The key role played by microbiological testing within a cocoa manufacturing plant

Food microorganisms and humans have had a long association whose origins pre-date recorded history. Food, providing nutrients for us, is an ideal culture medium for the growth of microorganisms. Microbial growth is controlled by factors related to the food itself, and also to the environment where the food is being stored and processed. The food-related factors include pH, moisture content, water activity (aw), oxidation-reduction potential, physical structure of the food and available nutrients. Food composition is also a critical factor influencing microbial growth. The environmental ones include temperature, relative humidity, gases present, and the types and numbers of microorganisms present in the food.

Contamination by disease-causing micro – organisms can occur at any point in the food-handling sequence. The incapacity to control or inhibit the growth of pathogens and other harmful microbes along the food chain can lead to foodborne illness. The consequences that may arise from a foodborne disease outbreak can be severe for both consumers and food companies1. The so-called ‘farm to fork’ control is now considered an essential approach to guarantee safe products to consumers. Consumers’ concerns about the increasing number of widely reported food poisoning outbreaks have caused the regulatory authorities to increase the level of regulation. Food manufacturers face increased scrutiny under the law, thus the food industry has made safety a top priority to ensure regulatory compliance2. Leading global food companies have developed different approaches to manage and improve product quality (i.e. ISO 9001-2000, ISO 22000, HACCP) to meet customers’ expectations. Food manufacturers are required to adhere to the principles of Good Manufacturing Practices (GMPs), creating a set of guidelines aimed at keeping food free of pathogens. To prevent, reduce and eliminate sources of food safety hazards, it is essential that companies put in place a Quality Assurance System.

Such a system is based on two strictly correlated parts. The Quality Control, which covers the procedures carried out in the laboratory on a regular basis to ensure that all aspects of the daily work are under control: monitoring of procedures, consumables, equipment, temperatures of incubators, refrigerators and staff performances; and the Quality Assessment, which evaluates the performance of the laboratory to guarantee the generation of reliable laboratory results. To achieve this goal, laboratories ensure that testing is purposeful and efficient through the auditing process, which helps to drive continuous improvement, by identifying weaknesses and recommending due changes. Furthermore, it is also mandatory for all accredited laboratories to participate successfully in a Proficiency Testing Program (PTP) to evaluate laboratory performances, assessing and demonstrating the reliability of data they are producing.

In the food industry, microbiological testing can be effective when used in coexistence with chemical and physical control systems. During production, important parameters are measured continuously and, when deviations occur, the processing is adjusted accordingly, all as part of a preventive approach based on the application of a Hazard Analysis Critical Control Point (HACCP) to identify hazards defining and implementing systems of control at all stages in the food supply system1. Microbiological examination can be used to help set critical limits in HACCP systems and to verify that these Critical Control Points (CCPs) remain under control.

In order to evaluate the chocolate manufacturing industry, it is important to begin from the very first step: the provisioning of raw material. Production of high quality chocolate and cocoa products starts with the selection of the raw materials in order to guarantee the quality of the final product. The cacao seeds must be fermented, dried and roasted to produce the chocolate; fermentation and drying are mainly done in technically underdeveloped or developing countries. Most of the dried beans are processed into cocoa, cocoa butter, and chocolate in more technically advanced countries. However, to increase local economic return, there is a trend towards processing an increasingly higher percentage of cocoa beans into intermediate products, such as chocolate liquor and cocoa butter, in the country of origin3. This practice may cause some concern because of the poor hygienic conditions of the facilities where these processes are performed.

The manufacturing process of chocolate starts on arrival at the factory of the cocoa beans where they are sorted and cleaned; the visual check at reception is a key tool to guarantee good quality. The presence of moisture condensation in containers and of damaged packaging material could represent a risk not to be underestimated. Extreme care must be taken to ensure that the reception area of bulk materials is maintained in a clean and hygienic condition, and regular microbiological checks should be performed on pipes and valves. Therefore, effective cleaning and sanitisation programmes need to be put in place to ensure that unacceptable levels of contamination are never reached. Critical Control Points such as tanks for chocolate masses, storage silos and the flow line circuits that deliver food products require precise cleaning procedures such as Cleaning in Place System (CIP) designed for automatic cleaning and disinfecting4.

