Testing Dietary Supplements in the Face of Unpredictable Microorganisms

Take a moment and think about the diversity of the human race. The world population is just under seven billion, and every day we encounter people who are incredibly unique in their appearance, disposition, and abilities. Like humans, microorganisms are diverse and constantly changing. Even though an organism may belong to a single bacterial strain, there is still diversity within the community.

Microorganisms, like us, are constantly rearranging their genetic code to survive-and, like us, microorganisms in stressed or injured states may act differently than expected.

All of this unpredictability can make testing a product for microorganisms quite challenging and perplexing at times. Seasoned microbiologists often field questions from puzzled customers, such as “Why are the results of my testing different this time?” or “We have tested this product hundreds of times and have never seen this issue before.” And, perhaps the most deviling question: “I thought this was scientific; why does it work one time and not work another time?”

If our environment remained static and unchanged, we could reasonably expect the same microbiological method to yield the same result for the same organism every time. But, as change is constantly occurring, this simply is not the case.

This article will explore some prevalent and common testing challenges often encountered in today’s microbiology laboratory.

Suspect Results

Let’s take a step back and consider how a nutritional product or dietary supplement is made and then tested. Every week, a manufacturing plant churns out several thousand pounds of product. From this production lot, a laboratory-sized sample is taken and sent off for testing. At the laboratory, the size of the sample is reduced further to an amount required for testing. This small sub-portion of product is most often treated with broth, in order to promote growth of target organisms.

Following incubation, a very small amount of this broth-often less than a milliliter-is transferred to different testing stations, each of which is specialized to detect a specific organism of interest. Depending on the test results, it’s this small sample of the original production lot that could determine whether a product will be deemed safe to release for sale-or contaminated and thus destroyed.

Imagine if, based on this small sample, a call from the testing lab said that a suspect result was generated and that additional testing is required. And, a few days later, additional testing rendered a negative result. This is a highly frustrating situation for a manufacturer. Naturally, the manufacturer will want to know what occurred to cause the initial result to be suspect. Let’s look at some of the reasons why negative results can occur.

The Element of Surprise

To start with, it’s not uncommon for another microorganism-aside from the microorganisms you expect and are testing for-to be present in a product sample. This unexpected microorganism may thrive under the same conditions as the target organisms and, as a result, will grow readily during the incubation period.

Going further, however, by a twist of fate, this “surprise” microorganism may produce the same reactions as the target organism; thus, during testing, effects that are due to the “surprise” microorganism may instead be attributed to the target organism. Why? Because although a test is generally meant to only detect specific organisms by “selecting” against other organisms, testing methods cannot possibly detect all of the millions of different microorganisms that exist. There are simply too many. Moreover, because microorganisms are diverse and constantly changing, test methods may not be able to detect new reactions that were previously unknown.

Here’s an example: several years ago, I was charged with investigating a microbiology issue wherein a screening test indicated the potential presence of pathogens in a plant sample. However, further investigation revealed that the organism identified was not a pathogen; rather, it was a microbe commonly found on the plant’s leaves. Obtaining this information allowed our company to act appropriately in handling and testing the product to ensure a safe product and reliable test results.

If you get a suspect result after testing, various methods, including cutting-edge DNA detection methods, can be employed to strenuously determine the cause. Once this information is obtained, additional research should be performed to find the source of the microorganism. This source could be a naturally occurring organism from a raw ingredient or an environmental contaminant. Collecting this information can be useful in reducing or eliminating the problem.

Inhibition of Microbial Growth

“Inhibition” is another prevalent area of concern in the microbiological laboratory. This can be caused by the sample itself or by other microbes in the product that inhibit the growth of the target organism. Many dietary supplement ingredients and finished formulations can inhibit microbiological growth under standard laboratory conditions.

You might ask, “If a product inhibits microbial growth, isn’t that a good thing?” Not necessarily. Some organisms can survive under harsh conditions and even form spores that wait for the right conditions to start growing and multiplying. If, under testing conditions, a product sample inhibits growth of these bacteria or spores, it could potentially mask the contamination that will eventually take place under the right conditions.

The only way to accurately determine whether your sample is inhibiting bacterial growth is through proper testing and verification to determine whether target organisms are able to be grown under the conditions of the test. If E. coli were present in a sample, for instance, would a test be able to detect its presence? Or, would the sample “inhibit” its detection? Proper testing of this sort can be time-consuming and requires proper laboratory techniques and training to ensure it is performed correctly. Verification testing can be done by preparing a sample in an appropriate broth. Then, the sample is inoculated with target organisms (Salmonella, Staphylococcus, E. coli, Pseudomonas, etc.).

The bottom line is that target organisms should be able to be detected; otherwise, it means that a sample is inhibiting their growth and could be masking the contamination present. And if your product is inhibiting microbial growth under standard testing conditions, costly contamination issues could go undetected.

Testing Methodologies

GMPs for dietary supplements specify that test methods should be scientifically valid. What makes a method scientifically valid is its ability to repeatedly deliver reliable results. There are many validated methods available to supplement manufacturers with which to test their products. However, many times these methods may be validated to food products, and not necessarily to dietary supplements. For instance, many microbial testing methods have been written for very specific products such as eggs, meat, milk, nuts, etc. These testing methods were not written with dietary supplements in mind. And “dietary supplements” encompass such a large scope and variety of products that it’s risky to simply assume that a method written for, say, eggs will also work for supplements. Dietary supplement manufacturers should choose from AOAC, pharmacopeia, compendium, or other such validated method sources-but they must, again, verify that the methods chosen do work for their products.

Often, one method will work for one product but not for a different product.  Petrifilm methodologies are excellent because they are relatively easy to set up and the interpretation of results is straightforward. However, Petrifilm should not be used on every sample type. For instance, it is difficult to see microbial growth on samples that are dark in color, and many common dietary ingredients are dark-colored.

Additionally, Petrifilm methods require that samples be within a specific pH range; otherwise, results will not be valid. Many dietary ingredients have either a naturally high or low pH. Minerals, such as calcium carbonate and magnesium oxide, have high pH levels, and a sample preparation may need to be pH-adjusted in order to use Petrifilm methods. (pH can be adjusted by adding acid or base to the sample in order to bring it into a neutral range.) Also, some of these ingredients are so extreme-high or low-in their natural pH value that Petrifilm should not be used for testing.

With this in mind, a variety of methods should be taken into consideration for testing dietary ingredients. Extensive testing expertise and experience are also needed to know which methods are appropriate for certain ingredients.

Expect the Unexpected

In a perfect a world, microbiological results would be a certainty. Testing advances have drastically reduced the element of surprise in microbiological testing. But in dealing with organisms that are constantly evolving and changing, the apple cart can be overturned at the most unexpected times. When this occurs, it is essential to have the knowledge and expertise to get back on solid ground.