Animal Research International (2015) 12(2): 2159 – 2165 2159

MICROBIOLOGICAL QUALITY OF RAW AND ROASTED AFRICAN PALM WEEVIL ( RHYNCHOPHORUS PHOENICIS ) CONSUMED IN THE SOUTH EASTERN

1EBENEBE, Cordelia Ifeyinwa and 2OKPOKO, Valentine Obinna 1Department of Animal Science and Technology, Nnamdi Azikiwe University, , , Nigeria. 2Department of Zoology, Nnamdi Azikiwe University, PMB 5025, Awka, Anambra State, Nigeria.

Corresponding Author: , C. I. Department of Animal Science and Technology, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. Email: [email protected] Phone: +234 8186306424

ABSTRACT

The level of microbial contamination of African palm weevil (Rhynchophorus phoenicis) was carried out to assess the health implications of consumption of the larva in raw and roasted forms. Raw Rhynchophorus phoenicis larva collected from rotting palm at Mgbo, Oba in Idemili Local Government Area and roasted Rhynchophorus phoenicis purchased along Onitsha-Owerri expressway all in Anambra State, Nigeria were used for the study. Streak method was used in the assessment of the microbial load in the raw Rhynchophorus phoenicis whereby fluid from intestinal content was inoculated to Nutrient and MacConkey agar and incubated at 37 oC for 48 hours, while those on Sabaroud agar were incubated at room temperature for five days. The roasted ones were milled before plate count method was applied. In this method one tenth milliliter (0.1ml) of the 4 th part of diluents produced after serial dilution to the concentration of 10 -6 was aseptically inoculated into MacConkey agar (3 plates), Nutrient agar (3 plates) and Sabaroud agar (3 plates) respectively. The result showed three species of bacteria: Staphylococcus aureus, Escherichia coli and Salmonella spp. in the live APW and three species of bacteria: Bacillus subtilis, Pseudomonas aeruginosa and Proteus vulgaris as well as two species of fungi: Cladosporum spp. and Aspergillus flavus in the roasted Rhynchophorus . Total bacterial count in the roasted Rhynchophorus phoenicis was 1.72 x 10 6 CFU/g while Total fungal count was 4.3 x10 2 CFU/g. Rhynchophorus phoenicis though reported to be highly nutritious in terms of amino acid profile and presence of unsaturated fatty acid can be a source of food poison if not properly handled in sanitary manner during collection, processing and post processing period.

Keywords: African palm weevil, Rhynchophorus phoenicis, Staphylococcus aureus, Escherichia coli, Salmonella spp., Bacillus subtilis, Pseudomonas aeruginosa, Proteus vulgaris, Cladosporum spp., Aspergillus flavus

INTRODUCTION Control over the occurrence of potential hazard in the food supply is a sine qua non in The concept of food safety has evolved in the increasing the consumer confidence in the developed worlds over the past few years owing safety of food. A food material adjudged to be a to food related diseases, but most of the delicacy can be a pot of poison if it contains regulations to ensure food safety are yet to be disease causing pathogens. It becomes adopted in many African countries including imperative to assess microbiological qualities of Nigeria. food relished by humans to ensure that the

ISSN: 1597 – 3115 ARI 2015 12(2): 2159 – 2165 www.zoo-unn.org Ebenebe and Okpoko 2160 nutritive quality not devalued by its microbial the prevention of arteriosclerosis and other content. heart related diseases. The African palm weevil (APW) – APW larval oil was also reported to have

