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

Microbiological Testing of Enzymes

What are Enzymes?

Enzymes are specialized proteins that accelerate the rates of chemical reactions by lowering the activation energy; the substrates are converted into products.  There are numerous different types of commercially available enzymes such as protease, amylase, cellulose and lipase.

Where are they used?

Many different industries use enzymes in the production of their products.  They range from animal nutrition, brewing industry, food processing, detergents, dairy industry, paper industry, biofuels, contact lens cleaners, photographic industry, carbohydrate processing and biochemicals.  Enzymes are also an integral part of the pharmaceutical and Nutraceutical industry.  In laundry detergents enzymes that degrade proteins causing stains, such as those found in grass stains, red wine and soil can be used.

Lipases are another useful class of enzymes that can be used to dissolve fat stains and clean grease traps or other fat-based cleaning applications.  Nutritional enzymes have risen to prominence during the past several years, driven by customers that would like to improve their digestive health. The biggest category for supplemental enzymes is still human digestion, an area that has become increasingly more important for maintaining optimum health. Supplementation of single enzymes, such as lactose to treat lactose intolerance, is probably the most familiar area of enzyme use. However, general dietary supplementation with a broad range of digestive enzymes is becoming increasingly more common. Supplemental enzymes can also be used to enhance the effect of herbs and botanicals. The active component of herbs and other botanicals can remain trapped within the cell wall matrix or may be in a form that is not easily broken down and absorbed. Select enzymes are added to these products to break open certain bonds to free the bioactive compounds.  The application of enzymes technology to pharmaceutical products is also a growing field.

How do they Work?

Amylases in the detergents can help by aiding in the breakdown of starch stains.  Also in the biofuel industry cellulases are used to break down cellulose into sugars that can be fermented, and then further processed to ethanol.  Trypsin is used in baby food to predigest the food for the babies.   Proteases can also be used in contact lens cleaners to remove proteins on the lens.  The figure below show a schematic diagram of how enzymes work:

Most enzymes require microbiological testing similar to other industrial products.   BioLumix has an easy method to complete this goal.  The versatility of the BioLumix system allows for a wide range of enzymes to be tested for a broad array of microorganisms; such as Total Aerobic Count, E. coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa, Lactic Acid Bacteria, Coliforms, Enterobacteriaceae, and Yeast and Molds. Liquid products allow a direct sample to be pipetted directly into the vial, while solid samples require a dilution step first.

The BioLumix Rapid Automated Method:

Enzymes traditionally can be very difficult to test due to their unique properties.  The two most common difficulties when working with enzymes is solubility and pH levels of the product.  When using the BioLumix system, pH is adjusted once the product has first been diluted at 1:10 in TSB or buffer during the preparation before the sample is placed into the vial.  Solubility issues can be solved in a few different ways. One way is by allowing the product to sit in solution and give it time to breakdown, and another way it to warm the product in a water bath or an incubator.

During enzyme testing at BioLumix, on plates the enzyme created a precipitate that looked very similar to colonies, which could lead to difficulties in reading the plate.  The plate needed to be  sub-cultured to determine if it was growth or just the precipitate.  The precipitate did not interfere with the BioLumix vials and provided a clear answer.  Additionally, the results were obtained two days sooner.  An oil based enzyme can also cause difficulties when using traditional plating methods versus the BioLumix system.  In the BioLumix system the oil does not have an effect on the result and can give a definitive answer.  While on plates, the oils can sometimes appear on top of the agar and give the appearance of colonies.

Another less common issue is gas production by the product when it is mixed with a liquid.  If the product is known to release gas the easy solution is to allow the product to off gas before adding it to the appropriate vial.  Then after an hour of being in the liquid, the sample can be stomached or mixed again to release the remaining gas.  While in traditional methods, the gas production can give false positive results in tests with Durham tubes, which would then require additional days to complete.

BioLumix Advantage:

The BioLumix system is designed to accelerate product release with a simplified, automated approach. This yields fast, accurate, real-time results while reducing costs. The BioLumix offers real-time results of contaminated samples saving hours, possibly days. Completed Total Aerobic Count, Coliform or Enterobacteriaceae results are obtained within 18-22 hours; Yeast and Mold results are obtained within 48 hours; the objectionable organisms can be detected within 24 hours after pre-enrichment. Also these assays are simpler to perform than the standard methods saving time, labor and money. The system is unaffected by product interference, delivering accurate results with faster product release.  When referring to batches of product that will be released days early due to the BioLumix system, which allows the storage and production less time to run and hastens the turnaround time of the product, the overall process of enzyme production will decrease.

Tackling those challenging Nutraceutical Products with Rapid Automated Microbiology Testing!

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.  However, microbiology quality assurance requires any such product to be tested.  Nutraceutical products come in a wide variety of colors and compositions that can make it difficult to process the samples and count colonies on plates.  Some products cause the coloring on plates to be so dark that one cannot see the actual colonies, other samples have particulate matter that resembles colonies or hides them altogether making counting plates time consuming and inaccurate.  The BioLumix vials not only streamline microbiological testing, but also make the challenging samples easier to work with due to the fact that color, particulate matter, and sticky samples do not interfere with the ability of the system to detect growth of organisms. Below are a few examples of samples that many struggle with:


Psyllium is the husk of the seed of a Plantain and is used to improve digestion and cleanse the digestive system; it is an excellent dietary supplement.  However, before it reaches the shelves for sale, the product needs to be tested in the laboratory, to be sure it is free of harmful microorganisms and is safe for consumption.  This particular product is a spongy fiber, and when it is added to an enrichment broth or diluent, it begins to swell, and harden into a sticky substance that is very difficult to pipette and mix with the agar on the plate. Figure 1 shows the lack of mixing of 1.0 ml of a 1:10 dilution of the Psyllium with the agar.

To avoid this sticky situation, necessary dilutions can be performed quickly and added to the appropriate BioLumix vials before the product has a chance to harden.  Even though, the product will become a bit harder after being added to the BioLumix vial, the growth of organisms will not be inhibited.
Spirulina and turmeric – both colorful raw materials (and different dyes used to color capsules) may make it difficult or even impossible to see and count colonies on pour plates as can be seen in figure 2.  Because the BioLumix CO2 sensor is impermeable to liquids and colors, no product interference due to color of the product will impact the results.

Particulate matter is always a concern during sample preparation, and rightfully so.  As shown in Figure 3, it is sometimes impossible to tell the difference between product and colony.  The BioLumix vial is not affected by particulate matter because only CO2 gas can penetrate the CO2 sensor. As a result, there is no product interference from particulate matter present in the sample.

The Problem: The inability to achieve homogenous mixtures, excess color, and particulate matter are three major causes of inaccurate plate count results when testing Nutraceutical and dietary supplement products for microbiology.

The Solution: The BioLumix vial is able to support growth of organisms in a favorable broth environment, and these problems do not interfere with results.   The BioLumix System is an automated microbial testing system designed to accelerate product release with a simplified, approach.  This yields fast, accurate results while reducing costs.  No more waiting days for completed assays.  The BioLumix System brings automated microbiological testing in house with most assay results achieved over night.  It is simple enough to be operated by non-microbiologists, providing accurate data of raw materials, environmental samples, and finished products.

Biolumix Automation Drives Profits

BioLumix Automation Drives Profits!

No longer look at micro testing of raw materials and finished products as a “necessary evil” in remaining cGMP compliant.   Rather, with BioLumix Rapid, Automated Microbiology, you can view microbiology product testing as a position of company strength and competitive advantage!

TIME IS MONEY. . .  we have detailed financial models prepared by our current customers that demonstrate the dramatic impact BioLumix is having when you apply Multiple Days Saved to real profit drivers, such as:  

Biolumix System

  •   Savings as a result of early detection of contamination
  •   Lower cost of inventory
  •   Increase capacity of existing factory and lab
  •   Reduce cost of storage
  •   Lower cost of test related overhead
  •   Savings on cost of capital
  •   Potential improvement in account receivables. . .  all of which directly impact profits!

Automation Drives Profits
The enacted cGMP regulations require manufacturers to ensure that products are free of contamination by objectionable organisms, and that appropriate microbiological testing is conducted.

BioLumix Rapid Microbiology offers a high degree of automation, significant reduction in time to results, reduced staff involvement, and faster product release. . .  all which impact key “operational profit drivers,” such as:

  • Savings as a result of early detection of contamination
  • Lower cost of inventory
  • Increase capacity of existing factory and lab
  • Reduce cost of storage
  • Lower cost of test related overhead
  • Savings on cost of capital
  • Potential improvement in account receivables

 

Contact Us TODAY To Learn More About Our FREE DEMO PROGRAM!

Rapid Microbiological Testing of E. coli with the BioLumix Vial

Escherichia coli are Gram negative rod-shaped organisms found naturally in the lower intestines of warm blooded organisms. Most serotypes of this organism are relatively harmless, making up a small percentage of bacterial colonization in the gut. These serotypes prevent the establishment of pathogenic strains.

