Academia Journal of Scientific Research 6(7): 272-281, July 2018 DOI: 10.15413/ajsr.2018.0132 ISSN 2315-7712 ©2018 Academia Publishing

Research Paper

Microbial quality analysis at different stages of cocoa processing

Accepted 21st June, 2018

ABSTRACT

Microbiological quality assurance has been employed in food industries for many years, to guarantee the quality of food products for human consumption; however, foodborne diseases continue to occur. This study sought to assess the microbial quality of cocoa products at various processing stages as well as the processing environment, in a typical cocoa processing company. Products and environmental samples were collected at the various stages of processing within the production area and analysed for the presence of yeast and moulds, Enterobacteriaceae and Salmonella. Colonies of the Enterobacteriaceae were tested for E. coli and David Ewusi-Mensah1,2*, John A. Salmonella since they are hygienic indicators. Confirmatory tests yielded negative Larbi3 and Jingyu Huang1 results for these, thereby attesting to the fact that the test Enterobacteriaceae 1Hohai University, College Of showed the absence of E. coli and Salmonella and as a result, validating safety for Environment, Nanjing-Jiangsu, further processing. Yeast and mould, however, recorded the highest mean colony China. counts in all but the butter samples, thus indicating possible contamination at the 2No. 1 Xikang Road, Nanjing, 210098, China. point of packaging and poor storage conditions. The results also showed that 3Kwame Nkrumah University of microbial quality of production environment has a significant influence on the Science and Technology, College products due to various means of contamination. of Science, Kumasi-Ghana.

*Corresponding author. E-mail: Key words: Quality assurance, microbial contamination, cocoa processing, food [email protected]. safety, microbial quality.

Abbreviations: TPC, Total plate count; EMB, eosin methylene blue agar; GMP, good management practices; FSMS, food safety management system; HACCP, hazard analysis and critical control points; EMB, eosin methylene blue; cfu, colony forming count; NACMCF, national advisory committee on microbiological criteria for foods.

INTRODUCTION

Cocoa, seeds from fruit pods (Theobroma cacao), is production (Bittenbender et al., 2012; Papalexandratou et essential to about 40-50 million people worldwide, al., 2011; Nielsen, 2006). As a cash crop, it has the huge including over 5 million small-scale farmers who grow this contribution of the producing nations’ economy as it is on a valuable cash crop (Guehi et al., 2010; Bittenbender et al., high demand (Essegbey and Ofori-Gyamfi, 2012; World 2012). Cocoa beans are an important ingredient in food Cocoa Foundation, 2014) especially in food industry for its industry and continue to be enjoyed today in thousands of numerous products (Food Safety and Standard Authority of different forms globally (Bittenbender et al., 2012). Africa India, 2006). countries with top cocoa production include Cameroon, In the food industry, food safety is given maximum Cote d’Ivoire, Ghana and Nigeria, as they produce about 2.8 attention since it has been linked to foodborne hazards million tonnes annually forming about 73% of the world’s present in food at the point of consumption. Food safety production. Asia/Oceania (Indonesia, Malaysia, and Papua hazards can occur at any stage in the food chain and as such New Guinea) and the Americas (Brazil and Ecuador) it is essential that adequate control measures be put in account for 14 and 13% respectively of world’s cocoa place to avoid or minimise the menace (Safety and Academia Journal of Scientific Research; Ewusi-Mensah et al. 273