Rhynchophorus phoenicis Fabricius 1801 is high pharmaceutical potentials based on the highly cherished in many parts of the country to specific gravity and refractive index. According the extent that it is called by various descriptive to Ekpo and Onigbinde (2004), R. phoenicis names in many parts of the country; Akwa Ibom larval oil has specific gravity and refractive index State – Nten, Edo State ( Bini ) – Orhu , Edo State lower than arachis oil, linseed oil and olive oil, (Eshan ) – Okhin , Delta State ( Itsekiri ) – Ikolo , meaning that the larval oil more may be Delta ( Urhobo ) – Edon , Delta ( Isoko ) – Odo , valuable in the pharmaceutical industries. Oyo State ( Oyo ) – Awon , Osun State ( Ilesha ) – However the food value of APW can Ekuku , Anambra State (most parts) – Akpa only be appreciated if the hazard analysis in the ngwo , Anambra State ( LGA) – Nza , Abia process of its collection, processing and State – Eruru ngwo , Benue State ( Idoma ) – preparation underscores the larva as a safe food Eko-ali (Ekpo and Onigbinde, 2005). for human nutrition. Several reports have Several reports in literature expressed indicated that food borne pathogens are the nutritional and medicinal value of responsible for death of human and animals in Rhynchophorus phoenicis larvae (Ekpo and several food related diseases (ICSMF, 1986; Onigbinde, 2004; Ekpo and Onigbinde, 2005; Siame et al., 1996; Voetsch et al., 2004; Yu et Banjo et al., 2006; Edijala et al., 2009; Nzikou al., 2005; EFSA, 2008, Majawicz et al., 2010; et al ., 2010; Womeni et al ., 2012; Ebenebe and Hald et al., 2012; Rafal et al., 2012). It is Okpoko, 2014). Ebenebe and Okpoko (2014) therefore important to assess the reported that R. phoenicis compares favourably microbiological quality of the APW in two forms with other meat proteins as it contains 21.1% (raw and roasted) in which it is consumed in crude protein, 65.2 % lipid and 5.2% ash. Banjo Nigeria. Estimation of microbial numbers in food et al. (2006) reported crude protein content of is frequently used in the assessment of 28.42%. Amino acid profile of R. phoenicis microbiological quality of food or to validate the larvae reported by Womeni et al. (2012) showed presumptive “safety” of foods. This procedure that the larva contained all essential amino acids requires that samples are taken of the food, (EAA); lysine, valine, leucine, isoleucine, microbiological tests or analyses are performed phenylalanine, threonine and methionine. and the results evaluated to ascertain the health According to them EAA like lysine and threonine risks of the food. normally deficient in grain and cereals were in high concentration in the larvae but low when MATERIALS AND METHODS compared to reference value for humans (FAO/WHO, 1991), while the other with the Raw Rhynchophorus phoenicis: Live R. exception of tyrosine and phenylalanine were in phoenicis larva used in the study were collected high concentrations. The presence of essential from rotting raffia palm at Mgbo (swampy land fatty acids linoleic and linolenic acids further with a stretch of river and small tributaries elucidate the fact R. phoenicis is a highly running through its surrounding), Oba in Idemili nutritious food material. Igwe et al. (2011) also Local Government of Anambra State. The larva reported appreciable amounts of Vitamin B3 were collected together with the frass (chewed (Niacin) and B1 (Thiamine) in addition to up palm pith) from three different rotting raffia Vitamin A and C in the termite ( Macrotermes palm and they were placed in a well aerated nigeriensis ). Apart from the nutritional value, plastic bucket before being transported to the Ekpo and Onigbinde (2004) reported the Entomological Unit of the Department of medicinal value of R. phoenicis on the basis of Zoology for identification using the key of Giblin- the high iodine value of the larval oil, which Davis et al. (2013). The samples were later they stated is an index of the degree of un- taken to Microbiological Laboratory for saturation of the larval oil and its usefulness in microbiological analysis by Dr. I. F. Okonkwo a

Animal Research International (2015) 12(2): 2159 – 2165 Microbiological quality of raw and roasted African palm weevil 2161 microbiologist in Department of Agricultural colony forming unit per gram (CFU/g) of the Engineering and Bioresources. Only streak sample (Cheesebrough, 2000). method was applied for the live larva. Nine larvae were randomly picked from the samples Characterization of Bacterial Isolates: from each of the three different rotting palms. Colonies of bacterial isolates were subjected to Each of the larva was cut open with lancets and colonial characterization using the methods the fluids from the intestinal contents aseptically described by Pelczar et al. (1993) and inoculated into nine prepared plates, three Cheesebrough (2000). Further characterization containing MacConkey agar, three Nutrient agar was based on microscopic and biochemical and three Sabaroud agar respectively using wire methods (Pelczar et al., 1993; Cheesebrough, loop. The plates containing Nutrient agar and 2000). Cultures of fungi isolated only in the the inoculums, as well as those with MacConkey roasted Rhynchophorus larva were identified on agar and inoculums were incubated at 37 oC for the basis of macro and micromorphology, 48 hours while the remaining three plates with reverse and surface colouration of colonies in Sabaroud agar and inoculums were incubated at accordance with McCance (1990) and Abbey room temperature for five days. (2007).