E. coli is one of the most common bacteria found in the gut of animals. This includes humans. There are other animal species that contain this organism in the gut including reptiles and fish. E. coli colonizes the gut and can cause infection in the urinary tract and brain stem (meningitis) as well as intestinal diseases referred to as gastroenteritis. There are five classes of E. coli that produce disease. The most serious disease is the Enterohemorrhagic (EHEC) class. These organisms can cause diarrhea distinct from some others (including Shigella) in that there is copious bloody discharge and no fever. The life threatening situation is its toxic effects on the kidneys (hemolytic uremia).

Why Test for the Presence of E. coli?

E. coli is often used as indicator organisms to test the effectiveness of effluent disinfection in a wastewater treatment plant, on animal products as well as in nutraceutical and pharmaceutical products. While these organisms are generally harmless, they do live under the same conditions that human pathogens live. Since we cannot test for every pathogen, we test for easily detectable indicator organisms. The assumption is that if we kill the indicator organisms then we most likely kill the pathogens during effluent disinfection. E. coli has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli.

Current Methodology

The current methodology can take anywhere from 3-7 days and includes multiple broths, agars,
transfers and temperatures. Different methodologies are utilized by the various industries. Below
are some examples:

Nutraceutical and Dietary supplements: The protocol described in USP <2022> requires
absence of the organisms in 10 grams of product. A 1:10 dilution of product is made into TSB or
another enrichment medium. This mixture is incubated for 24 to 48 hours at 30°C to 35°C, and
then 1.0 mL is transferred to 10 mL of MacConkey broth. This mixture is incubated for 24 to
48 hours at 42°C to 44°C. A loop from the MacConkey broth is transferred to MacConkey agar
and the plate is incubated for 18 to 24 hours at 30°C to 35°C. If typical colonies appear, these
colonies are then transferred to Levine Eosin Methylene Blue agar and incubated 24 to 48 hours
at 30°C to 35°C. If none of the colonies exhibit green metallic sheen under reflected light or if
none of the colonies exhibit a blue-black appearance under transmitted light, the sample meets
the requirement for the absence of Escherichia coli. Because results can sometimes be misread
due to interpretation of plates, identification may be run on the sample(s) adding another 2-3
days of testing.

Pharmaceutical: The protocol described in USP <62> again requires the absence of E. coli in
10 grams of product. A 1:10 dilution is made and incubated for 18 to 24 hours at 30°C to 35°C.
From this mixture, 1.0 mL is transferred to 100 mL of MacConkey broth and incubated 24 to 48
hours at 42°C to 44°C. A loop is transferred to MacConkey agar and this plate is incubated 18 to
72 hours at 30°C to 35°C. Growth of typical colonies indicates the presence of E. coli which is
confirmed with an identification test.

Food Testing: The food industry follows Bacteriological Analytical Manual (BAM). In most
cases, the Most Probable Number (MPN) method is utilized. This is labor intensive, multi-
step assay consists of presumptive (in LST tubes), confirmed (in BGLB tubes), and completed
phases (in EC tubes). In the assay, serial dilutions of a sample are inoculated into broth media.
Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other 2
phases of the assay are performed and then uses the combinations of positive results to consult a
statistical table. From this table, the analyst is able to estimate the number of organisms present.
The 3-tube MPN test is used for testing most foods. The 5-tube MPN is used for water, shellfish
and shellfish harvest water testing, and there is also a 10-tube MPN method that is used to test
bottled water or samples that are not expected to be highly contaminated. Positive EC tubes
must be transferred to L-EMB agar and if typical colonies are observed, they must be further
identified.

The BioLumix E. coli Vial

The E. coli vial, or EC vial, is a membrane vial that is monitor by the fluorescent
signal in the instrument. The membrane separates the incubation zone from the
reading zone. If E. coli is present, it utilizes MUG (4-Methylumbelliferyl-3-D-
Glucuronide) through an enzymatic reaction to create fluorescence.
A 1:10 dilution is made in TSB or a similar growth medium and this mixture is
incubated for 18-24 hours at 35°C. From the TSB dilution, 0.1 mL is added to an
EC vial containing the MUG supplement and then tested in the BioLumix instrument. The EC
assay runs for 18 hours in the instrument. An
example of E. coli curves is shown in the graph. If
detection occurs, a confirmation test is performed.
From the time the sample is prepared to the time
the confirmation is complete, the EC test takes
only 2 days, saving valuable time.

The BioLumix assay is much simpler to perform, requiring less labor and disposables as any to
the standard method. It is faster (completed in 30 hours), accurate, automated, and it eliminates
any product interference due to the two zone vial.

References:

USP <2021> MICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY
SUPPLEMENTS
USP <2022> MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED
MICROORGANISMS—NUTRITIONAL AND DIETARY SUPPLEMENTS
USP <62> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TESTSFOR SPECIFIED MICROORGANISMS
Peter Feng, Stephen D. Weagant, Michael A. Grant, 2002. Bacteriological AnalyticalManual BAM). Chapter 4: Enumeration of Escherichia coli and the Coliform Bacteria

BioLumix Staphylococcus aureus Vial Assay

Organism of Interest

Staphylococcus aureus is a major pathogen of concern in infectious disease. This organism group may include drug resistant S. aureus, often defined as methicillin resistant Staphylococcus aureus (MRSA). S. aureus is also an objectionable organism for the dietary supplement and nutraceutical industries.

Backgroud

Wikipedia’s description is as follows: S. aureus can cause a range of illnesses fromminor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis,carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases suchas pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS),bacteremia, and sepsis. It is implicated in skin, soft tissue, respiratory, bone, joint,endovascular and wound infections. It is still one of the five most common causes ofnosocomial infections, often causing postsurgical wound infections. Each year, some 500,000 patients in American hospitals contract a staphylococcal infection.

Methicillin-resistant S. aureus, abbreviated MRSA and often pronounced “mer-sa” (in North America), is one of a number of greatly-feared strainsof S. aureus which have become resistant to most antibiotics. MRSA strains are most often found associated with institutions such as hospitals, but are becoming increasingly prevalent in community-acquired infections. Arecent study by the Translational Genomics Research Institute showed that nearly half (47%) of the meat and poultry in U.S. grocery stores were contaminated with S. aureus, withmore than half (52%) of those bacteria resistant to antibiotics.

Current Methodology for S. aureus

Nutraceutical and Dietary Supplement Products: USP <1222> describes the methodrequired to test for the absence of S. aureus (typically in 10 grams) in nutraceutical and dietary supplement products. The method involves mixing of the sample in TSB and pre-incubating the TSB containing product at 30 to 35 degrees for 18 to 24 hours. Followed by streaking a loopful from TSB onto the surface of one or more of the following media:Vogel–Johnson Agar Medium (VJ Agar), Mannitol–Salt–Agar Medium (MS-Agar),and Baird-Parker Agar Medium (BP Agar). If no plates contain colonies having the characteristics described, the test specimen meets the requirement for the absence of Staphylococcus aureus. If characteristic colonies are present, a coagulase test is performed.

Pharmaceutical Products: USP <62> describes a similar method to test for the absence of S. aureus. After the pre-incubation in TSB, Mannitol–Salt–Agar Medium isused for plating.

Food Testing: FDA Bacteriological Analytical Manual (BAM) describes several methods for testing of S. aureus in foods. Methods used to detect and enumerate S. aureus maybe dependent for testing of foods and on the past history of the test material. Processed foods may contain relatively small numbers of debilitated viable cells, whose presence must be demonstrated by appropriate means. Finding food contaminated with S. aureusmay lead to legal action against the party or parties responsible for a contaminated food product. For S. aureus specified levels > 100 cfu/g of S. aureus plating on Baird-Parker agar is recommended. The method involves spreading 1.0 ml on 3 plates and looking for typical colonies. At least 20 colonies must be present on the lowest dilution for reliable results. Typical colonies need to be tested for coagulase. The most probable number (MPN) method is recommended for products in which small numbers of S.aureus are expected to be low and in foods expected to contain a large population of competing species. MPN can be performed in TSB containing 10% NaCl and 1% sodium pyruvate. From each tube showing growth (turbidity) a loopful is transferred to a plate of Baird-Parker medium.

The BioLumix Methodology

Pre-Incubation step Objectionable organisms including S. aureus may be present in very low numbers andmay also be “damaged”. Recovery of these organisms may require growth enrichmentin simple broth media prior to testing in selective media. Enrichment in a media suchas Trypticase soy broth (TSB) is often used as the first step in testing for the presenceor absence of S aureus. In testing of dietary supplement and pharmaceutical samples,enrichment occurs for approximately 18-20 hours. This step is similar to the procedure recommended by USP <1222> and USP <62>. After the pre-incubation in TSB a small amount (0.1 ml, typically) is transferred into the selective media BioLumix STA Vial and inserted into the instrument for 22 hours.

