2023 Volume 71 Issue 1 Pages 74-77
Propan-1,3-diol (PD) and propan-1,2-diol (propylene glycol, PG) are very similar compounds because their structures, safety data, and anti-microbial activities are almost the same. Actually, both compounds are made up of three carbon atoms and two hydroxyl groups. Regarding their safety, they do not have serious hazard data for animals, and LD50 values (in rats) of both are similar. As for the anti-microbial activity, minimum inhibitory concentration (MIC) values of both PD and PG are approximately 10% (v/v). In this study, we used the preservatives-effectiveness test (PET) to evaluate the anti-microbial activities of PD and PG, because both compounds are used in cosmetics as preservatives. The results indicated that PD was more effective as an anti-microbial agent compared with PG, and the effect of PD was marked against Escherichia coli and Pseudomonas aeruginosa. Scanning electron microscopy (SEM) images showed that the membrane of Escherichia coli was injured by PD and PG, but the damage by PD was more marked. The damage of the cell membrane may be the cause of high anti-microbial activity of PD in PET. These results suggest that PD has greater potential as a preservative, and PD should be recommended as an additive for food and medicine.
Propan-1,3-diol (PD) and propan-1,2-diol (propylene glycol, PG) are alkanediols, with three carbon atoms and two hydroxyl groups. There is a difference in the hydroxyl group position between PD and PG, but the use of both compounds is very similar in industry and cosmetics, such as antifreeze, moisturizer, and preservative. Moreover, PD and PG have similar safety data. They have no genetic toxicity and high LD50 values (14.9 mL/kg body weight (b.w.) for PD and 21.0 mL/kg b.w. for PG) in animal studies (rats).1,2) The no observed adverse effect level (NOAEL) or no observed effect level (NOEL) when administered orally (single or multiple times) is high.3,4) Also, neither compounds causes eye or skin irritation with almost no adverse event in terms of sexuality and sensitization in animals and humans.1,2) As a similarity other than additive use and safety data, their anti-microbial activities can be mentioned. The activities of PD and PG have been reported as minimum inhibitory concentration (MIC). The values of MIC against Gram-negative bacteria and Gram-positive bacteria are equivalent: MIC50 values of PD and PG are 7.0–10.2% (v/v) and 5.0–10.5% (v/v), respectively.5)
Recently, microbiological production methods have been industrialized in PD manufacture, in which PD is produced by microbial fermentation of plant-derived glucose and glycerin.6) Therefore, PD is attracting attention as an environmentally friendly material, while PG is presently manufactured by chemical synthesis.
In this study, we discovered an advantage of PD as compared with PG as a preservative additive. To evaluate the anti-microbial effect of agents, the preservatives-effectiveness test (PET) described in the Japanese Pharmacopoeia7) was performed. The results indicated that PD has greater anti-microbial activity than PG. Therefore, PD should be viewed as an effective preservative additive for use instead of PG.
PD (purity: 99.4%) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). PG (purity: >99.0%) was purchased from ADEKA Co., Ltd. (Tokyo, Japan). Boric acid was purchased from Kozakai Pharmaceutical Co., Ltd. (Tokyo, Japan). All other chemicals were of analytical grade.
StrainsStaphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and Pseudomonas aeruginosa ATCC 9027 were used in all experiments. Before inoculation, these strains were pre-incubated. Appropriate amounts of pre-incubated S. aureus, E. coli, and P. aeruginosa were taken and suspended in sterile physiological saline to 107 to 108 CFU/mL to prepare microbial suspensions.
MethodsPreservatives-Effectiveness Test (PET)PD and PG solutions were prepared as final concentrations of 16 mM (0.12 % (v/v)), 48 mM (0.36 % (v/v)), 80 mM (0.6 % (v/v)), and 160 mM (1.22 % (v/v)), in 10 mM sodium phosphate buffer, pH 8.0, or 16 mM sodium borate buffer, pH 8.0. Sodium chloride was added to adjust their osmolality. Each microbial suspension was used to inoculate the PD and PG solutions, respectively, so that the final concentration was about 105 CFU/mL. They were stored in the dark at 22.5 °C for an appropriate period. Then, 1 mL of samples was collected 7, 14, and 28 d after the start of storage. The 1 mL of samples was incubated for 3 to 5 d under 32.5 °C to calculate the number of viable bacteria in the solution, according to the pour-plate method.7) The difference between theoretical inoculate amount and each viable amount is then expressed as a Log reduction value.
In a preliminary PET experiment for Candida albicans and Aspergillus brasiliensis, both fungi were not increased in the presence of PD. Since PET criterion for fungus was that it does not increase from inoculated amount, we investigated the anti-microbial effect of PD only for bacteria in this study.
Scanning Electron Microscope (SEM)Each microorganisms was used to inoculate physiological saline with 1% PD or 1% PG. They were stored at 22.5 °C for 7 d to simulate the PET condition. These strains were also used to inoculate Mullar–Hinton II media (MHM) with 1% PD and stored at 32.5 °C for 2 d to simulate the MIC condition. Then, all samples were collected by centrifugation (3000 rpm, 10–20 min) and washed with physiological saline. Treatments with 4% glutaraldehyde for 2 h and 2% osmium tetroxide for 1.5 h were performed for fixation. After washing the cells with physiological saline, a dehydration step using ethanol and a replacement step using t-butyl alcohol were performed sequentially. Samples were freeze-dried to be powdered, and then metal vapor deposition (Ag+) was performed by magnetron sputtering.