Workers, no date; Food and Agricultural organization of the Reed, 2010; Jürgen Fischer, 2012; Costa et al., 2015). In United Nation and World Health Organization, 2001; most cocoa processing companies, cocoa beans are Mamalis et al., 2009; Meador and Jie, 2013). At the processed into finished and semi-finished products for international level, standards have been made of high further processing and/or export. These products include priority to put the industry on the check (Gilmour, 2009). cocoa liquor (natural cocoa mass), cocoa butter (pure prime These include International Organization for pressed cocoa butter) and cocoa powder (natural/alkalized Standardization (ISO)-(ISO 22000) (Færgemand and cocoa powder). A typical production process as shown in Jespersen, 2005; Mamalis et al., 2009) Hazard Analysis Figure 1 (A, B, and C) is divided into three main phases Critical Control Points (Cordier, 1994; Afoakwa et al., depending on the process involved. These include: (i) the 2013), National Advisory Committee on Microbiological cleaning phase, where the beans are cleaned to remove Criteria for Foods (NACMCF), Good Manufacturing Practices stones, fibres and other foreign materials (Figure 1 (A)), (ii) (GMP) (Eastern Research Group Inc, 2004; National pre-processing phase, where cocoa beans are roasted, Confectioners Association (NCA), 2012), Codex winnowed and milled (Figure 1 (B)), and (iii) the Alimentarius Commission and Standard Operating processing phase; main processing stages where milled Procedures (SOP’s). Microorganisms play a vital role in food cocoa mass (liquor) is pressed in cake and butter for industry as food ingredients or in food spoilage and exist further processing and packaging (Figure 1 (C)). basically as moulds, yeasts and bacteria in food (Lasztity, no Packaging area is highly vulnerable to microbial date; Costa et al., 2015). Cocoa processing as a food contamination. Since operators have direct contact with the industry is no exception; microbes are the significant products, there is a possibility of contaminating the product reasons for fermentation (Mathis et al., no date; Nielsen, with human pathogens and others such as coliform group 2006; Guehi and Koffisouma, 2010) and contamination bacteria: E. coli, Campylobacter, Clostridium, Listeria, (food safety) (Lammers, 1998; Beckett, 2008; Sánchez- Staphylococcus aureus, Yeast and Mould, and Salmonella Hervás et al., 2008; Mikkelsen, 2010; Lima et al., 2012). As (Meursing, 2008; Afoakwa et al., 2013). Additionally, part of the cocoa value chain, standards aid in determining mycotoxins, such as ochratoxin A (OTA), are toxins the quality of cocoa beans such that beans should also be produced by a fungus under favourable conditions of well fermented, have a moisture content of maximum 7.5%, moisture and temperature which end up contaminating a free fatty acid content of maximum 1.5% and a cocoa products, making them unsafe for consumption (Lyndberg, butter content between 45 and 60% (Guehi et al., 2008). 2008; Gilmour, 2009). Marina et al. (2014), in their study, Meanwhile, one of the challenges facing the industry has to showed that fungi and mycotoxins exist in beans and it is do with the quality of beans as the raw material. The quality important to eliminate them throughout processing. The of beans does not just form the baseline for processing but cocoa processing industry is alarmed at the influence of also affects the quality of finished products (Pillars, 2008; Yeasts and moulds, E. coli, Salmonella and other pathogenic Smulders et al., 2009; Karlovsky et al., 2016). microbes (Lyndberg, 2008). Moulds and fungi, though used During fermentation of cocoa beans after harvesting, as ingredients in food industries, are linked with foods microbial activities lead to the formation of a range of spoilage as their activities cause food mildew which is metabolic end-products, such as alcohols, acetic acid and considered unfit to eat. They reproduce asexually through other organic acids, which diffuse into and destroys the spores (mycotoxin formation) in a large number which beans (Mathis et al., not dated; Eskes et al., 2006; Payne et spreads easily in the air (Afoakwa et al., 2013; Copetti et al., al., 2010; Lowor et al., 2012; Kresnowati, Suryani and 2014). The industry uses total Enterobacteriaceaeas an Affifah, 2013). These compounds formed significantly indicator of hygiene practices since they are resistant to influence the steer taste, cocoa flavour precursors and common disinfectants (Cordier, 1994), and are able to other sensory qualities of the beans (Eskes et al., 2006; colonise several niches in food processing plants (da Silva Smulders et al., 2009; Guehi and Koffisouma, 2010; Reed, do Nascimento et al., 2010; Krapf and Gantenbein- 2010; Jawad et al., 2013; Costa et al., 2015). Demarchi, 2010; National Confectioners Association, 2010; Microorganisms of all species may find their way into Kačániová and Juhaniaková, 2011; National Confectioners harvested beans through the hands of workers, unwashed Association’s Chocolate Council, 2011). In a study, it was baskets, knives and dried mucilage on fermentation reported that the sources of hazards of contamination could surfaces (Kostinek et al., 2008; Lambert, 2009; Kresnowati be microbial, physical or chemical at different stages of et al., 2013). The actual bean processing begins with processing and how HACCP measures can control them cleaning, roasting and winnowing (into nibs). The nibs (Afoakwa et al., 2013). (which were contaminated during post-harvest processes) As a matter of fact, though quality assurance stakeholders are sterilised in a batch or a continuous process by wetting are doing their best to keep their products within allowable and heating with steam at 125-130°C for 7-11 min. The nibs range as proposed by FSMSs microbial quality during go through further processing till the liquor is pressed into theirdaily routine checks (APIC, 1996; Eastern Research cake and butter as products (Lammers, 1998; Food Safety Group Inc, 2004; Færgemand and Jespersen, 2005; and Standard Authority of India, 2006; Meursing, 2008; International Cocoa Organization (ICCO), 2009; Afoakwa et Academia Journal of Scientific Research; Ewusi-Mensah et al. 274

