A simple, rapid, automated solution for microbiological testing.

Meeting the challenges of the new US Food and Drug Administration (FDA) regulations requires simpler, faster and more streamlined microbiological tests. The BioLumix System enables users to perform the entire microbial testing operation in-house rather than sending their samples to external laboratories for testing. Accordingly, the system allows companies to greatly reduce the amount of time and money required to have their samples tested by independent contract laboratories. Consequently, it enables the earlier release of raw materials and finished products. The BioLumix System can help to reduce quarantine times of 5-6 days to approximately 35-48 hours for microbiology testing. In addition, the system is fully automated.

The new FDA regulations regarding cGMPs (current Good Manufacturing Practices) will require the nutraceutical and dietary supplement industries to streamline and develop faster and more effective microbiological assays. Currently, many manufacturers send their samples to external testing laboratories. This is expensive and takes many days to obtain results. Internal laboratories are a better option but many manufacturers do not have the infrastructure or the personnel to internalize microbiological testing. The BioLumix System offers a fully automated microbiological system that can deliver much faster results, without the requirement of a full microbiological laboratory. It comprises an instrument with the capacity to test 32 individual assay vials, software and disposable vials.

Each instrument serves as an incubator. Up to 32 instruments can be connected to one computer. The software enables rapid, real-time results to be transferred to where they are needed most, without any operator involvement. A variety of disposable vials can be used to perform several microbiological assays, including total aerobic count (TAC), Yeast and Mould (YM), coliform (CM), E. coli (EC), Pseudomonas (PSE), Staphylococcus (SA) and probiotics. All assay vials are provided with ready-to-use sterile media and a certificate of analysis

Technology

The technology enables the simultaneous detection of changes in either colour or fluorescence as a result of microbial growth and metabolism (Figure 1).

Methods

Samples Used: The 29 products listed below were tested for total aerobic count, yeast and mould, Pseudomonas, E. coli and Staphylococcus (Table I).

[FIGURE 1 OMITTED]

Vials

Total Aerobic Count (TAC): These vials contain a C[O.sub.2] sensor at the bottom of the vial and nutrient growth medium above. The carbon dioxide generated by bacterial metabolism diffuses into the sensor and reacts with the reagents in the sensor, providing an indication of their presence. Only gases can penetrate the sensor, blocking liquids, micro-organisms and particulate matter. Results are obtained after an "overnight" (18-22 h) incubation. Yeast and Moulds: These vials contain the C[O.sub.2] sensor at the bottom of the vial and have selective medium above for the selective detection of yeast and moulds. Results are obtained after 48 hours of incubation. Pseudomonas: These vials contain the C[O.sub.2] sensor at the bottom of the vial and have selective medium above for the detection of Pseudomonas strains. The results are available after an overnight (18-22 h) incubation. Positive results can be verified with the oxidase reaction. Staphylococcus: These vials contain the membrane filter and selective and differential growth medium above containing mannitol and a pH indicator (Phenol Red). Growth of Staphylococcus results in a colour change from red to yellow. The results are available after overnight (18-22 hours) incubation. The coagulase reaction can be used to verify the presence of S. aureus.

E. coli: These vials contain a membrane filter to separate the incubation zone--where the media, sample and micro-organism are present--from the measuring zone in the bottom of the vial where the detector reads the results. This design allows for the optical testing of opaque samples. The E. coli assay is based on the detection of the microbial cleavage of 4-methylumbelliferyl-[beta]-D-glucuronide (MUG) using the fluorescence sensor. The results are available after an overnight (18-22 h) incubation. At the end of the assay, the indole reaction can be utilized to verify the presence of E. coli in approximately 10 minutes.

Schematic Protocol for BioLumix

The Dilute-to-Specification protocol was used (Figure 2), requiring dilution of the sample to the specification limit required for product action or release. If there is microbial growth above the specification limit, the sample fails; if there is no microbial growth or growth below the specification limit, the sample passes.