BioLumix Staph Vial Selectivity
The BioLumix STA vial utilizes a combination of inhibitors. These inhibitors target both unrelated organisms such as gram negatives and unrelated gram positives. Theuse of specific carbon sources (Mannitol) to selectively permit growth of primarily only staphylococcal species is also used. In addition, high salt concentrations also inhibitnon-S. aureus organisms including gram negatives and other gram positives. The goal is to slow or inhibit growth of these unrelated organisms including the inhibition of aclosely related organism Staphylococcus epidermidis in this BioLumix media vial.
Growth in the BioLumix STA Vial
A representative BioLumix STA Vial showing growth of S. aureus is shown in the accompanying Figure. The presence of S. aureus causes a detection time (DT) in thecurve (shown as a blue triangle). The figure also includes an un-related organism (Ecoli) tested under the same growth conditions in the BioLumix STA Vial.
Assay Endpoint
If there is no growth and no DT the sample is negative and does notcontain S. aureus. Growth in the
BioLumix STA Vial presumes the presence of S aureus. Confirmationusing a secondary test such ascoagulase is required to verify the presence of S aureus. The coagulase test can be performed directly from the vial.

Organism of Interest

Staphylococcus aureus is a major pathogen of concern in infectious disease. This organism group may include drug resistant S. aureus, often defined as methicillin resistant Staphylococcus aureus (MRSA). S. aureus is also an objectionable organism for the dietary supplement and nutraceutical industries.

Backgroud

Wikipedia’s description is as follows: S. aureus can cause a range of illnesses fromminor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis,carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases suchas pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS),bacteremia, and sepsis. It is implicated in skin, soft tissue, respiratory, bone, joint,endovascular and wound infections. It is still one of the five most common causes ofnosocomial infections, often causing postsurgical wound infections. Each year, some 500,000 patients in American hospitals contract a staphylococcal infection.

Methicillin-resistant S. aureus, abbreviated MRSA and often pronounced “mer-sa” (in North America), is one of a number of greatly-feared strainsof S. aureus which have become resistant to most antibiotics. MRSA strains are most often found associated with institutions such as hospitals, but are becoming increasingly prevalent in community-acquired infections. Arecent study by the Translational Genomics Research Institute showed that nearly half (47%) of the meat and poultry in U.S. grocery stores were contaminated with S. aureus, withmore than half (52%) of those bacteria resistant to antibiotics.

Current Methodology for S. aureus

Nutraceutical and Dietary Supplement Products: USP <1222> describes the methodrequired to test for the absence of S. aureus (typically in 10 grams) in nutraceutical and dietary supplement products. The method involves mixing of the sample in TSB and pre-incubating the TSB containing product at 30 to 35 degrees for 18 to 24 hours. Followed by streaking a loopful from TSB onto the surface of one or more of the following media:Vogel–Johnson Agar Medium (VJ Agar), Mannitol–Salt–Agar Medium (MS-Agar),and Baird-Parker Agar Medium (BP Agar). If no plates contain colonies having the characteristics described, the test specimen meets the requirement for the absence of Staphylococcus aureus. If characteristic colonies are present, a coagulase test is performed.

Pharmaceutical Products: USP <62> describes a similar method to test for the absence of S. aureus. After the pre-incubation in TSB, Mannitol–Salt–Agar Medium isused for plating.

Food Testing: FDA Bacteriological Analytical Manual (BAM) describes several methods for testing of S. aureus in foods. Methods used to detect and enumerate S. aureus maybe dependent for testing of foods and on the past history of the test material. Processed foods may contain relatively small numbers of debilitated viable cells, whose presence must be demonstrated by appropriate means. Finding food contaminated with S. aureusmay lead to legal action against the party or parties responsible for a contaminated food product. For S. aureus specified levels > 100 cfu/g of S. aureus plating on Baird-Parker agar is recommended. The method involves spreading 1.0 ml on 3 plates and looking for typical colonies. At least 20 colonies must be present on the lowest dilution for reliable results. Typical colonies need to be tested for coagulase. The most probable number (MPN) method is recommended for products in which small numbers of S.aureus are expected to be low and in foods expected to contain a large population of competing species. MPN can be performed in TSB containing 10% NaCl and 1% sodium pyruvate. From each tube showing growth (turbidity) a loopful is transferred to a plate of Baird-Parker medium.

The BioLumix Methodology

Pre-Incubation step Objectionable organisms including S. aureus may be present in very low numbers andmay also be “damaged”. Recovery of these organisms may require growth enrichmentin simple broth media prior to testing in selective media. Enrichment in a media suchas Trypticase soy broth (TSB) is often used as the first step in testing for the presenceor absence of S aureus. In testing of dietary supplement and pharmaceutical samples,enrichment occurs for approximately 18-20 hours. This step is similar to the procedure recommended by USP <1222> and USP <62>. After the pre-incubation in TSB a small amount (0.1 ml, typically) is transferred into the selective media BioLumix STA Vial and inserted into the instrument for 22 hours.

BioLumix Staph Vial SelectivityThe BioLumix STA vial utilizes a combination of inhibitors. These inhibitors target both unrelated organisms such as gram negatives and unrelated gram positives. Theuse of specific carbon sources (Mannitol) to selectively permit growth of primarily only staphylococcal species is also used. In addition, high salt concentrations also inhibitnon-S. aureus organisms including gram negatives and other gram positives. The goal is to slow or inhibit growth of these unrelated organisms including the inhibition of aclosely related organism Staphylococcus epidermidis in this BioLumix media vial.Growth in the BioLumix STA VialA representative BioLumix STA Vial showing growth of S. aureus is shown in the accompanying Figure. The presence of S. aureus causes a detection time (DT) in thecurve (shown as a blue triangle). The figure also includes an un-related organism (Ecoli) tested under the same growth conditions in the BioLumix STA Vial.Assay EndpointIf there is no growth and no DT the sample is negative and does notcontain S. aureus. Growth in theBioLumix STA Vial presumes the presence of S aureus. Confirmationusing a secondary test such ascoagulase is required to verify the presence of S aureus. The coagulase test can be performed directly from the vial.

Testing Nutraceutical Products in the cGMP Era: Questions and Answers

Questions to Dr. Ruth Eden

With the full implementation of cGMP’s for dietary supplements, what are the changes that you have seen in microbiology testing and inquiries regarding how to comply with the regulations?

The implementation of cGMP for all manufacturers (large and small) of dietary supplements and Nutraceutical products means that all manufacturers must test their products for microbiology quality assurance and generate a Certificate of Analysis for each batch. The products need to be tested in accordance with the methods described in USP <2021> and <2022>. Products need to be tested for Total Aerobic Microbial Count, Total Combined Mold and Yeast, Bile-Tolerant-Gram-Negative bacteria, and objectionable organisms (absent in 10 grams of organisms such as Salmonella, E. coli, and sometimes S. aureus). To do these tests companies either send product samples to contract laboratories for microbiological testing or test them internally. Many outside laboratories utilize BAM and AOAC methods that were never validated against the USP methodology for Dietary supplement products.

Any method that is different from the USP methodology (manual or equipment) used for microbiology testing needs to be validated prior to its use. The validation assures equivalency of the new method to the reference method. This means the new technique or device is giving us equivalent results to the USP methodology, the results are reproducible and can be trusted. USP <1223> Validation of Alternative Microbiological Methods describes the tests that need to be conducted.

Some manufactures were slow to implement the new regulations and the new methodology required for dietary supplement product testing. Others adopted methodologies from the food industry, without proper understanding of the difference between these methodologies and the USP methodologies. We get many inquiries from manufacturers that would like to save money, speed up time to microbiological results by internalizing their microbiological testing, while complying with the regulations.

What are some of the key issues surrounding microbiology testing that a manufacturer should consider when determining equipment and personnel needs for their laboratory?

Internal testing of products for microbiology gives the manufacturer much more control over the operation offering faster time to results, faster product release, and is more cost effective. Some manufacturers are hesitant to bring the microbiology testing in house for fear of the complexity of testing, the need for trained microbiologist, etc.

However, manufacturer share some common concerns when considering the internalization of microbiology testing: 1. Am I going to comply with FDA cGMP regulations? 2. How am I going to validate my testing? 3. Do I need to hire a microbiologist to internalize the testing? 4. Will this offer a return on my investment?

Also daunting is the need for a full validation package which is required for any method that deviates from the USP methodology. The validation package must properly document that the Installation Qualification (IQ) and Operational Qualification (OQ) were completed, to verify that the equipment was properly installed, calibrated and operational and that its performance are qualified. The Performance Qualification includes performance criteria such as: side-by-side comparison to the USP methodology, accuracy and precision, specificity, and range, limits of detection and quantification, along with ruggedness and robustness, reproducibility of the results, false positivity rate and false negative rate. Generating such data requires many resources.

BioLumix brings along a system that is fully validated during the system installation. With a simplified method such as the BioLumix System we can alleviate these fears and provide a simple cost effective method to test products for microbiology.

How does BioLumix plan to meet the changing requirements of dietary supplement manufacturers in implementing more precise, faster and cheaper microbiological testing in their laboratories?

