Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 1085-1094

ISSN: 2319-7706 Volume 3 Number 9 (2014) pp. 1085-1094 http://www.ijcmas.com

Original Research Article Microbiological quality of belina processed under different traditional practices in Gwanda,

Felix M. Mujuru*, Raphael Kwiri, Clarice Nyambi, Clive Winini and Dinga N. Moyo

Department of Food Processing Technology, School of Industrial Science & Technology, Harare Institute of Technology, Ganges Rd, Box BE 277, Belvedere, Harare, Zimbabwe. *Corresponding author

A B S T R A C T

The aim of this study was to investigate the microbiological quality of Mopani Worms (MW) () processed under different traditional practices in Gwanda, Zimbabwe.Freshly harvested worms (MW) were degutted using bare hands or gloved hands. The degutted worms were sub-divided into 4 equal portions and subjected to different traditional drying methods namely, boiling K e y w o r d s in salted water (5% w/w salt) for 30 minutes and solar drying, open pan roasting, drum roasting and hot-ash drying to approximately 15% moisture content. Contamination, Thereafter, dried MWs were microbiologically analysed against indicator micro- Microbiological organisms (total bacteria counts, coliforms and Escherichia coli) and pathogens quality, (Salmonella spp. and Staphylococcus aureus) as well as spoilage organisms (yeast Mopani worm, and molds).Microbiological tests conducted showed that, total bacteria counts -1 drying, (TBC) were relatively low with values ranging from 10-2500 CFUg . indicator Significantly, degutting MWs using bare hands followed by drum roasting and - microorganism boiling in salt water coupled with open pan roasting, had the least TBC (10 CFUg 1 -1 pathogens, and 30 CFUg ) respectively. Coliforms and E. coli were also detected in MWs -1 -1 spoilage with values ranging from 0-43 CFU g and 0-30 CFU g respectively (bare hands -1 organisms. degutted) whilst in gloves degutted MWs coliforms counts were 0-45 CFU g and E. coli counts were 0-35 CFU g-1. Furthermore, some MWs especially ash in dried samples that contained varying levels and types of yeasts and molds that included Fusarium and Penicillium. No Salmonella specie were detected in all sample tested. The study showed that, MW processing method and subsequent handlingare important parameters in determining the type and levels of their contamination. As such, it is imperative for harvesters and processors to observe good harvesting and manufacturing practices and follow protocols that do not result in re-contamination of produce as this might present a danger to the public consumers.

Introduction

In Africa, most people living in rural areas offer diet diversity and opportunities for are dependent on non-timber forest products income generation amongst the rural poor in for nutrition and income generation (FAO, developing countries (Illgner and Nel, 2000; 2012a). It has been reported that Sunderland et al., 2004; Bukkens, 2005). 1085

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Insects are a highly nutritious and healthy security value, it is of paramount importance food source with high fat, protein, vitamin, that the MW value chain should ensure fibre and mineral content (DeFoliart, 1989, production of good quality and safe products 1990; Barker et al., 1998; Ayieko and (FAO, 2010a). In practice, MW processing, Oriaro, 2008). Dufour (1987) noted that packaging and storage are generally insects provide valuable buffers against considered as basic and primarily poor seasonal shortages of food. For instance, as leading to spoilage by pests and well as being food, insects do provide for microorganisms (Gardiner, 2005). disposable incomes which can be used on Traditionally, MWs are processed by hand food, farming inputs etc (Ageaet al., 2008; degutting followed by sun or hot-ash or sand Hope et al., 2009; Yen, 2009a). drying (Nonaka, 1996; Mulhaneet al., 2001; Allotey and Mpuchane, 2003). Although in Given their rich nutritional profiles, insects dry environments, the drying operation can do provide favourable environments for be effective in limiting microbial microbial growth and survival (Klunder et contamination and spoilage, in humid areas al., 2012). However, microbial growth and outgrowth of microbes can be stimulated. In survival is also influenced by processing and addition, contamination through the air, soil storage conditions afforded to them along and packaging material used could result in the value chain (Belluco et al., 2013). In poor quality and unsafe products for human most instances, the insects are subjected to consumption (Taylor, 2003; Banjo et al., traditional processing methods, such as 2006b). boiling, roasting and sun drying (Glew, 1998). These are done to improve the taste, As noted by Mulhaneet al (2001), edible storability and palatability of edible insects insects, including MWs could be with an implied assumption of ensuring contaminated by bacteria, fungi and production of safe food product (van Huis et residues. As such, regulatory al., 2013). guidelines, which are based on scientific data should be formulated to protect Gonimbrasia belina, commonly referred to consumers along the MW value chain as Mopane Worms or amacimbi or madora (Zhou, 2004). However, lack of specific are an important source of nutrition and scientific information on microbiological income for most rural households in content hinders the development of Zimbabwe (Stack et al., 2003; Gardiner, appropriate legislative framework to 2005). MWs are an excellent source of consider at various stages of protein and minerals with a reported crude the value chain (Belluco et al., 2013). protein value of approximately 50% (Headings, 2002). MWs are the To this effect a study was conducted to stage of the emperor and feeds evaluate the effect of various processing and exclusively on Colophospermum mopane hygiene regimes on the microbial quality of (Timberlake, 1996). As noted by Gardiner mopane worms harvested and processed by (2005), MWs are a key resource for poor local communities in Gwanda, Zimbabwe. farmers and landless locals, which they exploit for improvement of their livelihoods Materials and Methods (Styles and Skinner, 1996). Site and sample selection

