Containing an Oxidoreductase enzyme, Mycosinate glucose Oxidase pathway (GOx), eliminates microbial growth

in a time frame of 24hours by introducing a continuous sustained release of low levels of Hydrogen Peroxide as a

product. Due to this sustained release, Mycosinate consists of anti-microbial and preservation properties, which is

advantageous not only in the purification of water but also in the preservation of milk.

The results found that 1g Mycosinate eliminated microbial growth at a rate of 0.011% in spiked e-coli deionised

water after 96hrs incubation at 37°C.

For milk samples containing 1g Mycosinate incubated at 37°C for 144hrs microbial growth was retarded by 1.25%,

increasing the shelf life of the product.

Ask yourself: Would you drink water from a dirty well or a hole in the ground, that’s contaminated with microbial

pathogens?

Unfortunately, thousands of people in the developing parts of the world do not have the luxury of choice when it

comes to hydrating their bodies. For most, their water supply is a water hole or river, where both human and ani-

mals water, wash and even excrete bodily fluids.

approximate 2.3 billion people suffering from waterborne related illnesses

annual dead count at 2.2 million with 60% infant mortality,

1 billion people lack basic necessities for sanitation.

With Mycosinate our objective was to offer people like those living in the 3rd World, a choice in the drinking water

available to them free from microbial growth, especially waterborne microorganisms such as Cryptosporidium,

Campylobacter and Pseudomonas.

Mycosinate thereby, demonstrates real potential as an alternative in the decontamination of water and preserva-

tion of milk.

Oxidoreductase Enzyme and Glucose Oxidase Reaction;

Oxidoreductase enzyme and Oxygen and glucose (honey) undergo a reaction called glucose oxidase, in which it catalyses

the oxidation of glucose in an oxidative ½ reaction to Hydrogen Peroxide and Gluconic Acid. It is this fast reaction that

enables Mycosinate to have the advantageous properties of Hydrogen Peroxide immediately within 24hrs and its anti-

bacterial disinfectant benefits.

Serial Dilutions

References

Summary

Discussion

Water Results; Colorimetric H2O2 Levels mg/l

Figure 2.0, 3.0 and 4.0 (L-R) represents the three water samples used to confer the anti-microbial properties of Mycosinate and

its Oxidoreductase enzymatic development of H2O2. Figure 2.0 is a colorimetric indication for deionised water, the results

shows Mycosinate activity to 0.023%. Further confirmation in Figure 3.0, sterile deionised water spiked with E-coli pathogen. My-

cosinate eliminated E-coli growth as far as 0.005% with 2mg/l present. Figure 4.0 Tap water, due to chlorination a purification

method already used for water treatments, its presence inhibited the Oxidoreductase enzyme activity therefore preventing the

GOx pathway and the production of H2O2. Mycosinate was shown to be in active with pre-treated waters. These similarities and

differences are distinguished further in the graphs below.

Milk Results; Colorimetric H2O2 Levels mg/l

Figure 4.0,5.0 and 6.0 (L-R), represents colorimetric results from milk samples. Figure 1.0, results from pasteurized pre-treated

milk, GOx pathway from its Oxidoreductase enzyme sustained microbial growth at 0.375% but was inactive at lower concentra-

tions. Figures 5.0 and 6.0, represents results from raw untreated milk direct from a dairy cow, enzymatic activity was present

0.2mg/l H2O2 levels 0.187% concentration. The results are a positive indication that Mycosinate can and may be used to pro-

long the shelf life of milk treated or untreated at high temperature within the 1st 24hrs of introduction. The GOx activity reaction

increases the Lactoperoxidase and its H2O2 levels naturally present in Milk, therefore it would be effective in developing coun-

tries where climatic temperatures are high and spoilage levels of milk especially upon transportation to processing plant, by re-

ducing spoilage levels and preserve milk until further preservation methods are applied. Graphs below emphasis the differences

in Mycosinate activity in the two milk sample types.

