Microbiology of Dietary Supplements

Official Methods for Dietary Supplements

The USP methods for dietary supplements include:

USP <2750> Manufacturing Practices for Dietary Supplements: Contains the official FDA ruling on dietary supplement cGMPs (21 CFR Part 111).

USP <2021> Microbial Enumeration Tests-Nutritional and Dietary Supplements: Enumeration of the total number of aerobic bacteria, enumeration of the total number of combined yeast and molds, and the presence of bile-tolerant gram negative bacteria present in all nutritional supplements, from raw materials to the finished product.

USP <2022> Microbiological Procedures for Absence of Specified Microorganisms-Nutritional and Dietary Supplements: Procedure for determining the absence of specified objectionable organisms. Good manufacturing practices require that objectionable organisms be absent from non-sterile nutritional and dietary products. Objectionable organisms are defined as microorganisms that may cause potential health hazards to the user of the product and would adversely affect the product safety.

USP <2023> Microbiological Attributes of Non-sterile Nutritional and Dietary Supplements: contains information about frequency of microbiological sampling and testing, recommended microbial limits for botanical ingredients and products, and finally, recommended microbial limits for dietary supplement ingredients and products. However, specifications are typically are developed by the manufacturer with guidance from USP <2023>.

USP <1223> Validation of Alternative Microbiological Methods:  Any alternative method must be validated against these UPS methods using the methodology described in USP <1223> including side-by-side testing, determination of the accuracy, specificity, limit of detection, robustness, ruggedness, etc.

Many methods validated for food products, are not necessarily validated or appropriate for dietary supplements. For example, many methods have been written for very specific products such as eggs, meat, milk, nuts, etc. These methods were not written with dietary supplements in mind. And “dietary supplements” encompass such a large scope and variety of products therefore, it is risky to simply assume that a method written for example for eggs will also work for supplements.  Therefore, a method having AOAC certification is not necessarily appropriate for dietary supplements.  AOAC had not certified any microbiological method for dietary supplements, all their methods are certified for food only.

Often, one method will work for one product but not for a different product. Petrifilm methodologies are excellent methodology for many food products. However, Petrifilm should not be used on dietary supplement samples without thorough validation. For instance, samples that are dark in color and many common dietary ingredients might make it difficult to see growth on the Petrifilm. Viscous products such as Psyllium begins to harden before an appropriate spread on a Petrifilm can be obtained.

Product Interference or Matrix Effect

Spirulina, turmeric, Psyllium, honey, Calcium carbonate, dyes, and oils: these are just a few of the many products that the Nutraceutical industry works with, and struggles with. Many botanicals, herbals, enzymes or protein powders create challenges for microbiological testing (Tackling those challenging Nutraceutical Products with Rapid Automated Microbiology Testing!, C. Ockerman 2013). Granules (which can often be mistaken for a bacterial colony), extreme pH, viscous products can all interfere with the plate count methodology.

Microbial Testing of Dietary Supplement

Microbiological testing in the dietary supplement industry consists according to the USP chapters, of monitoring levels of naturally occurring indicator organisms and spoilage organisms, as well as the absence of objectionable organisms (e.g., Salmonella, Staphylococcus aureus, and E. coli) in finished products.

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

• Total Plate Count

• Yeast and Molds

• Enterobacteriaceae (gram negative bile tolerant bacteria)
• E. coli
• S. aureus

• Salmonella

• Pseudomonas (for liquid products or products with high water activity, or topical/cosmetic products)

How to Test for Microbiology

Many companies outsource the microbiology testing to third party commercial testing labs instead of performing it in house.  Some companies fell that whenever products are sent out for testing quality results cannot be questioned, but time is sacrificed and a premium price is paid for the service. Therefore, companies tend to only bring their microbiological 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.

In house Laboratories can perform using the USP methodology.  In preparing its own media and reagents the lab needs to pay attention to USP <1117> (Good Microbiological Laboratory Practices).  Because in an internal laboratory the quality of the results depends on the quality of the culture media prepared.  This includes accurate weighing of dehydrated components, the use of high quality (USP Purified) water, completely dissolving the dehydrated media or individual ingredients, and the need to control the heating of the media to avoid damaging heat-labile components of the media. The quality control of each batch of the media is a critical concern.