BioLumix has developed a Rapid Microbiological Method that has been validated according to the FDA requirements. The validated BioLumix system is an alternative to the classic USP methodology. It is a streamlined testing system which is far less time consuming and requires less skilled technical personnel to operate. The BioLumix system offers a Certificate of Analysis for all relevant USP assays within just 48 hours. These include the FDA required tests such as total aerobic count, yeast and mold count, Bile-Tolerant-Gram -negative count, and objectionable organisms such as E. coli, S. aureus, and Salmonella. This means quarantine time for microbial testing is cut from 5-6 days to 24-48 hours for finished products, in-process and raw materials and environmental work surfaces.

Our system is fully validated during installation we perform the IQ, OQ and PQ required to fully validate the system in the customer location. We help write the appropriate SOPs, help generate the training records, and do not leave our customers until they are fully compliant.

The BioLumix testing system allows for faster sample preparation plus provides automated real-time communication, fast product release and early warning of contamination via USP microbiological testing. Because the BioLumix system is automated and its software generates the results, it is not necessary to have an employee dedicated solely to running the microbiology quality assurance tests. This makes the BioLumix system a cost-effective way for dietary supplement manufacturers to meet the new FDA cGMP requirements for microbiological testing.

A Novel Rapid Automated Method for Suitability Testing

Suitability Testing by USP Methodology

Suitability testing is performed in order to verify that the method utilized eliminates the effect of any
antimicrobial properties of the product. Therefore, the media diluent combination does not inhibit the
recovery and growth of microorganisms, if present in the sample. The goal of the suitability testing is to
establish the ability of the test to detect microorganisms in the presence of product.

The suitability described in USP <61> verifies the
validity of the testing method by showing the
recovery of microorganisms in presence of the
product. Total Aerobic Microbial Count and Total
combined Yeast and Mold can be carried out by
membrane filtration, pour plating or spread plate
method.

Suitability using USP <62> can use selective media to
detect various organisms such as: Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli,
bile-tolerant gram-negative bacteria, Clostridia,
Salmonella and Candida albicans. The samples are
first enriched by incubating in Trypticase Soy Broth
(TSB) or another appropriate neutralizing media, and then streaked onto selective agars for the
determination of presence of specified or the objectionable microorganisms.

The new USP <61> and USP <62> tests also provide harmonization to existing European Pharmacopeia
methods for testing non-sterile pharmaceutical products. Additional in order to verify the testing
conditions, a negative control is performed using the chosen diluent that shows no growth of
the microorganisms. While conducting the suitability testing precautions must be taken to avoid
contamination so they do not affect the microorganisms that are being tested. The procedure involves
the inoculation of the neutralized sample with low (not more than 100 cfu) and detecting the organisms
by the prescribed method.

Even though USP uses traditional microbiology methods, from the USP <61> and <62> states
that “Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopeial method has been demonstrated” and “any validated method,
including, Rapid Methods can be used”.

The New BioLumix Method

A study was recently conducted to show the utilization of BioLumix system (see figure below) in
suitability tests using a variety of products from both the pharmaceutical and cosmetic industries.

Sampling was conducted by taking ten gram of the product
and placing it into 90ml of M Letheen broth (or another
appropriate neutralizing broth) for a final dilution of 1/10.
An overnight culture of the target organism was diluted to
not exceed 100 cfu and the inoculum not exceeds 1% of the
volume of the diluted product. Then 1.0 mL of the
neutralized sample containing organism was placed into the
appropriate vial and a side by side comparison was done
with the appropriate USP method.

The products tested included Aloe, Hand Sanitizer, Lip Balm, Flavored Lip Balm, Medicated Lip Balm,
Breath Spray, Medicated ointment, and Sun Screen. A variety of different types of each product were
tested.

32 product samples were tested for suitability. Four bacteria (Staphylococcus aureus ATCC 6538;
Pseudomonas aeruginosa ATCC 9027; Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 8739; a
yeast (Candida albicans ATCC 10231), and a mold (Aspergillus niger ATCC 16404) were used to show the
effectiveness of the neutralization step. Thirty one
products were properly neutralized by the M Letheen
Broth as evidenced by detection time in the vials and
colonies on the plates. Only one product tested contained
a high level of ethanol, which required a 1:100 dilution in
M Letheen Broth to obtain neutralization. There was 100%
correlation between the two methods.

Typical data obtained by the system is shown in the figure:

The product was inoculated with ~ 100 cfu/g of three organisms: Staphylococcus aureus ATCC 6538 (light blue); Bacillus subtilis ATCC 6633 (Dark blue) and Escherichia coli ATCC 8739 (green). The Detection times obtained (shown as triangles on the curves) are comparable to data obtained without product.

What are the advantages of the BioLumix system?

Time Saving: The results are available much faster, for example, the results of the Yeast and Mold vialsoccurred in less than 48 hours, while the Aspergillus took nearly five days for countable colonies. All
products tested with bacteria using the automated BioLumix assay yielded results typically 10-13 hours,
instead of 48 hours for the plate count methodology. The advantage is that you can see results an
entire day early if the product is exhibiting any sort of inhibition. Data generation is slower using classic
microbiology so it can also slow down production improvements as well.
Labor Saving: The setup of the assay can be done much faster using the BioLumix system as opposed
to traditional plating methods, saving significant hands-on labor due to its automation and simplicity of
use. BioLumix can make the microbiological testing simpler, faster, and automated- saving significant
time and labor. It is paperless, increasing efficiency and saving on disposables, time and space, and best
of all the system is unaffected by product interference. The System is extremely easy to operate, with
its straightforward, streamlined testing design it offers accurate results leading to reduced material-
holding time for faster product release.

High Correlation with USP: The BioLumix System showed a high correlation between the instrument
results and the USP methodology. The system is fully automated with automated data archiving,
data maintenance in databases, and automated report generation. Regulators encourage rapid
microbiology methods for improved process control and product release. The BioLumix system
is validated as being at least equivalent to the compendial method. Under general notices of the
USP states that “Automated procedures employing the same basic chemistry as those assay and
test procedures given in the monograph are recognized as being equivalent in their suitability for
determining compliance.”

References:

USP <61> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: MICROBIAL ENUMERATION TESTS

USP <62>: MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TEST FOR SPECIFICIED
MICROORGANISMS

Rapid Yeast and Mold Testing yielding Results in 48 hours

Why test for Yeast and Molds?

Molds are agents which can be responsible for the contamination and spoilage of a variety of products. Some molds develop mycotoxins which diffuse into the product and may, if present in sufficient quantity, cause acute or chronic poisoning. The presence of yeasts may produce changes affecting product quality and salability, leading to significant financial implications for food producers.

Both yeast and mold can cause various degrees of deterioration and decomposition of products such as cosmetics, and foods. Products containing yeast and mold cells do not usually cause human illness, but high levels of these organisms can cause products to look, smell, or taste bad. This not only diminishes the appeal of the product and brand, but can also result in substantial economic losses to the producer, processor and consumer.

Yeasts are facultative anaerobe, mono-cellular fungi, fermenting sugar substrate to CO2 and H2O. Under anaerobic conditions yeasts ferment sugar to alcohol and CO2. The term “mold” is commonly used for the visible part of the fungi present on the surface of contaminated products. Fungal spoilage of food remains a serious problem, one that has been estimated to account for between 5% and 10% of all losses in global food production.

Plate Count Method for Yeast and Molds

Most industrial products are required to be tested for yeast and mold. The pate count methodology for testing yeast and molds are described in various reference publications, including Foods (Bacteriological Analytical Manual, 8th Edition, 1998. Chapter 18); Cosmetic and non-sterile Pharmaceutical products (USP) and Nutritional and Dietary supplement products (USP).

The methods involve mixing the sample with a diluent followed by decimal dilutions and the addition of the diluted sample into duplicate Petri dishes, the addition of selective culture media (such as Potato Dextrose Agar, Sabouraud Dextrose Agar). The plates are incubated for 5-7 days at 20-250C, after which plates with 25-250 colonies are counted. Due to the ability of some molds to spread rapidly on the plates, the interpretation of conventional colony counts can be difficult, requiring considerable staff input. Automated methods can help reduce the possibility of human error and free up skilled technicians for other tasks.

BioLumix Rapid Simplified Assay

Principle of Operation

The BioLumix Yeast and mold vial (YM) includes a CO2 Sensor. Carbon dioxide (CO2) is a universal metabolite produced by all microorganisms. For yeast and molds, the Krebs cycle is part of a metabolic pathway involved in the chemical conversion of carbohydrates, fats, and proteins into CO2, water and energy, and as a result all aerobic organisms generate CO2. The sensor is located at the bottom of the vial. The sensor vial contains a transparent solid sensor that changes color whenever CO2 diffuses into it. The color of the sensor is dark in sterile vials. As microorganisms grow the sensor turns yellow, indicating CO2 production and metabolic growth.