Roasted R. phoenicis: The roasted R. RESULTS phoenicis were purchased from stationed sellers along Oba – Nnewi new road around Oba The result obtained from the microbiological junction in Anambra State, Nigeria. Extraneous analysis of the raw/live R. phoenicis larva materials including pepper and other showed only bacterial contamination including condiments used in the preparation were Staphylococus aureus, Escherichia coli and removed from the sample and they were spread Salmonella species . No fungus was detected. out to sundry to some extent to make it crispy The colonial characteristics of the isolates are before milling. Twenty grammes of the milled presented in Table 1. larva was measured out and mixed with 180 ml sterile distilled water. One milliliter portion was Table 1: Colonial characteristic of bacteria aseptically transferred into 9ml distilled water isolated in the fresh Rhynchophorus and serial dilution continued until concentration phoenicis larva of 10 -6 was obtained in accordance with Braide Colony Agar Colonial morphology Plate and Nwaoguikpe (2011). One tenth milliliter A Nutrient Circular, moderately large, th (0.1ml) of the 4 part was then aseptically agar convex, shiny, light yellow inoculated into the MacConkey agar (3 plates), colonies Nutrient agar (3 plates) and Sabaroud agar (3 B Nutrient Circular, small, raised, agar smooth, shiny white colonies plates), respectively. Inoculums were spread C MacConkey Smooth colourless colonies evenly over the surface of the agar plates using agar a sterile spreader (Braide and Nwaoguikpe, 2011). The nutrient and MacConkey agar plates The microscopic and biochemical (three for each) were incubated at 37 oC for 48 characterization of the bacteria isolated are hours while the Sabaroud agar plates were presented in Tables 2 and 3, respectively. incubated at room temperature for seven days. Microbiological analysis of the roasted R. phoenicis larva on the other hand showed both Microbial Counting: On establishment of bacterial and fungal contamination. Bacteria colonies in the plates containing roasted APW, species isolated included Bacillus subtilis, the number of colonies was counted using an Pseudomonas aeruginosa and Proteus vulgaris , electronic counter. The mean count for each while the two species of fungi isolated were triplicate plate was obtained multiplied with the Cladosporum sp. and Aspergillus flavus. Total dilution factor to obtain the total viable cells per bacterial count was 1.72 x10 6 CFU/g and Total unit weight of the sample expressed as the fungal count 4.3 x 10 2 CFU/g respectively.

Animal Research International (2015) 12(2): 2159 – 2165 Ebenebe and Okpoko 2162

Table 2: Microscopic characteristics of Microscopic characteristics of bacterial isolates bacterial isolates in fresh Rhynchophorus in roasted Rhynchophorus phoenicis larva phoenicis larva showed the presence of oval cells in clusters of Colony Mot Gram Flagella Bacillus spp. and short rods of Pseudomonas rxn A - + - spp. and Proteus spp., respectively (Table 5). B + - + The biochemical characteristics of bacterial C + - + isolates in roasted Rhynchophorus phoenicis Cell Most probable larva indicated the catalse and H S in all the morphology identity 2 A Oval cells in Staphylococcus bacterial isolates, citrate in Bacillus subtilis and clusters aureus Pseudomonas aeruginosa and urease and indole B Short rods Escherichia coli only in Proteus vulgaris (Table 6). The C Short rods Salmonella spp. microscopic characteristic of fungal isolates in

the roasted Rhynchophorus phoenicis Table 3: Biochemical characterization of bacterial Larva indicated the presence of isolates in fresh Rhynchophorus phoenicis larva Cladosporum spp. and Aspergillus Colony Catalase Oxidase Coagulase H2S A + - + - flavus (Table 7). B - - - - C + - - + DISCUSSION Urease Indole Citrate ID of Isolate

A - - + Staphylococcus aureus The result obtained from the B - + - Escherichia coli microbiological analysis of live and C - - + Salmonella spp. roasted African Palm weevil (APW) Rhynchophorus phoenicis indicates the presence Table 4: Total heterotrophic counts and of three species of bacteria; Staphylococcus colonial characteristic of bacteria isolated aureus , Escherichia coli and Salmonella spp. in in the roasted Rhynchophorus phoenicis larva the live APW and three species of bacteria; Colony Agar Plate Colonial MPI Bacillus subtilis, Pseudomonas aeruginosa and morphology Proteus vulgaris as well as two species of fungi; D Nutrient Large but Bacillus spp. Cladosporum spp. and Aspergillus flavus in the agar short, wavy, roasted Rhynchophorus . Total bacterial count in rough, dull 6 white, single the roasted APW was 1.72 x 10 CFU/g while rods Total fungal count was 4.3 x10 2 CFU/g. E Nutrient Small, short, Pseudomonas Staphylococcus aureus in ready to eat food is /MacConkey single rods, spp. agar bluish green usually as a result of human contamination on nutrient through improper handling during preparation. agar, pale Salmonella presence in food is a useful indicator yellowish on of poor hygiene and post processing MacConkey agar contamination of food. Although the total viable F Nutrient Circular, Proteus spp. count was not obtained in this study, ICMFS /MacConkey Smooth (1986) noted that a level greater than 104 per agar cream gram in ready to eat foods is unacceptable as it coloured raised is an indication that contamination has Total heterotrophic Count 1.72 X 10 6CFU/g MPI = Most occurred. Absence of Salmonella in the roasted Probable Identity APW may be due to the effect of heat

