacteriological Analysis

2.2.1. Preparation of the Cassava Sample

The different cassava samples were prepared by weighing one gram of each of the granulated cassava (ground in sterile mortar and pistil) product in 9 mL of peptone water. These were the stocks used for the inoculation of the different isolation media below. The stocks were appropriately diluted prior to use for inoculation.

2.2.2. Nutrient Agar

Nutrient Agar (NA) was used for the determination of Total Viable Bacterial Counts (TVBC). Nutrient Agar (HiMedia Laboratories Pvt. Ltd, India) was prepared by weighing 14.0 g of NA powder into a 500 mL conical flask and dissolving in distilled water. This was brought to 500 mL volume and was heated and shaken intermittently to mix. Thereafter, it was tightly plugged with quality cotton wool, and was covered with aluminum foil and sterilized by autoclaving at 121 oC for 15 minutes. This was allowed to cool to 40 - 50 oC before dispensing into Petri dishes. The medium was allowed to solidify on the plate. A 0.1 mL of appropriately diluted sample was introduced into the sterile medium and evenly spread using a sterile glass spreader. This was subsequently incubated at 37 oC for 24 h. Colonies that form on the incubated plates were counted using a digital colony counter and results recorded.

2.2.3. MacConkey Agar

Total Coliform Counts (TCC) was determined using MacConkey Agar (MA). MacConkey Agar (HiMedia Laboratories Pvt. Ltd, India) was prepared by dissolving 27.6 g of MA powder in 500 mL of distilled water until a homogenous mixture was achieved. This was possible through gentle heating with continuous shaking. The medium was sterilized by autoclaving at 121 oC for 15 minutes. Thereafter, the sterile MA was allowed to cool to 40 – 50 oC before dispensing to sterile Petri dishes. The medium was allowed to solidify and a 0.1 mL of appropriately diluted sample was introduced into the sterile medium and evenly using a sterile glass spreader. This was subsequently incubated at 37 oC for 48 h. Colonies that form on the incubated plates were counted using a digital colony counter and results recorded.

2.2.4. Xylose Lysine Deoxycholate Agar (XLDA)

Confirmation and enumeration of Salmonella and Shigella spp were done on XLDA. Xylose Lysine Deoxycholate Agar (XLDA) was prepared according to the manufacturer’s specifications. A 500 mL XLDA (27.7 g of powder) medium was sterilized in a 1 L Erlenmeyer flask by bringing to boil over a heater. This was allowed to cool to 40 – 50 oC before dispensing to Petri dishes. Prepared medium was inoculated with a 0.1 mL of appropriately diluted sample and was spread evenly on the plate. This was subsequently incubated at 37 oC for 24 h and emergent colonies were counted using a digital colony counter.

2.2.5. Eosin Methylene Blue Agar

The Escherichia coli content of the sample was determined using Eosin Methylene Blue Agar (EMBA). Eosin Methylene Blue Agar (TM Media, Titan Biotech Ltd, BHIWADI, Rajasthan, India) was prepared by dissolving 18.0 g of EMBA in 500 mL of distilled water. The dissolution was accompanied by gentle heating and was thereafter sterilized by autoclaving at 15 psi (121 oC) for 15 minutes. Upon cooling to 45 – 50 oC, the medium was dispensed into sterile Petri dishes. A 0.1 mL of appropriately diluted sample was introduced into the sterile medium and was evenly spread. This was subsequently incubated at 37 oC for 24 h. Colonies that form on the incubated plates were counted using a digital colony counter and results recorded.

2.2.6. Mannitol Salt Agar

Staphylococcus aureus (SA) was isolated in Mannitol Salt Agar (MSA). Mannitol Salt Agar (HiMedia Laboratories Pvt. Ltd, India) was prepared by suspending 55.6 g MSA in distilled water. It was homogenized with frequent heating and agitation until completely dissolved. The MSA was sterilized by autoclaving at 121 oC for 15 minutes. This was allowed to cool to 50 oC before pouring into sterile Petri dishes. A 0.1 mL of appropriately diluted sample was introduced into the sterile medium and evenly spread using a sterile glass spreader. This was subsequently incubated at 37 oC for 24 h. Colonies that form on the incubated plates were counted using a digital colony counter and results recorded.

