Exploring Microbiological Control: Testing Methods for Preservative and Antimicrobial Effectiveness
What are Preservatives and Why are They Important?
Preservatives play a crucial role in ensuring food safety and extending shelf life. They help prevent the growth of microbial pathogens, thereby reducing the risk of foodborne illnesses and ensuring compliance with food safety regulations. By maintaining the quality of food over time, preservatives minimize spoilage and waste, which in turn enhances the marketability of food products. Without preservatives, the food industry would face significant challenges in maintaining both the safety and quality of finished products.
Preservatives can be broadly categorized into artificial and natural types. Artificial preservatives, such as sodium benzoate, potassium sorbate, and nitrates, are synthetically manufactured chemicals widely utilized to extend the shelf life and ensure the safety of processed foods, beverages, and baked goods. These substances work by inhibiting microbial growth and preventing spoilage, thereby prolonging the shelf life of food products.
Natural preservatives, on the other hand, are derived from natural sources and include a range of substances such as plant extracts (e.g., rosemary extract, green tea extract), vinegar, essential oils (e.g., clove oil, oregano oil), microbial extracts (e.g., bacteriocins), and specific secondary metabolites (e.g., chiber). These natural preservatives are increasingly popular in the food industry, particularly in organic and natural products, health foods, and fresh produce. They are valued for their perceived safety, minimal processing, and alignment with consumer preferences for clean labels and environmentally friendly options.
In addition to their antimicrobial properties, natural preservatives may offer additional health benefits, such as antioxidant activity, which can contribute to overall product stability and nutritional value. Their adoption is driven by growing consumer demand for natural and minimally processed ingredients, as well as regulatory trends favoring transparency and sustainability in food production.
Common Target Microorganisms of Concern for Preservatives
The primary target organisms may include pathogenic microorganisms like Escherichia coli, Listeria monocytogenes, Salmonella, spore forming pathogens such as Clostridium botulinum, and spoilage organisms like lactic acid bacteria, yeast, and mold. The specific microorganisms targeted depend on the type of food product, its expected shelf life, and storage conditions. For non-food related products, other target organisms may be considered based on the product itself. For example, environmental products may focus on organisms prevalent in water systems or industrial environments, such as Pseudomonas species. For oral care products, the primary target may be bacteria that are associated with dental diseases, such as Streptococcus mutans.
Understanding the specific target organisms is the key to selecting appropriate preservatives for the final product.
Testing Methods to Evaluate Effectiveness of Preservatives and Antimicrobial Products
There are several testing approaches which may be used to examine the efficacy of the preservatives and antimicrobial products, including: the agar diffusion method, broth dilution method, time-kill test, and challenge studies. The agar diffusion method and the broth dilution method are types of growth studies; their purpose is to determine if growth of the target organism can be inhibited by the preservatives. Time-kill studies are a type of die-off study, which may be used to evaluate if the preservative can provide lethality to the target organism. Challenge studies can be considered growth studies, die-off studies, or even a combination of both, depending on the study design. Each method has its own set of advantages and limitations and should be carefully designed based on your specific objectives.
Agar Diffusion Method
The agar diffusion method involves diffusion of antimicrobial products through an agar medium in order to determine if the antimicrobial products are capable of inhibiting microbial growth. There are two primary techniques: the agar disc-diffusion method and the agar well-diffusion method.
For the agar disc-diffusion method, a small paper disc soaked with antimicrobial solution is placed on the surface of the agar plate, which has been previously inoculated with the target organism. For the agar well-diffusion method, wells are created in the agar that was previously inoculated with the test organism, and then the antimicrobial solution is pooled into each well. Agar well-diffusion allows for larger volumes of the product to be tested and may be applicable for circumstances in which the product is not easily absorbed by the paper disc. The choice between these two methods depends on the form of the product and other specific requirements for the study.
The potential presence of antimicrobial compounds in the inoculated plates will be determined by evaluating the microbial growth on the plates for zones of clearing. The zone of clearing indicates that no microbial growth occurred due to antimicrobial activity. This clear zone can be measured and compared to other zones of clearing. For example, the area around the perimeter of the soaked disks placed on the plate in Figure 1 show a measurable zone of clearing.
Agar diffusion studies can be a quick and direct screening tool in order to determine if a given antimicrobial may have potential antimicrobial activity for further applications. In addition, the agar diffusion methods are simple and generally cost-effective and are therefore particularly useful when conducting initial investigations to evaluate potential antimicrobials. Results are visible and easy to interpret. However, this method is not appropriate for antimicrobials that cannot diffuse through the agar. In addition, quantitative results are frequently required during further analysis.
