2021 Volume 27 Issue 4 Pages 681-684
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the current COVID-19 pandemic, remains infectious for hours to days on surfaces. As SARS-CoV-2 continues to infect millions of people worldwide, it is imperative that safe and effective disinfecting agents are identified to help prevent its spread. We found that white distilled vinegar and 5%, 6% acetic acid exhibited a virucidal effect against SARS-CoV-2 within a short period of contact. However, grain vinegar (4% acetic acid concentration) did not show the effect over the same contact time. The addition of 0.01% ethyl acetate enhanced the virucidal effect of the 4% acetic acid. This study is the first to indicate the virucidal effects of acetic acid and vinegar solutions on SARS-CoV-2, but we do not recommend consuming vinegar as a means of prevention or treatment.
The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a pressing threat to global health. As of March 16, 2021, SARS-CoV-2 has infected more than 119 million people worldwide, causing more than 2.64 million deathsi). The transmission of SARS-CoV-2 occurs directly through respiratory droplets and droplet nuclei/aerosols, and indirectly through contaminated environmental surfaces (Ong et al., 2020). SARS-CoV-2 remains infectious for hours or even days on contaminated surfaces that are frequently touched by people (Chin et al., 2020; van Doremalen et al., 2020). Since enveloped viruses such as SARS-CoV-2 can be inactivated by ethanol and hand washing agents, hand washing and the use of sanitizers are effective in preventing the spread of infection (Kratzel et al., 2020; Patterson et al., 2020). Additionally, it is crucial to identify safe virucidal substances capable of inactivating SARS-CoV-2 on surfaces in order to prevent the spread of infection.
Vinegar has been known for its antibacterial properties since ancient times and is still used for preserving food today (Medina et al., 2007). Vinegar not only lowers pericellular pH, but also penetrates cell membranes as an un-dissociated molecule and lowers intracellular pH (Ishii et al., 2009). Thus, vinegar has strong antibacterial properties. Vinegar was reported to effectively inactivate SARS-CoV in 2003 (Rabenau et al., 2005), and vinegar inhalation has been included as adjunctive therapy in some cases of non-severe COVID-19 (Pianta et al., 2020). Moreover, the avian influenza virus can be effectively inactivated using acetic acid, the main ingredient of vinegar (Alphin et al., 2009). However, there is no information available on the ability of vinegar to inactivate SARS-CoV-2.
In this study, we evaluated the potential virucidal effects of vinegar and acetic acid, which is the principal component of vinegar, against SARS-CoV-2 in vitro. In addition, we evaluated a candidate substance capable of improving the virucidal effect of acetic acid against SARS-CoV-2.
SARS-CoV-2 (Hu/DP/Kng/19-020 strain) stock with an infectious titer of 2 × 107 50% tissue culture infective dose (TCID50)/mL was incubated with aqueous solutions of 4%, 5%, and 6% acetic acid (Kanto Chemical Co., Inc., Tokyo, Japan), grain vinegar (GV; 4% acetic acid concentration; Mizkan Co., Ltd., Aichi, Japan), and white distilled vinegar (WDV) diluted with water (WDV4; 4% acetic acid concentration; Mizkan Co., Ltd.) at a 1:9 ratio for 1 min and 5 min each.
In addition, two concentrations of WDV diluted with water (WDV5; 5% acetic acid concentration, and WDV6; 6% acetic acid concentration) and 4% acetic acid solutions with added ethyl acetate (0.001%, 0.01%, and 0.1%; Kanto Chemical Co., Inc.) were tested in contact with SARS-CoV-2 for 1 min as described above. As a control substance, 70% ethyl alcohol and phosphate-buffered saline were tested in contact with SARS-CoV-2 for 1 min in the same manner. The reaction was stopped by diluting the mixture 100-fold with Dulbecco's modified Eagle medium (DMEM) and the infectious titers were determined by the TCID50. In brief, the mixtures were inoculated onto VeroE6-TMPRSS2 cells in 96-well plates (1 × 104 cells/well, culture volume: 100 µl/well) after ten-fold serial dilution with DMEM containing 2% FBS, and the infectious titers were determined at 72 hours post-infection by staining with 2% crystal violet solution. The TCID50 was calculated by the Behrens–Karber method.