Abbreviations:

Glucose Oxidative Pathway; GOx Hydrogen Peroxide; H2O2 Codex Alimentarius Commission; CA

Food and Drugs Administration; FDA

Introduction

Results/ Findings

Conclusion

Materials & Methods

Acknowledgements

Glucose +O2 Glucose Oxidase H2O2 +D-Gluconic Acid

10-6 10-7 10-8 10-9 Figure 1.0, Represents an illustration of the serial dilutions carried

out and the plate count agar for quantitative results. Each dilution

tube comprised of 9ml of analyte to 1g of Mycosinate. Incubated

at 37°C for an initial 24 hrs. followed by additional 24hr periods.

Analytes consisted of Water; Tap water, Deionised and Sterile De-

ionised water spiked with E-coli, as-well as Milk; Pasteurised and

Raw untreated dairy milk. Agar plate counts were done with sam-

ples of sterile deionised water spiked with E-coli pathogen, in-

order to calculate cfu/ml of E-coli growth in water when introduced

to Mycosinate.

Figure 8.0,9.0 and 10.0, (L-R) represents a non-specific sterile agar media with spiked e-coli introduced at con-

centrations 1ml: 3ml sterile de-ionised water with 1 g of Oxidoreductase Enzyme (Mycosinate)/ 100ml H2O. The

image shows e-coli growth at low concentrations of 10-6 Figure 8.0 from one week incubation at 22°C, with a

bacterial count of 0227. A non-specific agar plate with a CFU count (Figure 8.0) at 171.5 x 10-6cfu/ml. Figure 9.0

represents concentrations of 10-8(Left two plates), 10-9(Right two plates). 10-8 agar counts for e-coli growth at low

levels of 0003 and 0002, a CFU count of 2.5 x 10-8cfu/ml. Where-as 10-9 for e-coli growth was counted in at

0000, therefore with a cfu count of 0 x 10-9cfu/ml i.e. no growth. Whereas 10.0 represents E-coli growth without

Mycosinate added.

Mycosinate was developed following studies into the anti-microbial mode of action of manuka honey. Its

antimicrobial effect is based on the continuous sustained release of low levels of H2O2. Mycosinate demonstrated

real potential in the decontamination of water and the preservation of milk. While the direct addition of H2O2 to food

and food products is not recommeneded by CAC or the FDA, mycosinate enzymatically generates H2O2 through the

action of GOx a widely used food and drink preservation method. For this reason it is believed that the use of

mycosinate for the purpose of decontamination or preservation will not present any regulatory issues.

Mycosinate demonstrated excellent anti-microbial efficacy against E-coli. The GOx pathway results in H2O2 production. GOx is

considered a natural preservative and is recognized by CAC (appendix 8) as an acceptable food additive

Therefore, by applying the Oxidoreductase enzymatic anti-microbial activity of Mycosinate directly or indirectly to a water sup-

ply for purification of drinking water especially in developing countries, or accompanied with Ozone treatments or chlorination

treatments in the elimination of microbial growth and improving water quality or whether it is used as a preservative in milk dur-

ing transportation purposes. Further research into this new technology derived from a natural product manuka honey, will con-

sequently benefit and improve standards of living and lifestyles to those less fortunate. As a result, this will lead to a reduction

in waterborne diseases that contribute to high death rates in particularly infant mortality.

Mycosinate, with further research can be an alternative in the purification and preservation of water and milk.

I WOULD LIKE TO THANK THE FOLLOWING PEOPLE

FOR ALL THEIR HELP THROUGH MY PROJECT;

JOHN BARRETT

JAMES BRENNAN

TOM PATTON

NOEL MORAN

CONNOR AND HARRY O MARA

DEREK RICHARDSON

MARITA MIMNAGE

PUTERI BALQIUS RAMELI

Antimicrobial and Immunostimualtory system comprising of

oxidoreductase enzyme (14-03-2012). Available at :

John.Barrett@healinghoneyinternational.com/Patent EP206894B1