Once the cocoa beans are cleaned of twigs, stones and dust, they are roasted in special ovens at temperatures ranging between 105- 120°C, to reduce acidity, remove dry taste, lower moisture content, deepen colour and facilitate shell removal. During roasting, the cocoa beans develop their characteristic flavour and aroma. Cocoa beans have a thin shell that must be removed before extrusion of the cocoa butter can be done. During this process, called winnowing, shells are cracked mechanically and then removed by air blown from a fan. The centre of the bean left is known as the nib. The roasted nibs are grinded in mills until the heat and friction from the grinding process will reduce them into a thick chocolatecoloured liquid, known as cocoa mass; part of this liquor is used as an ingredient of chocolate, while another part is further processed into cocoa butter and cocoa powder. To extract the cocoa butter, vital to making chocolate, the cocoa mass is pressed. After the extraction, the remaining solid blocks of compressed cocoa, also named cocoa cake, are grinded and sifted to produce cocoa powder. Depending upon the amount of cocoa butter pressed out of the chocolate liquor, the resulting cocoa powder can be differentiated according to the percentage of fat content – a parameter which determines its end-use, ranging from drinking chocolate to use in bakery products and fillings3.

The peculiarity of the cocoa manufacturing facilities is that no two units are likely to be the same, and consequently, a widely diffuse exposure to potential contaminants during the different production stages is expected and represents a unique challenge for an efficient quality control system. The operational techniques and activities that are used to fulfil the quality requirements encompass the full spectrum of the manufacturing phases. The laboratory is responsible for monitoring not only in line samples and final products, but also for guaranteeing that the working environment (i.e. items of equipment and utensils) is appropriately clean.

A rapid test used to determine the cleanliness of food processing equipment and manufacturing areas is the Adenosine Tri- Phosphate Test (ATP). The ATP is a compound found in all types of plants, animals and microbial cells. The nature of this stable compound, which persists long after a cell has died, makes it a perfect indicator to help assess standards of hygiene and cleaning procedures. The technique involves taking a sample of the ATP present on the surface being tested by swabbing; afterwards the reaction that occurs between ATP, derived from the food residue and bacteria and an oxidative enzymes, will result in a bioluminescence, measured in Relative Light Units (RLU) by a luminometer. ATP tests are not intended to replace microbial ones; such testing should still be carried out for monitoring purposes or for checking the presence of specific spoilage or pathogenic organisms5.

The total microbial population and the presence of indicator organisms are generally controlled, as they are part of the characteristics needed to meet the purchase specifications. Indicator organisms are used as important components of the microbiological testing programs of both industry and regulatory agencies and, functionally, they are indicator of safety and quality. Safety indicators suggest the presence of conditions associated with increased risk of exposure to a pathogen, while quality indicators assess the effectiveness of the sanitation programmes.

One of the methods used to indicate the quality of a food is the Total Viable Count (TVC). It can indicate whether a product has been contaminated or spoilt. Enumeration by this method involves inoculation of a non-selective agar medium with a specified volume of the test sample; usually the incubation regime is 30°C for 48, or 72 hours for aerobic mesophilic organisms. The results are recorded as the number of Colony Forming Units per gram (CFU/g). Other common causes of food spoilage are the presence of yeasts, particularly in foods of reduced water activity such as cocoa powders, and moulds that can spoil chocolate by hydrolysing lipids, thus resulting in fatty acids that cause rancid tastes. Selective agars are commonly used for the detection of both yeasts and moulds (incubation at 25°C normally take five days to obtain a result). The surface inoculation methods are more desirable than pour plate methods because of the need to maximise aerobic conditions for mould growth and to enhance colony morphology for confirmatory identification purposes, especially because some strains are able to produce mycotoxins which can cause serious chronic illness if consumed6.

As with any other ingredient or raw material, the water used within the cocoa manufacturing processes requires a programme to assure its quality. Water can be a source of spoilage bacteria, which can cause taints in food products and can also facilitate the spread of pathogenic microorganisms. Water testing is not only good practice, but it is required by national law and auditing bodies. The monitoring programme should cover its source, treatment and distribution within the factory, and include regular checks for compliance with legislative requirements. The presence of indicator as E. Coli in water samples might be the result of possible contamination, which might have adverse effect on the health of the consumers. E. Coli is used to indicate recent faecal contamination or unsanitary condition in the food-processing environment. Presumptive E. Coli colonies detected with a qualitative method must be confirmed using a simple and rapid confirmatory method.

Another indicator of faecal pollution is the Streptococcus Faecalis. Very high levels of Enterococci may cause illness due to the production of pressor amines such as Histamine. The calculation of presumptive Enterococci, expressed in CFU/g, is made from the number of typical colonies obtained on and within the selective medium after an incubation period of 48 ± 4 hours at 37 ±1°C. Indicators are usually present in numbers higher than pathogens and indicate unsatisfactory conditions when levels increase significantly. Since levels can be monitored, the trend analysis can identify situations before they get out of control.

