Sanitary quality of food ingredients displayed for sale in the local markets in rivers state, Nigeria

Food ingredient made from various plant are very important for every average Nigerian home. The study investigated the sanitary quality of Egusi, Achi, Offor, Cameroon pepper and red pepper sold in markets. The analysis was done using standard microbiological methods for microbial isolation and characterization. The total heterotrophic bacteria bacterial count done on samples studied revealed counts ranging from 4.1x105 to 6.4x106, 1.03x105 to 1.02x107, 5.4x105 to 7.2x106, 3.2x104 to6.4x106, and 4.3x104 to 5.0x106 cfu/g for Achi, Egusi, Offor, grinded pepper and Cameroon pepper respectively. Total coliform count ranging from 1.0x14x103 to 8.2x104 in all the samples studied, Total Staphylococcus count ranged from 3.35x103 to 6.4x105 cfu/g. Fungal counts ranged from 3.6x103 to 6.8x104 cfu/g. Six bacterial genera were isolated in this study including Bacillus sp. (25.5%), Micrococcus sp. (23.5%), Staphylococcus sp. (23.5%), Proteus sp. (13.8%), Enterobacter sp. (11.8%) and Enterococcus sp. (1.9%) while the fungal species isolated includes Saccharomyces sp., Aspergillus niger, Candida sp., Penicillium sp and Rhizopus sp. Antimicrobial susceptibility testing showed that all Gram Positive Isolates (Bacillus, Staphylococcus and Micrococcus sp were resistant to Ceftazidime, Erythromycin, Augmentin and Cloxacillin while the Grams negative (Proteus, Enterococcus and Enterobacter) showed resistance against Cefixime and cefuroxime. Proper handling and storage of food ingredient is recommended.

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Introduction

Plants are a cheap and reliable source of food. Certain nutrients like proteins and vitamins, which are important to make a balanced meal or diet, are cheaper to get from plants than from animals [1,2] . Owing to the heritage of Africa and the fact that resources are very scarce in developing countries, cheaper options of meal and are often preferred by a majority of the population in these countries. Wild legumes are among the plants which have been commonly used in the developing world as a cheap and reliable source of nutrition. Notable among these nutritious plants are egusi (Colocynthis citrillus L) Achi (Brachystogia euryc oma), Offor, (Diaterium microcarpum) and pepper (Capsinum annum). From the earliest times, man has derived food, shelter, medicine and decoration from plants. In most of the ancient civilization, one can find some references to plants [3] . In Nigeria, dietary pattern varies and is influenced by the vegetation belt. For example, in the northern parts of Nigeria, cereals dominate, while in the south, legumes, nuts, seeds and starchy roots or tubers are the main food components [4] . Among the legumes used in soups (mainly for emulsification and stabilization of soups) are Brachystegia eurycoma (achi), Detarium microcapum (ofor). Each of the soup thickeners differ in species from the others and so have their individual characteristic flavours, which they impart to soups. Often, choice depends on individuals, but Brachystegia eurycoma and Detarium microcarpum are favorite soup thickeners in South Eastern Nigeria [4] . Soups made from Egusi on the other hand are among the most popular delicacies in Nigeria. Melon seeds contain a fairly high amount of unsaturated fatty acid, linoleic acid (suggesting a possible hypocholesteronic effect [2] . Thickening, usually improves the taste, but most important is the nutritional value of foods. In fact, every time the soup is thickened, its nutritive value is determined by the ingredients added to it [3] . Thickening agents are often used as food additives and in cosmetics and personal hygiene products.

