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    Microbial Testing

    The nature of dietary supplements presents a variety of microbial testing challenges.

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    Jason Lilly & Tony Crosariol05.01.07

    Microbial Testing



    The nature of dietary supplements presents a variety of microbial testing challenges.



    By Jason Lilly, PhD, MBA, Director of Market Development
    & Tony Crosariol, Sales Development Manager—Nutraceuticals Market
    Neogen Corporation
    Lansing, MI



    The recent recall of spinach, peanut butter, and associated media fervor, shows what can happen to manufacturers of consumables when contaminated product reaches consumers. Although regulators currently treat the dietary supplement industry differently than the food industry, consumers hold manufacturers of all consumables to the same safety standard—unerringly safe.

    To reach this safety standard, the dietary supplement industry’s vast and varied array of raw ingredients presents challenges unseen in the food industry. To meet consumers’ demand of unerringly safe products, dietary supplement manufacturers must adopt safety and testing strategies that address and overcome these challenges.

    Testing Challenges



    While many conventional chemistry methods work just as well for dietary supplements as they do in food commodities, this is not the case for microbiological tests. The nature of the product to be tested (also called “matrix effect”) is a huge issue for manufacturers of botanicals, herbals, enzymes or protein powders.

    The term “matrix effect” can be broken down into two distinct categories:

    • Matrix inhibition. Matrix inhibition refers to the inherent anti-microbial nature of some products. This can cause inhibition of microbial growth at low dilutions during conventional plating analysis, resulting in artificially low counts.

    • Matrix interference. Matrix interference presents a situation where a sample’s opacity or dark color can obscure the visibility of colonies on the lower dilutions. Conventional microbiology cannot mitigate the issues surrounding heavily dyed compounds, granules (which can often be mistaken for a bacterial colony), extreme pH, or other anti-microbial components of a sample. Even rapid “ready-to-use” plates or agar films, while simplifying the users workflow, do not work with many of the botanicals, minerals and extracts that must undergo analysis.

    “We were working with one customer who was using a ready-plate method for a red-wine extract, and we quickly determined that this method was not suitable due to the fact that even when highly diluted, the matrix colored the agar so dark we could not count individual colonies,” said Jeff Demey, marketing manager at Neogen Corporation.

    It is also important for quality control managers to recognize that when performing validations not to simply just do spike and recovery studies. Using naturally contaminated products is best because that specific microorganism has adapted to the product and the environment and will respond differently than a lab strain under study.

    Dietary Supplement Testing Strategies



    Microbiological testing in the dietary supplement industry, whether it is performed in-house or by a third party, typically consists of monitoring levels of naturally occurring indicator and spoilage organisms, as well as ensuring that no food-borne pathogens (e.g., Salmonella, Listeria, and E. coli) are present in finished products.

    The typical battery of microbiological analyses for a dietary supplement includes:



    • Total Plate Count (a.k.a., Total Viable Count)
    • Coliforms (European and Asia/Pacific regions use a broader classification of Enterobactericeae)
    • Total E. coli
    • Yeast and Molds
    • Staphylococcus
    • Salmonella
    • Pseudomonas (for products with high water activity, or topical/cosmetic products)

    Microbiological testing typically involves:



    1. Sampling of raw materials or individual lots of finished product;
    2. Dilution of samples to reduce possible matrix effects and prepare samples for microbiological analysis; 3. If the specification for the organism to be tested is “absence in a specified quantity,” then a pre-enrichment is conducted to allow possible contaminating organisms to recover, or mitigate any preservatives that may inhibit organism growth; and
    4. Some form of endpoint analysis, which may include conventional plating, rapid agar films, automated optical readers, or ELISA- or DNA-based diagnostic detection systems.

    “The foundation of industrial microbiology is to utilize a methodology that not only fits your workflow and budget, but ensures the detection of the proverbial ‘needle in the haystack.’ That is the essence of dietary supplement microbiological testing,” said Mr. Demey. “Due to the anti-microbial nature of many of the dietary supplements, it is especially essential with these products to provide any injured organisms the time to recover and proper environment for growth, so a potentially contaminated product can be detected before it is released to consumers.”