Solution pH was measured using a pH meter (F-52; Horiba, Kyoto, Japan) equipped with a glass electrode (9615S-10D; Horiba). The ethyl acetate concentration was quantified by a gas chromatography system (GC2014; Shimadzu, Kyoto, Japan) equipped with a packed column (3.1 m × 3.2 mm, PEG-1000 25%; Shimalite 60/80 BT, Shimadzu).
As shown in Table 1, WDV5 (5% acetic acid concentration) and WDV6 (6% acetic acid concentration) showed a virucidal effect (> 3-log reduction) when in contact with the virus for 1 min. The virucidal effect was reduced in WDV4 (4% acetic acid concentration) when in contact with the virus for 1 min (> 1-log reduction) and 5 min (> 2-log reduction). GV exhibited no virucidal effect (< 1-log reduction) for either 1 min or 5 min. Since the main component of vinegar is acetic acid, we confirmed the effect of aqueous acetic acid solutions. The results showed that both the 5% and 6% aqueous acetic acid solutions exhibited strong virucidal effects when in contact with the virus for 5 min, resulting in a > 4-log reduction (below the detection limit), but the virucidal effect was lower (> 2-log reduction) when in contact for 1 min. The effect was further reduced (> 1-log reduction) when 4% aqueous acetic acid was used in contact with the virus for 5 min, and no virucidal effect (< 1-log reduction) was exhibited in the 1-min treatment.
| Solution | Acetic acid concentration in contact with the virus (w/w%) | Ethyl acetate concentration in contact with the virus (w/w%) | pH in contact with the virus | Contact time | Virus infectious titer (TCID50/ml) |
|---|---|---|---|---|---|
| PBS | – | – | – | 1 min | 3.4 ± 2.0 × 105 |
| 70% ethyl alcohol | – | – | – | 1 min | ≤6.3 ± 0.0 × 101 |
| WDV4 (4% acetic acid) | 3.6 | 0.013 | 2.72 | 1 min 5 min |
1.7 ± 0.4 × 104 5.6 ± 4.0 × 102 |
| WDV5 (5% acetic acid) | 4.5 | 0.016 | 2.70 | 1 min | 4.6 ± 4.7 × 102 |
| WDV6 (6% acetic acid) | 5.4 | 0.018 | 2.67 | 1 min | 1.5 ± 0.6 × 102 |
| GV (4% acetic acid) | 3.6 | 0.009 | 2.72 | 1 min 5 min |
1.1 ± 1.2 × 105 6.3 ± 3.2 × 104 |
| 4% acetic acid solution | 3.6 | – | 2.68 | 1 min 5 min |
8.0 ± 8.6 × 104 3.8 ± 5.2 × 103 |
| 5% acetic acid solution | 4.5 | – | 2.55 | 1 min 5 min |
2.8 ± 2.6 × 103 ≤6.3 ± 0.0 × 101 |
| 6% acetic acid solution | 5.4 | – | 2.46 | 1 min 5 min |
1.2 ± 1.6 × 103 ≤6.3 ± 0.0 × 101 |
Virus infectious titer is from three independent experiments (n = 3). Mean ± S.D.
PBS; phosphate-buffered saline, WDV; white distilled vinegar, GV; grain vinegar
Since WDV showed a stronger virucidal effect than the acetic acid solution, we focused on ethyl acetate, which is another substance besides acetic acid contained in WDV.
As shown in Tables 1 and 2, the addition of 0.01% ethyl acetate to the 4% acetic acid solution produced similar virucidal activity to WDV4 (containing 0.013% ethyl acetate). Hence, the addition of 0.01% ethyl acetate was found to enhance the virucidal activity of the 4% acetic acid solution (> 1-log reduction) However, the addition of lower or higher concentrations of 0.001% or 0.1% ethyl acetate, respectively, did not enhance the virus killing activity of the 4% acetic acid solution.
| Solution | Acetic acid concentration in contact with the virus (w/w%) | pH in contact with the virus | Contact time | Virus infectious titer (TCID50/ml) |
|---|---|---|---|---|
| 4% acetic acid solution | 3.6 | 2.68 | 1 min | 8.0 ± 8.6 × 104 |
| 4% acetic acid solution added 0.001% ethyl acetate | 3.6 | 2.68 | 1 min | 7.1 ± 9.1 × 104 |
| 4% acetic acid solution added 0.01% ethyl acetate | 3.6 | 2.68 | 1 min | 2.5 ± 2.3 × 103 |
| 4% acetic acid solution added 0.1% ethyl acetate | 3.6 | 2.69 | 1 min | 1.1 ± 0.8 × 104 |
Virus infectious titer is from three independent experiments (n = 3). Mean ± S.D.