It is important to take production timelines into consideration when determining what methods fit each company individual needs.  A major issue with microbiological testing is the hidden costs in labor and inventory hold time.  The one assay that often keeps a quality control manager up at night is their yeast and mold testing—which takes five days to obtain a result.

Why Automated Rapid Microbiology?

Internalizing the microbiology testing and especially adopting rapid microbiological methods (RMM) can speed up the time to results from 7-10 days to 24-48 hours. The time saved has a direct and REAL impact on many key operational profit drivers.  The Advantages of RMM can include: Greater accuracy, better sensitivity, and increased sample throughput, automated data capturing and allowing easier data handling, and reduced cost for product release.  Rapid methods allow test results to be obtained faster. More importantly, if a product is above a desired microbiological specification, the results are obtained a lot sooner, and corrective action can be taken days before traditional methodology would allow.

All analytical methods need to be validated prior to their introduction into routine use, and this is especially true for novel technology platforms, such as rapid microbiological methods (RMMs). Because many RMM technologies consist of a combination of instrumentation, software, consumables and reagents, in addition to specific detection, quantitative or identification methodologies, it is important to develop a comprehensive and holistic approach to the validation process to ensure that the entire RMM system is suitable for its intended use.

The BioLumix Advantage
The BioLumix System is a system that fits a need in the Dietary supplement industry in terms of speed, working with all product matrices, and automatically documenting the results in real-time, thereby reducing the labor requirement for microbiological testing.

The BioLumix system was extensively validated for dietary products and compared to the methodology described in USP <2021> and <2022>.  The validation methodology followed USP <1223> “Validation of Alternative Microbiological Methods”.  This included performance criteria such as: side-by-side comparison to the USP methodology, accuracy and precision, specificity,  limits of detection and quantification, along with ruggedness and robustness, reproducibility of the results, false positivity rate and false negative rate.

A full validation package including Installation Qualification (IQ), Operational qualifications (OQ) and Performance qualification (PQ) is included with the BioLumix system
The BioLumix Advantages include:

  • The only system with all assays on a single platform
  • Real-time communication – early warning of contamination
  • 48-hour Automated Certificate of Analysis
  • Simplified testing with ready-to-use vials
  • Each vial comes with a certificate of analysis, negating the need for QC of the vials
  • Validated for dietary supplement products – IQ, OQ, PQ
  • No product interference
  • 21 CFR Part 11–compliant software
  • Remote-access customer support 24/7/365

BioLumix RMM platform featured in Latest Edition of NutraCos Journal

Introduction

The BioLumix platforms Rapid Microbiological Method (RMM) was recently featured in NutraCos journal for its ability to offer a multitude of unprecedented advantages to companies, as well as its versatility across various industries. NutraCos is an international journal that features articles on everything from nutraceuticals and probiotics, to regulations and policy.1 Given the rapidity of technological advancement, and the complex structural nature of the cosmetic, OTC and nutraceutical industries, many companies and individuals utilize NutraCos as a way to stay current with the latest developments throughout the world. In the journals January/April 2013 edition, our President, Dr. Ruth Eden, and Senior Research Microbiologist, Roger Brideau, explain the technology, as well as the benefits of the BioLumix automated microbial testing system to an international audience.2

 

Why Use Rapid Microbiological Methods?
Rapid microbiological methods are technologies that allow their users to get microbiological test results faster compared to traditional USP methodologies, which use plates and require a minimum of two to seven days in order to achieve complete results.  The BioLumix system can report results within hours, and all final results are available within 48 hours. This can dramatically impact key profit drivers by eliminating the wait for microbial results that can tie up working capital and often results in storage expenses and delays in supplying products to the market. Essentially, inventory stays on the floor instead of going out the door to the customer/patient population.  A shortening of the length of the manufacturing cycle means less required working capital investments, and simplifies rapid microbiology.