A crucial element of the technology is the creation of two zones in each vial:

  • An incubation zone (upper part) for the sample and microorganisms. This zone tends to contain product debris and turbidity due to microbial growth. A reading zone (bottom part) that remains optically clear.
  • This two-zone vial eliminates interference of the optical pathway by the product and microbial turbidity. Since changes of color are monitored in the reading zone, results are not influenced by the presence of the sample or the growing microorganisms. Only gases can penetrate into the sensor that blocks liquids, microorganisms and particulate matter. The user introduces the sample by simply opening the screw cap and dropping the sample into the incubation zone.

Typical Results

The typical procedure involves the addition of the sample to a diluent followed by the addition of the sample to the ready-to-use vial. The vials are introduced into the BioLumix instrument and the rest of the operation is fully automated, including report generation.

Variety of yeast and molds where tested with the YM vial. High numbers of yeast can detect in a few hours where as single numbers detect normally in 30 hours. Mold grow a little slower, while high numbers can detect in 6-8 hours, low numbers might take up to 48 hours to detect.

variety of products were tested for yeast and molds including: Nutraceutical and Dietary supplements (multi vitamins, amino acids, enzymes, Psyllium, Spirulina, fish oil, and numerous other supplements); Over-the-Counter Pharmaceutical (soft and liquid capsules, cold remedies, vitamins and minerals, eye care products, oral hygiene products, laxatives, antacids, pain relives, to name a few); Cosmetics and toiletry products ( hand cream, Aloe concentrate, hair dye, shampoos and conditioners, tooth pastes and rinses, soaps, mascara, lipstick, foundation, and many more); Foods (cheese, yogurt, ice-cream, salad dressing, fruit pulp, etc.)

Advantages

The first noticeable advantage is that BioLumix provides results in 48 hours, compared to traditional methods, which take 5-7 days. The rapid results provided enables manufacturers to release their products into the supply chain much earlier than when testing with traditional methods.

BioLumix can make the microbiological testing simpler, faster, and automated- saving significant time, labor and money. The YM vial accelerates product release with a simplified, automated approach. This yields fast, accurate results while reducing costs.

  • Vial design prevents product interference
  • Automation and connectivity allows faster product release
  • Real-time communication for immediate action
  • Expedite results: Yeast and mold assay in 48 hours
  • Early warning of contaminated samples
  • No decimal dilutions required- direct sample introduction to vials
  • Automated data archiving and audit trail
  • Streamline testing increases laboratory efficiencies
  • 21 CFR Part 11 Compliant Software
  • Paperless laboratory: centralized test date automatically stored and protected
  • Barcode capability for automated sample entry
  • Environmental made easy

BioLumix to Exhibit its Rapid Automated Microbiology System for Pharmaceutical Testing

BioLumix will be exhibiting at the PDA’s 6th Annual Global Conference on Pharmaceutical Microbiology held at the Marriott Bethesda North Hotel & Conference Center October 17th and 18th. Please come by the BioLumix exhibit (booth #19) and see how we are innovating and streamlining microbial testing for the Pharmaceutical Industry.  Our Rapid Microbiological Method (RMM) will simplify and accelerate the testing process with all assays completed on a single platform.  The automated BioLumix System provides real-time communication for immediate action; most completed assay results in 12-18 hrs – Yeast and Mold within 48 hrs.

A poster entitled: “Case Study of a New Growth-Based Rapid Microbiological Method (RMM) That Detects the Presence of Specific Organisms and Provides an Estimation of Viable Cell Count“  describing the scientific data behind the BioLumix technology and its validation is being presented.  The poster will show a technology overview and validation case studies for the BioLumix System, the new RMM technology that is capable of simultaneously detecting microbial growth, providing an estimation of viable cell counts, and identifying the presence of specific microorganisms, using a novel and automated growth-based format is presented. The case studies demonstrate the ability of the technology to detect total aerobic microorganisms, total yeast and molds, and the presence of E. coli, Staphylococcus, Pseudomonas, Salmonella, as well as additional microorganisms.  The data shown in the study indicates that the BioLumix system can be used as a RMM with comparable results to USP <61>.   The method has good specificity in detecting target organisms and excluding non-target flora.  The detection limit for the BioLumix system equals or is slightly better that the limit for the plate count method. High precision or repeatability was obtained for all three assays tested.  The system is capable of performing multiple assays in a streamlined protocol

FDA’s Support and Encouragement of New Technologies

Dr. David Hussong, FDA’s Associate Director for New Drug Microbiology, Office of Pharmaceutical Science, CDER stated that “FDA actively encourages use of new technologies including rapid microbiology methods” (RMM).  Drs. David Hussong and Robert Mello (New Drug Microbiology Staff at CDER) published a paper and stated the following: “New microbiology methods can offer advantages of speed and precision for solving microbiological problems associated with materials or environmental influences.  Neither Corporate economics nor regulatory attitudes should be a barrier to the use of new testing technologies or different measurement parameters.  In fact, if we are to increase our understanding of quality-based products and processes, then quality by design principles and risk analysis methods must be extended to the development of new microbiological technologies. This approach will drive process engineering to yield real, measurable gains in microbiological product quality assurance.”

The FDA has accepted the BioLumix Drug Master File (DMF):  The DMF is helpful for all Pharmaceutical companies producing prescription drugs.  The DMF is a technical document that contains support data for specificity, limit of detection, robustness, ruggedness, and equivalence to USP methodology.  The DMF can be used to streamline the validation of the BioLumix system, saving companies that wish to adopt the technology time and money.  For over the counter drugs the documents can serve as the basis for the validation package.

The current validation package will allow OTC users to utilize the system immediately, since during the system installation all validation elements will be completed by our trainers and your microbiologists.

Fast Return on Investment

Streamlining your microbial testing with our reliable, easy-to-use system will save you time and money.   In most cases a very quick return on investment can be proved.  We invite you to contact us and inquire about our Return on Investment calculation.  We have shown in many cases the BioLumix System will pay for itself within the first 6 months of ownership. 

Hear What Our Customers Are Saying

Don’t just take our word for it, talk to our customers.  We have recently conducted a customer survey and are proud to share we have 100% customer satisfaction and that all of our customers would recommend the BioLumix.  Unanimously, our customers agree – the main features of the BioLumix system are:  Speed to results, simplicity of use, accuracy and clarity of data presentation. You are free to contact our customers to discuss their first hand experience.

Free Product Trial

Give us your most difficult samples and we will test them for free.  We will provide you with a detailed report which includes a side-by-side comparison to USP methodology along with a sample of the BioLumix 48 hour automated Certificate of Analysis.  The data generated is strictly confidential and is only used to help validate the system for your company should you decide to purchase.

Come to our booth #19 and speak to an expert or contact us directly (734-984-3100) to learn more about the exciting new developments at BioLumix; such as:  our space, time, labor and material saving technology, 48hr Certificate of Analysis, as well as our environmental monitoring and water testing solutions.

We look forward to working with you and earning another satisfied customer!

Are cGMP Regulations driving up your Costs? Find out how to reduce your Microbiological testing Costs

For answers Visit BioLumix at booth 21017 @ SupplySide West 2011 in Las Vegas

Since the completion of implementation of the cGMP’s for Nutraceutical Companies, all manufacturers must test their products for microbiology quality assurance and generate a certificate of analysis for each batch.  The products need to be tested in accordance with the methods described in USP <2021> and <2022>. Products need to be tested for Total Aerobic Microbial Count, Total Combined Mold and Yeast, Bile-Tolerant-Gram-Negative bacteria, and objectionable organisms (absent in 10 grams of organism such as Salmonella, E. coli, and sometimes S. aureus).  The state of the art BioLumix system helps streamline microbial testing, and allows for easy compliance with cGMP. The System is designed to accelerate product release with a simplified, automated approach. This yields fast, accurate, real-time results while reducing costs.  No more waiting days for completed assays. 

The streamlined all-in-one system is capable of testing raw materials, finished product, environmental swabs and processing water, all on a single platform.  Additionally, the system offers simplified sample preparation, real-time communication and a complete automated Certificate of Analysis, including the completion of all assays in 48 hours.   Dealing with difficult to read plates or manual data transfer is a thing of the past. With automated monitoring of ready-to-use assay vials and automated data processing and archiving (paperless), the microbiologist‘s job got a lot easier with the same accurate results in less than half the time.  Real-time data communication: Early warning of contaminated samples as well as sample release information could be automatically communicated through your intranet, significantly improving your company’s efficiencies.  BioLumix system can be used to test powders, oils, enzymes, botanical material, tablets, capsules, powders and liquids easily and without product interference.

BioLumix trainers will provide each customer with a full and customized Validation book, during the two and a half days of on-site training and installation of the system, including installation qualification, Operation Qualification and Performance qualification.  A sample Validation book will be available for viewing while visiting the BioLumix booth during SupplySide West 2011.

