Viro2Syl Mobile Technical University Munich Institute for Brewery

1. Subject of Analysis

The disinfectant, VirO2Syl, was presented to examine the mortality effect towards brewery yeast as well as inevitable beer vermin. It was dealt with a liquid, colorless and a slightly pungent smelling disinfectant, which according to the producer contains hydrogen peroxide and silver as active agents. Hydrogen peroxide sets oxygen free. This may irreversibly oxidize organic substances as well as vital enzymes of micro-organism cells. Silver has an oligodynamic effect, which means it even works bactericide in “traces”. The producer recommends a concentration between 1.0 – 2.0% for the disinfection of containers, pipes, pumps, equipment and surfaces. This customary solution showed slightly troubled in standard hard water.

2. Method of Analysis

2.1 Final Stage Method

2.1.1 Test Performance

The final stage test was performed, and the test pathogen suspension is adjusted to 20 million cells per ml. One ml (1ml) is taken and added to 29 ml disinfection solution. After a certain action time (1, 3, 5 min, etc.), exactly 1 drop is withdrawn and inoculated into the liquid media of the appointed test pathogen which then becomes incubated at 28°C. At the

same time, an 0-test is being done to examine the growth of the test pathogen without any noxious influence. The 0-test must show a positive result in any case.

2.1.2 Test Pathogen

As culture yeast is defined as the indicator pathogen, with a source occurring from insufficient cleaning and disinfecting, brewery yeast with lower fermentation was used as test pathogen. Furthermore, beer verminous micro-organisms were included in the tests. Experience shows that test results with yeast as test pathogen are not always transferable onto other micro-organisms. For this reason, final stage tests were done with micro-organisms at 20° and 4°C as follows:

Saccaromyces cerevisiae, var, uvarum, ssp. Carlsbergensis.

Lactobacillus brevis

Pediococcus damnosus

To test the yeast, beer wort was used as the culture medium. NBB-broth was taken for Lactobacillus brevis as the secondary culture medium, and Pediococcus damnosus was used for S-beer for incubation as an anaerobic media. The incubation time in the beer wort had a duration period of 7 days at 28°C and the anaerobic cultures had a duration period of 14 days at 28°C. The visual control for turbidity, residuum or fermentation took place daily and the final control when necessary were additionally performed under a dark field microscope with magnification of 640 times.

These micro-organisms are to be classified as follows:

a. Culture yeast Saccharomyces cerevisiae, var. uvarum, ssp. Carlsbergensis

If only one virulent test pathogen survives the disinfection process, it will multiply after a certain incubation time to the extent where the naked eye can make out the turbidity i.e., residuum. To determine at what point all pathogen are killed, the concentration, temperature and contact time were monitored daily . Consideration of the total number of pathogens used for the secondary culture medium, not the total pathogen used for the whole disinfection process were compared to the conditions under which at least 99,995% of the test pathogen were destroyed.

The final stage method evaluation shows positive with the growth of the pathogen (residuum) and negative with non-growth. Under the conditions of the final stage test, approximately 20,000 test pathogen exposed to the disinfection process, reached the secondary culture medium. No sign of growth was evident. The destruction rate was at least 99,995% (<1 survivor of 20,000).

For each concentration, a point in time was chosen when the test pathogen was still growing and the evaluation still positive. A longer contact time caused a negative result.

Under the test conditions, a customary solution was made to destroy at least 99,995% of the test pathogens within 1 to 3 minutes contact time.

This requirement is necessary as for laboratory tests, the conditions are much more favorable (homogenous pathogen distribution and shaking) than in shop use. Experience shows that in companies with biological problems, the disinfection customary solution does not meet these requirements.

3. Test Results

3.1 Surface Tension and pH-Value

The following pH and surface tension values were determined in 1,0 2,0 and 3,0- % customary solutions:

Concentration % Surface Tension mN/m pH

1 2 3

67.5 66.5 66.4

7.6 7.5 7.4

The agent contains no surface-active substances which would lower the surface tension. A lower surface tension is of advantage, for disinfection, as it allows the agent to seep in easier. Alternatively, a lower surface tension impedes rinsing of the disinfectant solution so that under any circumstances, a residue of the disinfectant may get into the product. Also surface-active solutions tend to foam which may impact the effect of CIP installations.

The pH values of customary solutions move about the neutral to slightly alkaline range. After adding 2ml of a 2% solution to 1L, beer and an immediate consumption thereafter, no changes in smell or taste of a beer could be noted, but for the sake of the customer, it must basically be prevented that disinfection residues get into the beer even if they are found to be harmless to the health of consumers.

3.2 Microbicide Effectiveness Against Culture Yeast

The final stage test results with yeast as the test pathogen are summed up. It showed that at 20°C action temperature, a concentration of under 1% is sufficient to destroy culture yeast within 3 minutes contact time yielding up to 99,995% destruction rate after 1 minute with a higher concentration. Single resistant yeast cells survived after such short contact times. Although the destruction rates shows a quick and reliable effectiveness of VirO2Syl against test pathogens, using the product with longer contact times , such as thirty minutes as opposed to fifteen minutes in lower concentrations is highly recommended.

3.3 Microbicide Effectiveness Against Lactobacillus Brevis

The destruction effectiveness against Lactobacillus Brevis at a test temperature of 20°C is to be marked as excellent. With a concentration of 0.5% and a 3 minute action time, growth was no longer to be noted. As expected, a 4°C test temperature required a higher concentration or longer action time for the disinfection success. Therefore, with lower temperatures, as far as they cannot be prevented in shop use, the customary concentration should be adjusted according to the upper value given by the producer and the action time prolonged. With a 10% addition of beer wort to the initial test solution, a deterioration of the disinfection effect was noted. Compared with yeast as test pathogen, the loss in effectiveness was lower with Lactobacillus Brevis.

3.4 Microbicide Effectiveness Against Pediococcus Damnosus

The effectiveness of VirO2Syl, against Pediococcus Damnosus was almost the same as against Lactobacillus Brevis. This applies for 20°C as well as 4°C test temperatures. Thereby, the range of application concentration stated by the producer guaranties a safe destruction of the tested beer vermin after a short contact time. In cold Pediococcus Damnosus also requires application concentrations in the upper range as recommended by the producer.

4.Summary

The disinfectant, VirO2Syl, based on hydrogen peroxide solution was tested through the final stage test in regards to its effectiveness against lower fermentation brewery yeast, as well as the obligatory beer vermin Pediococcus Damnosus and Lactobacillus Brevis. The agent contains no surface-active substances and the pH value of 1-3% customary solutions lies between 7,8 and 7,3. Corrosion tests on the equipment that were exposed within the experiments were not conducted, so there is no statement possible, however there was no evidence of any corrosive activity that were visually noted. The time test results were described graphically so that the curves reflected the threshold values where the secondary culture still showed growth. Regarding the application concentrations (1- 3%) recommended by the producer with contact times of thirty minutes. A quick destructive effect arises against all test pathogen in cold (4°C) with the effect of the agent as expected. Using a 5% concentration was recommended when contact times are lowered to fifteen minutes yielding the same results. The beer vermin were killed much faster than the culture yeast. Generally, disinfection in low temperatures should be prevented, as disinfectants in cold need higher disinfectant concentrations.