PBS; phosphate-buffered saline
In this study, we evaluated the potential virucidal effect of vinegar and acetic acid against SARS-CoV-2, and demonstrated that WDV could inactivate SARS-CoV-2 within a short period of contact. This effect was thought to be due to acetic acid, the main component of vinegar. Since SARS-Cov2 was not inactivated for 1 hour at pH 3 (Chan et al., 2020), our results suggested that the un-dissociated acetic acid might have penetrated the virus and caused severe damage. It was somewhat surprising that, despite the virucidal effect of 4% acetic acid aqueous solution in contact with SARS-CoV2 for 5 min, GV (4% acetic acid concentration) did not show the effect over the same contact time. This suggests that some impurities contained in GV, such as trace amounts of amino acids or sugars, may protect SARS-CoV-2 from inactivation (Table 3). By contrast, WDV5 (5% acetic acid concentration) and WDV6 (6% acetic acid concentration) exerted a stronger virucidal effect while in contact with the virus for 1 min than that of the 5% and 6% acetic acid aqueous solutions. WDV, unlike GV, contains far fewer amino acids and sugars (Table 3). Therefore, this might explain why WDVs were able to maintain their virucidal effect. Next, to identify substances that enhance the virucidal effect of WDV against SARS-CoV-2, we focused on ethyl acetate, which is the second most abundant volatile component after acetic acid in vinegar. Coincidentally, the virucidal effect was enhanced at a concentration of 0.01%, which is close to the concentration usually found in WDV. However, contrary to expectations, at higher concentrations (0.1%), the virucidal effect was reduced. We have not been able to establish a definite hypothesis about this phenomenon yet. Future research should therefore focus on the mechanism of how acetic acid inactivates SARS-CoV-2 and how ethyl acetate enhances the virucidal effect. To our knowledge, this study is the first to indicate the virucidal effects of acetic acid and vinegar solutions on SARS-CoV-2. It should be noted that although this study presents acetic acid solutions and WDV as virucidal agents to inactivate SARSCoV-2 present on surfaces, we do not recommend consuming vinegar as a means of prevention or treatment.
| Acids (g/100ml) | GV | WDV |
| Acetic acid | 4.02 | 15.13 |
| Citric acid | ND | ND |
| Ester (mg/100ml) | GV | WDV |
| Ethyl acetate | 0.01 | 0.05 |
| Amino acids (mg/100ml) | GV | WDV |
| Aspartic Acid | 3.76 | 0.60 |
| Threonine | 1.96 | 0.88 |
| Serine | 2.72 | 0.79 |
| Glutamic Acid | 3.40 | 1.11 |
| Proline | 2.18 | 0.30 |
| Glycine | 1.14 | 0.53 |
| Alanine | 2.95 | 1.28 |
| Valine | 3.19 | 1.28 |
| Cysteine | 0.17 | 0.26 |
| Methionine | 0.95 | 0.26 |
| Isoleucine | 2.88 | 1.05 |
| Leucine | 7.58 | 1.77 |
| Tyrosine | 2.99 | 0.99 |
| Phenylalanine | 4.37 | 0.99 |
| Histidine | 1.29 | 0.38 |
| Lysine | 2.11 | 1.77 |
| Tryptophan | ND | ND |
| Arginine | 5.79 | 2.37 |
| Asparagine | 0.91 | 0.60 |
| Glutamine | 0.06 | 0.15 |
| Total amino acids | 50.98 | 17.10 |
| Sugars (g/100ml) | ||
| Fructose | 0.01 | ND |
| Glucose | 0.29 | ND |
| Sucrose | ND | ND |
| Maltose | ND | ND |
| Total sugars | 0.30 | ND |
ND; Not Detected
Acknowledgements The SARS-CoV-2 strain (Hu/DP/Kng /19-020 strain) was provided by the Kanagawa Prefectural Institute of Public Health.
Conflict of interest This research was funded by Mizkan Holdings, Co., Ltd. J.Y., Y.T., S.K., and M.K. are employees of Mizkan Holdings, Co., Ltd. None of the principal investigators involved in this study or their family members are shareholders in Mizkan Holdings Co., Ltd., or are company officers, directors, or advisors. C.O. and Y.M. state that they have no conflicts of interest.