Results

Total Aerobic Count: Five of the 29 products tested produced counts above 1000 cfu/g and were all detected by the instrument within 22 hours. Twenty vials were inoculated with a variety of bacteria including Acinetobacter, Bacillus, Citrobacter, Enterobacter, Proteus, Pseudomonas, Micrococcus, Staphylococcus, Streptococcus and Yersinia at a level of 5000-20,000 cfu/g. All species were detected in the instrument within 18 hours.

Yeast and Moulds: Six samples had moulds above the specified level of 100 cfu/g and were all detected in the instrument within 40 hours. Twenty vials were inoculated with a variety of yeast and moulds, including the yeasts Candida albicans, Candida tropicalis, Geotrichum candidum, Rhodotorula mucilaginosa, and Zygosaccharomyces and the moulds Aspergillus niger, Aspergillus oryzae, Rhizopus stolonifer and a variety of Penicillium species at a level of 500-4000 cfu/g. All species were detected in the instrument within 35 hours.

Pseudomonas: Seven of the 21 samples tested using the Pseudomonas vials were positive for this contaminant. Most of the samples were detected very quickly (2-6 hours). Two samples were positive for Pseudomonas at a level >1 cfu in 10 g of product and were detected using the instrument within 16 hours, following an overnight incubation in TSB. There was no detection in the samples that did not contain Pseudomonas. Ten samples were inoculated with 4 different strains of Pseudomonas aeruginosa, P. fluorescens and P. putida at a level of 10-100 cfu/10 g of product. After 16 hours of pre-incubation, all species were detected in the system within 12 hours.

E. coli: Only one of the 21 samples tested was naturally contaminated with E. coli at a level of <1 in 10 g of product. Ten different samples were inoculated with E. coli at a level of 1-20 cfu/10 g. All were detected in the system in 18 hours after an overnight incubation in either TSB or lactose broth. Ten different strains of E. coli were separately inoculated into 10 vials at a level of 10-100 cfu/10 g of product. After 16 hours of pre-incubation, all species were detected in the system within 14 hours.

Staphylococcus: Three samples were positive for Staphylococcus at a level of >1 in 10 g of product. They were detected within 20 hours in the system after an overnight incubation in TSB. Eight vials were inoculated using 8 different strains of S. aureus at a level of 10-100 cfu/10 g of product. After 16 hours of pre-incubation, all were detected in the system within 14 hours.

[FIGURE 2 OMITTED]

Conclusion

Twenty nine nutraceutical products were selected for this study. They were analysed for microbial quality using the BioLumix instrument and the standard methodology. Samples were diluted so that only samples with counts above the specified level would detect in the system. The results showed that the BioLumix System was capable of quickly distinguishing samples with counts above the allowed levels from clean (below spec.) samples. All samples that contained micro-organisms above the specified level did detect in the BioLumix System, showing a high correlation between the instrument results and the standard methodology. The BioLumix instrument offers a significant reduction in time to obtain results and reduces hands-on labour owing to its automation and simplicity of use. The BioLumix System can be used to internalize microbiological testing without the requirement of a microbiologist. It simplifies testing, expedites time to results, reduces testing costs and accelerates product release.

For more information

Dr Ruth Firstenberg-Eden

President

BioLumix Inc.

3830 Packard Road, Suite 180

Ann Arbor, Michigan 48108, USA.

Tel. [phone]

[e-mail]

www.myBioLumix.com

Table I: Tested nutraceutical products.

Alfalfa Tablets                         Hoodia gordomi
Apricot Powder                            Iron Tablets
Carrageenan Powder                      Joint Advanced
Citrus Bioflavonoid              Joint Shield Advanced
Devil's Claw Powder                 Lemon Bioflavonoid
Echinacea Purpurea Powder       Multivitamin for Woman
Empty Gelatin Blue Capsules     Psyllium Husk Capsules
Empty Gelatin Red Capsules           Strawberry Powder
Fibre Tablets                      Rauwolfia vomitoria
Flaxseed Oil Capsules                Spirulina Tablets
Flax Tablets                  Vitamin: One-A-Day (Men)
Garlic Parsley Tablets         Wheat Germ Oil Capsules
Ginkgo Biloba Powder                       Whey Powder
Golden Seal                             Zinc Gluconate
Grape Skin and Blueberry