With this internal automated microbiological system, customers have control over their testing, leading to accurate results and accelerated product release while reducing costs.  The BioLumix Advantage: 

-          All assays can be performed on one Automated Microbial testing system

-          Automated Certificate of Analysis within 48 hours including results of all assays

-          Can be operated by non-Microbiologist

-          Complies with FDA cGMP Regulations

-          System is fully Validated during system installation

-          Designed to accelerate product release

-          Fully automated data archiving

You can find BioLumix on social media pages Linked In, Twitter and Facebook.  Follow us, become a Fan or join a discussion on one of our pages.

October 2011 marks the 5th year of BioLumix to exhibit at SupplySide West in Las Vegas, NV.  This yearly convention is held at the Sands Expo Center in The Venetian Hotel & Casino.  The Exhibit show hall will open beginning Wednesday, October 12th from 10am – 5:30pm, and Thursday, October 13th from 10am – 5pm.  BioLumix will be exhibiting in booth #21017.  Be sure to stop by booth #21017 to inquire about our free product trial and current Show Special! Pick up literature or leave your contact information to find out more about how the system can help your company save.

Rapid Microbiological Testing of Capsules, Softgels and Pills Encapsulation of Pharmaceuticals

Pharmaceutical products may be encapsulated in a relatively stable shell for oral consumption.  These shells are called capsules and can either be hard-shelled or soft-shelled.  Hard-shell capsules are commonly filled with dry powdered ingredients, pellets, or granules.  Soft-shell capsules (softgels) are made from gelatin and primarily contain oils or active ingredients that are dissolved or suspended in oil.  Both hard and soft-shell capsules may contain colorants, dyes, opaquing, dispersing, or hardening agents, and preservatives.  Tablets are solid doses of medicinal substances and may be soluble effervescent, chewable, molded, or compressed.

Traditional methods for testing

Plate count methodology as described in USP <61> is regularly used to test capsules and tablets.  Using this methodology, it takes two days for Aerobic Count results, and five days for Yeast and Mold results.  When testing for objectionable organisms, it may take several days using selective broths or agars to determine the absence or presence of these bacteria.   The colors of the shells and the products and the viscosity of the 1:10 dilution sometimes interfere with the reading of the plates.

Rapid Detection with the BioLumix System

The BioLumix System simplifies testing, expedites time to results, reduces the testing cost and accelerates product release while providing better control of microbial contamination.  The system can be used to automate microbial testing with a more cost effective and streamline manner, and reduces the error rates produced by paper-based activity recording and batch data entry.  The BioLumix system also helps automate microbiological quality control processes.

To validate the equivalency of the BioLumix system to USP <61> or USP <2021>, over 100 types of capsules, softgels, and tablets were tested with the BioLumix system and plate count methodology at various specified levels.  The products were tested for total aerobic microbial count (TAMC), Total yeast and mold count (TYMC), and the absence of E. coli, P. aeruginosa, S. aureus, and Salmonella (objectionable organisms) in 10 grams of product.  For each assay, a 1:10 dilution was created by adding 10 grams of product to 90 mL of TSB and further dilutions were performed depending on the desired specified level.  Some of the samples were inoculated with various bacteria.


Figure 1 shows the BioLumix curves obtained for the Total Aerobic assay.  The purple curve represents an inoculated colored capsule.  All the curves along the baseline represent different kind of capsules that were not contaminated.


Figure 2 shows the BioLumix curves for the Yeast and Mold assay.  The dark blue curve represents an inoculated product. All the curves along the baseline represent different kind of capsules that were not contaminated.

 

As seen in Figure 3, the BioLumix vial design is separated into two zones: the incubation zone where the sample is present and the detection zone where the reading takes place.  Therefore, the system prevents any product interference.

Figure 3 shows two vials that contained dissolved blue capsules.  The sample on the right contained bacteria while the sample on the left was clean.

Final results are seen in the BioLumix system roughly 25-40 hours faster than the plate count method in the Total Aerobic Count assay and 40-72 hours faster in the Yeast and Mold assay.

When testing for objectionable organisms, results may be obtained several days sooner than the plate count method.  Following the BioLumix protocol, 0.1 mL of the incubated sample in TSB is added to a selective vial and data is collected in the BioLumix instrument for 18-24 hours, depending on the assay.  If detection occurs, the sample may be verified with a confirmation assay.  This procedure eliminates hours or days waiting for plate results.

The BioLumix system is faster, less labor-intensive, and more sensitive than the plate count method.  With an automated certificate of analysis generated within 48 hours, the time-to-results is reduced and allows for quick release of products.

Caron Ockerman

References:

USP <61> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: MICROBIAL ENUMERATION TESTS

USP <2021> MICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY SUPPLEMENTS

USP <2022> MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MICROORGANISMS—NUTRITIONAL AND DIETARY SUPPLEMENTS 

Rapid Detection of Heterotropic Bacteria in Water

What categorizes Heterotrophic organisms?

Heterotrophic organisms are categorized by the requirement of organic carbon sources to produce energy.  These organisms can include bacteria, yeasts and molds, and can be found throughout the environment in both natural and treated water (including tap water).  They occur normally and are unique in their ability to proliferate in the nutrient-poor environment of water systems at various temperatures. 

Why Test Water for Heterotrophs?


The concentration of Heterotrophs in treated water is an indicator of water treatment efficacy.  The presence of these microorganisms does not necessarily indicate concentrations high enough to cause disease.  However, the concentration of these microbes is important when testing for coliforms in water.  If Heterotrophic organisms are found at a concentration of approximately 500 colony forming units (cfu) per mL or higher, then the detection of coliforms may be compromised.  An increase of Heterotrophic counts may indicate the presence of coliforms in the water being tested.

Traditional methods for testing water

Heterotrophic Plate Count (HPC) is the procedure traditionally used to estimate the number of viable organisms in a sample of water.  Three different methods – (i) spread plate; (ii) pour plate; and (iii) membrane filtration – are utilized with different types of media.  Although these methods are widely accepted, there are some disadvantages.

  • In the pour plate technique, organisms are suspended in agar until the media cools.  During this time, damaged organisms are susceptible to heat shock from the higher temperature of the agar.  Also, because the colonies are surrounded by agar, the count of obligate anaerobes becomes compromised.
  • If an automated spreader is used in the spread plate method, heavy growth of colonies may make counting of colonies difficult, rendering counts non-applicable.
  • When filtering water, samples may contain suspended solids that cannot pass through the filter, interfering with accurate counts.

Due to different techniques and different types of media used, the counts achieved may vary significantly.  In addition to multiple protocols and media, temperature and incubation time vary.  Samples may be incubated anywhere from a few hours to 7 days or even a few weeks at temperatures ranging from 20°C to 40°C.  This makes processing water samples laborious with much data collection.

Rapid Detection with the BioLumix System 

BioLumix is the most advanced microbiological testing system of its kind. This automated, all-in-one microbial testing system is extremely easy to operate. The system is both simple and cost-effective, revolutionizing your current testing methodology. A novel optical system sensing color and fluorescence in ready-to-use vials provides faster results, labor savings, automation, and connectivity. The system has a large repertoire of assays that it can perform including: Total Aerobic Count, Yeast and Mold, Coliforms, Enterobacteriaceae, E. coli, Pseudomonas, Staphylococcus, and Salmonella.

Recently a new assay for the detection of Heterotrophic bacteria in water was developed for the BioLumix system.  The developed assay was validated by testing 50 samples of multiple types of water that were tested by the BioLumix method and the plate count method side-by-side.  The BioLumix vials were directly inoculated with 0.1 mL of the water sample, or 1.0 mL of a 1:100 dilution (depending on the desired specified level), and a few water samples were inoculated with heterotrophic bacteria.  These samples were monitored in the BioLumix instrument for 35 hours.  Figure 1 shows the curves obtained.

Final results were seen in the BioLumix system roughly 13 hours faster than the plate count method using Standard Methods Agar.  These particular samples were tested at specified levels <10 cfu/mL and <100 cfu/mL, but the BioLumix method can detect organisms at a level of <1 cfu/mL of water.  The BioLumix system is faster, less labor-intensive, and more sensitive than the plate count method. 

  Caron Ockerman

Microbiological Testing of Gelatin Capsules

Introduction

Two-piece capsules have been used for almost a century in the pharmaceutical field, and gelatin has been adopted as the main material of these capsules due to its excellent characteristic as a gelling agent. The gelatin dissolves under high concentration into water of a high temperature and quickly gels in room temperature. The thickness of the film made by the gelatin becomes uniform.

The gelatin capsules consist of gelatin, plasticizers and water. Modern day shells may, in addition, consist of preservatives, colors, opacifying agents, flavors, sugars, acids, enteric materials etc. A mixture derived from pork skin and bones is used in capsules.  Pork skin gelatin contributes plasticity while bone gelatin gives firmness.