treatment. Salmonella is one of the most The total heterotrophic counts and colonial important food borne zoonotic pathogens with characteristic of bacteria isolated in the roasted significant health and economic impacts in Rhynchophorus phoenicis larva indicated the presence of Bacillus spp. Pseudomonas spp. and humans and animals (Voetsch et al., 2004). Proteus spp. on Nutrient and MacConkey agar Majawicz et al. (2010) reported that non typhoid (Table 4). Salmonella is the leading cause of food borne

Animal Research International (2015) 12(2): 2159 – 2165 Microbiological quality of raw and roasted African palm weevil 2163 illness, estimated to be implicated in 93.8 million purposes eat it raw, uncooked and unprocessed cases of gastroenteritis globally. Salmonella can in any form. ICSMF (1986) reported that E. coli be eliminated by heat treatment depending on is not supposed to be detected in foods, only treatment time, temperature and extent of levels less than 3 per gram is given satisfactory moisture content during cooking as considerable category. Escherichia coli infection is associated resistance to heat is observed in dry materials with eating improperly cooked or undercooked particularly if the material is wrapped by lipids food. Escherichia coli infection can cause as in the APW (EFSA, 2008). disease such as urinary tract infection, bacteraemia and meningitis. Table 5: Microscopic characteristics of Bacillus subtilis observed in roasted bacterial isolates in roasted Rhynchophorus APW is also associated with food borne disease. phoenicis larva Bacillus subtilis is an endophore that Colony Mot Gram Flagella rxn predominates in the soil (Braide and D + + + Nwaoguikpe, 2011). ICMFS (1986) showed that E + - + B. cereus in cooked food is as a result of F + - + inadequate temperature controls. Levels greater Cell Most probable 4 morphology identity than or equal to 10 CFU/g is considered D Oval cells in Bacillus subtilis potentially hazardous. clusters The presence of Pseudomonas E Short rods Pseudomonas aeruginosa aeruginosa in roasted APW is also undesirable, F Short rods Proteus vulgaris though Kruick (2013) noted that P. aeruginosa Mot = Motility, Gram rxn = Gram reaction is a common environmental organism that poses no health risk to healthy people, he Table 6: Biochemical characteristics of bacterial also indicated that most severe isolates in roasted Rhynchophorus phoenicis larva infection occurs in people who are Colony Catalase Oxidase Coagulase H S 2 already hospitalized with another D + - - + E + + - + disease condition. Besides, the F + - - + presence of P. aeruginosa will reduce Urease Indole Citrate ID of Isolate the nutritional and eating quality of the D - - + Bacillus subtilis E - - + Pseudomonas weevil as Nester et al. (1998) reported aeruginosa that P. aeruginosa produce protease F + + - Proteus vulgaris and lipases that can catalyze reactions leading to degradation of proteins and fats, thus Table 7: Microscopic characteristic of an undesirable flavor of the food product. fungal isolates in the roasted Proteus vulgaris are pathogens that Rhynchophorus phoenicis larva degrade food with high protein content. Braide Colony Colonial morphology Most Probable and Nwaoguikpe (2011) noted that they do not G Conidia borne of short Cladosporum give rise to food borne diseases but they lower branched clusters with spp. the nutritive value of foods. The total viable varying sizes and shapes. count of 1.72 x 10 6CFU/g suggests Conidiophores had no swelling. contamination and health risk to consumers. H Un-branched Aspergillus The observation of two species of fungi conidiophores, Swelling at flavus in the roasted APW; Cladosporum spp. and the apex, Conidia were borne on short chain of Aspergillus flavus, is not out of place because sterigma the weevils are sold uncovered in an open place Total heterotrophic Count 4.3 X 10 2CFU/g and the unsold larva are warmed for sale in subsequent days. Cladosporum is a Escherichia coli presence in the live APW is also cosmopolitan organism with its spores found in undesirable as many young people and some the air, water and soil. They cause deterioration communities that use the APW for medicinal and spoilage of foods. Rafal et al. (2012)