    The last major issue with microbiological testing is the hidden costs in labor and inventory holds. With so many advances in chemical analysis technology, the assay that often keeps a quality control manager up at night is their yeast and mold testing—which can take five days to produce a result. It is important to take production timelines into consideration when determining what methods fit your company’s individual needs.

    “These long holds can literally cost a company thousands of dollars per year,” said Mr. Demey. “Many companies try to save time by reading tests earlier and releasing, but we often receive phone calls from companies asking about new rapid microbiological methods because the cost of testing is minuscule compared to the cost of a recall or reworking a large volume of finished product.”

    Many companies outsource to third party commercial testing labs instead of performing microbiological and chemical analysis, determining optimum methods, and completing all the paperwork by themselves. While this practice is common in the food industry, it can be rate limiting in the dietary supplement space. Whenever products are sent out for testing quality results are guaranteed, but time is sacrificed and a premium price is paid for the service. So, companies tend to only bring their diagnostic testing in-house when they reach a critical mass such that the cost of sending out is equal to or more than starting up a laboratory.

    Bringing Microbiological Testing In-house



    Laboratories need to be cognizant of the needs when setting up a new lab. Some rapid methods for indicator organisms do not require large volumes of sterile media or special precautions; however this is different when dealing with pathogen testing. Salmonella and Listeria analysis of final producttypically involve a cultural broth pre-enrichment step, as the requirements for these harmful organisms are absence (e.g. zero bacteria) in a 25-gram sample.

    The enrichment scheme can be as short as 24 hours, but for the complex nature of nutraceuticals, it is recommended that customers stay with more traditional 48-hour methods, as too many false negative results can occur when trying to squeeze the pre-enrichment timelines. These enrichments typically involve large volumes (225 mL) of media that must be prepared by the customer, as well as the proper biosafety requirements.

    “We need to determine the need and comfort level by individual customer,” says Neogen’s Jake Knickerbocker, microbiology group leader. “By having worked with numerous customers our strength is in advising the customer on what makes both biological and economic sense given their situation, and recommending a variety of options. While we service the nutraceutical industry as a whole, we understand it is a customer of one when comes to fitting products to needs.”

    Neogen has made much progress in enabling dietary supplement companies to speed up their microbial analyses of complex materials with an assay system for indicator and spoilage organisms that has been widely adopted by numerous members of the elite, “Top 100” dietary supplement companies. The Soleris System (pictured at the top of this story) is something that fits a need in the industry in terms of speed, working across a variety of product matrices, and automatically documenting the results in real-time, thereby reducing the labor requirement for microbiological testing.

    Neogen has validated more than 400 different food, nutraceutical, and cosmetic commodities on the Soleris system across a variety of microbiological tests—only a half-dozen have not correlated with USP or FDA/BAM cultural methods. The system is also unique in the marketplace in that it utilizes traditional culture media coupled to a pH color indicator, in a patented vial system and reader which allows for rapid (often same shift) results for indicator and spoilage organisms.

    “This system was designed specifically to enhance supply chain efficiency,” said Neogen’s Mr. Crosariol. “Real time detection allows early alert to a positive, out of specification product, which allows your production to react faster.”

    Nearly all of Neogen’s clients are passing 95-99% of their products as negative to their product’s necessary specification and can be released quickly. Rapid methods allow that to happen faster. More importantly, if a product is above a desired microbiological specification, it can be known about sooner, and corrective action can be taken days before traditional methodology would allow.

    Conclusion



    In summary, microbiological analysis of nutraceutical products is more complex than traditional food microbiology; however, this does not mean it should be avoided. Traditional methodology or its variants must be closely examined for the applicability and limitations given the product mix to be tested. Likewise, rapid methods, which are a necessary for this ever-growing industry must be robust in their performance across a variety of matrixes.

    “As with all technical applications, the QC manager must exhibit some level of caveat emptor, as few diagnostics suppliers have experience and firsthand nutraceutical industry knowledge,” said Mr. Crosariol. “Neogen is lucky to have a deep customer base in dietary supplements, including NSF International, which has allowed us to first build our own tactical knowledge, and then share it with our clients.”

    About the authors: Jason Lilly is the director of market development for Neogen Corporation, Lansing, MI, and Tony Crosariol is sales development manager-Nutraceutical Market. They can be reached at 800-234-5333, E-mail:jlilly@neogen.com; Website: www.neogen.com.

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