RMM Using BIOLUMIX
The systems three components consist of a modular instrument, disposable vials, and a 21 CFR Part 11 compliant computerized software package, the latter of which provides the user with an audit trail, operator identification, a data management system, and multiple customizable report formats. This process functions by continually monitoring the changes of a broth medium in which target organisms grow. The BioLumix system uses a CO2 sensor at the top of each disposable vial to detect released CO2, which is produced by all microorganisms, as well as reagents at the bottom of each vial, which measure changes in color/fluorescence of the broth medium by way of light intensity units in response to microbial metabolism.

 

The BioLumix system is extremely versatile and is capable of performing most microbiological assays tests, such as those for microbial content (microbial enumeration of total counts and yeast and molds), objectionable organisms, suitability testing and preservative efficacy testing.

A suitability test, to show usage of an adequate neutralizer inactivates the preservative in the product being tested. This demonstrates that if organisms are in fact present they will be able to grow and be detected. The suitability test can be performed very simply and quickly with the BioLumix system.

Testing of any cosmetic, pharmaceutical or personal care product should include a survey for contamination often referred to as testing for Microbial Content, which uses assays designed to measure the growth of bacteria, yeasts and molds to flag products for the presence of contaminants.3, 4 For most cosmeceutical products, regulations state that total aerobic count should be <100 cfu/g, and that yeast and mold counts should be <10 cfu/g. The workflow of the testing of a personal care sample for the presence of microbial contamination is very simple and requires a fraction of the time and labor needed to perform the same tests using the USP methodology.  Each product can also be assayed specifically for the detection of E coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species.

Upon determination that a product has been properly neutralized and has very low levels of contamination, the user can conduct a Preservative Efficacy Test (PET), sometimes referred to as the Antimicrobial Efficacy Tests. This test is required for all cosmetic and personal care products in the United States and follows the guidelines set forth by USP 51.5 Briefly put, the product must be challenged within individual containers, each going through this process separately using one of the five required organisms, which include ATCC established strains of E. coli, S. aureus, P. auruginosa, C. albicans, and A. brasiliensis. The PET assay measures the reduction of a high inoculum (106-107 cfu/g) in the presence of a product containing preservative over 28 days, which also tests the ability of the preservative to prevent a rebound of an organism (regrowth). After these tests the BioLumix software produces a certificate of analysis with a succinct report of the microbiological properties of each product for use at your convenience. This equates to proactive detection of contaminated products, and ensures that minimal resources are spent on production and subsequent recall of said products if they are found to be contaminated.

Conclusion

The BioLumix Rapid Microbiology System can save multiple days of time to reach the assay endpoint versus traditional plate methods.  This time saving could dramatically impact key profit drivers, such as elimination of idle time with raw materials, work in progress, and speed to finished goods. Faster detection of microbial contamination also results in faster corrective action in the event that material is above the targeted specification level. Earlier awareness of contamination problems in production runs can result in less production of defective final finished goods. By significantly decreasing the manufacturing cycle, one can lower their required working capital investment.

The BioLumix instrument is a straightforward and easy to use system that allows for rapid and accurate results.  The system is fully automated including data archiving, data maintenance, and report generation. It is paperless and efficient; saving on disposables, time and space. In short, BioLumix streamlines microbiological testing, delivering a simpler, faster, and automated solution saving your company significant time, labor and money.

To read the whole article complete with direct comparison tables and figures, visit our scientific presentations page. (http://www.mybiolumix.com/scientific-presentations/)

 

References

  1. Lee, C. “NutraCos,” Vitafoods Europe. London, UK: Informa Exhibitions, 2013.
  2. Mori, F. L. “NutraCos,” Via M. Donati 6. Milano, Italy: B5 s.r.l., 2013.
  3. United States Pharmacopeia, Chapter <61>, 2009.
  4. United States Pharmacopeia, Chapter <62>, 2009.
  5. Moser, C.L.; Meyer B.K. AAPS PharmSciTech. 2009, 12 (1), 222-6