Figure 1: The three phases of Cocoa processing into semi-finished products; cleaning phrase, Pre-processing phase, processing phase.

al., 2013), various factors of processing contribute directly should be of much concern for effective elimination or and indirectly to the microbial quality of products which control. According to FSMSs, products whose parameters Academia Journal of Scientific Research; Ewusi-Mensah et al. 275

Table 1: Sampling points.

Stage of processing Sampling point Numbers of samples Sampling areas Before sterilization 2 Refinery area Refinery After sterilization, Liquor tank 2 (Surfaces, Floor)

Butter outlet point Press machine Press area Press Cake point Press machine (Surfaces, Floor)

Liquor packaging No liquor N/A N/A

Cake packaging room Cake packaging Kibbled cake 2 (Surfaces, Floor)

Butter filter press Filter press pump 2 None

Butter packaging Packaging point 2 Butter packaging room

Refrigerated warehouse. Finished product warehouse N/A N/A Normal warehouse.

do not meet or satisfy international standards (esp. environmental and product samples. Sterile swabs were microbial content) must be returned to the producers to be used to dab/swab the floor and equipment surfaces disposed of (destroyed) or reworked (National (environment) of the different areas of sampling and kept Confectioners Association (NCA), 2012). In such cases, in peptone water, as shown in Table 1 and Figure 1 with apart from the fact that companies may lose the trust of letters, to verify which process is vulnerable to microbial their clients, they suffer financial crisis as a result (Cordier, attack and as well contributes to the high microbial content 1994; U.S. Food and Drug Administration, 2013). In spite of in end products. In all, from 7 different sampling points, 9 these challenges, fewer studies have been carried out in this groups of samples were collected (18 environmental and 18 area and the few that do rather focus on the analysis of product samples; twice each at different shifts over a fewer microorganisms (thus either of; Enterobacteriaceae, month) for analysis. The product samples were also Salmonella spp, Yeast and Moulds together with Total Plate collected with sterile spatula and containers at different Counts) in food contamination and poor food quality, yet, sampling points in processing as shown in Table 1 and microbial contamination is still relevant in recent times Figure 1 (in blue shaded boxes). All samples were (Lammers, 1998; Camu et al., 2008; Ntiamoah and Afrane, refrigerated between temperatures of 2-5oC to inhibit 2008; Mounjouenpou et al., 2008; Nielsen, Snitkjaer and microbial growth and to keep them in their solid states. As van den Berg, 2008; da Silva do Nascimento et al., 2010; pre-requisite to the analysis, solidified liquor samples were Lefeber et al., 2010, 2011; Mikkelsen, 2010; Lima et al., scraped with a sterile spatula into smaller fragments which 2012; Lowor et al., 2012; Wessela, Q and Uist-Wessel, could easily mix in water, whiles cake was ground in a 2015). sterile mortar with a pestle to a fine texture which can This research, therefore, seeks to; (i) determine the easily dissolve in water. 1 g of the butter samples were stages of processing that are prone to microbial measured into a sterile beaker and mixed with sterile contamination, by conducting a thorough microbial analysis tween 60 (polyoxyethylene sorbitan monostearate) to of the environment (floor and surfaces) and product dissolve it well. Samples were also taken before and after samples, (ii) determine the distribution/presence of sterilisation to determine its efficiency. Two sets of samples microbes (Enterobacteriaceae, Salmonellaspp, Yeast and were taken on different occasions (two working shifts in a Moulds together with Total Plate Counts) at various day) to ensure accuracy. Table 1 shows the summary of the processing stages, and (iii) propose mitigation methods for sampling points and process. microbial quality assurance depending on the results.