Animal Research International (2015) 12(2): 2159 – 2165 Ebenebe and Okpoko 2164 showed that Cladosporum spp. dominate 80% Cambridge University Press, United of spores in the air in various parts of Europe. Kingdom. Cladosporum spp. can cause allergic reactions in EBENEBE, C. I. and OKPOKO, V. O. (2014). human which sometimes lead to asthma. Edible Insects in Southeastern Nigeria . Aspergillus flavus are well known Conference Paper Presented at the producers of aflatoxin whose primary target is Future Food Salon Group, Montreal, the liver and they are potent carcinogens, Canada, from August 26 - 28, 2014. mutagens and teratogens, thus they are acutely EDIJALA, T. K., EGHOGBO, O. and ANIGBORO, toxic to man and animals (Siame et al. 1996). A. A. (2009). Proximate composition Yu et al. (2005) reported that Aspergillus flavus and cholesterol concentrations of are the cause of invasive and non invasive Rhynchophorus phoenicis and Orytces aspergillosis in humans, animals and insects, monocerus larvae subjected to different and it causes allergic reactions in man. heat treatments. African Journal of Rhynchophorus phoenicis though rich Biotechnology, 8(10): 2346 – 2348. with proteins, essential unsaturated fats, EKPO, K. E. and ONIGBINDE, A. O. (2004). vitamins and minerals could pose health risks Pharmaceutical potentials of for consumers in the forms in which it is Rhynchophorus phoenicis larval oil. presently consumed, and this may negate the Nigerian Annals of Natural Sciences, plans for the promotion of entomophagy in this 9(2): 28 – 36. part of the world. The use of spices like garlic EKPO, K. E. and ONIGBINDE, A. O. (2005). (Allium sativum ) and Negro pepper ( Xylopia Nutritional potentials of the larva of aethiopica ) which have anti microbial properties Rhynchophorus phoenicis. Pakistan in the preparation of roasted APW though useful Journal of Nutrition, 4(5): 287 – 290. in controlling bacterial contamination, other EFSA (2008). Microbiological risk assessment in hygienic procedures like washing before feeding stuffs for food producing roasting, salting and use of preservatives that animals: Scientific opinion of the panel will control fungal growth, as well as adequate on biological hazards. The EFSA Journal packaging to prevent exposure to fungal spores pp.120-184. must be adopted to make the roasted APW fit FAO/WHO (1991). Protein Quality Evaluation. for human consumption. Report of Joint FAO/WHO Expert Consultation, FAO Food and Nutrition REFERENCES Paper, Number 51. GIBLIN-DAVIS, R. M., FALEIRO, J. R., JACAS, J. ABBEY, S. D. (2007). Foundation in Medical A. and VIDRYASSAGAW, J. E. (2013). Mycology . 4 th Edition, Kenalf Production, Biology and Management of Port Harcourt, Nigeria. Rhynchophorus Species. Repository BANJO, A. D., LAWAL, O. A. and SONGONUGA, document wwwcisr.ucr.edu/…/giblindav- E. A. (2006). The nutritional values of iesrobin-biology-an. Accessed 13 th June fourteen species of edible insects in the 2014. south western Nigeria. African Journal HALD, T., WINGSTRAND, A., PINES, S. M., of Biotechnology , 5(3): 208 – 301. VIERA, A., DOMINGUIES, A. R. BRAIDE, W. and NWAOGUIIKPE, R. N. (2011). LUNDSBY, K. and ANDERSON, V. D. Assessment of microbiological quality (2012). Assessment of Human Health and nutritional values of processed Impact of Salmonella in Animal Feed. 1 st edible weevil caterpillar (Rhynchophorus Edition, National Food Institute, phoenicis) in Port Harcourt, Southern Denmark. Nigeria. International Journal of ICMSF (1986). Sampling for Microbiological Biochemical Sciences, 5(2): 410 – 418. Analyses; Principles and Specification. CHEESEBROUGH, M. (2000). District Laboratory International Commission on Practice in Tropical Countries . Part 2, Microbiological Specifications for Food

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Animal Research International (2015) 12(2): 2159 – 2165