Given the economic importance and food This study was conducted in Samlodi ward 1086

Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 1085-1094 of Gwanda district located in the South with diameter of about 0.5m and length of Western part of Zimbabwe, which is one of 1.0m, fitted with rotating handle and placed the biggest sources of the MW. A total of 15 over burning charcoal. The same treatments women were selected to conduct the were applied for the other 14kg batch harvesting and processing of MWs. The degutted using bare hands. selection was done following community The dried mopane worms were then vacuum consultations for people involved in packaged in air tight high density MWharvesting, processing and polyethylene bags and transported to Harare trade.Harvesting and processing of MWs Institute of Technology Food Analysis was done on 5 separate days during the laboratory for microbiological tests where April-May season in they will be abundant. they were refrigerated prior to analyses. Approximately 28kg of fresh MW were harvested at various points confined to an Microbial reagents, equipment and area of 3km2. The harvested MWs were sample preparation divided into 2 equal portions and degutted at theharvesting site either using a) bare hands Chemicals, reagents and media used were all and b) by personnel wearing vinyl reusable of analytical grade. Ancillary equipment, hand gloves. The MWswas put in different namely refrigerators, thermometers, water plastic buckets and degutted within 4 hours baths, incubators, analytical balances and of harvesting before drying. were all well calibrated, blenders used were sourced by the Food Laboratory from Capri, Drying methods employed Limited, Zimbabwe and Incotherm Limited United Kingdom.Before analysis, each The two degutted worm samples sample individually underwent particle size wereinstantly sub-divided into 4 equal reduction by milling in a sterile blender portions and subjected to different drying (Hamilton Beach HBF500S, China) after methods namely, boiling in salted water (5% which, 10g of the sample powder was w/w salt) for 30 minutes and solar drying, homogenised in 90ml sterile peptone water open pan roasting, drum roasting and hot- (Oxoid Ltd, Basingstoke Hampshire ash drying to acceptable moisture levels of England). The sample homogenates approximately 15%. Of the 14kg of MWs underwent serial dilutions i.e. 10-1, 10-2, 10- that had been degutted using gloves and 3, 10-4 depending on the tests that were boiled in salted water, 3.5kg were dried in conducted. Thereafter 1ml aliquots from the an open pan roaster, with the other 3.5kg resultant dilutions were inoculated in dried in a solar drier. triplicate plates using the pour plate and spread plate techniques. The open pan roaster was basically a flat metal pan with a diameter of 60 cm with Microbial Analysis raised edges of about 15 cm, whilst the cabinet type solar drier was made up of a TBC, E.coli, Staphylococcus. aureus and (2m x1m x1m) metal and wooden frame Salmonella spp Coliforms, determination with green house plastic cover and wire mesh trays. In addition another 3.5kg was Plate Count Agar (PCA) (Oxoid Ltd, dried in hot ash while the remaining 3.5kg Basingstoke Hampshire England) was used was dried in a drum roaster. The roaster for Total Bacterial Count (TBC) and was comprise of a black, non-perforated drum done in conformity with Association of