To evaluate anti-microbial activity of PD and PG in phosphate buffer, both agents were inoculated with each microorganism and their anti-microbial activities were evaluated following the PET procedure. As shown in Fig. 1, PG showed no anti-microbial activity in any of the tested microorganisms because PG solution showed almost the same log reduction value as phosphate buffer alone. However, PD solution showed a higher log reduction value than the buffer alone; thus, it was considered that PD exhibits stronger anti-microbial activity than PG.
(A) S. aureus with PG, (B) S. aureus with PD, (C) E. coli with PG, (D) E. coli with PD, (E) P. aeruginosa with PG, (F) P. aeruginosa with PD. Bars represent the means ± S.D. (n = 3). 
, without agent; 
, PG or PD 0.12%; 
, PG or PD 0.36%; 
, PG or PD 0.6%; 
, PG or PD 1.2%.
There is a possibility of using PD and PG in ophthalmic solutions that include borate acid as a preservative agent. We, therefore, evaluated anti-microbial activity in borate buffer in the PET procedure, the same as in phosphate buffer. As shown in Fig. 2, PG solution did not show anti-microbial activity against E. coli and P. aeruginosa, but activity against S. aureus was observed in the 0.12 and 0.36% solution. Curiously, 0.6 and 1.2% PG solution showed a lower log reduction value than borate buffer without PG. However, PD solution showed higher log reduction than PG solution; thus, it was considered that PD also exhibits stronger anti-microbial activity than PG in borate buffer.
(A) S. aureus with PG, (B) S. aureus with PD, (C) E. coli with PG, (D) E. coli with PD, (E) P. aeruginosa with PG, (F) P. aeruginosa with PD. Bars represent the means ± S.D. (n = 3). , without agent; , PG or PD 0.12%; , PG or PD 0.36%; , PG or PD 0.6%; , PG or PD 1.2%.
To investigate the difference in the action of PD and PG on the bacterial membrane, SEM analysis was performed with microorganisms, which were treated with PD or PG in 1% solution. As shown in Fig. 3, S. aureus and P. aeruginosa treated with PD and PG did not show marked cell membrane destruction, while these treated E. coli cells showed clear damage in cell membranes, which were cracked or broken. Nevertheless, the PD treatment had more adverse affects on the cell membrane than PG treatment. In the above procedure, microorganisms did not obtain nutrition for 7 d from their environment because they were suspended in physical saline. It was considered that the malnutrition enhanced damage of the cell membrane; therefore, MHM was used instead of saline in PD treatment. Results of SEM for E. coli incubated in MHM as a dilution solvent are shown in Fig. 4. As expected, there was almost no damage of the cell membrane in either PD-treated or untreated bacterial cells.
PD or PG-treated: Each microorganism was treated with 1% PD or PG solution for 7 d. Untreated: Each microorganism was stored in saline for 7 d. Red arrows showed significant damages.
PD-treated: Each microorganism was treated with 1% PD solution in MHM medium for 2 d. Untreated: Each microorganism was stored in MHM for 2 d.
Generally, the MIC method is conventionally used to evaluate anti-microbial activity, and MIC values of PD and PG are almost the same. However, when we conducted this PET study with phosphate or borate buffer, we found a difference in anti-microbial activity between PD and PG in both buffers. In the PG solution, the reduction value of each bacterium was similar to phosphate buffer alone, and anti-microbial activity of PG was not noted. Conversely, the reductions of all bacteria in PD solution were clearly greater than in phosphate buffer alone, and the effect of PD was demonstrated even at a minimum concentration (0.12%). As with phosphate buffer, the anti-microbial activity of PD in borate buffer was stronger than that of PG. However, the effects of PD on E. coli and P. aeruginosa appeared at 0.12% concentration in phosphate buffer (Fig. 1), but a PD concentration of 0.36% was needed to affect those microorganisms in borate buffer (Fig. 2). It is often reported that borate ions in buffer interact with polyol.8) We therefore considered that borate ions and PD form a complex, respectively, and that higher concentration of PD is required to express the anti-microbial activity. Especially, attenuation of the PD effect in borate buffer was clear in E. coli.
In our observation of SEM, an effect of PD on the bacterial cell membrane was noted in E. coli, but damage to cell membranes of S. aureus (a Gram-positive bacterium) and P. aeruginosa (a Gram-negative bacterium) was not observed. The effect of PD on the membrane seems specific to E. coli, although PD is effective both on S. aureus and P. aeruginosa in PET (Figs. 1, 2). One possibility is that PD was incorporated into the bacteria and exhibits the anti-microbial effect by interacting with intracellular molecules. When MHM was used to simulate the MIC condition, the membrane of E. coli did not show injury. A decrease in anti-microbial activity of a silver ions was reported in the presence of medium components.9) Therefore, it was considered that the medium components and PD interact with each other, and this interaction leads to the absence of the injury.
We consider that the native anti-microbial activity of PD is higher than that of PG. However, PD might be more easily trapped by MHM components than PG for unknown reason, and, therefore, the concentration of effective PD is decreased. As the result, the apparent MIC values of PD and PG are equivalent. On the other hand, the results of PD and PG in PET were appeared as their native anti-microbial activities.
In conclusion, PD as a preservative agent is more effective than PG. Now, PG is being used as a preservative agent in medicine, food, etc., but the anti-microbial activity of PG was found to be not as effective as that of PD. In addition, safety data of PD and PG are similar and PD can be made from plants by microbial fermentation. Thus, PD is easy to use as a preservative and can be applied instead of PG.
The authors thank M. Ishikawa for laboratory assistance. This study was supported by members of Senju Pharmaceutical: T. Sekiya, A. Isowaki, H. Tokushige, T. Terai, A. Kawamura, and K. Ueda
The authors declare no conflict of interest.