Procedure MATERIALS AND METHODS FSMSs demands that food samples are tested for a wide All samples for microbial quality analysis were obtained range of pathogenic, spoilage and indicator organisms from a cocoa processing company, Real Products Ghana covering a vast range of foods and food groups. Two main Limited, Western Region-Ghana within a period of one methods adapted for this study were detection (qualitative) month in 2014. Samples for analysis were divided into two: and enumeration (quantitative). Detection method was Academia Journal of Scientific Research; Ewusi-Mensah et al. 276

done to determine the presence or absence of the Shigella (SS) agar were poured into sterile Petri dishes, and microorganisms, while enumeration method was to with a sterile inoculating loop, colonies were streaked on determine the number of the detected microorganisms the agar and incubated for 18-24 h for E. coli (at 37C) and present. These two methods made use of the pour plating Salmonella (at 45C) confirmatory test, respectively and spread plating (streaking) techniques to test for (Bartram and Pedley, 1996). General methodology was aerobic, mesophilic bacteria/total plate count (TPC), adopted to minimise potential errors in the analysis, as well moulds and yeasts, and Enterobacteriaceae (Salmonella and as a measure used to ensure the validity of individual E. coli) (Food and Agricultural organization of the United results. Sampling and sample storage procedures used Nation and World Health Organization, 2001; Dye et al., assured a true representation of what was taken from the 2013). Other sources of information checklists/quality site and that they reached the laboratory unaltered. assurance monitoring within the company (guided by GMP) Sampling and analysis were done in duplicates to ensure and interviewing of shift leads to basic operation activities. accuracy and precision, as well as blank samples. According to modern standards for effective processes, The graphs were drawn with Microsoft Excel, as well as significant processes, such as sterilization that reduces the mean count and standard deviation of the count of the bacteria counts to below 500 cfu/g, have minimal moisture microorganisms. Laboratory data were analysed using SPSS absorption and less or no impact on taste (FDA, 2013; windows version 16.0. A one-sample t-test was used to Duyvis Wiener, 2017). analyse transformed data. Furthermore, 1 g of each sample was weighed into a beaker and 9 ml of sterile water was added with a syringe, under a laminar horizontal flow table and stirred to form a RESULTS AND DISCUSSION uniform mixture. 1 ml of the solution was pipetted into sterilised test tubes and diluted with 9 ml sterile distilled In the present study, it was found that the sterilisation water (10-1 to 10-4), then into their respective well-labelled process was effective, however, this does not guarantee sterile petri dishes. 10 ml of prepared Nutrient Agar that any contamination will occur as the process proceeds ((LabM); for TPC test), Potato Dextrose Agar (for moulds and that pathogens and microbes will be inactive/present. and yeasts test; were inverted and incubated for 5 days at Related studies have shown that roasting and conching 30ºC), MacConkey Agar (Fluka 71043-for processes do not guarantee the thermal inactivation of Enterobacteriaceae test; were inverted and incubated for Salmonella and other microorganisms (Krapf and 24 h at 44C) and 3.8 g of Selenite Broth agar Gantenbein-Demarchi, 2010). Although there was a (Oxoid:CM0395- for Salmonella test; incubated for 48 h at significant difference in microbial count after sterilisation 44oC) were poured into each petri dish and swirled gently (p=0.001) which reduced the microbial content by 83%, to mix uniformly with the sample; the mixture was then and was not efficient as compared with the standards. allowed to solidify. For Enterobacteriaceae test, pink There was a gradual increase in TPC as the process colonies indicated the presence of lactose-fermenting proceeded as shown in Figure 2. In spite of the fact that colonies which produce acids to give the pink colour when counts were within the accepted ranges of 104-<107per ml observed (FDA, 2013). To confirm, the pink colonies were or g by International Standards Organisations (GS streaked on Eosin Methylene Blue (EMB) agar (Oxoid). A 740:2007; GS 738:2007; ISO 7218:1985(E)) (NSW Food negative outcome confirmed the absence of E. coli coliform. Authority, 2009; Centre for Food Safety, 2014; Jim and Counting was done using a colony counter after 24 h Mike, 2010), the increase in the count was due to (Meursing, 2008; PátKováa et al., 2010; Dye et al., 2013; contaminations in the tanks (especially the liquor). The Richard, 2013). Since all samples were taken 4 times at results showed that there was microbial growth in the different times, so were the test, using the average counts liquor tank. This may be due to the sufficient moisture as the final values. Total colonies were calculated using the (content) in the liquor to enhance microbial growth. Counts Equation 1 (Jackie, 2012): were identified by quantitative analyses of all the samples, products and environment. Colony count (CFU /ml) on an agar plate Total colonies= Mean values for total viable count recorded ranged from Total dilution of the tube (used to make a plate for colony count ) × Volume plated 0.00 to 4.80×102 cfu/ml in butter samples before (1) sterilisation. After the sterilization process, samples from the press room recorded the highest counts as 3.52×102 For the environmental samples, 10 ml of peptone water and cfu/ml. This high level may be attributed to the distilled water were measured into bottles and test tubes, environment in the press room as shown in Figure 2. respectively and sterilised. 1 ml of swab samples were Contamination during processing may be from pieces of pipetted into sterilised test tubes and diluted with 9 ml equipment with which food comes into contact with air, sterile distilled water (10-1 to 10-4), before the test for dust, water and other ingredients. Our results, as shown in Salmonella and E. coli was conducted. Figures 3 and 4, proves that the press is the most Eosin methylene blue (EMB) agar and Salmonella- vulnerable processing stage to microbial attack because it is Academia Journal of Scientific Research; Ewusi-Mensah et al. 277