Egusi melon is mainly a type of watermelon which many botanists think is not easily distinguishable, especially in appearance. In west Africa, where egusi melon is popular, its seed kernels are common components of daily meals and a major soup ingredient [5,6] . Egusi melon seed kernels have been reported to be a rich source of protein, essential nutrients, and oil [7] . The oil is edible and potentially useful for producing biofuel for automobiles the propagation of all varieties of melon, including egusi melon, is carried out using the seeds. (Brachystogia. Eurycoma) Achi is an economic tree that belongs to the family Caesalpiniaceae. It is a dicotyledonous legume that grows in the swaps or rain forests and well drained soil of South-Eastern Nigeria and Western Cameroun. It is a huge tree which has twisted and spreading branches with a bark that often exudes a buttery gum [8] . B. eurycoma is called Achi in Igbo, Ekalado or Eku in Yoruba, Okweri in Edo, Akpakpa or Taura in Hausa, Apaupan in Ijaw, and Odukpa in Ibibio [9] . The seed flour, which is a good source of carbohydrate and fiber, is used as flavoring and thickening agents for soups in Eastern Nigeria [2,10] . The seeds are used in folkloric medicine to maintain body temperature, soften stool, and protect against colon and rectal cancer [2] . A range of proximate, phytochemical, and pharmacological screening has been carried out in different parts of the plant following anecdotal account of its nutritious and medicinal value by local residents and traditional medical practitioners, respectively, in the localities where the plant predominately grows. Phytochemical screening has shown that B. eurycoma contains diverse bioactive compounds including flavonoids, phenolic compounds, alkaloids, saponins, and tannins. Nutritious compounds present in the plant include carbohydrate, proteins, lipids, and minerals [2] . Chilli pepper, a fruit and a spice, is used in small quantities to add flavour and taste to food. Commonly, chilli is dried and ground to use as a spice but is also used fresh in salads and curries. Chilli is thought to increase energy expenditure, so that over a long period of time (combined with less energy intake than energy expenditure) it may reduce body weight and obesity, Chilli is one of the richest sources of antioxidant vitamin C. The active ingredient of chilli, capsaicin, is also a potent antioxidant. The most important concerns are those ascribed to physiological harm and contamination with spoilage microorganisms [12] . Food ingredients such as Egusi, Achi, Offor and Pepper are easily perishable and requires to be used almost immediately after processing. However, in markets, these food condiments are grinded and kept in the markets sometimes for days without being sold out. Their constant exposure may encourage microorganisms to drop on them and proliferate. Traders with the mind of not running at loss still sell them after grinding for days to weeks. These may lead to potential public health risk. This study is aimed at determining the levels of contamination of grinded Egusi, Red Pepper, Achi, Offor and Cameroon pepper.

Materials and methods

A total of 45 samples grinded and exposed in the market (Egusi, Offor, Achi, red pepper and cameroon pepper). Samples were sourced from three markets within Obio-Akpor Local Government Area, of Rivers State. Each representative samples were grinded hygienically with a clean blender and properly packaged to avoid exposure and this was used as control and taken to the Microbiology laboratory for analysis in sterilized bags.

Isolation of Microorganisms

Twenty five grams (25g) of each sample (Egusi, offor, achi, red pepper and Cameroon pepper) were added into 225 mL of peptone water, swirled and allowed to stay for about 3hours on a laboratory shaker, serial dilution was 10 -4 . 0.1 ml of each last two prepared dilutions were transferred into sterile petri plates containing solid Plate count agar, MacConkey agar and Mannitol salt agar for bacteria and potato dextrose agar for fungi. The plates were incubated at 37 o C for 24 hours and 28 o C for 72hours for bacteria and fungi respectively. The number of colonies obtained was counted and multiply by inverse of the dilution factor to obtained the number of colony forming unit per milliliter (CFU/g) of the sample [25] . Discrete colonies on the different media were randomly selected based on their morphology and were sub cultured and incubated at 37 o C for 24 hours (h) to obtain pure colonies. Isolates were identified based on their morphological and cultural characteristics on growth media. Identification materials, reagents and protocols according to [22] were used to identify discrete colonies from the media of sub-cultured isolates. The identities of the isolates were confirmed using biochemical tests [22] . The cultural characteristics of each fungi isolates were identified according to their colour, shape and the cell morphology was done based on mycelia, hyphae, septate, spore formation using lactophenol blue. A piece of the mycelium from the Petri plates was mounted on a clean grease free slide using a sterile wire loop and covered with a cover slip, after which a drop of lactophenol cotton blue was added and examined with the microscope [22] .

