Determination of the bactericidal, fungicidal and virucidal activity in suspension tests
The suspension tests were performed according to the standards DIN EN 1040:2006 and DIN EN 1275:2006 (31,32) for bacteria and fungi and to the standard DIN EN 14476 (35) for viruses. All tests were performed under clean conditions, i.e. in presence of an organic challenge (0.3 g/ L albumine) at room temperature. As specified in the standards, the contact time was 5 min for bacteria and 15 min for yeasts. Likewise, the microorganisms suspension used ranged between 1.5 and 5 x 108 cfu/mL. Unlike described in DIN EN 1040 and DIN EN 1275 1 mL was used in the experiments instead of 10 mL. All products were tested at room temperature. Neutralization was carried out by dilution using an inactivator solution comprised of Tween 80 (30 g/L), Lecithin (3 g/L), L-histidine (1 g/L), Sodium-thiosulfate (5 g/L) and Saponin (30 g/L). The virucidal tests were carried out strictly according to DIN EN 14476. The calculation of the reduction factors was done as described in chapter 'Microbiological and statistical analysis'.
All strains were purchased at the German Collection of Microorganisms and Cell Cultures (DSMZ, Brunswick, Germany), except from the Methicillin resistant Staphylococcus aureus (MRSA), which was derived from the Culture Collection of the University of Gothenburg (CCUG). The corresponding strain code of the American Type Culture Collection (ATCC) is provided in Table 1 for information only.
Determination of the bactericidal, fungicidal and virucidal activity in surface tests
The determination of bactericidal an fungicidal activity on surfaces was performed according to DIN EN 13697 (33) and for virucidal activity according DIN EN 16777 (36). All tests using bacterial strains were executed at Rhine-Waal-University of Applied Sciences; for virucidal tests, an external lab (Dr. Brill und Dr. Steinmann Institute for Hygiene and Microbiology, Hamburg, Germany) was commissioned by the funder of this study.
All tests were performed under clean conditions, i.e. in presence of an organic challenge (0.3 g/L albumine) at room temperature. As specified in the standards, the contact time was 5 min for bacteria and 15 min for yeasts. Likewise, the microorganisms suspension used ranged between 1.5 and 5 x 108 cfu/mL. For the tests according to DIN EN 13697 P. aeruginosa, E. coli, S. aureus, E. hirae, A. brasiliensis and C. albicans were used; for the tests according to DIN EN 16777 the Modified Vaccinia Ankara virus (MVA) was used. The calculations of the reduction factors were performed as described in chapter 'Microbiological and statistical analysis'.
Determination of the antibacterial activity in laundering procedures using a laboratory washing machine
To assess the antimicrobial performance of products containing acetic acid for use in laundry detergents, a laboratory washing machine (Rotawash M228C, SDL Atlas, Rock Hill, SC, USA)) was used as described in Schages et al. (34). To simulate a normal household washing machine all quantities were downscaled adequately, i.e. a 1 L vessel was filled with 0.5 L of water in addition to the ballast load textiles, the soil ballast, the detergent and eight steel beads (to simulate the mechanics of a washing machine) as described below.
In this study, cotton (wfk 10 A, 170 g/m2, wfk testgewebe, Brüggen, Germany) was used as the ballast load. In addition to the ballast load, SBL2004 (SBL2004, wfk testgewebe, Brüggen, Germany) was used as a source of organic soil. All materials used are calculated based on the volume of water in a vessel of the laboratory washing machine:
Ballast load (100 g/vessel) consisted of 96.5 g textile ballast of standard cotton and of 3.5 g textile comprised by the SBL2004 swatches equalling approx. 1.2 g standard soil. A liquid heavy duty detergent (Ariel Actilift, Procter & Gamble, Germany) was dosed according to the detergent manufacturers’ instructions (120 mL/ 10 L) and adjusted to the volume of one vessel of the Rotawash (6 mL/0.5 L).
The duration of the wash cycle in the Rotawash was 60 min, and the temperature was adjusted to 30 °C, at a water inlet temperature of approx. 15 °C – 20 °C. In every test run five artificially contaminated biomonitors (one swatch per microorganism) are added to one vessel. In this series of experiments S. aureus, M. luteus, P. aeruginosa, E. coli and S. hominis were tested. All tests in the Rotawash were run in triplicates. The evaluation is performed as described in chapter 'Microbiological and statistical analysis'.
Microbiological and statistical analysis
The microbial count on each contaminated biomonitor was quantified by extraction with 1 mL TSB-TLH-thio (Tryptic Soy Broth with 30 g/L Tween 80, 0.3 g/L lecithin, 1 g/L histidine, 5 g/L sodium-thiosulfate) followed by investigating the colony forming units (cfu/mL) on surface culture on Tryptic Soy Agar (TSA) (Oxoid, Wesel, Germany; incubation at 37 °C for 24 h). Rotawash-tests were carried out in a 1.5 mL reaction tube (Sarstedt, Nürmbrecht, Germany) for 10 min at 15 °C and 1000 rpm in an orbital incubating shaker (Thermomix comfort, Eppendorf, Hamburg, Germany).
The colony forming units (cfu/mL) were investigated in surface culture either on TSA for bacteria (incubation at 37 °C for 24 h) or Malt Extract Agar (MEA) for C. albicans and A. brasiliensis (incubation at 30 °C for 48 h). After laundering, the microbial count on the test swatches is determined similarly. The number of colony forming units (cfu/mL) on plates was used to calculate the microbial load in the extraction liquid (cwei) (Eq. (1)):
See equation 1 in the supplementary files.
To calculate the LR, the logarithmic cfu value of the biomonitors was subtracted from the logarithmic mean of the initial microbial count of the respective biomonitors.
See equation 2 in the supplementary files.
Unless otherwise stated, the tests were performed in triplicates and statistically evaluated in the case of a non-Gaussian distribution using Students t-test, Kruskal-Wallis or in the case of a Gaussian distribution using a 2-way ANOVA.