2.3. Biochemical Identification of the Isolates

Triple sugar iron (TSI), urease test, oxidase, coagulase and catalase in addition to indole test, methyl red, Voges-Proskauer, and citrate utilization (IMVIC) were used to characterize the bacteria following the protocol documented by Cheesbrough [10].

2.4. Determination of Hydrogen Cyanide Concentration

Hydrogen cyanide concentration was determined by modifying the methods by Ezeh, et al. [11] andSawyerr, et al. [12] as presented below. Two grams of the cassava powder was added to 20 mL of sterile distilled water in a conical flask, shaken to form a paste, stoppered and allowed to stand at room temperature (28±2 oC). The paste was filtered (Whatman No.1) after addition of further sterile distilled water. The filtrate was made up to 50 mL volume. This was kept in the refrigerator (4 oC) until used.

2.4.1. Alkaline Picarate Preparation

Alkaline picarate was prepared by dissolving 1 g of picric acid and 2 g of sodium trioxo carbonate in a small volume of minimally warm water. This was thereafter brought to a 100 mL volume and stored in an amber-colored bottle in the fridge (4 oC) until used.

To determine the cyanide content, 4 mL of alkaline picarate was added to a 5 mL quantity of the filtered cassava solution. This was warmed for 5 min in a water bath set at 5 oC for brown color development. Thereafter, this was allowed to cool and read against a blank at 490 nm (UV-Spectrophotometer). The blank was prepared by adding 1 mL water to 4 mL of alkaline picarate solution. The cyanide concentration expressed as mg/kg was extrapolated from a standard curve.

2.5. Statistical Analysis

Descriptive statistics and one-way analysis of variance (ANOVA) were performed using SPSS (version 16.0).

3. RESULTS

Results of the bacteriological profiles of the different cassava products are shown below. Cassava chips, fufu, garri and cassava peels had numerically different bacterial loads. Table 1 presents the levels of bacterial load with respect to locality from where the products were sourced. The Table reveals that Ikyose of Katsina-Ala (KA) and Mbahaya of Makurdi Local Government had the highest TBVC value of 4.326 log10CFU/g. The TVBC of 3.215 got from samples from Ehurekpe in Oju was the lowest. However, most of the TVBC values were statistically homogeneous. The mean total coliform count (TCC) was highest (2.763 log10CFU/g) in Katsina-Ala (KA) (Ikyose) and lowest in Adaka (1.806 log10CFU/g) of Makurdi Local Government. Escherichia coli count (ECC) used as indicators of food quality was highest (1.681 log10CFU/g) in samples from KA area of KA local Government but was not isolated in some areas of Makurdi and Oju. Cassava peel samples from Makurdi had Salmonella sp (1.00 log10CFU/g) as opposed to all other locations where Salmonella and Shigella were not isolated.

Table-1. Influence of location on the bacterial quality (log10CFU/g) of cassava peels.

Local Govt.	Locations	TVBC	TCC	SAL	SHI	EC	SA
Katsina-Ala	Katsina-Ala	3.729	2.335	-	-	1.681	1.556
	Agasoma	4.050	2.505	-	-	1.415	1.681
	Agoasu	4.269	2.620	-	-	1.531	1.556
	Ikyose	4.326	2.763	-	-	1.505	1.748
Makurdi	Tionsha	4.215	2.964	1.000	-	1.663	2.924
	Mbahaya	4.326	2.256	-	-	1.505	1.681
	Yagba	4.107	2.601	-	-	1.204	1.707
	Adaka	3.982	1.806	-	-	-	-
	Mbayo	4.065	2.579	-	-	1.342	-
Oju	Obolori	3.934	2.756	-	-	1.204	1.672
	Ihiejwo	4.256	2.556	-	-	1.326	2.819
	Ehurekpe	3.215	2.065	-	-	-	1.833

Note: TVBC = Total Viable Bacterial Counts, TCC = Total Coliform Counts, SAL = Salmonella sp. SHI = Shigella sp. EC = E. coli, SA = S. aureus.

Table-2. Effect of location on the bacterial quality (log10CFU/g) of cassava chips.