Antimicrobial Sensitivity Testing

Sensitivity Testing: Antibiotic sensitivity patterns of all the confirmed isolates were performed by standard disk diffusion method according to Kirby-Bauer on Mueller-Hinton agar (Titan, Biotech Ltd, Indian) following the procedures recommended by CLSI (2012). Eight commonly used antibiotics (µg/disc) viz. amoxicillin-clavulanate or Augmentin (AUG), gentamycin (GEN), nitrofurantoin (NIT), cefuroxime (CRX), ofloxacin (OFL), cefixime (CXM), ciprofloxacin (CPR), ceftazidime (CAZ), Abtek, (UK) for Grams negative and Gram's positive bacteria were tested. From an overnight culture of all isolates, 0.5 MacFarland turbidity standards bacterial culture was prepared in sterile saline, from which 0.1mL was inoculated onto Mueller Hinton agar, after which antibiotic discs were carefully and aseptically placed on the surface of the agar. The plates were incubated at 37 o C for 24h. Zone of inhibition was measured in millimeter a ruler.

Results

Results

Discussion

Discussion

Microbial quality of Food ingredients displayed for sales

Food safety is a primary concern for everyone, especially in our country Nigeria where it forms part of the government 7-point agenda (3). Biochemical changes in foods when perceived undesirable are mediated by microorganisms that degrade them leading to their spoilage. Because food especially those of plant origin are excellent sources of nutrient, microorganisms find them favorable for their proliferation. The microbial population of food is an important indicator of food safety and quality. High microbial count in food poses a danger to the health of consumers, and their presence may also contribute to decomposition and depletion of nutrients. This study investigated the sanitary quality of some Food ingredients which includes Egusi, Achi, Offor, grinded pepper, and Cameroon Pepper sold in market places around Port Harcourt. Figure 1 presents the total heterotrophic bacterial count done on all the Food ingredients samples ranged from 4.3x104 to .2x106 cfu/g. The THBC for all control samples were lower indicating that exposure in market places increases the contamination levels of samples studied. The THBC counts observed in this study is however higher than the counts of 2.0x104 to 7.0x104 cfu/g reported by Tsado et al., [12] in Minna, Niger State. Our study is however in concomitance with the range published by Salari et al., [13] reported THBC of various food ingredients studied to be 1.0x102 to 4.0x106 cfu/g, This is comparable to the report of Salari [13] reported THBC of various samples studied to be 1.0x102 to 4.0x106 cfu/g from melon seeds. Filiz [14] . reported total heterotrophic bacteria the average numbers of 8.1 × 106 cfu/g in powdered red pepper. Elmali et al. [15] examined 15 powdered red peppers and found counts of 2.7 × 106 cfu/g. A study conducted by Winfred et al., [16] on Egusi powder sold in different markets had Total bacteria count ranged from Log 10 3.2 -4.4 cfu/g for the different markets in Ghana.

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Mean Total Heterotrophic Count of Samples Obtained from Different Food Ingredients

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Figure 2 shows high levels of coliform contamination ranging from 1.4x103 to 8.2x104 cfu/g for all the samples. This is comparable to the report of Salari et al., [13] who reported higher counts of 1.9x102 to 3.25x106 cfu/g. Elmali et al. [15] found the average number of 1.3 × 104 cfu/g of coliform in powdered red pepper. Filiz [14] reported the average number of 1.7 × 102 cfu/g of Coliforms in powdered red pepper. Schwab et al. [17] found very low level of Coliforms and E. coli (3-19 cfu/g) in powdered red pepper samples. Staphylococcus count was higher ranging from 3.35x103 to 6.4x105 cfu/g for all samples studied as shown in Figure 3.

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Mean Total Staphylococcus Count of Samples Obtained from Different Food Ingredient

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Mean Total Coliform Count of Samples Obtained From Different Food Ingredients

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The high Staphylococcus count could be as result of frequent contacts of food stuffs with the skin during storage and restorage after sales daily.