One important reason for the exclusive use of gelatin for making hard and soft capsules is its solubility characteristics in stomach fluids. It absorbs cold water readily, though the rate of absorption depends upon moisture content of gelatin. Bloom Strength is an empirical gel strength measure which gives an indication of the firmness of gel. The plasticizers used are glycerin, sorbitol etc.  Preservatives, if included, are generally a mixture of methylparaben (4 parts) and propylparaben (1 part) to the extent of 0.2%. Flavors, if added, should not exceed 2% and are generally ethyl vanillin or essential oils. Sugar, if included, may be up to 5% to give the gelatin shell desirable chewable characteristics.

Microbiology Testing

Each incoming lot of capsules needs to be tested using USP <61> and USP<62>. The total aerobic bacterial count should not exceed 3,000 cfu per gram, the combined yeast and mold counts along with Bile-Tolerant Gram-Negative Bacteria should not exceed 300 cfu per gram.  Material must also meet the requirements of the tests for absence in 10 grams of Salmonella species, Escherichia coli, and Staphylococcus aureus.

Challenges of Current Methodology

Testing gelatin capsules for microbiology might result in several challenges.  The 1:10 dilution of the product has high viscosity and is sometimes difficult to pipette. Many capsules have vivid colors that interfere the reading of the plates. 

The current methods used in microbiology originated over 100 years ago.  There have been limited improvements in methods used for microbiological testing in the last decade. The current USP methodology is slow, requiring up to 5-7 days for product release, is manual, and in many cases is inaccurate.  Paper-based QC laboratory processes can be expensive, error-prone, time and labor-intensive.  

Rapid microbiological methods (RMM) offer a cost effective alternative to USP methodology.  With an RMM’s high degree of automation, significant reduction in time to results, faster product release, and improved process control, while providing enhanced accuracy, better repeatability, and total automation

BioLumix Alternative

The BioLumix System simplifies testing, expedites time to results, reduces the testing cost and accelerates product release while providing better control of microbial contamination.  The system can be used to automate microbial testing with a more cost effective and streamline manner. The system reduces the error rates produced by paper-based activity recording and batch data entry.  The BioLumix system also helps automate microbiological quality control processes.

The BioLumix technology is based on continuous monitoring of changes in color or fluorescence as a result of microbial metabolism in ready to use test vials. The results are presented as soon as detection occurs without any involvement of the operator. The fully automated system offers a paperless operation with increase efficiencies.

The key to the technology is the two-zone ready to use vials which eliminates any product interference.  Many types of gelatin capsules were tested in the BioLumix system for total aerobic bacterial count, combined yeast and mold counts, Bile-Tolerant Gram-Negative Bacteria, and for absence in 10 grams of Salmonella species, Escherichia coli, and Staphylococcus aureus.  As can be seen in the figure, there is no product interference even with capsules containing the most vibrant colors.  All assays yielded clear results that correlated well with the USP methodology.

Key: Dark Blue-clear capsule;Green- Brilliant Red Capsule; Light Blue-Brown capsule;Red- Teal capsule; Purple-Inoculated Brilliant Red Capsule.

The BioLumix system is validated against the USP methodology.  All assay results are complete in 48 hours with an automated Certificate of Analysis; resulting in faster product release.

Rapid Microbiological Method or Conventional Method? What Should You Use?

In the get-it-done-yesterday environment companies are finding that the traditional microbiological methodologies and especially sending microbiological samples to outside labs cost them time, money and opportunities1.  Internalizing the microbiology testing and especially adopting rapid microbiological methods (RMM) can significantly speed up the time to results from 7-10 days to 24-48 hours.  The time saved creates opportunities for faster response to contamination, quicker product release, faster delivery of product to the marketplace, reduced warehouse space, and enhanced product quality.  Advantages of RMM can include: Greater accuracy, better sensitivity, increased sample throughput, automated data capturing allowing easier data handling, and reduced cost for product release.

However, like any other investment it competes for company resources.  Therefore, there is a need to calculate the financial benefits of the investment in RMM.

Dr. M. Miller, an authority on RMM, has written extensively on this topic and suggests that before adopting a new RMM or internalizing Microbiology the key steps required include2, 3, 4:

  1. Review of existing technologies
  2. Understand the technical and financial benefits of RMM
  3. Develop the business case for RMM

This will include the use of financial models that can compare the overall costs associated with the current microbiology method with the costs and savings associated with the purchase, qualification, and implementation of the RMM

Return on Investment (ROI)

ROI is the ratio of money gained or lost on an investment relative to the amount of money invested.  According to Dr. Miller3 for RMMs, the cost of performing the conventional method (CM) is compared with the cost (and savings) of using the new method. The information is reported as a percentage and usually represents an annual or annualized rate of return. The ROI is calculated using the following formula4:

CM costs can include:  cost of consumable; reagents and supplies; sample preparation time; data management documentation and record retention time; as well as cost of labor and overhead.

Potential investment in RMM can include:  Capital cost; and validation cost.

Potential cost savings of RMM can include:  Reduced time for product release; Lower headcount; lower re-processing fees; reduction in plant downtime; increased production yields; reduced raw materials, in-process and finished products inventory holding; reduction in back orders; increased production flexibility; and better protection of company image.

Payback Period (PP)

The PP is the time required for the return on an investment to “repay” the sum of the original investment. In the context of implementing an RMM, this would be the time (usually in years) required to realize sufficient cost savings to pay for the initial investment of the RMM capital equipment as well as for qualification and implementation activities3, 4.

Requirements from RMM

While Rapid Microbiological Methods (RMM) offer high degree of automation, significant reduction in time to results, faster product release, ability to employ non-microbiologists to operate the system, and improved control; there are three basic objectives that must be meet:

  1. Comply with FDA and cGMP regulations
  2. The RMM must be validated against the standard USP methodology using a protocol similar to USP <1223>.
  3. Ensure a swift return on investment.

Additionally, the ideal system should be capable of:

  1. Performing all the microbiological assays required to be performed by lab on a single platform.
  2. The technology should eliminate or at least minimize any product interference.
  3. Allow for a paperless microbiology laboratory-allowing the laboratory to operate without handwritten data entries
  4. Automate the system reporting
  5. Real-time communication to locations outside the lab to provide early warning of contamination

Required Commitment from RMM Vendors

Validation:

To make the transition to a rapid method the vendor should help in the system validation to make the transition to the new system smooth and “painless”. It should include Installation Qualification (IQ); Operational Qualification (OQ), and help with the Performance Qualification (PQ). PQ is the most extensive portion of the validation and sometimes companies considering RMM are deterred by the amount of work required for validation. Vendors can generated most of the data and submit it to the FDA in a form of a DMF.  The same information can be supplied to new users together with unique data pertaining to their products.

Software:

The software must be compliant with 21 CFR Part 11.  21 CFR Part 11 regulations are established requirements to ensure that electronic records and electronic signatures are trustworthy, reliable and generally equivalent substitutes for paper records and traditional handwritten signatures. Any instrument system containing software must be compliant with this regulation.

Technical Support:

Instruments suppliers should guaranty live technical service that is fast, knowledgeable, and readily available.

The BioLumix system is an example of a system that fulfills the requirements listed above.  Its straightforward streamlined design offers rapid accurate results leading to reduced material-holding time for faster product release.  Early warning of contaminated samples as well as sample release information could be automatically communicated through your intranet, significantly improving your company’s efficiencies.

To evaluate the value proposition of RMM systems BioLumix offers a free assessment of your specific ROI and Payback Period.  This will help you determine whether there are sufficient cost benefits of adopting a RMM system.  Past ROI calculations demonstrated that in many situations the ROI obtained showed that a RMM, like the BioLumix system, improved the company’s bottom line and satisfied the financial expectations of site management.

References

  1. 2009. Gadal, P.; Yvon, P. Rapid Microbio ROI – Calculating scientific benefits as return on investment dollars. Pharmaceutical Formulation & Quality. 11(3): 44-47.
  2. 2009. Miller, M.J. Breaking the rapid microbiological method financial barrier: A case study in RMM return on investment and economic justification. BioPharm International. 22(9): 44-53.
  3. 2009. Miller, M.J. Ensuring ROI from your RMM. Pharmaceutical Manufacturing. 8(6): 32-35.
  4. http://rapidmicromethods.com/files/roi.html
  5. USP <1223> Validation of Alternative Microbiological Methods.

Ruth Eden, PhD

President

BioLumix. Inc.

Environmental Monitoring in the Pharmaceutical & Nutraceutical Industries

FDA expects manufacturers to be in control of the environmental conditions within the manufacturing facility. Controlling the environmental condition is not only a regulatory requirement but also part of protecting and producing a quality product. Environmental monitoring of Pharmaceutical & Nutraceutical manufacturing facilities provides assurance that the environment is in control, and in compliance.  There is substantial evidence establishing a direct relationship between the level of environmental control and the final quality of the product.

A variety of methods are available to measure total particles in the air, Total Organic Carbon (TOC), and ATP (Adenosine Tri-phosphate).  These methods while very fast to perform do not correlate with total bacterial count or any specific group of organisms in many cases.  Therefore, these results do not tell us when we have viable organisms in the environment and on production lines. The standard plating methodologies can take several days for results.  Rapid microbiological methods can provide the solution

Why Not Monitor ATP?

