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How to Recognize and Avoid Non-Microbial Enzyme Activity in Foods

Report of Galactosidase Activity in Cheese Particulates in Inoculum Plated in a Chromogenic Enzyme Substrate Medium, with Additional Notes on Shellfish, Tomatillos, and Tart Cherries — Jonathan N. Roth, Ph.D., Micrology Laboratories, LLC, Goshen, IN.

Micrology Laboratories L.L.C. develops and manufactures rapid methods microbiological test media which are widely used for QA/QC analyses in the food and beverage industry. It is the original source of chromogenic media which contain a combination of more than one insoluble chromogenic enzyme substrate. Their patented Coliscan® media contain chromogenic enzyme substrates for β-glucuronidase (teal green chromogen) and β-galactosidase (pink/red chromogen). (Their ECA Check medium also contains these substrates as well as an additional substrate for Alpha-galactosidase.) Microbial colonies appear in these media as horizontally oriented circular colored dots.

During November 2008, a QA technician from a cheese-making company that was using Micrology Labs’ chromogenic media contacted Micrology Labs and indicated that an abundance of small pink spots developed upon incubation of the chromogenic medium that was inoculated with a Feta cheese sample. (The sample was homogenized by massaging in a diluent.) He was unsure of the composition of the spots that appeared in the medium, but since he was using the ECA Check® medium and the spots were pink, he presumed that they might be bacterial colonies of the genus Aeromonas. At our request, he over-nighted a plate containing the pink particles to Micrology Laboratories. Microscopic examination revealed that the pink spots, which ranged in size up to 2 mm length, were irregularly shaped and did not resemble microbial colonies. Subsequent testing by picking several of the pink particles, macerating them, and then adding them into MRS Easygel resulted in the emergence and growth of many CFUs which appeared to be of two types. Microscopic examination revealed gram positive rods as single or end to end double cells, and gram positive yeasts. These actively growing organisms were inoculated into plates of ECA Check® Easygel® to verify that the medium was inhibitory to their reproduction. The yeasts did not grow at all, and the gram positive bacillus exhibited very slight growth without any galactosidase production (the colonies did not exhibit any color.) These organisms were apparently active in the cheese-making process and remained alive in the finished cheese, but were inhibited by the ECA Check medium so that they did not grow when plated in the ECA Check®. However they were not killed by the ECA Check® and their high concentration in the cheese particles, along with their production of β-galactosidase apparently resulted in the cheese particles in the inoculum assuming a light pink color (positive for β-gal) which was confusing.

This is the first incident involving an apparent false positive reaction for β-galactosidase in cheese that has come to my attention in more than 30 years of working with chromogenic media, but this should not be construed as an indication that this type of reaction is rare. However, armed with the knowledge that β-galactosidase producing organisms such as yeasts and Lactobacillus species are commonly used in producing cheese and fermented milk products, it is surprising that this has not previously been more frequently observed and reported. It is well established that L. bulgaricus and other organisms used in making these products are producers of the enzyme β-galactosidase, which causes enzymatic hydrolysis of the lactose in the product. Therefore, it would be reasonable to expect that this would be a common occurrence when blended or stomached cheese samples containing noticeable particles are used in a pour plate chomogenic method for the detection of E. coli and coliform bacteria.

I am also aware of some additional food materials which can cause confusing, false positive appearing readings in chromogenic media that test for galactosidase or glucuronidase enzymes.

First is documentation that marine shellfish such as oysters and clams commonly possess endogenous glucuronidase in their tissues. This means that if a test sample containing such tissue is contacted with a chromogenic medium designed to indicate the presence of the enzyme glucuronidase, the tissure will give a positive color reaction.

A second example, which I encountered personally this past summer (2008), was a case where an organic vegetable grower observed teal/blue spots in plates of our Easygel® chromogenic media when assaying samples of tomatillos. The grower, upon seeing these blue spots, sent samples of the tomatillo to a very well known testing laboratory which reported gross contamination by E. coli.

 Upon further personal investigation of the tomatillos, I discovered that the teal/blue spots were particles (fragments) of tomatillo tissues (fruit and husk) that had been produced in the stomaching or blending of the sample preparation and that the fruit was negative for E. coli and other coliforms. In this case, the enzyme produced was β-galactosidase.

Finally, in October 2012, an email from a food processor was received that contained a photo attachment of a dish of our ECA Check Plus medium that contained many colored particles. The QA laboratory manager did not believe that the particles were bacterial colonies, but wondered if there was some plausible explanation for the appearance of the colored particles. The product that had been tested was Tart Cherry Puree. At our request, a sample of the product was sent to Micrology Laboratories, where it was plated on media containing individual chromogenic enzyme substrates. In our Coliscan Plus medium which contains only enzyme substrates for β-glucuronidase and β-galactosidase , there was no change in the normal red tissue particles. However, in our ECA Check Plus medium which contains enzyme substrates for β-glucuronidase, β-galactosidase, and α-galactosidase, the particles turned a dark green color within 1-4 hrs even at ambient temperature. This verified that the cherry tissue particles contained the enzyme α-galactosidase (see photo). This presence of this enzyme in a plant tissue is a new record for us as we have never had this brought to our attention previously.

These examples of unusual reactions from foods are no reason to abandon the very excellent rapid test methods built around the use of chromogenic enzyme substrates. First, this occurs among very few foods. Second, the problem is easily solved and neutralized.

Even if the situation occurs, it is obvious to the experienced technician that the colored particles are not microbial colonies. The colored particles are variously sized and irregularly shaped, while microbial colonies are generally similarly sized and regularly flattened with a round or oval profile. Examination at 10-40X further clarifies this observation. If such a situation is observed, it is simple to check your plates at different time intervals and ascertain that the color is not microbial.

First, observe your plates after they have been inoculated and solidified to see if there are any visible particles in the medium and, if so, whether they are colored and what is their shape and size. If there are visible particles check the plates after 2-4 hrs incubation time. If the particles have acquired color due to the chromogens in the medium, they cannot be microbial colonies as the colonies could not grow to a visible size that fast (2-4 hr). Also, the particles will still be the same shape and size.

Further, more defining, tests may be done such as those in the case of the cheese. In this case, there was no apparent microbial colony formation, but it was possible that there could be living organisms other than the E. coli or coliforms for which the media are specific. There could be other types of organisms in the tissue that did not grow due to inhibitors in the specialized medium. Therefore, the transfer of several macerated particles to MRS medium determined that in this case living lactobacillus and yeast types were present in the cheese, but did not grow in the first medium. However their presence would be considered normal in cheese and not a problem. Had the cheese particles contained E. coli or coliforms, there would have been bacteria released in the macerating of the cheese and typical colonies would have grown along with the coloring of the particles containing them. In this case, it is doubtful that the cheese itself contained the β-galactosidase, but that it was associated with the bacteria and/or yeasts used in the cheese-making and retained in the finished product.

Also, if you suspect that a product may contain endogenous enzymes, aseptically deposit thin slices or tiny samples of the product in a plate of the chromogenic medium you are using and observe them. If they do contain endogenous enzymes, they will generally turn color within 1-2 hours incubation, even at ambient temperature.

If you have a product which you know contains endogenous enzyme, but still wish to test for specific bacteria, you might also consider adding some of the inoculum into an enrichment broth and incubating for several hours, and then using some of the enriched material as an inoculum. This will dilute any effect of endogenous enzyme in the product itself while giving a larger population of the bacteria which were present in/on the product.

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