µ=269.167 600 SD= 147.62 butter Data set are ±2SD below and above the mean. 500 cake/liquour

400

300 Total plate Total count/cfu/ml 200

100

0 Before ster. After ster. Liquor tank Press 3 Press 4 Packaging

Sampling area

1. Figure 2: Total plate count in product samples.

9 Enterobacteriaceae Salmonella 8 Yeast and moulds

7

6

5

4 obial count/cfu/ml obial

Micr 3

2

1

0 Refinery Press room Cake room Butter room Warehouse cake Warehouse butter Processing area

Figure 3: Microbial quality in environmental samples (Faecal coliform and Salmonella).

the only processing stage which is an open system and 2008; Afoakwa et al., 2013; Copetti et al., 2014; Costa et al., exposed to the surrounding as other studies have 2015). The nearness of the press room to the cleaning area, confirmed (Eastern Research Group Inc, 2004; Meursing, as well as the cocoa warehouse can enhance easy migration Academia Journal of Scientific Research; Ewusi-Mensah et al. 278

100 Enterobacteriacea 90 Salmonella 80 Yeast and Moulds 70

60

50

40

icrobial icrobial count/cfu/ml M

30

20

10

0 Before steril After steril Liquor tank Press cake 3 Press cake 4 Bagged cake Presss butter 3 Press butter 4 Filtered butter Blocked butter Product samples

Figure 4: Microbial quality in product samples (Faecal coliform, Salmonella, yeasts and moulds).