Adenosine-Tri-Phosphate (ATP) is an energy molecule stored in all microorganisms and therefore an indicator of life.  ATP bioluminescence began to gain traction over plate count approaches in the late 1980s, especially in the food industry.  It is basically a “dirt” detector rather than a microorganism detector.   It is a common misconception that the results received from ATP testing systems in relative light units (RLUs) for surface samples should correlate with a microbial total plate count result.

The lack of correlation between ATP and plate counts means that samples can have high ATP readings and no bacteria or conversely have low ATP readings and high bacteria count.  For example, the inside of a tomato is sterile and doesn’t contain bacteria, yet it contains large amounts of ATP.  Likewise, it is possible to have bacteria and still get pass from an ATP based test, because 10,000 bacteria/swab are required for detection.  To complicate the matter further, there are different levels of ATP in different cells on the surface. Yeast, for example, contains 100 times the ATP amount of Coliform bacteria.

The standard used for post-cleaning surface contamination is typically 100 – 1,000 bacteria / 100cm2, and 10-50 yeast and molds which is lower than the detection limit for the ATP test.   Therefore, ATP results do not tell whether the surface is microbiologically acceptable.  In other words, it does not tell how many or what kind of microorganisms or chemical contaminants are present on the surface—only that there is organic matter present on the surface in which microorganisms might be able to grow.  Also, sanitizer residues on food contact surfaces or certain food components may interfere with the ATP reaction.

The BioLumix Advantage

BioLumix is an optical rapid, automated system that simplifies microbiological testing.  The system can be utilized for simplified monitoring of the manufacturing environment. Three assays were tested:  Total Aerobic Count, Total Combined Mold and Yeast Count, and Bile Tolerant Gram Negative Count (Enterobacterial Count).  A study was performed to compare and validate the BioLumix system as an alternative to the standard plate count method in detecting microbial contamination in manufacturers’ production surfaces.  Five different surfaces were used to simulate production surfaces.  10X10 cm coupons were inoculated with microorganisms, allowed to dry and then swabbed.  The swabs were inserted into a diluent (buffer or TSB) and analyzed by both the BioLumix and plate count assays.


A total of 550 coupons were tested, 290 were inoculated with various levels of contamination from <10 to <5,000 cfu/swab. The swabs containing microorganisms above the specified levels showed a very good correlation between the BioLumix and the various plate count results, with an overall agreement for samples above spec of 97.2%.  Almost all the discrepant swabs had plate counts very close to the specified level. None of remaining 260 swabs which contained counts below the specified levels did detect in the BioLumix system.  Consequently there was 100% agreement between the two methods.  The system also has the utility to detect for objectionable organisms such as E. coli, S. aureus and Salmonella.

Therefore, the BioLumix system offers:

  •  Automation of results
  •  Speed
  • Paperless
  • Detects microorganisms and not indicators
  • Can detect multiple types of organisms
  • Increases operation efficiency and consequently, improves quality and reduces costs                 
  • Enhanced reporting and ability to track trends

Rapid Microbiological Methods in the Era of Validations

Any new technique or equipment needs to be validated prior to entering its use in the commercial arena. The validation assures equivalency of the new method to the reference method. This means that the new technique or device is giving us “real results” that are reproducible and that can be trusted. USP <1223> Validation of alternative microbiological methods and ISO 16140 defines the general principle and the technical protocol for the validation of alternative methods in the field of microbiological analysis In the industrial world there is a pressing demand for rapid microbial detection technologies in order to improve the quality of products and their safety and speed up time to results. Their benefit could include significant reductions in time-to-result over conventional methods, improved sensitivity, specificity and accuracy, benefits of automation, reduced requirements for staff training, rapid product release, and lower inventory.

There are several new rising technologies that provide rapid microbial detection; such as the exceptional one developed by BioLumix. However, to be broadly used and accepted these new technologies need to comply with USP standards and fulfill the validation process. In order to get the validation assurances a series of pre-established analyses need to be done and compared the data generated by the new technology with the USP data to show equivalence. How valid the classical analyses are, it is a matter of historical use and common acceptance.

There are three required qualification components in a validation package; these are: installation, operation, and performance qualifications. The assurances for installation and operation qualifications deal with how reliable the setting and handling are; and that the proper conditions are given for the device or technique to work properly. To ascertain the reliability of performance qualification intuitively important are the internal positive and negative controls and well-known standards. Controls are the building blocks of any scientific approach to test hypotheses and to answer specific questions accurately. The controls and standards contain known amounts of the substance tested by the new technology. Under good laboratory practices isolated microbes are used in the manufacture of these controls and standards. Controls and standards are then used to validate the detection and quantification of unknown samples by the specific technique or device. In addition, controls and standards allow the validation of various important performance criteria, such as: accuracy and precision, specificity, and range, limits of detection and quantification, along with ruggedness and robustness. Accuracy is how close the results to the true value are. The precision deals with the agreement among individual tests. Specificity is given when detection of a particular microbe occurs even in a mix of different microbes without cross detections. The range is the upper and lower microbial count level limit that gives precise and accurate measurements. Limit of detection are the lowest microbial counts that can be detected. Ruggedness is related with the reproducibility of the results considering different equipment, personnel, laboratories, etc. And robustness indicates the capacity of the device or technique to absorb small variations such as: incubation conditions, media brands, reagents quality, etc.

The validated BioLumix system is an alternative to the classic USP methodology, which is highly time consuming and requires trained technical personnel. Using the BioLumix system, in just 48 hours a certificate of analysis is generated from samples that do not require special handling neither sophisticated processing. Hence, one of the best advantages of the BioLumix system is that does not require highly skilled personnel. BioLumix system ought to be used by a wide variety of manufacturing industries, which need to accurately certify the microbiologic status of their products in order to ensure their safety. Remarkably, the BioLumix system is capable of detecting a wide variety of microbes that raise health concerns such as: E. coli, Salmonella, Staphylococcus, yeast and molds, etc. BioLumix is committed to fulfilling the high demand for saving industries money and time in this fast pacing era of assurances and validations.   

A Rapid+Easy Method for Microbiological Testing of Dietary Supplements Complying w/cGMP & USP

Nutraceutical and dietary supplements consist of vitamin and mineral supplements, amino acids, herbs, essential oils, enzymes, homeopathic remedies and other natural products. For years dietary supplements have been unregulated by the U.S. Food and Drug Administration (FDA). In 1994 the Dietary Supplement Health and Education Act (DSHEA) was enacted, which stated that dietary supplement manufacturers must ensure supplements’ safety before marketing them.1

In 2007 the FDA issued the final rule for the implementation of current good manufacturing practices (cGMP) for supplements. 2 The FDA 21 Code of Federal Regulations (CFR) Part 111 required the implementation for large manufacturers in June 2008 and that by June 2010 all dietary supplement manufacturers test their products for identity, purity, contaminants and microbiology quality assurance, like pharmaceutical companies do. 3

To meet these new requirements, dietary supplement manufacturers must test their products for microbiology quality assurance and generate a certificate of analysis for each batch. To do this they either send product samples to contract laboratories for USP microbiological testing or test them internally. To reduce the time to get results, increase efficiency and reduce expenses, BioLumix offers a fast, automated, cost effective way for nutritional and dietary supplement companies to perform USP microbiological testing. The BioLumix system is an in-house method to easily perform accurate and automated microbiology quality assurance assays on finished products, in-process and raw materials and environmental work surfaces. This system can cut quarantine time for microbial testing from 5-6 days to 35-48 hours.

Samples are placed in ready-to-use, disposable vials inserted into an instrument that determine the microbial quality of the product. The system can perform all FDA-required tests such as total aerobic count, yeast and mold count, Enterobacterial count, and objectionable organisms such as E. coli, S. aureus, Salmonella and P. aeruginosa. The BioLumix testing system also includes a step-by-step process to optimize the installation, operation and performance so that it is easy for nutraceutical and dietary supplement manufacturers to do microbiology quality assurance.

The BioLumix testing system allows for faster sample preparation plus provides automated real-time communication, fast product release and early warning of contamination via USP microbiological testing. Because the BioLumix system is automated and its software generates the results, it is not necessary to have an employee dedicated solely to running the microbiology quality assurance tests. This makes the BioLumix system a cost-effective way for dietary supplement manufacturers to meet the new FDA cGMP requirements for microbiological testing. For more information on the BioLumix cGMP USP microbiological testing system, visit http://www.mybiolumix.com or call 1-734-984-3100.

References

1. Dietary supplements. FDA Web site. http://www.fda.gov/food/dietarysupplements/default.htm.

2. FDA Issues Dietary Supplements Final Rule. FDA Web site.

http://www.fda.gov/bbs/topics/NEWS/2007/NEW01657.html

3. FDA 21 CFR part 111. FDA Web site.

http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=111&showFR=1FDA

Karen F. Vieira, Ph.D.