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Official Analytical Chemists (AOAC) cooking (> 70°C)(Stannard, 1997)were used protocols, 1990; Maturin and Peeler, 2001. for comparison. Enumeration of total coliforms was done Klunder et al. (2012) noted that, insects like using Violet Red Bile Agar (VRBA) many products, are rich in nutrients (Scharlau Ltd, Spain) of which E. coli and moisture, providing a favourable colonies were distinguished among all the environment for microbial survival and coliform colonies on the VRBA by their growth.This was supported by Vega and increased bluish fluorescence around Kaya (2012), who highlighted that insects colonies under long wave UV light 366nm collected in nature and insects raised on on adding 100 µg of 4-methyl-umbelliferyl- farms may be infected with pathogenic -D-glucuronide (MUG) (Oxoid Ltd, micro-organisms, including bacteria, virus, Basingstoke Hampshire England) in the fungi and protozoa. Practically, most food VRBA overlay. Enumeration of S. aureus stuffs including processed MWs can be was done by transferring 1ml of the sample contaminated through the environment, from homogenate onto 3 plates of Baird-Parker human contact, processing and post handling Agar, (Oxoid Ltd, Basingstoke Hampshire practices which could potentially cause food England) as follows; 0.4ml, 0.3ml and 0.3ml borne diseases (Belluco et al., 2013). and incubated at 35oC for 45-48 hours in which colonies of S. aureus were Table 1show relatively low counts of total distinguished by their circular, smooth, bacteria count (TBC) in MWs degutted convex, moist, 2-3 mm appearance in using either bare hands or gloves compared conformity with Bacteriological Analytical to products such as cured and salted meat Manual, 2001, Bennett and Lancette, 2001. where TBCs of up to 106 CFU g-1 are within acceptable range or even in food stuff that Yeasts and molds determination require further cooking (> 70°C) (Stannard, 1997, foodsafety.govt.nz). TBC Dichloran 18% Glycerol (DG18) Agar rangeswere10-2500 CFUg-1with higher (Oxoid Ltd, Basingstoke Hampshire values observed inMWs degutted using England) was used for enumeration of yeast gloves compared to those which were and moulds and was done in conformity degutted using bare hands. TBCs in MWs with Bacteriological Analytical Manual degutted using gloves were ranging from (BAM) protocols, (Tournas et al.,2001). The 300-2500 CFU g-1 whereas those degutted o plates were incubated in the dark at 25 C for using bare hands ranged from 10-300 CFU 5 days and thereafter colonies were counted, g-1. Although the use of gloves was effective all in accordance with BAM (Tournas et al., in preventing personnel hand damages 2001) general methods for enumeration of during the degutting process (Kozanayi and yeast and moulds. Frost, 2002), but use of gloves increased microbiological counts on the product. This Results and Discussion could be attributed to the fact that the gloves used were not hygienically designed and Specific studies on the microbiological were considered difficult to clean and quality and safety of insects as food are rare sanitise. In addition, they did provide a in the scientific literature. As such, moisture rich environment which could be microbiological guidelines of similar favourable for bacterial growth and products derived from sources that multiplication (Klunder et al., 2012). In fact include cured or salted meat, miscellaneous Kozanayi and Frost (2002) stated that, dried foods and foods requiring further 1088

Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 1085-1094 overall function of using gloves during G. respectively. On the other hand in those belina degutting is for hand protection rather degutted using gloves coliforms ranged from than hygienic use, hence reason why the 0-45 CFU g-1 while E. coli were from 0-35 gloves might not hygienically designed. CFU g-1.Coliforms counts were within expected limits of less than 1000 CFU g-1 in It was also observed that degutting using food stuff that require further cooking (> bare hands followed by drum roasting and 70°C) or in cured and salted meat (Stannard, boiling in salt water coupled with open pan 1997; Lim et al.2012) for instance MWs, roasting, had the least TBCs (10 CFU g-1and while their presence including E.coli 30 CFU g-1) respectively. This is attributable signifies dangerto public health(Oranusi to exposure of the product to high and Braide, 2012). temperatures (during roasting) as noted by Klunder et al (2012), and high osmotic Relatively high levels of S. aureus were stress due to salt addition in boiling water, observed in hand degutted and ash dried which could kill microbes. MWs compared to other drying methods. The presence of S. aureus in food is a good According to Gardiner (2005), insects such indication of poor personal hygiene since its as MWs can be contaminated with both main sources are food handlers (via their pathogenic and spoilage microorganisms hands` direct contact with food or through during harvesting, processing, packaging respiratory secretions (Argudin et al., 2010), and/ or storage. This is mainly attributed to hence justifying higher counts in MWs the fact that such practices are often degutted using bare hands than when gloves considered crude and poor (DeFoliart, are used. Contrary to this, Taylor (2003) 1989).Microbiologically, insects can noted that degutting by hand is quite harbour different kinds of food pathogenic common and a faster practice in MWs bacteria (Belluco et al., 2013) as processing, hence the most preferred method demonstrated by the presence of S. aureus, (Kozanayi and Frost, 2002). However, E. coli and coliforms in the MW samples. In despite high temperatures being achieved addition, from the studies by Braide (2012), during ash drying and the presence in other microorganisms found in the diet could be samples, the subsequent handling of the found on the skin and gut contents of the product could be a source of caterpillar. As such, the MWs could be recontamination (Reijet al.,2004). contaminated with the microorganisms well before human handling and manipulation Although high levels of S. aureus and E.coli although at low levels (Belluco et al., 2013). were observed in samples that were ash dried, drum roasted and boiled and solar Indicator microorganisms such as coliforms dried, it should be noted that in general, and E. coli were both present in MWs insects themselves have been shown to degutted using both bare hands and gloves harbour intrinsically dangerous pathogens and followed by subsequent processing (ash for humans (Veldkampet al., 2012). drying, drum roasting and boiling in salted However, Giaccone (2005) could not water and solar drying) except in those that exclude the possibility of insects becoming were boiled in salted water and roasted on carriers of pathogenic microorganisms to an open pan. From Table 1,MWsdegutted humans hence recommended cooking or using bare hands,coliforms and E. coli pasteurisation as risk minimizing steps. This ranged from 0-43 CFU g-1and 0-30 CFU g-1 was supported by Amadiet al (2005) who

