Practical method for spore enumeration in cocoa powders

With a practical method for determining concentrations of bacterial spores in cocoa powders, NIZO supports industry to assess microbial spoilage risks in heat-treated dairy products. 

The presence of bacterial spores in cocoa powders is inevitable, due to the natural fermentation process of cocoa beans. These spores are heat resistant forms of bacterial cells that can potentially cause food spoilage when they survive heat treatments and are able to germinate and grow in the finished product.   

Usually, spore concentrations in cocoa powders are low, and spoilage incidents rare. However, a reliable method of determining spore concentrations is needed, to properly assess the risk of spoilage for finished heat-treated (e.g. UHT) dairy products containing cocoa powders. At the same time, the results will vary when different classical microbiological plating methods are used, making interpretation and reliable risk assessment difficult. Finally, cocoa powders pose added challenges for enumerating spores compared to other beverage ingredients, due to its antimicrobial effect, poor wettability and dark colour.  

NIZO and its industrial consortium partners Olam Cocoa, Cargill, Barry Callebaut, The Coca-Cola Company, FrieslandCampina, Abbott Nutrition and Tetra Pak, have published a paper providing an optimised and aligned method for reliable enumeration of spores in cocoa powders. It includes expert insights on interpreting the results in practice.  

This paper provides an important reference for cocoa producers and buyers worldwide to reach agreement on acceptable specifications of spores in cocoa powders for assessing spoilage risks of finished liquid products.    

The manuscript has been published in The Journal of Food Protection, via Open Access. There is also a summary explaining the highlights and results of the consortium available.

Innovative ways to decrease food waste in dairy

NIZO initiated, coordinated and executed the pre-competitive industrial consortium research project that included cocoa producing companies, cocoa buying companies and a process technology and packaging company.  

Cocoa offers unique challenges for counting bacterial spores, making classical plating methods challenging: 

• Poor hydration of cocoa powder can result in insufficient wetting or small lumps, increasing the survival rate of cells or spores.
• The relatively high detection limit of the classical plating method can miss low concentrations of the spores that may still cause issues in the finished product.
• It can be difficult to see the colonies of spores in the agar, due to the dark, heterogeneously distributed cocoa. This can result in inaccurately low spore numbers. 

Overcoming the challenges of spore enumeration in cocoa powders

The manuscript describes step by step the proposed method for spore enumeration in cocoa powders. It also offers important insights to support the real-life use of the method and the interpretation of the analytical results: 

• Concentrations of heat resistant spores in cocoa powders are usually below the limit of detection of classical plating methods (<100 CFU/g), leading to an underestimation of the risk.  
• The antimicrobial effects of cocoa can result in inaccurately low spore counts: this can be overcome by limiting the concentration of cocoa in agar plates to <2.5 mg/mL. 
• The main bacterial spore species found in cocoa powder are Bacillus subtilis and Bacillus licheniformis, which are both known to express great strain-to-strain variety in heat resistance. Swarming of these species on agar plates can result in an underestimation of spore concentrations; this can be overcome by adding a non-nutrient overlay.
• Median values of total spore concentrations are low (<400 CFU/g for total mesophilic spore formers and <75 CFU/g for total thermophilic spore formers) and concentrations of heat resistant spores (i.e. surviving 30 minutes at 100°C) are very low (<5 CFU/g). 

Importantly, the relation between concentrations of (heat–resistant) spores in cocoa powders and spoilage of heat-treated beverages containing cocoa is still unclear. This will require more detailed investigations, specifically on the role of cocoa hydration. For more information on our  continuing research in this area, please contact Robyn Eijlander, Senior Project Manager Microbiology and Food Safety.