BLIS K12 CLINICAL TRIALS

1. BLIS K12 SHOWN TO INCREASE INTERFERON (the body's protection mechanism)

Elevated levels of interferon gamma in human saliva following ingestion of Streptococcus salivarius K12
Chilcott CN, Crowley L, Kulkarni V, Jack RW, McLellan AD, Tagg JR BLIS Technologies Ltd, Centre for Innovation Department of Microbiology and Immunology, University of Otago, Dunedin

INTRODUCTION
Users of the oral probiotic, Streptococcus salivarius K12 (BLIS K12 Throat Guard), reported that taking the bacterial lozenges at the onset of flu-like symptoms appeared to help prevent further development of the symptoms. Interferon gamma (IFN- ) is known to contribute to non-specific immunity against many intracellular bacteria and viruses. In previous studies it has been reported that IFN- can be detected in saliva samples during viral infections at concentrations up to 25 pg/ml (1). Research in our lab has shown that human and murine leukocyte preparations produced IFN- when exposed to S. salivarius K12 cells.

MATERIALS AND METHODS
Dosage regime
Four adult subjects took twelve BLIS K12 Throat Guard lozenges (four lozenges every two hours)
Saliva collection and preparation
One ml of un stimulated saliva was collected from the subjects before taking the lozenges and 6, 8, 10, 14 and 24 hours later. Saliva samples were frozen until processed. Ten μl of complete protease inhibitor (Roche) was added per ml of saliva. The saliva samples were treated by adding 20 μl 2.5M NaCl and 20 μl 1.5 M sodium acetate to 200 μl of saliva. The samples were incubated on ice for 30 minutes. The saliva was then centrifuged for 5 min at 10,000 rpm, and the supernatant collected for testing.
Interferon gamma ELISA assay
Interferon gamma was detected in the saliva samples using an ELISA kit (BD Biosciences). One hundred μl of saliva sample supernatant was added to each well. The ELISA assay was conducted according to the manufacturer’s protocol.

RESULTS
Interferon was not detected in the saliva samples until after 8 h. Between 22- and 139 pg of interferon gamma was detected in the 24 h samples (Fig. 1).

Fig1: Detection of interferon gamma in saliva following ingestion of Streptococcus salivarius K12

During a viral infection the virus titre has been shown to increase during the first two days. An increase in the production of interferon during this time then leads to a decrease in the viral titre. The induction of interferon gamma by ingestion of S. salivarius K12 may help in preventing further development of the symptoms, by reducing the viral titre.

ACKNOWLEDGEMENTS AND REFERENCES
Spear GT, Alves MR, Cohen MH, Bremer J, Landay AL. (2005). Relationship of HIV RNA and cytokines in saliva from HIV infected individuals. FEMS Immunol. Med. Microbiol.45(2):129-136.
Special thanks go to the participants in this study.

2. JOURNAL OF CLINICAL MICROBIOLOGY - Feb 2003

To read the complete study in the Journal of Microbiology you can visit our Clinic Website www.australianbreathclinic.com
Extracts from the Journal of Clinical Microbiology, Feb 2003, p. 558-563 Authors: CE Kazor, PM Mitchell, AM Lee, LN Stokes, WJ Loesche, FE Dewhirst, BJ Paster.

"The purpose of the study was to compare the bacteria profiles of the tongue of healthy subjects and subjects with halitosis by using culture-independent molecular methods.

Tongue scrapings were analysed from healthy subjects with no complaints of halitosis and subjects with halitosis. Those species most associated with healthy subjects were Streptococcus salivarius, Rothia mucilaginosa, and an a species of Eubacterium (strain FTB41). Streptococcus salivarius was the predominant species in healthy subjects, as it represented 12 to 40% of the total clones analysed from each healthy subject.

Overall, the predominant bacteria on the tongue of healthy subjects was different from that on the tongue of subjects with halitosis. Those species most associated with halitosis were Atopobium parvulum, Eubacterium sulci, Solobacterium moorei."

We have summarized the results in an easy to read table below.

RESULTS AND DISCUSSION
It is noteworthy that S.salivarius was by far the most predominant species detected in healthy subjects: in one subject (subject H1), S. salivarius represented more than 40% of the detectable species. In contrast, S. salivarius was detected in only one of the subjects with halitosis and was detected at very low levels.

CONCLUSION
Whilst this study was being done by Dr Loesche in USA, Professor Tagg was doing a totally independent study in Otago, New Zealand. Both microbiologists came up with the same results and same conclusions.

Streptococcus salivarius is a normal bacteria of the mouth that incubates the subject at day one of birth. In healthy subjects, normal counts of Streptococcus salivarius is obtained. However in chronically sick people or people with chronic halitosis, Streptococcus salivarius is missing.