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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 1085-1094 noted that boiling insects for at least 5 boiling in salted water and roasted in an minutes was an efficient process for open pan or in a drum roaster. From Table eliminating Enterobacteriaceae but not 1, it can be noted that in ash dried MWs, sporeforming bacteria, which could pose although at low levels, fungi were present, spoilage and safety risks in cooked insects which were confirmed to be Fusarium and (Klunder et al., 2012). This explains the Penicillium types. This supported the presence of significant levels of E.coli in ash assertion made by Taligoola and Gashe, dried compared to the other 3 methods (1996) who noted that frequent fungal where boiling was done prior to drying of isolates in dried MWs were species of the MWs. Aspergillus, Penicillium, Fusarium, CladosporiumandPhycomycetes, of which Significantly, in all processing practices some may produce mycotoxins (Mpuchane pathogenic Salmonella spp.was not detected, et al., 2000). For example, (Frazier and indicating that the product was safe in as far Westhoff, 1978) highlighted that as this pathogenic species is concerned. In Aspergillus, Penicilliumand Rhizopusspecies research done on microbiological content of are known to produce various mycotoxins in fresh, processed, and stored edible insects food and feeds infield and under storage. (MWs excluded) by Klunder et al(2012) From table 1, it can be observed that yeasts and Giaccone (2005) pathogenic species and molds were most abundant in ash and such as Salmonella sppand Listeria solar dried MWs, with higher value in boiled monocytogenes were never isolated in the and solar dried samples. The relatively high tested samples. This led to the conclusion presence in solar dried samples could be that the insects concerned were unlikely to attributable to dust and air as the solar driers attract microbial flora that could pose risks used had a mesh cloth which allowed for air to human health (van Huis et al., 2013). It is and dust circulation. This was supported by also important to note that even S.aureus Frazier and Westhoff (1978) who stated that was present, but it was within expected fungi was ubiquitously distributed in soil limits of less than 1000 CFU g-1 in food and air and further isolated Aspergillus in stuff that require further cooking (> 70°C) or food products that are sun-dried owing to in cured and salted meat for instance MWs spores deposition. (Lim et al. 2012). Conclusion and recommendations According to Stannard (1997) yeasts and molds are a common cause of food spoilage, The microbiological profiles of the Mopane particularly foods of reduced water activity worms are determined to a large extent by (aw) such as dried MWs. Yeast have not the processing method and subsequent been implicated in food poisoning whilst handling of the product along the production molds and some of its strains are able are and supply chain. From the studies it can be able to produce mycotoxins which can cause concluded that the processing method and serious chronic illness if consumed subsequent handling are important (Mpuchane et al., 2000). Table 1, also parameters in determining the type and shows the presence of various yeasts and levels of contamination of the MWs. For it molds in MWs processedunder different was shown that regardless of degutting practices. The presence of yeasts and molds method employed, boiling MWs in salted was limited in MWs samples which were water followed by open pan drying produced degutted using bare hands followed by a microbiologically safe product. In

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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 1085-1094 addition, it can also be concluded that use of imperative for harvesters and processors to gloves does not reduce microbial load observe good harvesting and manufacturing effectively and that wider sanitary practices and follow protocols that do not conditions are of key consideration result in re-contamination of produce. particularly post processing. As such, it is

Table.1 Mean microbial examination (CFU g-1) of G. belina processed under different traditional methods

Salmonella Process TBC Coliforms E.coli S.aureus YM* *types spp Fusarium & Ash drying 60 43 30 25 0 35 Penicillium D e g u t t e d Drum u 10 28 8 5 0 0 s roaster - i n g b a r Boiled in e Yeasts & soil h salted water a 300 13 3 10 0 200 slime moulds n and solar d s drying

Boiled in salted water 30 0 0 0 0 0 - and open pan roasting

Ash drying 2500 45 35 6 0 40 yeasts

D Drum -

e 660 3 5 2 0 0 g roasting u t t e d u s i n Boiled in Yeasts, g

g salted water rhizopus, soil

l 450 14 2 2 0 150 o and solar slime moulds v e

s drying

Boiled in salted water 300 0 0 0 0 0 and open pan roasting YM* yeast and mould, *types yeasts and molds types, the values shown in Table 1 are mean values

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