of spores of microbes into the environment especially presence of Aspergillus suggests that spores of the fungi moulds. Low temperatures (which inhibits microbial may have escaped the entire heating processes (PátKováa et activities) in the transporter that carries pressed cake al., 2010; Afoakwa et al., 2013; Richard, 2013). through milling (into powder) to packaging might account The mean values Enterobacteriaceae recorded for for the decrease in the count at the packaging area (Eastern product samples ranged from 0.00±0.00 to 3.20×101cfu/ml Research Group Inc, 2004). Again, it was observed that after sterilisation in liquor tank and pressed cake, conveyor belts carrying pressed cake after pressing were respectively (Figure 4). Liquor, after sterilisation in liquor left open, which is a vulnerable point of exposure for tank, also showed no counts. All butter products recorded contamination. Experts suggest that conveyor belts should counts below the detection limits for Enterobacteriaceae. be covered and disinfected regularly and when necessary The microbial growth on MacConkey agar showed the (Eastern Research Group Inc, 2004; Lyndberg, 2008). The presence of coliforms in both the environment and product results showed the absence of microbes in all butter samples. The Salmonella-Shigella agar showed no growth of samples. This may be due to the fact that butter lacks microorganisms. This implies that Salmonella was absent in sufficient moisture to enhance microbial growth, which the samples. Salmonella is a unique pathogen in that it is should be less than 0.2aw for butter according to known to survive in low moisture foods. Possible sources of international standards (AOCs Ca 2e-84). Microorganisms contamination could be storage, transportation and post- individually have a range of moisture content for growth harvest handling. Studies on the history of Salmonella and survival. Bacteria dominate in foods with high moisture outbreaks in chocolate and cocoa products observed (minimum of about 0.90 aw) while yeasts and moulds causes, such as environment, cross-contamination, require less moisture of minimum water activity of 0.70 aw inadequate thermal processing and leaky wastewater, in to survive(Lasztity, no date; Cordier, 1994). Canada, UK and other European countries in the early For yeasts and moulds, the mean values recorded ranged 1980s and 2000s (da Silva do Nascimento et al., 2010; from 0.00±0.00 to 3.52×103 cfu/ml in all butter samples and National Confectioners Association, 2010). In the present bagged cake, respectively exceeding the limits, <102 per study, the absence of Salmonella could be as a result of good ml/g, according to Ghana Standards, as shown in Figure 4. heating processing, as well as some GMPs adopted in the The results showed a growth of yeast and moulds which processing. We observed from our routine monitoring that were mostly circular with entire edges. The greyish centres, 30% alcohol content solutions were available at the when isolated after microscopy analysis, were specifically entrance of all processing room, as well as driers for moulded. The round pea green colonies with entire edges, disinfecting the hands of operators. Also, washrooms were creamy round colonies, as well as round colonies with white kept clean aside few leakages of water sometimes, which entire edges were Aspergillus filamentous fungi species. The seems not to contaminate, according to the results. Academia Journal of Scientific Research; Ewusi-Mensah et al. 279

E. coli is a member of faecal coliform bacteria group, 2. The method of Batch Sterilisation, where sterilization is which are naturally found in the intestines of mammals. done in batches, should be adapted. This process will Therefore, their presence in the environmental analysis is significantly enhance the efficiency of the sterilisation mainly due to recent faecal contamination. Confirmatory process. analysis showed the absence of E. coli count which may be 3. Each pressing machines in the press room should have its attributed to good hygiene and sanitation practices. The own homogenizer that supplies it with liquor for pressing. confirmatory test also proves the fact that, though E. coli This is to make it easier to trace contamination in any form. were absence, there could have been other 4. Temperatures in the packaging rooms and conveyors Enterobacteriaceae present, but for the purpose of this should be kept low at all times by ensuring that the cooling study, they were not investigated since it is not the focus of systems (air conditions and cold water jackets) are the industry. Nevertheless, counts of Enterobacteriaceae in working properly. the press and bagged cake (3.20×10and 8.68×101 cfu/ml, 5. The warehouse for keeping finished products should be respectively) as shown in Figure 4, as well as some areas in kept well according to the FSMS requirements to prevent the processing, may be due to contamination from further contamination as products await shipping and operators especially maintenance team, as well as the exportation. Management may consider the temperature of environment. The mean values for the environmental the entire warehouse, relative humidity, stacking of the analysis recorded for Enterobacteriaceae ranged from 0.00 packaged products, cleaning of the warehouse, as well as to 7.81 cfu/ml with the highest being recorded at the press other GMP requirements. floor, followed by the refinery area and cake room respectively. The least count was recorded at the cake warehouse as shown in Figure 3. Salmonella was absent in ACKNOWLEDGEMENT all the areas of processing. The authors wish to acknowledge Real Products Ghana Limited (RPGL)-Ghana, a branch of Cafiesa (CACAOS FINOS CONCLUSION Ecuatorianos- Ecuador) for their support.

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