Broth Diffusion Method
The aim of the broth diffusion method is to determine the concentration of an antimicrobial that is necessary to inhibit microbial growth. This method also helps to identify the minimum inhibitory concentration (MIC) for the antimicrobial solution and is widely used for liquid antimicrobial products. The procedure includes dilution of the antimicrobial product to various concentrations in broth growth medium, followed by inoculation of each dilution with target microorganism(s). Inoculated broth is then incubated within tubes or in a 96-well plate, and then evaluated for growth by checking for turbidity or using a color indicator.
A variation of the broth dilution method is to monitor growth real-time during incubation using a spectrophotometer. For this approach, the optical density (OD) value can be continuously measured during the incubation period to allow for real-time monitoring of microbial growth. The equipment can record OD values every few seconds to generate a detailed growth curve. This variation of the broth dilution method is advantageous because it provides data in the form of growth curves, it is possible to efficiently test for multiple variables at the same time, and results may be more precise compared to traditional methods. The primary drawback to this method is that you cannot differentiate between live and dead cells with the spectrophotometer, so while you can generate growth curves you cannot quantify die-off of the target organism(s). In addition, some microorganisms may not be suitable candidates for this method due to their tendency to form cell aggregations or biofilms which interfere with accurate spectrophotometer readings.
The following is an example of a study utilizing the broth diffusion method. A total of twelve preservative variables were evaluated in their ability to prevent Escherichia coli O157:H7 growth using the broth dilution method with a spectrophotometer. The continuous measurement of optical density generated detailed growth curves in addition to providing OD values at the endpoint for each variable. The data was useful in comparing the efficacy of each variable to prevent the growth of E. coli O157:H7. Figure 2 demonstrates how some preservative variables resulted in delayed growth (longer lag phase) and lower OD values (less overall growth) as compared to the unpreserved control variables. These growth curves can provide valuable insight into which preservative variables may be most promising for future applications.
Time-Kill Test Method
The time-kill test method may be used to assess the ability of a given antimicrobial to reduce the population of a target organism over time. By incubating the target organism in the presence of the antimicrobial and then sampling at various time intervals, we can determine the numbers of viable cells present at each sampling time and generate a curve to visualize target organism “die-off”. For this reason, the method is often referred to as a “die-off” study.
For example, in a time-kill study assessing a preservative's ability to reduce populations of Salmonella and lactic acid bacteria, the method allowed for precise observation of population declines over time. The study demonstrated significant reductions in microbial populations for several preservative conditions, as shown in Figure 3.
Challenge Studies
Challenge studies are designed to evaluate the real-world effectiveness of a preservative in an actual food or nutritional supplement product. These studies involve formulating the product with the preservative, inoculating the product with the target organism, and then sampling at intervals over the storage period to determine if the target organism population declines or increases.
Challenge studies provide a realistic evaluation of the preservative's performance under practical conditions. Although they are often more expensive and time-consuming than the other methods described, they offer valuable insights into how the preservative will perform in the actual product. The product formulation, intrinsic product factors such as pH, and the manufacturing process of the product may all influence how effective a preservative is in an actual food product.
For example, a challenge study was conducted to evaluate the fate of yeast in various products formulated with the same preservative level (Figure 4). Yeast growth was inhibited in Product 1 and 3, but yeast growth was observed in Product 2 and 4 under the same conditions (Figure 4).
How to Customize Studies to Meet Specific Requirements and Objectives
Designing antimicrobial testing studies to align with the specific needs of a product requires a thorough understanding of the product itself, its manufacturing process, and the client's objectives. Screening tests using the agar diffusion method, broth dilution method, or time-kill curve method can be a useful initial approach to assessing antimicrobial activity of potential candidate preservatives. These screening tests are especially valuable when considering multiple preservatives or variations in usage levels, as they provide a preliminary assessment of antimicrobial efficacy.
Following successful screening results, a challenge study can be conducted to validate the preservative's effectiveness in the actual product. In order to obtain reliable challenge study results, each individual study should be designed considering the client objectives and the expected storage conditions for the product. Studies must be designed to be scientifically sound by adhering to established in-house developed methods and/or peer reviewed literature sourced methods and should include such details as the use of bacterial strains that were isolated from similar products, if possible, appropriate microbiological media, appropriate positive and negative controls, and the appropriate number of replicates for proper data analysis and comparable results across different studies. Moreover, a well-designed challenge study should incorporate flexibility, allowing the study design and testing parameters to be reassessed in response to initial observations. This adaptive approach ensures that unexpected findings are addressed appropriately, optimizing the study to achieve the best possible outcomes.
Questions on how to apply these tools to your food product evaluation?