Local Govt.	Locations	TVBC	TCC	SAL	SHI	EC	SA
Katsina-Ala	Katsina-Ala	3.729	2.193	-	-	1.301	1.204
	Agasoma	3.415	2.025	-	-	1.114	1.602
	Agoasu	3.672	2.305	-	-	1.079	1.477
	Ikyose	3.556	2.025	-	-	1.091	1.415
Makurdi	Tionsha	3.833	2.121	-	-	1.301	1.903
	Mbahaya	3.604	2.009	-	-	1.176	1.204
	Yagba	3.215	2.220	-	-	1.146	1.326
	Adaka	3.782	1.968	-	-	-	-
	Mbayo	3.256	2.204	-	-	1.231	0.954
Oju	Obolori	3.672	2.093	-	-	1.114	1.699
	Ihiejwo	3.156	2.162	-	-	1.301	1.778
	Ehurekpe	3.065	2.215	-	-	-	1.231

Note: TVBC = Total Viable Bacterial Counts, TCC = Total Coliform Counts, SAL = Salmonella sp. SHI = Shigella sp. EC = E. coli, SA = S. aureus.

Effect of location on the bacterial quality (log10CFU/g) of cassava chips is presented in Table 2. The TVBC was highest (3.833) in cassava chips from Tionsha in Makurdi Local Government Area and least (3.065) in samples from Ehurekpe in Oju. The TCC values obtained from cassava chips was highest (2.305) from Agossu in KA and lowest (1.968) in samples from Adaka in Makurdi. Salmonella and Shigella spp. were not isolated from the cassava chips from the locations at the time of the study. The three locations had similarly the highest (1.301) EC contaminations. Escherichia coli was not isolated in cassava chips from Adaka and Ehurekpe. Staphylococcus aureus was highest (1.903) in MA area of MA local Government Area.

Effect of location on the bacterial quality log10CFU/g of gari is shown in Table 3. The Table shows that the TVBC was highest (3.655) in gari from Agoasu district of KA local Government and lowest (3.070) in Ihejiwo in Oju locality. The TCC (log10CFU/g) was highest (2.361) in Agoasu district of KA local Government and lowest (1.903) isolated from Adaka area of Makurdi Locality. Salmonella and Shigella spp were not isolated. The Table also shows EC count to be highest at 1.397 log10CFU/g from samples isolated from KA in KA Local Government Area. Some localities had no E. coli whereas Staphylococcus aureus was highest (1.778og10CFU/g) in Tionsha area of Makurdi.

Mean bacterial load (log10CFU/g) of the cassava products irrespective of location is presented in Table 4. The Table shows that cassava peels used for animal feeds was the highest values of TVBC (4.456) TCC (3.025) EC (1.663) and SA counts. Gari had correspondingly the lowest values of TVBC (3.193) TCC (2.580) EC (not isolated) and SA counts. Salmonella sp. and Shigella sp. were not isolated (Table 3) from any of the cassava food product. The E. coli counts ranged from not isolated to 1.663 log10 CFU/g. Table 4 also presents the SA counts ranging from 1.505 log10CFU/g to 1.806 log10 CFU/g.

Table-3. Effect of location on the bacterial quality (log10CFU/g) of gari.

Local Govt.	Locations	TVBC	TCC	SAL	SHI	EC	SA
Katsina-Ala	Katsina-Ala	3.602	2.146	-	-	1.397	1.301
	Agasoma	3.301	2.000	-	-	1.079	1.161
	Agoasu	3.653	2.361	-	-	1.079	1.342
	Ikyose	3.344	2.079	-	-	1.000	1.301
Makurdi	Tionsha	3.778	2.113	-	-	1.000	1.778
	Mbahaya	3.544	2.000	-	-	-	1.000
	Yagba	3.176	2.161	-	-	1.176	1.113
	Adaka	3.778	1.903	-	-	-	-
	Mbayo	3.204	2.204	-	-	-	0.778
Oju	Obolori	3.602	2.113	-	-	1.100	1.477
	Ihiejwo	3.070	2.113	-	-	1.204	1.402
	Ehurekpe	3.079	2.161	-	-	-	1.000

Note: TVBC = Total Viable Bacterial Counts, TCC = Total Coliform Counts, SAL = Salmonella sp. SHI = Shigella sp. EC = E. coli, SA = S. aureus.

Table-4. Mean bacterial load (log10CFU/g) of the Cassava products irrespective of location.

Process Products	TVBC	TCC	SAL	SHI	EC	SA
Cassava peels	4.456	3.025	-	-	1.663	1.806
Cassava chips	4.310	2.623	-	-	1.415	1.579
Fufu	4.334	2.806	-	-	1.447	1.643
Gari	3.193*	2.580	-	-	-	1.505