Staphylococcus is considered the third most important cause of disease in the world amongst the reported food-borne illness [19,20] . Staphylococcal food poisoning is a persistent cause of gastroenteritis worldwide, especially in developed countries [21] . All the control samples had lower counts compared to the grinded samples sourced from the markets for all microbial parameters analyzed. This could be attributed to the fact that they have not been exposed and also not stored over time. Sanitary measures were taken during the grinding of the control samples. Our counts recorded for both samples sourced from the market and the controls are however high when compared to microbiological standards and acceptable range for food stuffs which is (1.0x102Cfu/g). The implication is that the samples are not fit for consumption as they harbor microorganisms above required standards. Consequently, these samples are not eaten raw and there are all chances that the pathogens will be denatured or killed during cooking or pasteurization. Fungal counts ranged from 3.6x103 to 6.8x104 cfu/g for all the samples as shown in Figure 4. This implies that most of the samples supported the proliferation of fungal species. Six bacterial genera were isolated in this study as shown in Evidently the vendors store the powders in flexible pouches, wooden or plastic boxes, and plastic bowls/buckets. This practice may easily expose the food product to microbial contamination and adversely compromise its safety. Most of the sellers indicated that a batch of these grinded food ingredient lasts a maximum of a week or even more, but this heavily depends on patronage. The ingredients, according to the women, is usually kept in HDPE bowls/buckets from which measured portions are scooped and sold to consumers. The powder is sold in unbranded, clear, and flexible polyethylene or polypropylene bags and secured with a knot. This practice is likely to increase the risk of rancidity occurring in the powder whiles in storage since it is often exposed to air and moisture. The result of the antibiogram conducted on the Gram-Positive Isolates (Bacillus, Staphylococcus and Micrococcus spp.) revealed resistance to Ceftazidime, Erythromycin, Augmentin and Cloxacillin This agrees with other findings. [22, 23] . The Gram-negative isolates (Proteus, Enterococcus and Enterobacter) showed resistance against Cefiixidime and cefuroxime. The antimicrobial susceptibility pattern observed for Enterococcus sp agrees with Shareef and Shara [24] who reported similar patterns from clinical samples. The resistance to the antibiotics tested may be attributed to the production of beta-lactamase, an enzyme that inactivates βlactam rings in β-lactam antibiotics and closely related antibiotics. Resistance in antibiotic pose risk as their ineffectiveness can result in mortality.

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Mean Total Fungi Count of Samples Obtained from Different Food Ingredient

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Conclusion

These Food ingredients were found to be potential vectors in the transmission of opportunistic pathogenic microorganisms. This study confirms the occurrence of different pathogens in samples of food ingredients and concludes that these pose potential public health risk if not adequately processed before consumption. If these food ingredients are processed and handled carefully it would minimize the possibility of contamination and ensure that the product is of good quality and is safe for consumption. Recommendations 1. Proper and hygienic storage food condiments in market places 2. Buyers should not be allowed to touch food stiffs before buying 3. Traders should blend amount of product they can sell off within the shortest time to avoid over storage.

Figures and tables from the paper

S/N	Antibiotics	Bacillus spp.	Staphylococcus spp.	Micrococcus spp.
1	OFL	8.5	26	24
2	AUG	R	R	R
3	CRX	18	14	15
4	CAZ	R	R	R
5	GEN	22	19	19
6	CTR	20	14	12
7	ERY	R	R	R
8	CXM	R	R	R

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Antimicrobial Susceptibility Pattern of Gram Positive Bacteria in millimeter (mm)

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S/No	Antibiotic	Proteus spp.	Enterococcus spp.	Enterobacter spp.
1	CRX	R	R	16
2	GEN	12	12	18
3	CXM	R	R	R
4	OFL	12	12	25
5	AUG	R	R	08
6	NIT	R	R	15
7	CPR	10	10	24
8	CAZ	15	12	32

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Antimicrobial Susceptibility Pattern of Gram-Negative Bacteria in millimeter (mm)

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Isolates	Frequency
Bacillus sp.	13 (25.5)
Micrococcus sp.	12 (23.5)
Staphylococcus sp.	12 (23.5)
Proteus sp.	7 (13.8)
Enterobacter sp.	6 (11.8)
Enterococcus sp.	1 (1.9)
Total	51(100)

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Frequency of Bacterial Isolates from Samples

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