For public health reasons, where exists evidence of a potential hazard to health, pathogen testing system is needed. The main foodborne hazard in cocoa and cocoa products is Salmonella, reason why it should be the target organism. Testing for this organism at specific control points provides the best means of quality control. The presence of Salmonella in low-moisture foods, such as cocoa products, is a concern, even if these products, due to the low aw, do not support the growth of Salmonella. It is established that even low numbers of Salmonella can cause illness; and very low infective doses may be linked to the high percentage of fat in chocolate, which may protect the pathogen against the acidic conditions of the stomach7, therefore increasing the probability for onset of foodborne disease.

The production of cocoa powder is a dry operation, which does not destroy Salmonella or other vegetative organisms. Apart from the roasting processes, there are no other barriers to assure the elimination of this pathogen. Since it is not possible to prevent Salmonella from entering the facility, it is necessary to take preventive measures during processing to avoid recontamination of the product, by introducing preventive measures based on the HACCP (i.e. establishing barriers to separate the raw materials handling area and the other areas prior to inactivation steps from the succeeding ones, controlling all traffic between the different areas including the movement of personnel and materials). The basic concept is to prevent the occurrence of recontamination by building safety into the process. Prevention is a key component to control Salmonella in lowmoisture products.

The ability of the organism to survive under dry and other adverse environmental conditions makes it difficult to control. A suitable indicator for Salmonella has not been identified8. However, testing with enumeration of Enterobacteriaceae, whose high levels could suggest an increased risk for the presence of Salmonella may help. Even though high levels of the indicator suggest an increased risk, low levels of Enterobacteriaceae do not guarantee the absence of the pathogen8,9.

The most widely adopted method for the detection of this pathogen is the real-time PCR (Polymerase Chain Reaction), a technique targeting the genetics of microorganisms. PCR has been shown to have a very high specificity and the capacity to cut testing time, allowing fastening the positive release of finished products, especially when coupled with a pooling samples technique that allows testing more samples at once, thus enabling companies to have accurate test results before the product is purchased.

Microbiological analyses are an essential part of a comprehensive food safety management system, made up of Good Manufacturing Practices (GMPs) and Sanitation Standard Operating Procedures (SSOPs) and a meticulously implemented Hazard Analysis and Critical Control Point (HACCP) programme. A Food Quality and Safety system should not be viewed as a one-time effort, but rather as part of an industry commitment to an ongoing process, aimed at minimising the risk of microbial contamination.

References

1. IFST Professional Food Microbiology Group. 1997. Development and use of microbiological criteria for foods. Food Science and Technology Today 11 (3), p.140

2. Dillon, M.; Griffith, C. 2001. Auditing in the Food Industry. From Safety and Quality to Environmental and Other Audits. Woodhead Publishing

3. UNCTAD secretariat United Nation. 2008. Cocoa Study: Industry Structures and Competition Study

4. Cordier J. L. HACCP in the chocolate industry. 1994. Food Control, 5 (3), pp.171-175

5. H.L.M. Lelieveld, M. A. Mostert, J. Holah and B. White. 2003. Hygiene in Food Processing: Principles and Practice. Woodhead Publishing pp.269-273

6. Jayeola C. O.; Oluwadun A. O. 2010. Mycoflora and nutritional components of cocoa powder samples in South West Nigeria. African Journal of Agricultural Research Vol. 5(19), pp.2694-2698

7. Waterman, S. R. and P. L. C. Small. 1998. Acid-sensitive enteric pathogens are protected from killing under extremely acidic conditions of pH 2.5 when they are inoculated onto certain solid food sources. Appl. Environ. Microbio. 64:3882-3886

8. European Food Safety Authority EFSA E. 2007. Opinion of the Scientific Panel on Biological Hazards (BIOHAZ) on the request for review of the opinion on microbiological risks in infant formulae and follow-on formulae with regard to Enterobacteriaceae as indicators. The EFSA J.444: 1-14

9. Cordier, J. L. 2008. Production of powdered infant formula and microbiological control measures. In J. M. Farber, and S. Forsythe (eds). Enterobacter sakazakii. ASM Press. Washington, DC. pp.145-185

About the Author

Maurizio Pagano was a Researcher with the NGO Legambiente active in environmental protection. He later joined an interdisciplinary team at the ISS – National Centre for Food Quality and Risk Assessment. He is now responsible for microbiological and analytical control in a chocolate manufacturing site at Cadbury Kraft Foods UK. The unit is responsible for the Quality Management System implementation process.